Aptamer Recognition Research Articles

Novel electrochemiluminescent assay for the aptamer-based detection of testosterone

This work presents a proof-of-concept assay for the detection and quantification of small molecules based on aptamer recognition and electrochemiluminescence (ECL) readout. The testosterone-binding (TESS.1) aptamer was used to demonstrate the novel methodology. Upon binding of the target, the TESS.1 aptamer is released from its complementary capture probe – previously immobilized at the surface of the electrode – producing a decrease in the ECL signal after a washing step removing the released (labeled) TESS.1 aptamer. The analytical capability of the ECL assay towards testosterone detection was investigated displaying a linear range from 0.39 to 1.56 μM with a limit of detection of 0.29 μM. The selectivity of the proposed assay was assessed by performing two different negative control experiments; i) detection of testosterone with a randomized ssDNA sequence and ii) detection of two other steroids, i.e. deoxycholic acid and hydrocortisone with the TESS.1 aptamer. In parallel, complementary analytical techniques were employed to confirm the suggested mechanism: i) native nano-electrospray ionization mass spectrometry (native nESI-MS) was used to determine the stoichiometry of the binding, and to characterize aptamer-target interactions; and, ii) isothermal titration calorimetry (ITC) was carried out to elucidate the dissociation constant (Kd) of the complex of testosterone and the TESS.1 aptamer. The combination of these techniques provided a complete understanding of the aptamer performance, the binding mechanism, affinity and selectivity. Furthermore, this important characterization carried out in parallel validates the real functionality of the aptamer (TESS.1) ensuring its use towards selective testosterone binding in further biosensors. This research will pave the way for the development of new aptamer-based assays coupled with ECL sensing for the detection of relevant small molecules.

Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection

Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their selection. Those aptamers having high G value and complex structure were chosen. In dot blot assay, aptamers recognized PAG protein in an animal after 42 days of artificial insemination which later given birth to a healthy calf. Further the cross reactivity with serum of 0th day animal (post AI) or with non pregnant animal serum was minimal. Aptamers have also shown interaction with PAG protein of buffalo origin. These selected aptamers have commercial application especially in development of biosensors for early detection of pregnancy in bovine.

A dual-screening electrochemiluminescent aptasensor based on a mesoporous silica nano-sieve for specific detection of amyloid-β monomer

Amyloid β-protein (Aβ) aggregation in the brain is a closely related symptom of Alzheimer’s disease (AD), while Aβ in blood might be a more accessible peripheral biomarker to predict AD much earlier due to its abnormal fluctuation. However, when developing an aptasensor for Aβ detection, the specificity of the sensor will be impaired by Aβ aggregates such as Aβ oligomers or fibers because they have the same unit of recognition. Herein, a novel dual-screening sensing strategy based on specific recognition by aptamers and nano-sieving by a mesoporous silica membrane (MSM) with electrochemiluminescence (ECL) as the output signal is reported, in which the MSM achieves a size-screening effect to eliminate the influence of those macromolecules, assisting the specificity screening of the aptamer. The ECL signal is determined by the steric hindrance of the specifically captured Aβ on the aptamer in the nanochannels of the MSM towards the probe (luminol), affording a highly sensitive and specific detection ability for Aβ in complex matrices. In general, this work develops an ECL sensor that integrates aptamer recognition with size-selective separation for blood Aβ detection, which positively helps to monitor the occurrence and development of AD.

High sensitive electrochemical methamphetamine detection in serum and urine via atom transfer radical polymerization signal amplification

Herein we designed a highly sensitive and selective biosensor for methamphetamine (METH) detection based on aptamer recognition probe and atom transfer radical polymerization (ATRP) signal amplification strategy. In this experiment, METH aptamer and its complementary DNA strand were first attached to the electrode surface. In the presence of METH, the prioritized conjugation between METH and the aptamer will take one strand of DNA from the double-stranded DNA, so that the third segment of azide-modified DNA could be successfully modified onto the electrode surface. Through click chemistry and ATRP polymerization, the monomers with ferrocene were polymerized into a long chain, and the signal was amplified, then high-sensitivity detection of METH can be carried out. The result showed that the sensor could detect METH as low as 17 fM, which is about two orders of magnitude lower than that by traditional METH detection methods. Moreover, when different concentrations of METH were added to serum and urine, the recovery rate of the biosensor was as high as 93%. Therefore, using nucleic acid aptamer as capture probe and ATRP as signal amplification strategy can provide a promising application platform for sensitive detection of low concentration toxicants.

