N-methyl Mesoporphyrin Research Articles

CRISPR/Cas13a-mediated triple signal amplification strategy for viral RNA detection

CRISPR/Cas13a system has been combined with different isothermal amplification strategies for RNA analysis. Nevertheless, expensive enzymes are usually indispensable in these isothermal amplification methods, which increases the detection cost. In this study, a CRISPR/Cas13a-mediated triple signal amplification strategy has been designed for selective, sensitive and inexpensive detection of viral RNA. SARS-CoV-2 S RNA activated CRISPR/Cas13a to cut hairpin DNA, triggering catalytic hairpin assembly (CHA) and subsequent hybridization chain reaction (HCR) to form a long-stranded Y-type DNA. G-quadruplexes were distributed on the branch of the long-stranded DNA and bound with N-methyl mesoporphyrin IX (NMM) to enhance the fluorescence signal. SARS-CoV-2 S RNA was specifically detected with a limit of detection (LOD) of 285 fM. Notably, no additional expensive enzymes were required in the entire detection process. The CRISPR/Cas13a-mediated triple signal amplification strategy holds great potential for specific and sensitive viral RNA detection with low cost.

Concentration and activation biresponsive strategy in one analysis system with simultaneous use of G4 structure-specific signal probe and enzyme-catalyzed reaction

BackgroundEnzymes with critical effects on life systems are regulated by expression and activation to modulate life processes. However, further insights into enzyme functions and mechanisms in various physiological processes are limited to concentration or activation analysis only. Currently, enzyme analysis has received notable attention, particularly simultaneous analysis of their concentration and activation in one system. Herein, N-methyl mesoporphyrin IX (NMM), a specific dye with notable structural selectivity for parallel G-quadruplex nucleic acid enzyme (G4h DNAzyme), is employed for the analysis of its concentration. In addition, the peroxidase activity of G4h DNAzyme is characterized based on G4h DNAzyme–catalyzed decomposition of H2O2 to continuously consume luminol. Accordingly, an increased fluorescence (FL) response of NMM and a decreased FL response of luminol could be simultaneously employed to analyze the concentration and activation of G4h DNAzyme. ResultHerein, a novel concentration and activation biresponsive strategy is proposed using a G4h DNAzyme–based model that simultaneously employs a G4h structure–specific signal probe for enzyme concentration analysis and G4h DNAzyme–catalyzed reactions for enzyme activation analysis. Under optimal conditions, the biresponsive strategy can be effectively used for the simultaneous analysis of G4h DNAzyme concentration and activation, with detection limits of 718.7 pM and 233.4 nM respectively, delivering acceptable performances both in cell and in vitro. SignificanceThis strategy can not only be applied to concentration and activation analyses of G4h DNAzyme but can also be easily extended to other enzymes by simultaneously combining concentration analysis via target-induced direct reaction and activation analysis via target-induced catalytic reaction, offering deeper insights into various enzymes and enabling their effective implementation in bioanalysis and biochemistry.

Nanoscale octopus guiding telomere entanglement: An innovative strategy for inducing apoptosis in cancer cells

Telomere length plays a crucial role in cellular aging and the risk of diseases. Unlike normal cells, cancer cells can extend their own survival by maintaining telomere stability through telomere maintenance mechanism. Therefore, regulating the lengths of telomeres have emerged as a promising approach for anti-cancer treatment. In this study, we introduce a nanoscale octopus-like structure designed to induce physical entangling of telomere, thereby efficiently triggering telomere dysfunction. The nanoscale octopus, composed of eight-armed PEG (8-arm-PEG), are functionalized with cell penetrating peptide (TAT) to facilitate nuclear entry and are covalently bound to N-Methyl Mesoporphyrin IX (NMM) to target G-quadruplexes (G4s) present in telomeres. The multi-armed configuration of the nanoscale octopus enables targeted binding to multiple G4s, physically disrupting and entangling numerous telomeres, thereby triggering telomere dysfunction. Both in vitro and in vivo experiments indicate that the nanoscale octopus significantly inhibits cancer cell proliferation, induces apoptosis through telomere entanglement, and ultimately suppresses tumor growth. This research offers a novel perspective for the development of innovative anti-cancer interventions and provides potential therapeutic options for targeting telomeres.

