T4 Polynucleotide Kinase Activity Research Articles

An electrochemical biosensor for T4 polynucleotide kinase activity assay based on host-guest recognition between phosphate pillar[5]arene@MWCNTs and thionine.

T4 polynucleotide kinase helps with DNA recombination and repair. In this study, an electrochemical biosensor was developed for a T4 polynucleotide kinase activity assay and inhibitor screening based on phosphate pillar[5]arene and multi-walled carbon nanotube nanocomposites. The water-soluble pillar[5]arene was employed as the host to complex thionine guest molecules. The substrate DNA with a 5'-hydroxyl group initially self-assembled on the gold electrode surface through chemical adsorption of the thiol group, which was phosphorylated in the presence of T4 polynucleotide kinase. Titanium dioxide nanoparticles served as a bridge to link phosphorylated DNA and phosphate pillar[5]arene and multi-walled carbon nanotube composite due to strong phosphate-Ti4+-phosphate chemistry. Through supramolecular host-guest recognition, thionine molecules were able to penetrate the pillar[5]arene cavity, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 polynucleotide kinase concentration in the range of 10-5 to 15 U mL-1 with a detection limit of 5 × 10-6 U mL-1. It was also effective in measuring HeLa cell lysate-related T4 polynucleotide kinase activity and inhibitor screening. The proposed method offers a unique sensing platform for kinase activity measurement, holding great potential in nucleotide kinase-target drug development, clinical diagnostics, and inhibitor screening.

An electrochemical biosensor for T4 polynucleotide kinase activity identification according to host-guest recognition among phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite and toluidine blue.

T4 polynucleotide kinase (T4 PNK) helps with DNA recombination and repair. In this work, a phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite (PP5@PdNPs@rGO)-based electrochemical biosensor was created to identify T4 PNK activities. The PP5 used to complex toluidine blue (TB) guest molecules is water-soluble. With T4 PNK and ATP, the substrate DNA, which included a 5'-hydroxyl group, initially self-assembled over the gold electrode surface by chemical adsorption of the thiol units. Strong phosphate-Zr4+-phosphate chemistry allowed Zr4+ to act as a bridge between phosphorylated DNA and PP5@PdNPs@rGO. Through a supramolecular host-guest recognition connection, TB molecules were able to penetrate the PP5 cavity, where they produced a stronger electrochemical response. With a 5 × 10-7 U mL-1 detection limit, the electrochemical signal is linear in the 10-6 to 1 U mL-1 T4 PNK concentration range. It was also effective in measuring HeLa cell lysate-related PNK activities and screening PNK inhibitors. Nucleotide kinase-target drug development, clinical diagnostics, and screening for inhibitors all stand to benefit greatly from the suggested technology, which offers a unique sensing mechanism for kinase activity measurement.

An electrochemical biosensor for detection of T4 polynucleotide kinase activity based on host-guest recognition between phosphate pillar[5]arene and methylene blue

T4 polynucleotide kinase (T4 PNK) is an important DNA repair-related enzyme that plays a crucial role in DNA recombination, replication and damage repair. Herein, an electrochemical biosensor was developed for detection of T4 PNK activity and inhibitor screening based on supramolecular host–guest recognition between phosphate pillar (Dumitrache and McKinnon, 2017) [5] arene (PP5) and methylene blue (MB). The water-soluble PP5 employed as the host for complexation of MB guest molecules. The substrate DNA with 5′-hydroxyl group was first self-assembled on the gold electrode surface through the chemical adsorption of the thiol group, which was phosphorylated in the presence of T4 PNK and adenosine triphosphate (ATP). TiO2 served as a bridge to link phosphorylated DNA and PP5 via the robust phosphate-Ti4+-phosphate chemistry. The immobilized PP5 captured the MB on electrode surface via the supramolecular host–guest recognition interaction, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 PNK concentration in the range of 2 × 10−4 to 5 U mL−1 with a detection limit of 1 × 10−4 U mL−1. It was also successfully used for PNK inhibitor screening and PNK activity assay in HeLa cell lysates sample. The proposed strategy provides a novel sensing platform for kinase activity assay and inhibitor screening, holding a great potential in clinical diagnostics, inhibitor screening, and nucleotide kinase-target drug discovery.

Label-Free Detection of T4 Polynucleotide Kinase Activity and Inhibition via Malachite Green Aptamer Generated from Ligation-Triggered Transcription.

