Dual-function paper-based biosensor for sensitive detection of hyaluronidase and human papillomavirus DNA using diffusion wet area as readout.

Herein, a novel dual-function paper-based biosensor using diffusion wet area as readout has been developed for simple and sensitive detection of hyaluronidase (HAase) and human papillomavirus (HPV) 16 DNA, respectively. The target-regulated-water absorption hydrogel synthesized by hyaluronic acid (HA) and single-stranded DNA (ssDNA) is chosen as an ideal material for diffusion wet area generation on paper. The hydrogel can be degraded through the enzymolysis of HA by HAase or the trans-cleavage of ssDNA by HPV DNA-activated CRISPR/cas12a system. Then the diffusion wet area generated by the broken hydrogel with the free water released from the hydrogel dropped on the glass fiber filter paper can be captured by a smart-phone and calculated by the product of the pixel ratio (pixels of wet area/pixels of glass fiber filter paper) and actual area of the filter paper. The diffusion wet area has good linear relationships with HAase (0-40 U/mL) and HPV 16 DNA (0.1-1000 pM), respectively. The biosensor provides a novel signal readout mode, shows satisfactory sensitivity with the detection limits of 0.15 U/mL for HAase and 66.95 fM for HPV 16 DNA, and has been successfully used for HPV 16 DNA and HAase detection in clinical urine and cervical brush samples, which is promising for early cervical cancer screening and HPV monitoring in HPV patients.