Aptasensing biosynthesized phosphatidylserine with a AuNPs nanozyme-based colorimetric aptasensor

Sensitive monitoring of the target products during the biosynthesis process is crucial,and facile analytical approaches are urgently needed.Herein,phosphatidylserine(PS)was chosen as the model target,a colorimetric aptasensor was developed for the rapid quantitation in biosynthesis samples.A chimeric aptamer was constructed with two homogeneous original PS aptamers.Specific recognition between the chimeric aptamer and PS results in the desorption of aptamer from the surface of the AuNPs nanozyme,and the peroxidase-like enzymatic activity of the AuNPs nanozyme was weakened in a relationship with the different concentrations.The developed aptasensor performed well when applied for analyzing PS in biosynthesis samples.The aptasensor offers good sensitivity and selectivity,under optimal conditions,achieving monitoring and quantitation of PS in the range of 2.5-80.0μmol/L,with a limit of detection at 536.2 nmol/L.Moreover,the aptasensor provides good accuracy,with comparison rates of 98.17%-106.40%,when compared with the HPLC-ELSD.This study provides a good reference for monitoring other biosynthesized products and promoting the development of aptamers and aptasensors in real-world applications.

Development of a Label-Free Colorimetric Aptasensor with Rationally Utilized Aptamer for Rapid Detection of Okadaic Acid

Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis.In this study,a label-free col-orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish.To exploit the binding capability of the anti-OA aptamer,the inherent molecular recognition mechanism of aptamer and OA was studied,based on molecular docking,fluorescent assay,and biolayer interferometry.Consistent results showed that the stem-loop near the 3’terminal of the aptamer exhibit dominate binding capacity.Based on the revealed recognition information,the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride(PDDA)for the development of the label-free colorimetric aptasensor,in which the 3’terminal was thoroughly exposed to OA.The aptasensor provided robust performance with a linear detection range of 100-1200 nmol L-1,a limit of detection of 41.30 nmol L-1,recovery rates of 91.6%-106.2%,as well as a high selectivity towards OA in shellfish samples.The whole detection process can be completed within 1 h.To our best knowledge,this is the first time that the anti-OA aptamer was thoroughly studied,and a label-free colorimetric aptasensor was rationally designed in this way.This study not only provides a rapid detection method for highly sensitive and specific detection of OA,but also serves as a reference for the design of efficient aptasensors in the future.

Electrochemical Aptasensor Based on Prussian Blue-Chitosan-Glutaraldehyde for the Sensitive Determination of Tetracycline

In this paper, a novel and sensitive electrochemical aptasensor for detecting tetracycline(TET)with prussian blue(PB) as the label-free signal was fabricated. A PB-chitosan-glutaraldehyde(PB-CS-GA)system acting as the signal indicator was developed to improve the sensitivity of the electrochemical aptasensor.Firstly, the PB-CS-GA was fixed onto the glass carbon electrode surface. Then, colloidal gold nanoparticles(Au NPs) were droped onto the electrode to immobilize the anti-TET aptamer for preparation of the aptasensor.The stepwise assembly process of the aptasensor was characterized by cyclic voltammetry(C-V) and scanning electron microscope(SEM). The target TET captured onto the electrode induced the current response of the electrode due to the non-conducting biomoleculars. Under the optimum operating conditions, the response of differential pulse voltammetry(DPV) was used for detecting the concentration of TET. The proposed aptasensor showed a high sensitivity and a wide linear range of 109-～ 105-M and 105-～ 102-M with the correlation coefficients of 0.994 and 0.992, respectively. The detection limit was 3.2×1010-M(RSD 4.12%). Due to its rapidity, sensitivity and low cost, the proposed aptasensor could be used as a pre-scanning method in TET determination for the analysis of livestock products.

