Functionalization strategies for graphene-based biomarker detection leverage the material’s exceptional electronic, chemical, and physical properties. Graphene’s high surface-to-volume ratio, tunable conductivity, and biocompatibility make it an ideal platform for biosensing applications. Key approaches include covalent and non-covalent modifications, aptamer immobilization, and antibody conjugation, each tailored to enhance selectivity and sensitivity toward specific biomarkers.
**Covalent Functionalization** involves the introduction of chemical groups (e.g., carboxyl, epoxy, or hydroxyl) onto graphene’s basal plane or edges via reactions such as Hummers’ oxidation or diazonium salt coupling. These groups serve as anchor points for biomolecule attachment. For instance, carboxylated graphene enables EDC-NHS chemistry to immobilize antibodies or aptamers, forming stable amide bonds. However, excessive covalent modification can disrupt graphene’s electronic properties, necessitating optimization to balance sensitivity and conductivity.
**Non-Covalent Functionalization** preserves graphene’s electronic structure while enabling biomolecule attachment through π-π stacking, hydrophobic interactions, or electrostatic binding. Pyrene derivatives with terminal functional groups (e.g., NHS esters) are widely used to link proteins or DNA probes. Similarly, polyethylene glycol (PEG) coatings reduce nonspecific adsorption, improving signal-to-noise ratios in complex biological fluids.
**Aptamer-Based Functionalization** is particularly effective due to aptamers’ high affinity and specificity for targets like proteins, small molecules, or cells. Thiol- or amine-terminated aptamers are grafted onto graphene via gold nanoparticle intermediaries or linker molecules. For example, a thrombin-binding aptamer immobilized on graphene field-effect transistors (GFETs) achieves sub-nanomolar detection limits by exploiting conformational changes upon target binding.
**Antibody Conjugation** relies on oriented immobilization techniques, such as Protein A/G-mediated binding or Fc-specific reactions, to ensure optimal antigen recognition. Graphene surfaces modified with streptavidin-biotin systems further enhance antibody loading efficiency, critical for detecting low-abundance biomarkers like cytokines or viral proteins.
### Sensitivity Mechanisms in Graphene Biosensors
The primary detection mechanisms in graphene-based biosensors include Dirac point shifts, conductance modulation, and electrochemical signal amplification.
**Dirac Point Shifts** in GFETs occur when charged biomarkers (e.g., proteins or nucleic acids) adsorb onto graphene, altering local carrier density. For instance, SARS-CoV-2 spike protein binding to an ACE2-functionalized GFET induces hole doping, shifting the Dirac point voltage by 10–50 mV at concentrations as low as 1 fg/mL. This electrostatic gating effect is highly sensitive to surface charge perturbations.
**Conductance Modulation** arises from biomolecular interactions that scatter charge carriers or modify graphene’s doping level. Aptamer-target binding can cause folding or unfolding, leading to measurable resistance changes. In glucose sensing, glucose oxidase-functionalized graphene exhibits conductance increases proportional to H₂O₂ production during enzymatic reactions, enabling real-time monitoring at <100 μM concentrations.
**Electrochemical Sensing** leverages graphene’s high electrocatalytic activity for redox reactions. Functionalized graphene electrodes detect biomarkers via direct electron transfer (e.g., hemoglobin oxidation) or mediated pathways (e.g., using ferrocene tags). Dopamine detection at graphene-modified electrodes achieves nanomolar limits of detection (LOD) due to enhanced electron transfer kinetics.
### Case Studies: COVID-19 and Glucose Monitoring
**COVID-19 Detection**
Graphene biosensors have demonstrated rapid, label-free detection of SARS-CoV-2 antigens and RNA. A GFET functionalized with anti-spike IgG antibodies detected viral proteins in nasopharyngeal samples with an LOD of 0.2 pM, outperforming conventional ELISA. Another approach utilized ssDNA aptamers targeting the nucleocapsid protein, yielding a 5-minute response time and 95% specificity in clinical validations. The sensor’s scalability and portability highlight its potential for point-of-care diagnostics.
**Glucose Monitoring**
Non-enzymatic graphene sensors exploit direct oxidation of glucose at defect-rich graphene surfaces, avoiding enzyme instability issues. A Ni(OH)₂-nanoparticle-decorated graphene electrode achieved a linear response range of 0.5–15 mM, covering physiological glucose levels. Alternatively, enzymatic sensors using graphene-platinum hybrids exhibited <5% error in human serum measurements, enabling continuous monitoring for diabetes management.
### Challenges and Future Directions
Despite progress, challenges remain in standardization, reproducibility, and interference mitigation (e.g., from nonspecific binding or biofouling). Advanced antifouling coatings (e.g., zwitterionic polymers) and machine learning-assisted signal processing are being explored to enhance reliability. Future developments may integrate multiplexed detection arrays or wearable formats for real-world applications.
In summary, graphene’s versatility in functionalization and transduction mechanisms positions it as a transformative platform for biomarker detection, with demonstrated success in critical applications like COVID-19 and glucose monitoring. Continued innovation in surface chemistry and device engineering will further unlock its potential in precision diagnostics.