Circular Exponential Amplification Research Articles

RETRACTED: A graphene oxide-based hairpin probe coupling duplex-specific nuclease signal amplification for detection and imaging of mRNA in living cells

In situ imaging of mRNA in living cells can help to monitor the real time mRNA expression and also useful for diagnosis and prognosis of the diseases. In this study, a new strategy was designed for simple, sensitive, and selective platform to detect the mRNA levels by combining a hairpin probe-graphene oxide (HP1/GO) and duplex-specific nuclease signal amplification (DSNSA). Initially, the DNA probe was adsorbed on the surface of GO to protect it from enzymatic digestion. Then, the target mRNA (T1) was hybridized with a partial hairpin probe which formed a duplex. Finally, under the action of DSN nuclease, the ssDNA in the DNA/RNA hybrid was selectively cleaved and produced small fragments. Then, T1 triggered the next reaction cycle, constituting a new circular exponential amplification. Here, we conclude that this assay is highly sensitive for the detection of target mRNA with the lower detection limit of 1 fM under optimal conditions. Furthermore, this strategy was successfully used for imaging of mRNA in living cells.

Circular exponential amplification of photoinduced electron transfer using hairpin probes, G-quadruplex DNAzyme and silver nanocluster-labeled DNA for ultrasensitive fluorometric determination of pathogenic bacteria.

The authors describe a fluorometric strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. This strategy relies on the combination of target-modulated photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA (labeled with silver nanoclusters) along with hairpin probe-based circular exponential amplification. The reaction system involves three hairpin probes (H1, H2 and H3). Probe H1 contains an aptamer against S. Typhimurium and the recognition sequence for nicking endonuclease. It is used to recognize S. Typhimurium and participates in polymerase-catalyzed target recycle amplification and secondary-target recycle amplification. Probe H2 contains an aptamer against hemin and is used to form the G-quadruplex DNAzyme in the presence of hemin and potassium ion. It acts as the electron acceptor and quenches the fluorescence of the labeled DNA. Fluorescence is best measured at excitation/emission wavelengths of 567/650nm. Probe H3 contains the template sequence for the synthesis of AgNCs and the H2-annealing sequence. Both H2 and H3 are utilized to perform a strand displacement reaction and to achieve PET between G-quadruplex DNAzyme and DNA/AgNCs. To the best of our knowledge, this is the first example of a PET between G-quadruplex DNAzyme and DNA/AgNCs coupled with circular exponential amplification. The assay has an ultra-low detection limit 8cfu·mL-1 of S. Typhimurium. The assay is rapid, accurate, inexpensive and simple. Hence, the strategy may represent a useful platform for ultrasensitive and highly specific detection of pathogenic bacteria as encountered in food analysis and clinical diagnosis. Graphical abstract The reaction system includes three hairpin probes (H1, H2 and H3), primer probe (P), Phi 29 DNA ploymerase (Phi 29) and nicking endonuclease Nt.AlwI (Nt.AlwI). Phi 29 and Nt.AlwI -assisted signal amplification leads to the recycling of target and produces numerous single stranded-DNAs (S). Strand displacement amplification leads to photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA/AgNCs. HAP-based circular exponential amplification of PET results in an ultrasensitive fluorometric assay.

Primer dephosphorylation-initiated circular exponential amplification for ultrasensitive detection of alkaline phosphatase.