QCM Aptasensor for Rapid and Specific Detection of Avian Influenza Virus

There has been a need for rapid detection of Avian Influenza virus (AIV) H5N1 due to it being a potential pandemic threat. Most of the current methods, including culture isolation and PCR, are very sensitive and specific but require specialized laboratories and trained personnel in order to complete the tests and are time-consuming. The goal of this study was to design a biosensor that would be able to rapidly detect AIV H5N1 using aptamers as biosensing material and a quartz crystal microbalance (QCM) for transducing method. Specific DNA aptamers against AIV H5N1 were immobilized, through biotin and streptavidin conjugation, onto the gold surface of QCM sensor to capture the target virus. Magnetic nanobeads (150 nm in diameter) were then added as amplifiers considering its large surface/volume ratio which allows for faster movement and a higher target molecule binding rate. The result showed that the captured AIV caused frequency change, and more change was observed when the AIV concentration increased. The nanobead amplification was effective at the lower concentrations of AIV, however, it was not significant when the AIV concentration was 1 HA or higher. The detection limit of the aptasensor was 1 HAU with a detection time of 1 h. The capture of the target virus on to the surface of QCM sensor and the binding of magnetic nanobeads with the virus was confirmed with electron microscopy. Aptamers have unlimited shelf life and are temperature stable which allows this aptasensor to give much more consistent results specifically for in field applications.

Fluorescent aptasensor for detection of live foodborne pathogens based on multicolor perovskite-quantum-dot-encoded DNA probes and dual-stirring-bar-assisted signal amplification

In this study,a fluorescent(FL)aptasensor was developed for on-site detection of live Salmonella typhimurium(S.T.)and Vibrio parahaemolyticus(V.P.).Complementary DNA(cDNA)of aptamer(Apt)-functionalized multicolor polyhedral oligomeric silsesquioxane-perovskite quantum dots(cDNA-POSSPQDs)were used as encoded probes and combined with dual-stirring-bar-assisted signal amplification for pathogen quantification.In this system,bar 1 was labeled with the S.T.and V.P.Apts,and then bar 2 was functionalized with cDNA-POSS-PQDs.When S.T.and V.P.were introduced,pathogen-Apt complexes would form and be released into the supernatant from bar 1.Under agitation,the two complexes reached bar 2 and subsequently reacted with cDNA-POSS-PQDs,which were immobilized on MXene.Then,the encoded probes would be detached from bar 2 to generate FL signals in the supernatant.Notably,the pathogens can resume their free state and initiate next cycle.They swim between the two bars,and the FL signals can be gradually enhanced to maximum after several cycles.The FL signals from released encoded probes can be used to detect the analytes.In particular,live pathogens can be distinguished from dead ones by using an assay.The detection limits and linear range for S.T.and V.P.were 30 and 10 CFU/mL and 10^(2) -10^(6) CFU/mL,respectively.Therefore,this assay has broad application potential for simultaneous on-site detection of various live pathogenic bacteria in water.

Extraordinary tunable dynamic range of electrochemical aptasensor for accurate detection of ochratoxin A in food samples

We report the design of a sensitive,electrochemical aptasensor for detection of ochratoxin A(OTA)with an extraordinary tunable dynamic sensing range.This electrochemical aptasensor is constructed based on the target induced aptamer-folding detection mechanism and the recognition between OTA and its aptamers results in the conformational change of the aptamer probe and thus signal changes for measurement.The dynamic sensing range of the electrochemical aptasensor is successfully tuned by introduction of free assistant aptamer probes in the sensing system.Our electrochemical aptasensor shows an extraordinary dynamic sensing range of 11-order magnitude of OTA concentration from 10^−8 to 10^2 ng/g.Of great significance,the signal response in all OTA concentration ranges is at the same current scale,demonstrating that our sensing protocol in this research could be applied for accurate detections of OTA in a broad range without using any complicated treatment of signal amplification.Finally,OTA spiked red wine and maize samples in different dynamic sensing ranges are determined with the electrochemical aptasensor under optimized sensing conditions.This tuning strategy of dynamic sensing range may offer a promising platform for electrochemical aptasensor optimizations in practical applications.

