Development Of Functional Material Scaffolds for Sensing Applications 
 
by 
 
Madhumati Ramanathan 
 
 
 
 
A dissertation submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Doctor of Philosophy 
 
Auburn, Alabama 
May 14, 2010 
 
 
 
 
Keywords: Array biosensor, titanium dioxide, silica, immobilization, lysozyme, florisil 
 
 
 
 
Approved by 
 
Aleksandr L. Simonian, Chair, Professor of Materials Engineering 
Jeffrey W. Fergus, Associate Professor of Materials Engineering 
ZhongYang Cheng, Associate Professor of Materials Engineering 
Curtis G. Shannon, Professor of Chemistry 
 
 
 
 
 
 
 
 
 
 ii 
Abstract 
 
 
Developments in the field of chemical and biological sensors call for sensitive and 
selective sensing surfaces to provide consistent information about target analytes. Since 
such sensors are mostly operating in complex multi-component environments, their 
interaction with interferent can have deleterious effects on their activity. Thus, stable 
sensing surfaces form the most critical aspect of a biosensor design and their 
development requires reliable surface morphology and chemistry for allocation of 
chemical and bio-recognition elements.  
Studies in this dissertation were aimed at developing modified titanium dioxide 
nanoparticles using techniques such as silanization and cross-linking to act as scaffolds 
for stable immobilization of proteins via direct covalent bonding or encapsulation in 
silica particles during its bio-inspired lysozyme mediation. Spatially defined presence of 
protein-reporter agents, on transparent titania nanoparticles coated on glass slides, 
enabled the development of fluorimetric array biosensors for simultaneous detection of 
multiple analytes. The different approaches used to develop the functional layers were 
compared in terms of biosensor sensitivity and stability. 
Organophosphorus hydrolase (OPH) was used as the model biosensing enzyme to 
show the potential application of the above chemistries for the detection of 
organophosphate (OP) neurotoxins. Reporter pH responsive fluorophores conjugated to 
enzyme transduced the catalytic hydrolysis of OPs by OPH, into a measurable optical 
 iii 
signal. The developed sensor has potential applications for the detection of OP pesticides 
in environmental samples. As an extension to OP detection, the principle of molecular 
recognition based on thermodynamic-complex formation of fluoride ions with 
aluminum(III) octaethylporphyrin contained in a plasticized film with chromoionophore 
was exploited for the detection of organophosphofluoridates like Diisopropyl 
Fluorophosphate (structural analogue of Sarin and Soman chemical nerve agents). 
Studies also included the activation of magnesium silicate (florisil) particles with 
reactive chemicals for the development chemical sensors. Modified Nash reagent was 
utilized in the form of fluoral-P and adsorbed onto florisil micro-particles.  These pre-
activated particles were attached to the glass slide surface via tape and via dispersion in 
polydimethyl siloxane (PDMS) matrix. Fluorimetric quantification of photoluminescent 
product of formaldehyde and fluoral-P activated florisil, namely, 3,5-diacetyl-1,4-
dihydrolutidine (DDL) allowed for the detection of formaldehyde. 
 iv 
Acknowledgements 
 
 
I would like to express my deep sense of gratitude to my mentor, Prof. Aleksandr 
L. Simonian, for his constant motivation and support during my entire course of study at 
Auburn. His wisdom, guidance and encouragement have been invaluable in enabling me 
to develop the confidence and aptitude necessary to realize my goals.  
I would like to thank my committee members, Profs. Jeff. Fergus, ZhongYang 
Cheng and Curtis Shannon for their useful discussions and suggestions in my research. I 
would also like to thank Dr. Virginia Davis for agreeing to be my external reader. None 
of this work would be accomplished without the enzyme OPH, generously provided by 
Prof. James Wild and his team at Texas A&M. I would like to thank Drs. Heather 
Luckarift, Mark Meyerhoff, Grigory Vertelov and John Lock for their support, 
discussions and suggestions with various aspects of the work presented in this 
dissertation.  
I would like to thank Maj. Leamon Viveros for introducing to me the basic 
biochemistry techniques when I first joined the lab. I would additionally like to thank Dr. 
Joel Golden, for his time to help me become acquainted with Array Biosensor, during my 
initial stages of study. Ms. Lin Wang (University of Michigan, Ann Arbor) prepared the 
aluminum octaethyl porphyrin presented in this work.  
A special note of appreciation goes to Roy and Alison, for their patience with my 
requests regarding various aspects of working in the department. I would like to thank 
 v 
our entire research group ?Valber, Shankar, Sista, Ainur, Sheetal, Tony, Saroja, 
Markhabat and Sarah, for their support during my research studies.  
I am grateful for being blessed with parents who have always encouraged me to 
pursue my dreams and have been supportive of my decisions. I would like to thank my 
Sister, Jeeju and Kaavya, for their support and encouragement throughout my studies and 
in all aspects of my life. Finally, I would like to thank my husband and parents-in-law, 
for their love, understanding and support, especially over the past months, while I have 
been busy with my dissertation writing.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 vi 
Table of Contents 
 
 
Abstract ............................................................................................................................... ii 
Acknowledgements  ........................................................................................................... iv 
List Of Tables .................................................................................................................... xi 
List Of Figures .................................................................................................................. xii 
1. Motivation of Research   ..................................................................................................1 
 1.1. Introduction   ........................................................................................................1 
 1.2. Dissertation organization .....................................................................................2 
2. Chemical Sensors .............................................................................................................4 
2.1. Introduction   ........................................................................................................4 
2.2. Classification of chemical sensors  ......................................................................6 
2.2.1. Optical transducers .....................................................................................7 
2.3. Sensing elements  .................................................................................................9 
2.3.1. Ionic recognition .......................................................................................10 
2.3.2. Molecular recognition ...............................................................................11 
2.3.3. Bio-molecular recognition ........................................................................13 
2.3.3.1. Affinity biosensors ........................................................................13 
2.3.3.2. Catalytic biosensors ......................................................................15 
2.3.4. Bio-Mimetic biosensors ............................................................................16
 vii 
2.4. Integration of sensing and transducing elements: Biosensors  ..........................17 
2.4.1. Adsorption.................................................................................................17 
2.4.2. Covalent binding .......................................................................................18 
2.4.3. Cross-Linking ...........................................................................................19 
2.4.4. Encapsulation and entrapment ..................................................................19 
2.5. Nanomaterials for sensing applications  ............................................................20 
3. Current Art in Sensors for the Detection of Organophosphates ....................................22 
3.1. Introduction  .......................................................................................................22 
3.2. Toxicity of Organophosphates  ..........................................................................23 
3.3. Need for detection  .............................................................................................25 
3.4. Detection of Organophosphates  ........................................................................29 
3.4.1. Conventional analytical techniques ..........................................................29 
3.4.2. Inhibition based biosensors .......................................................................30 
3.5. Biosensing Element: Direct detection using Organophosphorus Hydrolase  ....33 
3.6. Multi-Analyte Sensing Platform: Array Biosensor............................................35 
4. Array Biosensor Based on Enzyme Kinetics Monitoring By Fluorescence 
spectroscopy: Application for Neurotoxins Detection .......................................................37 
4.1. Introduction  .......................................................................................................37 
4.2. Materials and Methods  ......................................................................................38 
4.2.1. Chemicals and reagents ............................................................................38 
4.2.2. Preparation of glass slides  ........................................................................39 
4.2.3. Characterization of titanium dioxide nanoparticles  .................................43 
4.2.4. Sensing principle  ......................................................................................43 
 viii 
4.2.5. Experimental set-up ..................................................................................44 
4.3. Results and Discussions  ....................................................................................46 
4.3.1. Characterization of the metal oxide nanoparticles ....................................46 
4.3.2. pH response ...............................................................................................53 
4.3.3. Enzymatic reaction....................................................................................54 
4.3.4. Specificity of detection .............................................................................57 
4.3.5. Stability of the immobilized enzyme ........................................................59 
4.4. Conclusions  .......................................................................................................60 
5. Lysozyme Mediated Formation of Protein-Silica Nanocomposites for Biosensing 
Applications .......................................................................................................................62 
5.1. Introduction  .......................................................................................................62 
5.2. Materials and Methods  ......................................................................................64 
5.2.1. Chemicals and Reagents  ..........................................................................64 
5.2.2. Instrumentation .........................................................................................65 
5.2.3. Preparation of titanium dioxide coated glass slides ..................................65 
5.2.4. Functionalization of pretreated glass slides for lysozyme immobilization ......65 
5.2.5. Formation of OPH encapsulated silica particles on pretreated glass slides67 
5.2.6. Microscopic characterization of silica particles ........................................67 
5.2.7. Enzyme assays for OPH activity...............................................................68 
5.2.7. OPH activity assays on Array biosensors .................................................69 
5.3. Results and Discussions  ....................................................................................69 
5.3.1. Formation of silica particles for OPH encapsulation ................................69 
5.3.2. Covalent attachment of lysozyme for silica formation .............................75 
 ix 
5.3.3. Encapsulation of fluorescent OPH for studies on array biosensor ...........77 
5.4. Conclusions  .......................................................................................................80 
6. Monitoring of Diisopropyl Fluorophosphate Hydrolysis by Fluoride Selective 
Polymeric Films Using Absorbance Spectroscopy ............................................................81 
6.1. Introduction  .......................................................................................................81 
6.2. Materials and Methods  ......................................................................................85 
6.2.1. Chemicals and Reagents  ..........................................................................85 
6.2.2. Preparation of aluminum octaethyl porphyrin (Al[OEP]) ........................86 
6.2.3. Preparation of (Al[OEP])-ETH 7075 cocktail ..........................................86 
6.2.4. Preparation of slide coated with polymerized film ...................................86 
6.2.5. Instrumentation .........................................................................................87 
6.2.6. Procedure ..................................................................................................88 
6.3. Results and Discussions  ....................................................................................89 
6.4. Conclusions  .......................................................................................................92 
7. Formaldehyde Detection Using Activated Magnesium Silicate Particles .....................94 
7.1. Introduction  .......................................................................................................94 
7.2. Materials and Methods  ......................................................................................96 
7.2.1. Chemicals and Reagents  ..........................................................................96 
7.2.2. Florisil treatment with fluoral-P ...............................................................96 
7.2.3. Immobilization of activated florisil on glass slide ....................................97 
7.2.4. Instrumentation .........................................................................................98 
7.2.5. Sensor characterization .............................................................................99 
7.3. Results and Discussions  ..................................................................................100 
 x 
7.3.1. Formaldehyde detection using activated magnesium silicate particles ..100 
7.3.2. Activation of particles .............................................................................103 
7.3.3. Response curve .......................................................................................105 
7.4. Conclusions  .....................................................................................................110 
8. Overall Conclusions .....................................................................................................111 
9. Future work ..................................................................................................................115 
9.1. Bioinspired mediation of titanium dioxide for coating on glass slides  ...........115 
9.2. Enzyme stabilization at extreme pH conditions ...............................................117 
References   ......................................................................................................................120 
 
 xi 
List of Tables 
 
 
Table 2.1 Classification of evanescent wave sensors   ........................................................9 
Table 2.2 List of amino acids with ionizable side chains ..................................................18 
Table 3.1 List of Organophosphates ..................................................................................27 
Table 3.2 Some of the conventional analytical techniques to determine OP nerve agents 
????????? ?????????????????????????? 30 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 xii 
List of Figures 
 
 
Figure 2.1 Schematic representation of a chemical sensor  .................................................5 
Figure 2.2 Classification of chemical sensors based on mode of transduction ...................7 
Figure 2.3 Modes of sensing element interaction with analyte .........................................10 
Figure 2.4 Schematic mechanism of pH optode ................................................................12 
Figure 2.5 Schematic representation of a cross-linker .......................................................19 
Figure 3.1 Schematic representation of the general chemical structure of an 
organophosphorus compound ............................................................................................22 
 
Figure 3.2 Phosphorylation of AChE in the presence of OP .............................................24 
 
Figure 3.3 Annual agricultural use in million pounds for some of the OPs in US ............26 
 
Figure 3.4 Chemical structures of OP chemical nerve agents ...........................................28 
 
Figure 3.5 Schematic representation of the principle behind inhibition based OP 
detection?? ?????????????????????????????.31  
 
Figure 3.6 Molecular structure of OPH .............................................................................34 
 
Figure 4.1 OPH immobilization chemistry on titanium dioxide coated glass slide. R and 
R1 represent neutravidin and OPH/BSA respectively ........................................................42 
 
Figure 4.2 Schematic representation of paraoxon hydrolysis ............................................44 
 
Figure 4.3 Schematic representation of array biosensor development. A: Principle of 
protein patterning on glass slide. B: Photograph of the incubation holder used for 
patterning biomolecules with fluorophores. C: CCD image of array of biosensors 
developed on glass slide. D: Schematic representation of array biosensor .......................45
 
 
Figure 4.4 Transmission electron microscopy image of titanium dioxide particles of the 
paste within 6 months of being purchased from the manufacturer ....................................47 
 xiii 
 
Figure 4.5 X-ray diffraction studies of glass slide coated with titanium dioxide 
nanoparticles ......................................................................................................................48 
 
Figure 4.6 SEM image of plain glass slide (A) and glass slide coated with titanium 
dioxide nanoparticles .........................................................................................................50 
 
Figure 4.7 The changes in net fluorescent intensity of the working and reference/control 
spots n a particular channel of the glass slide with pH for 10 mM Ches buffer solution ..54 
 
Figure 4.8 Response of reference and working spots to 0.5 mM paraoxon, 1 mM Ches 
buffer, pH 8.2. Images were taken for every 5 s ................................................................55 
 
Figure 4.9 Response curve of sensor to paraoxon introduction. The standard errors were 
calculated for three samples introduced for every concentration. The inset shows the 
linear region of response curve ranging from 2.5 M to 0.1 mM paraoxon .....................57 
 
Figure 4.10 Response curves of paraoxon, parathion and DFP for concentrations ranging 
from 2.5 M to 0.8 mM prepared in 1 mM Ches, pH 8.2-8.3 ...........................................59 
 
Figure 4.11 Bar graph showing the percentage of retained signal value relative to the 
sensitivity present on day 1 of sensor preparation. ............................................................60 
 
Figure 5.1 Schematic representation of the chemistry used for lysozyme immobilization66 
 
Figure 5.2 Schematic representation of lysozyme mediated formation of silica particles  
and its encapsulation of OPH on glass slide coated with titanium dioxide 
nanoparticles???.... ......................................................................................................68 
 
Figure 5.3 Hydrolysis of paraoxon to para-nitrophenol by OPH encapsulated in silica 
using TMOS (?) and TEOS (?) precursors ......................................................................71 
 
Figure 5.4 Leaching test: Change in absorbance with time for a paraoxon solution after 
incubation with OPH encapsulated silica slides using TMOS (?) and TEOS (?) .............72 
 
 
Figure 5.5 SEM and optical images of silica particles using TEOS and TMOS with 
schematics. SEM images of Plain (a) and Pretreated (b) slides, SEM images of silica 
particles formed using TMOS precursor on plain (c) and pretreated (d) slides, SEM 
images of silica particles formed using TEOS precursor on plain (e) and pretreated (f) 
respectively. Optical microscopic images of OPH encapsulated silica particles formed in 
solution mediated by lysozyme using TEOS (g) and TMOS (h) precursors respectively .74 
Figure 5.6 Activity of OPH ecapsulated in silica particles with covalently immobilized 
lysozyme ............................................................................................................................76 
 
 xiv 
Figure 5.7 Response of pretreated glass slides with patterned encapsulation of OPH-CNF 
on array biosensor. A) Array of spots formed on glass slides B) pH response of working 
(OPH-W) and control (BSA-R) spots C) Paraoxon response of sensors over a period of 
nearly 2 months?.. ???? ???????????????????????7 9 
 
Figure 6.1 Schematic representation of the principle of DFP hydrolysis by OPH and 
subsequent detection by optical sensing film.....................................................................84 
 
Figure 6.2 Schematic of the experimental set-up of the sensing film in cuvette ...............87 
 
Figure 6.3 Absorbance spectra of film on quartz slide treated with various concentrations 
of DFP hydrolyzed by OPH ...............................................................................................90 
 
Figure 6.4 Response curve of the % change in absorbance at 538nm for various 
concentrations of hydrolyzed DFP referenced against non-hydrolyzed DFP. The data was 
fit for linear regression (y = 0.4086 x + 6.465, R2 = 0.966). The standard error was 
calculated for n=3 (3 different pairs of reference and working sensors). ..........................91 
 
Figure 7.1 Schematic representation of the instrumental set-up. A: Fluorimeter with the 
glass slide placed at an angle of 45o with respect to the incoming light source and 
outgoing fluorescence to the detector. B: Sensor involving cut glass slide with activated 
florisil particles attached using a double-sided adhesive tape or PDMS. The diameter of 
the area enclosing particles was 5 mm. C: Schematic of the gas phase sensor with a 
window made on the cuvette to place the slide with activated florisil particles attached via 
tape facing the solution inside cuvette ...............................................................................99 
 
Figure 7.2 Top: Original Nash reaction. Bottom: Hantzsch Reaction with fluoral p ......101 
 
Figure 7.3 Fluorescent and colorimetric response of fluoral P treated florisil particles. ?a? 
and ?b? correspond to untreated and activated  florisil particles under visible light 
respectively, ?c? and ?d? correspond to untreated and activated florisil particles 
respectively, when treated with formaldehyde under UV light  (350 nm). The figure on 
the right shows the calorimetric changes in activated florisil particles when treated with 
different concentrations of formaldehyde solutions ........................................................103 
 
Figure 7.4 Effect of florisil treatment with fluoral P. Time based intensities intensity of 
DDL product obtained on reaction between HCHO and fluoral P before its treatment with 
florisil and the reaction between HCHO and filterant of fluoral P solution obtained after 
its treatment with florisil particles. The inset shows the reduced scale of intensity for the 
treated fluoral P solution (FP activity after florisil treatment. .........................................104 
 
Figure 7.5 Influence of formaldehyde concentration on the fluorescence emission 
wavelength. The inset graph shows the variation of peak emission wavelength and 
intensity as a function of formaldehyde concentration. ...................................................106 
 
 xv 
Figure 7.6 Response curve obtained for the different concentrations of formaldehyde 
when treated with activated fluoral P when immobilized on glass slide using an adhesive 
tape and PDMS. Inset graph shows the linear range of response for either florisil support 
with linear regression plots. For tape: y= 0.9894 x + 6.8908, R = 0.983, for PDMS y = 
0.8520 x + 20.8076, R = 0.9643. .....................................................................................108 
 
Figure 7.7 Variation in the response time (?) and slope (?) of DDL fluorescence for 
different concentrations of formaldehyde for gas phase detection. The inset shows the 
linear range of response for formaldehyde of concentrations from 0.025 mM to 1 mM. 
(y=0.1108x + 9.1625, R = 0.9934). .................................................................................109 
 
