Signal Amplification Strategy Research Articles

Target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive and direct Pseudomonas aeruginosa detection

Pseudomonas aeruginosa (P. aeruginosa), a kind of gram-negative pathogenic bacteria, are causative agents of severe infections, such as lower respiratory tract infections in children and cancers. Detecting low levels of P. aeruginosa in clinical samples in an easy-to-operate manner is highly desired but still poses a problem. Herein, we established a target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive detection of P. aeruginosa in a wash-free way. This approach involves the allosteric probe’s accurate recognition and binding to target P. aeruginosa, leading to subsequent multiple-cycle amplification. Afterward, the amplified products were translated to induce the aggregation of gold nanoparticles (AuNPs), resulting in color variations. The utilization of the allosteric probe, which is integrated with the aptamer sequences, enables wash-free detection of P. aeruginosa. Taking the merit of multiple signal amplification process, the suggested method showed a strong linear response to the extracted P. aeruginosa within a concentration range of 10–105 cfu/mL, with a low limit of detection for individual P. aeruginosa detection. The proposed technique has considerable clinical promise for early disease diagnosis because to its high sensitivity and wash-free simplicity.

CRISPR-Cas12a-mediated colorimetric aptasensor of netilmicin based on enzymes-assisted signal amplification and nanozyme employing a rationally truncated aptamer

Netilmicin (NET) is a typical veterinary antibiotic used in aquaculture and animal husbandry, but its excessive accumulation poses a serious threat to the ecological environment and human health. Therefore, a novel and highly sensitive Crispr-Cas12a-mediated colorimetric aptasensor was developed by combining the enzyme-assisted signal amplification strategy and magnetic NMOF-Pt nanozyme composite material (NPSM) for the visual detection of veterinary antibiotic residues in real samples. A high-affinity truncated aptamer (N-20) targeting NET was evolved from a parental aptamer sequence with the help of comprehensive recognition mechanism studies. Subsequently, N-20 was elaborately designed into the isothermal enzyme-assisted strand displacement amplification reaction. The amplified products (Act-DNAs) activated the trans-cleavage activity in the Crispr Cas12a system. The NMOF-Pt nanozyme was released from NPSM after cleaving the single chain of DNA (ssDNA). The quantity of released nanozyme was positively correlated with the amount of NET to quantitatively achieve the detection with the linear range and limits of detection (LOD) of about 0.003–30 ng/mL and 1.15 pg/mL, respectively. This method was successfully applied to detect NET in Lake water, milk and pork samples with satisfactory recoveries ranging from 89.9 % to 104.3 %. The constructed aptasensor demonstrated superior analytical performance and exhibited enormous application prospects in food supervision, environment monitoring, and medical laboratory science.

Integration of high-entropy nanozyme and hollow In2S3 nanotube heterostructures decorated with WO3 for ultrasensitive PEC aptasensing of highly toxic mycotoxin

As a highly toxic mycotoxin, patulin threatens human and animal health. Herein, a new signal amplification strategy was designed for photoelectrochemical (PEC) analysis of patulin based on nanotube-like In2S3/WO3 heterostructures with the assistance of PtRuFeCoW high-entropy alloy nanorods (HEANRs). Specifically, the prominent photoactivity of the In2S3/WO3 was investigated by UV−vis diffuse reflectance and photoluminescence spectroscopy, coupled by elaborating the photoelectron transport mechanism in detail. Simultaneously, the excellent peroxidase-like activity of the PtRuFeCoW HEANRs was certificated mainly by UV–vis absorption spectroscopy. On such foundation, the PtRuFeCoW HEANRs modified capture DNA was cast onto the In2S3/WO3 based photoanode for catalyzing 4-chloro-1-naphthol (4-CN) oxidation to form insoluble benz-4-chloro-hexadienone (4-CD) precipitate in the presence of H2O2, ultimately weakening the PEC currents. The resulting PEC sensor exhibited a wider detection range from 0.1 pg mL−1 to 500 ng mL−1 and a lower detection limit was down to 0.063 pg mL−1 (S/N = 3). The HEANRs nanozyme assisted PEC method holds great promise for ultrasensitive detection of environmental pollutants in practice.