Engineering smart thioflavin T binders with switchable DNA topologies for constructing multiple label-free biosensors

Thioflavin T (ThT) has been used as a sensitive fluorescence probe for lighting up nucleic acids. The binding of ThT is dependent on the structural topologies of DNA sequences. To date, most oligonucleotides interact with ThT through only a single topology, and this limits the applications of ThT as a sensing agent. Here, we modulated an HIV-1 integrase aptamer, 93del, to have switchable topologies by optimizing its 3′ flanking residues to enhance its ThT-binding capacity. Our data revealed that the sequences formed stem-loop hairpins and I-motif/G-quadruplex (G4) hybrids in Na+ buffer under alkaline and acidic conditions, respectively. Both topologies could bind ThT with high affinity (Kd ∼ 0.5 μM) and dramatically enhance the fluorescent signal of ThT. However, in the presence of K+, the sequences could be assembled into interlocked G4s, which had a low affinity for ThT but a high affinity for N-methyl mesoporphyrin IX (NMM). This allowed us to build highly sensitive fluorescent K+ sensors that were insusceptible to interference from Na+. Additionally, we found that the stacking of homologous base pairs in the hairpin loops was essential for ThT recognition and could be used as an indicator in the construction of label-free molecular beacons, allowing monitoring of microRNAs effectively in human serum spiked test. By employing external stimuli to induce the transformation of these topologies, we were able to sensitively detect adenosine triphosphate (ATP) molecules or Hg2+in vitro under specific acidic conditions. Our study provides a strategy for exploiting undiscovered functions of existing aptamers.

Characterization of the conformational binding of N-methyl mesoporphyrin IX using fluorescent model DNA telomeric G-quadruplex forming sequences

Characterization of the conformational binding of N-methyl mesoporphyrin IX using fluorescent model DNA telomeric G-quadruplex forming sequences

Label-free ratiometric fluorescence detection of Pb2+via structure-specific fluorescent dyes and dual signal amplification.

Lead ions (Pb2+) are a widely distributed and highly toxic heavy metal pollutant, which seriously threatens the environment, economy and human safety. Here, a label-free ratiometric fluorescent biosensor was constructed for Pb2+ detection using DNAzyme-driven target cycling and exonuclease III (Exo III)-mediated DNA cycling as a dual signal amplification strategy. The SYBR Green I (SGI) and N-methyl mesoporphyrin IX (NMM) used in this study are characterized by low cost, storage resistance, and short preparation time compared with conventional signaling probes labeled with fluorescent groups. Unlike the single-emission fluorescence strategy, monitoring the fluorescence intensity ratio of SGI and NMM can effectively reduce external interference to achieve accurate detection of Pb2+. DNAzyme structures on the surface of magnetic beads (MBs) can recognize Pb2+ and activate the target circulatory system to cleave single-stranded DNA (ssDNA). The ssDNA further initiated the Exo III-assisted DNA circulatory system to digest double-stranded DNA (dsDNA) and release guanine-rich G1. Finally, the fluorescence signals of SGI and NMM were weakened and enhanced, respectively. The sensing strategy achieved a wide linear range from 0.5 to 500 nM and a low limit of detection (LOD) of 26.4 pM. Furthermore, its anti-interference ability and potential applicability for Pb2+ detection in actual samples were verified. This work ingeniously combines the dual signal amplification strategy with the ratiometric sensing strategy constructed by structure-specific fluorescent dyes, which provides a promising method for constructing sensitive and accurate fluorescent biosensors.

G-quadruplex in the TMV Genome Regulates Viral Proliferation and Acts as Antiviral Target of Photodynamic Therapy.

Plant viruses seriously disrupt crop growth and development, and classic protein-targeted antiviral drugs could not provide complete protection against them. It is urgent to develop antiviral compounds with novel targets. Photodynamic therapy shows potential in controlling agricultural pests, but nonselective damage from reactive oxygen species (ROS) unexpectedly affects healthy tissues. A G-quadruplex (G4)-forming sequence in the tobacco mosaic virus (TMV) genome was identified to interfere the RNA replication in vitro, and affect the proliferation of TMV in tobacco. N-methyl mesoporphyrin IX stabilizing the G4 structure exhibited inhibition against viral proliferation, which was comparable to the inhibition effect of ribavirin. This indicated that G4 could work as an antiviral target. The large conjugate planes shared by G4 ligands and photosensitizers (PSs) remind us that the PSs could work as antiviral agents by targeting G4 in the genome of TMV. Chlorin e6 (Ce6) was identified to stabilize the G4 structure in the dark and selectively cleave the G4 sequence by producing ROS upon LED-light irradiation, leading to 92.2% inhibition against TMV in vivo, which is higher than that of commercial ningnanmycin. The inhibition of Ce6 was lost against the mutant variants lacking the G4-forming sequence. These findings indicated that the G-quadruplex in the TMV genome worked as an important structural element regulating viral proliferation, and could act as the antiviral target of photodynamic therapy.

