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

Abstract

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 T_HBV, 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 T_HBV, self-assembly of T_HBV with H₁ allows the rest of the DNA sequence of H₁ to facilitate H₁–H₂ complex formation and releases T_HBV 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.