A novel aptasensor based on DNA hydrogel for sensitive visual detection of ochratoxin A.

Anovel visual detection mode isproposed to improve the detection sensitivityfor the determination of ochratoxin A (OTA). The mode is based on aptamer recognition and the signal amplification of rolling circle amplification (RCA) products self-assembled DNA hydrogel. Moreover, gold nanoparticles (AuNPs) were directly assembled inside the DNA hydrogel by adjusting the padlock probe sequences to achieve a stronger binding force between the DNA hydrogel and AuNPs; this avoids the need for modification of AuNPs with DNA sequences. In the presence of OTA, DNA hydrogel is formed. With higher concentrations of OTA, a larger amount of DNA hydrogel is formed. When AuNPs are added to the DNA hydrogel, AuNPs can be enclosed inside the DNA hydrogel. With more DNA hydrogel, there is less AuNPs in the supernatant. Thus, the absorbance of the supernatant is anti-correlated with the concentration of OTA. After optimization of the experimental conditions, the change in the absorbance of the supernatant was linearly correlated with the concentration of OTA, in the range 0.05 to 10ng/mL; the limit of detection was 0.005ng/mL. The good specificity of the developed biosensor was confirmed in the presence of other mycotoxins that are coexistent withor analogues of OTA. By comparing the developed method with the ELISA method, the accuracy and stability of this new method were also verified, with good performance obtained in real samples. Diagram of the principle of the colorimetric aptasensor for OTA detection based on rolling circle amplification product self-assembled DNA hydrogel.

Research of exosome in bone metastasis through dual aptamer recognition based entropy-driven amplification

Sensitive and accurate detection of exosome will greatly facilitate the early diagnosis of diverse diseases, such as cancers. Herein, a novel dual aptamer recognition based entropy-driven amplification was established for accurate analysis of exosomes. There are two main procedures in the proposed biosensor, including dual aptamer based recognition of exosome and entropy-driven catalytic system based signal recycling. In the recognition process, designed SMBs-S1 probe and S2–S4 probe complex, containing a CD63 aptamer and an EpCAM aptamer, respectively, are utilized for cooperated identification of exosomes. S4 probe was then released from S2–S4 probe complex through chain replacement of S5. The released S4 probe triggers entropy-driven catalytic system based signal recycling and endow the method a superior sensitivity. Impressively, owing to the cooperated identification of CD63 and EpCAM protein, the method exhibited a superior specificity and stayed stable under the interference of free CD63 and/or EpCAM protein. We believe that the sensitive, accurate strategy will provide a powerful tool for multiple biomarkers analysis and related clinical applications.

Ternary electrochemiluminescence biosensor based on black phosphorus quantum dots doped perylene derivative and metal organic frameworks as a coreaction accelerator for the detection of chloramphenicol

In this work, a sensitive and selective electrochemiluminescence (ECL) aptasensor was developed using an enhancing mechanism of the metal–organic frameworks (Co-Ni/MOF) in the perylene derivative (BP/PTC-NH2)/peroxydisulfate (S2O82-) system for chloramphenicol (CAP) assay. Firstly, black phosphorus quantum dots (BPQDs) were synthesized and added into the precursor solution of PTC-NH2 to synthesize BPQDs doped PTC-NH2 nanoparticles (BP/PTC-NH2), which were acted as the ECL emitters. Co-Ni/MOF with excellent encapsulation effect and catalytic effect, can react with co-reactants (K2S2O8) to produce more SO4•-, thus the ECL signal of the compound was obviously raised by 3.8 times greater than single BP/PTC-NH2, achieving signal amplification of the ECL system. In the presence of chloramphenicol, we can see a considerable enhancement of signal, it could be attributed to aptamer recognition resulted the aptamer pulling down from the sensing interface by CAP. Therefore, based on this enhancement effect and the specific recognition function of aptamer, a sensitive ECL aptamer sensor was designed for CAP detection. Under optimal conditions, the aptasensor can quantify the concentration of CAP from 1.0 × 10-13 to 1.0 × 10-6 M with low limit of detection of 2.9 × 10-14 M (S/N = 3). Besides, the developed ECL aptasensor could selectively detect CAP in a mixture containing CAP interference compounds, which demonstrated its potential to detect other antibiotics in aquatic environment.

