Microfluidic-based graphene field effect transistor for femtomolar detection of chlorpyrifos

Chlorpyrifos is one of the most widely used pesticides that acts on the nervous system by inhibiting acetylcholinesterase. Prolonged use of chlorpyrifos causes severe neurological, autoimmune, and persistent developmental disorders in humans. Therefore, in this study, a highly sensitive and robust biosensor platform was devised by fabricating graphene field effect transistors (graFET) on Si/SiO2 substrate for the detection of chlorpyrifos in real samples. Anti-chlorpyrifos antibodies were immobilized successfully on the graphene surface. Under optimal conditions, graFET sensor showed an excellent response for chlorpyrifos detection in the linear range of 1 fM to 1 µM with a limit of detection up to 1.8 fM in spiked samples. The developed graFET biosensor is highly stable, sensitive, and specific for chlorpyrifos as confirmed by its significant ability to detect changes in electrostatic potential. These findings signify useful efficacy of immunobiosensors for the detection of chlorpyrifos and other organophosphates in fruits and vegetables.

Here, we report the detection of chlorpyrifos pesticide on functionalized graphene-FET-based immunosensor. The graphene-FET is fabricated using exfoliation technique on Si/SiO 2 substrate followed by thermal evaporation of Cr/Au (5/50 nm) electrodes after electron beam lithography and has been used for the detection of chlorpyrifos (Chl) with a limit of detection (LOD) of 1.8 fM in standard samples that is 10-fold (an order of magnitude) less than the previous reports 31 , making them one of the most sensitive detectors of chlorpyrifos till date. Along with ease of fabrication, the developed devices are highly sensitive, cost effective, can detect minute changes in antigen concentrations, and can be easily integrated into electronic chips for real-time sensing for on-site applications.

Methods
Labeling and Immobilization of chlorpyrifos antibody on FET device. Chl-Ab was labelled with graphene via carbodiimide chemistry. For this, 0.5 mg of Ab was added dropwise to 75 μM EDC (1-ethyl-3-(3-dimethylaminopropyl) and 75 μM NHS (N-hydroxysuccinimide) solution. The reaction was incubated at RT for 2 h and centrifuged at 10,000 rpm for 15 min at 4 °C and pellet was redissolved in 1 mL of phosphate buffer (PB), pH 7.4. 100 μg of chl-Abs were added dropwise to freshly activated graphene and incubated at RT for 30 min followed by incubation O/N at 4 °C. The graphene (gra) labelled Abs were further characterized to confirm the binding by spectroscopic and microscopic techniques.
Characterization of chlorpyrifos antibody labelled with graphene. The morphological studies of graphene and gra-chl-Abs nanocomposites were done with scanning electron microscope (SEM) (Hitachi S-3400N). The samples for SEM imaging were made electroactive by coating with gold using Quorum SC7620 sputter coater. Atomic force microscopy (AFM) was performed on Bruker Dimension Icon. Fourier transformed Infra-red (FTIR) spectroscopy was carried out using Shimadzu 8700 FTIR. Optical characteristics were studied by UV-Vis spectrophotometer using (Shimadzu 2600).

Fabrication of FET device.
To fabricate FET devices, we have used mechanically exfoliated graphene, which is known to perform at very low noise levels [32][33][34][35] . Exfoliation was performed on Si/SiO 2 substrates using scotch tape (3 M magic tape) technique where 285 nm SiO 2 forms back-gate dielectric. Suitable graphene flakes were identified by an optical microscope (Olympus BX51) followed by spin coating and baking of one layer each of PMMA 495 and PMMA 950 at 150 °C that formed the positive resist for e-beam lithography performed in Raith Pioneer to pattern the contact pads. MIBK and IPA (1:3) were used to develop polymer layer post e-beam lithography. Afterwards 5/50 nm of Cr/Au were thermally evaporated to form the source-drain electrodes. The sample was then mounted on ceramic chip carrier using silver epoxy. The contact pads were ball bonded in TPT HB05 wire bonder machine.
Electrochemical characterization of immobilized chl-Abs on fabricated FET device. All electrical measurements were done using SR 830 Lock-In Amplifier. Keithley 2400 source meter was used to provide back gate voltage. The chl-Abs were immobilized via carbodiimide activation reaction that activates the carboxylic groups on graphene surface and binds via amine groups present on antibodies. The immobilization and hybridization of chl-Abs on FET electrode was studied at different stages by recording changes in resistance. The immobilization of chl-Abs (1 ng/mL) was done in 50 mM PB (pH 7.4). The washing with PB was done at each step to remove excess and unbound material. Chl-Ag was added at different concentrations (1 fM to 1 µM). Real-time sensing in liquid state was done by applying a constant current of 100 nA across the sample and monitoring the resistance change in the FET for various antigen (chl-Ag) concentrations.

