Highly Sensitive Detection of Protein Biomarkers Using Graphene Field-Effect Transistor Integrated with a Microfluidic Platform

In this work, we present a highly sensitive biosensing platform for the detection of protein biomarkers by utilizing aptamer-modified graphene field-effect transistors (GFETs) contained within a microfluidic chip. We have chosen thrombin as a model biomarker and its corresponding DNA-based aptamer as a target receptor which is anchored to the graphene surface to modulate the drain current of the GFET. The proposed biosensing platform has the potential to be used in early-stage disease diagnosis and in applications related to lab-on-a-chip-based health monitoring technology. Aptamer-based biosensing offers many advantages including low-cost, chemical and thermal stability, mass producibility, long shelf-life and reusability. Among different target analytes, protein biomarker detection is critical in early screening and diagnostics of diseases. Thrombin is an important protein biomarker which is produced by the body and plays a significant role in blood clotting or coagulation process or regulation of tumor growth [1]. Therefore, detection of thrombin is key to monitoring a number of cardiovascular diseases. Graphene-based FET (GFET) has become a highly promising candidate as next-generation biosensors due to the various inherent advantages it offers such as facile integration with the existing planar technology due to its 2-dimensional structure, label-free sensing, and high sensitivity. However, one of the major challenges with GFET-based biosensing is the lack of reproducibility due to each graphene layer having different physical dimensions and therefore electronic properties. Moreover, since GFET is highly sensitive to the charge disturbances at the FET channel, the sample liquid that is in contact with the graphene surface must be controlled. Therefore, integration of GFET biosensor with a microfluidic device is expected to enhance the sensing performances in terms of sensitivity and reproducibility. In this work, we have implemented a microfluidic chip integrated with GFET device to build a miniaturized platform for aptamer-based detection of a protein biomarker. The GFET devices were fabricated by transferring CVD (chemical vapor deposition) grown graphene onto the microfabricated gold electrodes on the Si/SiO 2 substrate where the electrodes form the source and the drainas schematically depicted in Figure 1. The FET devices were then sealed with a PDMS block containing a microfluidic channel as shown in Figure 2. The graphene channel was then functionalized with amine reactive 1-pyrene butyric acid N-hydroxysuccinimide ester (PBASE) by delivering the PBASE dissolved in dimethyl formamide through the microfluidic channel. The PBASE was immobilized on graphene surface via the π-π stacking interaction between graphene and the pyrene group of PBASE. The graphene channel was then further functionalized with the aptamers by flowing the amine terminated thrombin-binding aptamer solution through the microfluidic chip. The aptamers were crosslinked with the PBASE on graphene surface via the covalent linkage between the amine-reactive succinimide group of PBASE and the amine group of the aptamers [2]. For the biomarker detection, various concentrations of thrombin solutions were introduced to the GFET device through the microfluidic flow system. For a given concentration of thrombin, the drain current (I DS ) vs. gate voltage (V GS ) characteristics were measured in order to observe the shift in the Dirac voltage (V Dirac ). Figure 3 shows the Raman spectra of graphene on Si/SiO 2 substrate before and after PBASE functionalization. The additional peak at 1618 cm -1 in Figure 3(a) represents the pyrene group resonance peak due to π – π stacking interaction [3] between the aromatic pyrenyl group of PBASE and the basal plane of graphene, confirming the immobilization of PBASE on graphene. Figure 4 shows the UV-vis spectra of the device sample before and after the aptamer immobilization. The peak at 260 nm indicates the presence of aptamers on the device surface. The resulting measurements are presented in Figure 5. It can be seen that introduction of thrombin causes the Dirac voltage to shift to the right suggesting p-doping of the graphene surface [4]. This is consistent with the cationic nature of thrombin at neutral pH. The corresponding calibration curve is shown in Figure 6. The sensor shows a linear behavior over a wide range of concentrations from 1 nM to 1µM. References: [1] M. L. Nierodzik and S. Karpatkin, Cancer Cell , 10(5), pp. 355–362, 2006. [2] G. T. Hermanson, Bioconjugate Techniques (3rd Ed.) , Academic Press, pp. 299–339, 2013. [3] S. Xu et al. , Nat. Commun. , vol. 8, p. 14902, 2017. [4] S. Ghosh, N. I. Khan, J. G. Tsavalas, and E. Song, Front. Bioeng. Biotechnol. , vol. 6, 2018. Figure 1