Abstract
Electrochemical sensors based on antibody-antigen recognition events are commonly used for the rapid, label-free, and sensitive detection of various analytes. However, various parameters at the bioelectronic interface, i.e., before and after the probe (such as an antibody) assembly onto the electrode, have a dominant influence on the underlying detection performance of analytes (such as an antigen). In this work, we thoroughly investigate the dependence of the bioelectronic interface characteristics on parameters that have not been investigated in depth: the antibody density on the electrode’s surface and the antigen incubation time. For this important aim, we utilized the sensitive non-faradaic electrochemical impedance spectroscopy method. We showed that as the incubation time of the antigen-containing drop solution increased, a decrease was observed in both the solution resistance and the diffusional resistance with reflecting boundary elements, as well as the capacitive magnitude of a constant phase element, which decreased at a rate of 160 ± 30 kΩ/min, 800 ± 100 mΩ/min, and 520 ± 80 pF × s(α-1)/min, respectively. Using atomic force microscopy, we also showed that high antibody density led to thicker electrode coating than low antibody density, with root-mean-square roughness values of 2.2 ± 0.2 nm versus 1.28 ± 0.04 nm, respectively. Furthermore, we showed that as the antigen accumulated onto the electrode, the solution resistance increased for high antibody density and decreased for low antibody density. Finally, the antigen detection performance test yielded a better limit of detection for low antibody density than for high antibody density (0.26 μM vs 2.2 μM). Overall, we show here the importance of these two factors and how changing one parameter can drastically affect the desired outcome.Graphical abstract
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Electrochemical sensors based on antibody-antigen recognition events are commonly used for identifying many biological markers [1,2,3]
Among these three detection mechanisms, conductometric detection is known for its high sensitivity to physicochemical reactions at the bioelectronic interface and the ability to differentiate these reactions by removing background effects [7]
Antibody-conjugated electrochemical biosensors (‘immunosensors’) are used in many fields, including environmental protection, biotechnology, drug screening, food safety, security, veterinary medicine, and the monitoring and diagnosis of diseases [8]. These biosensors are considered to have several advantages such as short test times and low test costs [9]. These advantages are achieved by covalently binding the antibody probe to the electrode surface, and reducing the amount of antibody needed for the test and increasing its accessibility [10]
Summary
Electrochemical sensors based on antibody-antigen recognition events are commonly used for identifying many biological markers [1,2,3] These sensors utilize various detection mechanisms that are classified based on the output electrical signal: current (namely, amperometric detection), potential (namely, voltammetric detection), and impedance (namely, conductometric detection) [4,5,6]. Antibody-conjugated electrochemical biosensors (‘immunosensors’) are used in many fields, including environmental protection, biotechnology, drug screening, food safety, security, veterinary medicine, and the monitoring and diagnosis of diseases [8] These biosensors are considered to have several advantages such as short test times and low test costs [9]. Some electrochemical parameters of the bioelectronic interface, such as the electrode surface area [11], the electrode material [12], or the linker concentration [13], have already been reported to drastically affect the resulting sensitivity, only limited information has been reported on biological components, such as the antibody probe density and the antigen analyte incubation time
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