基于核酸适体的电化学生物传感器*

核酸适体是一类体外筛选的、可与目标分子高效、高特异亲合的RNA或DNA寡核苷酸片段,与常规识别分子（如抗体等）相比,核酸适体作为一类新型识别分子具有明显特色和优势,已被广泛应用于生物传感等分子识别和应用研究领域。本文就基于核酸适体的电化学生物传感器（标记型和非标记型）的近期进展作简要评述,包括适体简介、标记型（“信号衰减”型、“信号增强”型、酶标记型和纳米粒子标记型）和非标记型电化学适体生物传感器等内容。     

Aptamer‐Based Turn‐On Detection of Thrombin in Biological Fluids Based on Efficient Phosphorescence Energy Transfer from Mn‐Doped ZnS Quantum Dots to Carbon Nanodots

This paper presents the first example of a sensitive, selective, and stable phosphorescent sensor based on phosphorescence energy transfer (PET) for thrombin that functions through thrombin–aptamer recognition events. In this work, an efficient PET donor–acceptor pair using Mn‐doped ZnS quantum dots labeled with thrombin‐binding aptamers (TBA QDs) as donors, and carbon nanodots (CNDs) as acceptors has been constructed. Due to the π–π stacking interaction between aptamer and CNDs, the energy donor and acceptor are taken into close proximity, leading to the phosphorescence quenching of donors, TBA QDs. A maximum phosphorescence quenching efficiency as high as 95.9 % is acquired. With the introduction of thrombin to the “off state” of the TBA‐QDs‐CNDs system, the phosphorescence is “turned on” due to the formation of quadruplex‐thrombin complexes, which releases the energy acceptor CNDs from the energy donors. Based on the restored phosphorescence, an aptamer‐based turn‐on thrombin biosensor has been demonstrated by using the phosphorescence as a signal transduction method. The sensor displays a linear range of 0–40 nM for thrombin, with a detection limit as low as 0.013 nM in pure buffers. The proposed aptasensor has also been used to monitor thrombin in complex biological fluids, including serum and plasma, with satisfactory recovery ranging from 96.8 to 104.3 %. This is the first time that Mn‐doped ZnS quantum dots and CNDs have been employed as a donor–acceptor pair to construct PET‐based biosensors, which combines both the photophysical merits of phosphorescence QDs and the superquenching ability of CNDs and thus affords excellent analytical performance. We believe this proposed method could pave the way to a new design of biosensors using PET systems.     

Highly Sensitive Voltammetric Thrombin Aptamer Sensor Based on the Synergistic Effect of Doping/Depositing Gold Nanoparticles in Polydopamine Film

A highly sensitive square‐wave voltammetric thrombin (TB) aptamer sensor was developed using functional polydopamine (PD) film by doping and depositing gold nanoparticles into the bulk and the surface of PD. The aptamer sensor was fabricated by immobilizing a thiolated TB‐binding aptamer (TBA) on the AuNPs‐doped/deposited PD film. AuNPs‐supported methylene blue labels were used for the detection of human α‐TB. Under the optimized conditions, the aptamer sensor’s dynamic range and the detection limit were determined to be 2.0 pM–50 nM and 0.97±0.06 pM, respectively. Finally, the proposed aptamer sensor was successfully examined in human serum samples and satisfactory results were obtained.     

核酸适配体功能化的稀土上转换纳米材料在生物检测中的应用

稀土上转换纳米材料可以吸收近红外光并发射出可见光或紫外光,在生物传感领域得到了广泛研究。核酸适配体能高特异性和高亲和性地与靶标物结合,被广泛应用于生物传感、疾病诊断等领域。将稀土上转换纳米材料与核酸适配体结合构建的检测体系,可实现对目标物灵敏、高选择性的检测。本文介绍了近几年核酸适配体功能化的稀土上转换纳米材料在生物小分子、蛋白质、核酸、病原微生物、细胞等方面的应用,并展望了其在分析检测领域的发展前景。     

Phase Separation of DNA-Encoded Artificial Cells Boosts Signal Amplification for Biosensing

Life-like hierarchical architecture shows great potential for advancing intelligent biosensing, but modular expansion of its sensitivity and functionality remains a challenge. Drawing inspiration from intracellular liquid-liquid phase separation, we discovered that a DNA-encoded artificial cell with a liquid core (LAC) can enhance peroxidase-like activity of Hemin and its DNA G-quadruplex aptamer complex (DGAH) without substrate-selectivity, unlike its gelled core (GAC) counterpart. The LAC is easily engineered as an ultrasensitive biosensing system, benefiting from DNA′s high programmability and unique signal amplification capability mediated by liquid-liquid phase separation. As proof of concept, its versatility was successfully demonstrated by coupling with two molecular recognition elements to monitor tumor-related microRNA and profile cancer cell phenotypes. This scalable design philosophy offers new insights into the design of next generation of artificial cells-based biosensors.     

