Target-specific Aptamer Research Articles

Magnetic graphene oxide and vertically-ordered mesoporous silica film for universal and sensitive homogeneous electrochemiluminescence aptasensor platform

Herein, we demonstrated a convenient and highly sensitive homogeneous electrochemiluminescence (ECL) aptasensor platform by combination of magnetic composite probe and vertically-ordered mesoporous silica films (VMSF). Magnetic composite probe composed of biorecognition element (target-specific aptamer), ECL signal reporter (Ru(bpy)32+) and large-scale magnetic graphene oxide (M−GO), can be prepared by simple incubation, magnetic separation and redispersion prior to use. High-affinity and specific binding between target and aptamer can detach aptamer from M−GO and simultaneously release the slight amount of Ru(bpy)32+ into a solution, which can be sensitively detected by VMSF modified indium tin oxide (VMSF/ITO) due to the excellent electrostatic preconcentration capacity of VMSF. In this sensing system, detectable signal of Ru(bpy)32+ can be remarkably amplified by VMSF/ITO and meanwhile background signal can be reduced by the adsorption capacity of M−GO and size exclusion property of VMSF/ITO, effectively promoting the analytical performance of the present homogeneous ECL aptasensor. The applicability and universality of this sensing platform was confirmed by employing alpha fetoprotein (AFP) and prostate specific antigen (PSA) as the target models and their detection limits were 0.08 pg/mL and 9.6 pg/mL, respectively. Furthermore, the designed homogeneous ECL aptasensor platform is label-free, immobilization-free, low-cost, and free of additional electrode modification and sample pretreatment procedures, rendering a simple sensing strategy for quantitative determination of various biomarkers in clinical diagnosis by adapting the specific aptamer.

Polymeric integration of structure-switching aptamers on transistors for histamine sensing.

Aptamers that undergo large conformational rearrangements at the surface of electrolyte-gated field-effect transistor (EG-FETs)-based biosensors can overcome the Debye length limitation in physiological high ionic strength environments. For the sensitive detection of small molecules, carbon nanotubes (CNTs) that approach the dimensions of analytes of interest are promising channel materials for EG-FETs. However, functionalization of CNTs with bioreceptors using frequently reported surface modification strategies (e.g., π-π stacking), requires highly pristine CNTs deposited through methods that are incompatible with low-cost fabrication methods and flexible substrates. In this work, we explore alternative non-covalent surface chemistry to functionalize CNTs with aptamers. We harnessed the adhesive properties of poly-D-lysine (PDL), to coat the surface of CNTs and then grafted histamine-specific DNA aptamers electrostatically in close proximity to the CNT semiconducting channel. The layer-by-layer assembly was monitored by complementary techniques such as X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and fluorescence microscopy. Surface characterization confirmed histamine aptamer integration into PDL-coated CNTs and revealed ∼5-fold higher aptamer surface coverage when using CNT networks with high surface areas. Specific aptamers assembled on EG-CNTFETs enabled histamine detection in undiluted high ionic strength solutions in the concentration range of 10 nM to 100 μM. Sequence specificity was demonstrated via parallel measurements with control EG-CNTFETs functionalized with scrambled DNA. Histamine aptamer-modified EG-CNTFETs showed high selectivity vs. histidine, the closest structural analog and precursor to histamine. Taken together, these results implied that target-specific aptamer conformational changes on CNTs facilitate signal transduction, which was corroborated by circular dichroism spectroscopy. Our work suggests that layer-by-layer polymer chemistry enables integration of structure-switching aptamers into flexible EG-CNTFETs for small-molecule biosensing.

Electrochemical assisted enhanced nanozymatic activity of functionalized borophene for H2O2 and tetracycline detection

