A photonic crystal sensing array based on a tandem CRISPR/Cas13a system for ultra-sensitive and high-throughput detection of the CVA6 virus

Coxsackievirus A6 (CVA6) has emerged as a major cause of hand-foot-mouth disease (HFMD), yet no standardized detection method for it is currently available. Developing a simple, sensitive, and specific CVA6 test is crucial for HFMD control and safeguarding the health of at-risk children. Herein, a photonic crystal (PC) sensing array based on a tandem CRISPR/Cas13a system has been proposed for highly specific and ultra-sensitive analysis of CVA6 RNA, without the need for reverse transcription and amplification procedures. In this strategy, two crRNAs targeting CVA 6 RNA were designed and screened, and the fluorescence signal of the tandem CRISPR/Cas13a system was found to be up to 4.2 times higher than that of the non-tandem CRISPR system. The PC array with periodic nanostructures was prepared through self-deposition and further enhanced the fluorescent signal output from the tandem CRISPR system, owing to the match of the emission wavelength of the fluorescent dyes and the photonic band gap (PBG) of the PC. Benefitting from the synergistic effect of the tandem CRISPR system and PC array, as well as the high trans-cleavage activity of Cas13a protein, this engineered sensing array enables ultra-sensitive detection with a limit of detection (LOD) as low as 24.9 fM for CVA6. Meanwhile, this sensing strategy also achieved high-throughput and rapid analysis with a detection frequency of about 96 samples every 3.4 minutes. Therefore, the proposed strategy offers a simple workflow without reverse transcription or amplification, along with high sensitivity and high throughput, demonstrating strong potential for applications in biometrics and clinical diagnostics.