Structure-switching Aptamers Research Articles

A DNA Aptamer for Theophylline with Ultrahigh Selectivity Reminiscent of the Classic RNA Aptamer.

Since the report of the RNA aptamer for theophylline, theophylline has become a key molecule in chemical biology for designing RNA switches and riboswitches. In addition, theophylline is an important drug for treating airway diseases including asthma. The classic RNA aptamer with excellent selectivity for theophylline has been used to design biosensors, although DNA aptamers are more desirable for stability and cost considerations. In this work, we selected DNA aptamers for theophylline, and all the top sequences shared the same binding motifs. Binding was confirmed using isothermal titration calorimetry and a nuclease digestion assay, showing a dissociation constant (Kd) around 0.5 μM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a Kd of 9.8 μM. The selectivity for theophylline over caffeine is around 250,000-fold based on a strand-displacement assay, which was more than 20-fold higher compared to the classic RNA aptamer. For other tested analogs, the DNA aptamer also showed better selectivity. Using the structure-switching aptamer sensor design method, a detection limit of 17 nM theophylline was achieved in the selection buffer, and a detection limit of 31 nM was obtained in 10% serum.

Enzymatically Controlled Nanoflares for Specific Molecular Recognition and Biosensing.

In situ sensing of physiological and pathological species in cancer cells is of great importance to unravel their molecular and cellular processes. However, the biosensing with conventional probes is often limited by the undesired on-target off-tumor interference. Here, we report a novel strategy to design enzymatically controlled nanoflares for sensing and imaging molecular targets in tumor cells. The triggerable nanoflare was designed via rational engineering of structure-switching aptamers with the incorporation of an enzyme-activatable site and further conjugation on gold nanoparticles. The nanoflare sensors did not respond to target molecules in normal cells, but they could be catalytically activated by specific enzymes in cancer cells, thereby enabling cancer-specific sensing and imaging in vitro and in vivo with improved tumor specificity. Considering that diverse aptamers were selected, we expect that this strategy would facilitate the precise detection of a broad range of targets in tumors and may promote the development of smart probes for cancer diagnosis.

Structure-switching aptamer triggering signal amplification strategy for tobramycin detection based on hybridization chain reaction and fluorescence synergism.

Based on hybridization chain reaction (HCR) and fluorescence synergism, a novel aptasensor for tobramycin was successfully constructed. Tobramycin competed with cDNA-FAM to bind aptamers immobilized on magnetic beads. After magnetic separation, the released cDNA-FAM acted as initiator to trigger HCR amplification, thus the fluorescence was significantly enhanced due to binding of SYBR Green Ⅰ (SGI) to the formed long double-stranded DNA and the synergistic fluorescence of FAM. In the absence of tobramycin, the initiator was magnetically separated and no HCR occurred, more importantly, graphene oxide can quench the fluorescence of excessive hairpins/SGI and cDNA-FAM, so almost no background signal was detected. This aptasensor can monitor tobramycin in the range of 0.3-50μM with low detection limit of 17.37nM. Due to the potential generality of structure-switching aptamers and effectiveness of fluorescence synergism, this enzyme-free amplification strategy can be extended to other applications by rational design of nucleic acid sequences.

Label-free biosensing of mercury(II) in milk using an aptamer-gated graphene field-effect transistor