Fluorescence Sensing of Eclampsia Biomarkers via the Immunosorbent Atom Transfer Radical Polymerization Assay.

Accurate and quantitative detection of pre-eclampsia markers is crucial in reducing pregnancy mortality rates. This study introduces a novel approach utilizing a fluorescent biosensor by the immunosorbent atom transfer radical polymerization (immuno-ATRP) assay to detect the pre-eclampsia protein marker CD81. The critical step used in this sensor is the novel signal amplification strategy of fluorescein polymerization mediated by ferritin-enhanced controlled radical polymerization, which combines with a traditional enzyme-linked immunosorbent assay (ELISA) to further reduce the detection limit of the CD81 protein concentration. The fluorescence intensity was linear versus logarithmic CD81 protein concentration from 0.1 to 10,000 pg mL-1, and the detection limit was 0.067 pg mL-1. Surprisingly, in 30% normal human serum (NHS), the sensor can also detect target protein over 0.1-10,000 pg mL-1, with 0.083 pg mL-1 for the detection limit. Moreover, the proposed biosensor is designed to be cost-effective, making it accessible, particularly in resource-limited settings where expensive detection techniques may not be available. The affordability of this method enables widespread screening and monitoring of preeclampsia, ultimately benefiting many pregnant women by improving their healthcare outcomes. In short, developing of a low-cost and susceptible direct detection method for preeclampsia protein markers, such as CD81, through the use of the immuno-ATRP assay, has significant implications for reducing pregnancy mortality. This method holds promise for early detection, precise treatment, and improved management of preeclampsia, thereby contributing to better maternal and fetal health.

A novel electrochemical aptasensor based on AgPdNPs/PEI-GO and hollow nanobox-like Pt@Ni-CoHNBs for procymidone detection

Herein, an aptasensor based on a signal amplification strategy was developed for the sensitive detection of procymidone (PCM). AgPd nanoparticles/Polenimine Graphite oxide (AgPdNPs/PEI-GO) was weaned as electrode modification material to facilitate electron transport and increase the active sites on the electrode surface. Besides, Pt@Ni-Co nanoboxes (Pt@Ni-CoHNBs) were utilized to be carriers for signaling tags, after hollowing ZIF-67 and growing Pt, the resulting Pt@Ni-CoHNBs has a tremendous amounts of folds occurred on the surface, enables it to carry a larger quantity of thionine, thus amplify the detectable electrochemical signal. In the presence of PCM, the binding of PCM to the signal probe would trigger a change in electrical signal. The aptasensor was demonstrated with excellent sensitivity and a low detection limit of 0.98 pg·mL−1, along with a wide linear range of 1 μg·mL−1 to 1 pg·mL−1. Meanwhile, the specificity, stability and reproducibility of the constructed aptasensor were proved to be satisfactory.

An ultrasensitive photoelectrochemical biosensor based on AgBiS2/CdS photoanode and multiple signal amplification strategy for the detection of dibutyl phthalate plasticizer

Herein, a novel AgBiS2/CdS heterojunction was developed for the construction of sensitive biosensor for dibutyl phthalate (DBP) detection. Compared with the pristine CdS, the AgBiS2/CdS heterojunction exhibited enhanced photocurrent because of the promoted photo-carriers transfer due to the matched energy band arrangement between AgBiS2 and CdS. Based on this AgBiS2/CdS photoanode and a signal amplification strategy integrates DNAzyme-assisted walker reaction and hybridization chain reaction, a sensitive PEC sensing platform was designed for the detection of DBP. A wide detection ranges from 1 fM to 10 nM and a low detection limit of 0.3 fM were achieved. The proposed PEC biosensor with satisfactory sensitivity, selectivity, and stability shows potential application in environmental monitoring.

A Stable and Sensitive Engineering Bacterial Sensor via Physical Biocontainment and Two-Stage Signal Amplification.

