Electrochemical Aptasensors for Biological and Chemical Analyte Detection

Aptamers are short length, single-stranded DNA or RNA affinity molecules which interact with any desired targets such as biomarkers, cells, biological molecules, drugs or chemicals with high sensitivity. They have been extensively employed for medical applications due to having more advantages than the antibodies such as easier preparation and modification, higher stability, lower batch-to-batch variability and cost. Moreover, aptamers can be easily integrated efficiently with sensors, biosensors, actuators and other devices. In this review article, different applications of aptamers for biological and chemical molecules detection within the scope of electrochemical methods were presented with recent studies. In addition, the present status and future perspectives for highly-effective aptasensors for specific and selective analyte detection were discussed. As in stated throughout the review, combining of extraordinary properties of aptamers with the electrochemical-based biosensors could have improved the sensitivity of the assay and reduced limit of detection.     

电化学和电化学发光核酸适体传感器

核酸适体具有亲合力强、选择性高、稳定性好、易于修饰等优点,广泛用于对目标物如蛋白质、小分子等的灵敏检测.电化学具有成本低、灵敏度高、仪器小巧等优点.近年来,构建基于核酸适体的电化学传感器,已经成为一个热门的研究领域.本文重点评述了2005年以来核酸适体的电化学传感器的研究进展,并展望其发展前景.     

Aptasensors – the future of biosensing?

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplification. Aptamers, first reported in 1990, are attracting interest in the areas of therapeutics and diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors. The properties of aptamers, their applicability to biosensor technology, current research and future prospects are addressed in this short review.     

New trends in affinity sensing: aptamers for ligand binding

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and amplification. This review described the in vitro process to obtain aptamers (SELEX). It mentions the main characteristics of these molecules (i.e. affinity, specificity and stability). Moreover, it discusses advantages over antibodies. It reports potential applications of aptamers in analytical and diagnostic assays as biocomponents of biosensors (aptasensors) and allosteric ribozymes (aptazymes).     

细菌的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化技术(SELEX)筛选的能够以高亲和力和高特异性识别靶标分子或细胞的核糖核酸(RNA)和单链脱氧核糖核酸(ssDNA)。作为化学抗体,核酸适配体的制备和合成比抗体的成本更低。核酸适配体的靶标范围极其广泛,包括小分子、生物大分子、细菌和细胞等。针对细菌靶标筛选的适配体,目前主要应用于食品、医药和环境中的细菌检测。细菌的核酸适配体筛选可以通过离心法将菌体-适配体复合物与游离的适配体分离,并通过荧光成像、荧光光谱分析、流式细胞仪分选、DNA捕获元件、酶联适配体分析等方法表征适配体与靶标的相互作用。筛选出的适配体可结合生物、化学检测方法用于细菌检测。本文介绍了细菌适配体的筛选和表征方法以及基于适配体的检测方法的最新进展,分析了不同检测方法的利弊,并列出了2011~2015年筛选的细菌的核酸适配体。     

用于赭曲霉毒素A检测的电化学适配体生物传感器的研究进展

赭曲霉毒素（Ochratoxin）是一类主要由曲霉菌和青霉菌产生的次生代谢产物,其中赭曲霉毒素A（OTA）的毒性最强。OTA相当稳定,常规的食品加工难以去除,若摄入受OTA污染的食品或药物会对人类造成严重的危害。实现对OTA的灵敏和快速检测是及早发现和处置OTA污染的关键。近年来,核酸适配体因其独特的优点,被作为抗体的替代物用于构建OTA电化学生物传感器。本文介绍了经典的OTA检测方法和基于适配体的电化学生物传感检测方法,从OTA电化学适配体传感器的适配体优化、新型材料应用以及生物信号放大技术的应用等三个方面总结了该生物传感技术的研究现状,并对其未来的发展进行了展望     

核酸适配体在癌症诊断中的研究进展

核酸适配体是通过指数富集配体系统进化技术(SELEX)从体外合成的寡核苷酸文库中筛选得到的短的寡核苷酸分子(ssDNA或RNA).核酸适配体能够通过折叠成特定的空间结构与靶标分子进行特异性结合,与抗体相比,适配体具有高亲和力、易修饰、低成本、易于合成和低免疫原性等优势,可以针对细胞、蛋白质、组织、生长因子进行癌症生物标...     

