滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

通过捕获探针与纳米金膜之间的共价连接, 保证了滚环扩增(RCA)产物始终结合于金膜表面, Phi29 DNA聚合酶的高效扩增和Escherichia coli DNA链接酶的高度精确性使检测达到单碱基识别, 检测灵敏度达到10⁴ copies/mL. 实验结果表明, 与单碱基错配序列相比, RCA可明显增强检测的灵敏度. 该RCA基因传感器操作简单, 灵敏度和特异性较高, 在乙型肝炎病毒的快速检测方面具有一定的开发潜力.     

A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid

An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a “caged” inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.     

Chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms on a solid surface using target-primed rolling circle amplification

A new chemiluminescent (CL) method has been developed for the sensitive detection of DNA hybridization and single-nucleotide polymorphisms (SNPs) with target-primed rolling circle amplification (RCA). The capture oligonucleotide probe is firstly immobilized on a polystyrene well plate and then hybridized with the wild DNA target. A designed padlock probe is circularized after perfect hybridization to the DNA target. Then the RCA reaction can be initiated from the DNA target that acts as a primer and generates a long tandem single-strand of DNA with repeat sequences. In contrast, the mutant DNA target, which contains a mismatched base with the padlock probe, cannot initiate the RCA reaction and primes only a limited extension with the unligated padlock probe. Afterwards, a biotinylated oligonucleotide is used to hybridize with the RCA product in each repeat sequence and streptavidin-alkaline phosphatase (STV-AP) is employed to combine the anchored biotin. The DNA target is detected with the CL reaction of STV-AP and 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). With the RCA-based method, the sensitivity of DNA detection can be increased by about two orders of magnitude compared with that of direct DNA hybridization. A DNA target as low as 3.6 pM can be detected. Wild-type DNA and the one-base mutant DNA can be differentiated with high selectivity through this RCA reaction.     

A versatile platform for highly sensitive detection of protein: DNA enriching magnetic nanoparticles based rolling circle amplification immunoassay

A novel rolling circle amplification (RCA) immunoassay based on DNA enriching magnetic nanoparticles and assembled fluorescent DNA nanotags, magnetic nanoparticles-RCA immunoassay, is developed as a versatile fluorescence assay platform for highly sensitive proteins detection.     

Target-Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal-On Biosensing with Toehold-Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′-end protects it from 3′-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.     

Protein-Mediated Suppression of Rolling Circle Amplification for Biosensing with an Aptamer-Containing DNA Primer

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.     

DNA microarray fabricated on poly(acrylic acid) brushes-coated porous silicon by in situ rolling circle amplification

Microarrays hold considerable promise in large-scale biology on account of their analytical, massive and parallel nature. In a step toward further enabling such a capability, we describe the application of rolling circle amplification (RCA) for a sensitive and multiplex detection of nucleic acid targets on oligonucleotide-conjugated polymer brushes covalently grown from porous silicon. Both RCA and polymer brushes have been taken to increase the loading quantity of target molecules and thus improve the detection sensitivity without loss of multiplexing. Besides, polymer brushes were employed to protect porous silicon and to provide biologically simulated environments, making the attached biomolecules maintain bioactivity. This approach can reach a detection limit of 0.1 nM target analytes and three orders of magnitude dynamic range of 0.1-100 nM, with a fluorescence scanner. A two-colour DNA microarray was achieved via RCA of two kinds of circular DNA targets on one chip simultaneously. The porous silicon chip-based RCA technique is promising for the multiplex detection of deoxynucleic acids on microarrays.     

Comparative quantification of nucleic acids using single-molecule detection and molecular beacons

This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.     

Highly sensitive detection of cancer cells based on the DNA barcode assay and microcapillary electrophoretic analysis

Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (μCE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the μCE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF‐7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the μCE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF‐7 and SW620 cells in a volume of 200 μL, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the μCE analysis.     