Although engineering bacterial sensors have outstanding advantages in reflecting the actual bioavailability and continuous monitoring of pollutants, the potential escape risk of engineering microorganisms and lower detection sensitivity have always been one of the biggest challenges limiting their wider application. In this study, a core-shell hydrogel bead with functionalized silica as the core and alginate-polyacrylamide as the shell have been developed not only to realize zero escape of engineered bacteria but also to maintain cell activity in harsh environments, such as extremely acidic/alkaline pH, high salt concentration, and strong pressure. Particularly, after combining the selective preconcentration toward pollutants by functionalized core and the positive feedback signal amplification of engineering bacteria, biosensors have realized two-stage signal amplification, significantly improving the detection sensitivity and reducing the detection limit. In addition, this strategy was actually applied to the detection of As(III) and As(V) coexisting in environmental samples, and the detection sensitivity was increased by 3.23 and 4.39 times compared to sensors without signal amplification strategy, respectively, and the detection limits were as low as 0.39 and 0.86 ppb, respectively.

Recent Trends and Innovations in Bead-Based Biosensors for Cancer Detection.

Demand is strong for sensitive, reliable, and cost-effective diagnostic tools for cancer detection. Accordingly, bead-based biosensors have emerged in recent years as promising diagnostic platforms based on wide-ranging cancer biomarkers owing to the versatility, high sensitivity, and flexibility to perform the multiplexing of beads. This comprehensive review highlights recent trends and innovations in the development of bead-based biosensors for cancer-biomarker detection. We introduce various types of bead-based biosensors such as optical, electrochemical, and magnetic biosensors, along with their respective advantages and limitations. Moreover, the review summarizes the latest advancements, including fabrication techniques, signal-amplification strategies, and integration with microfluidics and nanotechnology. Additionally, the challenges and future perspectives in the field of bead-based biosensors for cancer-biomarker detection are discussed. Understanding these innovations in bead-based biosensors can greatly contribute to improvements in cancer diagnostics, thereby facilitating early detection and personalized treatments.

Aptamer-based protein molecule detection via cyclic reverse transcription coupling with self-priming hairpin-triggered CRISPR-Cas12a system

Protein biomarkers (e.g. thrombin) are of great significance for the biological process of the organism, and its aberrant expression is closely associated with the development of diseases. With thrombin, a serine protease that plays a crucial role in maintaining homeostasis and promoting blood clotting, as detection target, this study introduces a novel approach for sensitive and accurate measurement of protein biomarker expression by utilization of cyclic reverse transcription (CRT) in combination with the self-priming hairpin-triggered CRISPR-Cas12a system. In this method, an elegantly designed sensing probe is utilized to specifically bind with the thrombin protein and convert the protein signals to nucleic acids signals, following by the CRT and CRISPR-Cas12a system-based signal amplification strategy. Taking the merit of the two-stage amplification, this assay has the capability to detect thrombin at the fM level. In addition, due to the aptamer sequence’s strong selectivity to thrombin protein and the dual-check process in the signal amplification process (first in the CRT and second in the CRISPR system), the proposed test demonstrates exceptional specificity in detecting thrombin. By re-designing the sensing probe, the established method could be extended to various protein biomarker detection. Ultimately, this assay has successfully enabled the accurate evaluation of biomarker levels in constructed clinical samples, showing significant potential for application in the realm of clinical molecular diagnosis.

Biomimetic cascade intelligent paper chip sensor based on bimetallic porphyrin-based covalent organic framework with triple-enzyme mimetic activities

Enzyme-induced biological cascade catalysis can achieve selective and efficient transformations of substrates in vivo. Thus, the biomimetic cascade systems have attracted much attention in biological detection on account of their ingenious strategies for signal conduction and amplification. In this study, a porphyrin-based covalent organic framework (COFFe/CoP-Ph) with bimetal-nitrogen-coordinate centers (M − N4) were constructed via Schiff base reaction. The synergistic effect of the highly exposed M − N4 bimetallic active sites and the unique topology overcame the self-limitation of nanozyme, resulting in a significant enhancement of the triple-enzyme mimetic activities of COFFe/CoP-Ph, namely oxidase (OXD)-, peroxidase (POD)-, and catalase (CAT)-like activities. Simultaneously, a multiple enzyme-driven cascade catalysis strategy for signal amplification was proposed utilizing the OXD- and POD-like mimetic mechanisms of COFFe/CoP-Ph, enabling the colorimetric determination of malathion without the addition exogenous H2O2. The established colorimetric sensor exhibited an exceptional linear relationship within the range of 1–18 μM and a low detection limit of 11 nM. Subsequently, a portable smartphone platform was developed by integrating paper chip, facilitating successful malathion point-of-care testing (POCT) in real samples. This work not only creates a bimetallic COF nanozyme and establishes an efficient cascade amplification strategy, but also introduces a novel design approach for development of a low-cost and user-friendly POCT method.