Impedimetric Aptasensors Based on Carbon Nanotubes – Poly(methylene blue) Composite

The effect of aptamer structure and immobilization platform on the efficiency of thrombin binding and its detection using electrochemical impedance spectroscopy (EIS) characteristics was investigated with aptasensors based on glassy carbon electrodes covered with multiwalled carbon nanotubes (MWNTs). Aptamers with one or two binding sequences GGTTGGTGTGGTTGG specific for thrombin and poly(dA) and poly(dT) tags able to form dimeric products (aptabodies) were used to establish significance of steric and electrostatic factors in aptasensor performance. We have shown that electropolymerization of methylene blue onto MWNTs significantly improved electrochemical characteristics and sensitivity of thrombin detection against bare MWNTs. Charge transfer resistance and capacitance of the surface layer were measured in the presence of redox probe [Fe(CN)₆]^3?/4?. Aptasensors make it possible to detect thrombin in the concentration range 1 nM–1 µM with the limit of detection of 0.7 nM (monitoring resistance changes) and 0.5 nM (capacitance changes), respectively.     

Ferrocene-Labelled Electroactive Aptamer-Based Sensors (Aptasensors) for Glycated Haemoglobin

Glycated haemoglobin (HbA_1c) is a diagnostic biomarker for type 2 diabetes. Traditional analytical methods for haemoglobin (Hb) detection rely on chromatography, which requires significant instrumentation and is labour-intensive; consequently, miniaturized devices that can rapidly sense HbA_1c are urgently required. With this research, we report on an aptamer-based sensor (aptasensor) for the rapid and selective electrochemical detection of HbA_1c. Aptamers that specifically bind HbA_1c and Hb were modified with a sulfhydryl and ferrocene group at the 3′ and 5′-end, respectively. The modified aptamers were coated through sulfhydryl-gold self-assembly onto screen printed electrodes, producing aptasensors with built in electroactivity. When haemoglobin was added to the electrodes, the current intensity of the ferrocene in the sensor system was reduced in a concentration-dependent manner as determined by differential pulse voltammetry. In addition, electrochemical impedance spectroscopy confirmed selective binding of the analytes to the aptamer-coated electrode. This research offers new insight into the development of portable electrochemical sensors for the detection of HbA_1c     

Aptasensor for Thrombin Based on Carbon Nanotubes‐Methylene Blue Composites

The amperometric and EQCM aptasensors based on DNA aptamers immobilized by avidin‐biotin method or by electrostatic adsorption onto multiwalled carbon nanotube layer contained methylene blue (MB) have been developed and examined for thrombin detection in buffer and in spiked blood serum. The presence of MB increases the binding capacity of the surface layer and enhances the range of thrombin concentrations to be determined. This results in significant improvement of analytical characteristics of thrombin detection. The EQCM aptasensors allowed us to detect 0.3–100 nM and amperometric aptasensors 10–1000 nM of thrombin.     

Multiplex metal-detection based assay (MMDA) for COVID-19 diagnosis and identification of disease severity biomarkers

The ongoing COVID-19 pandemic caused by SARS-CoV-2 highlights the urgent need to develop sensitive methods for diagnosis and prognosis. To achieve this, multidimensional detection of SARS-CoV-2 related parameters including virus loads, immune response, and inflammation factors is crucial. Herein, by using metal-tagged antibodies as reporting probes, we developed a multiplex metal-detection based assay (MMDA) method as a general multiplex assay strategy for biofluids. This strategy provides extremely high multiplexing capability (theoretically over 100) compared with other reported biofluid assay methods. As a proof-of-concept, MMDA was used for serologic profiling of anti-SARS-CoV-2 antibodies. The MMDA exhibits significantly higher sensitivity and specificity than ELISA for the detection of anti-SARS-CoV-2 antibodies. By integrating the high dimensional data exploration/visualization tool (tSNE) and machine learning algorithms with in-depth analysis of multiplex data, we classified COVID-19 patients into different subgroups based on their distinct antibody landscape. We unbiasedly identified anti-SARS-CoV-2-nucleocapsid IgG and IgA as the most potently induced types of antibodies for COVID-19 diagnosis, and anti-SARS-CoV-2-spike IgA as a biomarker for disease severity stratification. MMDA represents a more accurate method for the diagnosis and disease severity stratification of the ongoing COVID-19 pandemic, as well as for biomarker discovery of other diseases.

A MMDA platform is developed by using metal-tagged antibodies as reporting probes combined with machine learning algorithms, as a general strategy for highly multiplexed biofluid assay.     

