Ultrasensitive Fluorescence Research Articles

Ultrasensitive inner filter effect fluorescence sensing platform for alkaline phosphatase based on arginine surface-engineered gold nanoclusters

Developing a simple yet powerful method for enzyme activity monitoring is of great significance in the early diagnosis of diseases. Herein, we designed a precise spectral overlap-triggered inner filter effect (IFE) fluorescence sensing platform for alkaline phosphatase (ALP) detection. ALP could catalyze hydrolysis of p-nitrophenylphosphate (PNPP) to generate p-nitrophenol (PNP) and PNP acts as a powerful acceptor in IFE reaction system to influence the excitation of fluorescent donors. Based on this principle, a standardized procedure was proposed for rational screening of high-performance donor/acceptor pairs to maximize IFE efficiency. The results proved that perfect spectral overlap and high quantum yield of fluorescent donors synergistically contribute to maximizing IFE efficiency. In this case, a high quantum yield of 51.7% of the arginine surface-engineered gold nanoclusters (Arg/ATT-AuNCs) produced perfect spectral overlap with PNP and achieved maximal IFE efficiency, which was eventually selected as fluorescent signal tag for ALP detection. The Arg/ATT-AuNCs-based approach achieved ultrasensitive detection of ALP with a detection limit of 0.0062 U/L, which exhibited 4.2–25.8-fold improvement compared to other donors. Furthermore, the accuracy and reliability of established method in real human serum samples were verified by spiking analysis and commercial kits, thus providing a promising platform for monitoring ALP. This work aims to provide a standardized procedure for rational screening high-performance donor/acceptor pairs and to develop an ultrasensitive IFE sensing platform for biomarkers detection, which will open up a promising horizon for other biomarkers detection in clinical diagnostics.

Ultrasensitive turn-off fluorescence detection of iodide using carbon dots/gold nanocluster as fluorescent nanoprobe

In this work, a novel fluorescence turn-off sensing strategy for I− ions detection was developed. The fluorescence of CDs/His-AuNCs hybrid probes was effectively turned off by I− ions. It was attributed to the internal filtration effect between the fluorescence emission peak of CDs and the absorption spectrum of His-AuNCs enhanced by I− ions. Quantitative analysis of I− ions can be achieved by analyzing the fluorescence quenching degree of CDs/His-AuNCs. Under the optimal experimental conditions, it exhibited good linearity in two concentration ranges of I− ions: 0.1–5.0 μM and 5.0–60.0 μM (low detection limit = 34 nM). The established sensing platform has been successfully exploited for the detection of I− ions in practical water samples. This work provided new insight into the determination of anions using fluorescent nanoparticles.

Fluorescence Signal Amplification: Red Carbon Dots@SiO2-Induced Ultra-sensitive Immunoassay for Diethyl Phthalate.

Carbon dots as new nanomaterials, have been widely used in rapid detection because of their nondestructive, real-time detection characteristics. Improving the sensitivity and selectivity of the method in complex real samples is new challenge and requirement for sensing technology. Here, we report an ultrasensitive fluorescence immunoassay (FIA) for trace diethyl phthalate (DEP) using red carbon dots@SiO2 (R-CDs@SiO2) as tags. SiO2 as a nanocarrier can effectively improve the bio-functionalization and utilization rate of carbon dots. Moreover, several R-CDs embedded in SiO2 nanospheres can magnify the fluorescence signal and improve sensitivity. R-CDs@SiO2 conjugate anti-DEP antibody (Ab) as fluorescent immunosensor, which can specifically recognize DEP. Under optimization conditions, the detection limit (LOD) of this FIA was calculated as 0.0011 ng/mL. In addition, the recoveries of this established FIA ranged from 96.8 to 108.5%, showing satisfactory accuracy. Compared with GC-MS/MS (LOD µg/mL), the sensitivity of the FIA was significantly improved. As a result, the FIA developed using R-CDs@SiO2 as tags has a high potential for determining trace DEP.

