Conformation Change Of Aptamer Research Articles

Adaptation of a Model Spike Aptamer for Isothermal Amplification-Based Sensing.

Isothermal amplification (IA) techniques like rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) have gained significant attention in recent years due to their ability to rapidly amplify DNA or RNA targets at a constant temperature without the need for complex thermal cycling equipment. Such technologies, combined with colorimetric systems rendering visual confirmation of the amplification event, are ideal for the development of point-of-need detection methods suitable for field settings where access to specialized laboratory equipment is limited. The utility of these technologies, thus far limited to DNA and RNA targets, could be broadened to a wide range of targets by using aptamers. Composed of DNA or RNA themselves, aptamers can bind to substances, including proteins, metabolites, and inorganic substances. Their nucleic acid nature can potentially allow them to serve as a bridge, extending the reach of DNA/RNA-centric technologies to the broader molecular world. Indeed, the change in aptamer conformation occurring during ligand interaction can be used to elaborate ligand-responding RCA or LAMP templates. By using an existing aptamer targeting SARS-CoV-2 Spike protein as a model, we explored the possibility of establishing ligand-responsive IA systems. Our study used aptamers with simple sequence modifications as templates in LAMP assays and hyperbranched RCA (HRCA) by exploiting the dynamic nature of the model aptamer to trigger these IA systems. Importantly, our work uniquely demonstrates that this aptamer's dynamic response to ligand binding can regulate both RCA and LAMP processes. This novel approach of using aptamer conformational changes to trigger LAMP paves the way for new aptamer-based detection assays. Our system detects 50 nM of Spike protein, with LAMP occurring within 30 min in the presence of Spike. The colorimetric readout showed clear results, allowing for the detection of Spike protein presence.

Thermodynamic compensation to temperature extremes in B. subtilis vs T. maritima lysine riboswitches

T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs. ∼40°C, yet employ similar lysine riboswitches to aid in the transcriptional regulation of the genes involved in the synthesis and transport of amino acids. Despite notable differences in G-C basepair frequency and primary sequence, the aptamer moieties of each riboswitch have striking similarities in tertiary structure, with several conserved motifs and long-range interactions. To explore genetic adaptation in extreme thermal environments, we compare the kinetic and thermodynamic behaviors in T. maritima and B. subtilis lysine riboswitches via single-molecule fluorescence resonance energy transfer analysis. Kinetic studies reveal that riboswitch folding rates increase with lysine concentration while the unfolding rates are independent of lysine. This indicates that both riboswitches bind lysine through an induced-fit (“bind-then-fold”) mechanism, with lysine binding necessarily preceding conformational changes. Temperature-dependent van’t Hoff studies reveal qualitative similarities in the thermodynamic landscapes for both riboswitches in which progression from the open, lysine-unbound state to both transition states (‡) and closed, lysine-bound conformations is enthalpically favored yet entropically penalized, with comparisons of enthalpic and entropic contributions extrapolated to a common [K+] = 100 mM in quantitative agreement. Finally, temperature-dependent Eyring analysis reveals the TMA and BSU riboswitches to have remarkably similar folding/unfolding rate constants when extrapolated to their respective (40 and 80°C) environmental temperatures. Such behavior suggests a shared strategy for ligand binding and aptamer conformational change in the two riboswitches, based on thermodynamic adaptations in number of G-C basepairs and/or modifications in tertiary structure that stabilize the ligand-unbound conformation to achieve biocompetence under both hyperthermophilic and mesothermophilic conditions.

Utilization of Spontaneous Alkyne-Gold Self-Assembly Chemistry as an Alternative Method for Fabricating Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (E-AB) sensors are a promising class of biosensors which use structure-switching redox-labeled oligonucleotides (aptamers) codeposited with passivating alkanethiol monolayers on electrode surfaces to specifically bind and detect target analytes. Signaling in E-AB sensors is an outcome of aptamer conformational changes upon target binding, with the sequence of the aptamer imparting specificity toward the analyte of interest. The change in conformation translates to a change in electron transfer between the redox label attached to the aptamer and the underlying electrode and is related to analyte concentration, allowing specific electrochemical detection of nonelectroactive analytes. E-AB sensor measurements are reagentless with time resolutions of seconds or less and may be miniaturized into the submicron range. Traditionally these sensors are fabricated using thiol-on-gold chemistry. Here we present an alternate immobilization chemistry, gold-alkyne binding, which results in an increase in sensor lifetimes under ideal conditions by up to ∼100%. We find that gold-alkyne binding is spontaneous and supports efficient E-AB sensor signaling with analytical performance characteristics similar to those of thiol generated monolayers. The surface modification differs from gold-thiol binding only in the time and aptamer concentration required to achieve similar aptamer surface coverages. In addition, alkynated aptamers differ from their thiolated analogues only by their chemical handle for surface attachment, so any existing aptamers can be easily adapted to utilize this attachment strategy.

