Across a wide range of biomarker detection schemes, carboxylterminated thiol self-assembled monolayers (COOH-SAMs) on Au are utilized to functionalize the sensing surface with a bioreceptor via amine coupling. However, commonly used COOH-SAM preparation methods result in large defect densities due to cooperative hydrogen bonding between carboxylic acid end groups, which in turn leads to large nonselective adsorption (NSA) of proteins to hydrophobic surfaces exposed by these defects. In this work, X-ray photoelectron spectroscopy is used to characterize the quality of COOH-SAMs by differentiating properly and improperly bound S groups. NSA of model small (lysozyme), medium (bovine serum albumin, BSA), and large (fibrinogen) proteins on COOH-SAMs is presented. Due to large NSA to COOH-SAMs, blocking is necessary for sensor reliability. However, conventional blocking techniques occur after functionalization (postblocking) and fail to prevent receptor NSA to the sensor surface during functionalization, which can cause receptor denaturation and allow the receptor to wash off the surface during later sensing. Here, a procedure is developed where the surface of COOH-SAMs is pretreated by blocking agents before functionalization. Preblocking can shield the COOH-SAM from oxidation, improve baseline stability, and prevent receptor denaturation. In this method, a preblocking protein orthogonal to the immunological system of interest is used to cover hydrophobic, nonselective sites on the sensor surface while still leaving carboxylic acid head groups available for covalent functionalization. Amine functionalization of BSA, antibody BSA, and antibody haptoglobin (aHp) is successfully completed after gelatin preblocking. Haptoglobin detection via surface plasmon resonance with a preblocked aHp sensor is shown to perform similarly to conventional postblocking, while demonstrating improved baseline stability and percentage of active receptors.

A fully integrated system that combines extended gate field-effect transistor (EGFET)-based potentiometric biosensors and electrochemical impedance spectroscopy (EIS)-based biosensors has been demonstrated. This integrated configuration enables the sequential measurement of the same immunological binding event on the same sensing surface and consequently sheds light on the fundamental origins of sensing signals produced by FET and EIS biosensors, as well as the correlation between the two. Detection of both the bovine serum albumin (BSA)/anti-BSA model system in buffer solution and bovine parainfluenza antibodies in complex blood plasma samples was demonstrated using the integrated biosensors. Comparison of the EGFET and EIS sensor responses reveals similar dynamic ranges, while equivalent circuit modeling of the EIS response shows that the commonly reported total impedance change (ΔZtotal) is dominated by the change in charge transfer resistance (Rct) rather than surface capacitance (Csurface). Using electrochemical kinetics and the Butler-Volmer equation, we unveil that the surface potential and charge transfer resistance, measured by potentiometric and impedance biosensors, respectively, are, in fact, intrinsically linked. This observation suggests that there is no significant gain in using the FET/EIS integrated system and leads to the demonstration that low-cost EGFET biosensors are sufficient as a detection tool to resolve the charge information of biomolecules for practical sensing applications.

Biosensors based on electrochemical impedance spectroscopy detect the binding of an analyte to a receptor-functionalized electrode by measuring the subsequent change in the extracted charge-transfer resistance (RCT). In this work, the stability of a long chain alkanethiol, 16-mercaptohexadecanoic acid was compared to that of a polymer-based surface linker, ortho-aminobenzoic acid (o-ABA). These two classes of surface linkers were selected due to the marked differences in their structural properties. The drift in RCT observed for the native SAM-functionalized gold electrodes was observed to correlate to the drift in the subsequent receptor functionalized SAM. This indicates the importance of the gold-molecule interface for reliable biosensing. Additionally, the magnitude of the baseline drift correlated to the percentage of thiol molecules improperly bound to the gold electrode as evaluated using X-ray photoelectron spectroscopy. Alternatively, the o-ABA-functionalized gold electrodes demonstrated negligible drift in the RCT drift. Furthermore, these polymer-functionalized gold electrodes do not require a stabilization period in the buffer solution prior to receptor functionalization. This work emphasizes the importance of understanding and leveraging the structural properties of various classes of surface linkers to ensure the stability of impedimetric measurements.

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Mu-opioid receptors (MORs) in the nucleus accumbens (NAc) can regulate reward-related behaviors that are dependent on mesolimbic dopamine, but the precise mechanism of this MOR regulation is unknown. We hypothesized that MORs within the NAc core regulate dopamine release. Specifically, we infused the MOR antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) into the NAc core while dopamine release was evoked by electrical stimulation of the ventral tegmental area and measured by fast-scan cyclic voltammetry. We report that CTAP dosedependently inhibited evoked dopamine release, with full blockade achieved with the 8 μg infusion. In contrast, evoked dopamine release increased after nomifensine infusion and was unchanged after vehicle infusion. These findings demonstrate profound local control of dopamine release by MORs within the NAc core, which has implications for regulation of reward processing.