• A specific Hg(II) biosensor based on aptamer-modified SGGT is developed. • The sensitivity and selectivity were improved by aptamer and BSA modification. • The biosensor had a high recovery rate for Hg(II) evaluation in real milk samples. Due to the environmental pollution by the growing urban, agricultural and industrial disposals, dairy products are susceptible to heavy metal pollution in the production process. As one of the most toxic heavy metals, mercury cause serious damage to the environment and even public health. Effective monitoring of Hg(II) in large number of diary samples requires the development of rapid assaying methods. In this research work, we report the preparation of a label-free biosensor for Hg(II) using an aptamer-gated graphene field-effect transistor (Apt-SGGT) with the gate electrode modified by a structure-switching aptamer against Hg(II) and blocked with bovine serum albumin. The aptamer immobilized on the gate electrode acts as both a receptor and a reporter for Hg(II) by specifically recognizing Hg(II) through stable T-Hg 2+ -T coordination complex, triggering the folding of the single-stranded aptamer into a duplex structure. The conformational change results in the redistribution of the surface charge on the gate electrode, and produces a large channel current response by the inherent field-effect amplification. This sensing mechanism can effectively enhance the selectivity of the SGGT-based biosensor to mercury ions by significantly reducing the unspecific responses of other interfering cations. The detection limit of Hg(II) by Apt-SGGT sensor was as low as 5 nM, and the linear detection range was from 5 to 200 nM. The sensor can realize the detection of mercury ion residues in milk with a high recovery rate, and has a good application prospect in the detection and analysis of heavy metal residues in complex systems.

High sensitivity detection of tumor cells in biological samples using a multivalent aptamer strand displacement strategy.

Monitoring the cell surface-expressed nucleolin facilitates early cancer diagnosis. Herein, we developed a multivalent aptamer displacement strand duplex strategy on cell membranes using a multi-receptor co-recognition design for improving the sensitivity and specificity of cancer cell recognition with an ultra-low background. The AS1411 aptamer labeled with the FAM fluorophore can be quenched using a partial complementary sequence modified with a BHQ1 tag which is partially hybridized with the AS1411 aptamer to create a receptor-activating aptamer. The multi-AS1411 activable probe based on the strand displacement strategy was constructed using multiple copies of the structure-switching AS1411 aptamer (bearing a short poly-A tail) linked together using the poly-T long chain (as a scaffold) which was synthesized by Terminal Deoxynucleotidyl Transferase (TDT)-mediated extension. We demonstrated the promising efficacy and sensitivity of our method in recognizing tumor cells in both cell mixtures and clinical cytology specimens. Due to its simple and fast operation with excellent cell recognition sensitivity and accuracy, it is expected to achieve the detection of low abundance target cells. Our approach will have broad application in clinical rapid detection and personalized medicine.

Label-free detection of Cu(II) in fish using a graphene field-effect transistor gated by structure-switching aptamer probes

Copper sulfate is a widely used agent to control insects, bacteria and algae for fishery. However, excess amount of copper ions in water accumulate in aquatic products through the ecological cycle system, highly threatening food safety and public health. Therefore, it is urgent to develop a rapid and efficient method for the determination of copper content in aquatic products. In this study, we developed a label-free biosensor for Cu(II) based on a graphene field-effect transistor gated by structure-switching aptamer probes (SSA-GFET) against Cu(II) we obtained before. The detection mechanism of the biosensor is attributed to the surface charge shift and the potential change of the gate electrode upon the specific binding of Cu(II). The SSA-GFET biosensor has a low detection limit of 10 nM and a linear range of 10 nM to 3 μM to Cu(II). In addition to the excellent selectivity to Cu(II), the biosensor also showes the advantage of high recovery rate for detection of Cu(II) in real fish samples. Because of the detection characteristics of label-free SSA-GFET, it has great advantages in the field of food safety and environmental detection.

Modulating the biofunctionality of enzyme-MOF nanobiocatalyst through structure-switching aptamer for continuous degradation of BPA