Cascaded autocatalytic hairpin assembly molecular circuit for amplified fluorescent aptamer luteinising hormone assay

The quantitative detection of luteinising hormone (LH) is critical for the study of the physiological mechanism of reproductive function and the assessment of infertility and the clinical treatment of reproductive disorders. However, conventional approaches for LH detection are mostly based on an antibody recognition module with the limitations of sensitivity, simplicity and cost. The development of robust LH sensing methods is therefore highly demanded for facilitating the diagnosis of LH-related diseases. We establish a convenient, amplified and sensitive fluorescent aptamer LH assay based on new target-triggered and cascaded autocatalytic hairpin assembly (C-aCHA) circuit amplification means via initiator sequence replication. Target LH molecules bind the aptamers in the aptamer/initiator duplexes to release the initiator sequences, which trigger CHA formation of DNA three-way junctions (TWJs) and the unfolding of fluorescently quenched signal hairpins to show amplified fluorescence. The TWJs further activate another CHA cycle for the yield of more initiator sequences to form the C-aCHA circuit amplification cycles, which lead to the unfolding of many signal hairpins to exhibit substantially magnified fluorescence recovery for detecting LH down to 8.56 pM in the range from 10 pM to 50 nM. In addition, the monitoring of trace LH in diluted serums by this sensing approach has been also verified. Our LH assay clearly outperforms current existing antibody-based methods and the C-aCHA signal amplification strategy can be easily extended as a robust means for sensitively monitoring various biomolecular markers with simple replacement of the corresponding aptamers for diverse applications.

Ultrasensitive detection of prostate-specific antigen using a SERS-based magnetic immunosensor

Concentrations of disease biomarkers and their changes are crucial for the diagnosis and monitoring of diseases, so detecting and capturing small changes in biomarkers is especially important. This study demonstrates a surface-enhanced Raman scattering (SERS)-active magnetic immunosensor using a signal amplification strategy for the ultrasensitive detection of prostate-specific antigen (PSA). The sensor consists of a biotin-modified aptamer (B-Apt) and a PSA antibody (Ab) labeled on magnetic beads (MBs), which can immobilize and anchor the target PSA when it is present. After sequential addition of streptavidin and a signal probe, the generated streptavidin-biotin complex reacts to form a three-dimensional network, thereby increasing the number of signal molecules to achieve signal amplification, with the detection limit of 0.87 pg/mL. In addition, this method was also performed to detect PSA in human serum samples. It is believed that this method provides a more sensitive tool for detecting PSA or other biomarkers in clinical samples.

MOF-derived sandwich-structured dual Z-Scheme Co9S8@ZnIn2S4/CdSe hollow nanocages heterojunction: Target-induced ultrasensitive photoelectrochemical sensing of chlorpyrifos

Exploring efficient photoactive material presents an intriguing opportunity to enhance the analytical performance of photoelectrochemical (PEC) sensor in the environmental analysis. In this work, a sandwich-structured multi-interface Co9S8@ZnIn2S4/CdSe QDs dual Z-Scheme heterojunction, derived from metal-organic framework (MOF), was synthesized as a sensing platform for chlorpyrifos detection, by integrating with enzyme-induced in situ insoluble precipitates strategy. The meticulously designed Co9S8@ZnIn2S4/CdSe QDs exhibited enhanced charge separation efficiency and was proved to be a highly effective sensing platform for the immobilization of biomolecules, attributing to the intrinsic dual Z-Scheme heterojunction and the distinctive hollow structure. The proposed PEC sensing platform combined with enzyme-induced in situ precipitate signal amplification strategy achieved superior performance for sensing of chlorpyrifos (CPF), showing in wide linear range (1.0 pg mL−1–100 ng mL−1), with a limit of detection (0.6 pg mL−1), excellent selectivity, and stability. This work offers valuable insights for the design of novel advanced photoactive materials aimed at detecting environmental pollutants with low level concentration.