Figure 9.1 Titanium dioxide deposition on glass slides via lysozyme mediation. 8L-100: 8 
alternative layers of lysozyme  (100 mg/ml) and titanium dioxide precursor. 8L-10: 8 
alternative layers of lysozyme  (10 mg/ml) and titanium dioxide precursor. 4L-100: 4 
alternative layers of lysozyme  (100 mg/ml) and titanium dioxide precursor. 4L-10: 4 
alternative layers of lysozyme  (10 mg/ml) and titanium dioxide precursor???? ..1 16 
 
Figure 9.2 Top figure shows the schematic of the experimental set-up for single pot 
reaction. The graph shows the response of the polymer film to different concentrations of 
DFP ..................................................................................................................................118 
 
 1 
1. MOTIVATION OF RESEARCH 
 
1.1. Introduction  
Employment of suitable chemistries and materials for the development of stable 
sensing surfaces is critical in a biosensor design as the recognition elements operate in 
complex environmental matrices that have harmful influence on their stability and 
sensitivity. In this study, in-situ functional material scaffolds for sensing elements using a 
combination of surface chemistries and morphologies for applications in biosensors and 
chemical sensors were developed. Particularly, the feasibility of titanium dioxide 
nanoparticles and magnesium silicate particles as scaffold surfaces with specific potential 
for signal amplification to enhance sensitivity were studied. 
Focus of my earlier studies involved the modification of array biosensor platform 
(a fluorimetric platform developed by the Naval Research Laboratories (NRL, 
Washington D.C.) for affinity-based assays), to allow for kinetic studies using enzymes 
as biorecognition elements. One of the main problems encountered was to implement an 
appropriate surface chemistry for enzyme immobilization on the planar waveguide of the 
array platform. Although preliminary results with aminosilane chemistry for OPH 
immobilization with application for neurotoxin detection were promising, the sensor 
showed poor reproducibility and relatively low sensitivity. It should be noted here that 
right choice of material surfaces and its appropriate functionalization for biomolecular 
 2 
immobilization is critical to the performance of biosensors, especially those involving 
enzyme kinetics, as they will otherwise require constant recalibration before detection, 
attributed to the loss of enzymatic activity over time, particularly when rebuilt or reused.
The goal of this research is to address the above issues by designing a 
combination of surface chemistry and morphology that act scaffold materials for stable 
immobilization with reporter structures, such that the final sensor is sensitive, specific, 
selective, reusable and portable. Extension of the methodology to pattern the 
biomolecular recognition elements on the material scaffolds to form an array biosensor 
has the potential for simultaneous detection of multiple analytes. The broader impacts of 
this research will include the understanding, development and application of 
biocompatible nanoparticles as smart material surfaces for biosensors attributed to their 
multifunctional role of providing a high surface area, chemically stable, transparent wave 
guiding support for protein immobilization.  
Specific use of titanium dioxide nanoparticles, polydimethylsiloxane and 
magnesium silicate particles (florisil) to act as a scaffold for chemical and biosensing of 
chemicals like Organophosphate neurotoxins and formaldehyde respectively is studied in 
this dissertation.  
1.2. Dissertation Organization 
The current chapter presents the motivation of research in the development of 
materials and their functionalization for the detection of chemical like Organophosphates 
and formaldehyde along with an overview of the dissertation. An introduction on 
concepts of sensing with the techniques used for immobilization of biomolecules on to 
various surfaces precedes literature review on organophosphates in Chapter 3.  Chapter 4 
 3 
reports on the development of an array biosensor using biocompatible titanium dioxide 
nanoparticles functionalized with an enzyme for the simultaneous detection of 
organophosphate neurotoxins. Chapter 5 details the development of an alternative 
strategy of OPH immobilization via encapsulation in lysozyme mediated silica particles 
on titanium dioxide coated glass slides. Chapter 6 discusses on the development of a 
biosensor based on the selective detection of fluoride ions resulting from 
organophosphofluoridate hydrolysis. Chapter 7 details on the development of chemical 
sensors based on the activation of magnesium silicate particles for formaldehyde 
detection. Chapter 8 summarizes the conclusions followed by the future work described 
in chapter 9. The references are provided after future work.  
 4 
2. CHEMICAL SENSORS 
 
2.1. Introduction 
Birth of chemical sensors and biosensors as alternative analytical tools can be 
largely attributed to the problems facing the analytical procedures such as the 
requirement of skilled personnel, sample pretreatment and conditioning; and bulky and 
expensive instrumentation required to determine the components of a substance termed as 
an ?analyte?. According to the International Union of Pure and Applied Chemistry 
(IUPAC) ?A chemical sensor is a device that transforms chemical information, ranging 
from the concentration of a specific sample component to total composition analysis, into 
an analytically useful signal. The chemical information, mentioned above, may originate 
from a chemical reaction of the analyte or from a physical property of the system 
investigated? [1]. Similarly, ?A biosensor is a device that uses specific biochemical 
reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole 
cells to detect chemical compounds usually by electrical, thermal or optical signals? [1, 
2]. Hence, chemical and biosensors are comprised of a receptor that interacts with the 
analyte of interest and a transducer that converts the receptor-analyte interaction into a 
measurable signal in real time, whose magnitude is proportional to the concentration of 
specific analyte. Figure 2.1 shows the schematic representation of a chemical/biosensor.  
 5 
 
 
 
 
Figure 2.1: Schematic representation of a chemical sensor 
 
The first chemical sensor, from a commercial viewpoint was a glass electrode, 
developed by Arnold Beckman in 1932 [3].  Nearly three decades later, Clark and Lyons 
built the first biosensor using glucose oxidase for glucose measurement [4]. Ever since, 
there has been tremendous progress in the field of chemical sensors and biosensors. With 
the technological advancements and integration of electronic miniaturization and 
nano/bio/material science and engineering; and constant need for better sensitivity, 
portability and ever increasing list of target analytes, the world market is projected to 
surpass ~ $ 11.5 billion for chemical sensors [5] by 2010 and ~ $6.1 billion for biosensors 
 6 
[6] by 2012. Chemical sensors and biosensors find applications chiefly in areas of health 
care, control of industrial processes, for environmental monitoring and homeland 
security. However, medical and environmental markets continue to remain the forefront 
users of biosensors. Biosensors though treated as a separate field, are generally accepted 
as a subclass of chemical sensors, in that the receptor is a biomolecular recognition 
element. Selectivity, sensitivity, accuracy, response times, recovery times and working 
lifetimes are some of the factors that affect the performance of chemical sensors and 
biosensors. 
2.2. Classification of chemical sensors 
Chemical sensors and biosensors are generally classified according to the type of 
transducer and sensing element employed to measure a particular signal parameter. 
Figures 2.2 shows the chemical sensor classification based on the transducer employed 
for detection. Other classifications include those based on the mode of delivery of the 
sample analyte as noted by Kissinger as single  use continuous and intermittent sensors 
[7]. Continuous/flow mode results in uninterrupted measurement of analyte while in the 
intermittent or batch mode, the analyte in introduced in the flow stream of carrier and 
measurement is done at regular intervals. Though the classification was originally meant 
for amperometric biosensors, it is also applicable to other transduction modes. Chemical 
sensors may also be classified according to the fundamental type of chemical recognition 
process [8]. Every transducer or recognition element has its advantages and 
disadvantages. For example, optical sensors normally do not require any reference unlike 
electrochemical transducers, however, electrochemical transducers are easy to 
miniaturize. Chemical recognition elements have better stability while the biomolecular 
 7 
receptors have better specificity and selectivity. Hence, the mode of recognition and 
detection employed is primarily governed by the analyte of interest and the demands of 
application.  
 
 
 
Figure 2.2: Classification of chemical sensors based on mode of transduction 
 
2.2.1. Optical transducers 
The sensors developed in this study use optical based transduction methods for 
detection. Here, a change in an optical property such as absorbance, fluorescence, 
luminescence and reflection that occurs upon the interaction of sensing layer with analyte 
forms the basis of detection. Absorption of photon by a molecule occurs when the 
difference in energy levels of the molecule is equal to the energy of the incident photon. 
 8 
This results in the excitation of the electron from the ground state to an excited state at a 
specific wavelength. The excited electron may return to the ground state giving out its 
excess energy in the form of fluorescence or luminescence. Luminescence occurring due 
to a chemical reaction is termed chemiluminescence and that by a living organism is 
termed bioluminescence.  
Reflectance based measurements involve studying the analyte on an optical 
surface generally using internal reflectance spectroscopy. Light striking the interface of 
two medium of refractive index n1 and n2 (n1>n2) undergoes total internal reflection when 
the angle of incidence in the denser medium is greater than the critical angle, which is 
defined by  
1
2s i n nnc   
Total internal reflection generates an evanescent wave field in the rarer medium 
that decays exponentially with distance.  Table 2.1 shows the optical sensing 
methodologies developed using the principle of evanescent waves. Excellent reviews on 
these methologies have been provided in the following references [9-11]. 
 9 
 
 
Table 2.1: Classification of evanescent wave sensors 
 
2.3. Sensing  elements  
The mode of sensing element and analyte interaction can be ionic, molecular or 
biological [12]. Figure 2.3 shows the various modes of sensing elements that can be 
employed for detection based on the type of interaction. The nature of interaction 
determines the sensor specificity and selectivity.  For example, interactions based on 
ionic recognition are generally selective, in that the sensing element interacts more 
selectively to one ion in a matrix sample. Biological interactions are more specific in 
nature.  
 10 
 
 
Figure 2.3: Modes of sensing element interaction with analyte [12]. 
 
2.3.1. Ionic Recognition 
Ionic recognition involves the selective detection of ions (guest) by a sensing 
membrane (host). This mode generally uses ion-selective electrodes [13], modified and 
screen printed electrodes [14-16]. Ion selective electrodes are mechanically stable, non-
porous, water insoluble membrane based devices that allow the permeability of analyte 
ions to produce a concentration dependent potential or current. Consequently, the signal 
 11 
parameter measured is electrochemical in nature. Selectivity may be enhanced in these 
electrode systems by introducing polymers either on the surface or matrix of electrode. 
  
2.3.2. Molecular Recognition 
 
Molecular recognition may be used as a principle of detection using chemical 
recognition agents and using spectroscopic analysis. In the case of chemical recognition 
elements, the thermodynamic feasibility, molecular size of analyte and rate of reacting 
species can form the basis of selectivity [12]. If the equilibrium constant (K) of a reaction 
for a specific analyte is high for one analyte-ligand complex when compared to others, 
selectivity is realized. This can be represented by the following equation. 
x
x
x
BA
ABKW here
ABxBA
]][[
][,
 
The signal parameter analyzed is usually optical or electrochemical. For example, a pH 
responsive indicator, when incorporated in a PVC membrane containing sodium 
tetraphenyl borate (Na+TPB-) to maintain electro-neutrality may change its absorbance  
(red/blue) over a certain pH range that can be measured using a spectrometer [12]. This 
forms the principle behind the development of many optodes or optical electrodes 
(Figure 2.4). An ideal optical sensor developed using this principle should have a 
material (ligand) that is sensitive and selective to the analyte, have good photo-stability, 
not leach in the reagent solution, have quick response times and be reversible in a 
reasonable time-frame, in its reaction to enable reusability.  
 
 12 
 
 
Figure 2.4: Schematic mechanism of pH optode [12] 
 
For sensing elements exhibiting same thermodynamic behavior towards analytes 
?A? and ?B?, selectivity towards ?A? or ?B? may be achieved if the kinetics of 
interaction is different. This is generally the case with enzyme based reactions, which 
will be detailed in the later sections. Molecular size as the basis of selectivity is achieved 
for analytes that have different sizes. Here, the host?s shape (cup-like or cavity) may play 
an important role in sieving out the interferent from analyte in a matrix sample for 
detection.   
Molecular recognition based on spectroscopic analysis exploit the principle of 
internal energy of molecules, which may be expressed as a sum of kinetic, electronic, 
vibrational, rotational and nuclear energies that are characteristic of particular regions of 
the electromagnetic spectrum. Methodologies employing this principle are shown in 
Figure 2.3.  
 
 
 13 
 
2.3.3. Biomolecular recognition 
Exploitation of biomolecular elements involved in biochemical processes 
occurring in living organisms for sensing applications generally provide greater 
selectivity and specificity in comparison to chemical recognition elements. These 
chemical sensors are known as biosensors and are regarded as sub-set of chemical 
sensors or as a separate class of their own. Depending on the type of biochemical 
interaction between the biomolecular element and the analyte, the sensors might be 
classified as affinity biosensors or catalytic biosensors. Affinity based biosensors employ 
antibodies, nucleic acids, receptors or molecularly imprinted polymers for detection 
purposes while catalytic biosensors employ enzymes, cells/tissues, microorganisms or 
organelles as recognition elements. 
 
2.3.3.1.  Affinity biosensors 
Antibodies 
Antibodies are proteins and can be developed against almost any antigen 
(target).Antibody based sensors are also called immunosensors as an organism develops 
antibodies in response to an attack on their immune system by antigens. The antibodies 
bind to the antigens and remove them. If ?Ab? is antibody and ?Ag? is antigen, then their 
binding can be represented by  
AgAbAgAb  
where, the affinity constant is given by  
 14 
]][[ AbAg AgAbK
 
This value of K is generally around 106 [12]. They are characterized by their 
selectivity, ultra-sensitivity and high affinity for binding with target antigens. 
Nucleic Acids 
In addition to antibodies, nucleic acids are affinity type biorecognition elements 
and hence fall under the category of affinity based systems. Their base-pairing 
characteristics attribute to their selectivity and specificity. DNA (Deoxyribonucleic acid) 
is the most commonly used nucleic acid for this purpose.  Recently, DNA based sensors 
have been termed ?genosensors? [17, 18]. With the advances in genetic engineering, they 
can be used in combination with other biomolecular elements  like enzymes and 
antibodies, to give rise to a broad range of biosensors for genetic testing, detection of 
biological agents and diagnosis of human diseases [19-21].  
Receptors 
Receptors are a special type of protein complexes with carbohydrates/ lipids that 
interact specifically with biological and chemical substances. The chemical substances 
are known as messengers, transmitters or ligands. The receptor-ligand interaction triggers 
a series of biochemical reactions, leading to an amplified physiological response in 
receptor bearing organism, which can be ion-channel opening, production of second 
messenger systems or activation of enzymes. They are selective and have affinity for a 
range of structurally related compounds. For example, phage displayed libraries can be 
created to contain specific receptors for targets and integrated with transducers to create 
biosensors [22].  
 15 
2.3.3.2. Catalytic biosensors 
In catalytic biosensors, the analyte, which is in a non-detectible form, on 
interaction with the biomolecules, undergoes a catalyzed chemical conversion to a form 
that can be detected by a transducer.  The steady state concentration of the species 
formed, lost or inhibited forms the principle of detection. Catalytic biosensors use 
enzymes, microbes, sub-cellular organelles, plant tissues or animal tissues as the 
biorecognition elements. 
Enzymes 
Enzymes are proteins that are characterized by their catalytic power, specificity 
and regulation. In biosensors, enzymes react with analytes of interest generally termed as 
substrates and catalyze reactions to form products that may be detected either directly or 
indirectly.  They are selective in both their interaction and rate of catalytic reaction. The 
catalytic activity of enzymes is attributed to its ability to reduce the energy of activation 
required for a reaction to take place. Besides the formation of detectable products, some 
enzyme-analyte interactions, result in the inhibition of their activity. The inhibition may 
be competitive or non-competitive, depending on the matrix of substrate. Accordingly, 
measuring the rate of species formed (product), lost (substrate) or inhibited (enzyme) 
from an enzyme catalyzed reaction forms the basis of detection. Most of the enzymes that 
are commonly used in the design of biosensors are oxidoreductases and hydrolases. 
Enzymes may be used in their wild form or may be purified and engineered to specific 
targets to improve the efficiency of the biosensor. 
 
 
 16 
Cells & Tissues  
Enzymes are generally isolated from cells and tissues and hence, are purified to 
provide selectivity in detection. However, in the absence of their natural environment, 
enzymes are known to degrade over time. Instead, cells and tissues containing a variety 
of enzymes may be utilized directly by their immobilization on transducer surfaces to 
develop biosensors. These biosensors have a number of advantages, in that; they are less 
expensive than using purified enzymes. As mentioned above, the enzymes are more 
stable in their natural environment. The major drawback of such biosensors is the loss of 
analyte selectivity, especially in complex matrices. 
Microbes  
Microbial biosensors use microorganisms like bacteria as biorecognition 
elements. The catalytic biorecognition is based on the biological processes such as 
consumption of metabolites, production of organic compounds or changes in respiration 
activity and their detection using appropriate transducers. For example, an ammonia 
biosensor can be constructed using nitrifying bacteria [23].  
2.3.4. Bio-mimetic sensors 
They form a separate class of sensors wherein the recognition elements are 
artificial synthetic systems that mimic the functioning of biological systems.  The 
artificial systems can mimic the function of biocatalyst, affinity receptors or nucleic 
acids. When considerable chemical modifications of the surface of working electrodes to 
enable electrocatalytic properties for voltametric and amperometric sensors is produced, 
artificial biocatalytic sensors are developed. The use of molecularly imprinted polymers 
[24], wherein the print molecule with functional monomers may be copolymerized to 
 17 
produce a groove in the polymer; can act as site mimics to play the role of affinity based 
biorecognition materials. In the case of nucleic acids, their synthetic counterparts are 
known as aptamers. The use of such artificial systems has several advantages. For 
example, they are easy to manipulate chemically, unlike biological systems, they are 
more stable and they do not require biological systems for their production, as in the case 
of antibodies. 
2.4. Integration of sensing and transducing elements: Biosensors 
The performance of a chemical sensor is largely governed by the integration 
methodology used for the sensing and transducing elements. In the case of biosensors, 
biological elements are immobilized onto various insoluble supports like glass, metal 
oxide particles, polystyrene etc. to stabilize the biomolecules and enable sensor 
regeneration. This can be achieved using different techniques [25-27] like adsorption, 
covalent binding, entrapment or encapsulation.  
2.4.1. Adsorption  
Adsorption is one of the earliest, simplest, fastest and most inexpensive technique 
used for the immobilization of proteins onto a support. Here, the protein molecules in 
solution, when in contact with the support, diffuse rapidly from the bulk liquid to the 
surface of contact, thereby becoming reversibly or irreversibly adsorbed. Adsorption 
takes place when interactions due to van der Waals forces, hydrophobic interactions, 
hydrogen bonding or those of ionic nature between the proteins and contact surface are 
involved. Some of the factors that affect the protein adsorption are pH, temperature, ionic 
strength and the nature of substrate.  
 
 18 
2.4.2. Covalent binding 
Nucleophillic attack at the activated functional group of the support material 
forms the basis of covalent bonding of proteins. Here the ionizable groups of amino acids 
in their unprotonated forms act as powerful nucleophiles. Among the 20 naturally 
occurring amino acids, only 7 amino acids have ionizable side chains, making them 
suitable for conjugation and modification purposes. These are listed in Table 2.2. 
 
