A Novel Approach for Colorimetric Detection of Glyphosate in Food Based on a Split Aptamer Nanostructure and DNAzyme Activity.

Glyphosate has become the most widely used herbicide worldwide in recent years. There are many concerns about toxicity and mutagenicity from long-term use of glyphosate in humans and animals. Therefore, the methods that can help in easy and quick detection of this chemical compound in food and water are critical. In this work, a biosensor was fabricated by combining the enzymatic properties of a specific DNA G-quadruplex and selectivity of a split aptamer to detect glyphosate in foods and water in a quick and simple colorimetric manner. The color change in this method is based on the oxidation of TMB by the G-quadruplex enzyme and the function of aptamer to trap glyphosate, which is visible to the naked eye in the presence and absence of the herbicide. The biosensor showed its high performance in various real samples of water and foods and provided a detection limit of 1.37 nM (R² = 0.9899) with a linear response range of 100 to 400 nM of glyphosate. This biosensor can provide an innovative, cheap and fast approach for the detection and monitoring of glyphosate in various foods and water.

Ratiometric electrochemical aptasensor based on split aptamer and Au-rGO for detection of aflatoxin M1.

A novel ratiometric electrochemical aptasensor based on split aptamer and Au-reduced graphene oxide (Au-rGO) nanomaterials was proposed to detect aflatoxin M1 (AFM1). In this work, Au-rGO nanomaterials were coated on the electrode through the electrodeposition method to increase the aptamer enrichment. We split the aptamer of AFM1 into 2 sequences (S1 and S2), where S1 was immobilized on the electrode due to the Au-S bond, and S2 was tagged with methylene blue (MB) and acted as a response signal. A complementary strand to S1 (CS1) labeled with ferrocene (Fc) was introduced as another reporter. In the presence of AFM1, CS1 was released from the electrode surface due to the formation of the S1-AFM1-S2 complex, leading to a decrease in Fc and an increase in the MB signal. The developed ratiometric aptasensor exhibited a linear range of 0.03 µg L-1 to 2.00 µg L-1, with a detection limit of 0.015 µg L-1 for AFM1 detection. The ratiometric aptasensor also showed a linear relationship from 0.2 µg L-1 to 1.00 µg L-1, with a detection limit of 0.05 µg L-1 in natural milk after sample pretreatment, indicating the successful application of the developed ratiometric aptasensor. Our proposed strategy provides a new way to construct aptasensors with high sensitivity and selectivity.

Split aptamer acquisition mechanisms and current application in antibiotics detection: a short review.

Antibiotic contamination is becoming a prominent global issue. Therefore, sensitive, specific and simple technology is desirable the demand for antibiotics detection. Biosensors based on split aptamer has gradually attracted extensive attention for antibiotic detection due to its higher sensitivity, lower cost, false positive/negative avoidance and flexibility in sensor design. Although many of the reported split aptamers are antibiotics aptamers, the acquisition and mechanism of splitting is still unknow. In this review, six reported split aptamers in antibiotics are outlined, including Enrofloxacin, Kanamycin, Tetracycline, Tobramycin, Neomycin, Streptomycin, which have contributed to promote interest, awareness and thoughts into this emerging research field. The study introduced the pros and cons of split aptamers, summarized the assembly principle of split aptamer and discussed the intermolecular binding of antibiotic-aptamer complexes. In addition, the recent application of split aptamers in antibiotic detection are introduced. Split aptamers have a promising future in the design and development of biosensors for antibiotic detection in food and other field. The development of the antibiotic split aptamer meets many challenges including mechanism discovery, stability improvement and new biosensor development. It is believed that split aptamer could be a powerful molecular probe and plays an important role in aptamer biosensor.

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy.

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF165 was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF165 was dependent on the concentration of VEGF165 . On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF165 complexes was revealed, which was similar to that of the intact aptamer/VEGF165 . Besides, the dissociation rate constant (koff ) of split aptamer/VEGF165 was close to that of intact aptamer/VEGF165 , and the interaction force of split aptamer/VEGF165 was higher than the force of intact aptamer/VEGF165 . It indicated that split aptamer also possessed high affinity with VEGF165 . The work can provide a new method for exploring the interaction of split aptamer/its targets at single-molecule level.