Bright orange-emissive carbon quantum dots as an ultrasensitive nanoprobe for new coccine determination in food samples

In this study, an ultrasensitive fluorescence method based on bright orange-emissive carbon quantum dots (OE-CQDs) was for the first time developed for the detection of new coccine (NC). The OE-CQDs were synthesised by a one-pot solvothermal method with saffron T as precursor. The detection mechanism was based on the interaction between OE-CQDs and NC, where the fluorescence of OE-CQDs was inhibited by NC via inner filter effect (IFE) and static interaction. The OE-CQDs probe exhibited high selectivity and good anti-interference ability towards NC sensing. A linear relationship with the concentration of NC in a range of 0.01–20.0 µmol L−1 and a detection limit as low as 1.8 nmol L−1 were obtained. The developed fluorescence method was successfully applied for NC determination in commercial food samples, including dried waxberry, haw flakes, watermelon frost lozenges, strawberry jam and energy drink, with satisfactory results were achieved. The recoveries were in a range of 97.0–104.0% and the relative standard deviations (RSDs) were less than 3.74%. This work highlights the great potential of using long-wavelength emissive CQDs as nanoprobe to achieve enhanced sensitivity in food additives sensing.

Ultrasensitive Fluorescence Detection and Imaging of MicroRNA in Cells Based on a Hyperbranched RCA-Assisted Multiposition SDR Signal Amplification Strategy.

Herein, an innovative fluorescent sensor was courageously empoldered for precise and ultrasensitive detection and imaging of target miRNA-21 through the agency of a dextrous target-motivated polymerization/nicking DNA nanomachineries based on a hyperbranched rolling circle amplification (HB-RCA)-assisted multiposition strand displacement reaction (SDR) signal amplification approach. Impressively, the ingenious technique not only realized target recycling via polymerization/nicking DNA nanomachineries but also involved HB-RCA amplification induced by the released transformation target as the repeated signal amplification. Most importantly, HB-RCA was firstly exploited to remarkably increase the local concentration and collision efficiency of the templates and primers, which could simultaneously generate multiple repeated DNA sequences as initiators to supply substantial banding positions for SDR, removing the massive fluorescence-resonance-energy-transfer (FRET) DNA duplexes from the repeated DNA sequences to remarkably avert the self-quenching of the fluorescence signal due to self-aggregation caused by the winding of the HB-RCA products, thereby leading to a conspicuously improved signal amplification multiplier. As proof of concept, an ingenious technique effectively and accurately distinguished target miRNA-21 even with a tiny change in cells compared to the conventional fluorescence in situ hybridization (FISH) approach. Moreover, the proposed fluorescent method apparently discriminated drug-manipulative miRNA expression level abnormities. Therefore, the proposed cascade nucleic acid amplification strategy could provide an epigamic avenue for ultrasensitive imaging of diverse biomarkers, which help researchers to better study the tumor mechanism, thereby unambiguously increasing cancer cure rates and reducing the risk of recurrence.

A specific sensor system based on in-situ synthesis fluorescent polymers by ARGET ATRP achieving sensitive exosome detection

Exosomes, as a kind of tumor biomarker, are often found at elevated levels above normal in the blood of cancer patients, making highly sensitive exosome testing important for monitoring the development and progression of cancer. In this work, an ultra-sensitive and specific fluorescence method for exosome assays was constructed on the basis of atom transfer radical polymerization (ATRP) technique. Firstly, two recognition units, the CD63 aptamer and the EGFR antibody, can specifically bind to exosomes to form a sandwich structure, which endows the proposed sensor with excellent selectivity through two specific recognitions. Secondly, activator regenerated by electron transfer ATRP (ARGET ATRP) polymerization amplification method can introduce a huge amount of the fluorescent monomers into the sensing system, leading to a marked rise in sensitivity. Under optimal experimental conditions, a clear linear relationship between the logarithm of the fluorescence intensity and the concentration of exosomes in the range of 5 × 104 exosomes/mL to 5 × 109 exosomes/mL was obtained. The limit of detection (LOD) was as low as 11,610 exosomes/mL. Furthermore, this sensor exhibited good selectivity and stability, and excellent immunity to interference, providing a new method for the detection of exosomes. The exosome detection strategy used in this system provides a new idea for effective detection of lung cancer at an early stage.