A Label-Free Colorimetric Aptasensor Containing DNA Triplex Molecular Switch and AuNP Nanozyme for Highly Sensitive Detection of Saxitoxin

Saxitoxin(STX),one of the most toxic paralytic shellfish poisons discovered to date,is listed as a required item of aquatic product safety inspection worldwide.However,conventional detection methods for STX are limited by various issues,such as low sensitivity,complicated operations,and ethical considerations.In this study,an aptamer-triplex molecular switch(APT-TMS)and gold nanoparticle(AuNP)nanozyme were combined to develop a label-free colorimetric aptasensor for the rapid and highly sensitive de-tection of STX.An anti-STX aptamer designed with pyrimidine arms and a purine chain was fabricated to form an APT-TMS.Specific binding between the aptamer and STX triggered the opening of the switch,which causes the purine chains to adsorb onto the surface of the AuNPs and enhances the peroxidase-like activity of the AuNP nanozyme toward 3,3’,5,5’-tetramethylbenzidine.Under optimized conditions,the proposed aptasensor showed high sensitivity and selectivity for STX,with a limit of detection of 335.6 pmol L^(−1) and a linear range of 0.59-150 nmol L^(−1).Moreover,good recoveries of 82.70%-92.66%for shellfish and 88.97%-106.5%for seawater were obtained.The analysis could be completed within 1 h.The proposed design also offers a robust strategy to achieve detection of other marine toxin targets by altering the corresponding aptamers.

An antifouling polydopamine-based fluorescent aptasensor for determination of arginine kinase

Simple yet efficient detection methods for food allergens are in urgent need to help people avoid the risks imposed by allergenic food.In this work,a polydopamine(PDA)-based fluorescent aptasensor was developed to detect arginine kinase(AK),one of the major allergens in shellfish.The aptamer towards AK was firstly selected via systematic evolution of ligands by exponential enrichment method and labeled with fluorescein amidite(FAM)to build a fluorescence resonance energy transfer(FRET)system with PDA particles.Polyethylene glycol(PEG)was employed to construct an antifouling surface for the aptasensor to eliminate food matrix interferences.With the presence of AK,the PDA-based aptasensor exhibited elevated fluorescent signals as the FAM-labeled aptamer bound to AK and detached from the PDA particles.The aptasensor showed great stability and resistance to nonspecific interference of background proteins and had a limit of detection(LOD)of 0.298μg/mL.The proposed aptasensor was further proved to be feasible for quantitative analysis of AK in nine species of shrimps and five commercial processed products,which indicated its high potential in tracing the presence of AK in complex aquatic products.

Fast Electrical Detection of Carcinoembryonic Antigen Based on AlGaN/GaN High Electron Mobility Transistor Aptasensor

As one of the most important tumor-associated antigens of colorectal adenocarcinoma, the carcinoembryonic antigen （CEA） threatens human health seriously ali over the globe. Fast electrical and highly sensitive detection of the CEA with A1GaN/GaN high electron mobility transistor is demonstrated experimentally. To achieve a low detection limit, the Au-gated sensing area of the sensor is functionalized with a CEA aptamer instead of the corresponding antibody. The proposed aptasensor has successfully detected different concentrations （ranging from 50picogram/milliliter （pg/ml） to 50 nanogram/milliliter （ng/ml）） of CEA and achieved a detection limit as low as 50pg/ml at Vas = 0.5 V. The drain-source current shows a c/ear increase of 11.5μA under this bias.

