Lab in a tube: a fast-assembled colorimetric sensor for highly sensitive detection of oligonucleotides based on a hybridization chain reaction

In this work, a one-pot signal amplified strategy was constructed based on generating a hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme as the color product for the detection of DNA sequences. A DNA sequence associated with the hepatitis B virus (HBV) was selected as a model target. The enzyme-free and label-free biosensor contained three oligonucleotide terms as target DNA, hairpin structures H1 and H2 which were partially complementary. It should be noted that the G-quadruplex structure was partially hidden in the hairpin structure H2. In the absence of THBV, hairpins H1 and H2 were stable enough due to the complementary sequences at the end of the oligodeoxynucleotides, which made it difficult to complete the self-assembly. Upon addition of THBV, self-assembly of THBV with H1 allows the rest of the DNA sequence of H1 to facilitate H1–H2 complex formation and releases THBV to the next cycle. In the presence of hemin and K+, the G-quadruplex at the end of the H1–H2 complex was liberated to form a hemin/G-quadruplex structure, which could catalyze achromatous tetramethylbenzidine (TMB) into a colored product. The color change of the solution could be quantitated by spectrophotometry and the naked eye. We also employed 20% ethanol in the buffer to accelerate H1–H2 complex formation in the DNA strand displacement reaction (DSDR), which cut down the reaction time of the detection process from 12 h to 1 h. The detection limit of the colorimetric sensor is 9.5 pM, and the linear range is 50 pM to 100 nM.