Research Progress on Rolling Circle Amplification (RCA)-Based Biomedical Sensing

Abstract

Enhancing the limit of detection (LOD) is significant for crucial diseases. Cancer development could take more than 10 years, from one mutant cell to a visible tumor. Early diagnosis facilitates more effective treatment and leads to higher survival rate for cancer patients. Rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic process that utilizes nuclease to generate long single stranded DNA (ssDNA) or RNA. The functional nucleic acid unit (aptamer, DNAzyme) could be replicated hundreds of times in a short period, and a lower LOD could be achieved if those units are combined with an enzymatic reaction, Surface Plasmon Resonance, electrochemical, or fluorescence detection, and other different kinds of biosensor. Multifarious RCA-based platforms have been developed to detect a variety of targets including DNA, RNA, SNP, proteins, pathogens, cytokines, micromolecules, and diseased cells. In this review, improvements in using the RCA technique for medical biosensors and biomedical applications were summarized and future trends in related research fields described.

Current and Emerging Methods for the Synthesis of Single-Stranded DNA

“…Rolling circle amplification (RCA) relies on an isothermal polymerase (typically the large fragment of Bsu, Bst, and E. coli DNA Polymerase I, or Φ29 DNA polymerase) to synthesize a long stretch of repeating ssDNA sequences in a single unit (Figure 2e) [83]. An RCA reaction requires four factors: A DNA polymerase, a corresponding DNA primer, a template, and deoxynucleotide triphosphates (dNTPs) [84]. In RCA reaction, nucleotides are added continuously to the annealed primer by the polymerase, which generates a long ssDNA with a repeating sequence [85].…”

“…In RCA reaction, nucleotides are added continuously to the annealed primer by the polymerase, which generates a long ssDNA with a repeating sequence [85]. RCA is a powerful induction system because it can produce large amounts of ssDNA at the scale of a micron and a detectable amplification of a single molecule [84]. Examples for microgram-scale ssDNA production include using cutter hairpins [86] or annealing of a complementary digestion splint to form double-stranded restriction sites [87,88].…”

Roadmap to the Bioanalytical Testing of COVID-19: From Sample Collection to Disease Surveillance

“… 45 These fragments act as a feeder sequence to bind to dye-labeled sequences for colorimetric detection on lateral flow strips (LFS). 63 , 64 Huang et al developed an assay for the colorimetric detection of SARS-CoV-2 using padlock probe RCA, which detected the RCA product by analyzing hydrogen ions released during the dNTPs addition in the synthesis of DNA strands using a pH indicator. The LOD of the assay was determined to be 2.5 pM (6 × 10 7 copies/reaction or 1.5 x10 9 copies/mL) using the synthetic glycoprotein gene for SARS-CoV-2 suspended in buffer with analysis time of 30 min at room temperature.…”

Room Temperature Isothermal Colorimetric Padlock Probe Rolling Circle Amplification for Viral DNA and RNA Detection

“…The circular probe then serves as a template for the chosen DNA polymerase to continually synthesize DNA strands and thereby amplifying the signal (Hamidi and Perreault, 2019) . If there is no complementary sequence to be targeted, this process would not occur (Gu et al, 2018) . Not only is this highly sensitive and specific, but the ability to modify the procedures of RCA to conduct direct RNA testing, room temperature reactions, and colorimetric visual detection makes RCA the preeminent method for viral detection.…”