Separation/Concentration‐signal‐amplification in‐One Method Based on Electrochemical Conversion of Magnetic Nanoparticles for Electrochemical Biosensing

We propose a separation/concentration‐signal‐amplification in‐one method based on electrochemical conversion (ECC) of magnetic nanoparticles (MNPs) to develop a facile and sensitive electrochemical biosensor for chloramphenicol (CAP) detection. Briefly, aptamer‐modified magnetic nanoparticles (MNPs‐Apt) was designed to capture CAP in sample, then the MNPs‐Apt composite was conjugated to Au electrode through the DNA hybridization between the unoccupied aptamer and a strand of complementary DNA. The ECC method was applied to transfer MNPs labels to electrochemically active Prussian blue (PB). The anodic and cathodic currents of PB were taken for signal readout. Comparing with conventional methods that require electrochemically active labels and related sophisticated labelling procedures, this method explored and integrated the magnetic and electrochemical properties of MNPs into one system, in turn realized magnetic capturing of CAP and signal generation without any additional conventional labels. Taking advantages of the high abundance of iron content in MNPs and the refreshing effect deriving from ECC process, the method significantly promoted the signal amplification. Therefore, the proposed biosensors exhibited linear detection range from 1 to 1000 ng mL⁻¹ and a limit of detection down to 1 ng mL⁻¹, which was better than or comparable with those of most analogues, as well as satisfactory specificity, storage stability and feasibility for real samples. The developed method may lead to new concept for rapid and facile biosensing in food safety, clinic diagnose/therapy and environmental monitoring fields.     

Flow—injection Chemiluminescence Determination of Reserpine in Medicine and Biological Fluids with Controlled—Reagent—Release Technology

A sensitive and rapid chemiluminescence(CL) flow injection with controlled-reagent-release technology for the determination of reserpine was proposed.The Cl reagents,luminol and dichromate,uses in this sensor,were all immobilized on anion-exchange resin.Through injection of 100μl of water,the reagents on the anion-ex-change resin column were eluted and in the presence of reserpine ,the CL intensity was decreased,by which reserpine could be sensed.Reserpine was quantified by measuring the decrement of CL intensity,which was observed linear with the logrithm of reserpine concentration in the rage of 1.0-500.0ng/mL,and the limit of detection was 0.4ng/mL(3σ)with a relative standard deviation of less than 3.0?The proposed procedure was applied in the assay of reserpine in pharmaceutical preparation and biological fhuids without any pre-treatment process and with sampling frequencies of 72 times per hour.     

Energy Landscape of Aptamer/Protein Complexes Studied by Single‐Molecule Force Spectroscopy

Aptamers are single‐stranded nucleic acid molecules selected in vitro to bind to a variety of target molecules. Aptamers bound to proteins are emerging as a new class of molecules that rival commonly used antibodies in both therapeutic and diagnostic applications. With the increasing application of aptamers as molecular probes for protein recognition, it is important to understand the molecular mechanism of aptamer–protein interaction. Recently, we developed a method of using atomic force microscopy (AFM) to study the single‐molecule rupture force of aptamer/protein complexes. In this work, we investigate further the unbinding dynamics of aptamer/protein complexes and their dissociation‐energy landscape by AFM. The dependence of single‐molecule force on the AFM loading rate was plotted for three aptamer/protein complexes and their dissociation rate constants, and other parameters characterizing their dissociation pathways were obtained. Furthermore, the single‐molecule force spectra of three aptamer/protein complexes were compared to those of the corresponding antibody/protein complexes in the same loading‐rate range. The results revealed two activation barriers and one intermediate state in the unbinding process of aptamer/protein complexes, which is different from the energy landscape of antibody/protein complexes. The results provide new information for the study of aptamer–protein interaction at the molecular level.     

