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

Zhaoli Gao,Jonathan Zauberman,Xinping Yang,Pedro Ducos,Li Ren,Maurizio Tomaiuolo,Fahmida Lubna,Qicheng Zhang,Sheng Wang,Alan T Charlie Johnson,Han Xia,Jinglei Ping,Huacheng Ye,Zhengtang Luo

doi:10.1021/acs.nanolett.8b00572

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.

Highlights

L abel-free and multiplexed nucleic acid testing is of interest for genetic screening and clinical diagnosis,[1,2] and nanobio-electronics have shown great promise for this application.[3]
Graphene field-effect transistors (GFETs) can be readily functionalized with single-stranded probe DNA for detection of specific target oligonucleotides with complementary sequences
By detecting the charge of target DNA hybridized with the probe, GFETs typically offer a limit of detection (LOD) ranging from 1 fM to 100 pM.[5−8] The broad range of sensitivities has been ascribed to the affinity-governed binding kinetics of target and probe

Summary

Nano Letters

Tests showed that hairpin probe DNA offered enhanced specificity against noncomplementary DNA with a single-base mismatch compared to the traditional single-strand probe. We demonstrated multiplexed detection of target DNAs T and T′ with a GFET sensor array, through the use of site-specific functionalization using two different hairpin probe DNAs. The second set of probe DNA (H1′) and helper DNA (H2′) was redesigned according to the base sequence of the second target DNA (T′) to trigger the self-assembly reaction in the presence of H3 and H4. We developed manufacturable GFET nucleic acid sensors based on hairpin probe DNA designed to enable signal amplification by target recycling and a hybridization chain reaction. Due to the target recycling and self-assembly amplification nature, the presence of low concentration of target is expected to generate a large number of annealed H1·H2·H3·H4 complexes, which can potentially result in a significant change of Dirac voltage for GFET detection of trace amount of target DNAs and RNAs. Electrical Measurement and Evaluation.

■ ACKNOWLEDGMENTS

■ REFERENCES