Lysozyme is an antimicrobial protein, often called the “body’s own antibiotic.” It is a ubiquitous enzyme widely available in diverse organisms, such as bacteria, bacteriophages, fungi, plants and mammals. Lysozyme is also extensively used in food industries for several purposes such as preserving meat and dairy products, as well as fruits and vegetables. Lysozyme has a molecular weight of 14,400 with a primary sequence containing 129 amino acids and an isoelectric point of 11.0 that causes lysozyme to behave as positively charged at neutral pH. Apart from its extensive use in food industry, lysozyme also plays a vital role as a biomarker for diagnosing various diseases such as breast cancer, Alzheimer’s disease, and rheumatoid arthritis.

Several analytical methods have been deployed for effective detection of lysozyme molecules. Some of these methods include chromatographic or antibody based techniques, sensitive colorimetric detection, surface plasmon resonance (SPR), and electrochemical impedance spectroscopy (EIS) measurement. In this work, we describe the graphene field effect transistor (GFET)-based selective detection of lysozyme molecules by utilizing a large area CVD-grown graphene.

To enhance the selective detection, the graphene is successively functionalized with aptamers in the form of single-stranded probe DNAs (pDNAs) which are specifically designed for lysozyme binding. These aptamers were anchored to the graphene surface through the cross-linking molecule 1-pyrenebutanoic acid succinimidyl ester (PBASE). The GFET devices were configured as electrolyte-gated FETs where the graphene is the conducting channel formed between the source and the drain electrodes on the SiO₂/Si substrate as schematically depicted in Figure 1 of the supplemented image. A PBS solution (0.01X) was used as the top gating dielectric. The pyrene group terminated PBASE is coupled to the graphene surface via the π-π stacking forces. The 5’-amine-modified pDNAs were attached to the amine-reactive succinimide group of PBASE by the conjugation reaction between the amine groups. The I_d−V_gs characteristics of the GFET devices were measured sequentially after each functionalization step and exposure to the target lysozyme molecules. The binding of the lysozyme molecules with the pDNAs induces changes in the charge carrier density in the graphene channel. This causes a detectable change in the Dirac voltage (V_Dirac) or the charge neutrality point (V_CNP) in the I_d−V_gs characteristics of the GFET. Our experimental results demonstrate detectable change in the V_CNP with varying concentrations of lysozyme indicating a selective detection capability of lysozyme up to 1 µM.

Figure 2 in the attached image shows the transfer process of large area CVD-grown graphene from SiO₂/Si substrate onto the pre-fabricated 4 independently addressable gold electrodes. The CVD grown graphene sample was purchased from Graphene Supermarket (NY, USA). The transfer process starts with spin coating of the graphene by a support layer of poly(methyl methacrylate) (PMMA) at 3000 RPM followed by immersion into 6M KOH solution for 30 minutes at 80^oC. This results in the etching of the underneath SiO₂ layer and separation of the top PMMA/graphene bilayer from the substrate. The PMMA protected graphene layer was then collected on top of the pre-fabricated gold electrodes and dried at room temperature. The electrode was then immersed into acetone for 8 hours to dissolve the top PMMA layer followed by annealing at 250°C for 2 h in argon-filled furnace. Figure 3 shows the I_d-V_gs characteristics of the GFET devices at different functionalization steps. A left-shift of V_CNP was observed after each step of functionalization with PBASE linker, Tween 20 and the pDNA attachment. This is due to the n-doping effect of graphene by the attached species on the surface. After exposing to 1 nM Lysozyme solution, a right-shift in V_CNP was observed due to the positive charge of lysozyme molecules at neutral pH of 7.0. The concentration dependent shifting of the FET measurements is demonstrated in Figure 4. With increasing concentration of lysozyme, the amount of right-shift in V_CNP increases and saturates at a lysozyme concentration of 1µM as shown in the calibration curve in Figure 5. The target selectivity of the GFET-based sensor, when investigated against the bovine serum albumin (BSA) is presented in Figure 6. The sensor shows reasonable selectivity toward lysozyme molecules and against the BSA molecules. Finally, the specificity of the aptamer modified sensor is tested against the unmodified GFET which shows negligible response to the lysozyme, verifying the effectiveness of the aptamers as bio-recognition elements for the GFET-based biosensor.