A colorimetric ATP assay based on the use of a magnesium(II)-dependent DNAzyme.

A colorimetric assay for ATP is described that uses a strategy that combines the concept of split Mg(II)-dependent DNAzyme, split aptamer, and hybridization-induced aggregation of gold nanoparticles (AuNPs). Both ATP aptamer and Mg(II)-dependent DNAzyme are split into two fragments which are allocated to two well-designed DNA probes. The probes also possess mutually complementary stem sequences and spacer sequences. In the presence of ATP, the separated DNAzyme sequences in the two probes assemble via the synchronous recognition of ATP with two fragments of the aptamer. Then, the activated DNAzyme catalyzes multiple cycles of the cleavage of its substrate DNA sequence. The latter acts as a linker and induces the aggregation of two types of ssDNA-modified AuNP through the hybridization between the complementary sequences. Thus, the color of the AuNP solution remains red. However, in the absence of ATP, the detached aptamer cannot induce the assembly of DNAzyme to cleave the linker DNA. This results in the aggregation of AuNP and a concomitant color transition from red to purple. This ATP assay, performed at a wavelength of 530 nm, has a linear detection range that extends from 10 pM to 100 nM, with a detection limit of 5.3 pM. It was applied to the detection of ATP in human serum. Conceivably, the strategy has a wide scope in that it may be applied to the colorimetric detection of various other analytes through the split aptamer configuration. Graphical abstract Schematic presentation of colorimetric assay for adenosine triphosphate (ATP) based on the use of a split Mg(II)-dependent DNAzyme, a split aptamer, and by exploiting the hybridization-induced aggregation of gold nanoparticles that leads to a color change from red to purple.

A sandwich dipstick assay for ATP detection based on split aptamer fragments.

Aptamer-based strip assay is an easy, highly efficient and low-cost detection method, which has been developed and easily applied to onsite detection. A new sensitive sandwich dipstick assay for adenosine triphosphate (ATP) detection was successfully developed based on specific recognition between split aptamer fragments and the target. In this method, the thiolated aptamer was first conjugated to the surface of gold nanoparticles (AuNPs), while the biotin-aptamer was immobilized on the surface of a nitrocellulose filter in the test line. In the presence of ATP, the thiol-aptamer/ATP/biotin-aptamer complexes were generated, which led to an obvious increase in optical signals at the test line. Under the optimal determination conditions, an excellent linear logarithmic response to the ATP concentration was obtained within the range of 0.5 µM to 5 mM. The limit of detection (LOD) of 0.5 µM was reached at a signal-to-noise ratio of 3. The dipstick assay showed a good average recovery of 96-108 % with the RSD of less than 20 % in urine samples. The proposed method exhibited high specificity against other nucleotides such as the uridine triphosphate (UTP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP). The results indicated that the dipstick strip may be considered as an inexpensive screening tool for onsite ATP determination. Graphical Abstract A simple split aptamer fragments based sandwich-type dipstick assay was developed for ATP detection.

One-pot assay using a target-driven split aptamer recognition and assembly strategy for convenient and rapid detection of gliotoxin.

An aptamer targeting gliotoxin (GTX) was optimized to increase the binding affinity by approximately 20 times and achieve higher structural stability and targeting specificity. Molecular dynamics simulations were used to explore the molecular mechanism and key action sites underlying the recognition of GTX by the optimized aptamer. Subsequently, the optimized aptamer was split into two fragments and a convenient and rapid one-pot assay for GTX detection was successfully established using a target-driven split aptamer recognition and assembly strategy. The method exhibited a good linear range of 0.128 nM to 2 µM, a low detection limit of 0.07 nM, and excellent selectivity for GTX. Furthermore, the method had good accuracy and stability in real sample analysis. Therefore, the developed one-pot method provides a reliable, convenient, and cost-effective approach for the widespread application of GTX detection.

Split aptamer-based sandwich-type ratiometric biosensor for dual-modal photoelectrochemical and electrochemical detection of 17ß-estradiol.