A potential forecast of carbon quantum dots (CQDs) as an ultrasensitive and selective fluorescence probe for Hg (II) ions sensing

Carbon quantum dots (CQDs) are remarkable nanomaterials owing to their auspicious aspects. Herein, we synthesized highly green fluorescent CQDs via a one-step facile hydrothermal method using a chicken feather as a carbon source. The synthesized CQDs have an average particle size of ∼ 5 ± 1 nm in diameter with a quantum yield of ∼ 77.2 %. As-prepared CQDs are directly used as a fluorescent probe for the sensitive and selective detection of Hg2+ ions with a detection limit of ∼ 6.2 nM. Interestingly, the above results suggested that the synthesized CQDs as a potential candidate for Hg2+ sensing application.

Facile synthesis of dual-labeling carbon dots for simultaneous visualization of lipid droplets and lysosomes

Single fluorescent probes (SFPs) that enable simultaneous and discriminative visualization of multiple organelles are highly desirable due to their great potential in deciphering spatial distributions and interplays among these organelles. However, carbon dots (CDs)-based SFPs possessing dual-labeling ability are extremely deficient and challenging. Herein, we report the first example of CDs-based lipid droplets (LDs)/lysosomes dual-targeted SFPs, LD-Ly-CDs, through rational design of covering lipophilic alkyl group (CH3) and hydrophilic amino residue (-NH2) on the surface. LD-Ly-CDs allow to discriminate LDs and lysosomes via the ultrasensitive fluorescence response towards their distinct polarity. With the probe LD-Ly-CDs, the distribution, size morphology and polarity of LDs and lysosomes in different cells are revealed. By the excellent dynamic recognition properties of LD-Ly-CDs, two fast and unpredictable LDs coalescence modes, adjacent LDs fusion and migration-mediated LDs coalescence, are discovered. Moreover, the dynamic imaging also uncovers a new model of LDs and lysosomes dynamic as well as an impressive lipolysis process during starvation model. The remarkable performances of LD-Ly-CDs not only provide a powerful tool to elucidate LDs-lysosomes related interactions, but also shed light on the development of dual-labeling carbon-based fluorescent nanomaterials.

Photoisomerization and ultrasensitive fluorescence detection of toxic deoxyguanosine adduct ANdG: a theoretical study

The toxic deoxyguanosine adduct (ANdG) is one of the most representative carcinogenic DNA adducts, resulting from human exposure to aromatic amine (AA). Accordingly, the ultrasensitive detection of AA-derived adduct in DNA with minimal disturbance to the native structures is key to elucidating carcinogenesis mechanisms and mitigating cancer risk. Considering the lack of adequate intrinsic optical emission in two obtained rotamers (ANG1 and ANG2), a set of quasi-intrinsic fluorescent probes based on the complementary cytosine are proposed to identify covalent G-adduct. It is found that the expanded C-analogues in solution could bring red-shifted absorption maxima and enhanced photoluminescence due to the additional π-conjugation. In particular, because PhpC possesses large Stokes shift (98 nm) and the highest fluorescence intensity (f flu. = 0.546) in emission, it is proposed as the biosensor to monitor the optical changes in the presence and absence of the considered ANG. Compared with native C base, the absorption maximum of PhpC is red-shifted by 145 nm, which contributes to the selective excitation after incorporating into the nucleic acids. More importantly, the fluorescence is insensitive to base pairing with natural guanine, while the efficient fluorescence quenching is observed after pairing with ANG1/ANG2 as a result of the obvious excited state intermolecular charge transfer. To evaluate the direct application of the bright C-analogues with a high selectivity for deoxyguanosine adduct ANdG in DNA, we further examined the effect of linking deoxyribose on absorption and fluorescence emission, which are consistent with the experimental data.

Simultaneous Determination of Antibiotics, Mycotoxins, and Hormones in Milk by an 8-17 DNAzyme-Based Enzyme-Linked Immunosorbent Assay.

