基于CRISPR/Cas系统的核酸生物传感器

近年来,CRISPR/Cas系统已经成为转录调控和基因组编辑的重要工具。除了在基因编辑领域的贡献,CRISPR/Cas系统独特的靶核酸顺式切割和非特异性单链核酸反式切割能力,在开发核酸检测的新型生物传感器方面展现出巨大潜力。构建基于CRISPR/Cas系统高灵敏度生物传感器的关键通常依赖其与不同信号扩增策略,诸如核酸扩增技术或特定信号转导方法的结合。基于此,本文旨在通过介绍不同类型的CRISPR/Cas系统,全面概述基于该系统的核酸检测生物传感器的研究进展,并重点对结合核酸扩增技术（PCR、LAMP、RCA、RPA和EXPAR）、灵敏的信号转导方法（电化学和表面增强拉曼光谱）和特殊结构设计生物传感的三大类型信号放大策略的CRISPR/Cas生物传感器进行总结和评论。最后,本文对目前的挑战以及未来的前景进行展望。     

Label‐free indicator‐free nucleic acid biosensors using carbon nanotubes

Carbon nanotubes (CNTs) are promising components for electrical biosensors due to their high surface‐to‐volume ratio and improved electron transfer properties. This review surveys CNT‐based label‐free indicator‐free biosensing strategies that have been demonstrated for the sensitive detection of nucleic acids. After an introduction to CNTs, the fabrication of biosensors and techniques for the immobilization of probe nucleic acids are outlined. Subsequently, two major label‐free strategies namely electrochemical transduction and field‐effect detection are presented. The focus is on direct detection methods that avoid labels, indicators, intercalating agents, mediators, and even secondary receptors. The review concludes with a comparison between the various biosensors and presents ways of engineering them so that they can be deployed in realistic diagnostic applications.     

Electrochemical molecular analysis without nucleic acid amplification

Electrochemical biosensors have revolutionized glucose monitoring but have not yet fulfilled their promise of a low cost, direct detection replacement for genetic amplification tests such as PCR [K. Kerman, M. Kobayashi, E. Tamiya, Recent trends in electrochemical DNA biosensor technology, Meas. Sci. Technol. 15 (2004) R1-R11; A. Chaubey, B.D. Malhotra, Mediated biosensors. Biosens. Bioelectron. 17 (6-7) (2002) 441-456]. It has been anticipated that the integration of nanoscale chemical structures such as self-assembled monolayers with electrochemical biosensors would increase sensitivity by decreasing inherent system noise. We have designed a novel biosensing approach incorporating such integration and achieved rapid, ultra-low concentration sensitivities without target amplification. Raw samples are mixed with lysis buffer to allow hybridization of nucleic acid targets with anchor and signal probes before immobilizing a signaling enzyme proximate to the biosensor surface. A bias potential is subsequently applied and the secondary byproduct of a cyclic peroxidase reaction measured. Further studies have demonstrated the application of our approach in protein, clinical chemistry, and ionic assays.     

DNA biosensors based on water-soluble conjugated polymers

Conjugated polymers (CPs) with large, delocalised molecular structures exhibit unique optical and electrochemical characteristics that can be used as excellent sensing elements. Recently, research on chemical and biological sensors that use water-soluble CPs as transducers has generated intense interest. Two main sensing mechanisms are used for the detection of DNA-related events, such as hybridisation, mismatch, single nucleotide polymorphism (SNP), SNP genotyping, conformational changes, and cleavage of the nucleic acids. One mechanism takes advantage of the fluorescence resonance energy transfer (FRET) between CPs and a chromophore label on the nucleic acid probes in which a series of cationic polyfluorene, polythiophene and polyarylene derivatives are frequently used. The other mechanism relies on the conformational effects of CPs, which is induced by combination of the specific targets in which cationic polythiophene derivatives are often used. The electron transfer property of CPs are always used to design high sensitive electrochemical DNA biosensors. Here we review recent progress in the development of optical and electrochemical DNA biosensors based on water-soluble CPs.     

Electrochemical study of the XNA on Gold microarray

A novel electrode array was developed based on the XNA on Gold trade mark microarray platform. The platform combines self-assembling monolayers, thick film patterning and streptavidin based immobilization to provide a robust, versatile platform capable of analysing virtually any biomolecule including nucleic acids, proteins, carbohydrates and lipids. Electrochemical analysis of the self-assembling monolayer/streptavidin (SAMS) XNA on Gold coating revealed that the ferrocene redox current for the SAMS modified electrode was greater than that with a bare Gold electrode. The electrochemical reaction of K4Fe(CN)6 was inhibited by the SAMS coating, but was reactivated upon addition of ferrocene. These results indicate that ferrocene is involved as a mediator in the electron transfer of K4Fe(CN)6 to the SAMS modified electrode. Addition of DNA to the SAMS resulted in only a minor change in the electrochemical signal, indicating that XNA on Gold can be used for electrochemical based bioanalysis. After cycling a SAMS electrode 50 times, no signs of deterioration were detected showing that coating has excellent stability. In addition to the biosensing applications, the scheme provides a non-invasive method for accessing the quality of the SAMS coatings which is of industrial interest. These studies show that the XNA on Gold microarray platform can be used for electrochemical studies, thus providing an additional alternative for developing multianalyte biosensors as well as expanding the range of detection methods available for microarray analysis.     

