集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

滚环扩增技术在病原微生物检测中的应用

滚环扩增（RCA）技术是一种简单的恒温DNA扩增技术,在DNA聚合酶的催化下通过扩增闭合环状模板产生成千上万的重复序列。相较于变温核酸扩增技术如聚合酶链式反应（PCR）,RCA无需昂贵的变温仪器,更适合现场检测。该文介绍了RCA技术的原理和分类,综述了其在细菌、病毒以及其它病原微生物检测方面的应用现状,并展望了RCA检测病原微生物的应用前景。RCA在检测病原微生物领域有着巨大潜力,同时可为新型冠状病毒（SARS－CoV－2）的快速检测提供思路和补充。     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

体积排阻色谱法定量检测9种型别人乳头瘤病毒原液中病毒样颗粒

据统计,5%以上的人类癌症由人乳头瘤病毒(HPV)导致。HPV疫苗的使用,尤其是多价HPV疫苗的使用,可有效预防HPV感染和肿瘤的发生。例如,9价HPV疫苗可有效预防90%以上HPV相关癌前病变。人乳头瘤病毒样颗粒(VLP)是HPV疫苗的唯一抗原。VLP由360份衣壳蛋白L1组成。VLP的含量测定对HPV原液和HPV疫苗的质量评价至关重要。该文发展了一种以体积排阻色谱(SEC)为基础的9种型别人乳头病毒样颗粒的定量方法。实验优化了包括色谱柱类型、色谱柱孔径、流动相离子强度和流动相pH值在内的色谱条件。经过考察,以SHIMSEN Ankylo SEC-300色谱柱(300 mm×7.8 mm, 3 μm)为固定相,以含有300 mmol/L NaCl和50 mmol/L磷酸盐(pH 7.0)的缓冲溶液为流动相时,VLP的色谱峰更窄,从而可获得更高的响应和更好的灵敏度,因此选择该色谱条件用于VLP与基质的分离。优化所得的方法具有较宽的线性范围,良好的重复性(峰面积的相对标准偏差不大于5.0%)和灵敏度(定量限为4.58~15.24 μg/mL)。将方法用于HPV原液中VLP的含量测定,监测VLP的稳定性。结果显示,HPV原液中VLP颗粒不稳定,于4 ℃放置一周后,VLP含量与生产后立即测得的含量相比存在一定程度的降解。此外,方法还可用于疫苗上清液中游离蛋白质的分析,监测铝佐剂对VLP的吸附情况。被测厂家的铝佐剂可较好的吸附VLP,无明显残余蛋白质检出。与传统的蛋白质定量方法相比,如Folin-酚法(Lowry法),该法具有操作简单、自动化程度高、分析通量高等优点,可实现VLP含量的批量化分析。     

靶标介导DNA自组装及催化电流放大的microRNA电化学检测研究

该研究报道了一种靶标介导的DNA自组装及催化信号放大免标记电化学传感器定量检测microRNA－21的分析方法。根据靶标序列,设计一条末端标记巯基且具有茎环结构的捕获探针以及两条与捕获探针和靶标部分互补的DNA单链,通过金－硫键作用将捕获探针固定在金电极表面。当靶标（microRNA－21）存在时,自组装形成一种H结构的DNA复合结构;利用核酸链中磷酸骨架静电吸附电解液中的钌氨离子（［Ru（NH3）6］3+,RuHex）以及DNA电子传递作用产生电化学信号;当无靶标时,不能形成DNA复合结构,电化学信号较弱。进一步利用铁氰根离子（［Fe（CN）6］3-）能够氧化电化学还原产物（［Ru（NH3）6］2+）,产生电化学－化学偶联,从而实现催化电流信号放大。采用电化学阻抗谱确证DNA复合结构的形成,采用计时电量法考察捕获探针密度对电化学信号的影响,并优化探针浓度、比例以及自组装时间,采用差示脉冲伏安法进行定量分析。结果显示,在0.1 fmol/L ~ 0.1 nmol/L范围内,峰电流与microRNA－21浓度具有良好的线性关系,检出限为12.8 amol/L。方法能有效区分其他microRNA以及单碱基错配核酸序列,成功用于多种细胞中microRNA－21的定量检测。该电化学传感器具有灵敏度高、选择性好、线性范围宽等优点,无需繁琐的电化学探针标记以及费时费力的PCR扩增、滚环扩增、链置换反应等分析策略,简化了操作流程,提高了方法的实用性。     

基于3D打印技术的集成核酸提取芯片制备

基于固相萃取原理,设计并制作了一种集成核酸提取功能的微流控芯片,用于人体全血中脱氧核糖核酸(DNA)的提纯。芯片主要包括:混合微通道、裂解腔、DNA提取腔、DNA存储腔。采用3D打印技术制作出芯片模板,通过模板注塑成型、氧等离子体键合等工艺制作出微流控芯片。利用该芯片,可以在20 min内完成血液和试剂的顺序加载、快速混合、血细胞裂解、DNA的提取等操作,且试剂消耗量少。通过对所提DNA链上稳定表达的内参基因甘油醛-3-磷酸脱氢酶(GAPDH)进行PCR扩增,并对扩增产物进行熔解曲线分析,以验证所提取DNA的质量。     

