Cascade Signal Amplification for DNA Detection

This paper reports a novel, isothermal DNA-detection method that integrates two steps of signal amplification: one by a protein enzyme and the other by a DNA enzyme (DNAzyme). Each analyte DNA molecule triggers a polymerase-mediated rollingcircle amplification (RCA) to produce a linear array of DNA peroxidases that catalyze a chemical oxidation and generate a colorimetric output. The current detection limit is 1 pm. DNA detection plays a critical role in biomedical research and clinical diagnostics. Extensive efforts have developed many sensitive methods based on modern biotechnology and nanotechnology, including the use of DNAzymes. DNAzymes can catalyze chemical reactions and amplify output signals. More importantly, they are more stable and robust than protein enzymes. Such properties lead to the wide use of DNAzymes in DNA detection. For instance, RNA-cleaving DNAzymes together with molecular beacons have been used in DNA detection, reaching a detection limit of 10 pm. Recently, Willner and co-workers have explored a method based on a preidentified DNA peroxidase that is a G-quadruplex–hemin complex. This DNAzyme catalyzes chemical reactions that generate chemiluminescence or produce colored products. If a DNA analyte molecule can only activate one DNAzyme, the detection will not be very sensitive. One way to overcome this problem is to introduce Au nanoparticles (AuNPs) as biobarACHTUNGTRENNUNGcodes. AuNPs are functionalized with many copies of DNAzymes. When an AuNP is immobilized to a solid surface by a DNA target molecule, multiple copies of DNAzymes will be immobilized and generate a strong output signal. DNAzymes can also be very efficiently produced by PCR, a thermal cycling technique. Based on the DNA peroxidase, we have developed an efficient way to amplify isothermal signals that greatly improves the detection limit. Our strategy contains two successive steps of isothermal, enzymatic amplification (Figure 1).