Encapsulating enzyme within MOF (enzyme-MOF) gives rise to new opportunity to improve the fragility of enzyme, but practical application of enzyme-MOF composite is far from being realized. The development of a novel enzyme-MOF composite system should simultaneously guarantee the enhanced activity and controllably complete recycling, and only in this way can we efficiently and economically utilize the enzyme-MOF composite. Herein, we addressed all these fundamental limitations of current enzyme-MOF composite by establishing aptamer-functionalized enzyme-MOF composite (HRP-ZIF-8@P1). HRP-ZIF-8@P1 relied on automatic structure switch of aptamer-target binding and aptamer-cDNA (complementary DNA) hybridization, achieving effectiveness in self-enriching substrate around HRP-ZIF-8@P1 to boost enzymatic activity first, subsequently hybridizing spontaneously with magnetically controllable cDNA sequence (Fe3O4@P3) to completely recover the HRP-ZIF-8@P1, where preferentially capturing substrate could further induce the release of the hybridized HRP-ZIF-8@P1 for automatically starting the cyclic enzyme catalysis. A 5.6-fold enhancement in the catalytic efficiency for BPA degradation was endowed, and 94.7% catalytic activity was retained for 8 consecutive degradations of BPA, both of which were even more significant than HRP-ZIF-8. Additionally, remarkable stability of HRP-ZIF-8@P1 was afforded by dual-layer protection of ZIF-8 and P1 in denaturing conditions. Taking the possibility of discovering an aptamer for any target into account, the aptamer-functionalized enzyme-MOF composites provide a generic and simple guide for simultaneously boosting enzymatic activity and controllably full recycling the enzyme-MOF systems, accelerating their commercial utilizations.

Aptamer-Based Fluorescence Quenching Approach for Detection of Aflatoxin M1 in Milk.

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, is a feed and food contaminant of global concern. In this study, we developed a fast and simple method for detection of AFM1 based on a structure-switching signaling aptamer. This aptasensor is based on the change in fluorescence signal due to formation of an AFM1/aptamer complex. To generate the aptasensor, the specific aptamer was modified with FAM (carboxyfluorescein), and their complementary DNAs (cDNA) were modified with a carboxytetramethylrhodamine (TAMRA) quenching group. In the absence of AFM1, the aptamers were hybridized with cDNA, resulting in quenching of the aptamer fluorescence due to the proximity of the aptamer’s fluorophore to the quenching group on the cDNA. On the other hand, in the presence of AFM1, a structural switch in the aptamer was induced by formation of an AFM1/aptamer complex. Changes in the structure of the aptamer led to the release of the cDNA, causing the generation of a fluorescence signal. Thus, AFM1 concentrations could be quantitatively monitored based on the changes in fluorescences. Under optimized conditions, this assay exhibited a linear response to AFM1 in the range of 1–100 ng/mL and a limit of detection of 0.5 ng/mL was calculated. This proposed aptasensor was applied to milk samples spiked with a dilution series of AFM1, yielding satisfactory recoveries from 93.4 to 101.3%. These results demonstrated that this detection technique could be useful for high-throughput and quantitative determination of mycotoxin levels in milk and dairy products.

Structure-switching aptamer sensors for the specific detection of piperaquine and mefloquine.

Tracking antimalarial drug use and efficacy is essential for monitoring the current spread of antimalarial drug resistance. However, available methods for determining tablet quality and patient drug use are often inaccessible, requiring well-equipped laboratories capable of performing liquid chromatography-mass spectrometry (LC-MS). Here, we report the development of aptamer-based fluorescent sensors for the rapid, specific detection of the antimalarial compounds piperaquine and mefloquine-two slow-clearing partner drugs in current first-line artemisinin-based combination therapies (ACTs). Highly selective DNA aptamers were identified that bind piperaquine and mefloquine with dissociation constants (K d's) measured in the low nanomolar range via two independent methods. The aptamers were isolated from a library of single-stranded DNA molecules using a capture-systematic evolution of ligands by exponential enrichment (SELEX) technique and then adapted into structure-switching aptamer fluorescent sensors. Sensor performance was optimized for the detection of drug from human serum and crushed tablets, resulting in two sensing platforms. The patient sample platform was validated against an LC-MS standard drug detection method in samples from healthy volunteers and patients with malaria. This assay provides a rapid and inexpensive method for tracking antimalarial drug use and quality for the containment and study of parasite resistance, a major priority for malaria elimination campaigns. This sensor platform allows for flexibility of sample matrix and can be easily adapted to detect other small-molecule drugs.