Trends in biosensing platforms for SARS-CoV-2 detection: A critical appraisal against standard detection tools

In this ongoing theme of coronavirus disease 2019 (COVID-19) pandemic, highly sensitive analytical testing platforms are extremely necessary to detect SARS-CoV-2 RNA and antiviral antibodies. To limit the viral spread, prompt and precise diagnosis is crucial to facilitate treatment and ensure effective isolation. Accurate detection of antibodies (IgG and IgM) is imperative to understand the prevalence of SARS-CoV-2 in public and to inspect the proportion of immune individuals. In this review, we demonstrate and evaluate some tests that have been used commonly to detect SARS-CoV-2. These include nucleic acid and serological tests for the detection of SARS-CoV-2 RNA and specific antibodies in infected people. Moreover, the vitality of biosensing technologies emphasizing on optical and electrochemical biosensors toward the detection of SARS-CoV-2 has also been discussed here. The early diagnosis of COVID-19 based on detection of reactive oxygen species overproduction because of virus-induced dysfunctioning of lung cells has also been highlighted.     

Ultrasensitive Fluorescence Polarization Aptasensors Based on Exonuclease Signal Amplification and Polystyrene Nanoparticle Amplification

Here, we combine T7 exonuclease (T7 Exo) signal amplification and polystyrene nanoparticle (PS NP) amplification to develop novel fluorescence polarization (FP) aptasensors. The binding of a target/open aptamer hairpin complex or a target/single‐stranded aptamer complex to dye‐labeled DNA bound to PS NPs, or the self‐assembly of two aptamer subunits (one of them labeled with a dye) into a target/aptamer complex on PS NPs leads to the cyclic T7 Exo‐catalyzed digestion of the dye‐labeled DNA or the dye‐labeled aptamer subunit. This results in a substantial decrease in the FP value for the amplified sensing process. Our newly developed aptasensors exhibit a sensitivity five orders of magnitude higher than that of traditional homogeneous aptasensors and a high specificity for the target molecules. These distinct advantages of our proposed assay protocol make it a generic platform for the design of amplified aptasensors for ultrasensitive detection of various target molecules.     

In Silico and In Vivo Evaluation of SARS-CoV-2 Predicted Epitopes-Based Candidate Vaccine

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, the causative agent of coronavirus disease (COVID-19)) has caused relatively high mortality rates in humans throughout the world since its first detection in late December 2019, leading to the most devastating pandemic of the current century. Consequently, SARS-CoV-2 therapeutic interventions have received high priority from public health authorities. Despite increased COVID-19 infections, a vaccine or therapy to cover all the population is not yet available. Herein, immunoinformatics and custommune tools were used to identify B and T-cells epitopes from the available SARS-CoV-2 sequences spike (S) protein. In the in silico predictions, six B cell epitopes QTGKIADYNYK, TEIYQASTPCNGVEG, LQSYGFQPT, IRGDEVRQIAPGQTGKIADYNYKLPD, FSQILPDPSKPSKRS and PFAMQMAYRFNG were cross-reacted with MHC-I and MHC-II T-cells binding epitopes and selected for vaccination in experimental animals for evaluation as candidate vaccine(s) due to their high antigenic matching and conserved score. The selected six peptides were used individually or in combinations to immunize female Balb/c mice. The immunized mice raised reactive antibodies against SARS-CoV-2 in two different short peptides located in receptor binding domain and S2 region. In combination groups, an additive effect was demonstrated in-comparison with single peptide immunized mice. This study provides novel epitope-based peptide vaccine candidates against SARS-CoV-2.     

Ultrasensitive Detection of COVID-19 Causative Virus (SARS-CoV-2) Spike Protein Using Laser Induced Graphene Field-Effect Transistor

COVID-19 is a highly contagious human infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the war with the virus is still underway. Since no specific drugs have been made available yet and there is an imbalance between supply and demand for vaccines, early diagnosis and isolation are essential to control the outbreak. Current nucleic acid testing methods require high sample quality and laboratory conditions, which cannot meet flexible applications. Here, we report a laser-induced graphene field-effect transistor (LIG-FET) for detecting SARS-CoV-2. The FET was manufactured by different reduction degree LIG, with an oyster reef-like porous graphene channel to enrich the binding point between the virus protein and sensing area. After immobilizing specific antibodies in the channel, the FET can detect the SARS-CoV-2 spike protein in 15 min at a concentration of 1 pg/mL in phosphate-buffered saline (PBS) and 1 ng/mL in human serum. In addition, the sensor shows great specificity to the spike protein of SARS-CoV-2. Our sensors can realize fast production for COVID-19 rapid testing, as each LIG-FET can be fabricated by a laser platform in seconds. It is the first time that LIG has realized a virus sensing FET without any sample pretreatment or labeling, which paves the way for low-cost and rapid detection of COVID-19.     

