Electrochemical biosensors and logic devices based on aptamers

Aptamers are molecular recognition elements with high specificity that are selected from deoxyribonucleic acid/ribonucleic acid (DNA/RNA) library. Compared with the traditional protein recognition elements,aptamers have excellent properties such as cost-effective,stable,easy for synthesis and modification. In recent years,electrochemistry plays an important role in biosensor field because of its high sensitivity,high stability, fast response and easy miniaturization. Through the combination of these two technologies and our rational design,we constructed a series of biosensors and biochips that are simple,fast,cheap and miniaturized. Firstly,we designed an adenosine triphosphate (ATP) electrochemical biosensor based on the strand displacement strategy. We can detect as low as 10 nmol/L of ATP both in pure solution and complicated cell lysates. Secondly,we creatively split the aptamers into two fragments and constructed the sandwich assay platform only based on single aptamer sequence. We successfully transferred this design on biochips with multiple micro electrodes (6×6) and accomplished multiplex detection. In the fields of biochips and biocomputers,we designed several DNA logic gates with electric (electrochemical) signal as output which paves a new way for the development of DNA computer.

Selecting small molecule DNA aptamers with significant conformational changes for constructing transcriptional switches and biosensors

Small molecule aptamers discovered by traditional selection methods usually lack conformational changes upon target binding.This limits the use of aptamers as molecular probes for small molecule detection and regulatory elements of genetic circuits.Here,we report a new method called capture and in vitro transcription-systematic evolution of ligands by exponential enrichment(CIVT-SELEX)to select DNA aptamers that can not only bind to small molecule ligands but also undergo significant conformational changes.Through this method,we select a structure-switching aptamer of uridine-5′-diphosphate(UDP).Taking advantage of its conformational changes,we first construct a UDP-responsive transcriptional switch by inserting the aptamer in a genetic circuit and demonstrate that it can respond to the addition of UDP and regulate the transcription of downstream genes.We also build a UDP aptamer-based biosensor that can be used for active glycosyltransferase screening.We believe this method can provide a universal platform for selecting small molecule aptamers with conformational changes and expand the use of aptamers in small molecule detection and genetic regulation.

Recent advances in quantum dots-based biosensors for antibiotics detection

Antibiotics are a category of chemical compounds used to treat bacterial infections and are widely applied in cultivation,animal husbandry,aquaculture,and pharmacy.Currently,residual antibiotics and their metabolites pose a potential risk of allergic reactions,bacterial resistance,and increased cancer incidence.Residual antibiotics and the resulting bacterial antibiotic resistance have been recognized as a global challenge that has attracted increasing attention.Therefore,monitoring antibiotics is a critical way to limit the ecological risks from antibiotic pollution.Accordingly,it is desirable to devise new analytical platforms to achieve efficient antibiotic detection with excellent sensitivity and specificity.Quantum dots(QDs)are regarded as an ideal material for use in the development of antibiotic detection biosensors.In this review,we characterize different types of QDs,such as silicon,chalcogenide,carbon,and other doped QDs,and summarize the trends in QD-based antibiotic detection.QD-based sensing applications are classified according to their recognition strategies,including molecularly imprinted polymers(MIPs),aptamers,and immunosensors.We discuss the advantages of QD-derived antibiotic sensors,including low cost,good sensitivity,excellent stability,and fast response,and illustrate the current challenges in this field.

Nucleic Acid Aptamers as Potential Therapeutic and Diagnostic Agents for Lymphoma

Lymphomas are cancers that arise from white blood cells and usually present as solid tumors. Treatment of lymphoma often involves chemotherapy, and can also include radiotherapy and/or bone marrow transplantation. There is an unquestioned need for more effective therapies and diagnostic tool for lymphoma. Aptamers are single stranded DNA or RNA oligonucleotides whose three-dimensional structures are dictated by their sequences. The immense diversity in function and structure of nucleic acids enable numerous aptamers to be generated through an iterative in vitro selection technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers have several biochemical properties that make them attractive tools for use as potential diagnostic and pharmacologic agents. Isolated aptamers may directly inhibit the function of target proteins, or they can also be formulated for use as delivery agents for other therapeutic or imaging cargoes. More complex aptamer identification methods, using whole cancer cells (Cell-SELEX), may identify novel targets and aptamers to affect them. This review focuses on recent advances in the use of nucleic acid aptamers as diagnostic and therapeutic agents and as targeted delivery carriers that are relevant to lymphoma. Some representative examples are also discussed.