Polynucleotide kinase (PNK) is a key enzyme that is necessary for ligation-based DNA repair. The activity assay and inhibitor screening for PNK may contribute to the prediction and improvement of tumor treatment sensitivity, respectively. Herein, we developed a simple, low-background, and label-free method for both T4 PNK activity detection and inhibitor screening by combining a designed ligation-triggered T7 transcriptional amplification system and a crafty light-up malachite green aptamer. Moreover, this method successfully detected PNK activity in the complex biological matrix with satisfactory outcomes, indicating its great potential in clinical practice.

TtAgo sensor for the sensitive and rapid detection of T4 polynucleotide kinase activity

As a next-generation biosensing technology, the application of Argonaute for analytical science research has attracted considerable attention. A Thermus thermophilus Argonaute (TtAgo)-based sensor is presented for detecting T4 polynucleotide kinase (PNK) activity considering that TtAgo’s multiple-turnover catalytic activity can be activated by binding to a 5′-phosphate termini of single-stranded DNA (ssDNA) for the cleavage of complementary strand. PNK catalyzes the phosphorylation of 5′-hydroxyl termini of ssDNA, and the phosphorylation product termed guide can activate the TtAgo-mediated complementary ssDNA reporter cleavage to release fluorescence signal. For the PNK activity monitoring, the developed method shows good analytic performance with a linear range of 0.0010–2 U mL−1 and a detection limit of 0.0010 U mL−1 without the use of a preamplification reaction. This method is successfully applied for the analysis of HeLa cells at a single-cell level. The developed method shows several advantages, including high sensitivity, low cost, facile operation, and has remarkable potential application for early disease diagnosis, drug screening, and clinical treatment.

Versatile fluorescence detection of T4 PNK and mRNA based on unique DNA nanomachine amplification

The development of DNA nanomachines provides a new strategy for the detection of tumor markers. In this work, an intelligent three-dimensional (3D) DNA walking machine with polynucleotide kinase (PNK) activator was designed, which was coupled with unique nanomachine formed by DNA nanowire cascade amplification reaction for versatile fluorescence detection of T4 PNK activity and messenger RNA (mRNA). When PNK exists, the free DNA walker was formed by hydrolysis cleavage of exonuclease, then the fluorophore-labeled report probe on the Au nanoparticles (NPs) was sheared during cycling cleavage reaction, thus the fluorescence signal was recovered for detection of PNK. Moreover, the DNA nanowires were produced by rolling ring amplification, then target mRNA sequentially initiated interval hybridization of hairpin probes through DNA nanowire, thus realizing DNA cascade reaction (DCR) with high “on” signal of DNA nanomachine for mRNA assay. This developed novel fluorescence nanomachine reported a new assay method with promising application for versatile targets and showed great potential for molecular-target therapies, and clinic diagnostics.

Kanamycin triggered nanozyme for homogeneous and amplified colorimetric detection of T4 polynucleotide kinase

It is of significance to develop efficient methods for detecting the activity of T4 polynucleotide kinase (T4 PNK) due to its essential role in the modulation of different life activities. In this work, we constructed a novel nanozyme using Kanamycin (KANA) as a trigger for the [Fe(CN)6]3- coordinated Cu2(OH)3NO3 (Cu2(OH)3NO3/[Fe(CN)6]3-) nanorods, and designed an amplified colorimetric method to detect T4 PNK. That was, the free KANA efficiently triggered the peroxidase-like activity of Cu2(OH)3NO3/[Fe(CN)6]3-, while the bound KANA by its aptamer lost the stimulative capability for the nanomaterials. On the basis of the bioreaction regulated generation of the KANA aptamer, a highly sensitive colorimetric assay aided by the rolling circle amplification (RCA) reaction for the detection of T4 PNK was realized. Results showed that this assay can detect T4 PNK from 1.0 × 10−3 to 10.0 U/mL, with a limit of detection (LOD) of 1.42 × 10−4 U/mL. The assay also showed acceptable performance in the detection of T4 PNK in serum samples. In addition to the satisfactory sensitivity and selectivity, the displayed T4 PNK assay also presented merits of operational convenience, without labeling or immobilization process and did not require costly instrument. It is expected that the KANA as a stimulator would have extended biosensing applications by coupling various bioreactions that can produce the KANA aptamer.

