DNAzyme Amplification Research Articles

Label-free colorimetric detection of divalent mercuric ions (Hg2+) based on T-Hg2+-T structure and exonuclease III dual-recycling and G-quadruplex-hemin DNAzyme amplification

ABSTRACT Detection of divalent mercury attracts great significance in most biological and environmental systems. In this study, a Thymine-divalent mercuric ions-Thymine (T-Hg2+-T) structure based colorimetric assay with Exonuclease III (Exo III) assisted recycling and G-quadruplex-hemin DNAzyme amplification is put forward for Hg2+ monitoring. The Hg2+ can combine with T-T mismatches of oligo-1 to form T-Hg2+-T base pairs, which brings forth a double-helix structure with a blunt terminus. Short mononucleotide fragment (trigger DNA) and Hg2+ could be released as the double-helix structure in oligo-1 is cleaved by Exo III. The released Hg2+ can be captured by another oligo-1, and generate the new cycles. At the same time, signal DNA (oligo-2) can hybridize with trigger DNA to form double-stranded DNA with a blunt terminus that can be cleaved by Exo III to accrue the trigger DNA and guanine (G)-quadruplex fragment. The trigger DNA can hybridize with another signal DNA, which implements the dual-recycling amplification. The G-quadruplex fragment can form the G-quadruplex-hemin DNAzymes, which can catalyze the oxidation of 2, 2ʹ-azino-bis(3-ethylbenzothiazo-line-6-sulfonate) disodium salt (ABTS2-) by H2O2. The color change, served as the signal output, can be measured with an ultraviolet spectrophotometer. The proposed assay allows Hg2+ to be detected in the range of 25 pM to 2000 pM with a detection limit of 7 pM. Moreover, it has been commendably applied to the determination of Hg2+ in environmental samples and biological samples.

Novel Fluorescence Switch for MicroRNA Imaging in Living Cells Based on DNAzyme Amplification Strategy.

MicroRNAs (miRNAs) play important roles in the regulation of target gene expression and cell development. Therefore, developing of accurate and visual detection methods for miRNAs is important for early diagnosis of cancer. In this study, we established a visual detection method for miRNA 155 based on DNAzyme amplification strategy in living cells. MnO2 nanosheets were employed to deliver locked DNAzyme and substrate DNA into cells. The gold nanoparticle (AuNP) probe was taken up by cells autonomously. Then, MnO2 nanosheets were reduced to Mn2+ by glutathione in cells and DNA modules were released. MiRNA 155 took away locker DNA by strand displacement reaction to activate the DNAzyme. Then, the DNAzyme cleaved the substrate DNA and released single-stranded DNA named key DNA. Then, Key DNAhybridized with the hairpin DNA, making cy5far away from AuNP and turning on its fluorescence. One target miRNA led to plenty of released key DNA when lots of substrate DNA was added. Thus, the visual detection of miRNA 155 in living cells would be initiated. Under confocal laser microscopy, the fluorescence was obviously observed in tumor cells but not in normal cells. The method has a linear range from 0.1 to 10 nM and a low detection limit of 44 pM on in vitro detection.

A visual and sensitive Hg2+ detection strategy based on split DNAzyme amplification and peroxidase-like activity of hemin-graphene composites

A visual and sensitive Hg2+ detection strategy was developed based on split DNAzyme amplification and hemin-graphene oxide composites (H-GNs). Two split DNAzyme sequences can form two entire enzyme-strands DNA (E-DNA) by T-Hg2+-T interaction. The E-DNA can bind with the loop of molecular beacon (MB) to form Mg2+-dependent DNAzyme structure. The formed DNAzyme can circularly cleave the loop of MB, resulting large amount of DNA fragments. The resultant DNA fragments can prevent H-GNs from aggregation by adsorbing on its surface. Consequently, the supernate with large amount of H-GNs shows dark blue color after chromogenic reaction. This strategy shows a linear range from 50 pM to 1200 pM. The limit detection can be low to 33 pM. This strategy provides a visual and enzyme-free amplification mode for quick and sensitive screen of Hg2+.

Amplified probing of protein/DNA interactions for sensitive fluorescence detection of transcription factors.

