Sample-in-answer-out centrifugal microfluidic chip reaction biosensor powered by Thermus thermophilus Argonaute (TtAgo) for rapid, highly sensitive and multiplexed molecular diagnostics of foodborne bacterial pathogens

被引：5

作者：

Wang, Nan ^{[1
]}

Zhang, Qiang ^{[1
,2
]}

Liu, Yajie ^{[1
]}

Huang, Baoyan ^{[1
]}

Man, Shuli ^{[1
]}

Ye, Shengying ^{[3
]}

Ma, Long ^{[1
]}

机构：

[1] Tianjin Univ Sci & Technol, Coll Biotechnol,China Int Sci & Technol Cooperat B, State Key Lab Food Nutr & Safety,Natl & Local Unit, Key Lab Ind Microbiol,Minist Educ,Tianjin Key Lab, Tianjin 300457, Peoples R China

[2] Branch Tianjin Third Cent Hosp, Tianjin, Peoples R China

[3] 983th Hosp Joint Logist Support Force, Chinese Peoples Liberat Army, Pharm Dept, Tianjin, Peoples R China

来源：

CHEMICAL ENGINEERING JOURNAL | 2024年 / 495卷

基金：

中国国家自然科学基金;

关键词：

Programmable nuclease; Centrifugal microfluidic chip (CMC); Loop-mediated isothermal amplification (LAMP); One-pot nucleic acid detection; Food safety; Thermus thermophilus Argonaute(TtAgo); NUCLEIC-ACID DETECTION; STAPHYLOCOCCUS-AUREUS; ABSCESS FORMATION; AMPLIFICATION; MICE;

D O I：

10.1016/j.cej.2024.153434

中图分类号：

X [环境科学、安全科学];

学科分类号：

08 ; 0830 ;

摘要：

Foodborne pathogens endanger public health and rapid, sensitive and accurate detection of them is vital. Argonaute stands in the frontline as the next generation nucleic acid detection tool, bearing a few comparable advantages. Centrifugal microfluidic chips (CMCs) use centrifugal force to achieve liquid flowing, mixing and reaction, eliminating complicated designs of valves and pumps. In this study, for the first time, we devised a Thermus thermophilus Argonaute (TtAgo)-powered centrifugal microfluidic chip reaction biosensor for Sample-inAnswer-out detection of Pathogen S. aureus (termed as ASAP) with ultrahigh rapidity and sensitivity in a one-pot fashion. The samples subjected to testing were mixed with a home-made nucleic acid fast extraction reagent and then the mixture was injected into the sample cell of CMC, which was centrifuged down into the reaction cell. The reaction cell was preloaded with both LAMP (Loop-mediated isothermal amplification) and TtAgo systems. As such, the species-specific nuc genes were amplified by LAMP, while the TtAgo performed site-specific cleavage to output fluorescent (FL) signals. The sample-to-answer time was 17 min, and the limit of detection (LOD) reached 1 CFU/mL. ASAP was able to perform simultaneous multiplexed detection and up to 16 samples can be detected at the same time. ASAP reduced reagent consumption and minimized the influence of carry-over and cross-contamination. ASAP was capable of detecting S. aureus in foods, but also detecting physiological samples from infect, holding great promise for practical applications. Overall, our work has enriched the Argonautepowered biosensing and CMC biosensor technology by providing a conceptually novel bacterial detection platform.

引用

页数：13