Smartphone-Based Free-to-Total Prostate Specific Antigen Ratio Detection System Using a Colorimetric Reaction Integrated with Proximity-Induced Bio-Barcode and CRISPR/Cas12a Assay.

The free-to-total prostate-specific antigen (f/t-PSA) ratio is of great significance in the accurate diagnosis of prostate cancer. Herein, a smartphone-based detection system is reported using a colorimetric reaction integrated with proximity-induced bio-barcode and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a assay for f/t-PSA ratio detection. DNA/antibody recognition probes are designed to bind f-PSA or t-PSA and induce the release of the DNA bio-barcode. The CRISPR/Cas12a system is activated by the DNA bio-barcode to release Ag+ from the C-Ag+-C structure of the hairpin DNA. The released Ag+ is used to affect the tetramethylbenzidine (TMB)-H2O2-based colorimetric reaction catalyzed by Pt nanoparticles (NPs), as the peroxidase-like activity of the Pt NPs can be efficiently inhibited by Ag+. A smartphone with a self-developed app is used as an image reader and analyzer to analyze the colorimetric reaction and provide the results. A limit of detection of 0.06 and 0.04 ng mL -1 is achieved for t-PSA and f-PSA, respectively. The smartphone-based method showed a linear response between 0.1 and 100 ng mL -1 of t-PSA or f-PSA. In tests with clinical samples, the smartphone-based method successfully diagnosed prostate cancer patients from benign prostatic hyperplasia patients and healthy cases with high sensitivity and specificity.