Aptamer based detection and separation platforms for ochratoxin A:A systematic review

Ochratoxin A(OTA),one of the most dangerous mycotoxins for human health,has been subjected to numerous studies for separation and detection in minimal amounts.Aptamers as novel recognition elements have been employed to fabricate ultrasensitive biosensors for the detection of OTA and designing delicate analytical tools.This review attempted to comprehensively examine all reported aptamer-based detection and separation platforms for ochratoxin.The most relevant databases were considered to discover all specific aptamers for dealing with OTA.Aptamer-based detection and separation devices specified for OTA were searched for,analyzed,discussed,and classified based on their specifications.The optical aptasensors have gathered a higher interest than electrochemical aptasensors,which can achieve a lower limit of detections.Moreover,some extraction platforms based on these aptamers were also found.However,aptamer-based devices seem to have some challenges in their application.

Aptamers—A Promising Approach for Sensing of Biothreats Using Different Bioinformatics Tools

Intentional release of pathogens or biotoxin against humans, plants, or animals is an impending threat all over the world. Continuous monitoring of environment is required for their detection. These signals can help to distinguish whether the bioattack has occurred or not. Biosensors utilise biological response including different biochemical reactions, antigen antibody reactions, electrochemical reactions, aptameric reactions etc. The currently available biosensors have a limit of detection, specificity and less linearity which affect their sensitivity. Aptamers are single stranded oligonucleotides binding species which are capable of tightly binding to their distinguishing targets. They are evolved from random oligonucleotides pools by using different strategies. These are capable of conscientiously distinguishing their target ligands. They have high sensitivity and a wide range of detection limit. The versatility of nucleic acid based methods allowed for the design of specific aptamer sequences, typically on the order of 10 to 30 base pairs in length, identifying the different biothreat agents in the environment. By using different bioinformatics tools we can design RNA aptamers for toxins of lectin family.

Cocaine Detection in Blood Serum Using Aptamer Biosensor on Gold Nanoparticles and Progressive Dilution

To fight against the illegal usage of cocaine it is necessary to develop various analytical methodologies. We report here an aptamer-based biosensor for the determination of cocaine using gold nanoparticles as the fluorescence quencher. By employing the progressive dilution （PD） strategy, simultaneous qualitative and quantitative analysis of cocaine in blood serum was achieved without pretreatment of the sample. The method described in this paper of- fers significant improvement in the detection accuracy and can be used to quantify cocaine levels in complex bio- logical samples such as serum with a simple procedure.

Signaling Kinetics of DNA and Aptamer Biosensors Revealing Graphene Oxide Surface Heterogeneity

Adsorption of fluorescently labeled DNA and aptamer probes to graphene oxide(GO)has been one of the most popular methods for developing biosensors.In the presence of target analytes,the quenched fluorescence would recover.In this work,we followed the kinetics of the reactions and found that the fluorescence would eventually drop after an initial increase,and this was attributed to the re-adsorption of the desorbed probe DNA molecules.Both a DNA probe and an aptamer for adenosine were used.This re-adsorption was attributed to the surface heterogeneity of GO,and the DNA probes desorbed from relatively weaker binding sites were re-adsorbed on the stronger binding sites.This re-adsorption can be avoided by extensive washing the samples,and also by blocking the GO surface or by heating.This fundamental understanding is important for achieving a stable signal of such biosensors.

