Designed Hairpin Probe Research Articles

Bidirectional Polymerization-Transcription Amplification-Encoded Dual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection of Multiple piRNAs.

PIWI-interacting RNAs (piRNAs) are a type of endogenous noncoding RNAs with a length of 24-31 nucleotides, and they can specifically bind with PIWI proteins to form the piRNA/PIWI complexes for regulating multiple physiological and pathological processes. Herein, we develop a bidirectional polymerization-transcription amplification-encoded dual-color fluorescent biosensor for label-free and primer-free measurements of multiple piRNAs. The designed hairpin probe contains a palindromic tail, and it can serve as the target recognition unit, polymerization primer, and transcription template. In the presence of target piRNAs, the hairpin probes are opened to expose a palindromic sequence that can trigger bidirectional polymerization and transcription reaction with the assistance of KF polymerase and T7 RNA polymerase for the production of numerous RNA aptamers. The aptamers subsequently bind with the corresponding fluorophores (DFHBI-1T/MG) to form the RNA aptamer-fluorophore complexes for the generation of enhanced fluorescence signals. This biosensor can sensitively detect piR-36026 with a limit of detection (LOD) of 82.08 aM and piR-36743 with a LOD of 44.44 aM. Moreover, it can quantify cellular piRNAs with single-cell sensitivity and distinguish cancer cells from normal cells. Furthermore, it has the capability of distinguishing the expression of piRNAs in the tissues of breast cancer patients and healthy individuals. By simply altering the target recognition site of the hairpin probe, this biosensor can be extended to detect various piRNAs, offering a powerful platform for piRNA-related clinical diagnostics and therapeutics.

Electrochemical Biosensing Platform Based on Toehold-Mediated Strand Displacement Reaction and DSN Enzyme-Assisted Amplification for Two-Target Detection.

Simultaneous detection of multiple targets is of great significance for accurate disease diagnosis. Herein, based on duplex-specific nuclease (DSN) assisted signal amplification and the toehold-mediated strand displacement reaction (TSDR), we constructed an electrochemical biosensor with high sensitivity and high specificity for dual-target detection. MiRNA-141 and miRNA-133a were used as the targets, and ferrocene (Fc) and methylene blue (MB) with significant peak potential differentiation were used as the electrochemical signal probes. The elaborately designed hairpin probe H1, which was fixed on the electrode surface, could be hybridized with the target miRNA-141 to perform signal amplification by the DSN-assisted enzyme cleavage cycle; thus, miRNA-141 could be detected by Fc signal changes at 0.41 V. The hairpin H1 can also combine with the MB-labeled signal probe (SP) output from miRNA-133a-induced TSDR, and the detection of miRNA-133a can be realized according to the response signal generated by MB at -0.26 V. The two sensing lines are independent of each other, and there is no mutual interference in the detection process. Therefore, two independent detection lines could be connected in series, and the simultaneous detection of two targets can be achieved on a single electrode. This novel detection strategy provides a new way to simultaneously detect different biomarkers.

Ultrasensitive strategy for OTA analysis by both recognition-activated multiple isothermal cyclic strand displacement amplification and DNAzyme-mediated cyclic signal reporting mechanism

We propose a recognition-driven ultrasensitive fluorescence sensing strategy for accurate analysis of ochratoxin A (OTA) by taking Mg2+-dependent DNAzyme based catalysis as the final signal output module. Recognition of target OTA destroys the simple double-strand recognition structure and contributes to the liberation of primer probe, which can trigger and activate the primer-mediated multiple isothermal cyclic strand displacement amplification (M-CSDA) process. The designed dual templates (T-P, T-A) can contribute to the generation of enormous nicked products (DNAzyme and primer), which will take effect in the triggering of M-CSDA. Meanwhile, one of the nicked products (DNAzyme) will also take part in the cyclically cleavage of designed hairpin probes (HP) with the presence of the cofactor Mg2+, inducing the recovery of fluorescent signal of quenched hairpin probes. Only the trace amount of target OTA could generate drastic fluorescent signal and ultrasensitive detection of OTA could be well achieved. Under optimized conditions, the detection limit of this method for OTA is 0.59 fg/mL. The characteristics of this method including the simplicity in dual signal amplification design, isothermal operation process and easy fluorescence measurement model exhibit the superior sensitivity and specificity for OTA detection. And this strategy can be further expanded for the design of ultrasensitive aptamer-sensing methods for series analytes.