Enhanced electrochemical detection of thrombin via a dual signal amplification strategy based on Au Nanoparticles@Carbon nanosheets and PtCu3 nanoparticles

Thrombin (TB) is an enzyme involved in blood coagulation and associated with various diseases such as thromboembolic disease, Alzheimer's disease, and cancer. This study introduces an electrochemical biosensor with a sandwich-type structure designed for the highly sensitive detection of TB. The working electrode (WE) was modified with gold nanoparticles loaded onto the carbonization product of two-dimensional Cu-bond tetrakis(4-carboxyphenyl)porphyrin (Cu-TCPP) nanosheets (referred to as Au@CNSs), enhancing the electrical conductivity and specific surface area of the WE. The modified electrode exhibited notable catalytic activity for H2O2 reduction. One aptamer of TB (Apt1), with a sulfhydryl group (-SH-) at the end, can tightly bound to Au@CNSs through a S-Au bond. Additionally, PtCu3 alloy nanocrystals, possessing high electrocatalytic activity towards H2O2 reduction, were prepared and modified with another TB aptamer (Apt2). In the presence of TB, a sandwich-type biosensing interface formed, resulting in an increased reduction current for H2O2. Under optimized conditions, the proposed aptasensor demonstrated a broad linear range from 0.01 pM to 32 nM and a low detection limit of 0.005 pM. Furthermore, the aptasensor exhibited excellent performance in terms of stability, reproducibility, and sensitivity, making it a promising tool for TB detection in human serum.

Dual-photoelectrode photocatalytic fuel cell-assisted self-powered biosensor: An ingenious and sensitive strategy for p53 gene detection

Self-powered sensors have the advantages of no external power supply, energy conversion in the environment and easy miniaturization, making it an attractive choice for building implantable, wearable and portable devices. However, it is difficult to sensitively detect low abundance targets owing to their poor output performance and interference from complicated settings. Herein, a new photocatalytic fuel cell-based self-powered electrochemical biosensor (PFC-SPEB) was proposed for the detection of p53 gene by combining high photocatalytic performance In2S3 and CuInS2 with magnetic nanobeads-assisted signal amplification strategy. In2S3 photoanode and CuInS2 photocathode were combined to form a dual photoelectrode system with an open-circuit potential up to 705.9 mV, which improved the photoelectric conversion efficiency by 1.4–6.9 folds compared with that of single photoelectrode system. Furthermore, the enzyme-assisted signal amplification strategy made a small number of p53 genes trigger the creation of abundant double-stranded DNA (dsDNA). Finally, manganese porphyrin was inserted into dsDNA to stimulate precipitation reactions on surfaces of the photoanode, leading to a sharp drop of signal. The constructed PFC-SPEB exhibited a wide linear detection range from 10 fM to 10 nM with a low detection limit of 1.40 fM, providing new creative inspiration for constructing SPEB for bioanalysis and clinical diagnostics.

"Two-in-One" PtPdCu Trimetallic Multifunctional Nanoparticles-Mediated Dual-Signal-Integrated Aptasensor for Ultradetection of Enrofloxacin.

Balancing the accuracy and simplicity of aptasensors is a challenge in their construction. This study addresses this issue by leveraging the remarkable loading capacity and peroxidase-like catalytic activity of PtPdCu trimetallic nanoparticles, which reduces the reliance on precious metals. A dual-signal readout aptasensor for enrofloxacin (ENR) detection is designed, incorporating DNA dynamic network cascade reactions to further amplify the output signal. Exploiting the strong loading capacity of PtPdCu nanoparticles, they are self-assembled with thionine (Thi) to form a signal label capable of generating signals in two independent modes. The label exhibits excellent enzyme-like catalytic activity and enhances electron transfer capabilities. Differential pulse voltammetry (DPV) and square-wave voltammetry (SWV) are employed to independently read signals from the oxidation-reduction reaction of Thi and the catalytic oxidation of hydroquinone (HQ) to benzoquinone (BQ) by H2O2. The introduced DNA dynamic network cascade reaction modularizes sample processing and electrode surface signal generation, avoiding electrode contamination and efficiently increasing the output of the catalyzed hairpin assembly (CHA) cycle. Under optimized conditions, the developed aptasensor demonstrates detection limits of 0.112 (DPV mode) and 0.0203 pg/mL (SWV mode). Additionally, the sensor successfully detected enrofloxacin in real samples, expanding avenues for designing dual-mode signal amplification strategies.

