BioanalysisVol. 6, No. 7 EditorialFree AccessCircular strand displacement polymerization reaction: a promising technique?Dou Wang, Chenchen Ge & Lingwen ZengDou WangGuangzhou Institutes of Biomedicine & Health, Chinese Academy of Sciences, Guangzhou 510530, China, Chenchen GeGuangzhou Institutes of Biomedicine & Health, Chinese Academy of Sciences, Guangzhou 510530, China & Lingwen Zeng* Author for correspondence: E-mail Address: lzeng8@126.comGuangzhou Institutes of Biomedicine & Health, Chinese Academy of Sciences, Guangzhou 510530, ChinaPublished Online:7 May 2014https://doi.org/10.4155/bio.14.28AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: circular strand displacement polymerization reactioncolorimetric inspection hairpin probeisothermal amplificationpoint-of-care diagnosisAs a leading method in detecting trace amount of nucleic acids, PCR has acted as an outstanding method for target amplification that offers high specificity and sensitivity. However, the need for a thermocycling apparatus has hindered its application in point-of-care (POC) diagnosis. Various nucleic acid isothermal amplification techniques based on new discoveries in DNA/RNA synthesis and auxiliary proteins or aptamers have demonstrated the possibility of DNA amplification in vitro, thereby eliminating the need for complex devices [1].A new isothermal amplification technologyCircular strand displacement polymerase reaction (CSDPR) is a new member of the family of isothermal amplification technologies, first introduced in 2009 by Guo et al.[2]. The designed reaction system initiates with a hairpin probe opening its stem segment, induced by a hybridization event between hairpin and target DNA. Next, a strand displacement polymerization reaction takes place in the presence of a primer and DNA polymerase, then the target DNA is displaced and triggers a new hybridization in a series of cascading reactions. This strategy has the advantage of using a hairpin probe as a template, as well as a fluorescence signal carrier to provide a simple detection method. CSDPR is a valuable tool in the detection of biological samples with single-stranded genomic DNA. Researchers have attached importance to this technology and developed various strategies and biochemical sensors to expand the application and scope of CSDPR. For example, the recently developed CSDPR technologies can not only detect single-stranded genomic DNA, but also small molecules and proteins with the aid of aptamers [3–5]. Notwithstanding these advances, further studies are still needed before we know for certain whether it is a promising technique as several weaknesses still exist in the technology (as discussed below), and the specificity, sensitivity and operability of CSDPR need to be improved.Specificity & sensitivityThe method's ability to discriminate base mismatches is directly related to the number of bases in the hairpin structure. The hairpin probe with a longer stem can improve mismatch discrimination ability, yet at the same time it decreases the hybridization rate and detection efficiency due to the larger activation barrier [6]. Therefore, careful design of the hairpin probe sequence is needed in order to decrease the nonspecific opening of the stem-loop structure. Meanwhile, in order to solve the specificity problem associated with a single hairpin probe, it is feasible to design multiple specific hairpin probes for a target with parallel processing. For the detection of a single nucleotide polymorphism, the combination of using high-fidelity T4 DNA ligase with CSDPR has shown good potential in personalized medicine; for example in the detection of cancer-related mutations and drug-resistant mutations of infectious agents [7].Despite the innovative design in which each primer extension requires a smaller time interval than PCR, the original CSDPR is still a linear amplification method and the amplification efficiency is not equivalent to an exponential amplification. Some researchers have tried to introduce nicking endonuclease with two molecular switches operating in series in order to drive an exponential amplification [8]. It enables CSDPR to hold a higher sensibility, thereby facilitating the detection of trace amounts of DNA and narrowing the window period before the pathogenic microorganisms can be detected.How to achieve colorimetric inspection?Most improved CSDPR techniques require costly fluorescence measurement systems or electrochemical transducer elements. Some modifications are required to achieve colorimetric inspection, such as the loop-mediated isothermal amplification producing large amounts of white precipitate. The extensively studied G-quadruplex DNAzyme would be a good candidate. Reverse complement of the G-quadruplex sequence can be added into the hairpin probe to catalyze a color change for a signal readout, as well as reducing the need for expensive labeling procedures [Wang D & Zeng L, Unpublished Data]. This homogeneous assay includes an amplification element, a detection transducer and a dye of acceptable specificity, as well as a format that eliminates the need for opening tube lids to avoid post-amplification manipulation and contamination. Lateral flow biosensor is another portable format to realize simple and visual readout. The use of CSDPR, coupled