Design of a Novel Electrochemical Aptasensor Based on Molybdenum Disulfide Nanosheets for Lysozyme Detection

The precise detection of lysozyme plays a crucial role in early diagnosis of kidney failure in leukemia patients.Herein,we designed an electrochemical aptasensor to detect lysozyme using a glassy carbon electrode(GCE)modified with molybdenum disulfide(MoS_(2))nanosheets decorated with chitosan(Ch)and gold nanoparticles(Au).In the next steps of aptasensor preparation,aptamer(Apt)strands were immobilized on MoS_(2)-Au-Ch/GCE surface via electrostatic interactions,and bovine serum albumin(BSA)solution was applied to cover unbonded areas on Apt/MoS_(2)-Au-Ch/GCE.The MoS_(2)-Au-Ch nanocomposite can warrant enhanced immobilization of aptamers and strong signals because of wrinkle-like structure and base planes in MoS nanosheets,presence of abundant amino and carboxyl functional groups in chitosan and Au nanoparticles.Differential pulse voltammetry(DPV)was used to evaluate the electrochemical behavior of the aptasensor at each preparation step and its detection performance.The developed electrochemical aptasensor based on MoS nanosheets showed remarkable performance for lysozyme detection in the range from 1 pmol/L to 10 nmol/L with a limit of detection of 2.2 fmol/L.The proposed aptasensor provided excellent selectivity against BSA,uric acid,cytochrome C,lysozyme and their mixture.In addition,it displayed great stability of about 99.58%of the initial signal upon 21-day storage.Tests on human serum and urine samples yielded promising recoveries ranging from 98 to 105%.Hence,MoS_(2)-Au-Ch nanocomposite-based aptasensor could be an outstanding candidate for medical device applications and protein quantification.

Electrochemical aptasensor for the detection of vascular endothelial growth factor(VEGF) based on DNA-templated Ag/Pt bimetallic nanoclusters

In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution. The DNA-Ag/Pt NCs show intrinsic peroxidase-mimicking activity and can effectively catalyze the H2O2-mediated oxidation of a substrate, 3,30,5,50-tetramethylbenzidine（TMB）, to produce a blue colored product. Based on this specific property, we employed the aptamer of VEGF to design a label-free electrochemical biosensor for VEGF detection. Under the optimized experimental conditions, a linear range from 6.0 pmol/L to 20 pmol/L was obtained with a detection limit of 4.6 pmol/L. The proposed biosensor demonstrated its high specificity for VEGF and could directly detect the VEGF concentration in human serum samples of breast cancer patients with satisfactory results. This novel electrochemical aptasensor was simple and convenient to use and was cost-effective and label-free in design, and would hold potential applications in medical diagnosis and treatment.

A Label-free Fluorescent Aptasensor for Turn-on Monitoring Ochratoxin A Based on AIE-active Probe and Graphene Oxide

A label-free fluorescent aptasensor for specific and ultrasensitive monitoring ochratoxin A（OTA） was developed using the specific aptamer of OTA（OSA） as recognition dement, an aggregation-induced emission（AIE） molecule（a 9,10-distyrylanthracene with two ammonium groups, DSAI） as a fluorescent probe, and graphene oxide（GO） as a quencher. In the absence of OTA, the AIE probe DSAI and OSA complex（DSAI/OSA） is adsorbed on the GO surface, and the fluorescence of DSAI will be quenched efficiently via the fluorescence resonance energy transfer（FRET） from DSAI to GO. Upon the OTA addition, a more stable complex（OSA-OTA） is formed and released from GO. Meanwhile, DSAI and OSA-OTA can form a new complex（DSAI/OSA-OTA）, then the fluorescent signal of DSAI recovers gradually. Therefore, by introducing GO and DSAI, the fluorescence signal of DSAI can be easily turned from ＂off＂ to ＂on＂ after the addition of OTA, and the ultrasensitive detection of OTA by monitoring the change of the fluorescence signal of DSAI can be readily realized. The detection limit of the assay can reach 0.324 nmol/L with a linear detection range of 10-200 nmol/L. And the aptasensor exhibits high selectivity for OTA against other analogues. Moreover, it has been successfully applied for the detection of OTA in red wine samples.