 
   
Table 2.2: List of amino acids with ionizable side chains 
 
The relative order of nucleophilicity in proteins is shown below [27].  
R-S-  > R-NH2  > R-COO-  = R-O- 
Thus, sulfyhydryls of cysteine are the strongest nucleophiles followed by amines 
from the lysines, histidines, arginines, the ? and N-terminals of a protein. The least potent 
nucleophiles are the carboxylate groups of glutamic and aspartic acids and phenolate of 
the tyrosine. Those amino acids, which are not essential for the functionality of a protein 
(such as catalysis in the case of enzymes and antigen binding in the case of antibodies) 
 19 
can be covalently bonded to support materials using various coupling reactions. Some of 
the reactions include diazotization, amide (peptide) bond formation, alkylation 
(arylation), Schiff?s base formation, Ugi reaction, amidination reactions, thiol-disulfide 
reactions, mercury-enzyme interactions and ?-irradiation induced coupling [25].  
2.4.3. Cross-linking 
Cross-linking techniques use cross-linkers to immobilize proteins onto various 
surfaces.  A cross linker is a molecule with a center spacer arm and reactive groups at 
either ends. This is shown in Figure 2.5. Depending on the type of functional groups 
present on ends, cross-linkers may be either homo-bifunctional or hetero-bifunctional. 
These cross-linkers react with the ionizable groups of amino acids to enable their 
immobilization onto various support materials.  
 
 
 
Figure 2.5: Schematic representation of a cross-linker [27] 
 
 
2.4.4. Encapsulation and entrapment 
 Encapsulation is a non-covalent technique in which the biomolecules are 
entrapped in the transducer using membranes such as cellulose acetate (a common 
dialyzing membrane), polycarbonate materials, polytetrafluoroethylene (Teflon), nafion 
and other polyurethanes. In the case of entrapment, the biomolecules, generally enzymes 
 20 
are mixed in a polymeric solution, which on gelation entraps the biomolecules, thereby 
immobilizing them. The type of immobilization technique chosen depends on the type of 
protein and support used for biosensor development.  
2.5. Nanomaterials for sensing applications 
Nanomaterials by definition include those objects and systems whose size, in at 
least one dimension is around 1-100 nm. They have interesting electronic, optical and 
magnetic properties, which emerge due to the quantum phenomena (confinement) at the 
nanoscale range. For example, noble metals at the nanoscale exhibit size dependent 
surface plasmon absorption phenomena in the visible region resulting from the oscillation 
of electrons at certain frequencies. These surface plasmons when excited by evanescent 
waves produce a surface sensitive analytical tool known as surface plasmon resonance 
(SPR) for measuring protein absorption [28] and this methodology has been used to 
develop biosensors for the detection of biological and chemical agents [10].  In addition 
to the interesting electronic properties, nanomaterials are characterized by very high 
specific surface areas, attributed to the inverse dependence of surface to volume ratio 
over length or radius (S/V ? l-1 and S/V = 3/R)[29, 30], thereby, making their surface 
favorable for adsorption of organic materials to lower the interfacial free energies [31]. 
These interesting properties are often exploited and integrated with the sensing and 
transducing components to enhance the performance of chemical sensors.  
Commonly used nanomaterials for sensing applications include metal oxide 
nanoparticles (like SiO2, TiO2, Al2O3, Fe2O3), metal nanoparticles (Au, Ag, Pt, Pd), 
fullerenes (carbon nano wires, nanotubes and fullerenes) and semiconductor 
nanoparticles (CdS, CdSe, CdTe, ZnSe) etc. Their role in enhancing the performance of 
 21 
sensors may be direct as in the case of stimuli responsive materials or indirect as in the 
case of providing a stable support for sensing layer, especially in the case for biosensing 
elements.  
 22 
3. CURRENT ART IN SENSORS FOR THE DETECTION OF ORGANOPHOSPHATES 
 
3.1. Introduction  
Organophosphates are a large family of phosphorus containing compounds that 
inhibit the activity of acetyl cholinesterase (AChE), an enzyme that is responsible for the 
hydrolysis of acetylcholine neurotransmitter [32, 33]. Their chemical structure has two 
alkyl, aryl, or alkoxyl side chains, a leaving group (which may be O, S, F, C and or N) 
and a characteristic covalent bond to sulfur or oxygen. Figure 3.1 shows the schematic 
representation of an OP compound.  
 
 
 
Figure 3.1: Schematic representation of the general chemical structure of an 
organophosphorus compound.  
 
The phosphorus atom is normally attached to four oxygen atoms in 
organophosphates. However, in the case of phosphonates, there are three P-O bonds and a 
P-C bond. In the case of phosphinates, there are two P-O bonds and two P-C bonds. The 
sulfur containing OPs with the P? S moiety are called the phosphorothionates. The other 
 23 
varieties of OPs are mainly derivatives of phosphoric, phosphinic or phosphonic acids 
[34]. Although these compounds were synthesized as early as the 1800s, their neurotoxic 
properties were not known until the early 1930s, following which, their application as 
pesticides became widespread [33]. The more toxic OP compounds are the notorious 
chemical warfare agents like Sarin, Soman, Vx and Tabun [35]. 
3.2. Toxicity of OPs  
AChE is an enzyme that hydrolyses the central and peripheral neurotransmitter, 
Acetylcholine (ACh) [33]. Normally, on reacting with ACh, the hydroxyl group of serine 
at the AChE active site is acetylated, resulting in the release of choline moiety subsequent 
to which, the AChE activity is recovered within 0.15 milliseconds from hydrolysis of 
acetylated AChE. However, an OP compound mimics ACh and thus, in their presence, 
trans-esterification or trans-phophorylation of AChE takes place. The regeneration of a 
phosphorylated AChE is extremely slow, taking more than 60 hours. Figure 3.2 
represents the schematic of the mechanism of OP inhibition of AChE. In the absence of 
active AChE, the ACh accumulation occurs, thereby affecting the sensory and motor 
organs. Uncontrolled convulsions, paralysis and death of the exposed organism follow.  
The level of toxicity of an OP compound is dependent on many factors such as its 
physical and chemical properties to name a few. The degree to which a protein 
phosphorylation occurs, determines the potency of OP. Proteins generally undergo 
phosphorylation in serine (OH-) since it has an unshared pair of electrons. The central 
phosphorus present in the OP structure is electrophillic, and hence, the presence of atoms 
or molecules in the OP side chains that donate electrons, greatly reduce its potency. 
Consequently, different OPs have different levels of toxicity. Dephosphorylation of 
 24 
AChE may be facilitated when certain hydroxylamine derivatives such as oximes are 
used [36]. However, in certain cases, when the enzyme is aged, irreversible binding of 
OP takes place and reactivation may not occur.  
 
 
 
Figure 3.2: Phosphorylation of AChE in the presence of OP (shown in red) 
 
OP may enter the body by multiple routes such as direct adsorption through the 
skin or by ingesting food contaminated by them, leading to additive toxicity. After they 
enter the body, they undergo oxidation or hydrolysis by a number of enzymes depending 
on the type of leaving group present. Some xenobiotic metabolizing enzymes, 
cytochromes P450 (CYPs) present in liver, play a major role in bio activating the 
phosphorothionates leading to the formation of an ?oxon? or oxygen analog of the parent 
 25 
OP, while some catalyze reduction reactions [37-39].  These oxons are highly potent and 
thus oxidation reactions of P=S to P=O bond may attribute to their toxicity [33].  OPs 
may also covalently bind to other Serine Esterases, namely, Butyryl Cholinesterase 
(BChE), Carboxyl Esterase (CaE), Neuropathy Target Esterase (NTE) Trypsin, 
Chymotrypsin and Tyrosine residue of Human Serum Albumin [37, 38, 40-44].  
Some organophosphates may result in delayed toxicity due to lipid solubility and 
fat storage in mammals.  Long-term exposures to these compounds in low doses may also 
result in Organophosphate Induced Delayed Neuro-toxicity (OPIDN) associated with 
inhibition of the Neuropathy Target Esterase (NTE) [45-47]. The afflictions of the 
affected human being with a variety of syndromes of central-peripheral, distal, sensory-
motor axonopathy are some of the characteristics of OPIDN. An intermediate syndrome 
may occur sometimes after 24-96 hours of exposure, characterized by acute respiratory 
paresis, muscular weakness, cranial nerve palsies and depressed tendon reflexes [48]. 
 
3.3. Need for Detection 
Approximately 50 million tons of OPs are produced for agricultural and non-
agricultural purposes in US alone [49]. Graph in Figure 3.3 shows the annual use of 
some of the OPs in US. A list of some of the OPs currently in use is provided in Table 
3.1 Although these chemicals are mainly used as pesticides and insecticides; they are 
toxic to non-target mammals as well. OPs may enter and spread in the environment by a 
number of processes such as volatilization into air, leaching into the ground soil, runoff 
from the fields etc. Due their potential afflictions on target and non-target organisms, the 
Food and Drug Administration (FDA) began employing safe levels of OP/pesticide 
 26 
residue tolerances in food. With the increased awareness of the effects of these pesticides, 
the United States Environmental Protection Agency (USEPA) in 1973 took over 
responsibilities in establishing limits for pesticide residues in environment [49]. The rate 
of introduction of new pesticides in the market decreased from 10-15 per year for every 
1800 OPs synthesized during 1955-70 to less than one of every 22000 synthesized per 
year currently.  
 
 
Figure 3.3: Annual agricultural use in million pounds for some of the OPs in US [49] 
 
 
 27 
 
 
 
Table 3.1: List of OP pesticides [49] 
 
 
The analytical procedures for the diagnosis OP exposure may include the 
determination of AChE and BChE activity, unbound nerve agent, decomposition 
products, analysis of fluoride induced reactivation of inhibited AChE and BChE with 
reconstitution of the inhibitor and phosphonyl-proteins-adducts in plasma [50]. However, 
these methods have poor specificity since, both OPs and carbamates inhibit the AChE 
and BChE enzymes. Depending on the severity of exposure, timely administration of 
atropine sulfate, glycopyrolate or prolidoxime may help reactivate the enzyme [51].  
In addition to their use as pesticides, OP compounds form the largest of the 
notorious group of chemical agents (V and G nerve agents) that are stockpiled in 
munitions and containers in various locations around the world [52-55].  For example, of 
the 40,000 metric tones of chemical agents stockpiled in Russia, 80% are nerve agents 
 28 
(Sarin, Soman and Vx), 2% is mustard and rest is lewsite-mustard mixture [53]. 
Similarly, 60% of the chemical agents stockpiled in US were Vx and Sarin (GB)[55]. 
Figure 3.4 shows the chemical structure of some of these nerve agents. To stall their 
further proliferation, actions due to the Geneva Convention has led to the process of 
destruction of these stockpiles and prohibit their development in the future [56, 57].  
 
 
 
Figure 3.4: Chemical structures of OP chemical nerve agents  
 
Despite the efforts by the USEPA and the world treaties, incidents due to OP 
compound exposure continue to take place. One such incident is the Mevinphos 
 29 
poisoning of 26 workers in apple orchard in Washington. (Mevinphos is an OP used to 
control apple aphids with an oral LD50 of 3.7 to 6.1mg/ml in rats) [58]. In 2002, there was 
an estimated 69,000 child cases of pesticides related poisoning incidents [59]. The OP 
nerve agents have been exploited for terrorist activities as well [60-62]. These events 
underscore the need for the development of quick, selective and sensitive sensors for 
these compounds in the environment to prevent further exposure to non-target organisms 
and loss of human life.  
 
3.4. Detection of OPs 
3.4.1. Conventional analytical techniques 
Several methods have been developed for the detection of organophosphates. 
These may include the use of conventional analytical techniques and chemical sensors. 
Some of the conventional analytical methods developed for nerve agent detection are 
listed in Table 3.2. In the case of chromatography techniques, they are generally coupled 
with element selective detectors like pulsed Flame Photometric Detectors (FPD), 
Electron Capture Devices (ECD), Nitrogen Phosphorus Detectors (NPD), electrolytic 
conductivity detectors and microwave induced pulsed plasma atomic wave detection. 
Although most of the above techniques are sensitive, they suffer from disadvantages of 
conventional analytical techniques, as discussed in the Section 2.1. 
 
 
 
 
 
 
 30 
 
 
Table 3.2: Some of the conventional analytical techniques to determine OP nerve 
agents. The references for the above are as follows. a: [63, 64], b: [65, 66], c: [67], 
d:[68], e: [69] f: [70] 
 
3.4.2. Inhibition based biosensors 
 Activity of the inhibited cholinesterase due to the presence of OPs against its 
initial activity forms the basis of detection behind these biosensors. Figure 3.5 gives the 
schematic representation of this principle. Several different types of electrochemical, 
piezoelectric, calorimetric and optical sensors have been developed employing this 
principle and a number of authors have extensively reviewed this [71-73]. Among these, 
the electrochemical sensors are the most widely studied and researched method. 
 
 
 31 
 
 
Figure 3.5: Schematic representation of the principle behind inhibiton based OP 
detection.  
 
Several amperometric [74-78] and potentiometric [79-82] methods have been 
devised using this principle. Insensitivity to environmental turbidity enables the use of 
amperometric method for detection of wastewater samples. Potentiometric biosensors are 
however less sensitive as they are easily influenced by the pH solution conditions and 
environmental factors. 
Conductometric biosensors exploit the principle of changes in ion concentration 
because of inhibition, for transduction purposes. Thin film planar electrodes immobilized 
 32 
with cholinesterase have been used to detect photo degradation products of methyl 
parathion using conductometric biosensor [83]. Enzyme inhibition resulting in changes in 
mass or acoustic wave at the surface of the piezoelectric material was used for building 
piezoelectric biosensor [84]. They have the advantages of chemical inertness, mass 
production, low cost and solid construction.  
In the case of less invasive and more sensitive optical based transduction 
methodologies, fiber optic biosensor based acetyl cholinesterase immobilization on 
Langmuir-Blodgett films by measuring the changes in absorbance of  para-nitrophenol 
[85], cholinesterase immobilization in polyvinylidenefluoride membrane in contact with 
sol-gel layer incorporated with bromocresol purple [86],  AChE conjugated with pH 
sensitive fluorophore in sol gel network [87], are a few of the many developments. 
Recently, using the principle of competitive and discriminative inhibition of acetyl 
cholinesterase on planar waveguide, detection of OP (100 ppt of Sarin in solution and 
250 picograms of Sarin in vapor) has been made possible [88].   
Selectivity and specificity of biosensors is one of its main features and 
requirements. In addition to knowing the general wide spectrum of compounds that may 
affect cholinesterases, it is critical to know the specific compound of the wide spectrum. 
One of the most disturbing and shocking civilian incidents stressing the importance of 
selectivity of biosensor was the use of the ?unknown knock out gas? by the Russian elite 
forces to counter the Moscow theatre siege by the Chechen terrorists in October 2002 
[89].  Apart from the complications of gas poisoning, its unknown nature made the 
treatment of the hostages even more difficult. Hundreds of hostages died because of gas 
poisoning and complications due to mistreatment.   
 33 
One of the chief disadvantages of inhibition based detection principle is that 
cholinesterases are inhibited by OPs and a wide spectrum of other compounds like 
carbamates, heavy metal ions etc. In addition, there are restrictions on sensor reusability 
due to the difficulties involving sensor regeneration. This can be overcome by the 
employment of sensing elements specific to OP compounds.  
 
3.5. Biosensing Element: Direct detection using Organophosphorus Hydrolase  
Although OPs are stable when kept in cool, dark, anhydrous conditions, in the 
presence of water, heat or light may result in its hydrolysis, oxidation or rearrangement of 
its structure. The hydrolysis rates may differ for different classes of Organophosphates. 
The second-generation biosensors use more direct approach by employing enzymes that 
act as catalysts to hydrolyze and detoxify OPs [90-92]. There are several enzymes like 
Human Paraoxonase [93], Squid DFPase [94]  and Organophosphorus Acid Anhydrolase 
OPAA-2 [95, 96] that hydrolyze OPs, however, Organophosphorus Hydrolase (OPH, EC 
3.1.8.1) is the only enzyme that can catalytically hydrolyze the P-S bond of OP substrates 
in addition to P-O, P-F and P-CN bonds [97-100].  
The current study employs OPH as the bio-sensing element for the detection of 
OPs. OPH is a remarkably stable metalloenzyme with 2 zinc ions present in its active 
site. Figure 3.6 shows the dimeric crystal structure of OPH [101]. Studies have shown 
that the replacement of zinc ions with cobalt ions can significantly increase its activity. 
Although OPH has hydrolysing capabilities towards a wide spectrum of OPs, it is highly 
specific towards those with oxygen as the leaving group. Studies show that OPH 
 34 
catalyses the hydrolysis of OPs through the nucleophilic attack of the central phosphorus 
atom.   
 
Figure 3.6: Molecular structure of OPH [101]. 
  
The first biosensor using OPH was developed by the group at Texas A&M. In 
their method, E-coli cells exhibiting OPH activity were cryoimmobilized in polyvinyl 
alcohol matrix [102]. A pH glass electrode was used to transduce the OP hydrolysis by 
OPH into a measureable signal. Another immobilization strategy involved the use of sol-
gel matrix for enzyme immobilization for the development of sol-gel modified field 
effect transistors[103, 104].  Discriminative detection of pesticides like OPs and 
 35 
carbamates was realized with the incorporation of AChE and OPH to develop an 
amperometric sensor [105, 106]. Immobilization within polyelectrolyte multi-layers on 
glass beads [107], nanocomposite formation by incorporation of OPH into silicone 
polymers  [108] and use of competitive inhibition of OPH immobilized on planar glass 
slides [109] are some OPH immobilization strategies developed using optical based 
spectroscopic methods of detection.  
Developments in fluorescent-based detection of OPs include the employment of 
pH responsive fluorophores to transduce the catalytic reaction into a measurable signal. 
Close proximity between the fluorophore and recognition element enables enhanced 
monitoring of enzyme-substrate reactions and detection of target OPs. This can be 
achieved through their conjugation and immobilization on suitable supports. Examples 
include the encapsulation of OPH-SNAFL [110] and OPH-CNF [111] conjugates in 
polyethylene glycol and polystyrene waveguides respectively. While these systems 
provide real-time target analysis apart from being highly sensitive towards different OPs, 
they suffer from the inability to provide multiple and simultaneous detection of several 
different analytes. 
 
