Exonuclease III-assisted Signal Amplification Research Articles

Colorimetric analysis of C-reactive protein via 'jellyfish' probe-based exonuclease III-assisted multiple-signal recycles.

C-reactive protein (CRP) is a potential biomarker for evaluating inflammatory responses in patients receiving coronary artery bypass graft surgery. Here, the authors depict a sensitive and reliable colorimetric approach for CRP analysis. In this method, an aptamer specifically binds with CRP and an initiator sequence is released from an arch probe to activate signal amplification. The released initiator sequence hybridizes with the toehold section in the 'jellyfish' probe to form a blunt terminus to induce exonuclease III-assisted signal amplification. The method exhibited a low limit of detection of 1.32 ng/ml and high intraday and interday precision for CRP detection. In summary, this colorimetric approach may provide a potential alternative tool for the evaluation of inflammation in patients receiving coronary artery bypass graft and clinical diagnostics of disease.

Facile and sensitive detection of mercury ions based on fluorescent structure-switching aptamer probe and exonuclease Ⅲ-assisted signal amplification

Hg2+ is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg2+-T mismatch structure and the signal amplification of exonuclease III (Exo III) assisted target cycle, a fluorescent probe for facile and sensitive detection of Hg2+ is constructed. The hairpin-looped DNA probe is rationally designed with 2-AP embedded in the stem and thymine-rich recognition overhangs extended at the termini. The cleavage of the double stranded DNA stem with stable T-Hg2+-T pairs catalyzed by Exo III is prompted to happen upon recognition of trace Hg2+. Under the optimal reaction conditions, there is an excellent linear relationship between Hg2+ concentration and fluorescence intensity in the range of 7.5–200 nM with a detection limit of 0.38 nM. In addition, the detection results of Hg2+ in Songhua River water and fish samples are satisfactory. The fluorescent probe avoids labeling additional quenchers or quenching materials and has strong anti-interference ability. Thus, the fluorescent probe has a broad prospect in practical application.

An electrochemical aptasensor based on exonuclease III-assisted signal amplification coupled with CRISPR-Cas12a for ochratoxin A detection

An electrochemical aptasensor based on exonuclease III-assisted signal amplification coupled with CRISPR-Cas12a for ochratoxin A detection

Sensitive and Portable Detection of Bacteria Using Exonuclease-III (Exo-III) Assisted Signal Amplification and Personal Glucose Meters.

Despite major advances in combating pathogenic bacteria caused infection, infectious diseases remain a major cause of death today. A variety of bacteria detection approaches have been established by exploiting the aptamer to specifically identify target. However, these methods require specific equipment or laboratory based instruments, which is expensive and not easily available to the public. To deal with this issue, we develop here a portable and sensitive pathogenic bacteria detection approach through integrating capture probe based recognition and Exo-III assisted signal amplification. In this method, a designed capture probe identifies the surface protein of target bacteria, leading to the release of blocker sequence. The released blocker sequence mediates Exo-III enzyme based signal amplification, generating a large amount of signal strand DNA (ssDNA) sequences. The obtained ssDNA works as a linker to conjugate streptavidin magnetic beads (SMBs) with DNA invertase. After magnet based enrichment and washing away of free DNA invertase, the SMBs-DNA invertase complex is used to catalyze the hydrolysis of sucrose into glucose for PGM readout. Based on this, the approach exhibits a 6 order of magnitudes in detecting Staphylococcus Aureus (S. aureus) with the limit of detection as low as 78cfu/mL. In addition, the capability to specifically detect target bacteria and high repeatability (relative standard deviation, 4.44%) endows the method wider applicable scenes. In all, the established shows a promising prospect in the detection of bacteria pathogen and the early-diagnosis of bacterial infections.