Exonuclease III-propelled DNAzyme walker: an electrochemical strategy for microRNA diagnostics.

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100pM and achieves a detection limit of 5 fM (3σcriterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics.

Catalytic Assembly of DNAzyme Integrates with Primer Exchange Reaction (CDiPER) for Highly Sensitive Detection of MicroRNA.

MicroRNAs (miRNAs) have significant regulatory functions in the modulation of gene expression, making them essential biomarkers for the diagnosis and prognosis of diseases. Nevertheless, the identification of miRNA poses significant difficulty in terms of its low abundance, necessitating sensitive and reliable approaches. Herein, we develop a simple approach, termed Catalytic assembly of DNAzyme integrates with Primer Exchange Reaction (CDiPER), for reliable and sensitive miRNA detection through the target recognition-triggered DNAzyme assembly and primer exchange reaction (PER) strategy. In this method, target miRNA can precisely bind with a specifically designed hairpin probe (H probe) to induce the conformation changes of the H probe, releasing DNAzyme sections to activate the PER process for signal amplification and fluorescence signal production. The established method displays a high dynamic range of over 6 orders of magnitude and a low detection limit of 312 aM. The created method has a number of unique advantages, such as (i) a better sensitivity than existing systems using PER for signal amplification as a result of its integration with the target recognition-triggered DNAzyme assembly and (ii) streamlined operating procedures. Further, the technology was used to detect the expression of miRNA in collected clinical samples from diabetes mellitus patients, revealing that miRNA was decreased in patients and demonstrating the significant clinical promise of the method.

Primer Exchange Reaction Coupled with DNA-Templated Silver Nanoclusters for Label-Free and Sensitive Detection of MicroRNA.

MicroRNAs (MiRNAs) play pivotal roles in regulating gene expression, and serve as crucial biomarkers for diagnosis of a variety of disease. However, label-free and sensitive miRNA detection remains a huge challenge due to the low abundance. Herein, we developed an approach through integrating primer exchange reaction (PER) with DNA-templated silver nanoclusters (AgNCs) for label-free and sensitive miRNA detection. In this method, PER was used to amplify miRNA signals and produce single-strand DNA (ssDNA) sequences. The produced ssDNA sequences mediated DNA-templated AgNCs based signal generation by unfolding the designed hairpin probe (HP). The generated AgNCs signal was correlated with the dosage of target miRNA. Eventually, the established approach exhibited a low detection of limit of 47 fM with a great dynamic range of more than five orders of magnitude. In addition, the method was also utilized to detect the miRNA-31 expression in collected clinical samples from pancreatitis patients and demonstrated that miRNA-31 was upregulated in patients, showing a great promising of the method in clinical application.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and β within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Highly sensitive detection of MUC1 by microchip electrophoresis combining with target recycling amplification and strand displacement amplification

Mucin 1 (MUC1) is usually overexpressed in a variety of malignant tumors, and quantitative analysis of MUC1 plays an important role in the early diagnosis of cancer. In this work, a highly sensitive MUC1 assay was developed by integrating microchip electrophoresis (MCE) with target recycling amplification (TRA) and strand displacement amplification (SDA). Specifically, the presence of MUC1 can trigger the exposure of the designed hairpin probe (HP) to initiate SDA and an amplified amount of ssDNA is produced finally. The amount of these ssDNA can be detected by MCE, then the concentration of MUC1 can be obtained through the correlation between MUC1 concentration and ssDNA concentration. The experimental results show that the MCE signal had a good linear relationship with MUC1 concentration in the range of 1.0 pg/mL - 1.0 × 103 pg/mL with a low limit of detection of 0.23 pg/mL under the optimal conditions (S/N = 3). Additionally, the assay had been successfully applied to detect MUC1 in biological samples with satisfactory results, providing an alternative assay for the detection of other tumor markers owing to the high sensitivity, high selectivity, simple operation and low sample consumption.