Application of split aptamers-based sensors for the detection of small moleculesEI北大核心CSCD

The split aptamers (SPAs)-based sensors is a novel kind of biosensors, assembled by two or more oligonucleotides in the presence of specific targets. To be successfully assembled, the sensor has to be induced by a specific target, which can aviod false-positive results and thus has a high degree of specificity and sensitivity. SPAs are suitable for the detection of various targets and show great advantages and potential in the development of aptasensors, especially for the detection of small molecules. However, the development and application of SPA-based sensors still remain challenging. Currently, the major difficulty is how to improve the stability of SPA-target complexes. Herein, this review summarizes the SPAs, strategies of splitting aptamers, and their applications in the detection of small molecules, aiming to provide new ideas for the development of novel, sensitive, and specific aptasensors. © 2023, Youke Publishing Co.,Ltd. All rights reserved.     

Alkaline Phosphatase-based Electrochemical Analysis for Point-of-Care Testing

Point-of-care testing (POCT) devices have evolved to provide beneficial information about an individual's health whenever needed. Enzyme-based analytical devices have facilitated the highly selective detection of numerous biological molecules and ions. Enzymes are commonly used as the tags of recognition components, such as antibodies, to generate and amplify detection signals. Particularly, alkaline phosphatase (ALP) is one of the most widely used enzymes because of its high turnover number and low cost. Rapid response time and the incorporation of many sensors fabricated by micro/nano processing technologies are the advantages in using electrochemical devices as analytical tools. Therefore, ALP-based electrochemical devices have potential applications for more practical POCT platforms. This review summarizes recent research progress of ALP-based electrochemical devices for POCT. In addition to ALP substrates, the application of ALP-based immunosensors, aptasensors, and DNAzyme sensors are discussed.     

Aptamers: Potential Diagnostic and Therapeutic Agents for Blood Diseases

Aptamers are RNA/DNA oligonucleotide molecules that specifically bind to a targeted complementary molecule. As potential recognition elements with promising diagnostic and therapeutic applications, aptamers, such as monoclonal antibodies, could provide many treatment and diagnostic options for blood diseases. Aptamers present several superior features over antibodies, including a simple in vitro selection and production, ease of modification and conjugation, high stability, and low immunogenicity. Emerging as promising alternatives to antibodies, aptamers could overcome the present limitations of monoclonal antibody therapy to provide novel diagnostic, therapeutic, and preventive treatments for blood diseases. Researchers in several biomedical areas, such as biomarker detection, diagnosis, imaging, and targeted therapy, have widely investigated aptamers, and several aptamers have been developed over the past two decades. One of these is the pegaptanib sodium injection, an aptamer-based therapeutic that functions as an anti-angiogenic medicine, and it is the first aptamer approved by the U.S. Food and Drug Administration (FDA) for therapeutic use. Several other aptamers are now in clinical trials. In this review, we highlight the current state of aptamers in the clinical trial program and introduce some promising aptamers currently in pre-clinical development for blood diseases.     

State-of-the-art colloidal particles and unique interfaces-based SARS-CoV-2 detection methods and COVID-19 diagnosis

In March 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-based infections were declared ‘COVID-19 pandemic’ by the World Health Organization. Pandemic raised the necessity to design and develop genuine and sensitive tests for precise specific SARS-CoV-2 infections detection. Nanotechnological methods offer new ways to fight COVID-19. Nanomaterials are ideal for unique sensor platforms because of their chemically versatile properties and they are easy to manufacture. In this context, selected examples for integrating nanomaterials and distinct biosensor platforms are given to detect SARS-CoV-2 biological materials and COVID-19 biomarkers, giving researchers and scientists more goals and a better forecast to design more relevant and novel sensor arrays for COVID-19 diagnosis.     

核酸适配体在多肽研究中的应用

多肽在生命体的生理过程中发挥着重要作用,其生理功能一直是生物学、药理学和医学等领域的重要研究内容.核酸适配体是经体外筛选获得的单链DNA或RNA,能与靶标高亲和力、高特异性地结合,有"化学抗体"或"化学家的抗体"之称.以多肽为靶标筛选获得的核酸适配体主要有两大用途:一是基于其识别功能,作为亲和试剂来建立分析检测方法或开展生物成像研究;二是基于它们的生物学活性,作为拮抗剂在活体水平影响靶标多肽的正常功能,阻碍下游信号通路,从而对疾病进行治疗.本文总结了近年来以多肽为靶标筛选的核酸适配体在体内及体外的用途,并探讨了其在筛选、表征及应用中存在的问题,并对其未来的发展趋势进行了展望.