Selection of a DNA aptamer for the development of fluorescent aptasensor for carbaryl detection

An improved ssDNA library immobilized systematic evolution of ligands by enrichment(SELEX)was applied to select aptamers against carbaryl.After nine selection rounds,a highly enriched ssDNA pool was obtained.The Apta3 was demonstrated as the optimal aptame r.In order to facilitate the modification of aptamer,the Apta3 was further truncated with the dissociation constant(K_(d))of 0.364±0.055μmol/L and a fluorescent aptasensor was developed.The linear range for carbaryl was from 100 nmol/L to1500 nmol/L,with the limit of detection was as low as 15.23 nmol/L.Besides,the biosensor was validated for the carbaryl spiked real samples,and the recoveries were between 97.7%and 107.3%.

Aptasensor Based on Screen-Printed Carbon Electrodes Modified with CS/AuNPs for Sensitive Detection of Okadaic Acid in Shellfish

Okadaic acid(OA),a small molecule substance derived from shellfish,is one of the most widely distributed marine toxins with acute symptoms of vomiting and diarrhea after accidental ingestion.For this,there is an urgently need for sensitive and reliable methods to detect OA in real shellfish samples.In this study,a simple aptasensor based on screen-printed carbon electrode(SPCE)with modification of chitosan(CS)and gold nanoparticles(Au NPs)was designed for electrochemical determination of OA,and the electrode surface was modified with Au NPs by potential-sweeping electrodeposition,which greatly improved the electrochemical response.The entire detection and characterization process were carried out by cyclic voltammetry(CV)with a linear correlation in the range of 0.01-100 ng/m L and a limit of detection(LOD)of 6.7 pg/m L.Furthermore,recovery rates of 92.3-116%were obtained demonstrating excellent accuracy through the recovery trial of mussel and scallop samples.

A simple AuNPs-based colorimetric aptasensor for chlorpyrifos detection

In this work, a simple gold nanoparticles(Au NPs) based colorimetric biosensor was developed for chlorpyrifos(Chl) detection using an aptamer as the capture probe. The Chl-aptamer with low dissociation constant(Kd) of 58.59 ± 6.08 nmol/L was selected by ss DNA library immobilized systematic evolution of ligands by enrichment(SELEX). In the absence of Chl, the Chl-aptamer acted as the stabilizer for Au NPs in salt solution. In the presence of Chl, the highly specific Chl-aptamer bound with Chl targets immediately,thus a self-aggregation of Au NPs induced by salt was displayed. The fabricated colorimetric aptasensor exhibited an excellent sensitivity for Chl detection with the LOD as low as 14.46 nmol/L. In addition, the aptasensor was applied to test Chl in tap water, cucumber and cabbage samples, the excellent recoveries with acceptable RSD values below 5% demonstrated that the method can be considered as a promising tool for simple, rapid Chl detection.

A novel aptasensor based on 3D-inorganic hybrid composite as immobilized substrate for sensitive detection of platelet-derived growth factor

A novel electrochemical detection approach for platelet-derived growth factor（PDGF） via ＂sandwich＂structure is reported in this paper. 3D-4MgCO3 Mg（OH）2 4H2O-Au NPs inorganic hybrid composite was utilized as immobilized substrate for sensitive PDGF detection and Pt-Au bimetallic nanoparticles were labelled on PDGF aptamer to indirectly detect PDGF for the first time. The proposed aptasensor exhibited a high catalytic efficiency towards reduction of H2O2, hence the sensitive detection of PDGF was achieved.Results showed that the aptasensor exhibited excellent linear response to PDGF, in the range of 0.1 pg/m L–10 ng/m L（4 fmol/L–400 pmol/L）, with detection limit of 0.03 pg/m L（1.2 fmol/L）.