A fluorescent aptasensor based on nitrogen-doped carbon supported palladium and exonuclease III-assisted signal amplification for sensitive detection of AFB1

Aflatoxin B1 (AFB1) has strong carcinogenicity and toxicity, so it is necessary to develop a highly sensitive detection method. In this paper, a novel nitrogen-doped carbon supported palladium (C–N–Pd) material was synthesized and firstly used as the energy receptor for fluorescent aptasensor. Using C–N–Pd as a novel quenching material and exonuclease Ⅲ (Exo III) as assisted signal amplification, a fluorescent aptasensor for AFB1 detection was constructed. Compared with the sensor without enzyme, the fluorescence intensity obtained by the sensor with Exo III increased by 74.7%. The fabricated fluorescent aptasensor exhibited a good selectivity toward AFB1 with a limit of detection (LOD) as low as 9 pg mL−1. Moreover, the designed aptasensor was successfully utilized to detect AFB1 in spiked corn, peanut and wine samples, and the LOD is 15 pg mL−1, 13 pg mL−1 and 18 pg mL−1, respectively. In addition, the aptasensor was also compared with HPLC method, and the good agreement was found between them.

Superior performance of a graphdiyne self-powered biosensor with exonuclease III-assisted signal amplification for sensitive detection of microRNAs.

Graphdiyne (GDY) is an sp and sp2 co-hydrocarbon allotrope whose particular structure endows it with many fascinating properties, including abundant chemical bonds, high conjugation, natural pores, high carrier mobility, high conductivity and stability, etc. In this work, two-dimensional graphdiyne is prepared as an electrode substrate material coupling with an exonuclease III-assisted amplification strategy to construct a superior-performance self-powered biosensor based on enzymatic biofuel cells for highly sensitive detection of the tumour marker miRNA-21. Glucose oxidase (GOD) is first immobilized on the GDY/AuNP composite to prepare a bioconjugate. GDY/AuNP modified carbon cloth is used as an enzyme biofuel cell electrode, which is then modified with bilirubin oxidase as a biocathode. The bioconjugate binds to GOD through specific binding to the bioanode. When miRNA-21 is present, specific recognition by exonuclease III in the system results in cleavage of the capture probe, and miRNA-21 is recovered and involved in the cycle. The target miRNA-21 then causes corresponding changes in the open-circuit voltage of the self-powered system. Based on this, a sensitive detection method was constructed, within the scope from 0.1 fM to 0.1 nM with a shallow detection limit of 55.2 aM (S/N = 3). The new approach triumphantly has been used to detect miRNA-21 in serum, which provides a compelling new way for early diagnosis of related cancers.

A Novel Colorimetric Nano Aptasensor for Ultrasensitive Detection of Aflatoxin B1 Based on the Exonuclease III-Assisted Signal Amplification Approach.

The detection of aflatoxin B1 (AFB1) has recently garnered much attention on the issue of food safety. In this study, a novel and sensitive aptasensor towards AFB1 is proposed using an Exonuclease III (Exo III)-integrated signal amplification strategy. This reported sensing strategy is regulated by aptamer-functionalized nanobeads that can target AFB1; furthermore, complementary DNA (cDNA) strands can lock the immobilized aptamer strands, preventing the signal amplification function of Exo III in the absence of AFB1. The presence of AFB1 triggers the displacement of cDNA, which will then activate the Exo III-integrated signal amplification procedure, resulting in the generation of a guanine (G)-rich sequence to form a G-4/hemin DNAzyme, which can catalyze the substrate of ABTS to produce a green color. Using this method, a practical detection limit of 0.0032 ng/mL and a dynamic range of detection from 0.0032 to 50 ng/mL were obtained. Additionally, the practical application of the established sensing method for AFB1 in complex matrices was demonstrated through recovery experiments. The recovery rate and relative standard deviations (RSD) in three kinds of cereal samples ranged from 93.83% to 111.58%, and 0.82% to 7.20%, respectively, which were comparable with or better than previously reported methods.