with a nicking endonuclease-assisted lateral flow biosensor, can lead to a DNA-detection system that operates without any qualified personnel or instruments [9]. However, any CSDPR-based detection format should not reduce the specificity and should give unequivocal results.Sample preparation is a bottleneckCSDPR technology also did not solve a key problem seen in other nucleic acid amplification methods. For example, sample preparation is a bottleneck, especially for trace DNA analysis, it is a cumbersome process involving specimen collection, DNA extraction and, for the case of double-stranded DNA, the need for heat denaturation to obtain single-stranded DNA for hybridization with the hairpin probe. In central laboratories, sample preparation is typically automated on large instruments. For POC nucleic acid tests, sample preparation is manually performed and requires an integrated design for all sample pretreatment steps in order to decrease time expenditure. Thus, it is advisable to synthesize all functional constituents on a disposable credit-card-sized microfluidic device. The device will reduce the consumption of samples and reagents, as well as reduce the operation complexity and the length of assay time.Still holds great promiseIn conclusion, this article briefly reviews the emergence and development of CSDPR as a promising new technology, but with some challenges and weaknesses that still need to be overcome. While this new emerging technology has some soft spots, the delicately designed hairpin structure still makes it hold great promise for biochemical assays. This technology will inspire the development of new techniques and instrumentation, such as the development of diagnostic devices for the detection of proteins, nucleic acids and toxins for biomedical applications. Future work should look at the application of this technology with an emphasis on the development of more convenient devices performed in a single tube or lateral flow biosensor without the need for complicated operations, combination of sample preparation, amplification and detection technologies that might facilitate fully integrated, low-cost POC nucleic acid test.This nucleic acid isothermal amplification technology should not only act as a vehicle to compete with the widely used non-isothermal biochemical sensors or to expand the state-of-art DNA nanotechnologies, but more importantly, should be used to expand the field of POC diagnostic applications making them more accessible and affordable for the masses without the need for complicated devices and highly qualified personnel.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1 Gill P, Ghaemi A. Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic Acids27(3),224–243 (2008).Crossref, Medline, CAS, Google Scholar2 Guo Q, Yang X, Wang K et al. Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction. Nucleic Acids Res.37(3),e20 (2009).Crossref, Medline, Google Scholar3 Ding C, Li X, Ge Y, Zhang S. Fluorescence detection of telomerase activity in cancer cells based on isothermal circular strand-displacement polymerization reaction. Anal. Chem.82(7),2850–2855 (2010).Crossref, Medline, CAS, Google Scholar4 Huang J, Chen Y, Yang L et al. Amplified detection of cocaine based on strand-displacement polymerization and fluorescence resonance energy transfer. Biosens. Bioelectron.28(1),450–453 (2011).Crossref, Medline, CAS, Google Scholar5 Lie P, Liu J, Fang Z, Dun B, Zeng L. A lateral flow biosensor for detection of nucleic acids with high sensitivity and selectivity. Chem. Commun.48(2),236–238 (2012).Crossref, Medline, CAS, Google Scholar6 Kolpashchikov D. Binary probes for nucleic acid analysis. Chem. Rev.110(8),4709–23 (2010).Crossref, Medline, CAS, Google Scholar7 Xiao Z, Lie P, Fang Z et al. A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction. Chem. Commun.48(68),8547–8549 (2012).Crossref, Medline, CAS, Google Scholar8 Connolly AR, Trau M. Isothermal detection of DNA by beacon-assisted detection amplification. Angew. Chem.49(15),2720–2723 (2010).Crossref, CAS, Google Scholar9 Liu J, Chen L, Lie P, Dun B, Zeng L. A universal biosensor for multiplex DNA detection based on hairpin probe assisted cascade signal amplification. Chem. Commun.49(45),5165–5167 (2013).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByComparison of chemiluminescent heterogeneous and homogeneous–heterogeneous assays coupled with isothermal circular strand-displacement polymerization reaction amplification for the quantification of miRNA-1411 January 2022 | The Analyst, Vol. 147, No. 19The Growing Interest in Development of Innovative Optical Aptasensors for the Detection of Antimicrobial Residues in Food Products3 March 2020 | Biosensors, Vol. 10, No. 3Ag(I)-coordinated hairpin DNA for homogenous electronic monitoring of hepatitis C virus accompanying isothermal cycling signal amplification strategyBiosensors and Bioelectronics, Vol. 73 Vol. 6, No. 7 Follow us on social media for the latest updates Metrics History Published online 7 May 2014 Published in print April 2014 Information© Future Science LtdKeywordscircular strand displacement polymerization reactioncolorimetric inspection hairpin probeisothermal amplificationpoint-of-care diagnosisFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Dou Wang