Research progress in the detection of common foodborne hazardous substances based on functional nucleic acids biosensors

With the further improvement of food safety requirements, the development of fast, highly sensitive, and portable methods for the determination of foodborne hazardous substances has become a new trend in the food industry. In recent years, biosensors and platforms based on functional nucleic acids, along with a range of signal amplification devices and methods, have been established to enable rapid and sensitive determination of specific substances in samples, opening up a new avenue of analysis and detection. In this paper, functional nucleic acid types including aptamers, deoxyribozymes, and G-quadruplexes which are commonly used in the detection of food source pollutants are introduced. Signal amplification elements include quantum dots, noble metal nanoparticles, magnetic nanoparticles, DNA walkers, and DNA logic gates. Signal amplification technologies including nucleic acid isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, biological barcodes, and microfluidic system are combined with functional nucleic acids sensors and applied to the detection of many foodborne hazardous substances, such as foodborne pathogens, mycotoxins, residual antibiotics, residual pesticides, industrial pollutants, heavy metals, and allergens. Finally, the potential opportunities and broad prospects of functional nucleic acids biosensors in the field of food analysis are discussed.     

Electrochemical biosensing with nanoparticles

This minireview looks at the latest trends in the use of nanoparticles (NPs) in electrochemical biosensing systems. It includes electrochemical characterization of NPs for use as labels in affinity biosensors and other applications. DNA analysis involving NPs is one of the most important topics of current research in bionanotechnology. The advantages of the use of NPs in designing novel electrochemical sensors for DNA analysis are reviewed. Electrochemical NPs can also be used in designing immunoassays, offering the possibility of easy, low cost and simultaneous detection of several proteins. Research into NP applications in electrochemical analysis is in its infancy. Several aspects related to sensitivity as well integration of all the assay steps into a single one need to be improved.     

Analytical potential of gold nanoparticles in functional aptamer-based biosensors

Gold nanoparticles (AuNPs) exhibit many predominant capabilities such as high biocompatibility, chemical stability, strong localized surface plasmon resonance absorption, and high extinction coefficient in the visible region. These properties have enabled the extensive use of AuNPs in optical and electrochemical biosensors. As a kind of functional nucleic acids, aptamers can be considered as a valid alternative to antibodies or other bio-receptors and have been widely employed to develop novel biosensors. We are summarizing here the state of the art of AuNP-based biosensors that use functional aptamers as molecular recognition elements. In many cases, AuNPs themselves can be used as a probe for detection, such as various colorimetric aptasensors and fluorescent aptasensors. They also can be used as probe vectors to enlarge detection signals and to increase the number of conceivable substrates in electrochemical aptasensors.     

Nucleic acids as a basis for creating biosensors

Here we present a brief conception of biosensors. Structural peculiarities and properties of single- and double-stranded nucleic acids that are to be taken into account when creating biosensors on the basis of these biomolecules are considered. On the example of two biologically active compounds a possibility is shown for constructing biosensors on the basis of liquid-crystalline dispersions of low molecular mass DNA and on the basis of liquid-crystalline DNA dispersions immobilized due to their inclusion into the synthetic polymeric matrix.     

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this "E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.     

Principles of creating biotransducers based on nucleic acid liquid crystals

A brief concept of biosensors is presented. Structural peculiarities and properties of single- and double-stranded nucleic acids that are to be taken into account when creating sensing units for biosensors using different principles of molecular recognition are considered. General schemes of sensing units based on liquid-crystalline dispersions formed from low-molecular mass DNA, its complexes with "cross-linking" agents and from circular superhelical DNA molecules are described.     

Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods

The present report reviews immobilisation techniques of purified oligonucleotides on electrochemical transducers and their corresponding detection techniques. Most of the literature reviewed was published in the 1990s. The immobilisation techniques of a DNA probe to the surface of an electrochemical transducer made from carbon, gold, platinum or polypyrrole, ranged from simple adsorption to covalent bonding. Recent efforts to couple the recognition layer containing the immobilised nucleic acid recognition layer with the electrochemical signal transducer are discussed. Special attention is given to hybridisation biosensing based on electroactive indicators.     

Nanopore sensors: From hybrid to abiotic systems

This paper provides an overview of different nanostructured architectures utilised in electrochemical devices and their application in biosensing and bioelectronics. Emphasis is placed on the fabrication of nanostructured films based on a layer-by-layer (LBL) films approach. We discuss the theory and the mechanism of charge transfer in polyelectrolyte multilayer films (PEM), as well as between biomolecules and redox centres, for the development of more sensitive and selective biosensors. Further, this paper presents an overview of topics involving the interaction between nanostructured materials, including metallic nanoparticles and carbon materials, and their effects on the preservation of the activity of biological molecules immobilised on electrode surfaces. This paper also presents examples of biological molecules utilised in film fabrication, such as DNA, several kinds of proteins, and oligonucleotides, and of the role of molecular interaction in biosensing performance. Towards the utilisation of LBL films, examples of several architectures and different electrochemical approaches demonstrate the potential of nanostructured LBL films for several applications that include the diagnosis and monitoring of diseases. Our main aim in this review is to survey what can assist researchers by presenting various approaches currently used in the field of bioelectrochemistry utilising supramolecular architectures based on an LBL approach for application in electrochemical biosensing.     