Controlled-Release Electrochemiluminescence Biosensor with Strong Self-On Effect by a Multiple Signal Amplification Strategy for Trace Detection of Prostate-Specific Antigen.

The enhancement of sensitivity in biological analysis detection can reduce the probability of false positives of the biosensor. In this work, a novel self-on controlled-release electrochemiluminescence (CRE) biosensor was designed by multiple signal amplification and framework-enhanced stability strategies. As a result, the changes of the ECL signal were enhanced before and after the controlled-release process, achieving sensitive detection of prostate-specific antigen (PSA). Specifically, for one thing, Fe3O4@CeO2-NH2 with two paths for enhancing the generation of coreactant radicals was used as the coreaction accelerator to boost ECL performance. For another, due to the framework stability, zeolitic imidazolate framework-8-NH2 (ZIF-8-NH2) was combined with luminol to make the ECL signal more stable. Based on these strategies, the constructed CRE biosensor showed a strong self-on effect in the presence of PSA and high sensitivity in a series of tests. The detection range and limit of detection (LOD) were 5 fg/mL to 10 ng/mL and 2.8 fg/mL (S/N = 3), respectively, providing a feasible approach for clinical detection of PSA.

Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri.

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5CFU/mL in a time of 70min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.

Ratiometric fluorescence platform for the ultrasensitive detection of kanamycin based on split aptamer co-recognition triggers Mg2+-DNAzyme-driven DNA walker systems

In this work, a novel 3D-DNA walker signal amplification strategy was designed to construct a fluorescent aptasensor for the detection of kanamycin (KAN). The aptasensor utilizes split aptamers for the synergistic recognition of KAN. The presence of KAN induces the split aptamers recombination to form the Mg2+-DNAzyme structure, which is activated by Mg2+ to drive the 3D-DNA walker process for cascading signal amplification. Employing gold nanoflowers (AuNFs) as walking substrate material increases the local DNA concentration to enhance the walker efficiency. The prepared fluorescent aptasensor achieved efficient and sensitive detection of KAN with satisfactory results in the concentration range of 1 × 10−8 - 1 × 10−3 μg/kg and the detection limit of 5.63 fg/kg. Meanwhile, the designed fluorescent aptasensor exhibited favorable specificity, anti-interference, storage stability and reproducibility, and verified the feasibility of its application in milk samples. The present work provides an effective tool for the regulation of KAN contamination in animal-derived foods with promising prospects.

Antibody-Bridged DNAzyme Walker for Sensitive Detection of Small Molecules.

Sensitive detection of small molecules with biological and environmental interests is important for many applications, such as food safety, disease diagnosis, and environmental monitoring. Herein, we propose a highly selective antibody-bridged DNAzyme walker to sensitively detect small molecules. The antibody-bridged DNAzyme walker consists of a track, small-molecule-labeled DNAzyme walking strand, and antibody against small molecules. The track is built by co-modifying fluorophore-labeled substrates and small-molecule-labeled DNA linkers onto a gold nanoparticle (AuNP). In the absence of the target molecule, the antibody binds small molecule labels at the DNAzyme walking strand and the DNA linker, driving the DNAzyme walking strand on the surface of the AuNP. The attached DNAzyme walking strand moves along the track and cleaves substrates to generate high fluorescence signals to achieve signal amplification. As target molecules exist, they competitively bind with antibody to displace the small-molecule-labeled linker and DNAzyme walking strand, rendering the DNAzyme walker inactive in substrate cleavage and causing weak fluorescence. By using this antibody-bridged DNAzyme walker, we achieved sensitive detection of two biologically important small molecules, digoxin and folic acid. This work provides a new paradigm by combining the signal amplification strategy of a DNA walker and immunorecognition for sensitive and selective detection of small molecules.