Simultaneous detection of SARS-CoV-2 proteins and nucleic acids by coupling PNIPAM with circle-to-circle amplification.

In the past few years, the global uncontrollable spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has underscored the need for rapid, sensitive, and accurate detection methods. Given the continuous mutation characteristics of viruses, single-target detection carries the risk of false-positive or false-negative results, whereas dual-target detection can effectively mitigate such risks. Therefore, thermosensitive polymer poly(N-isopropylacrylamide) (PNIPAM) was integrated with an isothermal amplification method-circle-to-circle amplification (C2CA) in this study, enabling individual and simultaneous detection of the SARS-CoV-2 S1 protein and N gene, respectively. PNIPAM-capture probe conjugates enabled specific target recognition and thermal separation from complex matrices, while C2CA achieved exponential signal amplification by leveraging two cascaded rolling circle amplification (RCA) reactions. During single-target detection, the method exhibited linear ranges of 0.1-1000 pg/mL for S1 protein detection and 0.1-1000 pM for Ng detection, with the limit of detection (LOD) of 55 fg/mL and 11.2 fM, respectively. The spiked recovery experiment demonstrated that this method possesses high specificity and robust anti-interference capability. Furthermore, in order to improve the accuracy and efficiency, simultaneous detection was achieved by integrating S1 protein and Ng detection into a single reaction system and labeling differential fluorophores. The LOD was 68.3 fg/mL for S1 protein and 33.1 fM for Ng, respectively. This method combines the advantages of homogeneous reaction and isothermal amplification, eliminating the need for complex instruments and cumbersome procedures, thereby making it suitable for resource-limited settings.