A linear DNA probe as an alternative to a molecular beacon for improving the sensitivity of a homogenous fluorescence biosensing platform for DNA detection using target-primed rolling circle amplification

Abstract

Herein, we report a simple and homogenous fluorescence method for ultrasensitive DNA detection. It is based on rolling circle amplification (RCA) and fluorescence resonance energy transfer (FRET). As an alternative to a molecular beacon (MB), a linear single-labeled DNA probe was used in this RCA-based fluorescence strategy for DNA detection. The performance of linear DNA probes was compared with that of the MB probe in an RCA-based fluorescence strategy. The results showed that the linear DNA probes could effectively avoid the fluorescence quenching between neighboring signal probes, which would significantly improve the sensitivity of the RCA-based fluorescence strategy. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.7 aM, which was about 100-fold lower than that of the RCA-based fluorescence strategy with MB as the signal probe. This method provides a simple, isothermal, and low-cost approach for sensitive detection of DNA and holds great potential for early diagnosis in gene-related diseases.

Ultrasensitive DNA detection based on target-triggered rolling circle amplification and fluorescent poly(thymine)-templated copper nanoparticles

“…7.79 aM (3s/slope), a value that is signicantly lower or slightly higher than those from other RCA-based uorescent biosensors to detect the target DNA. 25,36,37 The ultra-high sensitivity of present method is attributed to the synergistic effect of RCA and highly uorescent poly T-CuNPs. Importantly, it should be emphasized that this method is more sensitive than RCA-based one that relies on DNA-AgNCs, which implies that CuNPs-based uorescent signaling is more effective than AgNCs.…”

Photoinduced Electron Transfer-Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA

“…The fluorescence response (F 0 -F) increased quickly with increasing reaction time, and nearly reached a plateau after 180 min ( Figure S3). This might be attributed to the fact that the RCA reaction was completed because of exhaust of RCA substrates or inactivation of phi29 DNA polymerase after 180 min [39]. Therefore, 180 min was used as the optimum time for RCA reaction.…”

“…This would decrease the sensitivity of the RCAbased biosensing platform. [39] So, MB is not an ideal signal probe for RCA-based biosensing platform.…”

“…Different from a short ssDNA, RCA product is tens of kilobases long single-stranded tandem repeated copies of the sequence, and folds into random coils in solution. [38,39,46] In the "signal-on" strategy, MB sequence is complementary to the RCA product in each repeat sequence, and the coiled RCA product can serve as a template for periodic binding of MB, resulting in open of numerous MBs. Because of effective spatial separation of FAM and "GGG" of intra MB, MBs can restore the fluorescence of FAM.…”

Combining magnetic nanoparticle capture and poly-enzyme nanobead amplification for ultrasensitive detection and discrimination of DNA single nucleotide polymorphisms

“…nM-pM sensitivity, see ESI, Table S2) without target amplification and more sophisticated designs. However, the 1 pM LOD is still uncompetitive against some of the recent ultrasensitive DNA assays (ESI , Table S3) [57][58][59][60] . Thus a more powerful signal amplification strategy was designed to further improve sensitivity (see later section).…”