BACKGROUND: As one of the most potent environmental estrogens, 17ß-estradiol (E2), which can be enriched into organisms through the food chain and cause harmful biological effects in humans, has been frequently detected in the water environment of the world. High performance liquid chromatography (HPLC) and gas chromatograohy-mass spectrometry (GC/MS) have been widely used for quantification of E2. Despite excellent accuracy, tedious pretreatment and expensive instruments result in their limited application. It is clear that there is an urgent need to establish simple, sensitive and accurate methods for the determination of E2. RESULTS: A split aptamer-based sandwich-type ratiometric biosensor based on split aptamer was developed by coupling photoelectrochemical and electrochemical assays for E2 detection. For analysis, the two fragments of split aptamer recognized E2 by forming sandwich structure, which triggered hybridization chain reaction (HCR) to produce double-stranded DNA (dsDNA) with CdTe quantum dots (QDs) labeled hairpin DNA. The resultant dsDNA can further absorb methylene blue (MB) to sensitize CdTe QDs for an enlarged photocurrent (IPEC) and output a redox current of IMB, and both of them acted as response signals for detection; [Fe(CN)6]3-/4- probe produced redox current of I[Fe(CN)6]3-/4- as reference signal. Using IMB/I[Fe(CN)6]3-/4- and IPEC/I[Fe(CN)6]3-/4- as yardsticks, the developed split aptamer-based sandwich-type ratiometric biosensor provides two linear ranges of 0.1-5000 pg mL-1 for IMB/I[Fe(CN)6]3-/4- and 0.1-10000 pg mL-1 for IPEC/I[Fe(CN)6]3-/4- with detection limits of 0.06 pg mL-1 and 0.02 pg mL-1, respectively. SIGNIFICANCE: These results of the biosensor are benefiting from the coupling of photoelectrochemical (PEC) and electrochemical (EC) assays as well as the unique cooperative recognition mechanism of split aptamer. This method not only enabled the biosensor to be successfully applied to the determination of E2 in lake water, but also broadens the prospects for the realization of sensitive and accurate detection of E2.

Enhanced anode electrochemiluminescence in split aptamer sensor for kanamycin trace monitoring.

Covalently modifying electrochemiluminescence (ECL) luminophores to alter their energy levels or generate energy/electron transfer processes for improved performance is hindered by the complex design and fabrication processes. In this study, non-covalent bond self-assembly was employed to enhance the ECL property of gold nanoclusters with tryptophan (Try) and mercaptopropionic acid (MPA) as ligands (Try-MPA-gold nanoclusters). Specifically, through the molecular recognition of Try by cucurbit[7]uril, some non-radiative transition channels of the charge carriers on the surface of the Try-MPA-gold nanoclusters were restricted, resulting in a significant enhancement of the ECL intensity of the nanoclusters. Furthermore, rigid macrocyclic molecules acted on the surface of the nanoclusters through self-assembly, forming a passive barrier that improved the physical stability of the nanoclusters in the water-phase and indirectly improved their luminescent stability. As an application, cucurbit[7]uril-treated Try-MPA-gold nanoclusters (cucurbit[7]uril@Try-MPA-gold nanoclusters) were used as signal probes, and Zn-doped SnO2 nanoflowers (Zn-SnO2 NFs) with high electron mobility were used as electrode modification material to establish an ECL sensor for kanamycin (KANA) detection, utilizing split aptamers as capture probes. The advanced split aptamer sensor demonstrated excellent sensitivity analysis for KANA in complex food substrates with a recovery rate of 96.2 to 106.0%.

Smartphone-based chemiluminescence detection of aflatoxin B1 via labelled and label-free dual sensing systems.

To develop a sensing platform for onsite determination of AFB1 in foodstuffs, we developed smartphone-based chemiluminescence detection of AFB1 via labelled and label-free dual modes. The labelled mode was characteristic of double streptavidin-biotin mediated signal amplification, obtaining limit of detection (LOD) of 0.04 ng/mL in the linear range of 1-100 ng/mL. To reduce the complexity in the labelled system, a label-free mode based on both split aptamer and split DNAzyme was fabricated. A satisfactory LOD of 0.33 ng/mL was generated in the linear range of 1-100 ng/mL. Both labelled and label-free sensing systems achieved outstanding recovery rate in AFB1-spiked maize and peanut kernel samples. Finally, two systems were successfully integrated into smartphone-based portable device based on custom-made components and android application, achieving comparable AFB1 detection ability to a commercial microplate reader. Our systems hold huge potential for AFB1 onsite detection in food supply chain.