Recent Progress on the Development of Biofuel Cells for Self‐Powered Electrochemical Biosensing and Logic Biosensing: A Review

Biofuel cells (BFCs) based on enzymes and microorganisms have been recently received considerable attention because they are recognized as an attractive type of energy conversion technology. In addition to the research activities related to the application of BFCs as power source, we have witnessed recently a growing interest in using BFCs for self‐powered electrochemical biosensing and electrochemical logic biosensing applications. Compared with traditional biosensors, one of the most significant advantages of the BFCs‐based self‐powered electrochemical biosensors and logic biosensors is their ability to detect targets integrated with chemical‐to‐electrochemical energy transformation, thus obviating the requirement of external power sources. Following my previous review (Electroanalysis­ 2012 , 24, 197–209), the present review summarizes, discusses and updates the most recent progress and latest advances on the design and construction of BFCs‐based self‐powered electrochemical biosensors and logic biosensors. In addition to the traditional approaches based on substrate effect, inhibition effect, blocking effect and gene regulation effect for BFCs‐based self‐powered electrochemical biosensors and logic biosensors design, some new principles including enzyme effect, co‐stabilization effect, competition effect and hybrid effect are summarized and discussed by me in details. The outlook and recommendation of future directions of BFCs‐based self‐powered electrochemical biosensors and logic biosensors are discussed in the end.     

A Paper Sensor Printed with Multifunctional Bio/Nano Materials

We report a paper‐based aptasensor platform that uses two reaction zones and a connecting bridge along with printed multifunctional bio/nano materials to achieve molecular recognition and signal amplification. Upon addition of analyte to the first zone, a fluorescently labelled DNA or RNA aptamer is desorbed from printed graphene oxide, rapidly producing an initial fluorescence signal. The released aptamer then flows to the second zone where it reacts with printed reagents to initiate rolling circle amplification, generating DNA amplicons containing a peroxidase‐mimicking DNAzyme, which produces a colorimetric readout that can be read in an equipment‐free manner or with a smartphone. The sensor was demonstrated using an RNA aptamer for adenosine triphosphate (a bacterial marker) and a DNA aptamer for glutamate dehydrogenase (Clostridium difficile marker) with excellent sensitivity and specificity. These targets could be detected in spiked serum or feacal samples, demonstrating the potential for testing clinical samples.     

RNA Aptamers That Reversibly Bind Photoresponsive Azobenzene‐Containing Peptides

Modulation of biological networks assembled by diverse interactions among biologically active molecules has provided a platform for innovative biotechnologies. Here, we report RNA aptamers that bind to a photoresponsive peptide (KRAzR; Lys‐Arg‐azobenzene‐Arg) containing azobenzene chromophore, which can change its structure by photoirradiation. Aptamers were identified after 10 cycles of an in vitro selection procedure starting with a DNA library containing a 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation to the SPR sensor chip. Upon irradiation of 360 nm on the KRAzR‐immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm restored the aptamer binding to the surface. We also observed that direct photoirradiation of the aptamer–peptide complex on a gold surface actively promoted dissociation of the complex. Furthermore, a doped reselection method was applied to acquire structural and sequence information of aptamer 66. From a data analysis of the conserved region and the mutation frequency, we were able to select a plausible secondary structure among three candidates predicted by computational folding simulation.     

Preparation of Epirubicin Aptasensor Using Curcumin as Hybridization Indicator: Competitive Binding Assay between Complementary Strand of Aptamer and Epirubicin

The present work explains the fabrication of a novel electrochemical aptasensor for identifying and measuring the epirubicin (Epi) by using curcumin (Cur) as an anticancer electrochemical indicator. The aptasensor prepared by immobilizing the thiolated aptamer on the surface of graphite screen‐printed electrode modified with gold nanoparticles/functionalized multiwall carbon nanotubes, ionic liquid and chitosan nanocomposite (AuNPs/FMWCNTs‐IL‐Chit/SPE). To evaluate the willingness of aptamer to interaction with Epi in the presence of complementary strand DNA, competitive binding assay between the complementary strand of aptamer and Epi were used. Cur tends to bound to the grooves of two strands DNA. With increasing the concentration of Epi in the range of 0.007–7.0 μM, the peak current of Cur decreased, due to the formation of aptamer‐Epi complex and decreasing the amount of complementary strand DNA. Through the control experiments, we examined the response of fabricated aptasensor for some anticancer drugs, which have a structure similar to the Epi. The results showed that using the thiol‐terminated aptamer as a recognition layer led to a sensor with a high tendency for Epi compared to other anticancer drugs.     