The simultaneous detection of three kinds of small-molecule contaminants (antibiotics, mycotoxins, and hormones) in milk was realized by using an 8-17 DNAzyme-based fluorescent enzyme-linked immunosorbent assay (ELISA), in which 8-17 DNAzyme was utilized as the catalytic enzyme for amplifying the signal. Compared with the conventional ELISA in which horseradish peroxidase is used as the catalyzing factor, this 8-17 DNAzyme-based ELISA could achieve multicolor signal output with lower detection limits. The linearities for chloramphenicol, 17β-estradiol, and aflatoxin M1 were in the range of 0.3 ng/mL-3 μg/mL, 3 ng/mL-3 μg/mL, and 3 pg/mL-3 ng/mL with quantitation limits of 0.3, 3, and 0.003 ng/mL, respectively. This proposed scheme demonstrated that the 8-17 DNAzyme might be an effective substitute for horseradish peroxidase in ELISA for the development of ultrasensitive and multicolor fluorescence immunoassay, which would stimulate the development of ELISA in a new orientation.

Significant quantum yield enhancement for near infrared fluorescence dyes by silica templated silver nanorods

Silver's inherent optical properties of low absorption cross section, over a broad wavelength range, makes it an attractive material for use in biosensing. We report herein a unique silica template method to synthesize cetrimonium bromide free silver nanorods (AgNRs). The application of AgNRs in metal enhanced fluorescence (MEF) was explored by immobilizing visible/near-infrared (NIR) fluorescent dyes on AgNR assembled substrates exhibiting broadband optical properties. Theoretical modeling was performed to analyze the nanoscale “chaotic nature” of the AgNR substrates, showing that the plasmon band shifts and broadening were attributed to the resonance coupling between assembled nanorods with different conformations. The quantum yield (QY) of NIR dye Alex Fluor 790 was increased dramatically from 4% to 84%, with total fluorescence enhancement up to two orders of magnitudes. In addition, AgNR substrates can also amplify fluorescence emissions from high quantum yield dyes, nearly 10-fold for Alexa Fluor 647 (QY:33%) and threefold for Alexa Fluor 488 (QY:92%). The AgNR demonstrates the capability of dramatically enhancing NIR dye's QY, holding great potential for building up an ultra-sensitive fluorescence bioassay platform for early diagnosis of diseases.

CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly for ultrasensitive fluorescence detection of HIV-1 DNA

Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA. After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment, direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.

Ultrasensitive near-infrared fluorescence probe activated by nitroreductase for in vivo hypoxia detection

The fluorescence probe has attracted much attention in the detection of hypoxia-related pathological conditions with unique advantage. However, in vivo imaging the early stages of diseases formation and development remains a great challenge, mainly due to the lack of highly sensitive and specific fluorescence probe. Herein, a high-efficiency near-infrared fluorescence probe YQ-NO2 was synthesized to detect hypoxia via responding to nitroreductase (NTR) in living subjects. In vitro confocal-mediated competitive binding inhibition and flow cytometry analyses indicated YQ-NO2 was of high specificity on hypoxic cells. In vivo near-infrared imaging showed that the probe had the laudable characteristics of a low background inference, fast response and prolonged imaging time in the tumor-bearing mice. Furthermore, it had the property of high affinity and potent permeability, allowing for continuously monitoring cell hypoxia to evaluate longitudinal and dynamic change in the tumor area. Meanwhile, ischemic liver and enteritis sites could be accurately and timely imaged in mice. In addition, it was observed that the fluorescence intensity of the probe in tumor was significantly higher than that in inflammation. The probe has failed to exhibit any bio-toxicity in cells and animals treated with high doses. Ultimately, this near-infrared fluorescence probe will offer an imaging tool for characterizing the hypoxia microenvironment at the tiny lesions in vivo.

A highly sensitive NIR fluorescence probe for hypoxia imaging in cells and ulcerative colitis

Ulcerative colitis, a kind of inflammatory bowel disease (IBD), is caused by dysregulated immune response of intestinal bacteria. This chronic disorder can lead to a deficiency of O2 (hypoxia) in the colon microenvironment. Nitroreductase (NTR) is a highly expressed endogenous enzyme under hypoxia, so the detection of NTR can provide diagnostic information about ulcerative colitis. Herein, an ultrasensitive NTR-triggered fluorescence probe (WS-1-NO2) is developed for hypoxia imaging in ulcerative colitis. The probe shows a significant fluorescence enhancement (45-fold) after reacting with NTR, with an extremely low detection limit of 0.096 ng/mL. Furthermore, we apply it for fluorescence imaging of hypoxia in living cells, tumors and dextran sulphate sodium (DSS)-induced ulcerative colitis mouse models. We believe that the probe may be investigated as an effective potential tool for gaining insight into the hypoxia-relevant diseases, such as cancer and ulcerative colitis.

