Biosensors have become a key research domain owing to their potential applications in numerous areas, such as food and water safety, human health management, and environmental monitoring. various carbon-based nanomaterials have been extensively studied in biosensor-related works and are considered the most promising candidates for producing sensitive, rapid, and low-cost biosensors. We had reviewed carbon nanotubes (CNTs), graphene, and graphene oxide (GO) for the development of various biosensors. Moreover, the design of biosensors in multiple areas was also examined and highlighted their possible applications and assorted fabrication techniques. After thoroughly and systematically studied the carbon-based materials and the fabrication technique for biosensors, we selected graphene as the sensing element for our sensor as its superb mechanical and electrical properties and predetermined the fabrication method of our biosensor as photolithography.

First, the existence of bacteria is a great threat to food safety. Volatile compounds secreted by bacteria during their metabolic process can be dissected to evaluate bacterial contamination. Indole, as a major volatile molecule released by Escherichia coli (E. coli), was chosen to examine the presence of E. coli in this research. In this work, a graphene field-effect transistor (G-FET) was employed to detect the volatile molecule-indole based on a π-π stacking interaction between the indole and the graphene. The exposure of G-FET devices to the indole provokes a change in the electrical signal, which is ascribed to the adsorption of the indole molecule onto the graphene surface via π-π stacking. The adsorption of the indole causes a charge rearrangement of the graphene-indole complex, which leads to changes in the electrical signal of G-FET biosensors with a different indole concentration. Currently, the indole biosensor can detect indole from 10 ppb to 250 ppb and reach a limit of detection of 10 ppb for indole solution detection. We believe that our detection strategy for detecting bacterial metabolic gas molecules will pave the way to developing an effective platform for bacteria detection in food safety monitoring.

Second, to improve the selectivity of G-FET biosensors, graphene, as the sensing material, needs to be modified by sensing elements such as aptamers, antibodies, and so on to realize the specific detection. Noncovalent functionalization by π-interactions is an attractive method because it offers the possibility of attaching functional groups to graphene without disturbing the electronic network. 1-Pyrenebutanoic acid succinimidyl ester (PBASE) is one of the most commonly used linker molecules to connect the sensing material (graphene) and sensing elements (aptamer, antibody). PBASE is an aromatic molecule that has both an aromatic ring and a reactive N-hydroxysuccinimide (NHS) ester group, in which the aromatic ring can modify on the graphene surface by π−π stacking and the NHS ester group can react with the sensing elements that contains a primary amine group. Various concentrations of PBASE and DNA have been employed to functionalize graphene at different times in the past research works. However, the exact functionalization time and concentration of PBASE and sensing elements are still not determined. By modifying the sensing elements with a fluorescent group, the amount of functionalized fluorescently labeled molecules can be tracked by a Confocal scanning laser microscope (CSLM) using fluorescent intensity. We investigated the functionalization of graphene by PBASE and DNA systematically, including the concentration of PBASE and DNA, as well as modifying time of them. The result showed that a concentration of 1 mM for PBASE and 0.01 μM for DNA, whereas a functionalization time of 4 hours for PABSE and 2 hours for DNA would be the ideal parameters for the G-FET modification.

Third, the continuously mutated beta coronavirus severe acute respiratory syndrome (SARS-Cov-2) virus has been outbreaks wave after wave, and the epidemic is bound to continue. Currently, it takes as long as hours or even days for getting the results of the SARS-CoV-2 test. To overcome the limitations of dependence on the facilities for fluorescence detection, we extended the application of the emerging gene-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system to the development of field-effect transistor (FET) based biosensing systems. The CRISPR/Cas12a-assisted graphene FET (G-FET) biosensor was fabricated first time for the detection of the SARS-CoV-2 gene. The monolayer graphene has been employed as the conductive channel between the source and drains electrodes in the G-FET sensors. By functionalizing the graphene surface with a layer of DNA reporter, the SARS-Cov-2 gene detection can be initiated by the recognition and cleavage of the target DNA by Cas12a-crRNA. Using our novel CRISPR/Cas12a assisted G-FET platform developed in this study, our biosensor can reach a detection limit of 50 pM for nucleic acid.

In summary, we developed several sensors for pathogens' metabolic volatile compounds and nucleic acid detection with high sensitivity and selectivity. The pristine G-FET biosensors including liquid gate and back gate were fabricated for indole solution and gas detection, respectively. Moreover, the functionalization parameters of G-FET were systematically investigated to improve the sensing performance of the G-FET biosensors. The CRISPR/Cas12a assisted G-FET biosensors were also developed for the detection of the SARS-Cov-2 gene. By combining the CRISPR/Cas system and the G-FET, our biosensor can reach a detection limit of 50 pM. We believe that our study provides a comprehensive understanding and consideration to develop G-FET biosensors for food safety monitoring and disease diagnosis.