A label-free fluorescent biosensor for amplified detection of T4 polynucleotide kinase activity based on rolling circle amplification and catalytic hairpin assembly

T4 polynucleotide kinase (PNK) plays a key role in maintaining genome integrity and repairing DNA damage. In this paper, we proposed a label-free fluorescent biosensor for amplified detection of T4 PNK activity based on rolling circle amplification (RCA) and catalytic hairpin assembly (CHA). Firstly, we designed a padlock probe with a 5′-hydroxyl terminus for phosphorylation reaction, a complementary sequence of the primer for initiating RCA, and a complementary sequence of the trigger for triggering CHA. T4 PNK catalyzed the phosphorylation reaction by adding a phosphate group to the 5′-hydroxyl terminus of padlock probe, generating a phosphorylated padlock probe. Then it hybridized with the primer to generate a circular probe under the action of ligase. Subsequently, the primer initiated an RCA reaction along the circular probe to synthesize a large molecular weight product with repetitive trigger sequences. The triggers then triggered the cyclic assembly reactions between hairpin probe 1 and hairpin probe 2 to generate a large amount of complexes with free G-rich sequences. The free G-rich sequences folded into G-quadruplex structures, and the N-methylmesoporphyrin IXs were inserted into them to produce an amplified fluorescent signal. Benefiting from high amplification efficiency of RCA and CHA, this fluorescent biosensor could detect T4 PNK as low as 6.63 × 10−4 U mL−1, and was successfully applied to detect its activity in HeLa cell lysates. Moreover, this fluorescent biosensor could effectively distinguish T4 PNK from other alternatives and evaluate the inhibitory effect of inhibitor, indicating that it had great potential in drug screening and disease treatment.

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

T4 polynucleotide kinase (T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle, the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope (TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4 PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.

Highly efficient incorporation of dATP in terminal transferase polymerization forming the ploy (A)n-DITO-1 fluorescent probe sensing terminal transferase and T4 polynucleotide kinase activity

Fluorescent dye DITO-1 has almost no fluorescence in the absence of nucleic acid. G bases in single strand DNA can induce maximum fluorescent enhancement followed by the A bases when it binds the DITO-1. However, the incorporation efficiency of the dATP was higher than dGTP in terminal transferase (TdT) polymerization. As a consequence, ploy (A)n, rather than ploy (G)n via TdT polymerization had the superior photoluminance when it binded DITO-1 fluorescent dye. Here, we developed a high selective and sensitive sensing strategy for assaying TdT and T4 polynucleotide kinase activity (T4 PNK) based on the ploy (A)n-DITO-1 fluorescent probe. An increasing amounts of TdT enzyme could promote the distinct incorporation of dATP on the DNA primer and form poly (A)n ssDNA with a difference in length. A good linear relationship between the ΔF and the concentrations of TdT in a range of 0.2–50 U/mL was obtained and the detection limit was 0.05 U/mL. Based on the experimental results for TdT, we further expanded the application of this method for detection of a series of concentrations of T4 PNK. The ΔF and the logarithm concentrations of T4 PNK in the range of 0.1–10 U/mL showed a good linear response and the detection limit of 0.02 U/mL was obtained. In addition, the detection of T4 PNK in Hela cell lysate was achieved, demonstrating that the proposed method had the potential application in complex system. The ploy (A)n-DITO-1 fluorescent probe had the excellent properties of one-step readout, robustness for target detection in complex system, and easiness operation, and showed the great potential in clinical diagnostics, inhibitor screening, and drug discovery.

Electrochemical detection of T4 polynucleotide kinase activity based on magnetic Fe3O4@TiO2 nanoparticles triggered by a rolling circle amplification strategy.

An ultrasensitive electrochemical detection of the activity and inhibition of T4 polynucleotide kinase (T4 PNK) was developed by using magnetic Fe3O4@TiO2 core-shell nanoparticles, which was triggered by a rolling circle amplification strategy (Fe3O4@TiO2-RCA). We used Fe3O4@TiO2 as a substrate to anchor a DNA primer. DNA S1 with 5'-OH termini was phosphorylated in the presence of T4 PNK and ATP, which was adsorbed on the surface of Fe3O4@TiO2 NPs and served as the primer for subsequent RCA reactions. After adding circular template DNA S2, RCA was initiated in the presence of phi29 DNA polymerase and dNTPs. Then, Fc-labeled DNA S3 (Fc-S3) was hybridized with RCA. The obtained Fe3O4@TiO2-RCA was adsorbed on the surface of a magnetic gold electrode (MGE) by magnetic enrichment, resulting in an enhanced electrochemical signal. The T4 PNK activity can be monitored by measuring the electrochemical signal generated. This electrochemical assay is sensitive to the activity of T4 PNK with a dynamic linear range of 0.00001-20 U/mL and a low detection limit of 3.0×10-6 U/mL. The proposed strategy can be used to screen the T4 PNK inhibitors, so it has great potential in the discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.