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.

A novel liquid crystal aptasensor via DNA aptamer conformational change for on-site detection of cocaine in sewage

A novel one-step liquid crystal (LC)-based aptasensor has been developed for on-site detection of trace substances in sewage analysis. The platform utilizes different mechanisms to simultaneously trigger the upper and bottom layers of LC orientation. By employing trace amounts of CTAB in Tris-HCl buffer, the upper layer of LC is aligned, while DNA aptamers modified on a single glass induce the orientation of the bottom layer. When targeting cocaine, the asymmetric single-stranded DNA aptamers transition to a symmetric Y-shape, causing the reorientation of LCs from planar to homeotropic. This results in an optical change from bright to dark in the LC image. The sensing procedure involves adding pre-buffered samples at pH 7.4 to the system in one step. The platform requires only 10 ng of DNA aptamers, which can be kept dry. It enables on-site detection of cocaine in sewage, exhibiting a linear calibration curve from 1 pM to 15 µM in water (R2 = 0.997) with a limit of detection of 0.5 pM. The aptasensor demonstrates quantitative recoveries ranging from 89.6% to 110.9% in sewage samples. This sensitive, robust, and portable LC-based aptasensor shows promise as a tool for on-site detection of trace substances.

Synergistic Multieffect Catalytic Amplified Cathodic Electrochemiluminescence Biosensor via Target Binding-Induced Aptamer Conformational Changes for the Ultrasensitive Detection of Synthetic Cathinone.

Signal amplification is a powerful approach to increasing the detection sensitivity of electrochemiluminescence (ECL). Here, we developed synergistic multieffect catalytic strategies based on CuCo2O4 nanorod combination of Ag NPs as coreaction accelerators to fabricate an efficient covalent organic framework (PTCA-COF)-based ternary ECL biosensor. Concretely, the high redox reversibility of Co3+/Co2+ and Cu2+/Cu+ would constantly promote the decomposition of S2O82- for ECL emission. Meanwhile, the introduction of Ag NPs with excellent electrocatalytic activity further realized multiple amplification of the ECL signal. Furthermore, the good hydrogen evolution reaction (HER) ability of Ag@CuCo2O4 nanorods could accelerate the proton transmission rate of the system to amplify ECL behavior. In the presence of the target synthetic cathinone 4-chloroethcathinone (4-CEC) as the quenching ECL signal-response probe, the Ferrocene (Fc)-labeled aptamer folded into the conformationally limited stem-loop structure, bringing Fc near the ECL luminophore and resulting in quenched ECL emission. The quenching effect was connected with target-induced aptamer conformational changes and consequently reflected the target concentration. Under optimum conditions, the proposed biosensor realized a highly sensitive assay for 4-CEC with a large dynamic range from 1.0 × 10-12 to 1.0 × 10-6 g/L and a detection limit as low as 2.5 × 10-13 g/L. This study integrated multiple amplification strategies for efficient ECL enhancement, which provided a novel approach to constructing highly bioactive and sensitive sensors.

Comparison of Aptamer Signaling Mechanisms Reveals Disparities in Sensor Response and Strategies to Eliminate False Signals.