3.6. Multi analyte sensing platform: Array Biosensor  
One of the first multi-analyte sensors employed immunobased assays for 
biomedical applications [112].  Ever since, there has been significant progress in 
sandwich-based immunoassay technology for simultaneous detection of multiple 
analytes. A good number of the methods use the principle of evanescent waves to 
develop highly surface selective sensors for real-time analysis of biomolecular 
 36 
interactions. Some of the examples include the multi-analyte immunoassays based on 
surface plasmon resonance [113] and capillary flow systems [114].  Higher sensitivities 
have been obtained by increasing the optical path length as in the case of use of multiple 
internal reflection elements like planar waveguides [115-118], planar waveguide 
interferometers [119-121] and bundled optical fibers [122, 123]. Studies have also been 
performed to combine optical and electrochemical transduction methods [124]. Here, the 
planar waveguide is coated with Indium-Tin oxide (higher refractive index, 1.95-2.0) to 
improve the optical properties for total internal reflection and to electrically regenerate 
the immuno-sensing surface. There has been significant progress using Array Biosensor 
developed by the Naval Research Laboratory for simultaneous detection of multiple bio-
threat agents like Ricin, Cholera Toxin, B. Anthracis, Campylobacter Jejuni, 
Staphylococcus Enterotoxin B, Ochratoxin and many others [9, 125-132].  Array 
biosensor will be used in the current study as a fluorimeter to develop an enzyme based 
sensor for simultaneous detection of multiple OPs.  
 
 
 
 
 
 
 
 
 
 37 
 
4. ARRAY BIOSENSOR BASED ON ENZYME KINETICS MONITORING BY FLUORESCENCE 
SPECTROSCOPY: APPLICATION FOR NEUROTOXINS DETECTION 
 
4.1. Introduction 
The aim of the current study was to develop a biosensor array for the detection of 
OP neurotoxins like paraoxon (P-O), parathion (P-O) and DFP (P-F), using OPH as the 
bio-recognition element, conjugated with a pH-dependent fluorophore 
carboxynaphthofluorescein (CNF). The enzyme catalyzes the breakdown of OP 
neurotoxins and the generation of protons decreases the micro-environmental pH and the 
measured fluorescence intensity. BSA, that does not show any enzymatic activity towards 
OP, acts as the scaffold protein for fluorophore immobilization at the reference spots, 
which allows the realization of a differential mode measurement, by subtracting signals 
from the ?working?, and ?reference? spots. The main advantage of the differential mode 
measurement is that, common mode variations such as temperature and bulk pH changes 
are eliminated while local pH changes due to the enzymatic action at the OPH-loaded 
spots are amplified. 
An array biosensor unit developed at the Naval Research Laboratories (NRL) was 
adopted as the detection platform and appropriately modified for enzyme-based 
 38 
measurements. A planar multi-mode waveguide was covered with an optically 
transparent TiO2 layer to increase the surface area available for immobilization. The 
nanoparticles modified slides were functionalized with silane to enable covalent 
immobilization of OPH. Aminosilane (APTS)-gluteraldehyde (Glu) chemistry is one of 
the most widely utilized glass functionalization techniques for protein immobilization. 
Earlier studies [133] showed the development of a biosensor for the detection of 
paraoxon based on covalent immobilization of OPH using APTS-glu chemistry on glass 
slides coated with titanium dioxide nanoparticles. However, stringent conditions of 
moisture free environments are required as the use of aminosilane suffers from competing 
surface reactions and self-polymerization even in the presence of minute quantities of 
water. Accordingly, the different batches of sensors prepared when checked for paraoxon 
detection showed poor reproducibility with lower detection limits of 100 M. Hence, an 
alternative functionalization technique involving mercaptosilanes with GMBS crosslinker 
followed by avidin-biotin-NHS was developed for OPH immobilization on titanium 
dioxide coated glass slides 
4.2. Materials and Methods 
4.2.1. Chemicals and Reagents 
Ti-nanoxide ?T? paste was purchased from Solaronix Inc., (Aubonne, SA), 
absolute ethyl alcohol was purchased from Florida Distillers Co. (Lake Alfred, FL) , 
Sodium/potassium phosphate buffer, dimethyl sulfoxide (DMSO), N,N,-dimethyl 
formamide(DMF) and bovine serum albumin (BSA, fract V, cold alcohol precipitated)  
was purchased from Fisher Scientific, Pittsburgh, PA),.  Ches (2-[N-cyclohexylamino] 
ethanesulfonic Acid) was purchased from  Alfa Aesar (Ward Hill, MA), toluene 
 39 
(anhydrous, driSolv,) was purchased from EMD (Gibbstown, NJ), 3-(mercaptopropyl)-
trimethoxysilane (MPTS, 98%,) and diisopropylfluorophosphate (DFP) was purchased 
from Sigma Aldrich (St. Louis, MO), N-?-maleimidobutyryloxy succinimide ester 
(GMBS),  neutravidin was purchased from Thermo Fisher Scientific Inc., (Rockford, IL) 
CNF (5-(and-6-)-carboxynaphthofluorescein-succinimidyl ester, mixed isomers) and 
biotin-XX, SE ((2-[N-cyclohexylamino] ethanesul 6 ((6 ((biotinoyl) amino) hexanoyl) 
amino) hexanoic acid, succinimidyl ester) was purchased from Molecular Probes 
(Carlsbad, CA), glass slides were purchased from VWR scientific Inc., ( West Chester, 
PA).   
Wild-Type OPH (E.C.3.1.8.1) isolated from recombinant Escherichia coli strain 
was obtained from Texas A&M (Dr. Wild's Lab at TAMU). Paraoxon (diethyl-p-nitro 
phenyl phosphate) and parathion (O,O-diethyl-p-nitro phenyl phosphorothioate) was 
purchased from Chem. Service (West Chester, PA).  DI (de-ionized water, 18.2 M? cm) 
was used throughout and was obtained using a Millipore water purification system. 
4.2.2. Preparation of glass slides 
In order to remove any organic contaminants, the glass slides were incubated in 
concentrated hydrochloric acid (12.1N) for 30 min followed by a thorough water rinse. 
They were further dried under nitrogen atmosphere. The clean surface of the glass slide 
was coated with Ti-nanoxide paste, to boost the surface area. The composition of the Ti-
nanoxide paste obtained from the manufacturer (Solaronix, Switzerland) has 11% 
weight of nanocrystalline titanium dioxide anatase particles, water, less than 20% ethanol 
and polyethylene oxide. The procedure followed for coating the glass slide was ?Dr. 
 40 
Blade squeegee printing? method wherein, the glass slide was strapped on to a flat 
platform by its sides using an adhesive tape and the Ti-nanoxide paste was spread evenly 
on the slide with the help of a glass rod. The coated slides were sintered at 450 ?C for 
30 min, cooled down to room temperature, and cleaned with 1N hydrochloric acid. After 
cleaning, the slides were thoroughly rinsed in DI water and dried under nitrogen 
atmosphere. This was followed by silanization with 2% MPTS in dry toluene for 2 h in 
nitrogen atmosphere. The slides were then thoroughly rinsed in dry toluene. This was 
followed by incubation in 1 mM GMBS in absolute ethanol (dimethyl sulfoxide was used 
as the organic solvent to dissolve GMBS). Bonding occurs due to the covalent thioether 
linkage between the maleimide group of the cross-linker and sulfhydryl group of the 
silane. Subsequently, the slides were thoroughly rinsed in DI water and incubated in 
1 mg/ml of neutravidin (prepared in DI water) at 4 ?C for 12 h. This was followed by a 
thorough water rinse. The neutravidin-coated slides were incubated in freshly prepared 
1 mg/ml biotin-XXSE (1 mg biotin in 0.1 ml dimethyl formamide and 10 mM Phosphate 
buffer of pH 6.5) at room temperature for 1 h. After this, the slides were again rinsed in 
DI water. Before the immobilization of the enzyme, its activity was checked using the 
UV spectrophotometer. The extinction coefficient of the product of enzymatic reaction of 
paraoxon, namely, para-nitrophenol was 17,000 at an absorbance of 405 nm. The 
concentration of bovine serum albumin prepared in 0.1 M phosphate buffer with pH 8.0 
was maintained similar to that of OPH. The immobilization procedure employed (by 
NRL) to pattern the proteins and fluorophores on the glass slide involved the use of the 
physically isolated patterning (PIP) method. This method was used to generate 1 mm2 
array of biorecognition elements (antibodies) on the planar waveguide. Here, the 
 41 
polydimethylysiloxane made physically isolated patterning flow cell, was placed on the 
surface of the waveguide, and the antibodies were introduced in the channels. The 
problems faced with the above method for our study were the formation of bubbles that 
resulted in the non-uniform immobilization of the biomolecules and response variations 
among different arrays. To avoid these problems; simple, reproducible and long-lasting, 
multiple-use incubation holders (IH) were designed and engineered using the principle of 
capillary action and employed as an alternative to the PIP flow chambers [134].  
In this study, the principal of capillary action was used as shown in Figure 4.3 to 
direct the fluid flow and form rows of proteins and fluorophores on the waveguide 
surface. This is described in details in the earlier work [133]. The distance of hematocrit 
tubes from the glass slide surface determined the amount of solution that was needed to 
be placed on the slide-tube interface. This method of immobilization required 5?10 ?l of 
solution per row of protein/fluorophore array in contrast to the PIP method that required 
at least 50 ?l of solution. The whole incubation holder with the slide was placed in a Petri 
dish with moistened tissue to maintain humid conditions and prevent evaporation of 
applied solutions. The activated waveguide surface was incubated for at least 36 h at 4?C 
with OPH (4 mg/ml, phosphate buffer, pH 8.0) and BSA (4 mg/ml, phosphate buffer, pH 
8.0) After protein incubation, the slides were thoroughly rinsed in DI water and air-dried, 
for the immobilization of fluorophore. The pH sensitive fluorophore used in the study 
was CNF, functionalized with NHS ester. CNF solution of 1 mg/ml was prepared in 
10 mM phosphate buffer, pH 8.0 and DMF was used as the organic solvent to dissolve 
the CNF. The slides were incubated with CNF for 8 h at 4 ?C. After incubation, the slides 
 42 
were rinsed with DI water and stored in phosphate buffer at 4 ?C with cobalt chloride 
present to maintain the activity of OPH. Figure 4.1 gives the schematic representation of 
the slide chemistry.  
 
 
Figure 4.1: OPH immobilization chemistry on titanium dioxide coated glass slide. R 
and R1 represent neutravidin and OPH/BSA respectively.  
 
 43 
4.2.3. Characterization of titanium dioxide nanoparticles 
Transmission electron microscopy (TEM) was used to study the colloidal particle 
shape and morphology using Zeiss EM10 transmission electron microscope. The size and 
shape of the particles coated on the glass slides after heat treatment at 450 oC were 
studied using JEOL JSM 7000F field emission scanning electron microscope (JEOL 
USA, Inc., Peabody, MA) at 15-20KV in vacuum mode (< 7.75x 10-4 Torr). A thin layer 
of gold was sputtered on the oxide layer to make its surface conductive. The thickness of 
the coating on glass slide was measured using a profilometer (Tencor Instruments). X-ray 
diffraction studies were performed using Rigaku DMAX-B vertical diffractometer. The 
samples were exposed to X-rays (40KV, 40 mA) from a copper target and the intensity of 
the diffracted X-rays was measured. UV-visible spectroscopy was used to study the 
transmission characteristics of the coating on glass slide.  
4.2.4. Sensing principle  
OP neurotoxins undergo catalysed hydrolysis by OPH to generate protons that 
result in a change in the microenvironmental pH. The presence of a pH responsive 
fluorophore conjugated to OPH, serves to transduce the pH changes due to hydrolysis 
with a corresponding change in fluorescent intensity. This forms the basis of detection in 
the developed array biosensor.   
 44 
 
 
Figure 4.2: Schematic representation of paraoxon hydrolysis 
  
The developed sensor was tested for its sensing characteristics by measuring the 
pH response, enzyme action and stability.  Paraoxon was used as the model OP substrate.  
The hydrolysis of paraoxon is shown in Figure 4.3. For characterization using UV/Vis 
spectroscopy, the formation of para-nitrophenol was measured at 405 nm for detection 
purposes. 
4.2.5. Experimental Set-up 
The slide patterned with biorecognition elements conjugated with fluorophores 
was rinsed with DI water, air-dried and placed in the flow chamber of array biosensor. 
The flow chamber is made up of PDMS with six channel wells, each channel having a 
sample inlet and outlet [134]. As the direction of flow of the samples through the 
channels is in the direction perpendicular to that of the rows of biomolecules conjugated 
to fluorophores on the slide, multi-analyte sensing is made possible. A 54 mm, long, 
hollow, cylindrical aluminum tube with an 8 mm diameter opening was fit onto the laser 
diode in order to restrict the laser beam to fall only on the line generator. From the line-
generator, the laser-beam (diode laser, 635 nm) was directed to the edge of the slide to 
 45 
create evanescent waves that were used to excite the fluorophores on the waveguide 
surface, creating a fluorescence array. A CCD camera was used to monitor the changes in 
florescence intensity during the assay performed under room temperature conditions 
(Figure D).  
 
Figure 4.3: Schematic representation of array biosensor development. A: Principle 
of protein patterning on glass slide. B: Photograph of the incubation holder used for 
patterning biomolecules with fluorophores. C: CCD image of array of biosensors 
developed on glass slide. D: Schematic representation of array biosensor. 
 46 
 
Q-Capture Suite software (Q-Imaging, USA) was used for taking the images, and 
the TIFF analysis program developed at NRL, was used for data analysis. The rinsing 
buffer, 1 mM Ches, pH 8.2 was introduced at the flow rate of 4 ml/min using an Ismatec 
peristaltic pump, and the fluorescence array pattern was monitored with an exposure time 
of 2 s. After the background signal level was established, the OP samples prepared in 
buffer, were allowed to flow for 15 s until it filled the flow system. In order to give the 
true representation of the catalytic reaction, the maximum slope (change in 
intensity/time) was calculated for every concentration of the analyte introduced. The 
above-mentioned monitoring procedure was based on more than 100 experiments 
performed to optimize the measurement process. 
4.3. Results and discussions 
4.3.1. Characterization of the metal oxide nanoparticles 
The titanium dioxide nanoparticles were obtained as a colloidal paste containing 
11wt. % of nanocrystalline anatase particles with less than 20% ethanol and some 
polyethylene oxide. Over months of use, it was realized that these particles often 
aggregated resulting in cracked, non-adherent films when coated on glass slides. Hence, 
the sonication of the paste for 30 minutes was performed prior coating step. These 
particles were characterized using various techniques to ensure the development of 
sensitive biosensors. Figure 4.4 shows the transmission electron microscopy image of the 
titanium dioxide nanoparticles dried over carbon formvar grids. The particles were 
 47 
distributed between having a spherical and ellipsoidal morphology. Also, the average 
particle size was found to be 12-13 nm.  
 
Figure 4.4: Transmission electron microscopy image of titanium dioxide particles of 
the paste within 6 months of being purchased from the manufacturer  
To enable good adhesion between the titanium dioxide nanoparticles and the glass 
slide and to remove the organic solvents present in the paste, a sintering treatment at 
450oC was performed after the coated slides were air-dried. Figure 4.5 shows the X-ray 
diffraction of the plain and titanium dioxide coated glass slide. The peaks were plotted 
using JCPDS data (78-2486).  
 
 48 
 
Figure 4.5: X-ray diffraction studies of glass slide coated with titanium dioxide 
nanoparticles 
Peaks show that the nanoparticles are of anatase phase and that the heat treatment at 
450oC does not lead to rutile phase transformation. The particle size was calculated using 
the Scherrer equation. 
cosKD  
where, K is a dimensionless constant ~0.9,  is 1.541 ?, 2? is the diffraction (Bragg) 
angle and  is the full width at half maximum (fwhm) of the diffraction peak. The 
 49 
average crystallite size calculated from (101) peak was found to be 13.3 nm. This is 
consistent with the TEM approximation of the particle size. Hence, it can be concluded 
that sintering does not result in coarsening of the particles and this should explain the 
possible retention of the anatase phase as observed from the XRD peaks. Absence of 
coarsening is critical from the standpoint that coarsening will decrease the surface area 
from mechanisms due to increase in particle size and decrease in available pore volume 
of the film. 
The average weight of titanium dioxide nanoparticles coated on glass slide was 
found to be 7.08 + 0.7 mg. The thickness of the film measured using a profilometer was 
around 2.2 + m. The surface area and the pore size of the particles as provided by 
the manufacturer were 120 m2/g and 11 nm respectively.  Figure 4.6 shows the SEM 
image of plain and the nanoparticles coated on glass slides. The titanium dioxide 
nanoparticles appear to have a uniform coating with visible pores in-between particles. In 
addition, the average particle size of what appeared to be aggregated in the film was 
estimated to be 37 nm.  
 