Label‐free Electrochemical Aptasensor for the Determination of Serotonin

AbstractA label‐free electrochemical aptasensor was fabricated for the determination of serotonin (5‐HT) by the employment of specific binding between 5‐HT and aptamer and directly electrochemical measurement of 5‐HT oxidation. The comparison between two aptasensors and two strategies was studied. An electrochemical aptasensor fabricated using 57‐mer aptamer was facilely used to determine 5‐HT in the range from 1 μM to 100 μM with detection limit of 0.3 μM via one step recognition and detection. This work demonstrates that electrochemical method combing aptamer recognition and 5‐HT oxidation provides an alternative and promising method for the determination of 5‐HT with good selectivity and simplicity.

Detection of multiple mycotoxins based on catalytic hairpin assembly coupled with pregnancy test strip

Various mycotoxins are widely present in crop products, which cause serious harm to human and animals. In this study, combining the specific recognition of aptamer with catalytic hairpin assembly (CHA) strategy, the pregnancy test strip is used to detect multiple mycotoxins. The signal amplification can be further achieved through following enzymatic reaction to increase sensitivity. In the experiment, there is no need to assemble test strip as well as prepare and purify antibody/hapten. It only requires to couple human chorionic gonadotropin (hCG) with DNA probe to realize the target determination via CHA process and enzymatic signal amplification on the test strip. Under optimal conditions, using the CHA based pregnancy test strip detection platform, the visual limits of detection for aflatoxin B1, ochratoxin and zearalenone can be obtained within 15 min, which are 20, 50 and 20 pg/mL, respectively. The detection results of the spiked samples show that the platform possesses satisfied accuracy, reproducibility and practicability. The method has facile scheme, low cost and good design versatility, and reveals potential application prospects in high sensitivity, low cost, and convenient detection of mycotoxins.

A Glucose-Powered Activatable Nanozyme Breaking pH and H2 O2 Limitations for Treating Diabetic Infections.

The peroxidase-like activity of nanozymes is promising for chemodynamic therapy by catalyzing H2 O2 into . OH. However, for most nanozymes, this activity is optimal just in acidic solutions, while the pH of most physiological systems is beyond 7.0 (even >8.0 in chronic wounds) with inadequate H2 O2 . We herein communicate an activatable nanozyme with targeting capability to simultaneously break the local pH and H2 O2 limitations under physiological conditions. As a proof of concept, aptamer-functionalized nanozymes, glucose oxidase, and hyaluronic acid constitute an activatable nanocapsule "APGH", which can be activated by bacteria-secreted hyaluronidase in infected wounds. Nanozymes bind onto bacteria through aptamer recognition, and glucose oxidation tunes the local pH down and supplements H2 O2 for the in-situ generation of . OH on bacteria surfaces. The activity switching and enhanced antibacterial effect of the nanocapsule were verified in vitro and in diabetic wounds. This strategy for directly regulating local microenvironment is generally accessible for nanozymes, and significant for facilitating biological applications of nanozymes.

A Glucose‐Powered Activatable Nanozyme Breaking pH and H2O2 Limitations for Treating Diabetic Infections

AbstractThe peroxidase‐like activity of nanozymes is promising for chemodynamic therapy by catalyzing H2O2 into .OH. However, for most nanozymes, this activity is optimal just in acidic solutions, while the pH of most physiological systems is beyond 7.0 (even >8.0 in chronic wounds) with inadequate H2O2. We herein communicate an activatable nanozyme with targeting capability to simultaneously break the local pH and H2O2 limitations under physiological conditions. As a proof of concept, aptamer‐functionalized nanozymes, glucose oxidase, and hyaluronic acid constitute an activatable nanocapsule “APGH”, which can be activated by bacteria‐secreted hyaluronidase in infected wounds. Nanozymes bind onto bacteria through aptamer recognition, and glucose oxidation tunes the local pH down and supplements H2O2 for the in‐situ generation of .OH on bacteria surfaces. The activity switching and enhanced antibacterial effect of the nanocapsule were verified in vitro and in diabetic wounds. This strategy for directly regulating local microenvironment is generally accessible for nanozymes, and significant for facilitating biological applications of nanozymes.

Signal‐off Photoelectrochemical Aptasensor for S. aureus Detection Based on Graphite‐like Carbon Nitride Decorated with Nickel Oxide

AbstractA signal‐off photoelectrochemical (PEC) aptasensor was proposed for pathogenic bacteria detection based on graphite‐like carbon nitride decorated with NiO. The S. aureus be captured by the specific recognition of DNA aptamer, which was immobilized on the PEC electrode surface according to the specific effect of carboxylic group and NiO. The PEC signal would be obviously suppressed due to the transfer blocking of electron donor to PEC active material surface by steric hindrance and electrostatic repulsion. The PEC aptasensor showed excellent specificity and high sensitivity for S. aureus, and this provides simple strategy to construct point‐of‐care testing aptasensor for bacteria analysis.