Fluorescence biosensing of the leukemia gene by combining Target-Programmed controllable signal inspiring engineering

Clinical diagnosis urgently requires ultrasensitive, accurate and rapid monitoring of low-abundance biomarkers. A biosensing strategy capable of detecting target genes at the femtomolar scale was designed in this work. In the biosensing strategy, the target can induce the specially designed hairpin probe H1 to self-fold and form a 3′ blunt-ended structure. When there are the hybrid double-stranded P1-T1, ligase, polymerase and nickase, the target gene was recycled, and at the same time the system produces a lot of T1 and T2. T1 and T2 can simultaneously trigger HCR, causing the modified fluorophore FAM on the DNA strand to move away from the quencher group BHQ. The amplified fluorescent signal can be captured by a fluorescence instrument. It is exciting for us that three signal amplifications are involved to achieve femtomolar detection of target genes, namely target recycling, dual-triggered HCR of T1 and T2, and HCR. In addition, it still has good detection ability in actual samples simulated by serum. We expect that the sensing strategy proposed in this paper offers great potential for biomarker detection of leukemia for early clinical diagnosis.

Development of a CRISPR-Cas-Based Biosensor for Rapid and Sensitive Detection of 8-Oxoguanine DNA Glycosylase.

8-Oxoguanine DNA glycosylase is essential for maintaining genomic integrity and stability, while its abnormal activity may lead to the disturbance in the normal DNA damage repair and the occurrence of carcinogenicity and teratogenicity. Herein, we construct a CRISPR-Cas-based biosensor for rapid and sensitive measurement of 8-oxoguanine DNA glycosylases. This biosensor involves a hairpin probe and integrates quadratic strand displacement amplification (SDA) with a CRISPR/Cas12a effector with the characteristics of rapidity (within 40 min) and isothermal assay. The presence of 8-oxoguanine DNA glycosylase can initiate the quadratic SDA to produce large amounts of activators with the assistance of polynucleotide kinase (PNK). Subsequently, the activators can bind with crRNA to activate Cas12a, cleaving signal probes and recovering Cy5 fluorescence, which can be accurately quantified by single-molecule imaging. Notably, the designed hairpin probes can effectively block the hybridization of the generated activators with free hairpin probes, endowing this biosensor with high sensitivity. In addition, the utilization of PNK instead of apurinic/apyrimidinic endonuclease (APE1) greatly simplifies the experimental procedure to only a one-step reaction. The introduction of a single-molecule detection further reduces the sample consumption and improves the sensitivity. This biosensor displays a detection limit of 4.24 × 10-9 U μL-1, and it can accurately quantify cellular human 8-oxoguanine DNA glycosylase at a single-cell level. Furthermore, this biosensor can be applied for the screening of inhibitors, the analysis of kinetic parameters, and the discrimination of cancer cells from normal cells, with potential applications in molecular diagnostic and point-of-care testing.

Sensitive detection of hepatitis C virus using a catalytic hairpin assembly coupled with a lateral flow immunoassay test strip

Currently, PCR is the gold standard for the detection of hepatitis C virus (HCV). However, the PCR technique is complicated and time-consuming, which prevents its application and, clinical point-of-care testing (POCT). Herein, we report a POCT method with simplicity, high sensitivity and specificity, which consists of a catalytic hairpin assembly (CHA) signal amplification system coupled with a lateral flow immunochromatographic (LFIA) test strip for the detection of HCV. Two ingeniously designed hairpin probes were hybridized to form the H1–H2 duplex in the presence of the target DNA. The catalytic hairpin assembly which was characterized of isothermal and enzyme-free, was accomplished within 40 min and the reaction was then applied to a LFIA test strip. Only the H1–H2 duplex labeled with both digoxin and biotin could be captured by the test strip, and the fluorescence value was determined. In addition, we evaluated the application potential for the detection of clinical samples. The reported method demonstrated high sensitivity with a detectable minimum concentration at 1 fM and showed a good linear range from 10 nM to 10pM, and high specificity for various mismatched sequences. The results demonstrated that clinically positive samples could be successfully detected. In conclusion, the reported method is simple, rapid, and free of large-scale equipment. POCT is expected to be useful for HCV detection in clinic.