Solid-state electrochemiluminescence protein biosensor with aptamer substitution strategy

One solid-state electrochemiluminescence(ECL) protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed.Additionally,the biosensor was based on ECL photo-quenching effect of ferrocene(Fc) to tris(2,2'-bipyridyl)ruthenium(II)(Ru(bpy)32+).It was built up by modification of Au nanoparticles(AuNPs) and Ru(bpy)3 2+ on one Au electrode firstly,and then self-assembly of one special double-stranded DNA(dsDNA) onto the electrode.This dsDNA was prepared by hybridization of one Fc labeled molecular beacon single-stranded DNA(ssDNA) and one anti-thrombin aptamer ssDNA.Without the target protein,this Fc-dsDNA/Ru(bpy)3 2+-AuNPs/Au electrode trigged strong ECL signal,so we called it ECL "signal on" state.When thrombin was present in the sensing solution,the protein reacted with its aptamer from the Fc-dsDNA/Ru(bpy)3 2+-AuNPs/Au electrode.Then the left molecular beacon ssDNA on the electrode recovered to its normal stem-loop structure and consequently its Fc labeler was close enough to the electrode surface to quench the ECL signal from Ru(bpy)3 2+.It was in ECL "signal off" state.We measured the decrease in ECL intensity to sense the target protein.This was one endeavour to sense protein by using un-labeling target or probe strategy,which gave higher sensitivity and selectivity due to the better combination efficiency of protein and the un-labeled aptamer.6.25 fmol/L thrombin was detected out.

Aptamer-Based Antibiotic Electrochemical Detection Platform Using Portable Plastic Gold Electrode and Multichannel Chip

The development of accurate,rapid and practical electrochemical sensing technology for antibiotic detection has an important application prospect in many fields such as food safety,environmental monitoring,medical and health care.In this research,kanamycin specific DNA aptamer and its partially complementary short chain were self-assembled onto the surface of an integrated portable plastic gold electrode(PGE)by Au-s bond,and a simple kanamycin electrochemical biosensor interface was constructed.At the same time,the epoxy resin channel layer was designed,and the auxiliary instrument was set up independently in the laboratory,which could help to measure and analyze eight groups of samples automatically in turn.The quantitative analysis results showed that in 20 min,the sensor had a good linear relationship between the peak current change of square wave voltammetry and the negative logarithm of kanamycin concentration when kanamycin concentration was in the range of 1-10o0μmol/L,and the detection limit could reach 0.40μmol/L.In addition,the discrimination could be achieved within 5 min in the real-time analysis mode.The sensor had a simple construction method,good selectivity and stability,and could be used for rapid or real-time detection of kanamycin residues in actual water samples,which provided a new direction for the practical detection of kanamycin in environmental watersamples.

Aptamer-induced in-situ growth of acetylcholinesterase-Cu_(3)(PO_(4))_(2)hybrid nanoflowers for electrochemical detection of organophosphorus inhibitors

Enzyme-inorganic hybrid nanoflowers(HNFs)have shown excellent sensing capabilities due to their large specific surface area as well as the simplicity and mildness of the preparation process.However,coupling HNFs to electrodes to fabricate a uniform and controllable enzymatic electrochemical sensing interface remains a challenge.Here,we proposed an aptamer-induced insitu fabrication strategy for preparing an HNF-based electrochemical sensor with ideal performance.Central to this strategy is the introduction of acetylcholinesterase(AChE)-specific binding aptamer(Apt),which induces the in-situ growth of AChE-copper phosphate(Cu_(3)(PO_(4))_(2))HNFs on the surface of carbon paper(CP).In addition,a dense gold nanoparticle(AuNP)layer was electrodeposited on the CP for anchoring Apt and further extending the electroactive surface area.The prepared AChECu_(3)(PO_(4))_(2)HNF/Apt/AuNP/CP biosensor exhibited a wide detection range from 1 to 107 pM for the four organophosphorus inhibitors,including isocarbophos,dichlorvos,methamidophos,and parathion,with detection limits down to 0.016,0.028,0.071,and 0.113 pM,respectively.With the reactivation of pralidoxime chloride,the electrode can still recover 98.1%of the response after five times of repeated use.In real sample detection,the biosensor achieved high recoveries from 96.45%to 100.13%.The detection target may be extendable to other AChE inhibitors(e.g.,drugs for Alzheimer’s disease).This study demonstrates for the first time the feasibility of using aptamers as an inducer to fabricate an electrochemical enzyme sensing interface in-situ.This strategy can be used to fabricate other enzyme-based biosensors and therefore has broader applications.

