Abstract
Antibody-antigen (Ab-Ag) recognition is the primary event at the basis of many biosensing platforms. In label-free biosensors, these events occurring at solid-liquid interfaces are complex and often difficult to control technologically across the smallest length scales down to the molecular scale. Here a molecular-scale technique, such as single-molecule force spectroscopy, is performed across areas of a real electrode functionalized for the immunodetection of an inflammatory cytokine, viz. interleukin-4 (IL4). The statistical analysis of force-distance curves allows us to quantify the probability, the characteristic length scales, the adhesion energy, and the time scales of specific recognition. These results enable us to rationalize the response of an electrolyte-gated organic field-effect transistor (EGOFET) operated as an IL4 immunosensor. Two different strategies for the immobilization of IL4 antibodies on the Au gate electrode have been compared: antibodies are bound to (i) a smooth film of His-tagged protein G (PG)/Au; (ii) a 6-aminohexanethiol (HSC6NH2) self-assembled monolayer on Au through glutaraldehyde. The most sensitive EGOFET (concentration minimum detection level down to 5 nM of IL4) is obtained with the first functionalization strategy. This result is correlated to the highest probability (30%) of specific binding events detected by force spectroscopy on Ab/PG/Au electrodes, compared to 10% probability on electrodes with the second functionalization. Specifically, this demonstrates that Ab/PG/Au yields the largest areal density of oriented antibodies available for recognition. More in general, this work shows that specific recognition events in multiscale biosensors can be assessed, quantified, and optimized by means of a nanoscale technique.
Highlights
Immunosensing exploits one of nature’s most optimized molecular recognition mechanisms, namely the interaction between an antigen (Ag) and its specific antibody (Ab)[1]
Detection involves change of electrostatic charge due to about 1012 local recognition events per cm[2], single molecule force spectroscopy (SFS) involves the detection of a few biorecognition events occurring at nm length-scales
The success of electrochemically-gated organic field-effect transistor (EGOFET) in detecting nanomolar concentrations of IL4 in the case of protein G mediated Ab immobilization correlates with the larger areal density of available anti-IL4 antibodies as evidenced by SFS measurements, corresponding to 30% coverage with respect to just 10% in the case glutaraldehyde-based immobilization
Summary
Immunosensing exploits one of nature’s most optimized molecular recognition mechanisms, namely the interaction between an antigen (Ag) and its specific antibody (Ab)[1]. Detecting Ab-Ag interactions with a label-free sensing scheme requires the integration of biorecognition moieties at a solid-liquid interface and their coupling with the transducer. The transduction of the molecular binding event should occur with minimum, if not any, further chemical amplification or development steps[3]. This is relevant for point-of-care applications and in-field deployed sensors. Sensitivity and specificity both depend on how the bio-recognition groups are made available to the target and on the coupling between environment and transducer
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