Hairpin Probe Research Articles

Isothermal and enzyme-free MicroRNA assay based on catalytic hairpin assembly and rare earth element labeled probes

MicroRNAs (miRNAs) are a category of short non-coding RNA molecules which play essential roles in regulating gene expression. Correspondingly, the aberrant expression of miRNAs is commonly related to the development of various diseases including cancers and cardiovascular diseases. Hence, miRNA is considered as an ideal biomarker for disease diagnosis and its quantification is highly demanded. Herein, an isothermal and enzyme-free miRNA assay based on catalytic hairpin assembly (CHA) amplification coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection is proposed. Two hairpin probes are employed in CHA reaction with rare earth element tag on H1 for ICP-MS detection and biotin on H2 for immobilization on magnetic beads. In the presence of target miRNA, the two independent probes open up their stem-loop structure and hybridize. The target miRNA will be released after the hybridization to trigger another CHA reaction. Ultimately, one target miRNA molecule promotes a number of CHA cycles and produces several copies of hybridized complex leading to the enhanced ICP-MS signal. Based on this approach, miRNA-141 was successfully analyzed with high sensitivity and specificity. The limit of detection (LOD) was 88 fmol with a linear range 0.1–2 pmol. Compared with fluorescence mediated CHA method and ICP-MS mediated basic sandwich method, the current strategy displayed the lowest LOD. The developed method was also applied to analyze miRNA-141 and miRNA-21 simultaneously to demonstrate its multiple detecting capability and practical applicability. Furthermore, this miRNA assay has the potential to become a high-throughput method by employing more distinct rare earth elements.

Combining functional hairpin probes with disordered cleavage of CRISPR/Cas12a protease to screen for B lymphocytic leukemia

One of the causes of B lymphocytic leukemia is abnormal expression of the Pax-5a gene. Detection of the Pax-5a gene can provide effective technical means for early screening of B lymphocytic leukemia. In this work, we designed a sensing scheme to detect the Pax-5a gene based on the signal amplification system, which is based on dual-enzyme assisted target gene circulation, and the disordered cleavage of CRISPR/Cas12a protease. The hairpin probe (HP) in this scheme not only contains the binding sites of the target gene and the primer, but also cleverly contains half of the Nt.BbvCI splicing sites. When the target gene is present, through the synergistic effect of KF and Nt.BbvCI, a large number of single strands of a specific sequence can be produced. At the same time, the target gene falls off from the first hairpin and opens the other hairpin to realize the cycle of the target gene. The resulting single-strand can bind to the Cas12a/crRNA binary complex and unlock the anti-cleavage activity of the CRISPR/Cas12a protease. The single strands labeled with the fluorescent group (FAM) and the quenching group (BHQ) around the solution are cleaved, the fluorescence signal of FAM is restored, and a detectable fluorescence signal is generated. The detection limit is as low as 6.77 fM, and the target gene and the mismatch sequence can also be distinguished well. Therefore, the sensing scheme provides a new detection direction for the early diagnosis and screening of B lymphocytic leukemia.

Ultrasensitive detection of DNA methyltransferase activity: a novel dual-amplification fluorescence technique.

DNA methyltransferase (MTase) is an important regulatory enzyme in various biological processes. However, current methods for investigating MTase activity are still limited in terms of sensitivity and/or generality. Herein, we proposed a dual amplification fluorescence strategy for the ultrasensitive detection of DNA adenine methylation methyltransferase (Dam MTase) activity based on strand displacement amplification (SDA) coupled with rolling circle amplification (RCA). In this study, the hairpin probe could not be cleaved by Nt.AlwI nicking endonuclease (Nt.AlwI) in the presence of Dam MTase, and the subsequent SDA-RCA reaction was blocked, resulting in a weak fluorescence signal. Moreover, the blocking effect was more pronounced at a higher concentration of Dam MTase. This assay provides a very low detection limit (down to 0.0067 U ml-1), as well as good selectivity against other types of MTases (e.g., CpG methyltransferase (M.SssI MTase)). In addition, the analytical mode improves the generality and can be extended to the detection of other types of DNA MTases.

Ultrasensitive multiplexed miRNA detection based on a self-priming hairpin-triggered isothermal cascade reaction.