生命分析化学的重要领域——电化学微阵列芯片

生命分析化学是研究与生命活动相关的化学分子检测原理与方法的新兴交叉科学领域。将电化学分析方法与生物芯片相结合形成的电化学生物芯片正在向集成化、通量化、微型化和自动化等方向迅速发展。本文介绍近年来电化学检测在DNA芯片、蛋白质芯片以及适配体芯片等方面有代表性的研究进展。文中阐述了高、低密度电化学DNA芯片的制备与多种伏安检测方法;讨论了多酶电极芯片结构与测定干扰之间的关系,介绍了伏安法、阻抗法等电化学方法在蛋白芯片中的应用进展。此外,还简要介绍了电化学适配体芯片的制备及检测方法,并展望了电化学生物芯片的发展前景。     

应用纳米金／TiO2中空微球复合膜高灵敏检测转基因植物外源基因特定DNA序列

在碳糊电极表面制备的纳米金／TiO2中空微球复合膜可以极大地提高DNA检测的灵敏度．应用循环伏安法和电化学交流阻抗谱法研究了纳米金和TiO2中空微球在碳糊电极上的固载,并用[Fe（CN）6]^3-/4-作为指示剂以电化学交流阻抗谱法表征了DNA的杂交．此DNA电化学生物传感器成功检测了来自于花椰菜花叶病毒的35S启动子基因的DNA特定序列,其动力学检测范围为1．0×10^-12～1．0×10^-8mol／L。检测限为2．3×10^-13mol／L;同时对从一种转基因大豆中提取的外源基因胭脂碱合成酶基因终止子的聚合酶链式反应（PCR）扩增产物进行了检测,得到了满意的结果．     

一种用于核酸高灵敏检测的液滴式数字聚合酶链式反应芯片

为了实现对核酸的高灵敏度检测,构建了一种新型的液滴式数字聚合酶链式反应(dd PCR)芯片.该芯片由产生液滴的聚二甲基硅氧烷(PDMS)模块和储存液滴的玻璃腔室构成.实验结果表明,该芯片可以在25 min内产生2×106个直径为20μm的微液滴(体积4.187 p L).利用该芯片定量检测了表皮生长因子受体(EGFR)基因第19号外显子,在DNA浓度为106~101copies/μL范围内呈现良好的线性关系(R2=0.9998);在浓度为106copies/μL的19号外显子野生型DNA中检测105~100copies/μL的突变型DNA,其检测敏感度可达到0.0001%.该方法在同一芯片上实现了液滴产生、核酸扩增和荧光信号读取的功能,在核酸绝对定量及痕量突变基因的检测中具有潜在应用前景.     

一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

为满足液滴式数字聚合酶链式反应（PCR）技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求,构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21 μm的液滴.利用“玻璃天花板”的方式构建了独立于芯片主体材料的液滴收集腔,为液滴提供稳定的保存与反应环境;还构建了过滤结构,可有效过滤混入液相中的空气,提高芯片鲁棒性.同时,在液滴收集腔中引入反射层,增强荧光信号,使单个视野荧光成像时间缩短约40%,提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子,检测信号与DNA浓度在10¹~10⁵ copies/μL范围内呈现良好的线性关系（R²=0.998）.该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取,并具有较高的鲁棒性与检测效率,在核酸检测等方面具有应用潜力.     

Miniaturized isothermal nucleic acid amplification, a review

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.     

High-throughput DNA analysis by microchip electrophoresis

DNA analysis plays a great role in genetic and medical research, and clinical diagnosis of inherited diseases and particular cancers. Development of new methods for high throughput DNA analysis is necessitated with incoming of post human genome era. A new powerful analytical technology, called microchip capillary electrophoresis (MCE), can be integrated with some experimental units and is characterized by high-speed, small sample and reagent requirements and high-throughput. This new technology, which has been applied successfully to the separation of DNA fragments, analysis of polymerase chain reaction (PCR) products, DNA sequencing, and mutation detection, for example, will become an attractive alternative to conventional methods such as slab gel electrophoresis, Southern blotting and Northern blotting for DNA analysis. This review is focused on some basic issues about DNA analysis by MCE, such as fabrication methods for microchips, detection system and separation schemes, and several key applications are summarized.     

Coupling Sensitive Nucleic Acid Amplification with Commercial Pregnancy Test Strips

The detection of nucleic acid biomarkers for point‐of‐care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop‐mediated isothermal amplification (LAMP), programmable toehold‐mediated strand‐exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG–SNAP fusion reporter protein (human chorionic gonadotropin‐O⁶‐alkylguanine‐DNA alkyltransferase) led to LAMP‐to‐hCG signal transduction on low‐cost, commercially available pregnancy test strips. Our assay reliably detected as few as 20 copies of Ebola virus templates in both human serum and saliva and could be adapted to distinguish a common melanoma‐associated SNP allele (BRAF V600E) from the wild‐type sequence. The methods described are completely generalizable to many nucleic acid biomarkers, and could be adapted to provide POC diagnostics for a range of pathogens.     