A novel fluorescent aptasensor based on exonuclease-assisted triple recycling amplification for sensitive and label-free detection of aflatoxin B1

Aflatoxins are the most toxic type of mycotoxins, which may cause serious carcinogenesis, teratogenesis, and mutagenesis to humans and animals. In this work, we demonstrate a novel label-free fluorescent aptasensor based on exonuclease-assisted triple recycling amplification for the sensitive detection of aflatoxin B1 (AFB1). With the close cooperation of T7 exonuclease and three elaborately designed hairpin probes, the target AFB1 can perform three consecutive cycles of amplification reactions. In this process, each hairpin probe is fully utilized, and the target AFB1, the secondary target and the tertiary target are recycled, thereby achieving a high amplification. Interestingly and importantly, the secondary and tertiary targets generated by amplification are also excellent DNA template sequences for silver nanoclusters (AgNCs). In the presence of NaBH4 and AgNO3, a great number of DNA-AgNCs are synthesized, thereby producing a strong fluorescent signal. Under optimal conditions, the developed aptasensor exhibited high sensitivity to AFB1 with a low detection limit of 0.19 pg mL−1 and a wide dynamic range of 1 × 10−6–1 μg mL−1. In addition, the aptasensor also performed well in the determination of AFB1 in real samples.

Self-priming phosphorothioated hairpin-mediated isothermal amplification

We herein describe a novel technology, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP), enabling target nucleic acid detection. Isothermal amplification strategies are a simple process that efficiently raises the amount of nucleic acid at a constant temperature, but still has lots of problems such as the requirement of multiple exogenous primers and enzymes, which trigger non-specific background signal and increase the complexity of procedures. The key component for overcoming the above-mentioned limitations is the designed hairpin probe (HP) consisting of self-priming region along the 3′ stem and the 3′ overhang and phosphorothioate modifications at the 5′ overhang and the specific loop part. The HP was designed to open through binding to target nucleic acid. Upon opening of HP, its self-priming (SP) region is rearranged to form a smaller hairpin whose 3′ end could serve as a primer. The following extension produces the extended HP and displaces the bound target nucleic acid, which is then recycled to open another HP. Due to the reduced stability caused by the specific two phosphorothioate (PS) modifications, the 3′ end of EP1 is readily rearranged to form the foldback hairpin structure, which would promote the foldback extension to produce once more extended HP. Since the two PS modifications are always located at the same positions along the 5’ stem within the further extended HPs, the foldback reaction followed by the extension would be continuously repeated, consequently producing a large number of the long hairpin concatamers. Based on this unique design principle, we successfully detected even a single copy of target DNA with outstanding discrimination capability under an isothermal condition by employing only a single HP without the requirement for the complicated multiple primers. In conclusion, the sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling short target nucleic acid detection such as microRNAs or any target which is less than 200 mer.

Nucleic acid-functionalized metal-organic framework for ultrasensitive immobilization-free photoelectrochemical biosensing

This work established an immobilization-free photoelectrochemical (PEC) biosensor for ultrasensitive determination of carcinoembryonic antigen (CEA) based on the DNA-functionalized metal-organic frameworks (MOFs) and T7 exonuclease-aided recycling amplification. In this proposal, MOFs were served as nanocarriers for efficient encapsulation of electron donors, while an ingeniously designed hairpin probe (HP) employed as the recognition element. The recognition of CEA by its aptamer sequence in HP triggered the conformational change and the T7 exonuclease-aided recycling amplification, which opened the pore of MOFs to release a large number of electron donors, producing a significantly increased photocurrent. Benefitting from the high loading ability of MOFs and the excellent amplification efficiency of the T7 exonuclease-assisted recycling process, the proposed biosensor is capable of ultrasensitive and highly selective determination of CEA with a detection limit down to 0.36 fg mL-1 and a wide linear range from 1.0 fg mL-1 to 10 ng mL-1. Moreover, the proposed biosensor can also apply to measure CEA in spiked serum samples, indicating that this PEC biosensor holds excellent potential for application in bioanalysis and early disease diagnosis.

