芯片毛细管电泳用于人乳头瘤病毒分析的研究进展

Research progress on analysis of human papillomavirus by microchip capillary electrophoresis

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。

Human papillomavirus(HPV),is a common spherical DNA virus that can lead to six types of cancers later in life,which has recently garnered human’s attention.Microchip capillary electrophoresis(MCE)has provided simple,fast,portable,and sensitive HPV typing assay assisted by a variety of signal amplification technologies.This review presents the latest research progress of MCE in routine HPV typing assays,including both of the MCE techniques and MCE combined with the nucleic acid amplification techniques for HPV assay.The introduction on the former part concerns the MCE system,the MCE chips design and electrophoretic separation methods.The typical MCE system includes high voltage power supply,microfluidic chip of separation,sample injection,electrolyte cell,detection unit and so on.Four different chips are reviewed,containing straight separation channel,T-channel,serpentine channel and dual channel,because these microchips are the most used in the last decade.Furthermore,the high integration and high throughput on a single chip are often integrated the sample preparation unit on a chip.The advantages and disadvantages of different designed microchips are introduced at the same time.The separation methods of chip electrophoresis are briefly introduced.With the development and application of MCE for HPV detection,the separation time is greatly shortened from a few hours to several minutes.The review on the second part gives the comments on various kinds of nucleic acid amplification technologies coupled with MCE for HPV assay.Firstly,the comparative analysis is given on the polymerase chain reaction(PCR)combined with MCE,loop-mediated isothermal amplification(LAMP),PCR combined with restriction fragment length polymorphism(RFLP)for HPV DNA detection,and nucleic acid sequence based amplification(NASBA)for the detection of HPV mRNA,nested PCR and so on.Secondly,the reviews on the other methods beside MCE are also summarized,including the PCR coupled with Fourier transform-infrared spectroscopy(FT-IR spectroscopy),nanotechnology,DNA probes combined with electrochemical methods,reductive Cu(Ⅰ)particles catalyzed Zn-doped MoS2 quantum dots and T7 exonuclease with electrochemiluminescence,LAMP with CRISPR/Cas12a based lateral.In these non-MCE methods,the electrochemical sensing,e.g.,impedimetric detection,pulse voltammetry method and flow biosensor,is an ideal method due to its low background signal and excellent time control ability.Finally,although MCE technologies have been developed and the developed instruments are applied in recent years,there are still some challenges in MCE techniques,methods and applications.The first challenge faced in the application of MCE technique in HPV typing assay is that the MCE device can not be well utilized for the detection of HPV with high resolution and high sensitivity,because MCE can not do signal amplification of HPV nucleic acid.The second challenge is that even though some researchers have successfully integrated PCR and MCE on one chip,the technique still faces difficulty for wide application and there is still no really integrated PCR-MCE chip for HPV detection.The third one is the MCE technique is lack for the manufacture of miniaturized and automatic instrument.At the end of review,the authors’insights are given on the development of automatic,fast,high stable and reliable detection in the HPV typing via the portable MCE device.