 
 
 
 
 50 
 
 
Figure 4.6: SEM image of plain glass slide (A) and glass slide coated with titanium 
dioxide nanoparticles (B). 
The transparency of glass slides in the visible region of the electromagnetic 
spectrum along with its low intrinsic fluorescence, chemical inertness and resistance to 
high temperatures makes it a versatile support for fluorescence-based bioassays. In the 
case of array biosensor, in addition to being a protein support, the plain microscopic glass 
slide also acts as a planar waveguide to generate evanescent waves that in turn excites the 
patterned fluorophores present on the surface and create an array of sensing surfaces for 
simultaneous detection of multiple analytes. The generation of evanescent waves 
however requires that the following two conditions be met. 
a) Light is incident from a denser (waveguide) to a thinner medium  
b) Angle of incidence is greater than the critical angle which is defined by  
 51 
1
21s i n nnc  
where, n2 is the refractive index of the surrounding solution (analyte, buffer, water etc.) 
and n1 is the refractive index of biomolecular support (glass slide, polystyrene etc).  
For glass-water interface, the critical angle is 66.5 to 67o. The resulting 
evanescent field has a penetration depth (Dp)characterized by  
21221 /s in2 nnnD p
 
Here,  is the wavelength of incident light and ? is the angle of incidence. In an 
array biosensor, for an excitation wavelength of 635 nm (diode source), the penetration 
depth is ~ 760 nm. From the equation, one can modify the parameters such as the 
wavelength, angle of incidence and refractive indices of materials used to increase or 
decrease the penetration depth.  
The ?n? of non-porous titanium dioxide is 2.52. However, since the film prepared 
in this study has a pore size of 11 nm, the actual value is expected to be much lower. The 
current array biosensor configuration does not allow for modifications in ?. In order to 
understand if the titanium dioxide sensing film is the medium or the waveguide, refractive 
index of the thin film is of paramount. Since the conventional methods of determining the 
refractive index were unsuccessful due to the scattering of light by the films, an 
 52 
alternative technique using porosity was explored to determine the refractive index. 
According to the following equation, 
)( so li dnonso li dv o l u m etotal v o l u m eso li dN o nP o r o si t y
 
Given that the surface area of the anatase paste is 120 m2/ g, the average weight of 
particles coated on glass slide is 0.007.08 + 0.7 mg, and the average crystal size (d) is 13 
nm, assuming that the particles are spherical, the solid volume of anatase on glass can be 
found by rearranging the following equation.  
)( 6 a r e as u r f a c es p e c i f i cd  
where, ? is the density (mass/volume) of particles on slide. The relationship between 
porosity and refractive index is given by the following equation [135].  
( % )1 0 0111 22
dn
nP o r o s i ty  
where, n is the refractive index of porous anatase and nd is the refractive index of non-
porous anatase (2.52)[136]. The value of refractive index of the titanium dioxide film 
coated on glass slide was found to be 1.91 using this method. Ideally, for the developed 
thin film to act as a waveguide, the angle to incidence should be greater than 44o. The 
limitations of the array biosensor configuration only allowed for an angle of incidence for 
glass-water interface, which was ~ 67o. However, possible coupling of light from the 
 53 
slide into the film and its subsequent scattering by the high refractive index particles 
through the film could act as an excitation source for the patterned fluorophore on film, 
and thus explain the fluorescence array viewed via CCD camera. Further studies are 
required to investigate on to the optical properties of the developed film to act as 
waveguides. However, taking the above results into account, experiments were carried 
out to investigate on the sensing characteristics of the developed array biosensor.  
4.3.2. pH Response 
CNF is a pH sensitive fluorophore with an excitation and emission wavelength of 
598 nm and 668 nm, respectively at a pH 10.0. As the excitation source used in the array 
biosensor has a wavelength of 635 nm instead of 598 nm, it was necessary to investigate 
the effect of excitation wavelength on fluorescent intensity. Studies on the fluorimeter 
(Photon Technology International) showed more than 50% loss in fluorescent intensity 
due to excitation at 635 nm instead of 598 nm [133]. Figure 4.7 shows the pH response 
of the glass slide with immobilized proteins and fluorophore. The graph depicts that the 
changes in the intensity of the signal in both working and control channels are positively 
correlated with the changes in pH. The spot to spot coefficient of variance (CV) was 
found to be less than 20%. 
 54 
Figure 4.7: The changes in net fluorescent intensity of the working and 
reference/control spots in a particular channel of the glass slide with pH for 10 mM 
Ches buffer solution 
 
4.3.3. Enzymatic Reaction 
Figure 4.8 shows the response of the reference and the working spots to the 
introduction of 0.5 mM paraoxon in 1 mM Ches buffer. As detailed in the graph, the 
working spot has a huge drop in signal when compared to that of the reference spot. This 
drop in signal is typical for an enzymatic reaction that occurs due to the catalytic 
hydrolysis of paraoxon. The drop in intensity identified for the control spot can be 
attributed to the variations in sample buffer pH. In addition, it can be seen that the 
 55 
catalytic hydrolysis for this particular concentration of paraoxon is realized during the 
first 10 s, after sample introduction. Normally, the time taken for the hydrolysis depends 
on the concentration of paraoxon or target analyte. Thus, for all experiments the 
maximum slope was calculated for every concentration of paraoxon introduced.  
 
 
Figure 4.8: Response of reference and working spots to 0.5 mM paraoxon, 1 mM 
Ches buffer, pH 8.2. Images were taken for every 5 s. 
 
 
 
 56 
Figure 4.9 shows a concentration-signal relationship for paraoxon by the sensor 
taking a single pair of working and reference spot into consideration. The presented data 
was averaged and the standard error was determined for three samples for every 
concentration of 0.00125 mM, 0.0025 mM, 0.005 mM, 0.01 mM, 0.02 mM, 0.05 mM, 
0.1 mM, 0.2 mM, 0.4 mM and 0.8 mM paraoxon. Each point in the graph is calculated by 
the subtraction of the signal of the working spot from the control spot according the 
equation 
t r e fIwIS  
Where,  
S = slope, ?I w is the change in intensity of the working spot, ?I ref is the change in 
intensity of the reference spot and ?t is the change in time. 
The graph has the linear region ranging from concentrations of 0.00125 mM to 
0.1 mM and the signal saturates at concentrations above 0.35 mM. The inset in Figure 
4.9 shows the linear region of the response curve. Even though changes in intensity for 
concentrations as low as 0.00125 mM was obtained, it did not meet the statistical criteria 
of being three times greater than the standard deviation. Thus, the developed sensor was 
able to detect concentrations as low as 0.0025 mM. In spite of the good sensitivity, only 
54% of the potentially available fluorescence is used, due to excitation at 635 nm (laser 
source wavelength) instead of 598 nm. The system performance should improve 
significantly by exploiting a light source with 598 nm wavelength. 
 57 
 
Figure 4.9: Response curve of sensor to paraoxon introduction. The standard errors 
were calculated for three samples introduced for every concentration. The inset 
shows the linear region of response curve ranging from 2.5 M to 0.1 mM paraoxon.  
 
4.3.4. Specificity of Detection 
As mentioned at the introduction, there are several enzymes like Human paraoxonases 
[93], squid DFPase [94] and organophosphorus acid anhydrolase OPAA-2 [95, 96] that 
hydrolyses OPs, but, OPH (OPH, E.C. 3. 1. 8. 1) is the only enzyme that can catalytically 
hydrolyze the P-S bond of OP substrates apart from P-O, P-F, P-CN bonds [97-100]. The 
wild type OPH has different catalytic activities towards the P-O, P-F and P-S bonded OPs 
 58 
[90, 91] with a turn over number for paraoxon around 6000 s-1, DFP around 75 s-1 and 
Demeton-S around 4.3 s-1 [137]. 
  The specificity of the biosensor under study was examined using two P-O bond 
substrates namely paraoxon and parathion, and a P-F bond substrate DFP. It can be seen 
from figure 4.10 that the highest response is obtained for paraoxon followed by parathion 
that has 75 % less sensitivity than paraoxon, with DFP exhibiting the lowest (93% < 
paraoxon) sensitivity. This is similar to other investigations that have been made on the 
substrate specificity of OPH-WT with parathion and DFP showing turn over number less 
than 80% and 97 % respectively, when compared to paraoxon [138]. The lowest 
concentration the biosensor could detect for DFP and parathion based on the statistical 
criteria of being three times greater than standard deviation was 10 M. By using an 
array of enzymes with different substrate specificities such as OPH mutants and OPAA, 
this biosensor should be capable of discriminating different OP agents. 
 
 59 
 
Figure 4.10: Response curves of paraoxon, parathion and DFP for concentrations 
ranging from 2.5 M to 0.8 mM prepared in 1 mM Ches, pH 8.2-8.3.  
 
4.3.5. Stability of the immobilized enzyme 
The stability of the sensor was investigated by obtaining response curves for the 
linear region of paraoxon on various days after the waveguide preparation. Figure 4.11 
shows the changes in signal of the sensor relative to day 1 that is considered to be 100%. 
It can be seen that there is a drastic drop in signal level on the first 18 days after sensor 
preparation. On the 59th day, only 5% of the initial signal level is present; however, it has 
been proved enough for 0.01 mM paraoxon detection. The waveguide with patterned 
 60 
biomolecules and fluorophores prepared was stored in 10 mM phosphate buffer with 
cobalt chloride at 4 ?C. 
 
Figure 4.11: Bar graph showing the percentage of retained signal value relative to 
the sensitivity present on day 1 of sensor preparation.  
 
4.4. Conclusions 
A sensitive and rapid enzyme based array biosensor for OP detection was 
developed using the innate ability of OPH to catalyze the hydrolysis of OP neurotoxins. 
Ti-nanoxide coating that was used to boost the surface properties of the waveguide 
provided good characterization for the immobilization of the biorecognition elements and 
fluorophores. SEM images of the surface of the coated glass slides showed a uniform 
 61 
coating of Ti-nanoxide. The pH response of reference and working spots on the glass 
slide were similar allowing the discrimination between the catalytic and non-catalytic 
based pH changes. The lowest concentration of paraoxon that the biosensor could detect 
was 0.0025 mM, which is far below the lethal dosage level (0.007 mM in rats and 
mouse). The specificity of the sensor was validated by the simultaneous detection of 
parathion (P-O bond) and DFP (P-F bond) along with paraoxon. The sensor showed 
lower sensitivity for parathion and DFP, with detection limits of around 0.010 mM for 
parathion and DFP, respectively than paraoxon. Good storage conditions of the 
waveguide in 10 mM phosphate buffer with cobalt chloride under refrigerated conditions 
enabled enough retention of OPH activity (though less than initial 10%) to detect 
paraoxon concentration after more than 8 weeks. 
 62 
5. LYSOZYME-MEDIATED FORMATION OF PROTEIN-SILICA NANO-COMPOSITES FOR 
BIOSENSING APPLICATIONS 
 
5.1. Introduction 
The in situ encapsulation of biomolecules directly at a waveguide surface 
provides a potentially versatile tool with specific application to biosensor development. 
Various surfaces coated with titania nanoparticles, for example, have found increasing 
application in fields as diverse as photocatalysis, photovoltaics, photoelectrochromics and 
sensors due to the high photocatalytic activities of titania coatings [139-142]. Enzymes 
have been immobilized onto titania-coated waveguides via physical adsorption and 
covalent immobilization for biosensing applications and these techniques have been 
shown to enhance protein loading and retention of biomolecular activity [133, 143]. 
Apart from photocatalytic activity, interesting optical properties of titania nanoparticles, 
i.e. high refractive index and dielectric constants has enabled their use as waveguides 
when coated on glass slides and optical fibers[144-146]. Interfacing additional inorganic 
oxides, such as silica, in layers or core shell configurations is also known to tailor 
photocatalytic properties and enhance stability [147]. In addition, studies show that 
specific surface modifications of titania coatings provides appreciable biocompatibility 
[148]. 
 
 63 
For biosensor applications, however, complications arise when enzymes are 
directly introduced into oxide structures due to the extreme conditions required for 
inorganic oxide synthesis that are unfavorable for retention of activity in biomolecules. 
Recent research in biomineralization reactions, however, offers an alternative paradigm 
for the formation of silica nanoparticles under mild conditions that provides stability and 
retention of activity of the encapsulated biomolecules[149]. Studies on mineralization of 
silica in biological systems, for example, have led to the isolation of polycationic species 
that catalyze silica formation including silaffins and silicatein proteins, from marine 
diatoms and sponges respectively [150-152]. Recently the lysozyme-mediated formation 
of silica particles has also been demonstrated [153, 154]. Lysozyme as a template for 
biomineralization is advantageous as it is a ubiquitous protein and commercially 
available as opposed to specific silicification proteins that are expensive and labor 
intensive to purify. The resulting bio-nanocomposites of lysozyme with amorphous silica 
have been shown to retain the proteins native antibacterial activity [155]. Aside from 
being a benign method for oxide synthesis, the method also provides an effective method 
for enzyme immobilization that is amenable to encapsulation and stabilization of 
enzymes at a surface. For example, lysozyme-mediated silica formation was successfully 
utilized to encapsulate organophosphate hydrolase (OPH) on a gold surface plasmon 
resonance waveguide [156]. Similarly, a silica-precipitating peptide mediated silica 
formation and encapsulation of ?-Galactosidase in situ on functionalized silicon wafers 
[157]. In both cases, the encapsulated enzymes retained catalytic activity detectable by 
their respective analytes; namely paraoxon (for OPH) and lactose (for ?-Galactosidase) 
and provides a basis for biosensing applications. 
 64 
In this study, we extended the versatile mineralization reactions of lysozyme to 
coat silica particles on waveguides formed on glass slides, pretreated with a titanium 
dioxide coating. The choice of precursor and immobilization chemistry was studied in 
order to optimize encapsulation of OPH as a model enzyme. The stabilized OPH 
composites were patterned to form an array of sensors on the waveguide surface and 
tested on a commercially available array biosensor to demonstrate the versatility of the 
enzyme encapsulation methodology for biosensor development. 
5.2. Materials and Methods 
5.2.1. Chemicals and Reagents 
Lysozyme (from hen egg white), tetramethyl orthosilicate (TMOS) and tetraethyl 
orthosilicate (TEOS) were purchased from Sigma Aldrich (St. Louis, MO). 3-
mercaptopropyl trimethoxy silane (MPTS) was purchased from Gelest Inc (Morrisville, 
PA).  Potassium phosphate buffer (0.1 N NaOH, 0.1 M KH2PO4, pH 8.2), Ches buffer for 
spectrophotometer assays (0.05 M CHES, 0.05 mM CoCl2, pH 9.0), Ches buffer for the 
array biosensor (0.01 M CHES, 0.05 mM CoCl2, 2.7 mM KCl, 120 mM NaCl, pH 8.5) 
and phosphate buffered saline (PBS, 10 mM, pH 8.1) were prepared using reagents from 
standard commercial sources. Hydrochloric acid, acetone, bovine serum albumin (BSA, 
fract V, cold alcohol precipitated) and dimethyl sulfoxide (DMSO) were all purchased 
from Fisher Scientific (Pittsburgh, PA). Titanium dioxide nanopaste ?T? was purchased 
from Solaronix Inc, (Aubonne, Switzerland) and plain pre-cleaned glass slides were 
purchased from VWR Chemicals (West Chester, PA). The solvents, absolute ethanol 
(Florida Distillers Co.) and anhydrous drisolv toluene were purchased from EMD 
Biosciences Inc. (Gibbstown, NJ). N-?-maleimidobutyryloxy succinimide ester (GMBS) 
 65 
was purchased from Pierce (Rockford, IL) and CNF (5-(and-6-)-
carboxynaphthofluorescein-succinimidyl ester, mixed isomers) was purchased from 
Invitrogen (Carlsbad, CA).  Wild-Type OPH (E.C.3.1.8.1) from recombinant Escherichia 
coli was purified at Texas A&M as described elsewhere[158] .  
5.2.2. Instrumentation 
A standard UV/Vis spectrophotometer (Ultraspec? 2100 pro, Amersham 
Biosciences, Piscataway, NJ) was used for all absorbance-based measurements.  An 
Array Biosensor portable unit from the Naval Research Laboratories (Washington, DC) 
was used for all fluorescence-based assays with modifications as  described previously 
[159].  
5.2.3. Preparation of titanium dioxide coated glass slides 
A glass microscope slide forms the basis of the biosensor array and was pre-
treated with an initial coating of titanium dioxide nanoparticles in a manner described 
previously [159]. To differentiate between the initial titania paste coating and subsequent 
modifications, the titania-coated glass slides as prepared above will be referred to simply 
as ?pretreated glass slides?. 
5.2.4. Functionalization of pretreated glass slides for lysozyme immobilization 
The pretreated glass slides were chemically functionalized with MPTS and 
GMBS crosslinker in order to covalently immobilize lysozyme. Pretreated glass slides 
were treated with 2 % MPTS (in anhydrous toluene) for 45 minutes, and then rinsed in 
toluene. Silanated slides, after rinsing, were dried and incubated with 10 mM GMBS 
(prepared in absolute ethanol, diluted from a stock solution prepared in 10 % DMSO v/v) 
for 45 minutes. GMBS-treated slides were rinsed sequentially in ethanol and water and 
 66 
incubated with lysozyme (5 mg/ml in 10 mM PBS, pH 8.1) for 5 hours. Figure 5.1 shows 
the schematic representation of the chemistry used for lysozyme immobilization. In a 
control experiment, physical adsorption was used to immobilize lysozyme on pretreated 
glass slides by incubating with lysozyme (5 mg/ml in 10 mM PBS, pH 8.1) for 5 hours 
before rinsing with water as before. 
 
 
 
 
Figure 5.1: Schematic representation of the chemistry used for lysozyme 
immobilization.  
 
 
 
 67 
 
5.2.5. Formation of OPH-encapsulated silica particles on pretreated glass slides 
Two precursors were investigated for silica formation; in the first method, TMOS 
(30 l hydrolyzed with 170 l of 1mM HCl) was mixed with 820 l potassium phosphate 
buffer, (of 0.1 M, pH 8.1) containing 100 l of OPH (~1.5 mg/ml). A similar solution 
was prepared using TEOS in place of TMOS. 10 l of the above reaction mixtures were 
spotted to the pretreated glass slides and incubated for 5 hours at room temperature.  
 For fluorescence-based assays, immobilization of lysozyme was carried out using 
the MPTS-GMBS functionalization described in the previous section. OPH and BSA 
were conjugated with the fluorophore, CNF, using a protocol provided by the 
manufacturer. A centrifugal filter unit with a 30 kDa molecular weight cut-off was used 
to separate the conjugated protein from any unreacted dye. BSA was used as a scaffold 
protein for CNF immobilization as a negative control with no paraoxon-hydrolyzing 
activity. The methodology for pattering slides with the OPH/BSA-CNF conjugate 
involved the use of capillary tubes as described in a previous study [159]. Figure 5.2 
shows the lysozyme mediated formation of silica particles on glass slides.  
5.2.6. Microscopic characterization of silica particles 
Treated slides were sputtered with a thin layer of gold and visualized by Scanning 
Electron Microscopy (SEM, JEOL, model 840) in order to study and characterize the 
silica particles formed. No gold coating was required for characterization using optical 
microscopy (Aetos Technologies Inc, AL). 
 
 
 68 
 
 
 
 
 
Figure 5.2: Schematic representation of lysozyme mediated formation of silica 
particles and its encapsulation of OPH on glass slide coated with titanium dioxide 
nanoparticles 
 
5.2.7. Enzyme assays for OPH activity 
Activity of OPH was determined from the rate of catalytic hydrolysis of paraoxon 
to produce p-nitrophenol (PNP), which has a characteristic absorption peak at 405 nm. 
The conversion of paraoxon was investigated at a range of paraoxon concentrations after 
incubation of OPH-immobilized slides for 1 min and measurement of the respective 
absorbance change at 405 nm. 
 69 
 
 
5.2.8. OPH activity assays on the array biosensor 
Array biosensor assays were performed to test the efficiency of immobilization of 
OPH on the portable fluorimeter platform. Here, a red diode laser ( =635 nm) directed 
over the edge of the glass slide undergoes total internal reflection producing evanescent 
waves that act as an excitation source (and direct measurement) for the labeled 
biomolecules immobilized onto the slide surface. A CCD camera was used to monitor the 
resulting fluorescent intensity. 
 The sensor was characterized for pH variation by passing 10 mM CHES buffer (at 
a pH ranging from 8.0 to 10.0) at a fixed flow rate of 1.5 ml/min for 30 seconds. The 
flow was stopped when the images were captured for data analysis. For paraoxon 
response, different concentrations of paraoxon were prepared in buffer (1 mM CHES, pH 
8.4), and flowed through the channels of the sensor to pass through regions containing 
immobilized OPH and BSA and thereby form an array of sensing spots. Images 
containing the array of fluorescent spots were taken for exposure times of 3 seconds 
using Q capture pro software (Q Imaging, Canada). TIFF analyze software developed at 
NRL was used to quantify the fluorescent intensity of the captured images. 
5.3. Results and Discussions 
5.3.1. Formation of silica particles for OPH encapsulation 
Lysozyme catalyzes the formation of silica from its precursor solutions. Initially, 
the formation of silica particles and their ability to encapsulate OPH was investigated to 
ensure that the lysozyme-mediated formation of particles was not hindered when directed 
 70 
onto a pretreated surface. Lysozyme immobilization was achieved by physical adsorption 
and silica particles were formed in situ. OPH became encapsulated as the silica particles 
formed. The efficacy of the procedure was evaluated by monitoring the catalytic activity 
of encapsulated OPH for the hydrolysis of paraoxon. The graph in Figure 5.3 shows the 
absorbance of PNP, obtained as a result of paraoxon hydrolysis by OPH encapsulated 
within silica composites formed directly on the pretreated glass slides. The response of 
OPH activity was comparable and the encapsulation efficiency showed little variation 
between the two silicate precursors investigated (TEOS and TMOS). Quantification of 
the amount of immobilized lysozyme template was determined by measuring the 
concentrations of lysozyme in solution before and after immobilization and in the 
subsequent wash steps. Under the reaction conditions described, approximately 30% of 
the initial lysozyme was immobilized on the pretreated glass slide by adsorption alone. 
Interestingly, similar studies performed for OPH adsorption on the pretreated glass slides 
resulted in no retention of OPH and is attributed to poor electrostatic and/or hydrophobic 
interactions with the pretreated glass slide.  
 71 
 
Figure 5.3: Hydrolysis of paraoxon to para-nitrophenol by OPH encapsulated in 
silica using TMOS (?) and TEOS (?) precursors 
 
For silica encapsulation of OPH, TMOS as silicate precursor appeared to result in 
a silica matrix with increased sensitivity at low concentrations compared to TEOS but the 
standard error of measurements was more variable with TMOS as the precursor. It was 
considered that variability in the measurements may be due to OPH leaching from the 
silica particle matrix during analysis. In order to test this observation, the change in 
absorbance of the reaction solution was monitored after the functionalized slides had 
been removed from the reaction solution. After the removal of slides that contained OPH 
in silica prepared from TMOS, the hydrolysis of paraoxon continued, as evidenced by an 
 72 
increase in absorbance at 405 nm, indicating that OPH had leached from the surface of 
this preparation (Figure 5.4). In contrast, silica formation from TEOS produced a stable 
composite that prevented OPH from leaching over time.   
 