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.

A Sensitive Electrochemical Assay for T4 Polynucleotide Kinase Activity Based on Fe3O4@TiO2 And Gold Nanoparticles Hybrid Probe Modified Magnetic Electrode

An ultrasensitive electrochemical assay for T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on magnetic Fe3O4@TiO2 core–shell nanoparticles and gold nanoparticles hybrid probe (Fe3O4@TiO2-dsDNA-AuNPs) modified magnetic gold electrode (MGE). Fe3O4@TiO2-dsDNA-AuNPs hybrid probe was formed via DNA hybridization between phosphorylated DNA S1 modified Fe3O4@TiO2 and complementary DNA S2 assembled AuNPs. [Ru(NH3)6]3+ was used as an electrochemically active indicator to enhance the electrochemical signal. The proposed method is sensitive for T4 PNK activity assay with a linear range of 0.0001 to 10 U ml−1 and low detection limit of 0.00003 U ml−1. This strategy can be used to screen the T4 PNK inhibitors, holding a great potential for discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.

Hierarchical Metal-Organic Framework-Confined CsPbBr3 Quantum Dots and Aminated Carbon Dots: A New Self-Sustaining Suprastructure for Electrochemiluminescence Bioanalysis.

All-inorganic lead halide perovskites have become promising alternatives to traditional semiconductor electrochemiluminescence (ECL) emitters because of their appealing optoelectronic attributes, but major challenges remain in improving their stability and enhancing charge injection/transfer capacities. Herein, a self-sustaining suprastructure was constructed by successively loading aminated carbon dots (NCDs) and CsPbBr3 perovskite quantum dots (PeQDs) in situ into hierarchical zeolite imidazole framework-8 (HZIF-8). The elaborated architecture guarantees not only improved stability via the peripheral HZIF-8 protective barrier but also accelerated charge transport and efficient self-enhanced ECL between PeQDs and the surrounding NCDs in a confined structure. As a result, the ternary nanocomposite is endowed with greatly improved stability and ECL efficiency. Based on this ternary nanocomposite as an electrode substrate, a novel ECL sensing strategy is further proposed for the first time to evaluate T4 polynucleotide kinase activity and screen its inhibitors. This work opens an avenue for the advancement of perovskite-based ECL emitters as well as the development of corresponding applications in the ECL domain.

Electrochemical DNA Sensors Based on MoS2-AuNPs for Polynucleotide Kinase Activity and Inhibition Assay.

The determination of T4 polynucleotide kinase (PNK) activity and the screening of PNK inhibitors are critical to disease diagnosis and drug discovery. Numerous electrochemical strategies have been developed for the sensitive measurement of PNK activity and inhibition. However, they often suffer from additional labels and multiple steps of the detection process for the electrochemical readout. Herein, we have demonstrated an electrochemical DNA (E-DNA) sensor for the one-step detection of PNK with "signal-on" readout with no need for additional labels. In our design, the highly switchable double-stranded DNA (dsDNA) probes are immobilized on the gold nanoparticle-decorated molybdenum disulfide nanomaterial (MoS2-AuNPs), which possesses large surface area and high conductivity for elevating the signal gain in the PNK detection. This signal-on E-DNA sensor integrated with MoS2-AuNPs exhibits a much higher sensitivity than that without MoS2-AuNPs, showing a detection limit of 2.18 × 10-4 U/mL. Furthermore, this assay shows high selectivity, with the ability to discriminate PNK from other enzymes and proteins, and can be utilized to screen inhibitors. The proposed sensor is easy to operate with one-step readout and robust for PNK detection in the biological matrix and shows great potential for point-of-care in clinical diagnostics and drug screening.

