"All-U-Want" Strand Displacement Amplification: A Versatile Signal Amplification Method for Nucleic Acid Biosensing.

Strand displacement amplification (SDA) is an isothermal DNA amplification technique. Herein, we developed a novel SDA system, designated All-U-Want SDA (AUW-SDA), which was used as a signal amplification strategy for the construction of nucleic acid detection biosensors. AUW-SDA is capable of target turnover and can be utilized for detection of nucleic acid sequences without available 3'-ends. Of particular significance is the ability of AUW-SDA to generate a substantial number of programmable sequences in accordance with the specifications of the sensor signal output methods, irrespective of the sequence of the target nucleic acid. We used the gene of SARS-CoV-2 as a model target to develop a sensing platform with dual signal outputs. The colorimetric signals were generated by the G-quadruplex/hemin DNAzyme, in which the G-rich sequences were produced by AUW-SDA with a C-rich primer. On the other hand, by altering the sequence within the replaceable region of the primer, an activator sequence was obtained from AUW-SDA, which could trigger the activity of CRISPR/Cas12a, cleaving the probes modified with a fluorophore and quencher at each end and subsequently yielding the fluorescent signals. After the DNA sequences and reaction conditions were optimized, the limit of detection (LOD) values of the fluorescent and colorimetric assays were estimated to be 0.672 fM and 13.3 fM, respectively. The biosensors were utilized for biological sample detection. The reliability of the proposed method was validated against RT-qPCR results. In addition, a portable scanner-assisted high-throughput RGB analysis (PSHRA) method was developed. This method was applied to our biosensor for multilocus detection of SARS-CoV-2. The results obtained were satisfactory, indicating the potential of this approach for field testing or point-of-care (POC) diagnostics.