N-methyl mesoporphyrin IX (NMM) as electrochemical probe for detection of guanine quadruplexes

G-quadruplexes (G4) are stable alternative secondary structures of nucleic acids. With increasing understanding of their roles in biological processes and their application in bio- and nanotechnology, the exploration of novel methods for the analysis of these structures is becoming important. In this work, N-methyl mesoporphyrin IX (NMM) was used as a voltammetric probe for an easy electrochemical detection of G4s. Cyclic voltammetry on a hanging mercury drop electrode (HMDE) was used to detect NMM with a limit of detection (LOD) of 40 nM. Characteristic reduction signal of NMM was found to be substantially higher in the presence of G4 oligodeoxynucleotides (ODNs) than in the presence of single- or double-stranded ODNs and even ODNs susceptible to form G4s but in their unfolded, single-stranded forms. Gradual transition from unstructured single strand to G4, induced by increasing concentrations of the G4 stabilizing K+ ions, was detected by an electrochemical method for the first time. All obtained results were supported by circular dichroism spectroscopy. This work expands on the concept of electrochemical probes utilization in DNA secondary structure recognition and offers a proof of principle that can be potentially employed in the development of novel electroanalytical methods for nucleic acid structure studies.

Analysis of genetically modified soya and soya products using a fluorescence probe based on PCR-initiated isothermal amplification of G-quadruplex DNA

We proposed a novel strategy for the analysis of genetically modified (GM) soya samples by combining PCR-initiated generation and isothermal amplification of G-quadruplex DNA. PCR primers were designed to consist of a target-specific sequence, a G-quadruplex complement sequence, and a nicking endonuclease recognition site. After the PCR step, the nicking enzyme cut the recognition site, and single stranded G-quadruplex sequences were displaced and further amplified by polymerase during the isothermal amplification reaction. Then, the G-quadruplex sequences combined with N-methyl mesoporphyrin IX to generate strong fluorescence. This method has a detection limit of 0.2% and a linear range from 50% to 0.5% GM content. When exponential isothermal amplification was coupled to the preceding amplification step, the detection limit was further improved to 0.02% GM soya. GM contents were successfully detected at 0.1% GM percentage in soy milk and 0.9% GM percentage in tofu and dried tofu samples. The method may be applied to the analysis of various GM species simply by changing the target sequence in the primers.

Solution Structure of Poly(UG) RNA

Poly(UG) or “pUG” RNAs are UG or GU dinucleotide repeat sequences which are highly abundant in eukaryotes. Post-transcriptional addition of pUGs to RNA 3′ ends marks mRNAs as vectors for gene silencing in C. elegans. We previously determined the crystal structure of pUG RNA bound to the ligand N-methyl mesoporphyrin IX (NMM), but the structure of free pUG RNA is unknown. Here we report the solution structure of the free pUG RNA (GU)12, as determined by nuclear magnetic resonance spectroscopy and small and wide-angle x-ray scattering (NMR-SAXS-WAXS). The low complexity sequence and 4-fold symmetry of the structure result in overlapped NMR signals that complicate chemical shift assignment. We therefore utilized single site-specific deoxyribose modifications which did not perturb the structure and introduced well-resolved methylene signals that are easily identified in NMR spectra. The solution structure ensemble has a root mean squared deviation (RMSD) of 0.62 Å and is a compact, left-handed quadruplex with a Z-form backbone, or “pUG fold.” Overall, the structure agrees with the crystal structure of (GU)12 bound to NMM, indicating the pUG fold is unaltered by docking of the NMM ligand. The solution structure reveals conformational details that could not be resolved by x-ray crystallography, which explain how the pUG fold can form within longer RNAs.

Hybrid chain reaction and selective recognition-based homogeneous dual-fluorescence analysis of circulating tumor cells in clinical ovarian cancer samples

BackgroundOncological analysis is important in tumor diagnosis. We constructed a dual-fluorescence and binary visual analysis system for circulating tumor cells (CTCs) using the folate receptor as a biomarker, combined with hybridization chain reaction and nanomaterial amplification. This strategy integrates terminal protection, selective recognition properties of N-methyl mesoporphyrin IX and CdTe quantum dots for Cu2+ and double-stranded templated copper nanoparticles, and inkjet printing technology. ResultsIn fluorescence mode, folate receptor and A2780 ovarian cancer cells were specifically detected with a limit of detection of 0.1 fg mL−1, and 10 cells mL−1 were observed. The detection limits of both the color and distance reading modes were comparable to those obtained in fluorescence mode. The applicability of the method for quantifying CTCs was validated using 27 (6 negative and 21 positive) clinical ovarian cancer samples; the results agreed with those of both the clinical folate receptor-polymerase chain reaction kit and radiological and pathological results. SignificanceThis dual-fluorescence and binary visual CTCs detection method provides multiple options for clinical tumor liquid biopsy.