Systematic bio‑fabrication of aptamers and their applications in engineering biology

Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets.Compared to antibodies,aptamers are easy to in vitro synthesize with low cost,and exhibit excellent thermal stability and programmability.With these features,aptamers have been widely used in biology and medicine-related fields.In the meantime,a variety of systematic evolution of ligands by exponential enrichment(SELEX)technologies have been developed to screen aptamers for various targets.According to the characteristics of targets,customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity.In this review,we first summarize the latest research on the systematic bio-fabrication of aptamers,including various SELEX technologies,post-SELEX optimization,and aptamer modification technology.These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers.The latter provides a new perspective for the systems bio-fabrication of aptamers.Furthermore,on this basis,we review the applications of aptamers,particularly in the fields of engineering biology,including industrial biotechnology,medical and health engineering,and environmental and food safety monitoring.And the encountered challenges and prospects are discussed,providing an outlook for the future development of aptamers.

Capture-SELEX for Chloramphenicol Binding Aptamers for Labeled and Label-Free Fluorescence Sensing

Chloramphenicol(CAP)is a potent antibiotic.Due to its side effects,CAP is currently banned in most countries,but it is still found in many food products and in the environment.Developing aptamer-based biosensors for the detection of CAP has interested many researchers.While both RNA and DNA aptamers were previously reported for CAP,they were all obtained by immobilization of the CAP base,which omitted the two chlorine atoms.In this work,DNA aptamers were selected using the libraryimmobilized method and free unmodified CAP.Three families of aptamers were obtained,and the best one named CAP1 showed a dissociation constant(K_(d))of 9.8μM using isothermal titration calorimetry(ITC).A fluorescent strand-displacement sensor showed a limit of detection(LOD)of 14μM CAP.Thioflavin T(ThT)staining allowed label-free detection of CAP with a LOD of 1μM in buffer,1.8μM in Lake Ontario water,and 3.6μM in a wastewater sample.Comparisons were made with previously reported aptamers,and ITC failed to show binding of a previously reported 80-mer aptamer.Due to the small size and well-defined secondary structures of CAP1,this aptamer will find analytical applications for environmental and food monitoring.

Recent Progress on Highly Selective and Sensitive Electrochemical Aptamer-based Sensors

Highly selective,sensitive,and stable biosensors are essential for the molecular level understanding of many physiological activities and diseases.Electrochemical aptamer-based(E-AB)sensor is an appealing platform for measurement in biological system,attributing to the combined advantages of high selectivity of the aptamer and high sensitivity of electrochemical analysis.This review summarizes the latest development of E-AB sensors,focuses on the modification strategies used in the fabrication of sensors and the sensing strategies for analytes of different sizes in biological system,and then looks forward to the challenges and prospects of the future development of electrochemical aptamerbased sensors.

Peptide aptamers:The versatile role of specific protein function inhibitors in plant biotechnology

In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad- based virus resistance in crops, ＂reverse genetics＂ and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization.

Programmable Analytical Feature of Ratiometric Electrochemical Biosensor by Alternating the Binding Site of Ferrocene to DNA Duplex for the Detection of Aflatoxin B1

Ratiometric biosensor is among the promising strategies for the advanced analysis;its applicability in multiple scenarios,however,is seriously limited for the fixed analytical feature.Here,we reported ratiometric electrochemical biosensors with programmable analytical feature by alternating the binding site of ferrocene(Fc)to DNA duplex for the detection of aflatoxin B1(AFB1).DNA duplex was synthesized with AFB1 aptamer and its complementary DNA(cDNA)with Fc molecular tagged on aptamer or cDNA.For analysis,aptamer-AFB1 complex stripped from the electrode,and free cDNA triggered hybridization chain reaction to produce double-stranded DNA for methylene blue(MB)absorption.In this way,redox currents of Fc(I_(Fc))and absorbed MB(I_(MB))at the electrode were collected to produce the ratiometric signal of I_(MB)/I_(Fc)as a yardstick for AFB1.When Fc was tagged on aptamer,IFc decreased with higher concentration of AFB1(C_(AFB1)),the resultant biosensor allowed detection with an On/Off(I_(MB)/I_(Fc))mode;when Fc was tagged on cDNA,I_(Fc) kept constant as C_(AFB1)varying,offering an On/Constant(I_(MB)/I_(Fc))mode for the detection.Consequently,biosensor with On/Off mode showed a linear range of 0.1—100 pg/mL with a limit of detection(LOD)of 0.0650 pg/mL,while biosensor with On/Constant mode offered a wider linear rang of 1—10000 pg/mL but a relatively poor LOD of 0.330 pg/mL.Our work revealed the vital importance of binding site of probes to DNA,and this principle can endow ratiometric biosensor with programmable analytical feature(e.g.,linear range,LOD),allowing detections in various scenarios.