A CdSe@CdS quantum dots based electrochemiluminescence aptasensor for sensitive detection of ochratoxin A

In this work, a CdSe@CdS quantum dots (QDs) based label-free electrochemiluminescence (ECL) aptasensor was developed for the specific and sensitive detection of ochratoxin A (OTA). Chitosan (CHI) could immobilize abundant QDs on the surface of an Au electrode as the luminescent nanomaterials. Glutaraldehyde was used as the crosslinking agent for coupling a large number of OTA aptamers. Thanks to the excellent stability, good biocompatibility, and strong ECL intensity of CdSe@CdS QDs, as well as the quick reactions of the generated SO4•− in the electrolyte, strong ECL signals were measured. Because of the specific recognition of aptamer toward OTA, the reduced ECL signals caused by OTA in the samples were recorded for quantify the content of OTA. After optimizing a series of crucial conditions, the ECL aptasensor displayed superior sensitivity for OTA with a detection limit of 0.89 ng/mL and a wide linear concentration range of 1–100 ng/mL. The practicability and viability were verified through the rapid and facile analysis of OTA in real Lily and Rhubarb samples with recovery rates (n = 3) of 98.1–105.6% and 97.3–101.5%, respectively. The newly-developed QDs-based ECL aptasensor provided a new universal analytical tool for more mycotoxins in safety assessment of foods and feeds, environmental monitoring, and clinical diagnostics.

A SERS-LFA biosensor combined with aptamer recognition for simultaneous detection of thrombin and PDGF-BB in prostate cancer plasma

An innovative surface-enhanced Raman spectroscopy and lateral flow assay (SERS-LFA) biosensor combined with aptamer recognition had been developed for the convenient, rapid, sensitive and accurate detection of thrombin and platelet-derived growth factor-BB (PDGF-BB) associated with prostate cancer simultaneously. During the biosensor operation, thrombin and PDGF-BB in the sample were recognized and combined by thiol-modified aptamers immobilized on Au–Ag hollow nanoparticles (Au–Ag HNPs) surface and biotinylated aptamers immobilized on the test lines of the biosensor. Thus, thrombin and PDGF-BB were simultaneously captured between detection aptamers and capture aptamers in a sandwich structure. Finite difference time domain simulation confirmed that ‘hot spots’ appeared at the gaps of Au–Ag HNPs dimer in the enhanced electromagnetic field compared to that of a single Au–Ag HNP, indicating that the aggregated Au–Ag HNPs owned a good SERS signal amplification effect. The detection limits of thrombin and PDGF-BB in human plasma were as low as 4.837 pg ml−1 and 3.802 pg ml−1, respectively. Moreover, the accuracy of the biosensor which was applied to detect thrombin and PDGF-BB in prostate cancer plasma had been verified. This designed biosensor had broad application prospects in the clinical diagnosis of prostate cancer.

Generating aptamers towards human sperm cells using massively parallel sequencing

Determining the presence of sperm cells on an item or swab is often a crucial component of sexual offence investigation. However, traditional histological staining techniques used for the morphological identification of spermatozoa lack both specificity and sensitivity, making analysis a complex and time-consuming process. New methods for the detection of sperm cells based on aptamer recognition may be able to overcome these issues. In this work, we present the selection of ssDNA aptamers against human sperm cells using Cell-SELEX and massively parallel sequencing technologies. A total of 14 rounds of selection were performed following a modified Cell-SELEX protocol, which included additional steps for the isolation of spermatozoa from seminal fluid. Massively parallel sequencing using the Illumina Miseq platform was conducted on enriched aptamer pools to elucidate the structure of potential binders. A custom bioinformatics pipeline was also developed using Galaxy for the automated processing of sequencing datasets. This data revealed several promising aptamer candidates, which were shown to selectively bind sperm cells through both microscale thermophoresis and enzyme-linked oligonucleotide assays. These aptamers have the potential to increase the efficiency of sexual offence casework by facilitating sperm detection.Graphical abstract

Photoelectrochemical biosensor for lead ion determination based on complementary strand aptamers