Nucleic acid aptamers as aptasensors for plant biology

Our knowledge of cell- and tissue-specific quantification of phytohormones is heavily reliant on laborious mass spectrometry techniques. Genetically encoded biosensors have allowed spatial and some temporal quantification of phytohormones intracellularly, but there is still limited information on their intercellular distributions. Here, we review nucleic acid aptamers as an emerging biosensing platform for the detection and quantification of analytes with high affinity and specificity. Options for DNA aptamer technology are explained through selection, sequencing analysis and techniques for evaluating affinity and specificity, and we focus on previously developed DNA aptamers against various plant analytes. We suggest how these tools might be applied in planta for quantification of molecules of interest both intracellularly and intercellularly.     

Electrochemical DNA biosensors based on thin gold films sputtered on capacitive nanoporous niobium oxide

Electrochemical DNA biosensors based on a thin gold film sputtered on anodic porous niobium oxide (Au@Nb(2)O(5)) are studied in detail here. We found that the novel DNA biosensor based on Au@Nb(2)O(5) is superior to those based on the bulk gold electrode or niobium oxide electrode. For example, the novel method does not require any time-consuming cleaning step in order to obtain reproducible results. The adhesion of gold films on the substrate is very stable during electrochemical biosensing, when the thin gold films are deposited on anodically prepared nanoporous niobium oxide. In particular, the novel biosensor shows enhanced biosensing performance with a 2.4 times higher resolution and a three times higher sensitivity. The signal enhancement is in part attributed to capacitive interface between gold films and nanoporous niobium oxide, where charges are accumulated during the anodic and cathodic scanning, and is in part ascribed to the structural stability of DNA immobilized at the sputtered gold films. The method allows for the detection of single-base mismatch DNA as well as for the discrimination of mismatch positions.     

Recent advances in polyaniline based biosensors

The present paper contains a detailed overview of recent advances relating to polyaniline (PANI) as a transducer material for biosensor applications. This conducting polymer provides enormous opportunities for binding biomolecules, tuning their bio-catalytic properties, rapid electron transfer and direct communication to produce a range of analytical signals and new analytical applications. Merging the specific nature of different biomolecules (enzymes, nucleic acids, antibodies, etc.) and the key properties of this modern conducting matrix, possible biosensor designs and their biosensing characteristics have been discussed. Efforts have been made to discuss and explore various characteristics of PANI responsible for direct electron transfer leading towards fabrication of mediator-less biosensors.     

Allosteric enzymes as biosensors for molecular diagnosis

Biosensors are hybrid analytical devices that amplify signals generated from the specific interaction between a receptor and the analyte, through a biochemical mechanism. Biosensors use tissues, whole cells, artificial membranes or cell components like proteins or nucleic acids as receptors, coupled to a physicochemical signal transducer. Allosteric enzymes exhibit a catalytic activity that is modulated by specific effectors, through binding to receptor sites that are distinct from the active site. Several enzymes, catalyzing easily measurable reactions, have been engineered to allosterically respond to specific ligands, being themselves the main constituent of new-generation biosensors. The molecular basis, robustness and application of allosteric enzymatic biosensing are revised here.     

DNA生物传感器:缓慢的传感器?

近几年来,酶传感器、免疫传感器及微生物传感器等发展较为成熟,而DNA生物传感器的研究相对较少。文章从核酸杂交的原理出发介绍了DNA生物传感器的工作原理,举例说明了电化学、光学和声学等几种典型的DNA生物传感器,指出了其固有的优缺点,肯定了DNA传感器发展前景。     

DNA生物传感器进展

近几年来,酶传感器、免疫传感器及微生物传感器等发展较为成熟,而DNA生物传感器的研究相对较少.文章从核酸杂交的原理出发介绍了DNA生物传感器的工作原理,举例说明了电化学、光学和声学等几种典型的DNA生物传感器,指出了其固有的优缺点,肯定了DNA传感器发展前景.     

DNA生物传感器在环境污染监测中的应用

基于生物催化和免疫原理的生物传感器在环境领域中获得了广泛应用.近年来,随着分子生物学和生物技术的发展,人们开发了以核酸探针为识别元件,基于核酸相互作用原理的DNA生物传感器.该传感器可用于受感染微生物的核酸序列分析、优先控制污染物的检测以及污染物与DNA之间相互作用的研究,在环境污染监测中具有潜在的巨大应用前景.简要介绍了核酸杂交生物传感器的基本原理及其在环境微生物和优先控制污染物（priority pollutant）检测中的应用研究进展.