Catalytic Hairpin Assembly-Enhanced Graphene Transistor for Ultrasensitive miRNA Detection

Abstract

MicroRNAs (miRNAs) have emerged as powerful biomarkers for disease diagnosis and screening. Traditional miRNA analytical techniques are inadequate for point-of-care testing due to their reliance on specialized expertise and instruments. Graphene field-effect transistors (GFETs) offer the prospect of simple and label-free diagnostics. Herein, a GFET biosensor based on tetrahedral DNA nanostructure (TDN)-assisted catalytic hairpin assembly (CHA) reaction (TCHA) has been fabricated and applied to the sensitive and specific detection of miRNA-21. TDN structures are assembled to construct the biosensing interface, facilitating CHA reaction by providing free space and preventing unwanted entanglements, aggregation, and adsorption of probes on the graphene channel. Owing to synergistic effects of TDN-assisted in situ nucleic acid amplification on the sensing surface, as well as inherent signal sensitization of GFETs, the biosensor exhibits ultrasensitive detection of miRNA-21 down to 5.67 × 10 −19 M, approximately three orders of magnitude lower than that normally achieved by graphene transistors with channel functionalization of single-stranded DNA probes. In addition, the biosensor demonstrates excellent analytical performance regarding selectivity, stability, and reproducibility. Furthermore, the practicability of the biosensor is verified by analyzing targets in a complex serum environment and cell lysates, showing tremendous potential in bioanalysis and clinical diagnosis.

Sensitivity-Enhancing Strategies of Graphene Field-Effect Transistor Biosensors for Biomarker Detection

“…These molecules have an aromatic pyrene group on one end that enables π–π stacking with the graphene surface, and a carbonyl or succinimide ester group on the other end for activating biomolecules. The research team led by Wei Da Cheng utilized PASE-functionalized GFET biosensors for detecting biomarkers related to cancer, infectious diseases, and SARS-CoV-2 − (Figure c). To date, various types of biomolecules, including peptides, antibodies, enzymes, aptamers, and DNA probes, have been successfully immobilized onto graphene via PBASE linkers, exhibiting excellent electrical properties. ,, Its highly flexible alkyl chain end may lead to various orientations of antibodies on the graphene surface, potentially impacting the completeness of immune reactions (Figure d).…”

“…This range is significantly lower by 3 and 7 orders of magnitude compared to the single probe TDF dimer sensor and single-stranded DNA (ssDNA) sensor. Yang and colleagues reported a study on the use of tetrahedral nanostructures (TDNs) in catalytic hairpin assembly (CHA) reactions within GFET sensors for miRNA-21 detection. This well-designed probe avoids nonspecific adsorption issues typically associated with traditional ssDNA probes, such as tangling, aggregation, and adsorption, lowering the detection limit to 5.67 × 10 –19 M. Compared to graphene transistor channels typically functionalized with ssDNA probes, this technology’s sensitivity is enhanced by approximately 3 orders of magnitude.…”