Aptamers are nucleic acid-based affinity reagents that have been incorporated into a variety of molecular sensor formats. However, many aptamer sensors exhibit insufficient sensitivity and specificity for real-world applications, and although considerable effort has been dedicated to improving sensitivity, sensor specificity has remained largely neglected and understudied. In this work, we have developed a series of sensors using aptamers for the small-molecule drugs flunixin, fentanyl, and furanyl fentanyl and compare their performance─in particular, focusing on their specificity. Contrary to expectations, we observe that sensors using the same aptamer operating under the same physicochemical conditions produce divergent responses to interferents depending on their signal transduction mechanism. For instance, aptamer beacon sensors are susceptible to false-positives from interferents that weakly associate with DNA, while strand-displacement sensors suffer from false-negatives due to interferent-associated signal suppression when both the target and interferent are present. Biophysical analyses suggest that these effects arise from aptamer-interferent interactions that are either nonspecific or induce aptamer conformational changes that are distinct from those induced by true target-binding events. We also demonstrate strategies for improving the sensitivity and specificity of aptamer sensors with the development of a "hybrid beacon," wherein the incorporation of a complementary DNA competitor into an aptamer beacon selectively hinders interferent─but not target─binding and signaling, while simultaneously overcoming signal suppression by interferents. Our results highlight the need for systematic and thorough testing of aptamer sensor response and new aptamer selection methods that optimize specificity more effectively than traditional counter-SELEX.

Label-Free Electrogenerated Chemiluminescence Aptasensing Method for Highly Sensitive Determination of Dopamine via Target-Induced DNA Conformational Change.

A label-free electrogenerated chemiluminescence (ECL) aptasensing method for highly sensitive determination of dopamine (DA) was developed based on target-induced DNA conformational change. After anti-DA specific aptamer, as molecular recognition element, was hybridized with a capture ss-DNA (complementary with the aptamer), the formed double-strand DNA (ds-DNA) was self-assembled onto the surface of a gold electrode, and then Ru(phen)32+, as ECL reagent, was intercalated into ds-DNA to form an ECL biosensing platform. In the presence of DA, DA bound with its aptamer and target-induced DNA conformational change occurred, resulting in the dissociation of ds-DNA, the release of intercalated Ru(phen)32+ from the electrode surface, and the decrease of ECL intensity. For comparison, an ECL aptamer-based biosensing method using an ECL reagent-labeled aptamer was also developed for DA assay based on target-induced DNA conformational change. Because of the increase in the amount of ECL reagent into ds-DNA over that of the single-site ECL reagent-labeled aptamer, an obvious increase of ECL intensity was found at the ds-DNA modified electrode over the aptamer modified electrode. DA can be sensitively detected with a lower detection limit of 0.05 nM in the range from 0.1 to 100 nM. With the recognition of the aptamer for DA, DA can be selectively and sensitively detected in artificial cerebrospinal fluid and serum samples without interference from common small molecules. This work demonstrates that the combination of the direct transduction of specific recognition of DA and its aptamer into an ECL signal with Ru(phen)32+ intercalated ds-DNA amplification provides a promising strategy for the development of a simple, sensitive, and selective method for DA assay, which is of great importance in neurochemical assays and clinical diagnosis.

Selection of aptamer targeting levamisole and development of a colorimetric and SERS dual-mode aptasensor based on AuNPs/Cu-TCPP(Fe) nanosheets

Levamisole (LEV) is a veterinary drug that often remains in animal food. Consuming products containing high levels of LEV will cause a series of harmful reactions to human health. This work describes the Capture-SELEX (Capture-systematic evolution of ligands by exponential enrichment) screening strategy of LEV aptamers, using streptavidin modified agarose beads as a solid phase medium to separate target-bound and unbound ssDNA. The affinity and specificity of candidate aptamers were determined by SYBR Green I (SGI) dye and isothermal titration calorimetry (ITC), in which LEV-5 showed good binding affinity and specificity, and the dissociation constant was 66.15 ± 11.86 nM. Circular dichroism (CD) was used to characterize aptamer conformational changes before and after target binding, including increased helicity and enhanced base stacking. To evaluate whether this aptamer can be used for LEV detection, a colorimetric-surface-enhanced Raman spectroscopy (colorimetric-SERS) dual-mode aptasensor was constructed based on the peroxidase-like activity and SERS effect of AuNPs/Cu-TCPP(Fe) nanosheets. The detection limits of this dual-mode aptasensor for LEV were 5 nM and 1.12 nM, respectively. This aptamer-based method was further successfully used to detect LEV in milk, with recoveries ranging from 94.95% to 111.2%, providing a potential application for the detection of harmful substances in food.

