Rolling circle amplification (RCA) is an isothermal enzymatic process in which a short nucleic acid primer is amplified to form a long single-stranded nucleic acid using a circular template and special nucleic acid polymerases. Furthermore, this approach can be further engineered into a device for point-of-need monitoring of environmental pollutants.
In recent years, the discharge of contaminants from industrial and agricultural activities and urban wastewater has caused serious contamination of the aqueous system, posing a great potential threat to human health and aquatic life. These contaminants can be divided into three categories: (i) inorganic chemical substances, (ii) organic pollutants and (iii) microorganisms. These substances can cause adverse effects on the environment , for example, the disruption of hormones and the endocrine system and the induction of cytotoxicity and/or genotoxicity and carcinogenesis . The variable composition of pollutants and their location in aqueous environments over time have resulted in increasing focus on new technologies that use cheap and real-time strategies to monitor pollutants. Most of these strategies are based on laboratory platforms, such as inductively coupled plasma mass spectrometry (ICP-MS) for the detection of heavy metal ions, liquid chromatography-tandem mass spectrometry (LC-MS) for the detection of small organic chemicals or their metabolites, and polymerase chain reaction (PCR) for the detection of nucleic acids and genetic information, which require preprocessing and frequent data sampling, which means that they are both expensive and slow. These aspects highlight the need to develop a new strategy that is more sensitive, portable, and efficient for on-site detection of pollutants composed of multiple substances .
Recently, rolling circle amplification (RCA)-based analytical methods have received increasing attention in environmental monitoring. RCA is an uncomplicated and efficient isothermal enzymatic process using unique DNA and RNA polymerases to produce long single-stranded DNA (ssDNA) and RNA . In RCA, the polymerase will spontaneously and continuously add nucleotides to the primers that bind to the circular template, generating long ssDNA with tandem repeats of tens to hundreds of orders of magnitude. Unlike PCR, which requires a thermal cycler and thermostable DNA polymerase. RCA can be in solution, on a solid support, or in a complex biological environment at a constant temperature (room temperature to 37 °C). The ability of RCA to grow a long DNA chain on a solid support or inside a cell from one molecular binding event enables the detection of targets at a single molecule level . In addition, an RCA product comprising repeating cyclic sequences complementary to template DNA can be customized by template design. By designing the template, the customizable DNA product includes functional sequences, including aptamers, DNAzymes, spacer domains, and restriction endonuclease sites. Of course, by hybridizing the RCA product with a complementary nucleic acid linked to a functional part including biotin , fluorophores , antibodies , and nanoparticles , it is easy to synthesize a multifunctional material with a variety of properties, including biorecognition and biosensing. Collectively, the properties of high-efficiency isothermal amplification, single-molecule sensitivity, versatility of structure and composition, and multivalences make RCA a powerful tool in aqueous environments . Currently, RCA has been extensively studied to develop sensitive methods for detecting DNA, RNA, DNA methylation, single nucleotide polymorphisms, small molecules, proteins, and cells. In addition to diagnosis, RCA has also been proven to be effective for cell-free cloning and sequencing , in situ genotyping and genome-wide analysis of cells and tissues . Recently, RCA has received widespread attention for its use in the production of DNA nanostructures such as origami, nanoribbons, nanotubes, DNA nanoscaffolds, and DNA metamaterials for periodic nanocomponents . Importantly, these materials have high prospects in a wide range of applications, including environmental monitoring, drug delivery, and in vivo imaging of manufacturing electronic circuits, including DNA-based materials.
|Features||Conventional PCR Assay||Real Time-PCR Assay||RCA Assay|
|Sensitivity||Sensitive||Highly sensitive||Highly sensitive|
|Temperature conditions||Thermal cycle||Thermal cycle||Isothermal|
|Inhibition by biological samples||Yes||Yes||No|
|Instruments required||Thermocycler||Thermocycler||Not required|
|Post-assay analysis||Required||Required||Generally not required|
|Amplicon detection methods||Gel electrophoresis||Real-time detection/amplification graph||Gel electrophoresis, Turbidity measurement by visual inspection or using a real-time turbidimeter; dye-based visual detection|
|Overall assay time||3–5 h||2.5–4 h||1–1.5 h|
|Cost effectiveness||Less expensive||Expensive||Less expensive|