A fluorescent assay for sensitive detection of kanamycin by split aptamers and DNA-based copper/silver nanoclusters.

Kanamycin was a group of essential antibiotics generally served in treating infections of animals which leached into the environment residual in food, causing health concerns. Thus, selective and sensitive monitoring of kanamycin was significant for food safety. In this work, split aptamers were used as templates to prepare fluorescent Cu/Ag NCs for detection of kanamycin. According to the impressive affinity of the aptamer to kanamycin, two different detection modes were designed using kanamycin aptamer as a recognition molecule, in which one was to combine split aptamer Apt-1 with Apt-2 to form an entangled DNA as a Cu/Ag NCs template, the other was to associate the normal aptamer after encirclement to form Cu/Ag NCs templates. After the addition of kanamycin, the fluorescence signals of the Cu/Ag NCs synthesized in the two modes were both enhanced, but the approach with split aptamer exhibited a superior observable sensitivity than that of the normal type. The detection range showed a well linear relationship between 80 nM and 10 µM when the emission wavelength was 560 nm, and the detection limit was 13.3 nM. In addition, when streptomycin, oxytetracycline, chloramphenicol and chlortetracycline were involved in the selective interference experiment under the same conditions, the fluorescence intensity of the system performed no significant changes. The results demonstrated that this method possessed favorable specificity and selectivity for the assay of kanamycin, proficiently achieving efficient, rapid and sensitive evaluation of kanamycin in the milk samples.

Split-aptamer mediated regenerable temperature-sensitive electrochemical biosensor for the detection of tumour exosomes.

In this paper, a split-aptamer mediated regenerable temperature-sensitive (SMRT) electrochemical biosensor was constructed for the detection of exosomes. The split-aptamer used in this SMRT biosensor was composed of two fragments, one of which was immobilized on the surface of an electrode via sulfhydryl groups and named split-a and the other was labelled with methylene blue and named split-b. The two fragments could form sandwich structures at the electrode surface via target-induced self-assembly in the presence of target exosomes at 4 °C in PBS, and then realizing the detection of exosomes via voltammetry. In addition, due to the temperature sensitivity of the split-aptamer, the electrode could be regenerated through temperature-induced disassembly of the sandwich structures. Consequently, the SMRT biosensor realized sensitive and specific analysis of target exosomes with a limit of detection of 1.5 × 106 particles/mL and could be quickly and easily regenerated by washing with PBS at 37 °C for 30 s without any additives. This is the first study on the construction of a reproducible electrochemical biosensor using a split-aptamer for the specific detection of tumour exosomes, and may provide an innovative strategy for the economical and efficient design of regenerable electrochemical biosensors.

Split aptamer remodeling-initiated target-self-service 3D-DNA walker for ultrasensitive detection of 17ß-estradiol.

DNA walker machines, as one of the dynamic DNA nanodevices, have attracted extensive interest in the field of analysis due to their inherent superiority. Herein, we reported a split aptamer remodeling-initiated target-self-service 3D-DNA walker for ultrasensitive, specific, and high-signal-background ratio determination of 17ß-estradiol (E2) in food samples. Two split probes (STWS-a and STWS-b) were rationally designed that can undergo structural reassembled to serve as walking strands (STWS) under the induction of the target. Meanwhile, an intact E6-DNAzyme region was formed and activated at the tail of STWS. The activated E6-DNAzyme then continuously drives the 3D-DNA walker for signal amplification and specific detection of E2. Under optimal conditions, the proposed DNA walker-based biosensor exhibited excellent linearity in the range of 1 pM to 50 nM with a low limit of detection (LOD) of 0.28 pM, and good precision (2.7%) for 11 replicate determinations of 1 nM of E2. Furthermore, the developed DNA walker-based biosensor achieved excellent sensitive analysis of E2 in the complex food matrix with recoveries of 95.6-106.5%. This newly proposed split aptamer-based strategy has the advantages of ultrasensitive, high signal-to-background ratio, and high stability. Noteworthy, the successful operation of the DNA walker initiated by the split aptamer expands the principles of DNA walker design and provides a universal signal amplification platform for trace analysis.

A label-free colorimetric aptasensor based on split aptamers-chitosan oligosaccharide-AuNPs nanocomposites for sensitive and selective detection of kanamycin.