We herein describe an ultrasensitive isothermal strategy to detect miRNAs in a multiplexed manner by utilizing a self-priming hairpin-triggered cascade reaction and the adsorption properties of graphene oxide (GO). In principle, a self-priming hairpin probe (SHP) was designed to be opened through binding to the target miRNA and rearranged to serve as a primer. The following extension displaced the target miRNA to be recycled for opening another SHP and produced a double-stranded (ds) SHP with a longer stem region. The nicking enzyme recognition site within the ds SHP was then subjected to continuous repeated nicking and extension reactions, consequently producing a large amount of the trigger sequence. In the second reaction phase, the trigger also transformed another single-stranded (ss) target template probe (TTP) into ds TTP and simultaneously produced numerous target mimic strands (Target') in the same manner, which could activate the first reaction phase, mimicking the target miRNA. Since the ss portions of the two probes were all transformed to the ds forms (ds SHP and ds TTP), they are resistant to the adsorption by graphene oxide (GO) and then emitted intense fluorescence after the application of GO while the ss forms of the two probes produced a negligible fluorescence signal without the target miRNAs. Based on this unique design principle, we were able to simultaneously identify multiple target miRNAs very sensitively down to attomolar levels (42.63 aM for miRNA let-7a, 13.08 aM for miRNA-141, and 10.14 aM for miRNA-98) within 30 min.

Target-driven assembly of DNAzyme probes for simultaneous electrochemical detection of multiplex microRNAs.

In this work, we employed target-driven assembly of a Mg2+-dependent DNAzyme to develop an ultrasensitive electrochemical biosensor for the simultaneous detection of miRNA-21 and miRNA-141. The target miRNAs could hybridize with two partial DNAzymes, facilitating the formation of a stable and active Mg2+-dependent DNAzyme. With the help of the Mg2+ cofactor, the DNAzyme could circularly cleave the ferrocene (Fc) or methylene blue (MB) labelled hairpin probes and release Fc and MB labels from the electrode surface, which could significantly amplify the current suppression to achieve multiple detection of small amounts of miRNA-21 and miRNA-141. This electrochemical biosensor showed high sensitivity and selectivity for the simultaneous detection of miRNA-21 and miRNA-141. Furthermore, the proposed method was also successfully applied for the determination of miRNA-21 and miRNA-141 from diluted serum samples. Overall, the proposed sensor showed several considerable advantages including simple preparation, high sensitivity, and enzyme-free signal amplification. Therefore, the proposed electrochemical biosensor could be used as a highly efficient amplification strategy for simultaneous detection of various miRNA biomarkers in bioanalysis and clinical diagnostics.

Portable electrochemical biosensor based on laser-induced graphene and MnO2 switch-bridged DNA signal amplification for sensitive detection of pesticide

Developing portable, quantitative, and user-friendly analytical tools for sensitive pesticide assay is of significant importance for guaranteeing food safety. Herein, a novel electrochemical biosensor was constructed by integrating laser-induced graphene (LIG) electrode on polyimide (PI) foil and MnO2 nanosheets loaded on the paper for point-of-care test (POCT) of organophosphorus (OPs) residues. The principle of this biosensor relied on acetylcholinesterase (AChE)-catalyzed hydrolytic product-triggered disintegration of MnO2 nanosheets for releasing assistant DNA to initiate nicking enzyme-aided recycling amplification. In the presence of OPs, the activity of AChE was inhibited and could not initiate the cleavage of the electroactive molecules-labeled hairpin probe on the electrode, resulting in the maintenance of the electrochemical response to realize a “sign-on” determination of OPs. The proposed biosensor exhibited satisfactory analytical performance for OPs assay with a linear range from 3 to 4000 ng/mL and a limit of detection down to 1.2 ng/mL. Moreover, the biosensor was useful for evaluating the residual level of pesticides in the vegetables. Therefore, this novel biosensor holds great promise for OPs assay and opens a new avenue on the development of higher-performance POCT device for sensing applications in the environment and food safety fields.

Ultrasensitive Electrochemical Detection of cancer-Related Point Mutations Based on Surface-Initiated Three-Dimensionally Self-Assembled DNA Nanostructures from Only Two Palindromic Probes.