基于计时库仑技术的可再生型三磷酸腺苷适配体电化学传感器的研究

构建了一种可再生型三磷酸腺苷(ATP)适配体计时库仑电化学传感器.将一条短链DNA通过AuS键自组装固定在电极表面, ATP的核酸适配体与该短链DNA杂交而结合在电极表面.带负电的DNA通过静电吸引结合电解液中的六氨合钌(RuHex)阳离子.当传感器和靶分子ATP孵育后,ATP与核酸适配体结合,使适配体链从电极表面解离,电极表面吸附的DNA量减少,结合RuHex的量随之降低.通过计时库仑技术检测RuHex响应信号降低的量 ,可以对ATP进行定量测定.此传感器的电化学响应信号与ATP浓度对数值呈线性关系,线性检测范围为0.001~100 μmol/L,检出限(S/N=3)为0.5 nmol/L.此传感器检测靶分子ATP后,可以通过简单的操作步骤再生,再生5次后的响应信号为初始信号的90%以上.采用此传感器检测大鼠脑透析液中ATP的含量为(19.2±3.7) nmol/L (n=3).     

Impedimetric Aptasensor for Thrombin Recognition Based on CD Support

We report an aptamer biosensing array for thrombin detection by measuring the electrochemical impedance upon aptamer‐protein formation at the surface of CD‐trodes (GCDTs) in the presence of the redox couple [Fe(CN)₆]^3?/4?. GCDTs are fabricated from recordable compact discs that contain a fine gold layer. The biosensor is constructed by self‐assembling of a thiol‐modified thrombin binding aptamer (TBA) onto a GCDT surface. The sensor reveals good ligand specificity, recognition in a wide range of thrombin concentrations from 20 nM to 1 µM with a limit of detection of 5 nM.     

适配体生物传感器在病原微生物检测中的应用

适配体是通过指数富集系统进化技术(SELEX)体外筛选得到的一类能够特异性地结合小分子物质、蛋白,甚至整个细胞的寡核苷酸序列.由于具有制备简便、易于修饰、稳定性好等特点,适配体已广泛应用于构建生物传感器,实现对病原微生物的识别和检测.本文在阐述适配体基本原理的基础之上,结合近年来病原微生物适配体研究领域的最新研究成果,综述以病原微生物为目标的适配体筛选技术的最新进展;列举目前已经筛选获得的病原微生物(原生生物、病毒、细菌)适配体;综述适配体生物传感器在病原微生物检测中的应用.并展望了适配体生物传感器在病原微生物检测领域的发展趋势.     

Electrochemical aptamer sensor for highly sensitive detection of mercury ion with Au/Pt@carbon nanofiber‐modified electrode

In this paper, an electrochemical aptamer sensor was proposed for the highly sensitive detection of mercury ion (Hg²⁺). Carbon nanofiber (CNF) was prepared by electrospinning and high‐temperature carbonization, which was used for the loading of platinum nanoparticles (PtNPs) by the hydrothermal method. The Pt@CNF nanocomposite was modified on the surface of carbon ionic liquid electrode (CILE) to obtain Pt@CNF/CILE, which was further decorated by gold nanoparticles (AuNPs) through electrodeposition to get Au/Pt@CNF/CILE. Self‐assembling of the thiol‐based aptamer was further realized by the formation of Au‐S bond to get an electrochemical aptamer sensor (Aptamer/Au/Pt@CNF/CILE). Due to the specific binding of aptamer probe to Hg²⁺ with the formation of T‐Hg²⁺‐T structure, a highly sensitive quantitative detection of Hg²⁺ could be achieved by recording the changes of current signal after reacting with Hg²⁺ within the concentration range from 1.0 × 10^?15 mol/L to 1.0 × 10^?6 mol/L and the detection limit of 3.33 × 10^?16 mol/L (3σ). Real water samples were successfully analyzed by this method.     