Aptamer-Based Fluorescent Determination of Salmonella paratyphi A Using Phi29-DNA Polymerase-Assisted Cyclic Amplification

A rapid and ultrasensitive fluorescence aptasensor was developed for the detection of Salmonella paratyphi A based on aptamer and Phi29-DNA polymerase-assisted cyclic signal amplification. The method employed a designed arched probe, consisting of an aptamer and a primer, with a designed hairpin probe. The quenching groups and fluorescent groups were modified at the 3′ and 5′ ends of the hairpin probe, respectively. In the absence of the target, the primer was not released and the hairpin probe was not opened to produce fluorescence. The addition of target led to the release of the primer, which hybridized with the hairpin probe and triggered the chain-displacement polymerase reaction and produced a high fluorescence intensity. Under the optimized conditions, the linear range of this aptasensor was from 102 CFU·mL−1 to 108 CFU·mL−1 with a detection limit of 102 CFU·mL−1. Compared with other reported fluorescence detection methods, this approach has two advantages. First, this fluorescence aptasensor does not require nanomaterials as the quencher, which reduces the cost and saves time. Second, the chain-displacement polymerase reaction was used in this fluorescence aptasensor to amplify the signals, which further enhanced the sensitivity and lowered the detection limit. As this method was suitable for the detection of Salmonella paratyphi A in milk samples and potentially other bacteria, environmental monitoring and related food safety analysis should also be possible by this approach.

Label-free and enzyme-free sensitive fluorescent method for detection of viable Escherichia coli O157:H7

We have developed a label-free, enzyme-free, modification-free and DNA extraction-free fluorescent aptasensing (LEFA) method for detection of E. coli O157:H7 based on G-quadruplex formation using two ingeniously designed hairpin probes (GHP1 and GHP2). In the presence of E. coli O157:H7, it released the single stranded initiation sequence (IS) resulting in the toehold strand displacement between GHP1 and GHP2, which in turn led to the cyclic reuse of the production of DNA assemblies with numerous G-quadruplex structures and initiation sequences. Then these G-quadruplex structures can be recognized quickly by N-methyl mesoporphyrin IX (NMM) resulting in significantly enhanced fluorescence. The LEFA method was successfully implemented for detecting E. coli O157:H7 with a detection limit of 66 CFU/mL in pure culture, 10 CFU/mL and 1 CFU/mL after pre-incubation of the milk and tap water for 4 and 8 h, respectively. Moreover, the strategy could distinguish viable E. coli O157:H7 from dead E. coli O157:H7 and other species of pathogen cells. Furthermore, the whole process of the strategy is accomplished within 100 min. The results indicated that the approach may be used to effectively control potential microbial hazards in human health, food safety, and animal husbandry.

Enzyme-free isothermal target-recycled amplification combined with PAGE for direct detection of microRNA-21

MicroRNA-21 (miR-21) has been regarded as a kind of potential biomarker in several types of cancers. Herein, in this study, a simple, sensitive and cost-effective miR-21 approach was developed utilizing the isothermal target-recycled enzyme-free amplification strategy and polyacrylamide gel electrophoresis (PAGE). In the target-recycled enzyme-free amplification strategy, two rational designed hairpin probes (HPs, HP1 and HP2) can form into HP1-HP2 duplex in the presence of miR-21 under the isothermal condition, producing target signal in PAGE. The sensitivity and the linear range of miR-21 approach were demonstrated by the in vitro detection of miR-21, with the detection limit of 10 pM and the linear range of 50 pM to 8 nM. In particular, the contents of miR-21 in cell extractions of different cell lines were successfully detected through miR-21 approach and the relative expression was highly coincident with the results of stem-loop RT-PCR approach. In summary, the developed approach can detect miR-21 sensitively and easily.

Catalytic hairpin assembly gel assay for multiple and sensitive microRNA detection.