Upconversion Luminescence-Controlled DNA Computation for Spatiotemporally Resolved, Multiplexed Molecular Imaging.

DNA-based molecular circuits able to perform complex information processing in biological systems are highly desirable. However, conventional DNA circuits are constitutively always in an ON state and immediately operate when they meet the biomolecular inputs, precluding precise molecular computation at a desired time and in a desired site. In this work, we report a conceptual methodology for the construction of photonic nanocircuits that enable DNA molecular computation in vitro and in vivo with high spatial precision. Upon remote activation by spatially restricted NIR-light input, two types of cancer biomarker inputs can sequentially trigger conformational changes of the DNA circuit through a structure-switching aptamer and toehold-mediated strand exchange, leading to release of a signaling output. Of note, the NIR-light-gated nanocircuit allows for intended control over the specific timing and location of DNA computation, providing spatial and temporal capabilities for multiplexed imaging. Furthermore, an OR-AND-gated nanocircuit of higher complexity was designed to illustrate the versatility of our approach. The present work illustrates the potential of the use of upconversion nanotechnology as a regulatory tool for spatial and temporal control of DNA computation in cells and animals.

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors.

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

Selection and characterization of structure-switching DNA aptamers for the salivary peptide histatin 3

In this study, single-stranded DNA aptamers that switch structural conformation upon binding to the salivary peptide histatin 3 have been reported for the first time. Histatin 3 is an antimicrobial peptide that possesses the capability of being a therapeutic agent against oral candidiasis and has recently been linked as a novel biomarker for acute stress. The aptamers were identified through a library immobilization version of an iterative in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment (SELEX). Through the SELEX process, four unique aptamer candidates sharing a consensus sequence were identified. These selected sequences exhibited binding affinity and specificity to histatin 3 and in order to further characterize these aptamers, a direct format enzyme-linked aptamer sorbent assay (ELASA) was developed. The best performing candidate demonstrated an equilibrium dissociation constant (Kd) value of 1.97 ± 0.48 μM. These novel aptamers have the potential to lead to the further development of refined sensing assays and platforms for the detection and quantification of histatin 3 in human saliva and other biological media.

A multimode aptasensor based on hollow gold nanoparticles and structure switching of aptamer: Fast and sensitive detection of carcinoembryonic antigen

A fast and sensitive carcinoembryonic antigen (CEA) aptasensor with multiple detection modes was developed. It is based on hollow gold nanoparticles (HGNPs) and horse radish peroxidase (HRP) as both labels and signal amplification elements. In fabrication, capture DNA (cpDNA) was modified on magnetic beads, and reporter DNA (rpDNA) and HRP were co-modified on HGNPs. A combination DNA, containing a DNA aptamer (GAC-P) segment, hybridized with cpDNA and rpDNA, forming a satellite-structured biosensor. In response to CEA, the aptamer switches its structure, releasing rpDNA/HGNPs/HRP under magnetic separation. The surface plasmon resonance of HGNPs and the HRP enzyme activity of the supernatant were observed/detected with naked eyes, UV–vis spectroscopy, and electrochemistry techniques. The assay time is as short as ~45 min. CEA levels can be differentiated with naked eyes. The absorbance at 677 nm is linearly proportional to CEA concentration from 10 to 250 ng mL−1, with a limit of detection (LOD) of 8.6 ng mL−1. In contrast, the differential pulse voltammetry (DPV) peak current at -0.057 V is linearly correlated with CEA concentration in the range of 2−250 ng mL−1, with an LOD of 0.78 ng mL−1. The LOD values are lower than cancer patient threshold levels. The aptasensor exhibits negligible responses to interfering proteins. In addition, it displays good reproducibility, stability, accuracy and easiness of usage. The recovery rate for CEA spiked human serum samples are 100±5%. The developed assay strategy should provide a general platform for detecting other interested biomarkers.