Alkaline phosphatase (ALP) is an important diagnostic indicator for various human diseases including bone diseases, liver dysfunction, diabetes, breast and prostatic cancers. However, the conventional methods for ALP assay are usually cumbersome and time-consuming with low sensitivity. Here, we develop a new fluorescent method for ultrasensitive detection of ALP activity on the basis of primer dephosphorylation-initiated isothermal circular exponential amplification. We design two dual-functional hairpin probes (HP1 and HP2), which function as both the templates for exponential amplification reaction (EXPAR) and the generators for signal output. In the presence of ALP, the 3'-phosphorylated primer is dephosphorylated and subsequently hybridizes with the 3' protruding end of HP1 to initiate the first strand displacement amplification (SDA), producing trigger 1 and fluorescence signal. The released trigger 1 is complementary to the 3' protruding end of HP2 for the initiation of the second SDA, producing trigger 2 and fluorescence signal. Notably, trigger 2 is complementary to the 3' protruding end of HP1 and may subsequently initiate two consecutive SDAs, enabling circular EXPAR to generate an amplified fluorescence signal. This method exhibits high sensitivity with a detection limit of 2.0 × 10-10 U μL-1 and a large dynamic range of 5 orders of magnitude from 1.0 × 10-9 to 1.0 × 10-4 U μL-1, and it can measure ALP at the single-cell level. Importantly, this method can be applied for the measurement of kinetic parameters and the screening of potential inhibitors, providing a powerful tool for ALP-related biomedical research and clinical diagnosis.

Combining padlock exponential rolling circle amplification with CoFe2O4 magnetic nanoparticles for microRNA detection by nanoelectrocatalysis without a substrate

In this report, an ultrasensitive electrochemical biosensor for miR-21 detection was designed on the basis of a padlock exponential rolling circle amplification (P-ERCA) assay and CoFe2O4 magnetic nanoparticles (CoFe2O4 MNPs) by nanoelectrocatalysis without a substrate for signal amplification. Here, to improve catalytic efficiency, nanocatalyst (CoFe2O4 MNPs) and redox molecule (Tb) were co-immobilized onto the graphene (Gra) surface and formed Au@CoFe2O4/Tb-Gra probe. The obtained probe exhibits high-performance Tb catalysis by reducing the interaction distance between CoFe2O4 MNPs and Tb, and importantly, the detection sensitivity is significantly improved even in the absence of substrate (H2O2). Simultaneously, to further improve the sensitivity and specificity of the biosensor, P-ERCA assay was introduced. After multiple polymerization and nicking reactions, plenty of P-ERCA amplification product was produced and circular exponential signal amplification was achieved, giving an extreme sensitivity for miR-21 assay. The as-prepared biosensor shows a wide dynamic range of 1 fM to 2 nM with a low detection limit of 0.3 fM for miR-21 detection and exhibits high specificity and sensitivity.

Sensitive and rapid detection of microRNAs using hairpin probes-mediated exponential isothermal amplification

MicroRNAs (miRNAs) play significant roles in a diverse range of biological progress and have been regarded as biomarkers and therapeutic targets in cancer treatment. Here, we develop a rapid and sensitive miRNA assay on the basis of exponential isothermal amplification in combination with the hairpin probes, which was designed for sensitive detection of miRNA. The binding of target miRNA with a linear DNA template initiates exponential isothermal amplification reaction (EXPAR) and generates the universal triggers which are complementary to the 3′ protruding end of hairpin probe1(HP1). These triggers function not only as the primers to unfold the hairpin probes through catalysed hairpin assembly(CHA), generating distinct fluorescence signals, but also as the primer to initiate the next EXPAR. Moreover, CHA can release new triggers to initiate EXPAR or CHA. Thus this hairpin probes-mediated exponential isothermal amplification assay exhibits high sensitivity with a detection limit of 3.0×10−15M. More importantly, the isothermal condition and simple fluorescence measurement would greatly promote the development of a fast, point-of-care detection system. It can be completed in an hour which can effectively avoid miRNA from degradation. This hairpin probe-based circular exponential amplification assay holds a great promise for further application in biomedical research and early clinical diagnosis.

An autocatalytic DNA machine with autonomous target recycling and cascade circular exponential amplification for one-pot, isothermal and ultrasensitive nucleic acid detection.

An autonomous target recycling and cascade circular exponential amplification strategy was proposed to construct an autocatalytic DNA machine, which afforded one-pot, isothermal and ultrasensitive detection of nucleic acids. A low detection limit of 0.61 fM toward the target DNA with an excellent selectivity could be obtained.

Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide

MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches.

Graphene fluorescence switch-based cooperative amplification: a sensitive and accurate method to detection microRNA.