This study unveils the electrochemically-enhanced nanozymatic activity exhibited by borophene during the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2. Herein, the surface of the pristine borophene was first modified with the addition of thiocyanate groups to improve hydroxyl radical (•OH) scavenging activity. Then, the oxidation reaction of TMB was accelerated under applied electrochemical potential. Both factors significantly improved the detection limit and drastically decreased the detection time. DPPH testing revealed that the radical scavenging nature of borophene was more than 70%, boosting its catalytic activity. In the presence of H2O2, borophene catalyzed the oxidation of TMB and produced a blue-colored solution that was linearly correlated with the concentration of H2O2 and allowed for the detection of H2O2 up to 38 nM. The present finding was further extended to nanozymatic detection of tetracyclines (TCs) using a target-specific aptamer, and the results were colorimetrically quantifiable up to 1 μM with a LOD value of 150 nM. Moreover, transferring the principles of the discussed detection method to form a portable and disposable paper-based system enabled the quantification of TCs up to 0.2 μM. All the sensing experiments in this study indicate that the nanozymatic activity of borophene has significantly improved under electrochemical potential compared to conventional nanozyme-based colorimetric detection. Hence, the present discovery of electrochemically-enhanced nanozymatic activity would be promising for various sensitive and time-dependent colorimetric sensor development initiatives in the future.

A liquid crystal-based sensor exploiting the aptamer-mediated recognition at the aqueous/liquid crystal interface for sensitive detection of serotonin.

We report here a liquid crystal (LC)-based sensor for detecting serotonin (5-HT); the proposed sensor uses target-specific aptamer recognition at a cationic surfactant decorated-aqueous/LC interface. Our detection strategy focuses on the orientational transition of LCs upon biological interactions at the interface. In this sensing system, the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) forms a self-assembled monolayer at the aqueous/LC interface and triggers the homeotropic orientation of LCs. After introducing the 5-HT specific aptamer, an electrostatic attraction occurs between the cationic CTAB and anionic aptamer. This interaction destructs the surfactant monolayer at the interface, inducing reorganization of LC alignment from homeotropic to tilted conditions. In the increasing 5-HT levels, specific binding between 5-HT and the aptamer diminishes the interaction between the aptamer and CTAB, thereby maintaining the homeotropic alignment of LCs. The orientational transition of the LCs was observed under a polarized optical microscope. The developed biosensor has a linear detection range from 1 to 1000nM and a detection limit of 1.68nM. Moreover, the sensor was applied to a human urine sample and a detection limit of 2.25nM was obtained. Overall, the designed LC-based sensor is a sensitive, simple, cost effective, and selective platform for detecting 5-HT in aqueous solutions.

Culture-Free Quantification of Bacteria Using Digital Fluorescence Imaging in a Tunable Magnetic Capturing Cartridge for Onsite Food Testing.

Accurate, onsite detection of pathogenic bacteria from food matrices is required to rapidly respond to pathogen outbreaks. However, accurately detecting whole-cell bacteria in large sample volumes without an enrichment step remains a challenge. Therefore, bacterial samples must be concentrated, identified, and quantified. We developed a tunable magnetic capturing cartridge (TMCC) and combined it with a portable digital fluorescence reader for quick, onsite, quantitative detection of Staphylococcus aureus. The TMCC platform integrates an absorption pad impregnated with water-soluble polyvinyl alcohol (PVA) with an injection-molded polycarbonate (PC) plate that has a hard magnet on its back and an acrylonitrile-butadiene-styrene case. An S. aureus-specific antibody conjugated with magnetic nanoparticles was used to concentrate bacteria from a large-volume sample and capture bacteria within the TMCC. The retention time for capturing bacteria on the TMCC was adjusted by controlling the concentration and volume of the PVA solution. Concentrated bacterial samples bound to target-specific aptamer probes conjugated with quantum dots were loaded into the TMCC for a controlled time, followed by attachment of the bacteria to the PC plate and removal of unbound aptamer probes with wash buffer. The captured bacteria were quantified using a digital fluorescence reader equipped with an embedded program that automatically counts fluorescently tagged bacteria. The bacterial count made using the TMCC was comparable to a standard plate count (R2 = 0.9898), with assay sensitivity and specificity of 94.3 and 100%, respectively.