AbstractLead ion causes heavy metal pollution, which can cause serious damage to the ecological environment and human health. In this paper, a simple and sensitive photoelectrochemical method was designed for lead ion detection by using ZnO nanorods as the photoactive substrate and complementary strand aptamers as target recognition element. ZnO nanorods with favorable stability and photoelectric activity can provide a biocompatible microenvironment for aptamer loading. In the presence of lead ion, it would combine with the recognition aptamer S2 by competing with the aptamer S1, which led to changes in surface structure and charge density before and after the recognition of target, causing regular response of photocurrent. The developed method under optimized experimental conditions presented high sensitivity and good selectivity for lead ion detection with low detection limit as 3.5 × 10−11 mol/L. In addition, the proposed strategy was also further applied to the detection of lake water sample, which provided reliable experimental support and basis for further exploration and establishment of complex aptamer recognition and detection system.

Sulphur-doped graphene quantum dot based fluorescent turn-on aptasensor for selective and ultrasensitive detection of omethoate

Development of selective, ultra-sensitive, rapid and facile methods for the detection of chemical residues of toxic pesticides and hazardous chemicals are quite important in food safety, environmental monitoring and safeguarding public health. Herein, we presented a fluorescent turn-on aptasensor based on sulphur-doped graphene quantum dot (S-GQD) utilizing specific recognition and binding property of aptamer for the ultra-sensitive and selective detection of omethoate (OM) which is a systemic organophosphorus pesticide. The detection method is based on tuning aggregation-disaggregation mechanism of S-GQD by way of conformational alteration of the recognition probe. Fluorescence ‘turn-on’ process includes aggregation-induced quenching of S-GQD with aptamer via S-GQD-aptamer complex formation and its subsequent fluorescence recovery with the addition of OM by structural switching of S-GQD-aptamer complex to aptamer-omethoate complex. The reported ‘switch-on’ aptasensor has exhibited a low limit of detection of 0.001 ppm with high selectivity for OM over other pesticides.

A stable liquid crystals sensing platform decorated with cationic surfactant for detecting thrombin

A liquid crystals (LCs) sensing platform to detect thrombin was demonstrated based on high specificity of aptamer recognition. Initially, LCs doped with a cationic surfactant, octadecyl-trimethylammonium bromide (OTAB), adopted homeotropic alignment corresponding to dark optical image. Upon introduction of thrombin aptamer, the orientational transition of LCs from homeotropic to tilted state occurred due to the electrostatic interaction between it and OTAB. And accordingly a visual dark-to-bright alteration was observed for the polarized light microscope (POM) image of LCs. In the presence of thrombin with which the aptamer preferred to bind, OTAB released could induce the vertical arrangement of LCs and a dark optical image was regained. Therefore, by observing the changes of bright and dark optical appearances for LCs, thrombin could be analyzed with a detection limit of ~ 136 nM. The LCs sensing system, assisted with the specific combination of target molecule and its corresponding aptamer, reveals a simple and effective way to achieve label-free and low-cost detection of biomolecules in related clinic diagnosis.

Aqueous-phase synthesis of upconversion metal-organic frameworks for ATP-responsive in situ imaging and targeted combinational cancer therapy

Herein, the nanoscaled ATP-responsive upconversion metal-organic frameworks (UCMOFs) are aqueous-phase synthesized for co-delivery of therapeutic protein cytochrome c (Cyt c) and chemodrugs doxorubicin (DOX), achieving targeted combinational therapy of human cervical cancer. The UCMOFs are rationally fabricated by growing ZIF-90 on mesoporous silica-coated upconversion nanoparticles (UCNPs), in which the ZIF-90 layer attenuates the upconversion luminescence (UCL) and the rigid frameworks increase the stability of encapsulated proteins. Once the UCMOF@DOX/Cyt c are internalized into HeLa cells via specific recognition of sgc8 aptamers, the intracellular ATP triggers the dissolution of ZIF-90 into Zn2+, which facilitates not only the release of Cyt c and DOX but also the restoration of UCL for real-time monitoring of drug release. It has been demonstrated that the therapeutic efficacy is greatly improved by the combination of caspase-mediated apoptosis activated by Cyt c (protein therapeutics), DNA fragmentation induced by DOX (chemotherapy), and Zn2+-promoted generation of reactive oxygen species (ROS) (oxidative stress). Overall, our proposed multifunctional UCMOFs provide an effective platform for targeted combinational cancer therapy and in situ imaging, which hold great promise in biomedical and clinical applications.

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.