Methods to Improve the Stability and Selectivity of Electronic Biosensors
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Gezahagne, Hilena
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Abstract
Abstract
The enzyme-linked immunosorbent assay (ELISA) is a powerful diagnostic tool used for the accurate detection of serological diseases.1-4 Due to the high selectivity and sensitivity associated with this label-based approach, the ELISA remains one of the most prevalently used bioanalytical tools.1-4 Despite its advantages, the ELISA is a costly, time-consuming procedure that requires trained personnel to perform and interpret the assay, which delays treatment and increases morbidity and mortality rates.5 Consequently, label-free point of care technologies (PoCTs) are being developed to improve patient outcomes by delivering accurate and timely diagnostic results. Electronic biosensors, a type of label-free device, show great promise due to their high sensitivity and ease of miniaturization for use as a PoCT.6-10
Despite the promise of these label-free sensors, field deployable electronic biosensors are not commercially available. The successful commercialization of these label-free technologies relies on their ability to accurately detect target analytes in complex sample matrices, while demonstrating stability, selectivity, sensitivity, and reproducibility in the sensing response. Several factors influence the accuracy and reliability of the observed biosensing signal. Therefore, this work uses a systematic bottom-up research approach to investigate the correlation between the intrinsic properties of electronic biosensors, the external experimental factors and the observed biosensing response. This methodical approach advances the commercialization of electronic biosensors by improving the stability, selectivity, sensitivity, and reproducibility of these sensors.
Using the aforementioned bottom-up approach, the key components of electronic biosensors can be classified into three key categories: the sensor surface with the adsorbed surface linker, the bioreceptor and target biomolecules, and the transducer that converts the biochemical signal into an electronic response.11
The work presented in this thesis begins by examining the relationship between the structural properties of the adsorbed surface linker and the stability and reproducibility of impedimetric biosensors. Previous studies examine the relationship between the structural properties of adsorbed surface linkers and their end use applications.12-14 Carboxylic acid-terminated thiols are commonly used in electronic biosensors due to their ease of preparation, strong chemisorption on to gold (Au) sensor surfaces, and their ability to covalently bind receptor proteins via carbodiimide cross-linker chemistry.12-14 While the relationship between the structural properties and the uniformity of thiol-based self-assembled monolayers (SAMs) is well-documented, the impact of these properties on the stability of impedimetric biosensors remains unexplored in literature.12
This is crucial because drift in the impedimetric baseline response directly affects the stability of receptor-functionalized sensors, resulting in irreproducible impedimetric biosensing responses.15 Therefore, the first step in developing a stable and reproducible biosensor is to correlate the structural properties of adsorbed surface linkers to the magnitude of the impedimetric baseline drift. These structural properties can then be carefully selected to suppress the baseline drift, thereby improving the stability and reproducibility of impedimetric biosensing measurements.
The second step in this bottom-up approach involves optimizing the selectivity and sensitivity of these biosensors. The selectivity of biosensors is partially ascribed to the intrinsic specificity of receptor-analyte interactions.16-21 This work focuses on antigen-antibody interactions, with the receptor protein covalently immobilized using carbodiimide cross-linker chemistry. The immobilized receptor has specific binding sites that ensure the selective attachment of the target analyte. The sandwich ELISA is the most commonly used molecular diagnostic platform due to its high sensitivity and selectivity, making this sandwich approach particularly relevant for improving the selectivity and sensitivity of electronic biosensors.22-24
While previous literature has demonstrated the successful application of the sandwich approach in electronic biosensors, the fundamental differences in the sensing mechanisms between the ELISA, a label-based sensor, and electronic biosensors, which are label-free, have not been accounted for.25-28 To reliably adapt the sandwich approach to electronic biosensors, these differences must be studied. The work presented in this thesis studies the influence of charge on the translation of the sandwich approach to impedimetric and potentiometric biosensors. The Debye length was adjusted by diluting the measurement buffer. At biologically relevant ionic strengths, the sandwich approach could not be effectively used to improve the selectivity and sensitivity of the potentiometric sensors. In contrast, by selecting the appropriate experimental parameters, the impedimetric biosensors successfully used the sandwich approach to improve their selectivity and sensitivity at biologically relevant ionic strengths. The work presented in this thesis establishes a reliable method for translating the sandwich approach to impedimetric biosensors, thereby improving the selectivity and sensitivity of this label-free sensor.
Lastly, the electronic biosensing measurements studied rely on the specificity of antigen-antibody interactions. Consequently, biological buffers are necessary to maintain the stability and functionality of these proteins. Buffers are widely perceived to behave as spectators in these systems. As a result, buffers that have similar buffering capacities have been interchangeably used in several biosensing assays. Surface plasmon resonance (SPR) is used to study the influence that two commonly used buffers, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline-ethylenediaminetetraacetic acid polysorbate (HBS-EP) and phosphate-buffered saline (PBS), have on the real-time biomolecular interactions between two categories of antigenic proteins and their target antibodies. HBS-EP was found to improve the rate of antibody attachment to polyhistidine tagged capture antigens, which subsequently improved the analytical sensitivity of the sensor. Although buffers have been used interchangeably in biosensing literature, this work highlights the influence of buffers on antigen-antibody binding kinetics and the subsequent analytical sensitivity of these sensors. By using a bottom-up experimental approach, the work presented in this thesis systematically investigates and improves the experimental factors that influence the stability, selectivity, sensitivity and reproducibility of electronic biosensors.
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2024-09-06
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