Dual-color graphene quantum dots and carbon nanoparticles biosensing platform combined with Exonuclease III-assisted signal amplification for simultaneous detection of multiple DNA targets

Sensitive and simultaneous detection of multiple biomarkers such as target DNA or proteins using biocompatible materials with good analysis performance remains an important challenge. Herein, we successfully developed a signal “off-on” highly sensitive multiplex detection platform based on the combination of dual-color graphene quantum dots (blue GQDs and green GQDs) modified DNA probes with carbon nanoparticles (CNPs), which is a cheap, effective nonfluorescent quencher to simultaneously quench the fluorescence of both GQDs-DNA probes. The Exo III-assisted sequence-independent target recycling and signal amplification strategy was integrated into this sensing platform, which endows it with high sensitivity towards the multiplex detection of targets DNA. The detection limits of 6.6 pM for HIV and 9.5 pM for HBV were achieved respectively, which is about 60-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. Our proposed multiplex detecting platform is advantageous in both respective and simultaneous detection of multiple targets and can also discriminate perfectly matched targets from mismatched targets in both PBS buffer and 1% human serum samples, demonstrating its potential to be a reliable strategy for highly sensitive simultaneous detection of multiple target genes in practical diagnosis applications.

Mn2+-mediated magnetic relaxation switching for direct assay of ctDNA in whole blood via exonuclease III assisted amplification

Abstract Since circulating tumor DNA (ctDNA) has been regarded as a promising noninvasive biomarker for early cancer screening, cancer condition monitoring, and prognosis evaluation, the accurate and simple detection of ctDNA has been extensive explored. Up to now, there is still a lot of space for following-up the assay of ctDNA, especially simply direct analysis ctDNA in complex biological samples such as blood sample. In this work, we develop a Mn2+-mediated magnetic relaxation switching (MRS) assay for direct and simple detection of ctDNA via exonuclease III-assisted signal amplification. In the presence of target ctDNA, exonuclease III selectively cleaves the hybridization complex of between ctDNA and its complementary DNA labeled with magnetic beads and alkaline phosphatase (MB1000-DNA1-ALP) to release ALP and ctDNA, the former further generating Mn2+ and causing transverse relaxation time signal change while the later recycling to amplify the signal. The combination of efficient recognition of ctDNA, enzyme-assisted amplification, and the MRS assay could achieve direct and specific detection of ctDNA with a detection limit of 340 pM. In our opinion, this is the first successful direct detection of ctDNA in whole blood sample without ctDNA extraction from biospecimen, which make the assay for ctDNA much accurate and timely since a short half-life (2 h) of ctDNA in blood. The as-proposed method for ctDNA assay in this study shows potential tracking the evolution of tumor timely and serving as a liquid biopsy for diagnosis.

Sensitive and label-free chemiluminescence detection of malathion using exonuclease-assisted dual signal amplification and G-quadruplex/hemin DNAzyme

Malathion is one of the most commonly used organophosphorus pesticides that can cause serious harm to the ecological environment and human health. Herein, we demonstrated a label-free chemiluminescent aptasensor for the sensitive detection of malathion based on exonuclease-assisted dual signal amplification and G-quadruplex/hemin DNAzyme. Upon the addition of malathion, the aptamer probe specifically bound to the target to form a complex malathion-S3, leaving a duplex S1-S2. The complex malathion-S3 was digested by exonuclease I and the target was released. The released target was recycled to perform exonuclease I-assisted signal amplification. Furthermore, after treatment with exonuclease III, the duplex S1-S2 was converted into the secondary target ST. The secondary target ST interacted with the hairpin H1 to form a complex H1-ST, which was further digested by exonuclease III and released the secondary target. The released secondary target was recycled to perform exonuclease III-assisted signal amplification. After complete amplification, large numbers of G-quadruplex/hemin DNAzymes were generated. Under the optimal experimental conditions, the prepared aptasensor showed an excellent linear response to malathion with a detection limit of 0.47 pM. The relative standard deviations were in the range of 4.2–6.9%. Moreover, the aptasensor was successfully applied to detect malathion in spiked food and traditional Chinese medicine samples.

A gold nanoparticle-based colorimetric mercury(II) biosensor using a DNA probe with phosphorothioate RNA modification and exonuclease III-assisted signal amplification.