Gold nanoparticle-assisted SELEX as a visual monitoring platform for the development of small molecule-binding DNA aptasensors

To resolve time-consuming and imperceptible monitoring problems in the traditional systematic evolution of ligands by exponential enrichment (SELEX), we report gold nanoparticle-assisted SELEX (GNP-SELEX) as a visual, proofreading, and self-monitoring platform and its application to small molecule-binding single-stranded DNA (ssDNA) aptasensors. Through the colorimetric changes between rounds, GNP-SELEX enabled the rapid determination of target-specific aptamer library enrichment with neither target modification nor extra monitoring process. We identified ssDNA aptamers with high selectivity and binding affinity by targeting two small molecules (brassinolide; BL and bisphenol A; BPA) as a model. The rational design of selected aptamers by 3D molecular simulation increased their ability to detect BL or BPA in real samples as bioreceptors. These results suggest that GNP-SELEX is useful as a self-monitoring platform to discover ssDNA aptamers as well as to develop aptasensors for diverse targets in a rapid and simple way.

Aptamer-Conjugated Polydiacetylene Colorimetric Paper Chip for the Detection of Bacillus thuringiensis Spores.

A colorimetric polydiacetylene (PDA) paper strip sensor that can specifically recognize Bacillus thuringiensis (BT) HD-73 spores is described in this work. The target-specific aptamer was combined with PDA, and the aptamer-conjugated PDA vesicles were then coated on polyvinylidene fluoride (PVDF) paper strips by a simple solvent evaporation method. The PDA-aptamer paper strips can be used to detect the target without any pre-treatment. Using the paper strip, the presence of BT spores is directly observable by the naked eye based on the unique blue-to-red color transition of the PDA. Quantitative studies using the paper strip were also carried out by analyzing the color transitions of the PDA. The specificity of this PDA sensor was verified with a high concentration of Escherichia coli, and no discernable change was observed. The observable color change in the paper strip occurs in less than 1 h, and the limit of detection is 3 × 107 CFU/mL, much below the level harmful to humans. The PDA-based paper sensor, developed in this work, does not require a separate power or detection device, making the sensor strip highly transportable and suitable for spore analysis anytime and anywhere. Moreover, this paper sensor platform is easily fabricated, can be adapted to other targets, is highly portable, and is highly specific for the detection of BT spores.

Dual amplification in a fluorometric acetamiprid assay by using an aptamer, G-quadruplex/hemin DNAzyme, and graphene quantum dots functionalized with D-penicillamine and histidine.

D-penicillamine and histidine-functionalized graphene quantum dot (DPA-GQD-His) was synthesized and applied in a fluorometric method for determination of acetamiprid using a G-quadruplex DNAzyme. At first DNA probe (probe 1) consists of a target-specific aptamer with two arms of DNA segments. Probe 1 was hybridized with DNA probe 2 composed of a single DNA sequence with two split G-rich DNA sequences. This leads to the formation of a triplex-to-G-quadruplex (TPGQ). Next, acetamiprid was hybridized with the aptamer in the TPGQ to release free DNA probe 2. The released probe 2, in the presence of of K+, undergoes a structural change into a stem-loop structure (by self-complementary hybridization and Hoogsteen hydrogen bonding) that bears a G-quadruplex structure. This is followed by conjugation with hemin to form the G-quadruplex/hemin DNAzyme. The DNAzyme catalyzes the oxidation of o-phenylenediamine by H2O2 to produce a yellow fluorescent product with excitation/emission maxima at 420/560nm. The oxidation product interacts with DPA-GQD-His to achieve a rapid energy transfer between DPA-GQD-His and oxidation product. This increases the fluorescence of the oxidation product and quenches the fluorescence of DPA-GQD-His. DPA-GQD-His also improves the catalytic activity of DNAzyme towards oxidation of ophenylenediamine oxidization and enhances fluorometric response to acetamiprid. The assay works in the 1.0 fM to 1.0nM acetamiprid concentration range and has a 0.38 fM detection limit. It was successfully applied to the determination of acetamiprid in tea. Graphical abstractThe study reported one double amplification strategy for ultrasensitive fluorescence detection of acetamiprid in tea with D-penicillamine and histidine-functionalized graphene quantum dots and G-quadruplex/heminDNAzyme. The analtyical method exhibits ultra high sensitivity, selectivity and rapidity of fluorescenceresponse to acetamiprid.