Detection of single-molecule DNA hybridization using enzymatic amplification in an array of femtoliter-sized reaction vessels

Improving the sensitivity of DNA biosensors is extremely important in clinical diagnostics, gene therapy, and a variety of other biomedical studies. In this regard, we have developed a highly sensitive single molecule DNA assay platform with a 1fM experimental detection limit using enzymatic amplification in an array of femtoliter-sized reaction wells. To validate the utility of this technology in our study, we employed a fiber optic array to create thousands of femtoliter-sized reaction wells, each specifically functionalized with oligonucleotide probes capable of capturing biotinylated target DNA. After hybridization, the fiber was incubated with streptavidin-labeled enzyme solution. The bound single enzyme molecules were confined to individual reaction vessels containing excess fluorogenic substrate and catalyzed the production of a sufficient number of fluorescent product molecules to generate a detectable signal. At low target DNA concentrations with relatively short incubation times, only a small percentage of the capture sites bind target DNA, enabling a binary readout of target concentration from the high-density fiber array. This simple binary readout-based scheme is easy to perform and exhibits a high signal-to-noise ratio in the presence of trace amounts of DNA target. Furthermore, it also should be possible to extend this technology to protein detection by modifying the reaction wells with specific capture antibodies. We expect this assay to be useful in a number of biomedical applications where accurate and highly sensitive target analysis is critical.     

G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection

Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

Simple and Sensitive Electrochemical DNA Detection of Primer Generation‐Rolling Circle Amplification

A new electrochemical sequence‐specific DNA detection platform based on primer generation‐rolling circle amplification (PG‐RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG‐RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG‐RCA amplicons. End‐point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon‐bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon‐bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37 °C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point‐of‐care and on‐site testing. Real‐time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin.     

Aptamer-conjugated bio-bar-code Au–Fe3O4 nanoparticles as amplification station for electrochemiluminescence detection of tumor cells

An electrochemiluminescence (ECL) assay has been developed for highly sensitive and selective detection of tumor cells based on cell-SELEX aptamer-target cell interactions through a cascaded amplification process by using bio-bar-code Au–Fe₃O₄ as amplification station. Firstly, bio-bar-code toehold-aptamer/DNA primer/Au–Fe₃O₄ (TA/DP/Au–Fe₃O₄) nanoconjugates are fabricated with a ratio of 1:10 to efficiently avoid cross-linking reaction and recognize target cells, which are immobilized on the substrate by hybridizing aptamer to capture probe with 18-mer. Through strand displacement reaction (SDR), the TA/DP/Au–Fe₃O₄ composites further act as the amplification station to initiate rolling circle amplification (RCA). As a result, on the surface of TA/DP/Au–Fe₃O₄, a large number of Ru(bpy)₂(dcbpy)NHS-labeled probes hybridize to RCA products, which are easily trapped by magnetic electrode to perform the magnetic particle-based ECL platform. Under isothermal conditions, this powerful amplification strategy permits detection of Ramos cells as low as 16 cells with an excellent selectivity. Moreover, analysis of Ramos cells in complex samples and whole blood samples further show the great potential of this ultrasensitive approach in clinical application involving cancer cells-related biological processes.     

Target‐Induced and Equipment‐Free DNA Amplification with a Simple Paper Device

We report on a paper device capable of carrying out target‐induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA‐enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.     

Target-driven self-assembly of stacking deoxyribonucleic acids for highly sensitive assay of proteins

In this paper, we report a new signal amplification strategy for highly sensitive and enzyme-free method to assay proteins based on the target-driven self-assembly of stacking deoxyribonucleic acids (DNA) on an electrode surface. In the sensing procedure, binding of target protein with the aptamer probe is used as a starting point for a scheduled cycle of DNA hairpin assembly, which consists of hybridization, displacement and target regeneration. Following numbers of the assembly repeats, a great deal of DNA duplexes can accordingly be formed on the electrode surface, and then switch on a succeeding propagation of self-assembled DNA concatemers that provide further signal enhancement. In this way, each target binding event can bring out two cascaded DNA self-assembly processes, namely, stacking DNA self-assembly, and therefore can be converted into remarkably intensified electrochemical signals by associating with silver nanoparticle-based readout. Consequently, highly sensitive detection of target proteins can be achieved. Using interferon-gamma as a model, the assay method displays a linear range from 1 to 500 pM with a detection limit of 0.57 pM, which is comparable or even superior to other reported amplified assays. Moreover, the proposed method eliminates the involvement of any enzymes, thereby enhancing the feasibility in clinical diagnosis.     

Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.     

Multiplex quantitative foodborne pathogen detection using high resolution CE-SSCP coupled stuffer-free multiplex ligation-dependent probe amplification

Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen-specific genes are optimal markers. Although multiplex ligation-dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length-dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer-free MLPA using high-resolution CE-SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE-SSCP analysis was designed for species-specific detection. After careful optimization of each MLPA step, to ensure that CE-SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5-5 pg of genomic DNA, and more than 100-fold increase could be quantitatively determined. Thus, MLPA-CE-SSCP is a sensitive and reliable technique for pathogen detection.     

Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

?29 DNA polymerase (?29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ?29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ?29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.