Herein, we report a rapid and sensitive colorimetric detection of Hg2+ by designing a specific DNA probe with phosphorothioate RNA modification (PS-probe) for Hg2+ recognition and utilizing DNA-modified gold nanoparticles (DNA-AuNPs) as the transducer. The distance between two DNA-AuNPs is controlled by a linker DNA, providing the linker DNA-regulated aggregation or dispersion status of AuNPs in solution. Exonuclease III (Exo III) can trigger the recycled digestion of linker DNA strands, inhibiting the reformation of aggregated nanoparticles and hence leading to a color shift from purple to red. However, the Hg2+-induced cleavage of thePS-probe can efficiently prevent the digestion of linker DNA strands by Exo III and hence reassemble the modified AuNPs to form aggregates in purple color. Thus, a positive correlation between the linker DNA strands left and the addition of Hg2+ provides a quantitative basis for Hg2+ sensing. A linear range of A520/A700 versus Hg2+ concentration is achieved in the range 2-100nM associated with a detection limit as low as 1.30± 0.04nM. Moreover, the biosensor exhibits excellent selectivity for Hg2+. The strong selectivity behavior was confirmed by recoveries ranging from 96 to 114% in real water samples. Graphical abstractSchematic representation of sensing mechanism of Hg2+ using a DNA probe with phosphorothioate RNA modification (PS-probe) and Exo III-assisted signal amplification.

One-step and ultrasensitive ATP detection by using positively charged nano-gold@graphene oxide as a versatile nanocomposite.

A versatile nanocomposite was simply prepared based upon the electrostatic adsorption of positively charged gold nanoparticles with negatively charged graphene oxide (nano-gold@GO), and utilized as a novel fluorescence quenching platform for ultrasensitive detection of adenosine triphosphate (ATP). In the designed system, DNA-stabilized Ag nanoclusters (DNA/AgNCs) were used as fluorescent probes, DNA duplex was formed in the presence of ATP, and they can electrostatically adsorb onto the surface of nano-gold@GO to quench the fluorescence signal. Upon the addition of exonuclease III (Exo III), the DNA duplex would be hydrolyzed into DNA fragments and resulted in the recovery of the fluorescence signals due to the diffusion of AgNCs away from nano-gold@GO. Based on these, sensitive detection of ATP was realized with a detection range of 5.0pM-20nM. Notably, a good recovery in the range of 94-104% was obtained when detecting ATP in human serum samples, indicating a promising application value in early disease diagnosis. Graphical abstract A functional positively charged nano-gold@graphene oxide was fabricated and utilized as an enhanced fluorescence quenching platform for the detection of ATP, coupled with exonuclease III-assisted signal amplification.

A highly sensitive and selective fluorescence biosensor for hepatitis C virus DNA detection based on δ-FeOOH and exonuclease III-assisted signal amplification

Developing the high selectivity and sensitivity strategy for nucleic acid detection is crucial for early diagnosis and therapy of diseases. In this work, a novel low back-ground fluorescent sensor platform for the detection of nucleic acid has been developed based on δ-FeOOH nanosheets integrating with exonuclease III-assisted target-recycling signal amplification. Because of the strong binding ability between the single-strand DNA (ssDNA) and the δ-FeOOH nanosheets, the dye-labeled ssDNA probe would be quenched by δ-FeOOH nanosheets through fluorescence resonance energy transfer (FRET). By using magnetic separate properties of δ-FeOOH, the background signal was separated from the sensor system, and the low background sensor system was obtained. After adding the target DNA, a double-strand DNA complex (dsDNA) would be formed between the target DNA and dye-labeled ssDNA probe. Then, the dye-labeled ssDNA probe in the dsDNA complex would be stepwise hydrolyzed into short fragments from 3′-terminus by Exonuclease III, and the fluorescence signal was recovered due to the weak bind affinity between the short fragments and δ-FeOOH nanosheets. By using the fluorescence quenching ability of δ-FeOOH nanosheets and enzyme-assisted target-recycling signal amplification, this strategy could show an excellent selectivity toward hepatitis C virus DNA with a low detection limit of 10 pM. By simply changing the dye-labeled ssDNA probe sequence, this sensing platform can be developed as a universal approach for the simple, sensitive, and selective detection of different target DNA.