Ultra-sensitive and selective fluorescence approach for estimation of elagolix in real human plasma and content uniformity using boron-doped carbon quantum dots

Elagolix (ELX) is an orally administered non-peptidic GnRH antagonist that has been approved by the Food and Drug Administration in 2018 for the treatment of endometriosis pain. A sensitive and selective method for estimating elagolix (ELX) in human plasma and content uniformity was developed and validated. The spectrofluorimetric technique was used to investigate ELX utilizing boron-doped carbon quantum dots (B@CQDs). After gradually adding ELX, the quantum dots fluorescence was enhanced with LOQ of 1.74 ng mL−1, the calibration curve between ELX and corresponding fluorescence intensity was found over a range of 4–100 ng mL−1. The method was successfully applied in real human plasma with pharmacokinetic study and content uniformity test. The pharmacokinetic parameters as Cmax were found to be 570 ± 5.32 ng. mL−1 after 1 h, t1/2 was found to be 6.50 h, and AUC was found to be 1290 ± 30.33 ng. h. mL−1. B@CQDs were characterized using variety of instruments. The strategy is simple to implement in clinical labs and therapeutic drug monitoring systems.

Designing of a high-performance fluorescent small molecule enables dual-mode and ultra-sensitive fluorescence visualizing of HSO3− and HClO in dried fruit, beverage, and water samples

Herein, a novel hemicyanine derivative (E)-3-(1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate (BIS) has been reasonably designed. Compound BIS is long-wavelength emissive and water-soluble with a large Stokes shift. Intriguingly, probe BIS provides a dual-mode fluorescence response pattern for the sensing of bisulfite (HSO3−) and hypochlorous acid (HClO) with great limit of detections (3.6 and 57.4 nM). First, the 1,4-Michael addition of HSO3− on the conjugated double bond triggers a ratiometric response (I465/I575). Second, the rapid oxidation of HClO on the thioether moiety provides a turn-on response (I575). Evaluation of HSO3− and HClO levels in dried fruit, beverage, and water samples has been carried out with satisfactory results. Moreover, motivated by an impressive chromatic variation (red to blue), smartphone-assisted signal readout system and thin-film sensing platform are facilely constructed for real-time and on-site measurement of HSO3− levels. Furthermore, probe BIS is used for the in vivo imaging of HSO3− in edible fish models.

Ultra-Sensitive, Selective and Repeatable Fluorescence Sensor for Methanol Based on a Highly Emissive 0D Hybrid Lead-Free Perovskite.

A convenient and rapid detection method for methanol in ethanol remains a major challenge due to their indistinguishable physical properties. Herein, a novel fluorescence probe based on perovskite was successfully designed to overcome this bottleneck. We report a new zero-dimensional (0D) hybrid perovskite of [MP]2 Inx Sb1-x Cl7 ⋅ 6 H2 O (MP=2-methylpiperazine) displaying an unusual green light emission with near-unity photoluminescence quantum yield. Remarkably, this 0D perovskite exhibits reversible methanol-response luminescence switching between green and yellow color but fail in any other organic vapors. Even for blended alcohol solutions, the luminescent probe exhibits excellent sensing performance with multiple superiorities of rapid response time (30 s) and ultra-low detection limit (40 ppm), etc. Therefore, this 0D perovskite can be utilized as a perfect fluorescence probe to detect traces of methanol from ethanol with ultrahigh sensitivity, selectivity and repeatability. To the best of our knowledge, this work represents the first perovskite as fluorescence probe for methanol with wide potential in environmental monitoring and methanol detection, etc.

Construction of PEGylated boronate-affinity-oriented imprinting magnetic nanoparticles for ultrasensitive detection of ellagic acid from beverages.