Sensitive and selective detection of proto-oncogenes, especially recognition of point mutation, is of great importance in cancer diagnosis. Here, a ligation-mediated technique is demonstrated for the construction of an intertwined three-dimensional DNA nanosheet (3D SDN) on an electrode surface from only two palindromic hairpin probes (HP1 and HP2), creating a powerful electrochemical biosensor (E-biosensor) for the detection of the p53 gene. First, a capturing probe (CP) is immobilized on an electrode surface via Au-S chemistry, forming an electrochemical sensing interface. In the presence of the target p53 (T), the triggering probe is covalently linked to CP by a ligase. Moreover, target hybridization/ligation/dehybridization process is repeated, amplifying the target hybridization event and increasing the content of surface-confined triggering fragments. As a result, HP1 is opened and in turn interacts with HP2, forming intertwined 3D SDN where HP1 and HP2 are alternately arranged in parallel. Common hybridization and interaction between palindromic fragments are responsible for the assembly in the horizontal and vertical directions, respectively. An electrochemical indicator, methylene blue (MB), can be inserted into 3D SDN, generating a strong electrochemical signal. Utilizing the 3D SDN-based E-biosensor, the target DNA is detected down to 3 fM with a linear response range from 10 fM to 10 nM. Single point mutations are reliably identified even in fetal bovine serum and cellular homogenate. Because of the several advantages of simple design, good universality, inexpensive instrumentation, high assay specificity, and sensitivity, the 3D SDN-based E-biosensor is expected to provide a potential platform for screening point mutation required by early clinical diagnostics and medical research.

MUC1 detection and in situ imaging method based on aptamer conformational switch and hybridization chain reaction

Mucin 1 (MUC1) overexpression in tumor cells is related to various cancers, including breast, stomach, and lung cancer. MUC1 detection and imaging are important for cancer localization in tissue sections to support histopathological diagnosis. In this study, we developed a simple, enzyme-free MUC1 detection and in situ imaging method. Three hairpin probes, Apt-trigger, HP1-FAM, and HP2, were designed for MUC1 recognition and hybridization chain reaction (HCR). The Apt-trigger probe was composed of two sequences: the MUC1 aptamer and HCR trigger sequence. The 5’ end of the HP1-FAM probe was modified with a FAM signal molecule. In the presence of MUC1, the aptamer sequence is activated and bound to MUC1, which opens the hairpin structure. Then, the trigger sequence gets exposed and, complementary to HP1-FAM, triggers a continuous HCR process. This method was successfully used to detect MUC1 of 200 pM–25 nM and MUC1 in situ imaging in specific cells, such as human breast carcinoma (MCF-7) and human colon cancer (HT-29) cells.

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.

Nicking site enzyme assisted catalytic hairpin assembly based scaffold for sensitive monitoring of miRNA-21

As a class of non-coding RNA nucleotides, miRNAs has been widely regarded as an effective clinical biomarker for diagnosis and therapy of a variety of disease, such as colorectal cancer. However, development of an easy-to-operate and high-sensitive miRNA detection methods remains a huge challenge due to the low expression level, and high similarity with homologous RNA nucleotides. Herein, a novel CHA (catalytic hairpin assembly)-based sensing platform was established for sensitive, efficient and reliable in vitro detection of miRNA-21. The CHA based scaffold was constructed in a one-step self-assembly process of designed DNA probes (a S strand, a pair of hairpin probes). The presence of target miRNA initiates CHA process to form double-strand DNA (dsDNA) complex between the pair of hairpin probes, exposing nicking enzyme recognizing site. Nb.BbvCI identifies recognizing site in dsDNA section and generates a nick. With DNA polymerase based chain extension and replacement, liberated chain attends a next CHA process and a signal circle is formed. Compared with CHA, the method significant improves the sensitivity for miRNA-21 detection in vitro, showing a wider application for trace sample detection.

Radially-dependent ignition process of a pulsed capacitively coupled RF argon plasma over 300 mm-diameter electrodes: multi-fold experimental diagnostics

A synergistic combination of multi-diagnostic methods are proposed to investigate temporal evolution of electrical and plasma parameters at various radial positions over 300 mm-diameter electrodes during the pre-ignition, ignition, and post-ignition phases of a pulsed capacitively coupled radio-frequency (RF) argon discharge. The electron density, n e, and the optical emission intensity (OEI) of argon at 750.4 nm at different radial positions are measured time-resolved by using a hairpin probe and an optical probe, respectively. A B-dot probe is employed to determine the waveforms of the azimuthal magnetic field at different radii, from which the waveforms of the axial current density at corresponding radial positions are derived based on Ampere’s law. Then, the time evolution of the power density at various radii can be calculated, provided that the voltage drop between the electrodes is independent of radius. Meanwhile, the time-dependent total power deposited into the reactor is calculated with the voltage and the current waveforms measured by a voltage and a current probe at the power feeding point. It was found that during pre-ignition phase, the OEI and n e cannot be measurable due to extremely low power deposition when the system exhibits pure capacitive impedance. During the ignition phase, the OEI, the power density, and the current density exhibit the most significant increase at the electrode center, while the time evolution of n e measured by the hairpin probe seems to exhibit a relatively weak radial dependence during this phase. In particular, at r ⩽ 8 cm, the OEI at every radius was observed to change with time in the same manner as the power density during the ignition phase, because the RF power is absorbed primarily by electrons, which dissipate their energy via inelastic collisions. Shortly, the profile of n e becomes edge-high during the post-ignition phase and remains thereafter until the end of the pulse-on period. Methodologically, the synergistic diagnostics lay the foundation for extensive studies on spatiotemporal evolution of plasma ignition process under broader conditions, e.g. electronegative gas, lower working gas pressure and very high driving frequency, widely used by practical etching process.

