Integrated Approach from Sample-to-Answer for Grapevine Varietal Identification on a Portable Graphene Sensor Chip.

Identifying grape varieties in wine, related products, and raw materials is of great interest for enology and to ensure its authenticity. However, these matrices' complexity and low DNA content make this analysis particularly challenging. Integrating DNA analysis with 2D materials, such as graphene, offers an advantageous pathway toward ultrasensitive DNA detection. Here, we show that monolayer graphene provides an optimal test bed for nucleic acid detection with single-base resolution. Graphene's ultrathinness creates a large surface area with quantum confinement in the perpendicular direction that, upon functionalization, provides multiple sites for DNA immobilization and efficient detection. Its highly conjugated electronic structure, high carrier mobility, zero-energy band gap with the associated gating effect, and chemical inertness explain graphene's superior performance. For the first time, we present a DNA-based analytic tool for grapevine varietal discrimination using an integrated portable biosensor based on a monolayer graphene field-effect transistor array. The system comprises a wafer-scale fabricated graphene chip operated under liquid gating and connected to a miniaturized electronic readout. The platform can distinguish closely related grapevine varieties, thanks to specific DNA probes immobilized on the sensor, demonstrating high specificity even for discriminating single-nucleotide polymorphisms, which is hard to achieve with a classical end-point polymerase chain reaction or quantitative polymerase chain reaction. The sensor was operated in ultralow DNA concentrations, with a dynamic range of 1 aM to 0.1 nM and an attomolar detection limit of ∼0.19 aM. The reported biosensor provides a promising way toward developing decentralized analytical tools for tracking wine authenticity at different points of the food value chain, enabling data transmission and contributing to the digitalization of the agro-food industry.

Attomolar detection of hepatitis C virus core protein powered by molecular antenna-like effect in a graphene field-effect aptasensor.

Biosensors based on graphene field-effect transistors have become a promising tool for detecting a broad range of analytes. However, their performance is substantially affected by the functionalization protocol. In this work, we use a controlled in-vacuum physical method for the covalent functionalization of graphene to construct ultrasensitive aptamer-based biosensors (aptasensors) able to detect hepatitis C virus core protein. These devices are highly specific and robust, achieving attomolar detection of the viral protein in human blood plasma. Such an improved sensitivity is rationalized by theoretical calculations showing that induced polarization at the graphene interface, caused by the proximity of covalently bound molecular probe, modulates the charge balance at the graphene/aptamer interface. This charge balance causes a net shift of the Dirac cone providing enhanced sensitivity for the attomolar detection of the target proteins. Such an unexpected effect paves the way for using this kind of graphene-based functionalized platforms for ultrasensitive and real-time diagnostics of different diseases.

Ultrasensitive dopamine detection with graphene aptasensor multitransistor arrays.

Detecting physiological levels of neurotransmitters in biological samples can advance our understanding of brain disorders and lead to improved diagnostics and therapeutics. However, neurotransmitter sensors for real-world applications must reliably detect low concentrations of target analytes from small volume working samples. Herein, a platform for robust and ultrasensitive detection of dopamine, an essential neurotransmitter that underlies several brain disorders, based on graphene multitransistor arrays (gMTAs) functionalized with a selective DNA aptamer is presented. High-yield scalable methodologies optimized at the wafer level were employed to integrate multiple graphene transistors on small-size chips (4.5 × 4.5 mm). The multiple sensor array configuration permits independent and simultaneous replicate measurements of the same sample that produce robust average data, reducing sources of measurement variability. This procedure allowed sensitive and reproducible dopamine detection in ultra-low concentrations from small volume samples across physiological buffers and high ionic strength complex biological samples. The obtained limit-of-detection was 1 aM (10-18) with dynamic detection ranges spanning 10 orders of magnitude up to 100 µM (10-8), and a 22 mV/decade peak sensitivity in artificial cerebral spinal fluid. Dopamine detection in dopamine-depleted brain homogenates spiked with dopamine was also possible with a LOD of 1 aM, overcoming sensitivity losses typically observed in ion-sensitive sensors in complex biological samples. Furthermore, we show that our gMTAs platform can detect minimal changes in dopamine concentrations in small working volume samples (2 µL) of cerebral spinal fluid samples obtained from a mouse model of Parkinson's Disease. The platform presented in this work can lead the way to graphene-based neurotransmitter sensors suitable for real-world academic and pre-clinical pharmaceutical research as well as clinical diagnosis.

Optimizing PMMA solutions to suppress contamination in the transfer of CVD graphene for batch production.

Mass production and commercial adoption of graphene-based devices are held back by a few crucial technical challenges related to quality control. In the case of graphene produced by chemical vapor deposition, the transfer process represents a delicate step that can compromise device performance and reliability, thus hindering industrial production. In this context, the impact of poly(methyl methacrylate) (PMMA), the most common support material for transferring graphene from the Cu substrate to any target surface, can be decisive in obtaining reproducible sample batches. Although effective in mechanically supporting graphene during the transfer, PMMA solutions needs to be efficiently designed, deposited, and post-treated to serve their purpose while minimizing potential contaminations. Here, we prepared and tested PMMA solutions with different average molecular weight (AMW) and weight concentration in anisole, to be deposited by spin coating. Optical microscopy and Raman spectroscopy showed that the amount of PMMA residues on transferred graphene is proportional to the AMW and concentration in the solvent. At the same time, the mechanical strength of the PMMA layer is proportional to the AMW. These tests served to design an optimized PMMA solution made of a mixture of 550,000 (550k) and 15,000 (15k) AMW PMMA in anisole at 3% concentration. In this design, PMMA-550k provided suitable mechanical strength against breakage during the transfer cycles, while PMMA-15k promoted depolymerization, which allowed for a complete removal of PMMA residues without the need for any post-treatment. An XPS analysis confirmed the cleanness of the optimized process. We validated the impact of the optimized PMMA solution on the mass fabrication of arrays of electrolyte-gated graphene field-effect transistors operating as biosensors. On average, the transistor channel resistance decreased from 1860 to 690 Ω when using the optimized PMMA. Even more importantly, the vast majority of these resistance values are distributed within a narrow range (only ca. 300 Ω wide), in evident contrast with the scattered values obtained in non-optimized devices (about 30% of which showed values above 1 MΩ). These results prove that the optimized PMMA solution unlock the production of reproducible electronic devices at the batch scale, which is the key to industrial production.

Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors.

Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene's exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes' ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.

Clean-Room Lithographical Processes for the Fabrication of Graphene Biosensors.

This work is on developing clean-room processes for the fabrication of electrolyte-gate graphene field-effect transistors at the wafer scale for biosensing applications. Our fabrication process overcomes two main issues: removing surface residues after graphene patterning and the dielectric passivation of metallic contacts. A graphene residue-free transfer process is achieved by using a pre-transfer, sacrificial metallic mask that protects the entire wafer except the areas around the channel, source, and drain, onto which the graphene film is transferred and later patterned. After the dissolution of the mask, clean gate electrodes are obtained. The multilayer SiO2/SiNx dielectric passivation takes advantage of the excellent adhesion of SiO2 to graphene and the substrate materials and the superior impermeability of SiNx. It hinders native nucleation centers and breaks the propagation of defects through the layers, protecting from prolonged exposition to all common solvents found in biochemistry work, contrary to commonly used polymeric passivation. Since wet etch does not allow the required level of control over the lithographic process, a reactive ion etching process using a sacrificial metallic stopping layer is developed and used for patterning the passivation layer. The process achieves devices with high reproducibility at the wafer scale.