Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors

Abstract

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity.

Keywords: DNA biosensor; DNA self-assembly amplification; Graphene field-effect transistor; sub-fM limit of detection.

Figure 1

GFET biosensor arrays. (a) Optical image of a graphene field-effect transistor (GFET) array fabricated on 250 nm SiO₂ chip. (b) Schematic of hairpin probe DNA bound to a back-gated GFET using a pyrene linker (purple). (c) Current–gate voltage curve evolution of GFET following different chemical treatment steps.

Figure 2

Schematic showing the principle of the triggered self-assembly amplification for DNA detection on GFET. The GFET was functionalized by hairpin probe DNA H1 through the PBASE linker. The target DNA (T) opens the hairpin probe to form the complex H1·T. T is then displaced by helper DNA H2 through the toehold-mediated strand displacement reaction, leading to the formation of the H1·H2 complex and enabling target recycling. Hybridization chain reaction (HCR) was triggered by H1·H2 in the presence of two additional helper DNAs, H3 and H4. Amplified HCR products are then detected through a shift of the GFET Dirac voltage.

Figure 3

Sensing results and kinetic model. (a) Sensor response as a function of target DNA concentrations. Error bars are standard deviation of the mean. The limit of detection is <10 fM. (b) Sensor response as a function of incubation time for different target concentrations. (c) Simulation results of H1·H2 complex examined for different concentrations of target DNA show good qualitative agreement with panel a. (d) Simulation results of H1·H2 concentrations as a function of time with different target concentrations, showing trends similar to panel b.

Figure 4

Biosensor response to positive and negative controls. (a) Relative Dirac voltage shifts for various positive control experiments based upon concentrations of 1 μM for the target DNA and the specified helper DNAs. (b) Relative Dirac voltage shifts for 10 nM target DNA and negative controls with base mismatches at the ends; detailed DNA sequences are listed in Table S1. Error bars are standard deviation of the mean. Helper DNAs were of 1 μM.