Exponential Amplification Reaction Research Articles

The cysteine-induced BiOBr as a novel photoactive material with high photoelectric efficiency for ultrasensitive detection of microRNA

In this work, cysteine-induced BiOBr (cys-BiOBr) was prepared as a novel photoactive material with high photoelectric efficiency to construct a photochemical (PEC) biosensor for ultrasensitive detection of microRNA-155 (miRNA-155). Impressively, the photocurrent signal of cys-BiOBr showed an increase of 6 times compared to that of individual BiOBr, since the doping of S not only narrowed the band gap to enhance light absorption and accelerate carrier separation, but also augmented the surface oxygen vacancy concentration for preventing electron-hole pairs recombination, resulting in the obvious improvement of photoelectric efficiency. Furthermore, through exponential amplification reaction (EXPAR), a few miRNA-155 could be derived into abundant steady G-quadruplexes for immobilizing hemin to form the G-quadruplex/hemin complex for catalyzing H2O2 decomposition to significantly amplify photocurrent signal. The proposed PEC biosensor performed a wide range from 0.5 fM to 1 nM with a detection limit of 0.13 fM. This strategy provided a new method to prepare the highly efficient photoactive material for sensitive detection of biomolecules and exhibited enormous potential in early clinical diagnosis.

HpaII-assisted and linear amplification-enhanced isothermal exponential amplification fluorescent strategy for rapid and sensitive detection of DNA methyltransferase activity.

The detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, a HpaII-assisted and linear amplification-enhanced exponential amplification strategy is proposed for sensitive and label-free detection of M.SssI MTase activity. The P1 probe contains self-complementary sequence 5'-CTAGCCGGCTAG-3' at 3'-terminal. After denaturation and annealing, P1 probes hybridize with itself to generate P1 duplexes. M.SssI MTase induces methylation of cytosine at 5'-CG-3' in P1 duplexes, and thus, HpaII fails to cleave at 5'-CCGG-3' due to methylation sensitivity, leaving P1 duplex intact. Then, these intact P1 duplexes are extended along 3'-terminal through Vent (exo-) DNA polymerase to generate dsDNA, which is recognized and nicked at the recognition sites by Nt.BstNBI, releasing two copies of primer X. Primer X hybridizes with X' at the amplification template T1 (X'-Y'-X') and then serves as primers to trigger the exponential amplification reaction (EXPAR). The point of inflection (POI) values of real-time fluorescence curves is linearly correlated with the logarithm of M.SssI MTase concentration in the range of 0.125 [Formula: see text] 8 U mL-1 with a low detection limit of 0.034 U mL-1. In the absence of M.SssI, P1 duplexes are cut by HpaII and separated into ssDNA under the executed temperature of EXPAR and thus unable to trigger the amplification. The strategy provides good selectivity against other types of MTases and protein and is able to detect M.SssI activity in human serum. Furthermore, the analytical method has the generality and can be extended to the analysis of other types of DNA MTases.

GlaI cleavage assistant isothermal exponential amplification coupling with CRISPR/Cas12a for ultrasensitive detection of CLDN11 methylation: A potential marker for lung adenocarcinoma

DNA methylation is closely associated with cancer, and sensitive detection of DNA methylation is immensely significant for early cancer screening. In this study, for the first time, we identified CLDN11 methylation as a valuable marker for lung adenocarcinoma diagnosis and prognosis based on database analysis and experimental verification using clinical tissues and sera. Then, a flexible platform was proposed for ultrasensitive detection of CLDN11 methylation by integrating methylation-dependent restriction endonuclease GlaI cleavage with exponential amplification reaction (EXPAR) and CRISPR/Cas12a (GlaI–EXPAR–Cas12a platform). GlaI only recognizes and cleaves methylated target sites with excellent selectivity; the released fragments are primers for triggering EXPAR–CRISPR/Cas12a. Using Cas12a, false positives generated by EXPAR can be weakened to a negligible level. The proposed platform for detecting CLDN11 methylation achieved excellent performance, with a low limit of detection (1.25 × 10–15 M), and reliably identified CLDN11 methylation at an abundance of 0.1 % even in the presence of a large number of unmethylated fragments. More importantly, the proposed platform can be used for the quantification of human serum and genomic DNA, as well as to distinguish normal cells (HEK293) from cancer cells (H1299, A549 and H358). Through this integration platform, all 20 lung adenocarcinoma tissues exhibited significantly higher levels of CLDN11 methylation than a total of 16 paracancerous tissues. Taken together, the GlaI–EXPAR–Cas12a platform is ultrasensitive and convenient, and has perfect specificity. Further, it may function as a flexible platform for early screening of lung adenocarcinoma.

Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions.

Animproved electrochemical sensor has been developed for sensitive detection of the p53 genebased on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signalsby a factor of about 15.Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10pM and 10pM to 1nM, as well as a relatively low limit detection of 0.39 fM, which is aboutan order of magnitude lowerthan that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.

Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase.

The demethylase of fat mass and obesity related protein (FTO) is critical to regulate the dynamic N6-methyladenosine (m6A) modification of eukaryotic mRNAs, and its overexpression has found to be closely related to the initiation of several cancers. On the basis of a target-promoted specific activation of DNAzyme strategy coupled with self-primer exponential amplification reaction (SPEXPAR) cycles and DNA supersandwich assemblies, the highly sensitive and label-free electrochemical FTO assay approach is established. The modification of the catalytic core nucleobase of the DNAzyme probe by m6A can inhibit its cleavage activity. The presence of target FTO catalyzes the elimination of the methyl group to restore the DNAzyme activity, which cleaves the hairpin substrates to trigger the SPEXPAR for yielding many ssDNAs. The capture of these DNAs on the sensor electrode leads to the initiation of supersandwich assembly formation of long dsDNAs. Tremendous electrochemical signal probe of [Ru(NH3)6]Cl3 are then absorbed on these dsDNAs to produce highly amplified catalytic currents with the assistance of K3[Fe(CN)6] for detecting trace FTO with 63.1fM detection limit. Furthermore, the sensor can be employed for selective assay of FTO in cell lysates, revealing the great potential of this sensing strategy for biomedical and biological study applications.

CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction

Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3′-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.

Isothermal exponential amplification reactions triggered by circular templates (cEXPAR) targeting miRNA.

Isothermal exponential amplification reaction (EXPAR) is an emerging amplification technique that is most frequently used to amplify microRNA (miRNA). However, EXPAR also exhibits non-specific background amplification in the absence of the targeted sequence, which limits the attainable assay sensitivity of EXPAR. A novel modified isothermal EXPAR based on circular amplification templates (cEXPAR) was developed in this study. The circular template consists of two same linear fragments that complement the target sequence, and these two linear fragments are separated by two nicking agent recognition sequences (NARS). Compared with the linear structure template, this circular template allows DNA or RNA fragments to be randomly paired with two repeated sequences and can be successfully amplified. This reaction system developed in this study could rapidly synthesize short oligonucleotide fragments (12-22bp) through simultaneous nicking and displacement reactions. Highly sensitive chain reactions can be specifically triggered by as low as a single copy of target molecule, and non-specific amplification can be effectively eliminated in this optimized system. Moreover, the proposed approach applied to miRNA test can discriminate single-nucleotide variations between miRNAs. The newly developed cEXPAR assay provides a useful alternative tool for rapid, sensitive, and highly specific detection of miRNAs.

Suitability evaluation of toehold switch and EXPAR for cell-free MicroRNA biosensor development

The development of a robust and cost-effective sensing platform for microRNA (miRNA) is of paramount importance in detecting and monitoring various diseases. Current miRNA detection methods are marred by low accuracy, high cost, and instability. The toehold switch riboregulator has shown promising results in detecting viral RNAs integrated with the freeze-dried cell-free system (CFS). This study aimed to leverage the toehold switch technology and portability to detect miRNA in the CFS and to incorporate the exponential amplification reaction (EXPAR) to bring the detection to clinically relevant levels. We assessed various EXPAR DNA templates under different conditions to enhance the accuracy of the sensing platform. Furthermore, different structures of toehold switches were tested with either high-concentration synthetic miRNA or EXPAR product to assess sensitivity. Herein, we elucidated the mechanisms of the toehold switch and EXPAR, presented the findings of these optimizations, and discussed the potential benefits and drawbacks of their combined use.

A hairpin probe-mediated exponential amplification reaction for highly sensitive and specific detection of microRNAs.

In this work, we propose a hairpin probe-mediated exponential amplification reaction (HEAR) strategy that combines DNA strand displacement with a "who triggers, who gets generated" mode, providing excellent single-base discrimination and a reduced background signal. The detection limit is 19 aM, which is reduced by 3 orders of magnitude compared to traditional exponential amplification approaches. This one-pot strategy also exhibits a wide dynamic range, high specificity and short detection time. It is expected to become a powerful tool for clinical diagnosis.

A fluorescent biosensor based on exponential amplification reaction-initiated CRISPR/Cas12a (EIC) strategy for ultrasensitive DNA methyltransferase detection

DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH2-modified 3’ end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10−4 U/mL and a broad detection range from 2 × 10−4 to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.

DNAzyme-templated exponential isothermal amplification for sensitive detection of lead pollution and high-throughput screening of microbial biosorbents

Very low concentrations of lead (Pb2+) pollution can have far-reaching adverse impacts on human health, due to the cumulative toxicity of Pb2+. Herein, we report a DNAzyme-templated exponential isothermal amplification strategy (termed DNAzymee) for the ultrasensitive detection of Pb2+ pollution and the high-throughput screening of microbial biosorbents to remove Pb2+ pollution. DNAzyme can specifically recognize Pb2+, and this recognition event can be amplified by the subsequent exponential isothermal amplification reaction (EXPAR) and monitored by a G-quadruplex specific dye. The proposed design showed a low limit of detection (95 pM) and could identify Pb2+ pollution in different real samples with high precision. In particular, the proposed assay was used to screen Pb2+ biosorbents, and the results showed that Leuconostoc mesenteroides is a promising microbial biosorbent for removing Pb2+ pollution. Thus, the DNAzymee assay can serve as a platform to monitor lead pollution in the environment and screen efficient biosorbents for the control of lead pollution.

Specific detection of antibiotic-resistant bacteria using CRISPR/Cas9 induced isothermal exponential amplification reaction (IEXPAR)

The prevalence of antibiotic-resistant bacterial infections has raised great social concern and even aggravated the burden of public healthcare systems worldwide. Simple, rapid, highly sensitive and specific detection of antibiotic-resistant bacteria is urgently needed for guiding proper antibiotic treatment. Herein, we demonstrate a novel strategy to specifically distinguish and detect antibiotic-resistant bacteria using CRISPR/Cas9 induced isothermal exponential amplification reaction (IEXPAR). Through the specific recognition and cleavage of CRISPR/Cas9 system, the antibiotic-resistant genes can be digested into two short segments with free 3ʹ-OH end. Using one of the cleaved DNA as target to design IEXPAR template (Xʹ-Y-Xʹ), highly efficient exponential amplification of IEXPAR can be triggered, resulting in the direct detection of antibiotic-resistant genes under isothermal condition with high accuracy and rapidness. While for antibiotic-sensitive bacteria, CRISPR/Cas9 system cannot cleave any DNA due to the absence of antibiotic-resistant genes and no subsequent amplification reaction occurs which ensures the high specificity of our method. Further applying our method to the detection of real biological samples, the specific discrimination of antibiotic-resistant bacteria from antibiotic-sensitive bacteria is realized even with large amounts of DNA interferents. In consideration of the distinct advantages of rapid, high specificity and facile operation, this CRISPR/Cas9-induced IEXPAR assay may serve as an efficient and convenient tool for the accurate detection of antibiotic-resistant bacteria in complex biological samples.

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction.

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 µM theophylline in as little as 15 min.

Click Chemistry Actuated Exponential Amplification Reaction Assisted CRISPR-Cas12a for the Electrochemical Detection of MicroRNAs.

MicroRNAs (miRNAs) play a very important role in biological processes and are used as biomarkers for the detection of a variety of diseases, including neurodegenerative diseases, chronic cardiovascular diseases, and cancers. A sensitive point-of-care (POC) method is crucial for detecting miRNAs. Herein, CRISPR–Cas12a combined with the click chemistry actuated exponential amplification reaction was introduced into an electrochemical biosensor for detecting miRNA-21. The target miRNA-21 initiated the click chemistry–exponential amplification reaction in the electrochemical biosensor to produce numerous nucleic acid fragments, which could stimulate the trans-cleavage ability of CRISPR–Cas12a to cleave hairpin DNA electrochemical reporters immobilized on the electrode surface. Under optimal conditions, the minimum detection limit for this electrochemical biosensor was as low as 1 fM. Thus, the proposed electrochemical biosensor allows sensitive and efficient miRNA detection and could be a potential analysis tool for POC test and field molecular diagnostics.