As important modulators of gene expression, microRNAs (miRNAs) have been identified as promising biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. Here we report a facile, multiple and sensitive miRNA detection method that uses conventional gel electrophoresis and catalytic hairpin assembly (CHA) system without any complex nanomaterials or enzymatic amplification.Methods: In this study, three pairs of hairpin probes are rationally designed with thermodynamically and kinetically preferable feasibility for the CHA process. In the present of target miRNA, the stem of the corresponding hairpin detection probe (HDP) will be unfolded and expose the concealed domain. The corresponding hairpin assistant probe (HAP) then replaces the hybridized target miRNA to form specific HDP/HAP complexes and releases miRNA based on thermodynamically driven entropy gain process, and the released miRNA triggers the next recycle to produce tremendous corresponding HDP/HAP complexes.Results: The results showed that the CHA gel assay can detect miRNA at fM levels and shows good capability of discriminating miRNA family members and base-mismatched miRNAs. It is able to analyze miRNAs extracted from cell lysates, which are consistent with the results of conventional polymerase chain reaction (PCR) method. Depending on the length of the designed hairpin probes, the CHA gel assay consisting of different hairpin probes effectively discriminated and simultaneously detected multiple miRNAs in homogenous solution and miRNAs extracted from cell lysates.Conclusion: The work highlights the practical use of a conventional gel electrophoresis for sensitive interesting nucleic acid sequences detection.

Ultrasensitive detection of DNA based on target-triggered hairpin assembly and exonuclease-assisted recycling amplification

An ultrasensitive electrochemical detection of target DNA was developed based on target-triggered hairpin assembly and exonuclease III (Exo III)-assisted recycling quadratic amplification strategy. The detection employed a gold nanoparticles (AuNPs) modified Au electrode and two specially designed hairpin probes P1 and P2. P1 probe contained G-quadruplex-forming sequence and target DNA recognition region, and was immobilized on the electrode. P2 probe was used as a secondary complementary sequence which can displace target DNA and hybridize with P1 probe. In the absence of target DNA, these hairpin structures of P1 and P2 can coexist. While in the presence of target DNA, it can trigger the self-assembly process of P1 and P2 and initiate the Exo III-assisted two recycling process, resulting in the formation of G-quadruplex structure on electrode surface. Finally, with the addition of hemin, numerous G-quadruplex-hemin complexes formed on the electrode surface and gave a pronounced electrochemical response in differential pulse voltammogram (DPV). Taking K-ras proto oncogene as an example, the proposed DNA biosensor exhibited a wide detection range from 10fM to 20nM, and an extremely low detection limit of 2.86fM. Moreover, it can clearly discriminate one-base difference in DNA sequence, thus can identify the mutation of the target gene. The proposed DNA biosensor has potential applications in the fields of clinic diagnosis, biomedicine, food and environment microbial monitoring.

Enzymatic repairing amplification-based versatile signal-on fluorescence sensing platform for detecting pathogenic bacteria

A novel fluorescence biosensing strategy for ultrasensitive and specific detection of pathogenic bacteria based on target-triggered enzymatic repairing amplification (ERA) has been developed. This strategy relies on target–aptamer binding mediated ERA reaction, which is carried out cyclically with the help of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) to produce amplified fluorescence signal. In our assay, the specially designed hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of reporter oligonucleotides and secondary primers, which can initiate a new cycle of polymerization-repairing amplification. Moreover, by the combination of polymerase-catalyzed incorporation of lesion bases with UDG and Endo IV-assisted ERA, multiple cycle of amplification of the recognition event is achieved, enabling ultrasensitive detection of pathogenic bacteria. Under optimal conditions, this biosensor exhibits ultrasensitivity toward target pathogenic bacteria with detection limits of 9.86cfumL−1 and a detection range of 5 orders of magnitude. Additionally, the biosensor has the ability of combating nonspecific background. Furthermore, an archer probe containing the anti-target aptamer sequence and a primer sequence is designed, which translates the binding of target to aptamer into the presence of primer sequence, enabling the detection of various targets, such as protein, DNA, small molecular, and any substance possessing its aptamer. Hence, the proposed target-triggered ERA-based signal-on fluorescence sensing strategy indeed create a versatile and useful platform for detection of pathogenic bacteria, related food safety analysis and clinical diagnosis.