Chemical oxidation-free site-specific 5-hydroxymethylcytosine assay

5-hydroxymethylcytosine (5hmC) shows a very close correlation with various diseases. Accurate quantification of 5hmC is of great significance for revealing its function in different tissues. Here we proposed a chemical oxidation-free and site-specific 5hmC assay. First, T4 β-glucosyltransferase (T4 β-GT) and AbaSI restriction endonuclease were used to cleave the double-strand DNA at the 3' end side away from the 5hmC to produce the sticky end. Then, a hairpin probe was ligated to the sticky end of target 5hmC as an adapter. Finally, polymerase chain reaction (PCR) amplification was employed to amplify the ligation products to achieve a high sensitivity determination of target 5hmC. The selectivity of the resulting assay is up to 4000-fold and 540-fold to distinguish 5hmC from cytosine (C) and 5-methylcytosine (5mC), respectively. The detection limit of the proposed 5hmC assay is 0.89 fM. This method can detect not only the content of site-specific 5hmC but also the percentage of site-specific 5hmC.

A magnetic separation-assisted cascade hybridization chain reaction amplification strategy for sensitive detection of flap endonuclease 1

Sensitive and accurate detection of flap endonuclease 1 (FEN1) is essential to understand its roles in DNA replication and repair and explore its functions in tumor diagnosis and prognosis. Here, we designed a magnetic separation-assisted cascade hybridization chain reaction (HCR) amplification strategy for enzyme-free and sensitive detection of FEN1. Firstly, the forked dsDNAs contained the recognition site (5′ overhanging DNA flap) of FEN1 were modified on magbeads surface to form magnetic recognition probes. By the recognition and catalytic cleavage of FEN1, the 5′ flaps were removed into solution and then purified by magnetic separation. Then, the obtained flaps served as triggers to activate the linear-assembly of hairpin probes H1 and H2 (primary HCR), forming nicked double helices with split fragments. Finally, the fragments acted as new triggers to activate the branched-assembly of hairpin probes H3 and H4 (secondary HCR), causing fluorophore/quencher labeled on H3 far away and fluorescence recovery. Benefiting from the purification of triggers by magnetic separation and the signal amplification effect of cascade HCR, this strategy achieved a low detection limit of 3.6 × 10−5 U μL−1 for FEN1, and enabled the assay of it in actual samples. The results demonstrate that our strategy will provide a potent approach for sensitive detection of FEN1 in early tumor diagnosis and prognosis.

Multiple amplified microRNAs monitoring in living cells based on fluorescence quenching of Mo2B and hybridization chain reaction

Imaging intracellular microRNAs (miRNAs) demonstrated an essential role in exposing their biological and pathological functions. However, the detection of sequence-specific miRNAs in living cells remains a key challenge. Herein, a facile amplified multiple intracellular miRNAs imaging platform was constructed based on Mo2B nanosheets (NSs) fluorescence (FL) quenching and hybridization chain reaction (HCR). The Mo2B NSs demonstrated strong interaction with the hairpin probes (HPs), ssDNA loop, and excellent multiple FL dyes quenching performance, achieving ultralow background signal. After transfection, the HPs recognized specific targets miRNAs, the corresponding HCR was triggered to produce tremendous DNA-miRNA duplex helixes, which dissociated from the surface of the Mo2B NSs to produce strong FL for miRNAs detection. It realized to image multiple miRNAs biomarkers in different cells to discriminate cancer cells from normal cells owing to the excellent sensitivity, and the regulated expression change of miRNAs in cancer cells was also successfully monitored. The facile and versatile Mo2B-based FL quenching platform open an avenue to profile miRNAs expression pattern in living cells, and has great applications in miRNAs based biological and biomedical research.

