Portable “dual-signal-on” colorimetric and photothermal biosensor for accurate detection of Hg2+ based on split Cas12a-mediated cascade strand displacement and phosphorothioate-modified G-quadruplex DNAzyme

Abstract

The split crRNA with Cas12a (SCas12a)-powered biosensor has shown sensitivity and accuracy in nucleic acid detection, but its application to metal ion target detection still needs to be explored. By integrating an SCas12a-mediated cascade strand displacement reaction (CSDR) with a phosphorothioate-modified hairpin G-quadruplex (psHG4) reporter, a portable “turn-on” colorimetric/photothermal system was engineered for the precise and sensitive detection of Hg²⁺ (termed SCas12a/CSDR-psHG4). Target Hg²⁺ triggers an SCas12a-mediated CSDR and then activates SCas12a's nuclease activity. Activated SCas12a cleaves the loop region of psHG4, thereby liberating the previously silenced psG4 sequence, which subsequently assembles into a psG4/hemin complex in the presence of K⁺ and hemin. This complex catalyzes the typical oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), generating the colorimetric and photothermal product TMBox. The RGB value of the solution was recorded with a portable smartphone and the temperature change was tracked using a thermometer. This colorimetric/photothermal biosensor displayed a linear range of 1 pmol L⁻¹ to 100 nmol L⁻¹ and 100 fmol L⁻¹ to 10 nmol L⁻¹, with a detection limit of 0.5 pmol L⁻¹ and 60 fmol L⁻¹, respectively. Utilizing the SCas12a technique, SCas12a/CSDR-psHG4 presents a fresh method for identifying metal ion targets, along with significant prospects for enhancing human health protection and environmental surveillance.