Herein, the split aptamers, chitosan oligosaccharide, and AuNPs were combined as nanocomposites that present different formations to develop a label-free colorimetric aptasensor for rapid detection of small molecules. Kanamycin was chosen as a model target. Computational studies were performed to assist in the design of orientated immobilization of the split aptamers onto the AuNPs surface. Chitosan oligosaccharide was initially applied as an aggregation inducer of AuNPs, and chitopentaose was screened as the optimal. Under optimized conditions, the proposed aptasensor showed high sensitivity and selectivity, with a limit of detection of 20.58 nM, a linear range of 25-800 nM, and good recoveries of 98.49-104.9% and 85.69-107.0% when employed to detect kanamycin in tap water and milk samples, respectively. Only 55 min was needed for the whole assay. More importantly, this study can serve as a novel and robust reference for the aptasensing detection of other small molecules.

A split aptamer (SPA)-based sandwich-type biosensor for facile and rapid detection of streptomycin.

As antibiotic pollution is gaining prominence as a global issue, the demand for detection of streptomycin (STR), which is a widely used antibiotic with potential human health and ecological risks, has attracted increasing attention. Aptamer-based biosensors have been developed for the detection of STR in buffers and samples, however, the non-target signals due to the conformational variation of free aptamers possibly affect their sensitivity and stability. In this study, by introducing the STR-specific split aptamer (SPA), a sensitive evanescent wave fluorescent (EWF) biosensor is developed for the sandwich-type based detection of STR. The standard calibration curve obtained for STR has a detection limit of 33 nM with a linear range of 60-526 nM. This biosensor exhibited good selectivity, reliable reusability for at least 100 times measurements, and high recovery rates for spiked water samples; moreover, all detection steps are easy-to-operate and can be completed in 5 min. Therefore, it exhibits great promise for actual on-site environmental monitoring. Additionally, without introducing any other oligonucleotides or auxiliary materials, this SPA-based biosensing method shows potential as a simple, sensitive, and low-cost manner for the detection of other small molecular targets.

Using Simple-Structured Split Aptamer for Gold Nanoparticle-based Colorimetric Detection of Estradiol.

Demand for the detection of estradiol, which is a naturally occurring hormone, has been increasing. Gold nanoparticle-based colorimetric aptasensors have been developed for estradiol detection; however, the long sequence of aptamers due to the formation of the secondary structure likely affects the sensitivity of the aptasensors. Herein, a sensitive colorimetric biosensor is developed for label-free detection of estradiol by using an estradiol-specific split aptamer. The results demonstrate that a superior response is observed when a split aptamer with a high free energy of the secondary structure (ΔG > -3 kcal/mol) is used, in comparison to that observed using a split aptamer with a low free energy of the secondary structure (ΔG < -3 kcal/mol) at 27°C. After selecting the appropriate split aptamer, the standard calibration curve obtained for estradiol has a detection limit of 6.7 nM, with a linear range of 6.7 nM - 66.7 µM in the logarithmic scale. Furthermore, this assay is sensitive, easy-to-operate, inexpensive, and non-time-consuming (provides results within 50 min), thereby showing potential for clinical applications (detection of other small molecular targets).

Electrochemical aptasensor for 17ß-estradiol using disposable laser scribed graphene electrodes.

17ß-Estradiol (E2), the strongest of the three major physiological estrogens in females, is an important factor in the female reproductive system. The abnormal level of E2 causes health issues, such as weak bones, urinary tract infections and even depression. Here, we present a novel, sensitive and selective, electrochemical aptasensor for detection of 17ß-estradiol (E2). The E2 recognition aptamer was split into two fragments: the first fragment, functionalised with adamantane, is attached to poly(ß-cyclodextrin) (poly(ß-CD))-modified electrode surface through host-guest interactions between the adamantane and poly(ß-CD). The second fragment, labelled with gold nanoparticles, forms the stem-loop structure with the first fragment only in the presence of E2. That specific recognition process triggers the change in the electrochemical signal (a change in the peak current from reduction of AuNPs), recorded by means of differential pulse voltammetry (DPV). The feasibility of the sensing design was firstly investigated on the commercially available glass carbon electrodes (GCE), with achieved a linear detection range of 1.0 × 10-13 to 1.0 × 10-8 M and a limit of detection (LoD) 0.7 fM. The sensing methodology was then translated onto single-use, disposable, laser-scribed graphene electrodes (LSGE) on a plastic substrate. The dynamic sensing range of E2 on LSGE was found to be 1.0 × 10-13 to 1.0 × 10-9 M, with a LoD of 63.1 fM, comparable to these of GCE. The successful translation of the developed E2 aptasensor from GCE to low-cost, disposable LSGE highlights a potential of this sensing platform in commercial, portable sensing detection systems for E2 and similar targets of biological interest.

