共查询到19条相似文献,搜索用时 109 毫秒
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信号放大是新型生物传感分析过程中重要的环节。核酸介导的信号放大技术凭借核酸材料灵活的结构设计、低成本和易于制备等特点,在生物传感快速检测技术的开发上逐渐发展成为一项重要的分支,广泛应用于食品、环境和医药等新型检测方法开发。介绍了传统和新型核酸扩增技术、生物条形码和DNA walker等信号放大机理和应用,同时进一步综述核酸信号放大技术结合光学生物传感在食品污染物中检测的应用,如化学污染物、毒素类污染物、和重金属污染物等,并对核酸介导的信号放大技术在食品污染物检测中的问题和前景进行讨论。 相似文献
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The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique. 相似文献
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This paper reports the design and implementation of a contactless conductivity detection system which combines a thermal control cell, a data processing system and an electrochemical (EC) cell for label-free isothermal nucleic acid amplification and real-time monitoring. The EC cell consists of a microchamber and interdigitated electrodes as the contactless conductivity biosensor with a cover slip as insulation. In our work, contactless EC measurements, the effects of trehalose on amplification, and chip surface treatment are investigated. With the superior performance of the biosensor, the device can detect the amount of pure DNA at concentrations less than 0.1 pg μl(-1). The EC cell, integrated with a heater and a temperature sensor, has successfully implemented nicking-based strand-displacement amplification at an initial concentration of 2.5 μM and the yields are monitored directly (dismissing the use of probes or labels) on-line. This contactless detector carries important advantages: high anti-interference capability, long detector life, high reusability and low cost. In addition, the small size, low power consumption and portability of the detection cell give the system the potential to be highly integrated for use in field service and point of care applications. 相似文献
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Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids
We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies. 相似文献
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Zaytseva NV Montagna RA Lee EM Baeumner AJ 《Analytical and bioanalytical chemistry》2004,380(1):46-53
A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442–1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin–streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture. 相似文献
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Miniaturized isothermal nucleic acid amplification, a review 总被引:1,自引:0,他引:1
Micro-Total Analysis Systems (μTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented. 相似文献
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人乳头瘤病毒(human papillomavirus,HPV)是一种常见的球形DNA病毒,目前已报道其可以导致6种类型的癌症发生,因此HPV病毒检测方法的研究引起了人们的重视。芯片毛细管电泳(MCE),作为一种芯片实验设备,结合各种信号放大技术为HPV分型检测提供了简单、快速、高灵敏度和易便携化的检测方法。该文综述了MCE在常规HPV分型检测中的最新研究进展,主要分为MCE技术和MCE结合核酸扩增技术两个部分。综述的第一部分介绍了MCE系统、MCE芯片结构设计和电泳分离方法。典型的MCE系统包含了高压电源、分离芯片、电解液池、进样系统、检测系统等。该文还介绍了近年来应用最广泛的4种芯片通道,包括分离直通道、T型通道、蛇形通道以及双通道,并分别对它们的优缺点进行了比较。第二部分主要介绍芯片电泳在HPV检测中的应用和发展。由于MCE技术的应用,HPV目标物的分离时间,从以前的几个小时缩短到几分钟,极大地提高了分离速度。重点介绍了各种核酸扩增技术结合MCE检测HPV的方法。对聚合酶链式反应(PCR)和MCE结合用于HPV的检测技术、环介导等温扩增(LAMP)技术的HPV检测方法、基于PCR结合限制性片段长度多态性(RFLP)技术用于HPV分型的DNA检测、基于核酸序列扩增(NASBA)技术检测HPV mRNA、巢式PCR等进行了比较分析。其次,对HPV其他检测方法进行了总结,其中包括PCR结合傅里叶变换红外光谱法(FT-IR)、纳米技术、DNA探针结合电化学方法、亚铜粒子氧化还原锌掺杂的二硫化钼量子点结合T7外切酶电化学发光法和基于CRISPR/Cas12a的环介导等温扩增法。在这些非MCE方法中,电化学传感法,如阻抗法、脉冲伏安法和流动生物传感器,由于背景信号低、时间控制能力强,是一种比较理想的方法。最后,虽然近年来MCE技术得到了发展,所开发的设备得到了应用,但目前在MCE技术、方法和应用方面仍然存在一些挑战。MCE技术在HPV分型检测应用中面临的第一个挑战是,MCE本身无法对HPV核酸进行信号放大,从而不能在HPV的高灵敏和高选择性分析中得到很好的应用。第二个挑战是,虽然有一些研究者已经成功地将PCR和MCE集成在一个芯片上,但该技术的广泛应用仍面临困难,目前仍然没有真正集成的PCR-MCE芯片用于HPV检测。第三个挑战是目前MCE技术无法实现小型化、自动化器件的制造。最后,文章就MCE在HPV分型检测中开发更自动化、更快速以及更稳定可靠的检测技术提出了一些观点和见解,希望能对感兴趣的读者提供一些启发。 相似文献
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In this work, we report the first electrochemistry-based real-time polymerase chain reaction technique for sequence-specific nucleic acid detection. This new technique builds upon the advantages of the well-established fluorescence-based counterpart, such as short assay time (simultaneous target DNA amplification and detection). In addition, this electrochemical approach could employ simple and miniaturizable instrumentation compared to the bulky and expensive optics required in the fluorescence-based schemes. We have demonstrated a proof-of-concept experiment showing that the utilization of solid-phase extension of the electrode surface-immobilized capture probe with Fc-dUTP during PCR resulted in the accumulation of the redox marker on the transducer surface. This new technique can be applied to a microfabricated PCR electrochemical device for point-of-care diagnostics as well as on-site environmental monitoring and biowarfare agent detection. 相似文献
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Exponential Isothermal Amplification of Nucleic Acids and Assays for Proteins,Cells, Small Molecules,and Enzyme Activities: An EXPAR Example 下载免费PDF全文
Michael S. Reid Dr. X. Chris Le Dr. Hongquan Zhang 《Angewandte Chemie (International ed. in English)》2018,57(37):11856-11866
Isothermal exponential amplification techniques, such as strand‐displacement amplification (SDA), rolling circle amplification (RCA), loop‐mediated isothermal amplification (LAMP), nucleic acid sequence based amplification (NASBA), helicase‐dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on‐site, point‐of‐care, and in situ assay applications. These amplification techniques eliminate the need for temperature cycling, as required for the polymerase chain reaction (PCR), while achieving comparable amplification yields. We highlight here recent advances in the exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. The incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables the highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from nonspecific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. 相似文献
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Lei He Rongrong Huang Pengfeng Xiao Yuan Liu Lian Jin Hongna Liu Song Li Yan Deng Zhu Chen Zhiyang Li Nongyue He 《中国化学快报》2021,32(5):1593-1602
Due to their high specificity and affinity towards various targets,along with other unique advantages such as stability and low cost,aptamers are widely applied in analytical techniques.A typical aptamerbased electrochemical biosensor is composed of a aptamer as the biological recognition element and transducer conve rting the biologic interaction into electrical signals for the quantitative measure ment of targets.Improvement of the sensitivity of a biosensor is significantly important in order to achieve the detection of biomolecules with low abundance,and different amplification strategies have been explored.The strategies either employ nanomaterials such as gold nanoparticles to construct electrodes which can trans fer the biological reactions more efficiently,or attempt to obtain enha nced signal through multi-labeled carriers or utilize enzyme mimics to catalyze redox cycling.This review discusses recent advances in signal amplification methods and their applications.Critical assessment of each method is also considered. 相似文献