Ultrasensitive Electrochemical Aptasensor Based on Surface-Initiated Enzymatic Polymerization

Coupled with the ability of fast and quantitative response, electrochemical aptasensors （EA） have great potential in the application of early diagnosis of cancer biomarker. In order to determine the rare hiomarkers in the compli- cate clinical samples, scientists are making unremitting efforts towards improving the sensitivity and selectivity of EA. Herein, a ＂sandwich＂-strucmre electrochemical aptasensor （SEA） is developed for analysis of cancer biomarker carcino-embryonic antigen （CEA）. Two DNA aptamers are employed, one of which is thiolated at Y-terminal and immobilized onto the gold electrode as a capture probe, while the other one is served as signal probe. The two ap- tamers could ＂sandwich＂ the target and signal probe is then subjected to the terminal deoxynucleotidyl transferase （TdT）-catalyzed incorporation of biotin labeled dNTPs into its 3＇-terminal. Thus the as-generated long DNA oligo tails allow specific binding of numerous avidin modified horseradish peroxidase （Av-HRP）, resulting in enhanced peroxidase catalyzed electrochemical signals. This signal amplification strategy is termed as surface initiated enzy- matic polymerization （SIEP）. This SIEP amplified SEA has a detection limit of 10 pg·mL^-1, indicating the out- standing amplification efficiency.

A novel aptasensor strategy for protein detection based on G-quadruplex and exonuclease Ⅲ-aided recycling amplification

The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.

Handheld Aptasensor for Sandwiched Detection of Chloramphenicol

Chloramphenicol(CAP)is an antifungal agent approved for use in aquarium.However,CAP residue imparts serious adverse effect on human health as well as the ecology.Thus,keeping track of CAP in aquatic products and its environmental metabolites are of great importance.Herein,we developed a novel and neat electrochemical aptasensor for the detection of simulated CAP residual in a sandwich fashion.The screen-printed carbon electrode was employed as the sensing platform and electrodeposited with peculiar nanogolds to enhance the electrochemical performance.For the specific recognition of such small target,a pair of chloramphenicol-binding aptamers,the primary Aptl and the secondary Apt2,were tailored as an artificial single-chained antibody couple,of which the thi-olated Aptl stood on the coated working substrate to capture the analyte,while the biotinylated Apt2 rallied the signal output.In the presence of CAP,Aptl at the lower place cooperated with the upper Apt2 pinched CAP together.After that,the reporter avidin-conjugated horseradish peroxidase associated atop the mounted biotin labels,resulting in an electrocatalytic amplification.In whole,this modular biosensing construction would set a paradigm not just for practicable checkpoint quarantine or point-of-care testing,but potentially adaptable for technological fusion with“Internet Plus”and“Internet of Things”.

An intriguing electrochemical impedance aptasensor based on a porous organic framework supported silver nanoparticles for ultrasensitively detecting theophylline

Porous organic frameworks(POFs)are excellently stable porous materials,which can be employed as host platforms to support metal nanoparticles as functional composites for various applications.Herein,a novel POF is successfully prepared via Friedel-Crafts reaction.Silver nanoparticles(AgNPs)are embedded in the prepared POF to generate an Ag@POF composite,which not only possesses high surface area,outstanding physicochemical stability and outstretchedπ-conjugation skeleton,but also exhibits preferable electrochemical stability and conductivity.This composite is able to immobilize a mass of aptamer strands to fabricate an intriguing electrochemical aptasensor.Electrochemical impedance spectroscopy(EIS)is a commonly used technology to analyze the electrochemical signal variation.The Ag@POF-based biosensor shows the excellent electrochemical detection behavior through analyzing EIS.For instance theophylline as a research mode,the Ag@POF based electrochemical aptasensor reveals ultra-sensitiveness,high selectivity,remarkable stability,good repeatability and simple operability even in various real samples.Notably,this aptasensor has the sensitive detection performance with the limit of detection of 0.191 pg/m L(1.06 pmol/L)in a wide concentration range of 5.0×10^(-4)–5.0 ng/m L(2.78×10^(-3)–27.8 nmol/L).