Design of a Fluorescence Turn-on and Label-free Aptasensor Using the Intrinsic Quenching Power of G-Quadruplex to AMT.

A novel fluorescent aptasensor based on the G-quadruplex induced fluorescent quenching of psoralen and the competitive interactions between 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), adenosine triphosphate (ATP) and G-rich DNA functionalized split ATP aptamer was proposed. The binding of ATP to the G-rich DNA functionalized split aptamer induced a significant enhancement in fluorescence emission intensity while undergoing excitation at 340 nm. Under the optimal conditions, the developed aptasensor showed high selectivity and good accuracy for detecting ATP. The practicality of the proposed aptasensor has been confirmed by successfully analyzing ATP in spiked human blood serum samples with satisfactory results. As far as we know, this is the first time that the intrinsic quenching ability of G-quadruplex was applied to simply construct a fluorescence turn-on and label-free aptasensor. On account of the superiority of the simplicity of the design strategy, more work is expected in the future to develop a variety of novel sensors for other important analytes using the quenching capability of G-quadruplex through reasonable designs.

A split aptamer sensing platform for highly sensitive detection of theophylline based on dual-color fluorescence colocalization and single molecule photobleaching.

A new split aptamer sensing platform is developed for highly sensitive and selective detection of theophylline based on single molecule photobleaching (SMPB) technique. The sensing system contains two probes. One is formed by one streptavidin and four biotinylated RNA fragments labelled with fluorescein isothiocyanate (FITC). Each biotinylated RNA fragment contains two repeating aptamer fragments. The other probe is the complementary aptamer fragment labelled with Cy5 dye. The existence of theophylline can trigger the first probe to bind as many as eight Cy5-labelled probes. The average combined number depends on the theophylline concentration and can be measured by SMPB technique. In the sensing system, the dual-color fluorescence colocalization is performed by the red fluorophore (Cy5) and green fluorophore (FITC), in which the red fluorophore is utilized for quantitative counting of photobleaching steps, while the green fluorophore serves as a counting reference to increase detection efficiency. On basis of the principle, an ultra-sensitive sensing platform of theophylline is created with a low limit of detection (LOD) of 0.092 nM. This work provides not only a highly sensitive method for theophylline detection but also a novel perspective for the applications of SMPB technology to construct biosensors.

Duplex featured polymerase-driven concurrent strategy for detecting of ATP based on endonuclease-fueled feedback amplification.

We have developed a novel amplification strategy termed Endo IV-assisted feedback amplification (EFA) taking advantages of rolling circular amplification (RCA) and Endo IV-assisted signal amplification (ESA) biosensing platform for detecting of adenosine triphosphate (ATP). Two kinds of specially programmed DNA complexes were employed into EFA system, one composed of a split aptamer fragment and a circular template, and the other composed of AP probe and the same circular template. Hence, ATP as a target induced the self-assembly of spilt aptamer fragments and initiated RCA reaction generating a linear DNA, which consists of hybridization elements with Complex II and formation elements of G-quadruplex. More importantly, the addition of endonuclease IV can cut the Complex II into two parts, and one of which can be trimming by phi29 DNA polymerase initiating the new round of RCA reaction producing more RCA products. Thus significantly enhanced fluorescent signal can be measured for ATP as expected, and our proposed strategy exhibits improved performances toward ATP ultrasensitive detection with a limit of detection (LOD) as low as 0.09 nM. Additionally, our developed biosensor demonstrates high selectivity and the superiority of simplicity towards ATP. Above these significant aspects, our proximity binding-induced RCA reaction-based fluorescent assay and Endo IV-fueled feedback signal amplification strategy presents an optimal detection performance towards ATP for potential application in related research and clinical diagnosis.