An enzyme-free and ratiometric biosensor for single nucleotide polymorphism with portable electrochemical system

Portable, low-cost and efficient biosensing systems are valuable for single nucleotide polymorphisms (SNPs) analysis, particularly in resource-limited fields. Herein, a rapid, enzyme-free and ratiometric SNPs biosensor with portable electrochemical system enabling simultaneous recognition and signal amplification is proposed. To demonstrate its versatility and effectiveness, we have selected the human cancer-related KRAS gene and the soybean mosaic virus resistance Rsv3 gene as our analysis models. Our biosensor is constructed based on a combination of strand displacement reactions and hybridization chain reactions (HCR). This unique design enables to differentiate between mutant (MT) and wild (WD) sequences based on the differential release of HCR products on the electrode surfaces. Importantly, this enzyme-free SNPs biosensor concurrently achieves both SNPs recognition and signal amplification, significantly reducing the reaction time to just 40 minutes. Furthermore, we have developed a miniaturized biosensor system that incorporates screen printed gold electrodes (SPGE) and smartphones. This system is integrated with a ratiometric strategy with an limit of detection as low as 1.2 fM, enhancing accuracy and eliminating external interference. This simple SNPs biosensing system possesses high reliability, positioning it as a versatile biosensor for the determination of various SNPs mutations.