A simple paper-based aptasensor for ultrasensitive detection of lead (II) ion

In this study, a simple paper-based aptasensor has been developed for the ultrasensitive detection of lead (Pb2+) ion within about 10 min. The aptasensor has been successfully designed by taking advantages of the Förster Resonance Energy Transfer (FRET) process and the super fluorescence quenching property of graphene oxide (GO) sheet. The sensing mechanism of the aptasensor is based on the conformational switch of the Pb2+-specific aptamer from a random coil to a G-quadruplex structure. An injection of Pb2+ on the paper-based platform induces the release of the specific aptamer from the GO surface that recovers the fluorescence emission. Under the optimal experimental conditions, there is a good linear relationship between the fluorescence recovery and the Pb2+concentration in the ranges of 5–70 pM and 0.07–20 nM. Moreover, the aptasensing array exhibits a high sensitivity to Pb2+ with an ultra-low detection limit of 0.5 pM. The developed aptasensor has been successfully applied to determine Pb2+ in tap water, lake water, milk, and human blood serum. The paper-based aptasensor can be efficiently utilized to detect other metal ions and biological molecules by substituting target specific aptamer.

In vitro selection of CD70 binding aptamer and its application in a biosensor design for sensitive detection of SKOV-3 ovarian cells

Single-stranded DNA aptamers recognizing CD70 molecule overexpressed in some tumor cell lines was isolated by means of systematic evolution of ligands by exponential enrichment (SELEX). Both purified CD70 protein and CD70-expressing cells were used to isolate target-specific aptamer. After certain rounds of protein-based SELEX and cell-SELEX (ten and seven rounds, respectively), Aptamer 928 (hereafter Apt928) with high affinity and specificity for CD70 was obtained. The specific binding of the aptamer to its target could block interaction of CD70 with CD27 (known as CD70 receptor). Dissociation constant of Apt928 was calculated to be 66 nmol L−1. Moreover, ATTO 674N-labeled Apt928 was applied as a fluorescent aptasensor for rapid and sensitive detection of SKOV-3 cells as CD70-positive cells. The detection limit of this aptasensor was 14 cells mL−1.

Multiplexed CVD Biomarker Detection in Human Serum Using Aptamer Immobilized High Electron Mobility Transistor

Cardiovascular diseases (CVDs) are the major causes of death in the world today. Identifying and managing CVD risk is of primary importance in efficient patient care. Proper risk assessment strategies can provide information to healthy as well as high risk individuals on how to better manage their health. This can greatly reduce the mortality rate of cardiovascular disease. Using protein based cardiac biomarkers for risk assessment can improve early diagnosis and aid in prognosis of CVDs. Semiconductor based electronic sensors are very attractive for rapid and sensitive protein detection. Highly Electron Mobility Transistor (HEMT) based sensors have been used in a variety of applications such as ion, gas and biomolecule sensing and possess desirable features such as high sensitivity and resistance to harsh chemical environment. In this research, A unique sensing methodology using AlGaN/GaN HEMT has been developed to directly detect target protein in high salt concentration environment. The target protein was captured on the surface of the gate electrode, which is separated from the transistor channel by a small gap, and functionalized with target specific aptamer. By forming an electrical double layer (EDL) structure, the solution capacitance modulates the FET drain current. In this way, highly sensitive determination of cardiac biomarkers in high ionic strength media such as 1X PBS and human serum can be realized. HEMT is fabricated using 4 mask processes: mesa isolation and defining active device area, ohmic contact deposition and annealing, metal interconnect deposition and device passivation. For electrical measurement, a pulsed gate electrode voltage is supplied to the gate electrode which creates a solution capacitance in the gap between the gate electrode and transistor dielectric. This solution capacitance modulates the potential drop across the transistor dielectric and hence the drain current. The solution capacitance changes when the device is functionalized with the target specific aptamer and when the target protein binds to the receptor. The aptamer based AlGaN/GaN HEMT biosensor can detect target proteins with high sensitivity, selectivity and a wide dynamic range of detection in 1X PBS (with 4% BSA) and clinical human serum samples.

Orchestrating a Symphony on a Single Conjugate: Aptamer Targeting, Gene Silencing, and Immunomodulation to Enhance Antitumor Response

Over the past decade, immunotherapy has rapidly changed the landscape of medicine, especially oncology. Checkpoint inhibitors and monoclonal antibodies capable of deploying the body’s own immune functions against diseased cells have been heralded as a soon-to-be standard of care for cancer. The excitement around immunotherapy has been accompanied by impressive results, although some are tailored only to a limited number of patients and others are accompanied by substantial toxicity profiles.1 Therefore, there are still unmet needs within the field.