Figure 5.4: Leaching test: Change in absorbance with time for a paraoxon solution 
after incubation with OPH encapsulated silica slides using TMOS (?) and TEOS (?) 
 
The activity and retention of the encapsulated OPH may depend upon a number of 
factors, one of them being the morphology of particles formed during encapsulation. 
SEM analysis was used in order to characterize the matrix morphology and the effect of 
precursor on the resulting composites. The initial pretreatment of glass slides with T-
paste (titanium dioxide coating) results in a homogeneous coverage of nanoparticles that 
 73 
can be clearly differentiated from a non-coated glass slide. As such, encapsulated OPH 
was visualized for both pretreated and untreated glass slides. Figure 5.5 shows that silica 
particles formed using TEOS appear pronounced and distinct with a wide particle size 
distribution (100-500 nm). This can be seen more clearly on plain glass slides than for 
pretreated glass slides due to the presence of the titania background coating. In the case 
of TMOS, the distribution of silica particles is more homogenous and uniform and 
produces a gel-like coating of silica particles. Optical images of the silica particles 
encapsulating OPH confirm that the TMOS precursor forms silica particles that are small 
and homogeneous with an even distribution of encapsulated enzyme, while the silica 
particles formed with the TEOS precursor again appear larger and possibly aggregated. 
This difference in particle morphology between the two silicate precursors requires 
further investigation as porosity of the matrix may contribute significantly to substrate 
interaction with the encapsulated enzyme and influence the stability of the matrix against 
leaching. In addition, preliminary observations suggest that the rate of silicification 
differs for the two precursors when catalyzed by lysozyme with silica formation from 
TEOS being a significantly more rapid process compared to TMOS (data not shown). 
The small particle distribution for TMOS-silica creates a large surface area that could 
explain the enhanced leaching of OPH as was observed in Figure 5.4.  Thus, despite the 
heterogeneity of TEOS-based silica particles, the structure provides optimal retention for 
encapsulating OPH under the given experimental conditions. As such, for further 
experimental studies, TEOS was used as the precursor for silica formation. 
 
 
 74 
 
 
 
Figure 5.5: SEM and optical images of silica particles using TEOS and TMOS with 
schematics. SEM images of Plain (a) and Pretreated (b) slides, SEM images of silica 
particles formed using TMOS precursor on plain (c) and pretreated (d) slides, SEM 
images of silica particles formed using TEOS precursor on plain (e) and pretreated 
(f) respectively. Optical microscopic images of OPH encapsulated silica particles 
formed in solution mediated by lysozyme using TEOS (g) and TMOS (h) precursors 
respectively. 
 
 75 
5.3.2. Covalent attachment of lysozyme for silica formation 
Preliminary studies confirmed that physical adsorption of lysozyme to pretreated 
glass slides did not hinder the formation of silica particles and OPH was successfully 
encapsulated in the resulting inorganic matrix. However, adsorption strategies of 
immobilization suffer from potential problems of protein leaching due to physiological 
changes in pH and ionic strength. For long term operation of a biosensor, the stability of 
the matrix and corresponding retention of enzyme activity (of the sensing component) is 
paramount. In the study described herein, the possibility of lysozyme leaching from the 
preformed structure could in turn destabilize the encapsulated OPH immobilized on the 
surface. An alternative is to anchor the template lysozyme in place using covalent 
attachment chemistries to potentially enhance the stability and reusability of the final 
biosensor.  
Covalent immobilization by MPTS modification allowed subsequent orientation 
of GMBS to preferentially bind the lysine residues of lysozyme. MPTS/GMBS-
functionalized glass slides provided a platform for silica formation indicating that the 
orientation of lysozyme using covalent attachment did not significantly hinder the 
silicification reaction of lysozyme (Figure 5.6). The protein immobilization efficiency 
indicated that ~90% of lysozyme was retained by MPTS/GMBS chemistry providing a 
significant increase to the loading capacity over physical adsorption alone (~30%). The 
efficiency of enzyme immobilization in silica was determined by measuring the activity 
of the encapsulated OPH. MPTS/GMBS-functionalized slides demonstrated linear 
detection rates (slope: 0.1945) corresponding to catalytic activity with increasing 
 76 
concentrations of paraoxon in the range 0.024 mM to 0.495 mM. The detection limit for 
the encapsulated OPH was approximately 0.05 mM paraoxon.  
 
Figure 5.6: Activity of OPH encapsulated in silica particles with covalently 
immobilized lysozyme 
 
 Protein immobilization in which the immobilization matrix is covalently 
anchored to the pretreated glass slide should theoretically improve the long-term stability 
and hence applicability for bio-analytical devices. In addition, the use of a 3-dimensional 
matrix of silica significantly increases the surface area for protein loading[143, 159]. In 
order to test the stability of the silica-encapsulated OPH, the retention of OPH activity 
was investigated following controlled changes in physiological conditions. High salt 
 77 
concentrations, for example, can be detrimental to protein stability in immobilized 
systems.  The presence of 0.5 M sodium chloride in buffer solutions, for example, results 
in complete desorption of cytochrome C (a cationic protein with an isoelectric point (pI) 
similar to lysozyme, pI=10 and 10.5 respectively) [160]. Pretreated glass slides were 
functionalized with MPTS/GMBS and lysozyme as described above and used to mediate 
the formation of silica containing OPH. The resulting functionalized slides were then 
treated with 0.5 M NaCl and retention of OPH activity was determined. No loss of OPH 
activity was observed following the high salt treatment, indicating that the silica particles 
provided stable encapsulation of OPH and offer protection against environmental 
changes. In contrast, a control slide in which OPH was attached in the absence of 
lysozyme for silica mediation resulted in 33% loss in activity. The possibility of OPH to 
mediate silica formation that could explain the presence of initial activity requires further 
studies.  
5.3.3. Encapsulation of fluorescent OPH for studies on array biosensor 
Array biosensor, developed at the Naval Research Laboratories was used to 
demonstrate the enzyme encapsulation described herein on a portable system. To directly 
integrate with the NRL array biosensor, the reporter for detection activity was 
fluorescent-based and detectable using fluorescent dyes that excite at 635nm and emit in 
the 665-680 nm range [161]. OPH was conjugated with the pH-dependent fluorophore, 
CNF, resulting in the utilization of  only 50 % of the actual fluorescent intensity of CNF 
due to the variations in available excitation wavelength (635 nm) from that required for 
CNF (598 nm) [133, 159]. The catalytic hydrolysis of organophosphates by OPH 
generates protons which directly influences the fluorescence intensity of CNF. 
 78 
Mercaptosilane chemistry was used for lysozyme immobilization in order to pattern 
biomolecules for the biosensor array. MPTS/GMBS chemistry allowed for direct 
comparison with previous studies [159] that involved covalent immobilization of OPH. 
The pretreated glass slides were patterned with immobilized lysozyme and incubated 
with a mixture of dye-conjugated OPH and silicate precursor solution. The lysozyme-
mediated silica formation encapsulates the fluorescently-labeled OPH directly at the 
sensor surface in a series of well-defined ?spots?. Control spots were similarly derived 
using fluorescently-labeled BSA which allowed for direct variations in physiological 
conditions and environmental pH changes to be evaluated in order to eliminate external 
variations. Figure 5.7 A shows the patterned slide with 2 rows of control (BSA) spots 
and 4 rows of working (OPH) spots. The presence of the fluorescent spots indicated that 
the planar waveguide properties were not affected by a layer of silica at the surface. 
Figure 5.7 B shows the pH response of OPH conjugated to the pH sensitive fluorophore, 
CNF, encapsulated and patterned on the pretreated glass slide. BSA-CNF provides a 
control to differentiate between any changes in pH that may occur from hydrolysis of 
paraoxon and those due to sample-buffer variations. The graph shows a good correlation 
in pH response between the BSA and OPH spots, confirming that BSA was encapsulated 
in silica and was a suitable control for our assay (Figure 5.7 B).  
Figure 5.7 C shows the paraoxon response, determined by calculating the slope 
for changes in fluorescent intensity over a period of time for specific concentrations of 
paraoxon. Though concentrations as low as 7 M paraoxon could be detected, the 
statistical detection limit was ~35 M. To determine the stability of the encapsulated 
enzymes, the measurements were repeated after 12 and 66 days of sensor preparation. 
 79 
The sensor retained more than 60% of its original activity even after 2 months of storage. 
By comparison, covalent attachment of OPH by MPTS/GMBS chemistry (to a similarly 
pretreated glass slide) lost ~80% of activity within 18 days and 95% of OPH enzyme 
activity over a period of 59 days[159].  
 
 
 
 
Figure 5.7: Response of pretreated glass slides with patterned encapsulation of 
OPH-CNF on array biosensor. A) Array of spots formed on glass slides B) pH 
response of working (OPH-W) and control (BSA-R) spots C) Paraoxon (prepared in 
1 mM Ches, pH 8.4) response of sensors over a period of nearly 2 months 
 
 80 
 
5.4. Conclusions 
In this study, the utility of lysozyme to template silica nanoparticle formation on 
glass slides that have been precoated with a titanium dioxide paste was demonstrated. 
OPH was introduced during the synthesis procedure along with the ceramic precursors 
and the encapsulated OPH retained catalytic activity towards paraoxon. Under the given 
experimental conditions, TEOS as a precursor for silica formation was superior in 
encapsulating OPH than TMOS. Covalent attachment of lysozyme prior to silicification 
resulted in the formation of a stable silica matrix for OPH encapsulation with 
reproducible linear detection rates for paraoxon. Extension of the methodology to pattern 
silica and encapsulate OPH that was conjugated with a fluorescent indicator enabled the 
development of a spectro-fluorimetric array biosensor with OPH. OPH activity was 
retained within the resulting sensor array for over 60 days demonstrating a significant 
improvement in stability over previously reported configurations [159]. In addition, 
encapsulation in silica using this method is rapid with preparation times of a few hours, 
compared to the several days for covalent immobilization of OPH as reported previously. 
OPH encapsulation was demonstrated herein as a model enzyme system, but the 
encapsulation methodology is applicable to a wide variety of biomolecules, providing the 
potential for multiple enzyme immobilizations and simultaneous analysis of multiple 
analytes [157].  
 81 
6. MONITORING OF DIISOPROPYL FLUOROPHOSPHATE HYDROLYSIS 
 BY FLUORIDE-SELECTIVE POLYMERIC FILMS USING ABSORBANCE SPECTROSCOPY 
 
6.1. Introduction 
Organophosphonofluoridates (OPFs) belong to a subclass of organophosphates 
(OPs) with anti-cholinesterase properties [33]. The presence of fluorine attached to the 
central phosphorus atom differentiates OPFs from other OPs with O, S, or CN as the 
leaving groups following chemical hydrolysis. Among the most notorious chemical 
warfare agents, Sarin and Soman both belong to this phosphonofluoridate subclass, 
known as GB and GD agents, respectively.  Consequently, significant effort is being 
directed into the research and development of relatively simple sensors for their detection 
in the environment.  Some of the conventional OFP detection systems include 
chromatographic techniques interfaced with mass spectrometers [162], surface acoustic 
wave sensors based on changes in properties of a piezoelectric crystal upon the 
adsorption of specific analyte [163] and molecular imprinting techniques in combination 
with spectrometers using a europium probe to measure the hydrolysis products of Sarin 
[66]. As such, these techniques offer good sensitivity; however, most of these 
sophisticated technologies have limited portability, and their complex operation requires 
skilled personnel.   
 82 
In lieu of these approaches, the employment of enzyme-based biosensing systems 
may be a less expensive alternative with better operational abilities for field detection 
purposes. In previous studies, the inhibition of acetyl-cholinesterase was used as a means 
to detect and quantify neurotoxic OPs providing good sensitivity [164]. However, their 
limits in application included poor selectivity and limited reusability attributed to the 
nearly irreversible AChE enzyme inhibition.  A more specific biorecognition element 
such as organophosphorus hydrolase (OPH, E.C. 3.1.8.1) that catalyses the hydrolysis of 
various OPs with dramatically different kinetic parameters has the potential to 
discriminate certain OPs against other cholinesterase inhibitors [97, 99, 165]. For 
example, the K cat values at 7.0 pH and 25 oC of OPH from Pseudomonas diminuta that 
hydrolyzes a variety of OPs such as paraoxon (P-O), DFP (P-F), Soman (P-F) and Sarin 
(P-F) was found to be 2070 s-1, 41 s-1, 4.8 s-1 and 56 s-1 respectively [97]. Extensive 
studies using site-directed mutagenesis have shown tailored catalytic activities towards 
substrates having different bonds [166]. There is a related enzyme, organophosphorus 
acid anhydrolase (OPAA, EC 3.1.8.2) that is specific towards OFP (type G) [95] 
hydrolysis, which has the potential to selectively detect OPFs over other OPs.   
The biosensors based on OPH or OPAA reported to date, which are specific for 
OFPs, chiefly rely on the generic detection of protons [111, 133, 159, 167] and/or the 
specific detection of fluoride ions [168] generated upon hydrolysis of the target species.  
Proton detection as a means for OFP quantification suffers from several disadvantages.  
First, pH-based detection systems are largely influenced by the buffering capacity of the 
target analyte sample. High buffering capacity of the sample may prevent the ability to 
monitor changes in proton activity, and poorly buffered samples may render the system 
 83 
unstable. Hence, extremely good referencing techniques are crucial in order to avoid false 
positive signals, and they are difficult to sustain.  Secondly, all OPs, regardless of their 
leaving groups or their application as pesticides or chemical warfare agents (CWAs), 
produce hydrogen ions upon hydrolysis, thereby creating challenging selectivity issues.  
Addressing the selectivity requirement of an OP biosensor is critical to differentiate the 
presence of a pesticide such as parathion or acephatae from a chemical nerve agent such 
as Sarin or Soman. This discrimination is critical in order for the local emergency 
planning committees (LEPC) to act appropriately for remediation and prevent public 
panic.  Thirdly, the reuse of enzyme-based sensors over extended periods is limited by 
the loss of their enzymatic activity requiring constant recalibration (and even during) 
detection analysis.  These issues may be addressed by developing a system that combines 
the advantages of immobilized enzymatic hydrolysis and selective fluoride ion sensing 
devices.  One such development involves the use of a commercially available pF ion-
selective electrode coupled to OPH immobilized onto a silica gel in a batch mode set-up 
[167]. The system however suffers from poor sensitivity.  
Recent studies of fluoride detection systems based on polymeric films have 
demonstrated that thin films prepared with Al(III) porphyrins in conjunction with a 
lipophillic pH indicator such as dibromofluorescein can be employed to create a highly 
sensitive and reusable fluoride ion sensor based on the coextraction principle [169]. In 
this preliminary study, we show that it is possible to develop a stable, enzyme-affiliated 
OPF sensing system by utilizing such a fluoride ion-selective optical film to transduce the 
OPF hydrolysis by OPH into a measureable signal.  The interfacing of an Al[OEP]-ETH 
7075 ion-selective membrane with an enzyme-based measurement greatly widens the 
 84 
window of dynamic range of detection.  The principle of OFP detection in the current 
study is shown in Figure 6.1. 
 
 
 
 
 
Figure 6.1: Schematic representation of the principle of DFP hydrolysis by OPH 
and subsequent detection by optical sensing film.  
 