SERS molecular-ruler based DNA aptamer single-molecule and its application to multi-level optical storage

Real-time analysis of DNA aptamer conformational changes is essential to understanding the function of DNA aptamers at the single-molecular level. Herein, we design core-satellite nanostructures as surface-enhanced Raman scattering (SERS) molecular-rulers to monitor the change process of single Hg2+ aptamer under dark-field microscopy (DFM-) correlated Raman spectroscopy. Applying the SERS molecular-ruler, the individual Hg2+ aptamer conformational change events triggered by Hg2+ that leads to the stepwise closing of the satellite to the core is continuously monitored by changes in SERS intensity. Moreover, individual Hg2+ aptamer conformational change events are confirmed by in-situ scanning electron microscopy (SEM) and finite difference time-domain (FDTD) calculation. Furthermore, the SERS molecular-ruler is a valid method for studying aptamer intermediate states, the binding sites in aptamer and the kinetics of Hg2+ aptamer configuration change at the single-molecule level. Such findings are promising for exploring the mechanism of DNA aptamer configuration change and providing the fundamental understanding for designing various DNA aptamer based biological applications. For future applications, SERS molecular-ruler has the potential to realize multi-level optical storage and presents a new practical and effective method for designing multi-level storage system.

Carbon dots-thionine modified aptamer-based biosensor for highly sensitive cocaine detection

Due to the application of aptasensors in biosensing, researchers are encouraged to design and develop sensing platforms with greater sensitivity and selectivity. This report introduces an aptasensor whose capture DNA is immobilized on the surface of an ITO electrode modified with a gold nanoparticle (prepared with physical vapor deposition technique). Here, target DNA combined with carbon dots that are modified with thionine to provide an oxidation–reduction redox probe to monitor cocaine. In the presence of cocaine, the aptamer conformational change, was take placed and reduced the distance between thionine and the electrode surface, and increased the charge transfer efficiency. Under optimal conditions our cocaine biosensor shows a dynamic range of 10–70 pM with a detection limit of 0.26 pM. Determination of cocaine in human serum and urine samples shows the practical application of the proposed aptasensor.

Aptamer-based Electrochemical Detection of Tyrosinamide Using Metal-organic Frameworks/silver Nanoparticles Modified Glassy Carbon Electrode

In this report, a new aptamer-based assay was presented reporting the electrochemical aptasensing for sensing tyrosinamide (Tyr-NH2). This strategy was relied on unbeatable conformational flexibility and specific recognition of aptamers. The tyrosinamide aptamer (Tyr-NH2-aptamer) was immobilized onto the metal-organic frameworks/silver nanoparticles modified glassy carbon electrode and hexacyanoferrate was selected as a probe to monitor interface variations during modification of the electrode and the aptamer conformational change generated by the Tyr-NH2 binding. Results showed that measurements by using electrochemical impedance spectroscopy had linear with the Tyr-NH2 concentrations in range of 0.01-0.25 nM and 0.25-1.15 nM. Detection limit of this system was found to be 2.3 pM. This method was also used to the Tyr-NH2 detection in serum samples successfully. Remarkable simplicity, ease of use and low-cost, make methodology as sensitive analytical system for sensing of the Tyr-NH2 that can be miniaturized. This strategy offers some promising advantages in reliable detection of the Tyr-NH2, which may be helpful in the routine analysis.

DNA walker-mediated biosensor for target-triggered triple-mode detection of Vibrio parahaemolyticus

Herein, we have constructed a target-triggered and DNA walker-mediated biosensor with triple signal (BTS) outputs mode for sensitive and reliable detection of pathogenic bacteria. Vibrio parahaemolyticus (VP) being the detection target model, the aptamer conformational changes induced by VP have been designed to activate the DNA walk on the modifiable and conductive surface of Fe3O4 nanoparticles to generate triple signal outputs, including electrochemiluminescence (ECL), fast scan cyclic voltammetry (FSCV) and fluorescent pixel counting (FLPC). Limits of quantification (LOQ) of VP were as low as 1 CFU⋅mL−1 by ECL with a linear range of 1–106 CFU⋅mL−1, 1 CFU⋅mL−1 by FSCV with a linear range of 1–106 CFU⋅mL−1, and 10 CFU⋅mL−1 by FLPC with a linear range of 10–107 CFU⋅mL−1 respectively, all squared correlation coefficients R2 being > 0.99. In addition, optical and electrochemical results, signal-on and signal-off results, electrode phase and solution phase results could be mutually verified by integrating of multiple detection techniques in one biosensor, greatly improving the accuracy and reliability. Therefore, the designed BTS has provided a powerful strategy for pathogenic bacteria detection considering its high detection sensitivity and accuracy, exhibiting great potential in food safety, water quality and biological contamination.