Aptamer contained triple-helix molecular switch for rapid fluorescent sensing of acetamiprid

In this study, an aptamer-based fluorescent sensing platform using triple-helix molecular switch (THMS) was developed for the pesticide screening represented by acetamiprid. The THMS was composed of two tailored DNA probes: a label-free central target specific aptamer sequence flanked by two arm segments acting as a recognition probe; a hairpin-shaped structure oligonucleotide serving as a signal transduction probe (STP), labeled with a fluorophore and a quencher at the 3′ and 5′-end, respectively. In the absence of acetamiprid, complementary bindings of two arm segments of the aptamers with the loop sequence of STP enforce the formation of THMS with the “open” configuration of STP, and the fluorescence of THMS is on. In the presence of target acetamiprid, the aptamer-target binding results in the formation of a structured aptamer/target complex, which disassembles the THMS and releases the STP. The free STP is folded to a stem loop structure, and the fluorescence is quenched. The quenched fluorescence intensity was proportional to the concentration of acetamiprid in the range from 100 to 1200nM, with the limit of detection (LOD) as low as 9.12nM. In addition, this THMS-based method has been successfully used to test and quantify acetamiprid in Chinese cabbage with satisfactory recoveries, and the results were in full agreement with those from LC-MS. The aptamer-based THMS presents distinct advantages, including high stability, remarkable sensitivity, and preservation of the affinity and specificity of the original aptamer. Most importantly, this strategy is convenient and generalizable by virtue of altering the aptamer sequence without changing the triple-helix structure. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.

A pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration

We present here a pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This pH-responsive activatable aptamer probe is composed of two single-stranded DNA. One was used for target recognition, containing a central, target specific aptamer sequence at the 3'-end and an extension sequence at the 5'-end with 5-carboxytetramethylrhodamine (TAMRA) label (denoted as strand A). The other (strand I), being competent to work on the formation of i-motif structure, contained four stretches of the cytosine (C) rich domain and was labeled with a Black Hole Quencher 2 (BHQ2) at the 3'-end. At neutral or slightly alkaline pH, strand I was hybridized to the extension sequence of strand A to form a double-stranded DNA probe, termed i-motif-based activatable aptamer probe (I-AAP). Because of proximityinduced energy transfer, the I-AAP was in a “signal off” state. The slightly acidic pH enforced the strand I to form an intramolecular i-motif and then initiated the dehybridization of I-AAP, leading to fluorescence readout in the target recognition. As a demonstration, AS1411 aptamer was used for MCF-7 cells imaging. It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment. The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues. These results implied the I-AAP strategy is promising as a novel approach for cancer imaging.

Sensitive colorimetric detection of K(I) using catalytically active gold nanoparticles triggered signal amplification

In this work, we report a simple, ultrasensitive, and feasible colorimetric assay for metal ion (K+, used as a model) via inherent peroxidase-like enzymatic amplification strategy of gold nanoparticles (AuNPs). It is shown that peroxidase-like activity of AuNPs can be improved dramatically by its surface activation with target-specific aptamer molecules. Whereas when the target exists, the aptamers leave the surface of AuNPs in a target concentration-dependent manner, resulting in a decrease of the nanoenzymatic catalytic ability of AuNPs. Thus, K+ can be quantified in the presence of AuNPs by using a colorimetric sensing probe (3,3′,5,5′-tetramethylbenzidine). The color change of the solution is relevant to the dose of the target, and this can be achieved with the naked eyes and monitored by UV–vis spectrometry. A linear dependence between the absorbance and target K+ concentration is obtained under optimal conditions in the range from 0. 1nM to 1μM with a detection limit (LOD) of 0.06nM estimated at the 3Sblank level. The sensitivity displays to be 2–9 orders of magnitude better than those of other K+ detection methods. This sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interest.

Label-free DNA Y junction for bisphenol A monitoring using exonuclease III-based signal protection strategy.

