An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA

Wang，Dou1; Wang，Xuedong1; Ye，Feidi2; Zou，Jin2; Qu，Jiuxin2; Jiang，Xingyu1

2023-04-25

10.1021/acsnano.2c10143

ACS Nano   影响因子和分区

1936-0851

1936-086X

Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.

amplification-free CRISPR/Cas13a digital detection microwell array SARS-CoV-2

SCI ; EI

英语

NI期刊 ; NI论文

第一 ; 通讯

National Key R&D Program of China["2022YFC2403500","2021YFF1200100","22234004","SZBL2019062801004"] ; National Natural Science Foundation of China["81730051","22204068","JCYJ20210324105006017"] ; Shenzhen Science and Technology Program["KQTD20190929172743294","JSGG20220606142200001","JSGG20220919091201003","KCXFZ20211020163544002","ZDSYS20200811144003009"] ; Shenzhen Key Laboratory of Smart Healthcare Engineering[JCYJ20190809115005628] ; Natural Science Foundation of Shenzhen City[2022B1212010003] ; Guangdong Provincial Key Laboratory of Advanced Biomaterials[2019ZT08Y191] ; Guangdong Innovative and Entrepreneurial Research Team Program[2019A1515110292] ; Guangdong Basic and Applied Basic Research Foundation["2021A1515220111","QYZDJ-SSW-SLH039"] ; Chinese Academy of Sciences[2020YFA0908900] ; null[2022YFB3804700] ; null[2018YFA0902600]

Chemistry ; Science & Technology - Other Topics ; Materials Science

Chemistry, Multidisciplinary ; Chemistry, Physical ; Nanoscience & Nanotechnology ; Materials Science, Multidisciplinary

WOS:000972243800001

AMER CHEMICAL SOC

20231814045118

Amplification ; Clinical research ; RNA

Biological Materials and Tissue Engineering:461.2 ; Medicine and Pharmacology:461.6

2-s2.0-85153803135

Scopus

1.Shenzhen Key Laboratory of Smart Healthcare Engineering,Guangdong Provincial Key Laboratory of Advanced Biomaterials,Department of Biomedical Engineering,Southern University of Science and Technology,Shenzhen,No. 1088, Xueyuan Road, Xili, Nanshan District, Guangdong,518055,China
2.Department of Clinical Laboratory,Shenzhen Third People’s Hospital,Second Hospital Affiliated to Southern University of Science and Technology,National Clinical Research Center for Infectious Diseases,Guangdong,518055,China

Wang，Dou,Wang，Xuedong,Ye，Feidi,et al. An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA[J]. ACS Nano,2023,17(8):7250-7256.

Wang，Dou,Wang，Xuedong,Ye，Feidi,Zou，Jin,Qu，Jiuxin,&Jiang，Xingyu.(2023).An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA.ACS Nano,17(8),7250-7256.

Wang，Dou,et al."An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA".ACS Nano 17.8(2023):7250-7256.