Results and Discussion
Characterization of chl-Abs immobilized gra-FET immunosensor. Functionalization of graphene was achieved by carbodiimide chemistry that resulted in the activation of carboxylic groups in graphene and helps in covalent bond formation with amine reactive chl-Abs. The fabrication of functionalized graphene for sensing chl-Ag was done with FET immunosensor. While blocking non-specific sites with BSA on the surface of graphene, chl-Abs was immobilized and specific Ag added on the electrode surface. Change in resistance (%R) was monitored w.r.t. increase in chl-Ag concentration (Fig. 1).
To check the suitability of graphene for chlorpyrifos detection, binding of graphene with chl-Abs was first studied (see supplementary information). UV-Vis spectra (Fig. 2a) showed the peak at 230 nm for graphene (blue line) while red shift observed at 235 nm after binding with chl-Abs (red line). FT-IR spectra showed (Fig. 2b) peaks at 1116 and 1635 cm −1 for C=O and C-C stretch, respectively. Presence of peak at 1372 cm −1 for C-N confirmed that binding occurred between graphene and chl-Abs. The resistance (R) vs gate voltage (Vg), measured in constant current mode, showed the characteristic bell-shaped curve with a maxima at ~8 V that corresponds to charge neutrality point or Dirac point (Fig. 2c). The transfer characteristics before and after antibody interaction clearly shows a shift in the graphene Dirac point 19 . The shift in the Dirac point indicates a change in the number density of the graphene channel, due to antibody-antigen interaction, which has led to charge redistribution of the graphene channel. The Raman spectra (Fig. 2d) displayed the characteristic sharp 2D peak for single layer graphene (SLG) with the ratio of I 2D /I G ~ 2. The mobility of device obtained from σ vs n was approximately 750 cm 2 /Vs Scanning electron micrograph (SEM) of SLG showed a clear graphene layer (Fig. 2e). Figure 2f showed morphology of (i) graphene after binding of (ii) chl-Abs with graphene (while globular structures), reconfirmed the immobilization on the graphene electrode. Further passivation of unobound sites with BSA can be seen in Fig. 2f-iii and binding with chlorpyrifos antigen with specific antibody (Fig. 2f-iv). Binding characteristics of chl-Abs. Figure 3a showed the gra-FET immunosensor platform. To develop a specific and sensitive assay for small molecules is an important aspect. The main step is to synthesize appropriate antibody that is specific and sensitive towards the target biomolecules. Therefore, chl-BSA conjugate was used to generate Abs in rabbit. The developed antibodies showed high specificity for chl upto the dilution of 1:640,000. From the binding assay, it was confirmed that the developed Abs are mainly produced against chl not against BSA (Fig. 3a). The phenomenon can be explained as there might be a uniform distribution of haptens conjugated with the carrier protein (BSA) that helped in the recognition of hapten (chl) by the immune system of animal 10,16 .
Electrochemical performance of fabricated gra-FET electrode system. Graphene was functionalized with EDC/NHS for activation of carboxylic groups for the development of grapheneFET-basedsensor 22 . Blocking unbound surface of graphene was done with BSA and the change in the resistance was monitored (Fig. 3b). To reconfirm the change in resistance obtained from graphene layer but not from gold surface. The biomolecules (Antibody-BSA (for blocking)-chl-Ag) were immobilized on gold surface and the signal was recorded that showed no change in resistance at each step of binding. This process again confirmed that the change in resistance was due to graphene but not from bare gold (Fig. 3c). For the quantitative estimation of detection capabilities of FET device, the graphene channel resistance was monitored continuously at each step to estimate the sensing action of FET for various chl-Ag concentrations. The graphene channel functions as a transducer that converted a chemical signal from the biomolecules and acted as receptor in this case, into a measurable electrical signal that could be read by the lock-in amplifier. The diffusive motion of carriers in the channel is captured by the Drude conductivity expression, which is given by σ = neμ, where σ, n, and μ are the conductivity, carrier density and mobility of the carriers respectively. The Ag-Ab interaction at the reactive site leads to doping of graphene channel due to heterogeneous electron transfer, which was manifested as a change in graphene channel resistance, since σ ∝ R 1/ . The sensitivity of the devices were determined by extracting the % change in R, which was obtained by taking the R of the channel with a buffer solution as the baseline. The calibration curve of the immunoassay developed at various chl-Ag concentrations is shown in Fig. 3d. A measurable change in R was recorded for concentrations as low as 1.8 fM. For chl-Ag concentration at 10 pM after that the resistance saturates, probably because all the sites in chl-Abs have been occupied with chl-Ag (CITE). Kinetic studies were performed, and exponential sensogram fit plotted for chl (Fig. 3e). The spikes in the resistance may originate due to changes in the local electrostatic environment, which is a transient state when the system is out of equilibrium. The spiked chlorpyrifos was prepared in linear range of 1 fM to 1 µM with detection range from 1.8 fM to 100 pM. The gra-FET-based sensor for chl detection showed a significant enhancement in the limit of detection (LOD) of 1.8 fM for chl-Ag-Ab complex, compared to other devices (Table 1), making it one of the most sensitive detectors till date. The LOD can be further enhanced, possibly by using large area chemical vapour deposition (CVD) grown graphene. The larger area can lead to an enhancement in the number of active sites on functionalization, and an increased LOD. Since graphene is highly stable and has a low environmental impact, such functionalized graphene FET-based devices can be easily integrated into an electronic chip for large-scale field applications for detection of not just chlorpyrifos but other specific biomolecules as well.

Conclusions
In conclusion, we have developed graphene-based FET immunosensor for the detection of widely used chlorpyrifos pesticide. The detection, which was performed by monitoring the change in resistance of the funtionalized-graphene channel, for several antibody resistance, yielded a label-free detection of chlorpyrifos with very low detection limit of 1.8 fM in standard buffer. The results of our measurements clearly indicate the choice of using graphene channel as a detector for real-time sensing of very low concentrations of chlorpyrifos, and should be further exploited for providing a low cost platform for on-field surveillance. The limit of detection   Table 1. Comparison of developed electrochemical sensors for chlorpyrifos with its limit of detection.