 
 
 85 
Diisopropylfluorophosphate (DFP), is a structural analogue of the class-G 
chemical warfare agents and is used as the model target analyte in these studies.  Here, 
DFP undergoes catalytic hydrolysis in the presence of OPH to give equimolar 
concentration of fluoride ions.  The generated products are exposed to fluoride sensing 
polymer film coated on quartz slide resulting in the coextraction of fluoride ions and 
protons from solution phase by the aluminum porphyrin and chromoionophore (ETH 
7075), respectively, into the film.  This coextraction results in the protonation of ETH 
7075 which is measured as a change (decrease) in absorbance at  . of the depronated 
form of the chomoionophore   The entire set-up of the sensing film with analyte solution 
was contained in a standard 1 cm cuvette (Figure 6.2). 
6.2. Materials and Methods 
6.2.1. Chemicals and reagents 
Octaethyl porphyrin (OEP) was purchased from Frontier Scientific (Logan, UT) 
and aluminum chloride (AlCl3) was purchased from Strem Chemicals, Inc. 
(Newburyport, MA). Diisopropyl fluorophosphates (DFP), 4?,5?-dibromofluorescein 
octadecyl ester (ETH 7075), 2-nitrophenyloctylether (o-NPOE), poly(vinyl chloride), 
sodium fluoride, benzonitrile and tetrahydrofuran was purchased from Sigma Aldrich (St. 
Louis, MO). L-Glycine, phosphoric acid and quartz slides were purchased from Fisher 
Scientific (Pittsburgh, PA).  Ches (N-cyclohexyl-2-aminoethanesulfonic acid) was 
purchased from Alfa Aesar (Ward Hill, MA). Wild-type OPH (E.C. 3.1.8.1) from 
recombinant Escherichia coli was purified at Texas A&M as described elsewhere [158]. 
De-Ionized water (18.2 M? cm) was used from Millipore water purification system for 
the preparation of buffer solutions. 
 86 
6.2.2. Preparation of Aluminum octaethyl porphyrin (ALOEP) 
The Al[OEP] was synthesized by refluxing 0.5 mmol OEP and 2 mmol aluminum 
chloride in 25 ml benzonitrile overnight under nitrogen atmosphere. Following the 
evaporation of benzonitrile, the crude product was dissolved in methanol and precipitated 
in 3 M hydrochloric acid solution.  The collected precipitate was further washed with 3 M 
hydrochloric acid. Purification of the crude product was achieved by flash 
chromatography on silica using dichloromethane as the solvent. The final product was 
characterized by mass spectrometry yielding a desired molecular weight peak at m/z = 
591.3 and using UV/Visible spectrometry. 
6.2.3. Preparation of Al[OEP]-ETH 7075 cocktail 
The ALOEP-ETH 7075 optical fluoride sensing film was prepared using the 
procedures reported by Badr and Meyerhoff [170]. The most effective formulation as 
discussed by Badr et. al. resulted from the mixture of 2.14 mg of Al[OEP], 2.08 mg ETH-
7075, 30 mg of 2 nitrophenyloctylether, and 15 mg of poly(vinyl chloride) with 1 ml of 
tetrahydrofuran. Complete dissolution of the reagents and mixing was ensured (Al[OEP]-
ETH 7075) using sonication. The cocktail was then sealed in a glass vial and stored at 
4oC until use.  Cocktails were used within 3 days of their preparation.   
6.2.4. Preparation of slide coated with polymerized film 
Quartz slide was cleaned using acetone prior coating with polymeric films. Doctor 
blade method was used to coat 20 l of Al[OEP]-ETH cocktail on each of the cleaned 
slides. The slides were with the optical sensing film were allowed to dry, and then stored 
in dark until use.  
 
 87 
6.2.5. Instrumentation 
UV/vis spectrophotometer (Ultraspec? 2100 pro, Amersham Biosciences, 
Piscataway, NJ) was used for all absorbance measurements. The schematic of the 
experimental set-up comprising of a cuvette with sensing film is shown in Figure 6.2. 
The slide coated with the polymer film is placed in a standard 1 cm cuvette containing the 
analyte solution.  A slot with inside groves made on the cuvette cap was used to hold the 
slide in a position such that it was perpendicular to the light path. The arrangement was 
sturdy enough to prevent the movement of slide during handling and experimentation. 
The whole system was in turn placed in the standard cuvette holder of the 
spectrophotometer for measurement purposes.  
 
   
Figure 6.2: Schematic of the experimental set-up of the sensing film in cuvette 
 
 
 88 
6.2.6. Procedure 
  For the detection of DFP, various concentrations of DFP were formulated in 
CHES buffer, pH 8.5, and were completely hydrolyzed by treatment with 10 l of ~ 7 
g/ml of OPH for 2 min.  The reaction solutions were then further diluted in glycine 
phosphate buffer (2.0 M, pH 3.0) such that the final concentrations of DFP in contact 
with Al[OEP]-ETH 7075 doped film were in the range of 0.1 M, 1 M, 10 M, 25 M, 
50 M, 75 M and 100 M.  The absorbance spectra of the polymer films in contact with 
the hydrolyzed/diluted DFP solutions for 10 min were recorded on a UV-vis 
spectrophotometer (Ultaspec 2100pro, Amersham Biosciences). The absorbance peaks 
corresponding to the chromoionophore?s deprotonated state were monitored for changes 
and plotted against the various DFP concentrations.  This two step procedure of 
hydrolysis and detection was similar to the studies performed for glucose sensing using 
the Al[OEP]-ETH 7075 polymer films [171]. To reference against possible non-
enzymatic hydrolysis of DFP over the same assay time, ALOEP-ETH 7075 films were 
also exposed to samples that were not hydrolyzed by OPH.  The percentage changes in 
the absorbance (%A) were calculated as follows: 
 
100%
D F Po
xo
O P HD F Po
xo A AAA AAA  
 
where, Ao is the absorbance of the polymer film coated slide in glycine phosphate buffer 
solution and Ax is the absorbance of the slide when treated with ?x? M DFP. 
 89 
6.3. Results and discussions 
The use of Al[OEP]-ETH 7075 polymeric film on quartz slide to detect fluoride 
ions has been discussed in detail by Badr et. al [170]. Studies have shown its application 
for glucose sensing by coupling enzyme-based reactions with these polymeric films on 
microtitre plate wells [171]. Since OPH is rendered inactive due to the protonation of its 
active site [172] at pH 3.0, the hydrolysis and detection of DFP is carried out in two steps 
under different pH conditions similar to the procedure followed by Robboh et. al for 
glucose detection [171].  
Although OPH activity has its highest substrate specificity with 
phosphotriesterase (P-O) bonds, its hydrolyzing capability towards OFPs is well 
established [106, 111, 159, 167]. As shown in Figure 6.1, complete hydrolysis of DFP 
results in the production of equimolar concentration of fluoride ions. Figure 6.3 shows 
the absorbance spectrum of the polymeric films treated with various concentrations of 
DFP.  From the figure, the absorbance peak corresponding to the de-protonated form of 
the pH chromophore was found to be 538 nm.  It should be noted that changes in the 380-
400 nm range, correspond to spectral changes of the Al[OEP] species due to dimerization 
of the Al[OEP] species upon interaction with the extracted fluoride ions [170]. This 
spectral region can also be utilized for analytical purposes, although there is potential for 
more interferences in this region when examining real-world samples. Figure 6.4 shows 
the percentage change in absorbance at 538 nm as calculated using equation 1 for various 
concentrations of DFP tested. A single optical polymer film was used to test the various 
 90 
concentrations of DFP samples hydrolyzed by OPH for a single experimental data set.  
 
Figure 6.3: Absorbance spectra of film on quartz slide treated with various 
concentrations of DFP hydrolyzed by OPH. 
 
Another fluoride sensing film was used as a control for DFP samples with similar 
concentrations that were not hydrolyzed by OPH. The error bars of mean values were 
calculated for 3 sets of experiments using three different fluoride sensing films. The 
response was nearly linear in the range of DFP concentrations (100 nM ? 100 M), over 
which the sensing system was tested (Figure 6.4). Based on the response of sensor to 
DFP samples not treated with OPH (considered blank), it was found that concentrations 
 91 
as little as 0.1 M were detectable, which are much lower than those obtained using 
earlier DFP biosensor studies with pH FETs (20-25 M DFP) [168] and optical 
waveguides (1-10 M DFP) [111, 159]. This level of DFP detection is below the oral 
lethal dosage limits in humans (~21 M) for DFP and dermal lethal dosage limits for GB 
(0.7 M) and GD (0.5 M) nerve agents. The sensor is however restricted by the slow 
response times governed by the fluoride detection step. 
 
Figure 6.4: Response curve of the % change in absorbance at 538 nm for various 
concentrations of hydrolyzed DFP referenced against non-hydrolyzed DFP. The 
data was fit for linear regression (y = 0.4086 x + 6.465, R2 = 0.966). The standard 
error was calculated for n=3 (3 different pairs of reference and working sensors). 
 
 92 
 
It should be noted that in the current configuration, the enzymatic reaction 
requires a high pH to be efficient (pH 8.5) while the sensing chemistry of the polymer 
film requires low pH (pH 3.0). Hence, optimal analytical capabilities are only achieved 
when the reaction and detection process is carried out in two separate steps. However, it 
may be possible to ultimately overcome this limitation by locally changing pH values 
near the sensing film using electrochemical methods to generate protons [173] or by 
encapsulating the enzyme in silica matrix with appropriate surfactants to protect it against 
extreme acid conditions [174]. 
6.4. Conclusions 
The utility of enzyme-based, catalytic hydrolysis of organophosphosphate 
neurotoxins that release fluoride ions in conjunction with an amplifying fluoride optical 
sensing film is demonstrated here as a potential approach to overcome the current 
shortcomings for the development of OFP-specific sensing systems. The combined 
sensing strategy (catalytic DFP hydrolysis monitoring by fluoride selective polymeric 
films) uses simple absorbance spectroscopy to enable the detection of sub-micromolar 
quantities of analyte, a significant improvement over earlier OFP detection systems. The 
introduction of chromoionophore for monitoring absorbance changes at ~ 538 nm for the 
presence of fluoride ions enables the potential use of less expensive optical substrates 
such as commercially available glass slides and those coated with metal oxides as 
opposed to the expensive quartz slides, as their absorption bands influence on the pH 
chromophore absorbance measurements become insignificant.  
 93 
This system can be readily adapted to portable spectrophotometers such as the 
commercially available Ocean Optics USB 2000 spectrometer (Denedin, FL). Further 
enhancements in sensitivity may be introduced by increasing the path length of light by 
using the thin fluoride sensing films in a wave-guide configuration [175]. The only 
limitation at present for integration with OPH/OPAA immobilized biosensors is the two-
step reaction conditions required for hydrolysis and detection. When overcome, the 
sensing approach described here has the potential for creating highly selective and simple 
detection system capable of screening for the presence of a wide spectrum of OPs. 
 94 
7. FORMALDEHYDE DETECTION USING ACTIVATED MAGNESIUM SILICATE PARTICLES 
 
7.1. Introduction 
Formaldehyde is one of most widely found aldehydes that may be introduced in 
the atmosphere via primary emissions from natural vegetation and industrial plants [176-
179]. The more sophisticated pathways of formaldehyde introduction may involve 
photochemical degradation of organic compounds such as methyl-tert-butyl ether 
(MTBE) [177, 180] and pollutants from mobile and stationary sources [181]. 
Formaldehyde being a possible health hazard is a matter of concern. Although its 
carcinogenic properties are controversial and questionable, the International Agency for 
Research on Cancer (IARC) identifies the chemical as a probable Class 2A carcinogen 
[182]. Some of the possible health effects of formaldehyde exposure may include eye and 
upper respiratory irritation, allergic skin reactions, drowsiness, headache and nausea 
[183]. In spite of its simple chemical structure, its toxicity and widespread applications 
have led to extensive studies to control and detect its presence in the environment. More 
recently, health issues related to the presence of formaldehyde and its release from 
trailers used for temporary housing following the Iowa Floods in 2008 [184]  and 
hurricanes Katrina and Rita in 2005 [185-187] have demanded the attention and 
investigation by organizations like FEMA.  
 95 
 
Importance to the research and development of formaldehyde detection methods 
has grown over years for reasons in addition to its toxicity. For example, quantification of 
formaldehyde when present as an end product is also used to detect carbohydrates (to 
study cigarette smoke [188] and acyl-gylcerides [188, 189].  Chromatographic methods 
are the most commonly used techniques for the detection of formaldehyde [190-196]. 
These methods typically require preliminary derivation of formaldehyde using 
hydrazines, such as 2,4-dinitrophenylhydrazine (DNP) before analysis. Despite their 
sensitivity (with detection limits up to 3 nM), these methods lack selectivity.  
An attractive alternative is the approach introduced by Nash  [197]- the 
condensation of ammonia and 2,4-pentane-dione with formaldehyde, which gives a 
fluorescent compound 3,5-diacetyl-1,4-dihydrolutidine (DDL). Subsequent modification 
of Nash?s original approach has enhanced the sensitivity of formaldehyde detection. 
Among the most significant was the introduction of fluoral-P reagent, which is a mixture 
of pre-reacted ammonia and 2,4-pentanedione [198]. Fluoral-P, when utilized in a flow-
injection mode in conjunction with high performance liquid chromatography analysis of 
the products, enabled sub-micromolar detection limits [199-201]. A more recent 
development involved the use of a solid support coupled with a flow-injection technique 
[202] wherein fluoral-P was absorbed onto C-18 beads and then placed onto the column 
for detection. Application of artificial neural networks for quantification of formaldehyde 
to optimize the dynamic range of detection has also improved analytical capabilities 
[203]. 
 96 
 The current study builds on Nash method to develop a simple and robust 
formaldehyde sensor.  Magnesium silicate (florisil) micro-particles were functionalized 
with Nash reagent in the form of Fluoral-P.  The pretreated particles were attached to the 
glass slide surface and exposed to various concentrations of formaldehyde. The intensity 
of fluorescence was monitored using a fluorimeter. The experimental conditions for 
particle attachment were optimized for maximum sensitivity. 
7.2. Methods and Materials 
7.2.1. Chemical reagents 
Formaldehyde solution (37% W/W), ammonium acetate and acetic acid were purchased 
from Fisher Scientific (Fair Lawn, NJ); Florisil particles 60-100/PR mesh size (280m2/g 
surface area), Silica gel 200-425 mesh size (480m2/g surface area) and aluminum oxide 
150 mesh size (155m2/g surface area) were purchased from Sigma Aldrich (St Louis, 
MO); 2,4-pentane-dione was purchased from Acros Organics (NJ, USA), De-Ionized (DI, 
18.2 M?.cm) water was obtained from Millipore water purification system and 
polydimethyl siloxane (PDMS) was purchased from Hisco (Atlanta, GA). 12.1 mM 
formaldehyde stock solution was prepared in DI water, from which further dilutions were 
made for assays. The sample solutions were tested within 24 hours of their preparation. 
Ammonium acetate was vacuum dried prior use. All chemicals were used as received 
unless otherwise mentioned. 
7.2.2. Florisil treatment with fluoral-P 
 The florisil particles were treated with fluoral-P using the procedure described 
below that was modified from the original Nash method. Fluoral P was prepared by 
weighing 15.4 g of ammonium acetate and mixing it with 300 L of acetic acid, 200 l of 
 97 
2,4-pentane-dione and 4.5 ml of DI water. The resulting mixture was slightly heated in a 
microwave for ~ 10 s to dissolve the chemicals and form a solution. 
 For treatment of the particles, to 1.1 g of florisil, 2 ml of the above fluoral P stock 
solution and 8 ml DI water was added and mixed thoroughly. After 20 minutes of the 
treatment, the solution was filtered using a Buchner funnel. The florisil particles were air-
dried for about 2 minutes, stored in containers and sealed with paraffin film. This was 
essential to preserve the reactivity of particles since it was inevitable to have 
formaldehyde emissions from cabinets present in the lab. We will refer to such prepared 
fluoral-P treated particles as ?activated florisil?. 
7.2.3. Immobilization of activated florisil on glass slide 
 The glass slides were cut (2.5 x 1.75 cm) using a diamond tip cutter to fit onto the 
slide holder of the fluorimeter. Two different techniques were used to attach the florisil 
particles on glass slides. The first technique involved the use of a double-sided adhesive 
tape (Permanent, Scotch 3M). A 5 mm hole drilled in plexiglass was used as a template to 
control and maintain the position, quantity and area of particles covering the slide. In the 
second method, PDMS was used as a matrix scaffold to enable robust attachment of 
activated florisil on glass slides. PDMS was prepared using the standard procedure in 
which parts A and B were weighed in the ratio of 10:1 and mixed thoroughly. The 
resulting air bubbles were degassed with a vacuum pump. The precut glass slides were 
plasma cleaned and spin coated with the above PDMS at 500 rpm for 2 minutes. The 
activated florisil was sprinkled onto the PMDS-coated surface and the slide was cured at 
100oC for 15 minutes. The slides with attached florisil were then cooled to room 
temperature and stored in sealed containers until use.  
 98 
7.2.4. Instrumentation 
 Figure 7.1 shows the schematic of the experimental setup. The fluorescence 
measurements were collected with a Photon Technology International fluorimeter.  The 
glass slide was placed at 45o to the detector and light source (Figure 7.1 A). The area of 
particles attached to the glass slide (? 5mm) was within the area exposed to the detector 
(approximately 10mm x 8mm) to prevent the DDL formation outside the window of 
detection (Figure 7.1 A&B).  
In the case of gas phase detection, the florisil area of coverage was equal to the 
area of window (approximately 10mm x 8mm) that allowed the incoming and emitting 
light travelling from source and to the detector respectively. The glass slide with these 
particles attached via tape was held with the activated florisil facing the inside walls of 
the 4-sided methacrylate cuvette on which another window was made to allow the 
interaction between the particles and gaseous HCHO present inside the cuvette (Figure 
7.1 C). 
 
 99 
 
 
Figure 7.1: Schematic representation of the instrumental set-up. A: Fluorimeter 
with the glass slide placed at an angle of 45o with respect to the incoming light 
source and outgoing fluorescence to the detector. B: Sensor involving cut glass slide 
with activated florisil particles attached using a double-sided adhesive tape or 
PDMS. The diameter of the area enclosing particles was 5 mm. C: Schematic of the 
gas phase sensor with a window made on the cuvette to place the slide with activated 
florisil particles attached via tape facing the solution inside cuvette. 
 
7.2.5. Sensor Characterization 
Formaldehyde detection was carried out by placing 10 L of formaldehyde 
solution on the florisil particles attached to the glass slide via tape/PDMS. Evaporation of 
HCHO was prevented by covering the glass slide with a watch glass. This was also done 
 100 
to avoid any cross contamination of HCHO that may be released from the cabinets and 
other secondary sources present in the laboratory. After 15 minutes of reaction, emission 
scans were performed in the range of 450 nm to 530 nm with an excitation wavelength of 
410 nm. To account for false positive signals, water was used as a reference. For 
detection of formaldehyde in air, various formulations of aqueous formaldehyde was 
placed in a closed cuvette with florisil particles positioned in the headspace above the 
samples (Figure 1C). Time based scans were obtained for excitation and emission 
wavelengths of 410 nm and 482 nm, respectively.  
7.3. Results and discussions 
7.3.1. Formaldehyde detection using activated magnesium silicate particles  
Though introduced a few decades ago, the Nash approach for formaldehyde 
detection remains among the most sensitive and selective methods available. The 
underlying mechanism depends on the reaction in the presence of ammonia and acidic 
media that progresses rapidly with formaldehyde, but has increasingly negligible rates of 
reaction for higher-order ketones. For example, the same reaction with acetaldehyde is 
about 1000 times slower [197]. In addition, considerable fluorescence is expected to 
result from the product, 3,5-diacetyl-1,4-dihydrolutidine (DDL), only when formaldehyde 
is involved in the reaction.  Therefore, fluorimetric detection is advantageous compared 
to absorbance spectroscopy. DDL has an emission peak around 510 nm when excited at 
410 nm [197].  Extending the conjugated chain length of the reaction intermediates could 
shift this emission into the red or infrared regions.   
 
 
 101 
 
 
 
 
 
Figure 7.2: Top: Original Nash reaction. Bottom: Hantzsch Reaction with fluoral p 
 
The Nash method is based on the well known Hantzsch pyridine synthesis 
(Figure 7.2). With the introduction of fluoral-P [198], which is a pretreated mixture of 
ammonium acetate, 2,4-pentane-dione and acetic acid, the reaction conditions are more 
convenient with only HCHO remaining to form fluorescence product. As an imine, 
fluoral P is unstable in acidic media and decomposes to 2,4-pentane dione and ammonia. 
Hence, they are normally stored under refrigerated conditions, once prepared.  
 
