Aptamer-based Field-Effect Biosensor for Tenofovir Detection

During medical treatment it is critical to maintain the circulatory concentration of drugs within their therapeutic range. A novel biosensor is presented in this work to address the lack of a reliable point-of-care drug monitoring system in the market. The biosensor incorporates high selectivity and sensitivity by integrating aptamers as the recognition element and field-effect transistors as the signal transducer. The drug tenofovir was used as a model small molecule. The biointerface of the sensor is a binary self-assembled monolayer of specific thiolated aptamer and 6-mercapto-1-hexanol (MCH), whose ratio was optimized by electrochemical impedance spectroscopy measurements to enhance the sensitivity towards the specific target. Surface plasmon resonance, performed under different buffer conditions, shows optimum specific and little non-specific binding in phosphate buffered saline. The dose-response behavior of the field-effect biosensor presents a linear range between 1 nM and 100 nM of tenofovir and a limit of detection of 1.2 nM. Two non-specific drugs and one non-specific aptamer, tested as stringent control candidates, caused negligible responses. The applications were successfully extended to the detection of the drug in human serum. As demonstrated by impedance measurements, the aptamer-based sensors can be used for real-time drug monitoring.


Investigating the sensing surface and TFV interaction
Square wave voltammetry (SWV) and intercalator, daunomycin, were exploited in order to support the data presented in this work by showing that the responses of EIS and FET experiments were caused by binding of drug and aptamer conformational changes. To this end, the SWV response of the sensing surface was recorded after daunomycin intercalation following three different orders: A) Daunomycin intercalation (no drug bound to aptamer) B) Sensing surface incubation in TFV solution, then daunomycin intercalation C) Daunomycin intercalation, then sensing surface incubation in TFV The recorded SWV responses ( Figure S1) were analyzed in Nova software and the peak amplitudes are illustrated in Figure  S2 for comparison. Intercalators bind to DNA due to two phenomena: first, intercalation to hydrophobic state of a double stranded DNA (dsDNA); second, electrostatic interactions with phosphate groups of the DNA backbone 1 . Therefore, intercalation happens to both single stranded DNA (ssDNA; aptamers before binding and conformational change) and dsDNA (part of aptamer after conformational change) to some extent. It has been reported in the literature that binding is more effective in dsDNA than in ssDNA 1,2 .
The conformational change of the TFV aptamers after the incubation with drug can be inferred from the data presented in Figure S2, suggesting the successful binding phenomena. The average of the oxidation peaks was higher in the case where daunomycin was intercalated with the aptamers that have already been incubated in specific drug solution (B) than in the case where they were not (A). Further proof is the SWV response showing that the oxidation peaks were increased when the same electrodes of case A were incubated in drug solution (case C). This shows that the binding reaction had caused a conformational change in aptamer structure that brought the daunomycin molecules, which are already intercalated to the structure, closer to the electrode surface.

Field Effect Transistor as signal transducer
FETs have attracted much attention as they offer rapid, label-free and cost effect detection of the analyte. In digital electronics MOSFET acts as a switch where two electrodes (source and drain) are used to connect a semiconductor material (channel). Current flowing through the channel between the source and drain is electrostatically controlled by a third electrode (gate), which is capacitively coupled through a dielectric layer covering the channel region 3 . In the case of an extended-gate MOSFET biosensor the physical gate is extended by connecting it to an external electrode functionalized with specific receptors for selective capturing of the desired biomolecules. In this work a home designed n-MOSFET device has been used as the signal transducer that in combination with the gold extended gates, biofunctionalized with TFV-aptamers, formed the AptaFET biosensor specific for TFV detection. A schematic of the biosensor is illustrated in Figure S3. The detail of the structure and MOSFET production is mentioned elsewhere 4 . The charged biomolecules when captured by probe molecules produce a gating (electrostatic) effect, which is transduced into a readable signal in the form of change in electrical characteristics of the MOSFET such as drain-to-source current or channel conductance 3,5 . Some applications where MOSFETs are employed for biosensing include cancer therapy 6 , drug screening 7,8 , POC disease detection 9 and diabetes management 10 .

Biosensing surface optimization
The biosensing surface used in this work was obtained after a range of optimization experiments. The focus of the optimization was to improve the performance of the tenofovir-aptamer interaction to enhance the specificity of the response. In addition, stability and reliability of the biosensor was tested using different chemistries for the biosensing surface. To do so, different sensing surfaces were formed and tested by EIS. Specific target of the aptamer (tenofovir) was used to examine the specific response and abiraterone was exploited to examine the nonspecific response. Final results of these experiment presented that the binary SAM of aptamer:MCH provides the best performance.
The following sensing surfaces were examined in this optimization step: 1. SAM layer: ternary SAM of aptamer, MCH, hexanedithiol (HDT) Sensing surface includes a SAM of thiolated aptamer: 1,6-hexanedithiol ( ratio: 1-3000) and backfilling with MCH. EIS responses to blank solution ( Figure S4, right) presented instabilities.  EIS responses to negative control ( Figure S6-Right-down) were null, and it responded to specific target approximately 10% of signal shift ( Figure S6-Right-top). This proved the stability and specificity of this sensing surface. However, it was very complex and time consuming to form this surface.

SAM layer: binary SAM of aptamer-MCH
Sensing surface includes a binary SAM of aptamer and MCH (tested ratios: 1:10, 1:50, 1:100, 1:150). EIS responses to negative control ( Figure S7-f) were null, and it responded to the specific target with ~15% of average signal shift ( Figure S7-b, c, d, e). This biosensing surface exhibited higher sensitivity, higher specificity, and it saved time and effort as well.