A label-free DNA Y junction sensing platform for the amplified detection of bisphenol A (BPA) has been constructed by the ingenious combination of toehold-mediated strand displacement and exonuclease III (Exo III)-based signal protection strategy. Three hairpin probes were utilized as the building blocks to fabricate the DNA Y junction with cascaded signal amplification via a series of toehold-mediated strand displacement reactions. Exo III was employed as a protecting agent for the first time to keep the Y-shaped molecular architecture intact, thereby greatly enhancing the fluorescence intensity of DNA intercalator SYBR Green I. The resulting biosensor exhibits ultrasensitivity towards BPA at low concentration (5 fM) without any labeling, modification, immobilization, or washing procedure. Our proposed sensing system also displays remarkable specificity to BPA against other possible interference molecules. Moreover, this DNA junction biosensor is robust and can be applied to the reliable monitoring of spiked BPA in environmental water samples with good recovery and accuracy. With the successful demonstration for BPA detection, the label-free DNA Y junction can be readily expanded to monitor other analytes in a simple, cost-effective, and ultrasensitive way by substituting the target-specific aptamer sequence.

Aptamer/target binding-induced triple helix forming for signal-on electrochemical biosensing

Owing to its diversified structures, high affinity, and specificity for binding a wide range of non-nucleic acid targets, aptamer is a useful molecular recognition tool for the design of various biosensors. Herein, we report a new signal-on electrochemical biosensing platform which is based on an aptamer/target binding-induced strand displacement and triple-helix forming. The biosensing platform is composed of a signal transduction probe (STP) modified with a methylene blue (MB) and a sulfhydryl group, a triplex-forming oligonucleotides probe (TFO) and a target specific aptamer probe (Apt). Through hybridization with the TFO probe and the Apt probe, the self-assembled STP on Au electrode via Au–S bonding keeps its rigid structure. The MB on the STP is distal to the Au electrode surface. It is eT off state. Target binding releases the Apt probe and liberates the end of the MB tagged STP to fold back and form a triplex-helix structure with TFO (STP/TFO/STP), allowing MB to approach the Au electrode surface and generating measurable electrochemical signals (eT ON). As test for the feasibility and universality of this signal-on electrochemical biosensing platform, two aptamers which bind to adenosine triphosphate (ATP) and human α-thrombin (Tmb), respectively, are selected as models. The detection limit of ATP was 7.2nM, whereas the detection limit of Tmb was 0.86nM.

Aptamer Biosensor for Selective and Rapid Determination of Insulin

Insulin plays an important role in glucose metabolism and its detection in biological fluids is of interest. In this study, a triple-helix molecular switch was employed for the simple, sensitive, and rapid determination of insulin. The triple-helix molecular switch was composed of a target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide acting as a signal transduction probe. This approach takes advantage of unique properties of aptamers and triple-helix molecular switches such as high affinity, selectivity, and stability. In the absence of insulin, the fluorescence of triple-helix molecular switch is on. Upon addition of insulin, the aptamer binds to its target, leading to the release of the signal transduction probe, folding of the signal transduction probe to a stem loop structure, and the quenching of the fluorescence. This sensor showed a high selectivity toward insulin and a limit of detection as low as 9.97 nM. The sensor was employed for the determination of insulin in biological samples. This platform may be generalizable for a variety of molecules.

Aptamer-controlled reversible inhibition of gold nanozyme activity for pesticide sensing.

This study addresses the need for rapid pesticide (acetamiprid) detection by reporting a new colorimetric biosensing assay. Our approach combines the inherent peroxidase-like nanozyme activity of gold nanoparticles (GNPs) with high affinity and specificity of an acetamiprid-specific S-18 aptamer to detect this neurotoxic pesticide in a highly rapid, specific, and sensitive manner. It is shown that the nanozyme activity of GNPs can be inhibited by its surface passivation with target-specific aptamer molecules. Similar to an enzymatic competitive inhibition process, in the presence of a cognate target, these aptamer molecules leave the GNP surface in a target concentration-dependent manner, reactivating GNP nanozyme activity. This reversible inhibition of the GNP nanozyme activity can either be directly visualized in the form of color change of the peroxidase reaction product or can be quantified using UV-visible absorbance spectroscopy. This approach allowed detection of 0.1 ppm acetamiprid within an assay time of 10 min. This reversible nanozyme activation/inhibition strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interest.

Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers

We have designed a single-stranded DNAzyme–aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3′ end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme–aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner.