Suppressing the background activity of hemin for boosting the sensitivity of DNAzyme-based biosensors by SYBR Green I

Peroxidase-like DNAzymes have been extensively used to replace horseradish peroxidase (HRP) for developing biosensors for signal amplification. However, the background activity from the cofactor (i.e., free hemin) has limited the sensitivity of such sensors. Herein, we aim to find an inhibitor for hemin to suppress the background signal, and a classic split DNAzyme-based sensor was used to detect a complementary DNA oligonucleotide. After screening a series of dyes, SYBR Green I (SG, one of the DNA stanning dyes) was selected for suppressing the background. Simply by adding 0.84 μM SG, the background from 50 nM hemin was suppressed over 30-fold. The suppression was caused by the interaction between SG and hemin. In the presence of the target DNA, the formed duplex region and G-quadruplex structure can better bind SG and hemin respectively, thus preventing the interaction between them and showing a high activity of the DNAzyme. The optimized sensor showed a detection limit of 3.8 pM for the target DNA (p53 gene). In addition, the backgrounds from chemiluminescence, colorimetric and fluorescence sensing modes can all be reduced by adding SG to the split DNAzyme system. The suppression of the background of peroxidase DNAzymes is a critical step towards practical use of related biosensors.