Molecularly imprinted polymers (MIPs) can exhibit antibody-level affinity for target molecules. However, the nonspecific adsorption of non-imprinted regions for non-target molecules limits the application range of MIPs. Herein, we fabricated PEGylated boronate-affinity-oriented ellagic acid-imprinting magnetic nanoparticles (PBEMN), which first integrated boronate-affinity-oriented surface imprinting and sequential PEGylation for small molecule-imprinted MIPs. The resultant PBEMN possess higher adsorption capacity and faster adsorption rate for template ellagic acid (EA) molecules than the non-PEGylated control. To prove the excellent performance, the PBEMN were linked with hydrophilic boronic acid-modified/fluorescein isothiocyanate-loaded graphene oxide (BFGO), because BFGO could selectively label cis-diol-containing substances by boronate-affinity and output ultrasensitive fluorescent signals. Based on a dual boronate-affinity synergy, the PBEMN first selectively captured EA molecules by boronate-affinity-oriented molecular imprinted recognition, and then the EA molecules were further labeled with BFGO through boronate-affinity. The PBEMN linked BFGO (PBPF) strategy provided ultrahigh sensitivity for EA molecules with a limit of detection of 39.1fgmL-1, resulting from the low nonspecific adsorption of PBEMN and the ultrasensitive fluorescence signal of BFGO. Lastly, the PBPF strategy was successfully employed in the determination of EA concentration in a spiked beverage sample with recovery and relative standard deviation in the range of 96.5 to 104.2% and 3.8 to 5.1%, respectively. This work demonstrates that the integration of boronate-affinity-oriented surface imprinting and sequential PEGylation may be a universal tool for improving the performance of MIPs.

Ultrasensitive Fluorescence Detection and Accurate Colocalization Visualization of Dual-miRNAs in Cancer Cells Based on the Conjugated Chain Reaction of Multifunctional Pentagon DNA Nanostructures.

Herein, a multifunctional pentagon DNA nanostructure (MPDN) was assembled by the hybridization of a circular DNA scaffold containing five different fragments with five diverse DNA oligonucleotides for simultaneous sensitive detection and accurate colocalization imaging of dual-miRNAs in cancer cells. Exactly, the MPDN could specifically and efficiently internalize into folate (FA) receptor-overexpressed cells via specific binding of FA and the FA receptor to distinguish cancer cells from normal cells and transform trace amounts of targets miRNA-21 and miRNA-155 into substantial FAM and Cy5-labeled DNA polymers as the signal probe to generate two remarkable fluorescence emissions, realizing simultaneously sensitive detection of dual-miRNAs. Impressively, compared with traditional small fragment DNA probes with high fluidity, the DNA copolymers with extremely low diffusivity kept it in the originally generated position to achieve the colocalization imaging of dual-miRNAs more accurately for revealing the spatial expression information of dual-miRNAs in tissues and cells. This strategy provided programmable tool to simultaneously detect and accurately colocate dual-miRNAs for understanding normal physiology and the tumor mechanism.

Construction of Fast-Walking Tetrahedral DNA Walker with Four Arms for Sensitive Detection and Intracellular Imaging of Apurinic/Apyrimidinic Endonuclease 1.

Herein, a novel tetrahedral DNA walker with four arms was engineered to travel efficiently on the 3D-tracks via catalyzed hairpin assembly autonomously, realizing the sensitive detection and activity assessment as well as intracellular imaging of apurinic/apyrimidinic endonuclease 1 (APE1). In contrast to traditional DNA walkers, the tetrahedral DNA walker with the rigid 3D framework structure and nonplanar multi-sites walking arms endowed with high collision efficiency, showing a fast walking rate and high nuclease resistance. Impressively, the initial rate of the tetrahedral DNA walker with four arms was 4.54 times faster than that of the free bipedal DNA walker and produced a significant fluorescence recovery in about 40 min, achieving a sensitive detection of APE1 with a low detection limit of 5.54× 10-6 U/μL as well as ultrasensitive intracellular APE1 fluorescence activation imaging. This strategy provides a novel DNA walker for accurate identification of low-abundance cancer biomarker and potential medical diagnosis.