Chenchen Ge

Lingwen Zeng

Bioanalysis

通过对生物样品中的异生物质和生物进行选择性、灵敏和可重复分析获得的可靠数据是每个成功的药物开发计划的基础和关键部分。同样的原则也适用于许多其他研究领域，例如法医学、毒理学和运动兴奋剂检测。生物分析领域结合了复杂的技术，将样品制备和高级分离与 MS 和 NMR 检测系统、自动化和机器人技术相结合。监管机构制定的方法开发和验证标准越来越多地定义了速度和质量之间的界限。生物分析是一门进步的学科，未来拥有许多令人兴奋的机会来进一步减少样品量、分析成本和环境影响，以及提高灵敏度、特异性、准确性、效率、测定通量、数据质量、数据处理和处理。 《生物分析》杂志侧重于用于检测或定量研究人类或动物生物样本中分析物的技术和方法。生物分析鼓励提交描述前瞻性应用的文章，包括生物传感器、微流体、小型化分析设备以及新的连字符和多维技术。生物分析以简洁、一目了然的文章格式提供基本信息。该领域的主要进展由国际专家报告和分析，为现代生物分析家提供了一个权威但易于访问的论坛。

Reliable data obtained through selective, sensitive, and reproducible analysis of xenobiotics and biomolecules in biological samples is fundamental to any successful drug development program. This principle also applies to many other research fields, such as forensic science, toxicology, and doping detection. The field of bioanalysis integrates complex technologies, combining sample preparation and advanced separation with mass spectrometry (MS) and nuclear magnetic resonance (NMR) detection systems, automation, and robotics. Regulatory agencies are increasingly defining the boundaries between speed and quality through method development and validation standards. Bioanalysis is an evolving discipline with many exciting opportunities in the future to further reduce sample volume, analysis costs, and environmental impact, while enhancing sensitivity, specificity, accuracy, efficiency, throughput, data quality, data processing, and handling capabilities. The journal "Bioanalysis" focuses on technologies and methods used for the detection or quantification of analytes in human or animal biological samples. Bioanalysis encourages the submission of articles describing forward-looking applications, including biosensors, microfluidics, miniaturized analytical devices, and novel hyphenated and multidimensional techniques. The journal presents essential information in a concise and clear article format, with major advancements in the field reported and analyzed by international experts, providing a credible yet accessible forum for modern bioanalysts.

生物样品中异生物质和生物的选择性、灵敏和可重复分析所获得的可靠数据，是成功药物开发计划的基础和关键部分。这一原则同样适用于法医学、毒理学和运动兴奋剂检测等多个研究领域。生物分析领域结合了复杂的技术，将样品制备、高级分离与质谱（MS）和核磁共振（NMR）检测系统、自动化和机器人技术相结合。监管机构制定的方法开发和验证标准，越来越多地定义了速度与质量之间的界限。生物分析是一门不断进步的学科，未来有许多令人兴奋的机会，可以进一步减少样品量、分析成本和环境影响，同时提高灵敏度、特异性、准确性、效率、测定通量、数据质量、数据处理和处理能力。《Bioanalysis》杂志专注于用于检测或定量研究人类或动物生物样本中分析物的技术和方法。生物分析鼓励提交描述前瞻性应用的文章，包括生物传感器、微流体、小型化分析设备以及新的连锁和多维技术。该杂志以简洁、一目了然的文章格式提供基本信息，领域内的主要进展由国际专家报告和分析，为现代生物分析家提供了一个权威但易于访问的论坛。

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Circular strand displacement polymerization reaction: a promising technique?

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