Split aptamer-based fluorescent biosensor for ultrasensitive detection of cocaine using N-methyl mesoporphyrin IX as fluorophore

Cocaine is an extensively used illicit drug worldwide. So, the development of a sensing method for cocaine detection is necessary for examination and lawful action against drug trafficking and abuse. A label-free and simple split aptamer-based fluorescent aptasensor was proposed for cocaine detection using the N-methyl mesoporphyrin IX (NMM)/G-quadruplex system as a fluorescent indicator. The working principle of the developed aptasensor is an increase in the emission intensity of fluorescence of the NMM in the attendance of cocaine due to the formation of split G-quadruplex structure as a suitable platform for attachment of NMM. Under optimized conditions, the designed fluorescent aptasensor exhibited a useful linear concentration range from 50 pM to 25 nM and a limit of detection (LOD) of 16 pM. The applied potential of the designed fluorescent aptasensor was demonstrated by cocaine detection in spiked human serum samples with satisfying recoveries.

Genome-wide analysis of G-quadruplex in Spodoptera frugiperda

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a globally distributed lepidopteran crop pest that has developed resistance to most insecticides. The G-quadruplex (G4) is a secondary structure in the genome enriched in the promoters for regulating gene expression. However, little is known about G4 in S. frugiperda, especially whether G4 is involved in insecticide resistance and pest control. In this study, 387,875 G4 motifs in the whole genome of S. frugiperda were identified by bioinformatics prediction. We found that 66.90 % of theseG4 structures were located in genic regions and highly enriched in the upstream regions of start codons. Functional and pathway analyses showed that the genes with G4 enriched in promoter regions participate in several metabolic processes. Further analyses showed that G4 structures occurred more frequently in the promoters of P450 and CarE gene families. It was also investigated that G4 ligand N-methyl mesoporphyrin IX (NMM) decreased P450 protein activity in larval midgut tissue. Cytotoxicity and bioassay results revealed that NMM and pesticides had synergistic effects on toxicity. In conclusion, our findings suggest that G4 motif could be a new potential target for pest control.

Homopurine guanine-rich sequences in complex with N-methyl mesoporphyrin IX form parallel G-quadruplex dimers and display a unique symmetry tetrad

DNA can fold into G-quadruplexes (GQs), non-canonical secondary structures formed by π-π stacking of G-tetrads. GQs are important in many biological processes, which makes them promising therapeutic targets. We identified a 42-nucleotide long, purine-only G-rich sequence from human genome, which contains eight G-stretches connected by A and AAAA loops. We divided this sequence into five unique segments, four guanine stretches each, named GA1-5. In order to investigate the role of adenines in GQ structure formation, we performed biophysical and X-ray crystallographic studies of GA1-5 and their complexes with a highly selective GQ ligand, N-methyl mesoporphyrin IX (NMM). Our data indicate that all variants form parallel GQs whose stability depends on the number of flexible AAAA loops. GA1-3 bind NMM with 1:1 stoichiometry. The Ka for GA1 and GA3 is modest, ∼0.3 μM −1, and that for GA2 is significantly higher, ∼1.2 μM −1. NMM stabilizes GA1-3 by 14.6, 13.1, and 7.0 °C, respectively, at 2 equivalents. We determined X-ray crystal structures of GA1-NMM (1.98 Å resolution) and GA3-NMM (2.01 Å). The structures confirm the parallel topology of GQs with all adenines forming loops and display NMM binding at the 3′ G-tetrad. Both complexes dimerize through the 5′ interface. We observe two novel structural features: 1) a ‘symmetry tetrad’ at the dimer interface, which is formed by two guanines from each GQ monomer and 2) a NMM dimer in GA1-NMM. Our structural work confirms great flexibility of adenines as structural elements in GQ formation and contributes greatly to our understanding of the structural diversity of GQs and their modes of interaction with small molecule ligands.