Protein/DNA interactions play important roles in many cellular activities, and sensitive monitoring of such interactions can potentially elucidate the regulation of gene expression. In this work, on the basis of specific protein/DNA interactions, we describe the establishment of a simple and sensitive method for detecting transcription factors by coupling the nuclease protection strategy with metal-ion dependent DNAzyme amplification. Nuclear factor κB (NF-κB) p50 target molecules specifically bind DNAzymes containing DNA duplex probes and protect them from being digested by exonuclease III. Consequently, the protected DNAzymes react with the fluorescently quenched substrate hairpin signal probes and cause cyclic cleavage of the signal probes to generate substantially amplified fluorescent readouts for sensitively detecting NF-κB p50. Our method exhibits a dynamic range of 0.01 to 50 nM and a detection limit of 0.54 pM, respectively, for NF-κB p50. Moreover, due to the specific interaction between the target protein and the duplex probes, high selectivity towards NF-κB p50 against other control proteins is observed. With the features of simplicity, sensitivity and selectivity, the developed method can thus be employed to monitor different types of protein/DNA interactions for biological studies and disease diagnosis.

Label-Free and Amplified Electrochemical Detection of Single Nucleotide Polymorphism in Folded Nucleic Acid Secondary Structures

Single nucleotide polymorphisms (SNPs) in folded nucleic acid secondary structures are effective biomarkers for the diagnosis of various gene related diseases. However, it is of major challenge to detect these special SNP sequences owing to their folded secondary structures. In this paper, we have constructed a simple and label-free electrochemical sensor to detect SNP in folded nucleic acid secondary structures with high sensitivity by integrating Mg2+-dependent DNAzyme amplification with the electrochemistry of hemin. The binding of two assistant ssDNAs with the stem-loop-structured target SNP sequences results in the generation of the Mg2+-dependent DNAzymes, which cyclically cleave the signal hairpin probes on the sensor surface to free the G-quadruplex sequences. Hemin subsequently binds these G-quadruplex sequences to form many G-quadruplex/hemin complexes, and electrochemical reduction of the surface confined hemin therefore shows substantially enhanced current signals for ultrasensitive detection toward the target SNP sequences in the dynamic range between 5 fM and 500 pM with a detection limit of 1.52 fM. High discrimination capability for single base-mismatched sequences and potential applicability for real samples of the sensor has also been demonstrated, indicating the promising application of the sensor for early diagnosis of different genetic diseases.

Colorimetric detection of genetically modified organisms based on exonuclease III-assisted target recycling and hemin/G-quadruplex DNAzyme amplification.

An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2- causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420nm) at concentrations as low as 0.23nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs. Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.

Ultrasensitive voltammetric determination of kanamycin using a target-triggered cascade enzymatic recycling couple along with DNAzyme amplification

The authors describe a label-free electrochemical aptasensor for ultrasensitive and highly specific detection of the antibiotic based on cascade enzymatic recycling couple with DNAzyme amplification. The assay involves two sequential reactions: the first reaction is a λ exonuclease-assisted cyclic digestion reaction triggered by target-aptamer binding. The second reaction is a nicking endonuclease-aided cyclic nicking reaction, which produces a large amount of G-rich nucleic acid segments. These form a G-quadruplex/hemin complex in the presence of K(I) ions and hemin. Because the G-quadruplex/hemin complex acts as a horseradish peroxidase-mimicking DNAzyme with excellent redox activity, the electrochemical signal transduction is accomplished due to the electroreduction of H2O2. It appears that this work is the first example that cascade enzymatic recycling coupled to DNAzyme amplification is used for antibiotic detection. The aptasensor was applied to the quantitation of kanamycin and gave a response that is linear in the 1 pM to 10 nM kanamycin concentration range, with a detection limit as low as 0.5 pM. The working voltage (vs. Ag/AgCl) at which data can be acquired best is −0.35 V. The assay offers the advantages of remarkably improved sensitivity, use of affordable instrumentation, and simplified operation without the need for electrochemical labeling or addition of labile reagents. Thus, cascade enzymatic recycling coupled to DNAzyme amplification represents a versatile platform for highly sensitive and specific antibiotics detection.

