Development of Analytical Systems for Detection of Biomarkers and Neurotoxic Exposure

Abstract

Exposure to the chemical and biological compounds in environment poses a serious health threat to humanity. Timely awareness of the chemicals in environment plays a vital role in exposure assessment, damage control, and diagnosis and treatment of diseases to minimized the harm caused by the ongoing exposure. The monitoring of these compounds can be achieved by in-situ environmental detection and biomonitoring- detection of biomarkers of exposure. To achieve biomarker detection in a more efficient and reliable fashion, a Multi-Parametric/ Multimodal apparatus System (MPMS) was developed in this study. MPMS combines electrochemical measurement, Surface Plasmon Resonance (SPR), and fluorescence spectroscopy into a SPR/fluorescence spectroelectrochemical technique. It enables not only quantitation of biomarker molecules with validation from the three signals, but also monitoring of the sensor’s assembly process. In addition to its use in the development of biosensors, MPMS could potentially be applied in many other areas such as drug delivery systems, coatings for biomedical implants, electrochemical photocatalysis, electroluminescence, surface enhanced chemiluminescence, etc. This will allow unprecedented understanding for those functional interfaces of interest. As an example, the unique capabilities of the MPMS system were utilized to develop the Electrochemical Proximity Assay (ECPA) of Platelet-Derived Growth Factor (PDGF). PDGF is a potential biomarker for the exposure to asbestos, and crystalline silica. PDGF is also an important latent biomarker in diagnosis and recognition of malignant diseases such as cancers, glioblastomas and sarcomas. Therefore, the sensitive and reliable detection of PDGF has been attracting considerable attention. In this study, ECPA, a recently developed protein recognition strategy for point-of-care test, was employed for PDGF detection due to its high detection sensitivity and design flexibility. To demonstrate and testify the capabilities of MPMS, ECPA model system was tested first. As a result, MPMS achieved not only recognition of binding components involved in ECPA model system, estimation of their thicknesses and surface coverages, but more importantly, highly reliable in-situ monitoring of dynamic changes of components involved in interfacial binding via cross-validation and confirmation from three simultaneously generated signals- SPR, fluorescence and electrochemistry. In addition, the obtained corresponding proportions among magnitudes of three signals provide crucial information for future studies on simultaneous characterization of multiple components in one step, and differentiation of non-specific binding events. Another advantage using this technique is, the excitation of fluorescence is not only confined by surface plasmons, but by photons, so the fluorescence information can be also gained as the distance of fluorophores from the surface exceeds the decay length of surface plasmons. Subsequently, we studied the ECPA of PDGF using SPR/fluorescence spectroelectrochemical technique. It was found the cations present in the buffer solution play a key role in the hybridization efficiency, the aptamer-protein interaction and the background signals. As proper number of complimentary pairs, and proper buffer used, the electrochemical signals corresponded linearly to various concentrations of PDGF. In addition, MPMS has been proven able to characterize the process of proteins’ covalent binding and layer-by-layer assembly in both sequential and simultaneous fashion. Tricresyl phosphate (TCP) is a class of neurotoxic organophosphate cholinesterase inhibitors, exposure to which may induce nausea, vomiting, diarrhea and abdominal pain. Despite of its acute toxicity, TCP has been widely used in industrial applications, including flame retardants, and plasticizers. In many commercial jet oils, TCP is blended as an anti-wear additive, which could be released into the cabin air in the case of oil leakage of aircraft hydraulic systems. The TCP in cabin air could enter passenger’s body through ingestion, inhalation of aerosols, and dermal sorption, and cause the symptoms above. To timely monitor the TCP leakage, an automatic TCP sampling system was coupled with an electrochemical flow injection analysis system. TCP was absorbed and hydrolyzed by an alkaline alumina column, and subsequently washed out of the column for direct electrochemical detection. After each detection, NaOH solution was used for column regeneration. In spite of efficient hydrolysis of TCP through alkaline catalyst, the alkaline catalyst itself is consumed constantly during reaction, so continuous addition of alkaline catalyst is in demand for in-situ monitoring of TCP in environment. This not only introduces more chemicals to environment, but also increases the reagent and cost of detection. Furthermore, the resulted extremely basic solution leads to some limitation and difficulty for the detection system. To avoid mentioned problems, we explored on the development of reagentless catalyst for TCP’s hydrolysis. Ru(OH)3 and Fe(OH)3 were proposed to be capable of catalyzing the alcoholysis and hydrolysis of TCP at room temperature.