Dataset Information

A Highly Sensitive CRISPR-Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar-Level Real-Time Quantification.

ABSTRACT: The clustered regularly interspaced short palindromic repeats (CRISPR) molecular system has emerged as a promising technology for the detection of nucleic acids. Herein, the development of a surface plasmon resonance (SPR) sensor that is functionalized with a layer of locally grown graphdiyne film, achieving excellent sensing performance when coupled with catalytically deactivated CRISPR-associated protein 9 (dCas9), is reported. dCas9 protein is immobilized on the sensor surface and complexed with a specific single-guide RNA, enabling the amplification-free detection of target sequences within genomic DNA. The sensor, termed CRISPR-SPR-Chip, is used to successfully analyze recombinant plasmids with only three-base mutations with a limit of detection as low as 1.3 fM. Real-time monitoring CRISPR-SPR-Chip is used to analyze clinical samples of patients with Duchenne muscular dystrophy with two exon deletions, which are detected without any pre-amplification step, yielding significantly positive results within 5 min. The ability of this novel CRISPR-empowered SPR (CRISPR-eSPR) sensing platform to rapidly, precisely, sensitively, and specifically detect a target gene sequence provides a new on-chip optic approach for clinical gene analysis.

SUBMITTER: Zheng F

PROVIDER: S-EPMC9108660 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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A Highly Sensitive CRISPR-Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar-Level Real-Time Quantification.

Zheng Fei F Chen Zhi Z Li Jingfeng J Wu Rui R Zhang Bin B Nie Guohui G Xie Zhongjian Z Zhang Han H

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20220327 14

The clustered regularly interspaced short palindromic repeats (CRISPR) molecular system has emerged as a promising technology for the detection of nucleic acids. Herein, the development of a surface plasmon resonance (SPR) sensor that is functionalized with a layer of locally grown graphdiyne film, achieving excellent sensing performance when coupled with catalytically deactivated CRISPR-associated protein 9 (dCas9), is reported. dCas9 protein is immobilized on the sensor surface and complexed w ...[more]

PMID: 35343100

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Project description:Ultrasensitive detection of proteins is of great importance to proteomics studies. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy by coupling a polymerization initiator to a biospecific interaction and inducing inline atom transfer radical polymerization (ATRP) for amplifying SPR response. Bacterial cholera toxin (CT) is chosen as the model protein that has been covalently immobilized on the surface for demonstrating the principle. The specific recognition is achieved by use of biotinylated anti-CT, which allows initiators with a biotin tag to be fixed at the protein binding site through a neutravidin bridge and triggers the localized growth of polymer brushes of poly(hydroxyl-ethyl methacrylate) (PHEMA) via an ATRP mechanism. To further enhance the signal, a second ATRP reaction is conducted that takes advantage of the hydroxyl groups of PHEMA brushes from the first step to form hyperbranched polymers onto the sensing surface. The two consecutive ATRP steps significantly improve SPR detection, allowing low amounts of CT that yield no direct measurement to be quantified with large signals. The resulting polymer film has been characterized by optical and atomic force microscopy. Ascorbic acid (AA) is employed as deoxygen reagent in the catalyst mixture that effectively suppresses oxygen interference, shortening the reaction time and making it possible for applying this ATRP approach to flow injection based SPR detection. A calibration curve of PHEMA amplification for CT detection based on surface coverage has been obtained that displays a correlation in a range from 8.23 x 10(-15) to 3.61 x 10(-12) mol/cm(2) with a limit of detection of 6.27 x 10(-15) mol/cm(2). The versatile biotin-neutravidin interaction used here should allow adaptation of ATRP enhancement to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules with flow-injection assay and SPR spectroscopy.

Dataset Information

A Highly Sensitive CRISPR-Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar-Level Real-Time Quantification.

Publications

A Highly Sensitive CRISPR-Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar-Level Real-Time Quantification.

Similar Datasets

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