Aptamer Conformational Change Enables Serotonin Biosensing with Nanopipettes.

We report artificial nanopores in the form of quartz nanopipettes with ca. 10 nm orifices functionalized with molecular recognition elements termed aptamers that reversibly recognize serotonin with high specificity and selectivity. Nanoscale confinement of ion fluxes, analyte-specific aptamer conformational changes, and related surface charge variations enable serotonin sensing. We demonstrate detection of physiologically relevant serotonin amounts in complex environments such as neurobasal media, in which neurons are cultured in vitro. In addition to sensing in physiologically relevant matrices with high sensitivity (picomolar detection limits), we interrogate the detection mechanism via complementary techniques such as quartz crystal microbalance with dissipation monitoring and electrochemical impedance spectroscopy. Moreover, we provide a novel theoretical model for structure-switching aptamer-modified nanopipette systems that supports experimental findings. Validation of specific and selective small-molecule detection, in parallel with mechanistic investigations, demonstrates the potential of conformationally changing aptamer-modified nanopipettes as rapid, label-free, and translatable nanotools for diverse biological systems.

Signal-on electrochemical aptasensors with different target-induced conformations for prostate specific antigen detection

Prostate specific antigen (PSA) has become a potential biomarker for detecting prostate cancer (PCa) in the early stage. Herein, we report a target-induced resolution for the detection of PSA sensitively and specifically by amperometric electrochemical measurements. To meet a satisfactory performance, three conformations of pre-design DNA aptamers including two stem-loop structures and a double strand structure have been investigated and compared. All of them are immobilized on gold electrode as capture probes with redox-active molecular. The mechanism of signal transduction depends on molecular recognition events involving aptamer conformational changes, thus influencing the charge transfer. A short, single-stranded DNA (ssDNA) pseudoknot forming two stem-loop structural aptamers with labeled MB at the 3′ -terminus was found to posse the highest signal variation than other structure when induced by PSA due to the strong conformational change. With the optimized capture strand, the aptasensor showed the peak current increase of MB by the binding relationship between PSA and the sensor over a wide concentration range of 4 magnitude orders. The proposed aptasensor exhibited a wide detection range from 10 pg/mL to 500 ng/mL with a low detection limit of 1.24 pg/mL (S/N = 3). Moreover, the electrochemical aptasensor demonstrated good reproducibility, sensitivity, selectivity, and reliability for the detection of PSA. We also found the aptasensor had a good response in the human serum samples, making this device easy to operate for the detection of the PSA physiological concentration.

Capacitive Aptasensor for 17β-Estradiol Detection in a Non-Faradaic Electrochemical System Based on NH <sub>2</sub>-GO/PPy

17s-estradiol (E2) is an endocrine-disrupting chemical (EDC) being a hot debate topic that has been discussed among researchers for a decade due to its unexpected harmful effects on humans and wildlife. In this study, we report facile methods to fabricated label-free aptasensor based on the non-faradic mode by using amine-functionalized graphene oxide polypyrrole (NH2-GO/PPy) modified gold interdigitated electrode (IDE) for direct capacitive detection of 17β-estradiol. The as-prepared NH2-GO/PPy nanocomposites have been successfully characterized by FESEM, AFM, FTIR, and electrochemical impedance spectroscopy (EIS). The results show that NH2-GO/PPy/IDE was able to increase the capacitive current as opposed to bare gold IDE. While E2 binding was detected by a decrease in the electrode capacitance due to aptamer's conformational change towards E2. In addition, the biosensor showed a dynamic range of detection from 100nM to 1 pM with a low detection limit of about 0.13 pM. The recoveries result also presented in this paper were validated using HPLC exhibited high sensitivity for BPA detection and were successfully applied to detect E2 in real samples with good recoveries.