Exploring the performance of multi-channel tetrahedral nucleic acid tweezers platforms for efficient and sensitive biosensing

The nucleic tweezers-based detection strategies have shown excellent application prospects in molecular detection and diagnosis thanks to the specific target responsiveness and good biocompatibility. However, the limited detection efficiency and unsatisfactory sensitivity make their applications in complex diagnosis difficult, and few reports focused on it. Here, we explored the molecular dynamics simulations and biosensing performance of the Tetrahedral Nucleic Acid Tweezers, including the Antennae like-Tetrahedron Nucleic Acid Tweezer (ATNAT) and the Covered-Tetrahedron Nucleic Acid Tweezer (CTNAT), revealing the molecular dynamics of ATNAT and CTNAT reporters visually and molecularly. After that, we compared the multi-target detection capabilities of DNA tetrahedral tweezers, and combined the CTNAT reporter with better multi-target detection performance with the aptamer and Exponential amplification reaction (EXPAR), developing an efficient and sensitive EXPAR-cDNA-CTNAT strategy. Then we applied the EXPAR-cDNA-CTNAT strategy to detect testosterone, cortisol, and creatine kinase isoenzymes, realizing sensitive and accurate fatigue diagnosis. Compared with the traditional detection strategies, the EXPAR-cDNA-CTNAT strategy showed improved sensitivity and detection efficiency with excellent specificity, and the limits of detection (LODs) for the multi-target detection were as low as 41, 68, and 8 pM, respectively. The EXPAR-cDNA-CTNAT strategy was reliable for multi-target detection, which had great potential in biological science, food safety, and medical diagnosis.

Sensitive naked-eye detection of telomerase activity based on exponential amplification reaction and lateral flow assay.

Telomerase is a promising diagnostic and prognostic biomarker for cancers. Sensitive, simple, and reliable telomerase activity detection is vital for cancer diagnosis. Herein, we developed an ultrasensitive visualized assay for telomerase activity that combined the exponential amplification reaction (EXPAR) and lateral flow assay for easy and quick signal readout, which we termed as a lateral flow readout-EXPAR (LFR-EXPAR) assay. In the LFR-EXPAR assay, telomerase elongation products initiate the exponential amplification reaction, the generated trigger hybridizes with the reporter to form the recognition site of the nicking enzyme, and the nicking enzyme cuts the reporter strand. The degradation of the reporter can be detected with a universal lateral flow dipstick and read out with the naked eye. After conducting a series of proof-of-concept investigations, the LFR-EXPAR assay was found to achieve a sensitivity comparable to that of a TRAP (telomere repeat amplification protocol) assay. The LFR-EXPAR assay can be used to realize ultrasensitive and point-of-care detection of telomerase without requiring specialized instruments, holding great promise for early cancer diagnosis.

Combining CRISPR/Cas12a with isothermal exponential amplification as an ultrasensitive sensing platform for microRNA detection

MicroRNA (miRNA) is considered as a potential biomarker in the diagnosis of cancer. Thus, a new diagnostic technique with high sensitivity and selectivity is crucial for miRNA detection in clinical testing. Herein, we presented a sensing platform based on CRISPR/Cas12a and isothermal exponential amplification reaction (EXPAR) to realize ultrasensitive analysis of miRNA. MiRNA-27a (miR-27a) was chosen as a model target. In this sensing platform, a miR-27a-triggered cascade EXPAR process was introduced to achieve a mass of activators. The resultant activators could hybrid specifically with crRNA of CRISPR/Cas12a system, triggering the multiple-turnover trans-cleavage activity of Cas12a for achieving signal amplification. The sensing platform rendered a wide detection (ranging from 100 aM to 10 nM) and a low limit of detection (90 aM). In particular, this strategy achieved ultrasensitive detection of the other microRNA biomarkers such as let-7a and miR-141 by the flexible design of the region of template, which demonstrated the universal detection ability. Furthermore, an excellent selectivity feature was obtained by the discrimination of microRNA family members and base mismatch analysis. The proposed sensing platform also was able to assess miR-27a levels in breast cancer cell and serum sample, revealing a promising application in clinical molecular diagnostics.