Multiple self-cleaning paper-based electrochemical ratiometric biosensor based on the inner reference probe and exonuclease III-assisted signal amplification strategy

Rapid and accurate detection of nucleic acids plays a major role in biological research and clinical diagnostics. Here, a 3D multiple self-cleaning electrochemical ratiometric microfluidic paper-based analytical device (SER-μPAD) has been constructed for manganese super oxide dismutase (MnSOD) gene detection on the basis of the inner reference probe and exonuclease Ш (Exo Ш)-assisted analytes recycling amplification method. To simplify manual operations, a multipath self-cleaning tab that could manipulate fluid transport was introduced into the paper-based device, realizing time-programmable self-cleaning of the electrode. For achieving sensitive detection of MnSOD gene, the methylene blue (MB)-modified capture probe (CP) as the inner reference element was first self-assembled on triangular Au nanosheets modified paper working electrode to provide a built-in correction and improve the detection accuracy. When MnSOD gene existed, it hybridized with the hairpin-structured signal probe, triggering the cyclic amplification with the assistance of Exo Ш selective digestion to engender numerous residual DNA labeled with ferrocene (Fc) that could be captured on electrode surface by CPs. Hence, the Fc tags were close to the electrode surface, resulting in the oxidation peak current of Fc (IFc) increase, while that of MB (IMB) was constant on account of the unchanged distance between the MB tags and the electrode. The value of IFc/IMB was linear with MnSOD gene concentration from 10 nM to 1200 nM, and the detection limit was 3.91 nM. This strategy provides an accurate, robust, and sensitive method for nucleic acids detection and shows great potential in the construction of portable devices.

Fluorometric determination of mercury(II) via a graphene oxide-based assay using exonuclease III-assisted signal amplification and thymidine-Hg(II)-thymidine interaction.

A highly sensitive and selective fluorometric method is described for determination of mercury(II). It is based on (a) the use of graphene oxide (GO) acting as a quencher of the fluoresence of the carboxy-fluorescein (FAM), and (b) of Hg(II)-triggered cleavage of the newly formed nucleic acid sequences harbored blunt 3'-hydroxyl termini by exonuclease III (Exo III) that leads to signal amplification. Two DNA probes are used, viz. a capture probe (CP) and a help probe; HP) that is partially complementary. In the absence of Hg(II), the FAM-labeled hairpin (signal probe, SP) is adsorbed onto the surface of GO via π-stacking interactions. CP blocks the release of the HP for binding to SP. This results in quenching of the green fluorescence of the label. Upon addition of Hg(II), the linear structure of CP is converted to a hairpin structure due to the formation of thymidine-Hg(II)-thymidine duplexes. HP is released from the CP/HP hybrids, and this causes SP to be released from from GO and fluorescence to be recovered. The signal is strongly amplified by using Exo III-assisted targeting and recycling of HP. Hence, Hg(II) can be detected via the strong increase in fluorescence. The method has a linear response in the 0.1 to 30nM Hg(II) concentration range and a 10 pM detection limit. It was applied to the determination of Hg(II) in three (spiked) Chinese medicines. Graphical abstract Schematic representation of fluorescence sensing strategy for Hg2+ by using graphene oxide as a quencher and exonuclease III-assisted signal amplification.

Turn-On Fluorescence Aptasensor on Magnetic Nanobeads for Aflatoxin M1 Detection Based on an Exonuclease III-Assisted Signal Amplification Strategy

In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg−1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.

Exonuclease III-assisted signal amplification strategy for sensitive fluorescence detection of polynucleotide kinase based on poly(thymine)-templated copper nanoparticles.

A sensitive and label-free fluorometric method has been developed for the determination of polynucleotide kinase (PNK) activity, by employing exonuclease III (Exo III)-assisted cyclic signal amplification and poly(thymine)-templated copper nanoparticles (polyT-CuNPs). In the presence of PNK, cDNA with 5'-hydroxyl termini was phosphorylated and then hybridized with tDNA to form the cDNA/tDNA duplex, which subsequently triggered the λ exonuclease cleavage reaction, eventually resulting in the release of tDNA. The released tDNA could unfold the hairpin structure of HP DNA to generate partially complementary duplex (tDNA/HP DNA), wherein the HP DNA possessed T-rich sequences (T30) and tDNA recognition sequence. With the help of Exo III digestion, the tDNA was able to initiate the cycle for the generation of T-rich sequences, the template for the formation of fluorescent CuNPs. Conversely, the cDNA could not be cleaved by λ exonuclease without PNK and individual HP DNA could not be hydrolyzed by Exo III. The T-rich sequence was caged in HP DNA, resulting in a weak fluorescence signal. Under optimized conditions, the fluorescence intensity was linearly correlated to a concentration range of 0.001 to 1 U mL-1 with a low detection limit of 2 × 10-4 U mL-1. Considering the intriguing analytical performance, this approach could be explored to screen T4 PNK inhibitors and hold promising applications in drug discovery and disease therapy.