Aptamers as Recognition Elements for Electrochemical Detection of Exosomes

Exosome analysis is emerging as an attractive noninvasive approach for disease diagnosis and treatment monitoring in the field of liquid biopsy.Aptamer is considered as a promising molecular probe for exosomes detection because of the high binding affinity,remarkable specificity,and low cost.Recently,many approaches have been developed to further improve the performance of electrochemical aptamer based(E-AB)sensors with a lower limit of detection.In this review,we focus on the development of using aptamer as a specific recognition element for exosomes detection in electrochemical sensors.We first introduce recent advances in evolving aptamers against exosomes.Then,we review methods of immobilization aptamers on electrode surfaces,followed by a summary of the main strategies of signal amplification.Finally,we present the insights of the challenges and future directions of E-AB sensors for exosomes analysis.

Aptamer-based molecular imaging

Molecular imaging has greatly advanced basic biology and translational medicine through visualization and quantification of single/multiple molecular events temporally and spatially in a cellular context and in living organisms.Aptamers,short single-stranded nucleic acids selected in vitro to bind a broad range of target molecules avidly and specifically,are ideal molecular recognition elements for probe development in molecular imaging.This review summarizes the current state of aptamer-based biosensor development(probe design and imaging modalities)and their application in imaging small molecules,nucleic acids and proteins mostly in a cellular context with some animal studies.The article is concluded with a brief discussion on the perspective of aptamer-based molecular imaging.

基于Exo Ⅲ信号放大的荧光适配体传感器检测Kunitz型大豆胰蛋白酶抑制因子

Kunitz型大豆胰蛋白酶抑制因子(Kunitz Soybean Trypsin Inhibitor,KTI)是一种很关键的抗营养因子,不仅对动物消化系统和生长发育有不良影响,还制约各个行业对大豆的利用率,因此快速有效的检测方法是非常必要的。该研究建立一种基于核酸外切酶Ⅲ(Exonuclease Ⅲ,Exo Ⅲ)和碳纳米颗粒(Carbon Nanoparticles,CNPs)的信号辅助放大荧光传感体系用于KTI的检测。具体体系包括KTI适配体(Aptamer,APT)、互补链(Complementary DNA,c DNA)、信号探针(Signal Probe,SP)、Exo Ⅲ和CNPs共5个部分。通过可行性分析和CNPs浓度优化试验,测得KTI在100~600 ng/mL范围内呈线性相关,检测限为12.59 ng/mL。以豆浆作为样品,采用加标回收测得回收率为97.42%~102.85%,RSD在0.61%~2.36%之间,该方法可对实际样品中的KTI进行测定。

Aptamer-based competitive electrochemical assay of small biomolecules

A convenient aptamer-based competitive electrochemical biosensor for a small biomolecule,adenosine,was described. The sensing surface was fabricated by self-assembly of an aptamer/mercaptohexanol monolayer on a gold disk electrode. The principle of this aptasensor is based on the competition between an adenosine target molecule and a ferrocene-conjugated signaling DNA strand for the aptamer binding site on the sensing surface. Due to the competitive nature of this assay,the electrochemical responses of the surface captured ferrocene are inversely proportional to log[adenosine] in the range from 0.05 to 3.2 μM,with a detection limit of 25 nM. Moreover,the aptasensor also shows high selectivity for adenosine. The proposed aptasensor thus holds great potential for the detection of other small biomolecules.