A DNAzyme sensor based on target-catalyzed hairpin assembly for enzyme-free and non-label single nucleotide polymorphism genotyping

Single nucleotide polymorphisms (SNPs) are widely existed in human genome and associated with many diseases. Traditional PCR-based methods for SNP genotyping require protein enzyme, precise control of temperature and removal of resultant products, making the whole process labor intensive and time consuming. Although G-quadruplex DNAzyme-based assays provide many advantages over traditional approaches, the relatively low catalytic activity of DNAzyme becomes an unfavorable factor in its application process. Therefore, amplification of DNAzyme for further determination is of great desire in bioanalysis. In this work, we have developed an enzyme-free and non-label DNAzyme sensor for SNP genotyping based on target-catalyzed hairpin assembly (CHA) for DNAzyme amplification. The proposed sensor, carried out on microfluidic chemiluminescence (CL) assay, can sensitively discriminate rs242557 hotspot-SNP, the A/G single-nucleotide variation on human chromosome associated with Alzheimer's disease, with an absolute detection limit of 0.3 fmol.

A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification

Owing to the intrinsic importance of nucleic acid as bio-targets, the achievement of its simple and sensitive detection with high confidence is very essential for biological studies and diagnostic purposes. Herein, a label-free, isothermal, and ultrasensitive electrochemical detection of target DNA was developed by using a tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme releasing amplification strategy. Upon sensing of the nucleic acid analyte for the assembled hairpin-like probe DNA on the electrode, the DNA polymerase guided the target recycling and simultaneously triggered the lambda exonuclease cleavage, accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme. The electrocatalytic reduction of H2O2 by the generated hemin/G-quadruplex DNAzyme was used for the signal readout and further amplification toward target response. Such tandem functional operation by DNA polymerase, lambda exonuclease and DNAzyme endows the developed biosensor with a high sensitivity and also a high confidence. A low detection limit of 5fM with an excellent selectivity toward target DNA could be achieved. It also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine.

Sensitive and Selective DNA Detection Based on Lambda Exonuclease Assisted Signal Amplification

Abstract A new versatile chemiluminescence biosensing platform with G-Quadruplexes/Hemin DNAzyme and lambda exonuclease (λexo) assisted signal amplification was designed for sensitive detection of DNA. The system involved target DNA, phosphate-DNA, auxiliary DNA and hairpin DNA. The selective digestion of λexo to 5′-phosphorylated strand of phosphate-DNA-auxiliary DNA-target DNA duplex sandwich structure resulted in the release of target DNA and auxiliary DNA. The released target DNA hybridized with another phosphate-DNA-auxiliary DNA duplex to trigger the target DNA recycling, while the released auxiliary DNA hybridized with hairpin DNA to activate DNAzyme segments. The activated DNAzyme segments interacted with hemin to form stable G-Quadruplexes/ Hemin DNAzymes that could catalyze H 2 O 2 -mediated oxidation luminol to produce chemiluminescence signals. In the presence of 10 units λexo, 1.0 × 10 −3 M luminol, 3.0 × 10 −2 M H 2 O 2 and pH 9.0 buffer solution, the relative chemiluminescence intensity of luminol was linearly related with the concentrations of target DNA in the range of 2.0 × 10 −12 M to 8.0 × 10 −9 M. This unique analysis strategy provided a detection limit down to 7.0 × 10 −14 M. The present method was successfully applied to the determination of target DNA in serum samples, which was also capable of discriminating mismatched DNA from perfectly matched target DNA with a high selectivity.

Label-free and highly sensitive electrochemical detection of E. coli based on rolling circle amplifications coupled peroxidase-mimicking DNAzyme amplification.

In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of Escherichia coli has been developed on the basis of rolling circle amplification (RCA) coupled peroxidase-mimicking DNAzyme amplification. A aptamer-primer probe (APP) containing anti-E. coli aptamer and a primer sequence complementary to a circular probe, which includes two G-quadruplex units, is used for recognizing target and triggering RCA-based polymerase elongation. Due to RCA coupled DNAzyme amplification strategy, the presence of target E. coli leads to the formation of numerous G-quadruplex oligomers on electrode, which folds into G-quadruplex/hemin complexs with the help of K(+) and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong electrochemical response. As far as we know, this work is the first time that RCA coupled peroxidase-mimicking DNAzyme amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward E. coli with detection limits of 8cfumL(-1) and a detection range of 5 orders of magnitude. Besides, our biosensor also shows high selectivity toward target E. coli and has the advantages in its rapidness, low cost, simplified operations without the need of electrochemical labeling steps and additional labile reagents. Hence, the RCA coupled peroxidase-mimicking DNAzyme amplification-based electrochemical method might create a useful and practical platform for detecting E. coli and related food safety analysis and clinical diagnosis.