Precise analysis of T4 polynucleotide kinase and inhibition by coupling personal glucose meter with split DNAzyme and ligation-triggered DNA walker

Monitoring the information of T4 polynucleotide kinase (T4 PNK) activity and its potential inhibitors is of great significance for the therapeutic protocols development, nucleic acid metabolism process investigating, and kinase-targeted drug discovery. In this study, we establish a split DNAzyme and ligation-triggered DNA walker for precise analysis of T4 PNK and inhibition by personal glucose meters (PGM). The DNAzyme is divided into two independent pieces, which can reduce the background signal. After being phosphorylated at 5′-hydroxyl termini by T4 PNK and hybridization with template DNA, a complete DNAzyme unit can be generated and released with the addition of DNA ligase and invasive DNA. It shows high catalytic cleavage toward the substrate strand of the sandwich structure. The releasing invertase of detection probe can catalyze the hydrolysis of sucrose into glucose, which is measured using a pocket PGM. Using this triple enzyme (ligase, DNAzyme, and invertase) assistance catalysis strategy, the T4 PNK detection limit is determined to be 10 mU mL−1. Additionally, the inhibition effects also have been satisfactorily investigated. Furthermore, the practicality of our method is tested toward target spiked in diluted human serum, which indicating that it is promising for biological process investigation and clinic diagnostics.

DNAzyme-driven DNA walker biosensor for amplified electrochemical detection of T4 polynucleotide kinase activity and inhibition

Abstract T4 polynucleotide kinase (T4 PNK), a bifunctional repair enzyme, catalyzes the transfer of γ-phosphate residues from adenosine triphosphate (ATP) to 5′- hydroxyl termini in nucleic acids，which plays an essential role in regulating DNA phosphorylation modes during the repair of damaged DNA. Herein, a sensitive DNAzyme-driven DNA walker biosensor was developed for amplified electrochemical detection of T4 PNK activity and inhibition based on T4 PNK-triggered phosphorylation reaction and λ exonuclease (λ exo) cleavage. A novel functional hairpin-shaped swing arm DNA (SA) containing a DNA sequence of the Pb2+-dependent DNAzyme is designed to specifically bind to and hydrolyze the substrate strand (rSS) sequence that can drive DNA walker. Upon the reaction of SA， T4 PNK and ATP, λ exo removes the mononucleotides from the stem of the phosphorylated SA, unfolding the hairpin structure and initiating DNAzyme-driven DNA walker. Subsequently, the DNA walker can repeatedly bind and cleave rSS with the aid of Pb2+, releasing the methylene blue (MB) - labeled fragment of rSS. The T4 PNK activity can be facilely monitored by recording the electrochemical signal change of MB. The proposed biosensor exhibited a broad linear range from 0.01 U mL−1 to 15 U mL−1 with a low detection limit of 0.001 U mL−1. The proposed biosensor has also been utilized to screen the T4 PNK inhibitors and exhibited good performance in real sample analysis, making such a strategy hold potential promise in biological fields and clinical diagnosis.

A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition.

A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, andX-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at themaximum emission wavelength of 524nm using the maximum excitation wavelength of 488nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0UmL-1 with a detection limit of 0.0089UmL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay forT4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

Label-free fluorescence assay coupled exonuclease reaction and SYBR Green I for the detection of T4 polynucleotide kinase activity

A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I.

Magnetic bead-gold nanoparticle hybrids probe based on optically countable gold nanoparticles with dark-field microscope for T4 polynucleotide kinase activity assay

T4 polynucleotide kinase (T4 PNK) plays an essential role in DNA phosphorylation during the DNA repair process. Therefore, the sensitive, selective and convenient detection of T4 PNK activity is of great significance. In this work, we proposed a sensitive non-amplification strategy for the sensing of T4 PNK activity via dark field microscope (DFM) based on magnetic bead (MB)-gold nanoparticle (AuNP) hybrids probe, MB-dsDNA-AuNP (MDA). In the presence of T4 PNK, the 5'-OH termini of DNA are phosphorylated and cleaved into oligonucleotides by lambda exonuclease (λexo), resulting in the destruction of the MDA probe and the separation of AuNP from the MB. Through automatic counting of AuNPs from DFM images, T4 PNK activity can be quantitatively measured. This strategy revealed a limit of detection (LOD) as low as 0.0058 U/mL and exhibited a dynamic range from 0.01 to 1 U/mL. The strategy presents an excellent ability to discriminate T4 PNK from the other proteins and enzymes. Notably, this strategy was applied to screen the T4 PNK inhibitors and test the activity in cell lysates, showing great potential for the discovery of new anticancer drugs and other related research field.