Application of Some Room Temperature Ionic Liquids in the Development of Biosensors at Carbon Film Electrodes

Two room temperature ionic liquids, 1‐butyl‐3‐methylimidazolium bistriflimide and 1‐butyl‐3‐methylimidazolium nitrate, were employed for enzyme immobilization in a new sol‐gel matrix and, for the first time, were successfully applied as electrolyte carriers in a biosensing system. The new sol‐gel matrix, based on 3‐aminopropyltrimethoxysilane and 1‐butyl‐3‐methylimidazolium bistriflimide mixtures, did not crack even after several weeks when kept dry, and exhibited similar analytical properties to aqueous sol‐gel based glucose biosensors. The linear range was up to 1.1 mM of glucose, sensitivity was 62 nA mM^?1 and the limit of detection was 28.8 μM. The optimum ionic liquid electrolyte carrier was found to be 1‐butyl‐3‐methylimidazolium nitrate, where the biosensor was made by electrodeposition of the redox mediator, poly(neutral red), and the enzyme was immobilized by cross‐linking with glutaraldehyde. The results showed that application of room temperature ionic liquids to biosensors is very promising and can be further exploited.     

Suitability of dispersive liquid–liquid microextraction for the in situ silylation of chlorophenols in water samples before gas chromatography with mass spectrometry

Trace analysis of chlorophenols in water was performed by simultaneous silylation and dispersive liquid–liquid microextraction followed by gas chromatography with mass spectrometry. Dispersive liquid–liquid microextraction was carried out using an organic solvent lighter than water (n‐hexane). The effect of different silylating reagents on the method efficiency was investigated. The influence of derivatization reagent volume, presence of catalyst and derivatization/extraction time on the yield of the derivatization reaction was studied. Different parameters affecting extraction efficiency such as kind and volume of extraction and disperser solvents, pH of the sample and addition of salt were also investigated and optimized. Under the optimum conditions, the calibration graphs were linear in the range of 0.05–100 ng/mL and the limit of detection was 0.01 ng/mL. The enrichment factors were 242, 351, and 363 for 4‐chlorophenol, 2,4‐dichlorophenol, and 2,4,6‐trichlorophenol, respectively. The values of intra‐ and inter‐day relative standard deviations were in the range of 3.0–6.4 and 6.1–9.9%, respectively. The applicability of the method was investigated by analyzing water and wastewater samples.     

Chitosan‐Glutamate Oxidase Gels: Synthesis,Characterization, and Glutamate Determination

The biopolymer chitosan (CHIT) was chemically modified with glutaric dialdehyde (GDI) and used for the covalent immobilization of enzyme glutamate oxidase (GmOx). The relationships between the loaded, retained, and active units of GmOx in the CHIT‐GDI‐GmOx gels were determined by electrochemical assays. The latter indicated that on average ca. 95% of the GmOx was retained in the CHIT‐GDI matrix that was loaded with 0.10–3.0 units of the enzyme. The maximum activity of the GmOx immobilized in the gels corresponded to ca. 5% of the activity of the free enzyme. Platinum electrodes coated with CHIT‐GDI‐GmOx gels (films) were used as amperometric biosensors for glutamate. Such biosensors displayed good operational and long‐term stability (at least 11 h and 100 days, respectively) in conjunction with low detection limit of 0.10 μM glutamate (S/N=3), linear range up to 0.5 mM (R²=0.991), sensitivity of 100 mA M^?1 cm^?2, and short response time (t_90%=2 s). This demonstrated an efficient signal transduction in the Pt/CHIT‐GDI‐GmOx+glutamate system. The CHIT‐GDI‐GmOx gels constitute a new biosensing element for the development of glutamate biosensors.     

Biofuel Cells for Self‐Powered Electrochemical Biosensing and Logic Biosensing: A Review

This article reviews recent advances and progress in developing electrochemical (EC) biosensing and logic biosensing systems based on self‐powered biofuel cells (BFCs). BFCs that exploit enzymes and microbes have attracted a considerable recent interest owing to their unique ability to provide sustainable energy from renewable fuel source under mild conditions. This review focuses on recently introduced novel concepts for using BFCs as the basic element for EC‐biosensing and especially EC‐logic biosensing applications. The fabrication and design of such self‐powered EC‐biosensing and EC‐logic biosensing are described along and different new approaches for BFCs‐based EC‐biosensing and EC‐logic biosensing involving substrate effects, inhibition effects, blocking effects and gene regulation effects. Latest advances in coupling a self‐powered diagnostic operation with logic‐activated drug release functionality are discussed. We conclude with the implications of the new self‐powered biosensing/logic‐biosensing platforms along with future prospects and challenges.