Three-fluorescence sensor for minute-time scale low-cost analysis of urinary oxalate in urolithiasis metabolic assessment

Since oxalate plays an important role in the metabolic assessment of urolithiasis, there is need for convenient and efficient methods for oxalate detection. Herein, we report a three-signal fluorescence strategy for oxalate analysis based on the ability of oxalate to reduce Cu2+ to Cu+, and the ability of pyrophosphate-cerium coordination polymeric networks (PPi-Ce CPNs), cadmium telluride quantum dots (CdTe QDs), and N-Methyl Mesoporphyrin (NMM) to selectively detect Cu2+ and Cu+. The detection range was 100 nM to 1 mM, the turnaround time was 6 min, while the limits of detections for PPi-Ce CPNs, QDs and NMM as reporters were 25 nM, 10 nM and 40 nM, respectively. Visual detection of oxalate relied on color change in the solution, which could be observed using the naked eye. The fluorescent system was used for oxalate analysis in 44 urine samples (32 calcium oxalate stone patients, 12 controls without urolithiasis), and the results were consistent with clinical diagnosis and imaging data. Moreover, the visual system was used to analyze 8 urine samples (4 patients and 4 controls), and showed good consistency with clinical diagnosis and computed tomography imaging results. These findings suggest that the method has potential application for the metabolic assessment of urolithiasis.

Split-DNAzyme cooperating primer exchange reaction for sensitive miRNA detection

Sensitive quantification of microRNA (miRNA) plays a crucial role in early diagnosis and precise therapy of osteosarcoma. Herein, we build a label-free and sensitive miRNA quantification approach based on the activation of split-DNAzyme initiated primer exchange reaction (PER). Target miRNA cooperates the activation of split-DNAzyme with Mg2+ through assisting the assembly of DNAzyme to correct conformation, which enables the performance of PER-based nucleic acids amplification to produce a large amount of single-strand DNA (ssDNA) sequences. The G-quadruplexes (G4) in ssDNA sequences products bind with N-methyl mesoporphyrin IX (NMM) to form G4-NMM complex with the enhanced fluorescence respond. The results demonstrate that miRNA-21 can assist the activation of split-DNAzyme, and the active DNAzyme exhibits a high specificity and efficiency in inducing the subsequent PER. Based on the split-DNAzyme-assisted signal recycle and PER, the method eventually shows a high sensitivity and selectivity, providing a promising prospect for the for early stage tumor diagnosis and more precise tumor therapy.

Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly.

Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag+ from the C-Ag+-C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag+, respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color- and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.

A fluorescence strategy for the rapid detection of miRNA-21 based on G-quadruplex and cyclic amplification signal

MicroRNAs (miRNAs) are commonly used as biomarkers for the diagnosis of tumors. Since miRNA expression is strongly correlated to carcinogenesis, the detection of miRNA concentration in cells would be valuable for the diagnosis and evaluation of tumors. In this study, we proposed a system using two strands of DNA, one modified by a phosphate group at the 5’ end, called Cap, and the other with a hairpin structure, called HP. The Cap chain could open the hairpin structure of HP and expose the sequences rich in G bases to form the G-quadruplex structure. Then, a strong fluorescence signal was emitted in the presence of N-methyl mesoporphyrin IX (NMM). However, with the addition of miRNA-21, Cap hybridized with it to form double chains, which were then cleaved by the digestion of lambda exonuclease, resulting in a weak fluorescent signal. The proposed method could detect miRNA-21 at a concentration of 1.4 pM with a broad dynamic linear range from 5 pM to 5 nM.

β-Galactosidase triggered dual-signal competitive selective recognition of copper ion for diagnosis of urinary tract infection by Escherichia coli

Here, we reported a simple dual-fluorescence homogeneous and binary visualization (color and distance reading) strategy for the analysis of β-galactosidase (β-Gal) and Escherichia coli (E. coli) in clinical urine samples. This system used CdTe quantum dots (QDs) and N-methyl mesoporphyrin IX (NMM) as signal reporters, combining with inkjet printing technology. This work is based on the fact that β-Gal can hydrolyze substrates to generate p-aminophenol (PAP), reducing Cu2+ to Cu+. Then, QDs and NMM can selectively recognize these two metal ions. Under optimized conditions, the limit of detections (LODs) of the dual fluorescence analysis strategy for E. coli were as low as 0.03 (QDs) colony-forming units (CFU)/mL and 0.04 (NMM) CFU/mL, and the sensitivity was improved by two orders of magnitude compared to the single-signal molecular system. 1 CFU/mL E. coli could be distinguished by reading the color and distance with the naked eye. This method was applied for the quantitative analysis of E. coli in 45 clinical urine samples, whose results were consistent with the results of clinical standard plate colony counting and the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).

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.