 102 
To demonstrate the retention of fluoral-P reactivity of the activated florisil 
particles towards HCHO, the prepared particles were tested visually for fluorescence 
when treated with intermediate concentrations of formaldehyde (Figure 7.3) by shining 
UV light (Acticure?, Quebec, Canada) onto them.  Figure 7.3 (d) shows the fluorescent 
nature of the DDL product upon exposure to UV light for activated particles. 
Fluorescence was not observed for particles that were not activated with fluoral-P when 
treated with formaldehyde (3 c). These results demonstrate the presence of fluoral-P on 
florisil surface to produce and detect DDL. To further optimize the solid support that 
would best retain the fluoral-P reactivity, we also chose alumina and silica in addition to 
florisil particles for use as potential adsorbents by treating them with fluoral-P for 
activation.  The activated florisil particles produced the best response in terms of 
fluorescence intensity resulting from DDL formation (data not shown) and were used for 
all future experimental work. 
 103 
 
 
 
Figure 7.3: Fluorescent and colorimetric response of fluoral-P treated florisil 
particles. ?a? and ?b? correspond to untreated and activated  florisil particles under 
visible light respectively, ?c? and ?d? correspond to untreated and activated florisil 
particles respectively, when treated with formaldehyde under UV light  (350 nm). 
The figure on the right shows the calorimetric changes in activated florisil particles 
when treated with different concentrations of formaldehyde solutions.  
 
7.3.2. Activation of florisil particles 
The objective behind treating the florisil particles with fluoral-P was to provide a 
high surface area support that could retain the fluoral-P reactivity towards formaldehyde. 
In addition, this treatment would preserve the fluoral-P activity on solid support. Figure 
7.4 shows the time based scans for DDL resulting from 12.1 mM HCHO reaction with 
 104 
fluoral-P solution before and after it is used to treat florisil particles for an excitation and 
emission wavelength of 410 and 490 nm, respectively. The reaction of HCHO with 
residual fluoral-P from the florisil treatment produced fluorescence with an intensity 
about one order of magnitude less than fresh fluoral-P, which indicated that the majority 
of fluoral-P was successfully adsorbed on to florisil to form ?activated? particles.   
 
Figure 7.4: Effect of florisil treatment with fluoral P. Time based intensities 
intensity of DDL product obtained on reaction between HCHO and fluoral-P before 
its treatment with florisil and the reaction between HCHO and filterant of fluoral-P 
solution obtained after its treatment with florisil particles. The inset shows the 
reduced scale of intensity for the treated fluoral-P solution (FP activity after florisil 
treatment. 
 
 105 
7.3.3. Response Curve 
Though the described method of activating florisil particles showed a large step 
towards simplicity for formaldehyde detection, it still requires a robust setup for sensor 
development.  A method was devised to use glass slides as test substrates, distribute 
activated florisil onto the surface, and then add analyte solution. The resulting 
fluorescence appeared on the treated side, which was monitored. We exploited two 
techniques to attach pretreated florisil particles on glass slides. The first method involved 
the use of a double-sided adhesive tape. Optical noise was one of the problems 
encountered while using a tape to immobilize the florisil particles on glass slides. 
Another problem encountered with the use of tape was loss of particle adhesion upon the 
addition of HCHO. To address these problems, polydimethyl siloxane (PDMS) was used 
as a matrix to support the florisil particles on the glass slide.  PDMS is optically 
transparent in the visible range and inert to most chemicals. 
The sensitivity of the activated particles on the glass slide was characterized by 
exposing them to increasingly dilute concentrations of HCHO solutions. Water was used 
as a control to account for fluorescence resulting from unknown HCHO that may be 
present in the environment. In the earlier section, it was stated that the emission peak was 
around 490nm. However, it was also found that lower concentrations of HCHO resulted 
in the shift of emission peaks to lower wavelengths with an average maximum at 485 nm 
for the micro molar concentration range (Figure 7.5). Thus, the response curve was 
plotted for the % change in fluorescence intensity of DDL product at 485 nm for varying 
concentrations of formaldehyde. 
 106 
100Iw IwIdI n t e n s i tyinc h a n g eP e r c e n t a g e  
 
where, Id is the intensity of DDL from reaction with HCHO solution and Iw is the 
intensity for control (water).  
 
 
Figure 7.5: Influence of formaldehyde concentration on the fluorescence emission 
wavelength. The inset graph shows the variation of peak emission wavelength and 
intensity as a function of formaldehyde concentration.  
 
 
 107 
Figure 7.6 shows the response curve obtained for the different concentrations of 
HCHO when treated with activated florisil particles attached to glass slides using an 
adhesive tape and PDMS. Although the response curves for tape and PDMS supports 
show similar trends, the lowest concentration that could be detected was about 5 M for 
PDMS and 2.5 M for adhesive tape immobilization. The range for linear response for 
formaldehyde was between 2 M and 50 M HCHO for both florisil supports. The lower 
sensitivity with the use of PDMS matrix may be attributed to decreased wetting of the 
PDMS surface by the aqueous HCHO solution due to the hydrophobic nature of PDMS. 
In addition, the exposed surface of the activated particles may have been reduced through 
the use of PDMS.  However, Figure 7.6 indicates that the surface area of the florisil 
particles masked by PDMS did not affect the trend of HCHO response that was obtained 
using tape. It should also be noted that the activity of fluoral-P in PDMS was retained 
despite the thermal cure during sample preparation. 
 108 
 
 
Figure 7.6: Response curve obtained for the different concentrations of 
formaldehyde when treated with activated fluoral-P when immobilized on glass slide 
using an adhesive tape and PDMS. Inset graph shows the linear range of response 
for either florisil support with linear regression plots. For tape: y= 0.9894 x + 
6.8908, R = 0.983, for PDMS y= 0.8520 x + 20.8076, R = 0.9643.  
 
Vapor detection of HCHO was tested by exposing the fluoral P activated fluorisil 
particles to the headspace of a sample cell loaded with varying concentrations of HCHO 
in water.  Here, the activated particles were attached to the glass slides using the adhesive 
tape configuration only. The resulting fluorescence response curve is shown in Figure 
7.7. The slope was calculated over a time interval of 200 s and subtracted from the 
 109 
response obtained for water. As seen in the graph, the time required to attain the 
maximum fluorescence intensity decreased from approximately 8 minutes (<1 mM 
HCHO) to less than 3 minutes with increasing formaldehyde concentrations. 
Concentrations above 1 mM HCHO took less than 5 minutes to attain saturation intensity. 
It was found that for concentrations above 1mM HCHO, DDL formation took place 
within the first 5 minutes while nearly 10 minutes were required for HCHO 
concentrations under 1 mM. 
 
 
Figure 7.7: Variation in the response time to achieve maximum fluorescent intensity 
(?) and slope (?) of DDL fluorescence for different concentrations of formaldehyde 
for gas phase detection. The inset shows the linear range of response for 
formaldehyde of concentrations from 0.025 mM to 1 mM. (y=0.1108x + 9.1625, R = 
0.9934).  
 
 110 
 
7.4. Conclusions 
In this study, a simple, yet robust fluorescence detection method for formaldehyde 
by utilizing magnesium silicate (florisil) micro particles activated with modified Nash 
reagent in the form of fluoral-P was developed. The presence of a solid support confined 
the reaction environment to these particles within the window of detector, thereby 
increasing the system sensitivity. The sample volume requirement of the developed 
sensor is within 10 microliters, which is a significant improvement over earlier detection 
systems [204] that require several hundreds of microliters. The robust characteristic of 
the sensor with the introduction of PDMS for the attachment of activated florisil to glass 
slides parallels the ease of litmus paper testing for acid/base analysis. The resemblance is 
stronger considering that the DDL product produces visible changes in color in addition 
to fluorescence observed under UV light. This method is compatible with on-site field 
analysis of trailer homes using a portable fluorimeter and a small-format light source and 
emission filters. 
 
 
 
 
 
 
 
 
 
 111 
8. OVERALL CONCLUSIONS 
 
Integration of sensing and transducing components using appropriate surface 
chemistries and morphologies are critical to the detection of analytes with good 
sensitivity, stability, selectivity and quick response times for applications in chemical and 
biological sensors. 
The studies presented in this dissertation investigate the feasibility of titanium 
dioxide nanoparticles and magnesium silicate particles as potential surfaces conducive for 
sensing elements with the specific aim to enhance sensitivity via signal amplification of 
reporter molecules that traduce target-recognition element interactions. To achieve this, a 
planar waveguide coated with titanium dioxide nanoparticles was used as a biomaterial 
scaffold to develop an enzyme based evanescent array biosensor. Characterization 
experiments showed the presence of ~ 13 nm anatase phase crystalline particles in the 
film of thickness ~ 2.2 m. Theoretical calculations for estimations on its refractive index 
showed the presence of a relatively low refractive index anatase film of ~ 1.9 as 
compared to 2.52 for a pore free anatase film. To make the surface amenable for covalent 
immobilization of proteins for biosensor development, modifications were made using 
mercaptosilane and cross-linkers. An enzyme organophosphorus hydrolase (OPH), 
conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF) was used 
as a biosensing element and immobilized on the titanium dioxide surface for the detection 
 112 
of organophosphate neurotoxins. Bovine serum albumin (BSA) was used as a non-
enzymatic scaffold protein for CNF attachment at the reference spots to allow for the 
discrimination of pH changes due to enzymatic and non-enzymatic reactions. An array 
biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the 
detection platform and appropriately modified for enzyme-based measurements. The 
developed sensor enabled the detection of ~2.5 ?M paraoxon, and ~10 ?M DFP and 
parathion, respectively. Very short response time of 30 s was achieved with a total 
analysis time of less than 2 min. When operated at room temperature and stored at 4 ?C, 
the waveguide retained reasonable activity for greater than 45 days. However, there was a 
significant drop in sensitivity of signal within 3 weeks of sensor preparation. Desorption 
of some portion of adsorbed biomolecules from porous substrates and loss of enzymatic 
activity over time due to its covalent immobilization are some of the possible reasons 
behind this loss in signal. The experiments conducted to measure the presence of OPH in 
the storage buffer to check for desorption over a period of 2 months did not result in 
measureable concentrations of OPH. Hence, the loss in enzymatic activity was attributed 
to drop in sensitivity for covalently immobilized enzyme on titanium dioxide glass slides.  
In light of the above obervations, an alternative strategy of protein immobilization 
via its encapsulation in silica particles was studied.  This involved rapid enzyme 
immobilization directly on a waveguide surface by encapsulation in a silica matrix. OPH 
was again used as a model enzyme to demonstrate the utility of lysozyme-mediated silica 
formation for enzyme stabilization. Silica morphology and the efficiency of OPH 
encapsulation were directly influenced by the precursor choice used in silica formation. 
Covalent attachment of the lysozyme template directly to the waveguide surface provided 
 113 
a stable basis for silica formation and significantly increased the surface area for OPH 
encapsulation. OPH conjugated to a pH-responsive fluorophore was encapsulated in 
silica and patterned to a waveguide surface to demonstrate the immobilization strategy 
for the development of an organophosphate array biodetector. Silica-encapsulated OPH 
retained its catalytic activity for nearly 60 days with a detection limit of paraoxon of 
35 ?M. The encapsulation technique provides a potentially versatile tool with specific 
application to biosensor development.  
From the two methodologies developed to immobilize enzyme on glass slides 
coated with commercially obtained titanium dioxide nanoparticles, it was concluded that 
the covalent immobilization technique required longer sensor preparation times (~ 3 
days) and provided better sensitivity for paraoxon detection while the encapsulation 
technique of immobilization required ~ 10 hours for sensor preparation and provided 
relatively superior OPH stability. As an extension to OP detection, a novel system for the 
detection and quantification of organofluorophosphonates (OFP) was developed by using 
an optical sensing polymeric membrane to detect the fluoride ions produced upon OFP 
hydrolysis.  Diisopropyl fluorophosphate (DFP), a structural analogue of Type G 
Chemical Warfare Agents such as Sarin (GB) and Soman (GD) nerve agents, was used as 
the surrogate target analyte.  An optical sensing fluoride-ion-selective polymeric film was 
formulated from plasticized PVC containing aluminum(III) octaethylporphyrin and ETH 
7075 chromoionophore (ALOEP-ETH 7075).  Selected formulations were used to detect 
the fluoride ions produced by the catalytic hydrolysis of DFP by the enzyme 
organophosphate hydrolase (OPH, EC 3.1.8.1).  The changes in absorbance that 
corresponded to the deprotonated state of chromoionophore within the film resulted from 
 114 
simultaneous co-extraction of fluoride and protons, as DFP hydrolysis occurred in the 
solution phase in contact with the film.  The developed sensing system demonstrated 
excellent sensitivity for concentrations as low as 0.1 M DFP. 
Finally, the feasibility of magnesium silicate particles as scaffolds for chemical 
sensing elements was tested for the development of chemical sensors. To test this, a 
sensing strategy for formaldehyde (HCHO) detection in liquid and vapor samples was 
designed. Preliminary results showed that the sensors, in addition to being sensitive and 
flexible sensor, was extremely simple to build. Modified Nash reagent is utilized in the 
form of fluoral-P and is absorbed onto magnesium silicate (florisil) micro-particles.  
These pre-activated particles were attached to the glass slide surface via tape and via 
dispersion in polydimethyl siloxane (PDMS) matrix. Fluorimetric quantification of a 
photoluminescent product of formaldehyde and fluoral-P activated florisil, namely, 3,5-
diacetyl-1,4-dihydrolutidine (DDL) allows detection of aqueous HCHO for 
concentrations as low as 2 M. The only stipulation with its implementation on portable 
devices for on-site detection was the availability of appropriate excitation sources for 
DDL product to emit fluorescence. 
 
 
 
 
 
 
 
 115 
9. FUTURE WORK 
 
9.1. Bioinspired mediation of titanium dioxide for coating on glass slides 
Studies have shown the capability of lsyzome to mediate silica and titanium 
dioxide nanoparticles under room temperature conditions [205].  The titanium dioxide 
nanoparticles used as a scaffold material for enzyme immobilization detailed in chapters 
4 and 5, was synthesized via hydrothermal method (obtained from the manufacturer). 
Silica encapsulation of proteins conjugated to reporter fluorophores during its mediation 
by lysozyme on glass slide surfaces coated with titanium dioxide nanoparticles was 
studied in chapter 5. As an extension to this study and to develop an alternative technique 
for titanium dioxide deposition, glass slide that has immobilized lysozyme, can be treated 
with titanium dioxide precursor such as Titanium (IV) bis(ammonium lactate) 
dihydroxide (TiBALDH) solution. This method has the potential for direct encapsulation 
of enzymes on glass slide surfaces with titanium dioxide nanoparticles.  
Figure 9.1 shows the preliminary results on the deposition of titanium dioxide 
nanoparticles on glass slide via lysozyme mediation in alternative layers. 4 layers and 8 
layers of deposition showed the presence of titanium dioxide covering the entire glass 
slide surface. Two different concentrations of lysozyme (10 mg/ml and 100 mg/ml) were 
used for comparison purposes. The layers appear to be porous for both the concentrations 
used. However, films prepared with 10 mg/ml lysozyme appeared produce a film of 
 116 
particles similar in size to the titanium dioxide used in the earlier studies. Further studies 
are required to control and determine the surface area, porosity and refractive index of the 
film to enable its wave guiding properties. Since the lysozyme is expected to get 
encapsulated during the mediation process, the developed films also have potential 
antibacterial applications due to the presence of lysozyme on their surface.  
 
 
Figure 9.1: Titanium dioxide deposition on glass slides via lysozyme mediation.  
8L-100: 8 alternative layers of lysozyme  (100 mg/ml) and titanium dioxide 
precursor. 8L-10: 8 alternative layers of lysozyme  (10 mg/ml) and titanium dioxide 
precursor. 4L-100: 4 alternative layers of lysozyme  (100 mg/ml) and titanium 
dioxide precursor. 4L-10: 4 alternative layers of lysozyme  (10 mg/ml) and titanium 
dioxide precursor.  
 
 117 
 
9.2. Enzyme stabilization in extreme pH conditions 
Enzymes are rendered inactive at extreme conditions of pH. For example, a basic 
enzyme like OPH, which has an isoelectric point of ~8.3, does not hydrolyze OPs at pH 
of 3.0, attributable to its protonation. These biomolecules are specifically inclined to 
undergo denaturing when present in solution. Immobilization of enzymes such as their 
encapsulation in sol gel matrices are known to make them stable attributed to the ceramic 
cage possibly preventing ?unfolding-refolding? motions thereby preserving enzyme 
activity. Studies by Frenkel-Mullerad et. al. have shown that alkaline phosphatase, that is 
active in a pH range of 9-10, can maintain its activity at pH as low as 0.9, when 
encapsulated in silica gel with an anionic surfactant [174]. According to Frenkel-
Mullerad et.al., the pH inside the cage with enzyme containing a reservoir of ~ 100 water 
molecules has only a few protons (implying greater pH) in comparison to the external 
reservoir containing many water molecules (implying lower pH). The higher internal pH 
is thus sufficient to maintain the enzyme activity.  
Chapter 6 in this dissertation shows the development of DFP sensor using a 
fluoride ion selective membrane that operates at pH 3.0. Here, the hydroxyl ions interfere 
with fluoride ions to complex with aluminum porphyrin membrane when the system is 
operated at high pH and OPH in inactived at low pH due to its protonation. Hence, a two 
step reaction system of DFP hydrolysis at high pH following which, the solutions were 
diluted to reduce the pH for fluoride ion detection at pH 3.0, was designed. To 
circumvent this two step reaction, OPH can be encapsulated within silica particles 
synthesized using sol-gel process as described by Frenked- Mullerad et.al. for alkaline 
 118 
phosphotase, to preserve its activity at pH 3.0.  Figure shows the modified experimental 
set-up with preliminary results for DFP detection carried out under single pot reaction 
conditions.  
  
 
 
Figure 9.2: Top figure shows the schematic of the experimental set-up for single pot 
reaction. The graph shows the response of the polymer film to different 
concentrations of DFP. 
 119 
By introducing biosensing enzymes in the titanium dioxide precursor solution 
during its mediation via lysozyme as was explained in section 9.1, along with appropriate 
surfactant during encapsulation as explained in section 9.2; the potential application of 
titanium dioxide nanoparticles to protect enzymes at extreme pH may be realized. 
Further, the influence of nanoparticle size to modulate the microenvironmental pH may 
be studied. With this, the possibility of titanium dioxide nanoparticles to act as smart 
passive materials that enhance the functional properties of sensing layer by providing a 
biocompatible, high surface area, waveguiding support with the capability for 
biomolecular stabilization at extreme conditions may be realized. 
 120 
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