A simple fluorescence anisotropy assay for detection of bisphenol A using fluorescently labeled aptamer

Bisphenol A (BPA) is one of the environmental endocrine disruptors (EDCs), and BPA contamination in environment can cause high risks to human health. Rapid determination of BPA on sites is in high demand in environmental analysis. Taking advantage of aptamers as affinity ligands and fluorescence anisotropy (FA) analysis, we developed a simple and rapid FA assay for BPA by employing a single tetramethylrhodamine (TMR) labeled short 35-mer DNA aptamer against BPA. The assay is based on the BPA-binding induced conformation change of TMR-labeled aptamer and alteration of interaction between TMR and guanine bases, resulting in change of FA signals. We screened the FA change of aptamer probes having TMR label on a specific site of the aptamer upon BPA addition. The aptamer with a TMR label on the 22nd T base showed large FA-decreasing response to BPA and maintained good binding affinity to BPA. By using this TMR-labeled aptamer, we achieved FA detection of BPA with a detection limit of 0.5 μmol/L under the optimized conditions. This assay was selective towards BPA and enabled the detection of BPA spiked in tap water sample, showing the potential applications on water samples.

Target-responsive ratiometric fluorescent aptasensor for OTA based on energy transfer between [Ru(bpy)3]2+ and silica quantum dots.

A ratiometric fluorescent aptasensor based on energy transfer between [Ru(bpy)3]2+ and silica quantum dots (silica QDs) for assaying OTA was fabricated. Theaptamer for OTA was used as the gate to shield the fluorescent reagent [Ru(bpy)3]2+ into mesoporous silica nanoparticle (MSN). In the presence of OTA, the constrained [Ru(bpy)3]2+ was released from MSN due to atarget-induced aptamer conformational change. The released [Ru(bpy)3]2+ adsorbed onto the negatively charged silica QDs through electrostatic interaction. This createsappearance of fluorescence from [Ru(bpy)3]2+ at 625nm and decrease of the fluorescence from silica QDs at 442nm owing to the energy transfer. The value of FL625nm/FL442nm was in proportion to the concentrationof OTA in the range 0.5~100ngmL-1 with aLOD of 0.08ngmL-1. Practical applicability of this method was validated by thedetermination ofOTA in flour samples. Graphical abstract The sensing principle of this sensor.

Engineering an aptamer-based recognition sensor for electrochemical opium alkaloid biosensing

Here we propose an electrochemical aptamer-based sensing strategy for sensitive detection of the codeine (COD). Platform construction was started by decoration NH2-functionalized Fe3O4 with gold nanoparticles (Fe3O4/AuNPs). Carbon nanotubes were then placed on a glassy carbon electrode and decorated with the Fe3O4/AuNPs to serve as a signal amplifier (Fe3O4/AuNPs/CNTs/GC). The proposed nanoaptasensor integrated the merits of the deposition of Fe3O4/AuNPs and CNTs and the covalent attachment of the detection probe at the surface of platform. In this concept, COD was captured at the surface of sensing interface due to the specific binding of aptamer and COD, which led to COD detection. The long tunnels on modified electrode surface were formed owning to the attachment of COD-aptamer at the surface of sensor, while aptamer acted as gate of the tunnels. The change in conformation of aptamer upon target binding caused the closure of aptamer gate. Coupling the “Off–On” electrochemical switching properties of the aptamer modified electrode with inherent capabilities of nanocomposite led to high sensitivity, simplicity, stability and reproducibility of aptasensor. The assay has a 3.2 pM detection limit, and the response is linear up to 900 nM concentration of COD.

Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins.

Basing on the conformation change of aptamer caused by proteins, a simple and sensitive protein fluorescent assay strategy is proposed, which is assisted by the isothermal amplification reaction of polymerase and nicking endonuclease. In the presence of platelet-derived growth factor (PDGF-BB), the natural conformation of a DNA aptamer would change into a Y-shaped complex, which could hybridize with a molecular beacon (MB) and form a DNA duplex, leading to the open state of the MB and generating a fluorescence signal. Subsequently, with further assistance of isothermal recycling amplification strategies, the designed aptamer sensing platform showed an increment of fluorescence. As a benefit of this amplified strategy, the limit of detection (LOD) was lowered to 0.74 ng/mL, which is much lower than previous reports. This strategy not only offers a new simple, specific, and efficient platform to quantify the target protein in low concentrations, but also shows a powerful approach without multiple washing steps, as well as a precious implementation that has the potential to be integrated into portable, low-cost, and simplified devices for diagnostic applications.