A novel signal-amplified electrochemical aptasensor based on supersandwich G-quadruplex DNAzyme for highly sensitive cancer cell detection

Abstract A novel electrochemical biosensor for cancer cell detection was developed based on aptamer-based competition and supersandwich G-quadruplex DNAzyme amplification strategy. Due to the stronger affinity between the aptamer and cancer cells than that with its complementary oligonucleotide, the complementary oligonucleotide will be facilely replaced. As a consequence, we can detect cancer cells indirectly by detecting the releasing DNA which is proportional to the concentration of K562 cells. Through the supersandwich G-quadruplex DNAzyme amplification strategy, the sensitivity can be dramatically enhanced with detection limit down to 14 cells.

Label-free colorimetric detection of Hg2+ based on Hg2+-triggered exonuclease III-assisted target recycling and DNAzyme amplification

This work reported a label-free colorimetric assay for sensitive detection of Hg2+ based on Hg2+-triggered hairpin DNA probe (H-DNA) termini-binding and exonuclease Ш (Exo Ш)-assisted target recycling, as well as hemin/G-quadruplex (DNAzyme) signal amplification. The specific binding of free Hg2+ with the thymine–thymine (T–T) mismatches termini of H-DNA could immediately trigger the Exo Ш digestion, and then set free G-quadruplex segments and Hg2+. The Exo Ш impellent recycling of ultratrace Hg2+ produced numerous G-quadruplexes. The corresponding DNAzymes catalyzed efficiently the H2O2-mediated oxidation of the ABTS2− to the colored product in the presence of hemin. Using the color change as the output signal, and the Exo Ш-aided Hg2+ recycling and DNAzyme as the signal amplifier, the ultrasensitive assay system successfully achieved visual detection of Hg2+ as low as 1.0nM by the naked eye, and was suitable for field monitoring. The calibration curve was linear in the range of 50.0pM to 20.0nM for Hg2+ (R=0.9962) with a detection limit of 10.0pM. Moreover, this proposed strategy showed excellent selectivity, portability and low-cost, and was successfully applied to colorimetric detection of Hg2+ in laboratory tap water and Jialing river water samples.

Two-stage nicking enzyme signal amplification combined with DNAzyme amplification for the detection of bone morphogenetic protein 6 mRNA.

Highly sensitive detection of bone morphogenetic protein 6 (BMP6) mRNA is essential to monitor bone regeneration in the regenerating defects. In this work, we proposed a quantitative approach based on two-stage nicking enzyme signal amplification (NESA) and DNAzyme amplification for highly sensitive detection of BMP6 mRNA. The two-stage NESA involves two templates and two-stage amplification reactions under isothermal conditions. The first template contains two repeat sequences that could hybridize to the target RNA, triggering an exponential amplification. The amplified product was a short single-stranded DNA with the same sequence as the target RNA. The single-stranded DNA can trigger another linear NESA and produces a large amount of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. This proposed assay showed a quantitative analysis of BMP6 mRNA in a wide range from 1 fM to 100 nM with a detection limit of 0.01 fM.

Binding-induced and label-free colorimetric method for protein detection based on autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy

In this work, a new binding-induced and label-free colorimetric method for protein detection has been developed on the basis of an autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy. The system consists of two proximity probes carrying two aptamer sequences as recognition elements for target, and two hairpin structures include three-fourths and one-fourth of the G-quadruplex sequences in inactive configuration as functional elements. In the presence of target protein, two proximity probes bind to the protein simultaneously, forming a stable DNA–protein complex. Then the complex triggers an autonomous cross-opening of the two functional hairpin structures, leading to the formation of numerous hemin/G-quadruplex DNAzymes. The resulting DNAzymes catalyze the oxidation of colorless 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the green-colored ABTS•− with the presence of H2O2, thus providing the amplified colorimetric detection of target. Using human α-thrombin as the protein target, this binding-induced DNAzyme amplification colorimetric method affords high sensitivity with a detection limit of 1.9pM. Furthermore, this method might be further extended to sensitive detection of other proteins by simply replacing recognition elements of proximity probes.