Rapid 16S rDNA electrochemical sensor for detection of bacteria based on the integration of target-triggered hairpin self-assembly and tripedal DNA walker amplification

Diseases caused by bacteria pose great challenges to human health. The key to reduce disease transmission and mortality is to develop accurate and rapid methods for the detection and identification of bacteria. Herein, a rapid bacteria 16S rDNA electrochemical sensor based on target-triggered hairpin self-assembly and tripedal DNA walker (TD walker) amplification strategy was constructed. Specific variable region of 16S rDNA fragment of bacteria was used as biomarker. The target-triggered hairpin self-assembly strategy was used to prepare a TD walker. The hairpin DNA probes labeled with ferrocene (Fc) were designed and modified on surface of electrode. The “legs” of TD walker hybridized with three hairpin probes and opened their hairpin structures. Exo III enzyme recognised hybrid duplexes and selectively digest hairpin probes. The “legs” of TD walker was released and hybridized with the other three hairpin probes. In this way, the enzyme drived the walkers to walk along electrode interface, until hairpin DNA probes were all removed from the electrode, the Fc was far away from electrode interface. A significantly current reduction signal was obtained and bacteria were detected by recording this response. This strategy was low-cost and scalable, it could continuously recycle low-concentration targets, thus enhanced the detection sensitivity. As the proof-of-concept work, the electrochemical sensor was utilized as detector. The limit of detection (LOD) of detecting Staphylococcus aureus (S. aureus) was 20 CFU mL−1 and detection time was less than 3 h. It was expected to be widely used in clinical early diagnosis.

Dual-labeling ratiometric electrochemical strategy initiated with ISDPR for accurate screening MecA gene

An outstanding dual-labeling ratiometric electrochemical biosensor based on isothermal strand displacement polymerization reaction (ISDPR) for highly sensitive and selective detection of mecA gene has been proposed. Concretely, in the presence of mecA gene, the addition of methylene blue (MB)-labeled primer and polymerase induced recycling amplification to change the structure of the ferrocene (Fc)-labeled hairpin probe, thereby releasing abundant target gene to realize the signal amplification and dual-signal output. Through this process, the electrochemical responses of Fc (IFc) and MB (IMB) were both substantially reduced and increased proportionally, ensuring that the value of IMB/IFc can accurately reflect the true detection level of mecA gene. Benefiting from the “signal-on/off” strategy, the fabricated biosensor exhibited outstanding sequence specificity to discriminate mismatched mecA gene, which verified to be 2.72 times that of single-label detection for perfect match/single base mismatch (PM/MM) discrimination ratio. This strategy effectively integrated the advantages of signal amplification and ratiometric modes, making the biosensor exhibit a broad working range with 10 fM - 3000 pM and a limit of detection (LOD) with 3.33 fM (S/N = 3). Moreover, the proposed biosensor has good feasibility for mecA gene determination in water samples due to acceptable recoveries (95–115%) and repeatability relative standard deviations (RSD) value of 4%. This will provide a powerful sensing platform for improving accuracy and decreasing background signal of sensor for ARGs screening in environmental monitoring.

Experimental Investigation of Nonlinear Standing Waves in DC/VHF Hybrid Capacitive Discharges

It is recognized that in very-high-frequency capacitively coupled plasma (VHF CCP) reactors, the standing wave effects can be enhanced by the presence of nonlinear higher harmonics self-excited by the plasma series resonance (PSR). In this work, we investigated the influence of the dc bias on the excitation of the nonlinear standing waves in a parallel-plate capacitively coupled dc/VHF (60 MHz) hybrid discharge sustained in argon, with both the dc source and the VHF source applied to the top electrode. A hairpin probe was employed to determine the axial/radial distribution of the electron density, while a high-frequency B-dot probe was used to measure the radial distributions of the harmonic magnetic field and the harmonic current density. With no dc bias applied, the nonlinear standing wave excitation was observed to be predominant; the high-order harmonic current density exhibited a main peak at the electrode center and one or more minor peaks between the electrode center and the edge, leading to a center-high electron density profile. With the addition of the dc source, the harmonic excitations and the corresponding nonlinear standing wave effect tend to be suppressed with the increase of dc bias, resulting in improved plasma uniformities. Underlying mechanisms of the suppression effect of the dc bias on the nonlinear standing wave excitation were analyzed.