Design and Analysis of a Sample-and-Hold CMOS Electrochemical Sensor for Aptamer-based Therapeutic Drug Monitoring.

In this paper, we present the design and the analysis of an electrochemical circuit for measuring the concentrations of therapeutic drugs using structure-switching aptamers. Aptamers are single-stranded nucleic acids, whose sequence is selected to exhibit high affinity and specificity toward a molecular target, and change its conformation upon binding. This property, when coupled with a redox reporter and electrochemical detection, enables reagent-free biosensing with a sub-minute temporal resolution for in vivo therapeutic drug monitoring. Specifically, we design a chronoamperometry-based electrochemical circuit that measures the direct changes in the electron transfer (ET) kinetics of a methylene blue reporter conjugated at the distal-end of the aptamer. To overcome the high-frequency noise amplification issue when interfacing with a large-size (> 0.25 mm2) implantable electrode, we present a sample-and-hold (S/H) circuit technique in which the desired electrode potentials are held onto noiseless capacitors during the recording of the redox currents. This allows disconnecting the feedback amplifiers to avoid its noise injection while reducing the total power consumption. A prototype circuit implemented in 65-nm CMOS demonstrates a cell-capacitance-insensitive input-referred noise (IRN) current of 15.2 pArms at a 2.5-kHz filtering bandwidth. We tested our system in human whole blood samples and measured the changes in the ET kinetics from the redox-labeled aptamers at different kanamycin concentrations. By employing principal component analysis (PCA) to compensate for the sampling errors, we report a molecular noise floor (at SNR = 1) of 3.1 µM with sub 1-sec acquisition time at 0.22-mW power consumption.

Engineering G-quadruplex aptamer to modulate its binding specificity.

The use of aptamers in bioanalytical and biomedical applications exploits their ability to recognize cell surface protein receptors. Targeted therapeutics and theranostics come to mind in this regard. However, protein receptors occur on both cancer and normal cells; as such, aptamers are now taxed with identifying high vs. low levels of protein expression. Inspired by the flexible template mechanism and elegant control of natural nucleic acid-based structures, we report an allosteric regulation strategy for constructing a structure-switching aptamer for enhanced target cell recognition by engineering aptamers with DNA intercalated motifs (i-motifs) responsive to the microenvironment, such as pH. Structure-switching sensitivity can be readily tuned by manipulating i-motif sequences. However, structure-switching sensitivity is difficult to estimate, making it equally difficult to effectively screen modified aptamers with the desired sensitivity. To address this problem, we selected a fluorescent probe capable of detecting G-quadruplex in complicated biological media.

A “turn-on” electrochemical aptasensor for ultrasensitive detection of Cd2+ using duplexed aptamer switch on electrochemically reduced graphene oxide electrode

We report a versatile “turn-on” sensing strategy for the ultratrace level detection of Cd2+ ion based on structure-switching aptamer duplex which is immobilized on electrochemically reduced graphene (ERGO) electrode. This approach utilizes a methylene blue (MB) tagged aptamer probe (Apt) and its partially complimentary DNA (cDNA), designed to form aptamer duplex (cDNA-Apt) which is immobilized on the ERGO electrode. The double helix structure of cDNA-Apt on the ERGO electrode impedes the efficient electron transfer of MB to the electrode, due to the far proximity of MB and electrode, resulting in the decreased oxidation peak current of MB. The presence of analytes causes cDNA-Apt duplex to unwind and release the cDNA which can quantitatively facilitate the efficiency of electron transfer of MB, leading to the enhancement of electrochemical signal. It is noted that we can successfully achieve ultrasensitivity of the sensor owing to the large conformational change of Apt from the proposed aptamer architecture and “turn-on” mode sensing upon target binding. This sensing platform can be used to monitor Cd2+ from 1 fM to 1 nM and very low detection limit of 0.65 fM (S/N = 3), which is among the best in all the reported electrochemical aptasensors.

