注射式微流控芯片全集成核酸分析系统与精准医疗应用

Fully Integrated Nucleic Acid Analysis System Based on Syringe-Microfluidic Chip and Application of Precision Medicine

核酸检测方法可快速鉴定特异基因指标,但其广泛应用受限于多种仪器设备串行使用以及对操作人员的高专业技术要求。本团队开发了一套注射式微流控芯片全集成核酸分析系统,该系统主要包含两大模块,分别是可以为不同类型临床样本提供多种核酸提取方法的全自动注射式核酸提取模块,以及基于微流控芯片的微纳体系多指标联合并行检测等温扩增核酸检测模块。这两大模块既可以单独发挥各自的功能,也可以组合成全集成注射式微流控芯片核酸分析系统,形成全集成自动化、微纳反应体系、快速、多指标联合并行检测的核酸检测分析平台。采用本团队开发的注射式微流控芯片全集成核酸分析系统,分别对热带念珠菌标准株培养菌液和64例外阴阴道念珠菌感染疾病的临床拭子样本进行检测。结果显示:本系统对菌液的最低检测限为3.95×102 CFU/mL,而且样品制备更方便快捷,仅需1次加样操作,核酸提取时间为10 min;64例临床样本检测效果与金标准培养法相比,卡方检验为1,Kappa值为0.950,说明两种方法无显著差异,且一致性很高。本团队开发的注射式微流控芯片全集成核酸分析系统,可以为临床多指标微纳体系核酸快速检测提供一个可靠的平台,为临床医疗应用提供精准快检技术与便捷分析仪器支撑。

Objective Nucleic acid detection methods enable the rapid identification of specific genetic indicators.However,their extensive application is limited by the sequential use of multiple instruments and the high technical requirements for operators.A fully integrated nucleic acid analysis system can automate the sample-to-answer detection process.However,nucleic acid amplification via PCR technology is time-consuming,and stringent requirements are placed on temperature control accuracy and heating/cooling speed,which increases the manufacturing cost of the corresponding instruments.These systems also lack the flexibility to accommodate variations in sample preprocessing methods and nucleic acid extraction protocols for different types of clinical samples.To address these challenges,we present a novel,fully integrated nucleic acid analysis system that contains a syringe-based sample processing and nucleic acid extraction module,a fully enclosed PC-based multiplexed detection microfluidic chip,and a nucleic acid amplification product detection module.We applied this system for the precise medical testing of various pathogenic infections in gynecology.The results indicate that our system can achieve precise medical molecular diagnosis with the fully integrated automation of a micro-nano reaction system,as well as speedy processing,high sensitivity and specificity,and multiplexed parallel detection.The system also shows potential applications in the in vitro identification of genetic and other nucleic acid-related diseases,holding significant social and economic value for the prevention and control of major diseases in China.Methods This paper presents a fully integrated nucleic acid analysis system based on a syringe microfluidic chip.The system consists of two modules.The first is an automated syringe nucleic acid extraction module that accommodates multiple nucleic acid extraction techniques based on different clinical sample types.This process involves four primary steps:sample loading,incubation,washing,and lysis by heating and shaking.The second module is an isothermally amplified nucleic acid detection module capable of simultaneous multivariate detection.The nucleic acid amplification and detection platform,based on microfluidic technology,includes a disk-based microfluidic chip that serves as a nucleic acid isothermal amplification carrier,a temperature control platform ensuring precise isothermal amplification of nucleic acids,a fluorescence detection platform for real-time monitoring of nucleic acid amplification,and a software analysis platform for controlling and analyzing the entire system.The two modules perform their respective functions independently and can also be combined into an integrated syringe-microfluidic chip nucleic acid analysis system,forming a fully integrated micro-nano detection platform that rapidly automates both nucleic acid extraction and detection and is capable ofdetecting multiple indicators in parallel.Results and Discussions The fully integrated system was evaluated using a standard Candida tropical culture strain and 64 clinical swab samples obtained from patients with vulvovaginal candidiasis.The results show that the detection concentration threshold of 3.95X 10°CFU/mL was comparable to that of the conventional nucleic acid detection method,which involves extracting nucleic acid with a reagent kit and amplifying it on a commercial PCR machine.The sample preparation is more convenient and fast,with only one sample loading step,and nucleic acid extraction takes 10 min,resulting in significant time and labor savings(Table 1).A chi-square value of 1 and a Kappa value of O.950 indicate a strong correlation with the gold standard method of clinical microbial culture(Table 2).Although a false-negative result with two samples containing Candida albicans was obtained,optimizing the primer sequences and reaction systems for isothermal amplification can address this problem.Therefore,this comprehensive integrated nucleic acid detection platform provides rapid and accurate multiplexed detection for the diagnosis of clinical microbial pathogen infections and has advanced technological capabilities.Conclusions In this study,a fully integrated nucleic acid analysis system based on a syringe microfluidic chip is developed,which consists of two modules.One is an automatic syringe nucleic acid extraction module,and the other is an isothermally amplified nucleic acid detection module based on a microfluidic chip.The system was tested using a standard Candida tropical culture strain and 64 clinical swab samples of vulvovaginal candidiasis.The results show that the minimum detection concentration of the bacterial solution in the system is 3.95X 10°CFU/mL,and a more convenient and rapid sample preparation is achieved with only one sample loading step with a nucleic acid extraction duration of 1o min.Compared with the gold standard culture method,a chi-square test and Kappa value of 1 and O.950,respectively,indicate that there is no significant difference between the two methods,with high consistency.The syringe-microfluidic chip-based,fully integrated nucleic acid analysis system detailed in this study provides a reliable platform for the rapid detection of nucleic acids using multiple indicators in a micro-nano system.The system offers precise detection and convenient analysis and supports applications in clinical medicine,grassroots screening for infectious diseases,health prevention programs,and basic biomedical research.This underscores the significance of this system in molecular diagnostics with in vitro precision in clinical settings,such as genetic disease identification and tumor molecular marker co-detection,as well as its potential uses in food safety and preventive healthcare applications.