Ultrasensitive Biosensor for Detection of Mercury(II) Ions Based on DNA-Cu Nanoclusters and Exonuclease III-assisted Signal Amplification.

This paper describes a novel method for label-free mercury(II) ion detection based on exonuclease III-induced target signal recycling amplification using double-stranded DNA templated copper nanoclusters. The synthesized DNA-Cu nanoclusters were used with exonuclease III loop amplification technology for ultra-high sensitivity detection of mercury(II) ions, which were detected by significantly decreased fluorescence intensity. Under the optimal experimental conditions, there was a clear linear relationship between Hg2+ concentration in the range of 0.04 to 8 nM and fluorescence intensity. The detection limit for Hg2+ was 4 pM. In addition, the interference of other metal ions on the mercury(II) ion detection was also studied. To confirm the application of the fluorescent sensor, it was applied to determine the concentrations of mercury(II) ions in tap water, and the results showed that the method can be used to detect mercury(II) ions in water samples successfully.

Polydopamine nanotube mediated fluorescent biosensor for Hg(ii) determination through exonuclease III-assisted signal amplification.

We describe a highly sensitive fluorescence biosensor incorporating polydopamine nanotubes (PDNTs) based on the mechanism of exonuclease III (Exo III) assisted signal amplification for the determination of Hg2+ in aqueous solution. Fluorescent probes of FAM labeled ssDNA (FAM-ssDNA) adsorbed on the PDNTs act as an efficient quencher. In the presence of Hg2+, the FAM-ssDNA can bind to Hg2+ to form double stranded DNA (dsDNA) via the formation of T-Hg2+-T base pairs. Then, the dsDNA was removed from the surface of the PDNTs to restore the fluorescence. The release of the dsDNA was triggered by Exo III digestion. At the same time, the liberated Hg2+ mediates a new cycle of digestion. This assay is ultrasensitive for the selective recognition of Hg2+, and a detection limit as low as 10 pM was achieved. In addition, the fluorescent biosensing system also displays remarkable specificity to Hg2+ in the presence of other possible competing ions. This approach was applied to the determination of Hg2+ in real water samples with good recovery and high efficiency for practical analysis.

Sensitive and Label-Free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-Assisted Signal Amplification.

Transcription factors (TFs) regulate gene expression by binding to regulatory regions, and their dysregulation is involved in numerous diseases. Thus, they are therapeutic targets and potential diagnostic markers. However, widely used methods for TFs detection are either cumbersome or costly. Herein, we first applied DNA-Ag nanoclusters molecular beacons (AgMBs) in TFs analysis and designed an assay based on the switchable fluorescence of AgMBs. In the absence of TFs, a single-stranded DNA functioned as a reporter is released from a double-stranded DNA probe (referred as dsTFs probe) under exonuclease III (Exo III) digestion. Then, the reporter triggers downstream Exo III-assisted signal amplification by continuously consuming the guanine-rich enhancer sequences in AgMBs, resulting in significant fluorescent decrease eventually. Conversely, the presence of TFs protects the dsTFs probe from digestion and blocks the downstream reaction to keep a highly fluorescent state. To testify this rationale, we utilized nuclear factor-kappa B p50 (NF-κB p50) as a model TFs. Owing to the amplification strategy, this method exhibited high sensitivity toward NF-κB p50 with a limit of detection of 10 pM, and a broad linear range from 30 pM to 1.5 nM. Furthermore, this method could detect multiple TFs in human colon cancer DLD-1 cells and reflect the variation in their cellular levels after stimulation. Finally, by conducting an inhibition assay we revealed the potential of this method for screening TFs-targeted drugs and calculating the IC50 of corresponding inhibitors.