Detection of ATP from “fluorescence” to “enhanced fluorescence” based on metal-enhanced fluorescence triggered by aptamer nanoswitch

Herein, we demonstrate a novel approach to detect intracellular adenosine triphosphate (ATP) from “fluorescence” to “enhanced fluorescence” by metal-enhanced fluorescence (MEF), which is an activatable means to amplify fluorescence signal. In the meantime, hybridization chain reaction (HCR) is employed to aggregate DNA on the substrate of MEF, and the length of generated DNA strands is controllable. ATP target molecules specifically bind to aptamer, which is designed as the recognition part of the nanoswitch, and the sticky ends are released. Then, HCR is triggered, bringing Cy7 modified on H1 and H2 close to the surface of gold nanobipyramids (AuNBPs) substrate. The fluorescence signal is amplified with the ATP concentration increasing owing to MEF. Thus, the strategy based on structure-switching aptamer triggering metal-enhanced fluorescence (SATMEF) for Cy7 is designed to detect ATP via “enhanced fluorescence” signals, and the maximum fluorescence enhancement by the approach of SATMEF reaches 5.63-fold. We applied this DNA nanoswitch for quantitative analysis to ATP, and the results show that the intensity of enhanced Cy7 fluorescence is positively correlated with the ATP concentration in the range of 0.2−10 μM, with a detection limit of 35 nM. We believe that the SATMEF for quantitative detection by signal from “fluorescence” to “enhanced fluorescence” provides a new approach for those fluorophores which are superior but difficult to be quenched to construct DNA nanoswitches.

Probing the Mechanism of Structure-Switching Aptamer Assembly by Super-Resolution Localization of Individual DNA Molecules.

Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, l-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring the rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.

Analysis of the role played by ligand-induced folding of the cocaine-binding aptamer in the photochrome aptamer switch assay

The Photochrome Aptamer Switch Assay (PHASA) relies on ligand binding by an aptamer to alter the local environment of a stilbene compound covalently attached to the 5’ end of the aptamer. We used the PHASA with both structure switching and non-structure switching versions of the cocaine-binding aptamer. We show that the largest change in fluorescence intensity and the lowest concentration limit of detection (CLooD) is obtained using the structure-switching cocaine-binding aptamer. Fluorescence anisotropy measurements were used to quantify the affinity of the conjugated aptamer to cocaine. We also used thermal melt analysis and Nuclear Magnetic Resonance (NMR) spectroscopy to show that the addition of the stilbene to the aptamer increases the melt temperature of the cocaine-bound structure-switching aptamer by (6.4 ± 0.3) °C compared to the unconjugated aptamer while the free form of the structure-switching aptamer-stilbene conjugate remains unfolded.

Multicolor colorimetric detection of ochratoxin A via structure-switching aptamer and enzyme-induced metallization of gold nanorods

Colorimetric aptasensors have been intensively studied for the ochratoxin A (OTA) detection, but they mostly exhibit just one-color change, resulting in poor visual resolution and limited use for semi-quantitative analysis. Thus, we designed a high-resolution colorimetric assay on the basis of aptamer structural switching and enzyme-induced metallization of gold nanorods (AuNRs). DNA-alkaline phosphatase (ALP)-immobilized magnetic beads were prepared. The aptamer bounded to OTA to form G-quadruplexes, releasing ALP-labelled complementary DNA (cDNA-ALP). After magnetic separation, cDNA-ALP catalyzed the decomposition of ascorbic acid 2-phosphate to ascorbic acid that reduced Ag+, forming an Ag shell on the surface of AuNRs. This caused a blue-shift of the longitudinal local surface plasmon resonance peak of the AuNRs and a naked eye visible multicolor change. Under optimal conditions, the assay exhibited a 9.0 nM detection limit for OTA, with high specificity. This method is promising for the on-site visual semi-quantitative detection of mycotoxins in foods.