Sandwich capture ultrasensitive sensor based on biohybrid interface for the detection of Cronobacter sakazakii.

A simple, rapid and ultrasensitive visual sensing method for the detection of Cronobacter sakazakii (C. sakazakii) based on a biohybrid interface was established. During the entire sensing process, quadruple-cascade amplification showed its superior sensing performance. First, the prepared immunomagnetic beads (IMB) were used to isolate and enrich specific targets from the food matrix. After adding the fusion aptamer, the aptamer sequence specifically recognized the target and formed the immune sandwich structure of antibody-target-fusion aptamer. In addition, the fusion aptamer also included the template sequence of exponential amplification reaction (EXPAR), which contained the antisense sequence of the G-rich sequence. Therefore, a large number of G-rich sequences can be generated after EXPAR can be triggered in the presence of Bst. DNA polymerase, nicking endonuclease, cDNA, and dNTP. They were self-assembled into G-quadruplex structures and then combined with hemin to form G4/hemin DNAzyme, resulting in visible coloration and measuring absorbance at 450nm for quantitative detection. The assay showed a limit of detection (LOD) of 2CFU/mL in pure culture and 12CFU/g in milk powder in optimal conditions. This method provides a promising strategy for rapid and point-of-care testing (POCT) since it does not require DNA extraction, medium culturing, and expensive instrumentation. KEY POINTS: •Single-cell level detection of C. sakazakii with ultrasensitive and rapidness •The fusion aptamer integrated recognition and amplification •Sensing analysis of C. sakazakii based on cascade amplification of biohybrid interface.

Exponential isothermal amplification coupled MALDI-TOF MS for microRNAs detection

MicroRNAs (miRNAs) have attracted significant attention in biomedical research and clinical diagnosis. However, due to their inherent characteristics of low abundance and the high complexity of corresponding biological matrices, simultaneous detection of multiple miRNAs at low abundance is still a challenge. In this work, a method coupling exponential amplification reaction (EXPAR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed for label-free and simultaneous detection of multiple miRNAs. The assay can be performed under isothermal conditions in a single reaction tube, and finished in less than 30 min. It exhibits good quantification ability and with attomolar-level sensitivity for miRNAs detection. It also shows high specificity to distinguish miRNAs at single-nucleotide resolution. We used the method to detect the miRNA-21, let-7a, miRNA-100, and miRNA-125b in samples of spiked human serum and breast cancer cells (i.e., MCF-7, MDA-MB-231 and SK-BR-3). The quantification results were well consistent with the standard real-time fluorescence EXPAR. Consequently, the label-free mass-spectrometric platform could be a potential tool for miRNAs analysis in complex biological samples, and may be used for clinical diagnosis.

A multiple primers-mediated exponential rolling circle amplification strategy for highly sensitive detection of T4 polynucleotide kinase and T4 DNA ligase activity

T4 polynucleotide kinase (T4 PNK) and T4 DNA ligase are two important repair enzymes that play a significant role in maintaining the stability and integrity of genome. Here, we proposed a multiple primers-mediated exponential rolling circle amplification strategy for label-free and highly sensitive fluorescent analysis of T4 PNK and T4 DNA ligase activity. Firstly, we designed a target strand that contained two C-rich sequences, a complementary sequence of the primer, and three cleavage sites of Nt.BbvCI scattered among the three sequences. The 5′-hydroxyl terminus of target strand was catalyzed by T4 PNK to form a phosphorylated target strand. After the phosphorylated target strand was combined with the primer, T4 DNA ligase catalyzed the formation of circular DNA. Then, the primer triggered an exponential rolling circle amplification reaction along the circular DNA template, exponentially generating G-rich sequences. N-methyl mesoporphyrin IX as signal molecules specifically bound to the G-quadruplexs folded by G-rich sequences, generating a detectable fluorescent signal. Benefiting from exponential signal growth, this strategy showed extremely high detection sensitivity with detection limits of 8.11 × 10−5 U mL−1 of T4 PNK and 3.07 × 10−4 U mL−1 of T4 DNA ligase, respectively. In addition, the proposed strategy exhibited excellent inhibitor evaluation effects for two repair enzymes, and could detect T4 PNK in HeLa cell lysate. The obtained results showed that the proposed strategy had excellent application prospects in disease diagnosis and treatment evaluation.