Micellar electrokinetic chromatography on microchips

This review highlights the methodological and instrumental developments in microchip micellar EKC (MCMEKC) from 1995. The combination of higher separation efficiencies in micellar EKC (MEKC) with high-speed separation in microchip electrophoresis (MCE) should provide high-throughput and high-performance analytical systems. The chip-based separation technique has received considerable attention due to its integration ability without any connector. This advantage allows the development of a multidimensional separation system. Several types of 2-D separation microchips are described in the review. Since complicated channel configurations can easily be fabricated on planar substrates, various sample manipulations can be carried out prior to MCMEKC separations. For example, mixing for on-chip reactions, on-line sample preconcentration, on-chip assay, etc., have been integrated on MEKC microchips. The application of on-line sample preconcentration to MCMEKC can provide not only sensitivity enhancement but also the elucidation of the preconcentration mechanism due to the visualization ability of MCE. The characteristics of these sample manipulations on MEKC microchips are presented in this review. The scope of applications in MCMEKC covers mainly biogenic compounds such as amino acids, peptides, proteins, biogenic amines, DNA, and oestrogens. This review provides a comprehensive table listing the applications in MCMEKC in relation to detection methods.     

负压驱动皮升级等温核酸精确定量微流控芯片

精准医疗急需更加精确、灵敏、方便快捷的核酸定量方法。设计开发了一种结合双荧光等温扩增的高密度皮升级核酸定量微流控芯片。芯片采用多层软光刻技术制成,有3个反应通道,每通道有40 000个反应小室,共120 576个皮升级反应小室,反应小室的密度达7 000/cm~2。芯片利用负压驱动样品精确分配,热凝固油相封闭小室,无需阀门及其他仪器辅助。芯片中嵌入的氟硅烷纳米涂层能有效地阻止反应过程中水蒸气的散失。以乙肝病毒(hepatitis B virus,HBV)质粒作为模板,进行等温多底物自配引发扩增(isothermal multiple self-matchinginitiated amplification,IMSA),测试芯片定量性能。芯片检测模板的动态范围达6个数量级,可检测的最大模板量为36μL中1.13×10~6拷贝数核酸。该装置具有定量精确、灵敏、快捷、操作简单等优势,可用于精准检测。     

Parallel nanoliter detection of cancer markers using polymer microchips

A general multipurpose microchip technology platform for point-of-care diagnostics has been developed. Real-time nucleic acid sequence-based amplification (NASBA) for detection of artificial human papilloma virus (HPV) 16 sequences and SiHa cell line samples was successfully performed in cyclic olefin copolymer (COC) microchips, incorporating supply channels and parallel reaction channels. Samples were distributed into 10 parallel reaction channels, and signals were simultaneously detected in 80 nl volumes. With a custom-made optical detection unit, the system reached a sensitivity limit of 10(-6) microM for artificial HPV 16 sequences, and 20 cells microl(-1) for the SiHa cell line. This is comparable to the detection limit of conventional readers, and clinical testing of biological samples in polymer microchips using NASBA is therefore possible.     

Exponential Isothermal Amplification of Nucleic Acids and Assays for Proteins,Cells, Small Molecules,and Enzyme Activities: An EXPAR Example

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.     

Detection of the food allergen celery via loop-mediated isothermal amplification technique

Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0 % for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.     

Sensitivity by combination: immuno-PCR and related technologies

The versatility of immunoassays for the detection of antigens can be combined with the signal amplification power of nucleic acid amplification techniques in a broad range of innovative detection strategies. This review summarizes the spectrum of both, DNA-modification techniques used for assay enhancement and the resulting key applications. In particular, it focuses on the highly sensitive immuno-PCR (IPCR) method. This technique is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR or related techniques for signal generation and read-out. Various strategies for the combination of antigen detection and nucleic acid amplification are discussed with regard to their laboratory analytic performance, including novel approaches to the conjugation of antibodies with DNA, and alternative pathways for signal amplification and detection. A critical assessment of advantages and drawbacks of these methods for a number of applications in clinical diagnostics and research is conducted. The examples include the detection of viral and bacterial antigens, tumor markers, toxins, pathogens, cytokines and other targets in different biological sample materials.     

Applications of Loop-Mediated Isothermal DNA Amplification

During the last 10 years, with the development of loop-mediated isothermal amplification (LAMP) method, it has been widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency, and outstanding specificity. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. Expensive equipment are not necessary to acquire a high level of precision, and there are fewer preparation steps compared to conventional PCR and real-time PCR assays. This paper briefly summarized the applications of LAMP method in pathogenic microorganisms, genetically modified ingredients, tumor detection, and embryo sex identification.