MicroRNAs (miRNAs) play significant roles in a diverse range of biological progress and have been regarded as biomarkers and therapeutic targets in cancer treatment. Sensitive and accurate detection of miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in clinical diagnosis. Here, we developed a stable, sensitive, and specific miRNAs detection method on the basis of cooperative amplification combining with the graphene oxide (GO) fluorescence switch-based circular exponential amplification and the multimolecules labeling of SYBR Green I (SG). First, the target miRNA is adsorbed on the surface of GO, which can protect the miRNA from enzyme digest. Next, the miRNA hybridizes with a partial hairpin probe and then acts as a primer to initiate a strand displacement reaction to form a complete duplex. Finally, under the action of nicking enzyme, universal DNA fragments are released and used as triggers to initiate next reaction cycle, constituting a new circular exponential amplification. In the proposed strategy, a small amount of target miRNA can be converted to a large number of stable DNA triggers, leading to a remarkable amplification for the target. Moreover, compared with labeling with a 1:1 stoichiometric ratio, multimolecules binding of intercalating dye SG to double-stranded DNA (dsDNA) can induce significant enhancement of fluorescence signal and further improve the detection sensitivity. The extraordinary fluorescence quenching of GO used here guarantees the high signal-to-noise ratio. Due to the protection for target miRNA by GO, the cooperative amplification, and low fluorescence background, sensitive and accurate detection of miRNAs has been achieved. The strategy proposed here will offer a new approach for reliable quantification of miRNAs in medical research and early clinical diagnostics.

High Specific and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based Exponential Rolling Circle Amplification

In this paper, a padlock probe-based exponential rolling circle amplification (P-ERCA) assay is developed for highly specific and sensitive detection of microRNA (miRNA). The padlock probe is composed of a hybridization sequence to miRNA and a nicking site for nicking endonuclease. Using the miRNA as a template, specific ligation to the padlock probe and linear rolling circle reaction (LRCA) are achieved under isothermal conditions. After multiple nicking reactions, many copies of short DNA products are successively produced and then used as triggers in next circle amplification. Thus, a small amount of miRNAs are converted to a large number of triggers to initiate the rolling circle amplification reaction, and circular exponential signal amplification is achieved. This padlock probe-based exponential rolling circle amplification assay exhibits a remarkable sensitivity of 0.24 zmol using optimized sequences of the padlock probe. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to single-nucleotide difference discrimination between miRNA family members. The miRNA analysis in human lung cells was performed with this method. The result indicates this highly sensitive P-ERCA strategy will become a promising miRNA quantification method in early clinical diagnostics.

Sensitive Detection of MicroRNAs with Hairpin Probe-Based Circular Exponential Amplification Assay

MicroRNAs (miRNAs) play important regulatory roles in a wide range of biological processes, and their aberrant expression is associated with cancer development and a variety of diseases. Here, we develop a simple, sensitive, and specific miRNA assay on the basis of circular exponential amplification in combination with the hairpin probes. The binding of target miRNA with a linear DNA template initiates the first strand displacement amplification (SDA) and generates the universal triggers which are complementary to the 3' protruding end of a hairpin probe. These universal triggers function not only as the primers to unfold the hairpin probes through an extension reaction, generating distinct fluorescence signals, but also as the amplification templates to initiate the second SDA reaction. Moreover, the second SDA reaction can release new triggers to initiate the above two consecutive SDA reactions, thus constituting a circular exponential amplification which enables the conversion of a small amount of miRNAs to a large number of universal triggers to unfold abundant hairpin probes. This hairpin probe-based circular exponential amplification assay exhibits high sensitivity with a detection limit of 3.80 × 10(-13) M and a detection range of 4 orders of magnitude. It can even discriminate single-nucleotide difference between miRNA family members and perform well in real sample analysis. Notably, in this assay, the long-stem hairpin probes are unfolded through an extension reaction rather than through a conventional hybridization reaction controlled by the thermodynamic equilibrium in the case of molecular beacons, making the design of hairpin probes very simple. This hairpin probe-based circular exponential amplification assay holds a great promise for further application in biomedical research and early clinical diagnosis.