Smartphone-assisted colorimetric detection of BRCA-1 gene based on catalytic hairpin assembly amplification and G-quadruplex DNAzyme

• An enzyme-free colorimetric biosensor for sensitive detection of BRCA-1 gene is developed. • The target-triggered CHA and the peroxidase-like activity of G-quadruplex DNAzyme are used for signal amplification. • The colorimetric biosensor exhibits a high sensitivity toward target DNA with a detection limit of 10 pM. • The proposed strategy has a great potential application in genetic analysis and clinical diagnosis. In this study, we develop an enzyme-free colorimetric biosensor for the determination of breast cancer susceptibility gene (BRCA-1), which integrates catalytic hairpin assembly (CHA) signal amplification with the peroxidase-like activity of G-quadruplex DNAzyme. With the addition of BRCA-1 gene, the hairpin probes (H1 and H2) are able to generate numerous duplex DNA assemblies with liberated G-quadruplex forming sequences. After binding with cofactor hemin, the G-quadruplex forming sequences fold into G-quadruplex DNAzymes, which effectively catalyze the H 2 O 2 -mediated oxidation of 2,2’-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) to free-radical cation (ABTS •+ ), accompanied by a color transition from colorless to green. The colorimetric readout can be recorded using a smartphone camera or measured by UV-vis spectrophotometer. The developed colorimetric biosensor can detect target DNA as low as 10 pM with a linear range from 50 pM to 200 nM. Meanwhile, the colorimetric biosensing platform allows the determination of target DNA at a concentration as low as 0.2 nM through smartphone applications. In addition, the colorimetric biosensor shows excellent selectivity and acceptable applicability for the detection of BRCA-1 gene in human serum samples with satisfactory recoveries of 93.47–104.14% and RSDs of 6.89–8.24%, which might hold great application potential in genetic analysis and clinical diagnosis.

CRISPR/Cas12a and immuno-RCA based electrochemical biosensor for detecting pathogenic bacteria

Scheme 1 Schematic illustration of immuno-RCA and Cas12a based electrochemical biosensor for quantitative analysis of E. coli O157:H7. • An electrochemical biosensor based on CRISPR was established to detect bacteria. • The electrochemical biosensor can be used in the detection of actual samples. • The immuno-RCA was used to amplify the activity of CRISPR/Cas12a system. • Have a wide detection range from 10 to 10 7 CFU•mL −1 , with a LOD of 10 CFU·mL −1 . Rapid, simple, and sensitive detection of food-borne pathogenic bacteria is extremely important for preventing and controlling food-borne diseases. Herein, an electrochemical biosensor based on CRISPR/Cas12a combined with immuno-rolling circle amplification (immuno-RCA) was developed for detecting the pathogenic bacterium, Escherichia coli O157:H7. Based on a sandwich-type immunoassay on magnetic beads, immuno-RCA generated long single-stranded DNA with abundant E. coli O157:H7-specific aptamers and target repeated sequences, which allowed recognition and binding of E. coli O157:H7 and complex formation by CRISPR/Cas12a and crRNA, respectively. Stimulation of Cas12a produced trans- cleavage activity toward a non-specific methylene blue labeled DNA hairpin probe on the electrode surface. In the presence of E. coli O157:H7, the CRISPR/Cas12a non-specific trans- cleavage activity was triggered, the hairpin DNA on the Au electrode was cleaved, and the peak current was altered. Under optimal conditions, the developed biosensor presented a broad dynamic detection range from 10 to 10 7 CFU·mL −1 , with a detection limit of 10 CFU·mL −1 . Moreover, the biosensor did not exhibit any cross-reactivity with other non-target bacteria. These results revealed that the developed biosensor is a simple, sensitivity, and specific platform for E. coli O157:H7 detection.

Dual-stage amplified fluorescent DNA sensor based on polymerase-Mediated strand displacement reactions

A sensitive fluorescent strategy for DNA detection is developed based on a dual stage amplification process mediated by DNA polymerase. In the sensing method, there are two kinds of DNA probes used, including a hairpin probe (HP) and a double-stranded DNA (DSD) tagged with a quencher and a fluorophore, respectively. The presence of the target DNA triggers the two-step cyclic amplification reactions with the assistance of DNA polymerase, thus leading to the considerable fluorescence restoration. The experiment conditions are carefully studied to achieve optimal performance, including the construction of DSD, the concentration of HP, the reaction time, and the amounts of polymerase. Under the optimal conditions, the strategy responded linearly to DNA target from 0.5 to 50.0 nM with a relative low detection limit of 0.38 nM (according to 3σ/S rule). Moreover, the method shows good detection performance in serum samples, confirming its practical usage for efficient DNA assay. The mix-and-measure simplicity and dual-stage signal amplification make the developed method a novel way for sensitive detection of DNA.