Amplification-free and label-free rapid detection of Staphylococcus aureus using solution-gated graphene transistor-based DNA biosensor with hybridization enhancement by interface engineering

Staphylococcus aureus (S. aureus) is one of the leading causes of foodborne illnesses worldwide, posing a significant risk to food quality and safety. However, conventional detection methods often require complex amplification and labeling procedures, which are time consuming and expensive. In this study, an extremely sensitive electrochemical biosensor using a solution-gated graphene transistor (SGGT) was constructed for the amplification-free and label-free rapid detection of S. aureus by utilizing specific single-stranded DNA (ssDNA) to modify the gold (Au) sensing gate. This modified ssDNA was designed to hybridize with the target S. aureus DNA sequence. Interface engineering of the ssDNA-modified sensing gate via optimization of specific target and probe sequences was proposed to improve its hybridization efficiency. This enabled the sensitive quantitative detection of S. aureus DNA. The resulting biosensor exhibited excellent specificity in distinguishing non-complementary, 3base/9-base DNA mismatch targets of S. aureus DNA and the specific DNA sequences of other common pathogenic bacteria. It also enabled the rapid detection of extracted target DNA sequences in solution (within 27 min) and had a low limit of detection (LoD) of 10-17 M over a wide linear detection range (10- 17-10- 8 M). Furthermore, the newly-developed biosensor facilitated the real-time and on-site detection of the S. aureus genome (ATCC 6538) with LoD of 10- 17 M (103 CFU/mL) in a portable smart-sensing system. This suggests a promising future for the development of rapid, label-free, and amplification-free detection of foodborne illnesses using low-cost, highly sensitive SGGT DNA biosensors with appropriate functionalization and interface engineering of the sensing electrode.