Highly sensitive fluorescence detection of target DNA by coupling exonuclease-assisted cascade target recycling and DNAzyme amplification

Because of the intrinsic importance of nucleic acid as bio-targets, the simple and sensitive detection of nucleic acid is very essential for biological studies and medical diagnostics. Herein, a simple, isothermal and highly sensitive fluorescence detection of target DNA was developed with the combination of exonuclease III (Exo III)-assisted cascade target recycling and DNAzyme amplification. A hairpin DNA probe was designed, which contained the 3′-protruding DNA fragment as target recognition unit, the caged DNA fragment in the stem region as target analogue, and the caged 8–17 DNAzyme sequence in the loop region as signal response unit. Upon sensing of target DNA, the 3′-strand of hairpin DNA probe could be stepwise removed by Exo III, accompanied by the releasing of target DNA and autonomous generation of new target analogues for the successive hybridization and cleavage process. Simultaneously, the 8–17 DNAzyme unit could be exponentially released from this hairpin DNA probe and activated for the cyclic cleavage toward the ribonucleotide-containing molecular beacon substrate, inducing a remarkable fluorescence signal amplification for target detection. A low detection limit of 20fM with an excellent selectivity toward target DNA could be achieved. The developed cascade amplification strategy may be further extended for the detection of a wide spectrum of analytes including protein and biological small molecules by combining DNA aptamer technology.

Label-Free Colorimetric Aptasensor Based on Nicking Enzyme Assisted Signal Amplification and DNAzyme Amplification for Highly Sensitive Detection of Protein

Highly sensitive detection of proteins is essential to biomedical research as well as clinical diagnosis. Here, we develped a novel label-free colorimetric aptasensor based on nicking enzyme assisted signal amplification and DNAzyme amplification for highly sensitive detection of protein. The system consists of a hairpin DNA probe carrying an aptamer sequence for target, a G-riched DNA probe containing two G-riched DNAzyme segments and the recognition sequence as well as cleavage site for nicking enzyme, a blocker DNA, and the nicking enzyme. The hybridization of the G-riched DNA with the blocker DNA prohibits the formation of the activated DNAzymes in the absence of target. Upon addition of target to the system, the hairpin probe is opened by the specific recognition of the target to its aptamer. The open hairpin probe hybridizes with a G-riched DNA and forms a DNA duplex, which triggers the selective cleavage of the G-riched DNA probe by nicking enzyme, leading to the release of the aptamer-target complex and the G-riched DNAzyme segments. The released open hairpin probe then hybridizes with another G-riched DNA probe, and the cycle starts anew, resulting in the continuous cleavage of the G-riched DNA probes, generating a much of G-riched DNAzyme segments. The G-riched DNAzyme segments interact with hemin and generates the activated DNAzyme that can catalyze the H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) to the colored ABTS(•-), thus providing the amplified colorimetric detection of target. With the use of thrombin (Tb) as a proof-of-principle analyte, this sensing platform can detect Tb specifically with a detection limit as low as 1.5 pM, which is at least 4 orders of magnitude lower over the unamplified colorimetric assay. Moreover, the assay does not involve any chemical modification of DNA, which is simple and low-cost. This sensing platform provides a promising approach for the amplified analysis of target molecules.

A folate receptor electrochemical sensor based on terminal protection and supersandwich DNAzyme amplification

Based on the protecting effect of folate receptor (FR) toward folic acid (FA) modified DNA and the signal amplification of supersandwich DNA structure, we designed an interesting electrochemical biosensor for FR. In the present system, with the increase of FR, protecting more FA bound DNA from hydrolysis by exonuclease I (Exo I), FA bound DNA will hybridize to form more supersandwich DNA structure resulting in an increased electrochemical signal. A relationship between the concentration of the target protein, FR, and the obtained electrochemical signal can be established. The signal was obtained by the catalysis on H2O2 in the system containing Fc and hemin/DNAzyme. The detection concentration range of FR was from 1.0to 20.0ng/mL with an achieved detection limit of 0.3ng/mL which approached clinically relevant concentrationsof FR.

A target-activated autocatalytic DNAzyme amplification strategy for the assay of base excision repair enzyme activity

Based on a target-activated autocatalytic DNAzyme amplification strategy, novel fluorescence sensing platforms were constructed for highly sensitive and selective assay of base excision repair enzyme activity. By using a rolling circle amplification (RCA)-coupled amplification cascade, an extremely low detection limit (0.002 U mL(-1)) was achieved.