Dynamic aqueous multiphase reaction system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis

AbstractRecently, CRISPR-Cas technology has opened a new era of nucleic acid-based molecular diagnostics. However, current CRISPR-Cas-based nucleic acid biosensing has largely a lack of the quantitative detection ability and typically requires separate manual operations. Herein, we reported a dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis by taking advantage of density difference of sucrose concentration. In the DAMR system, recombinase polymerase amplification (RPA) and CRISPR-Cas12a derived fluorescent detection occurred in spatially separated but connected aqueous phases. Our DAMR system was utilized to quantitatively detect human papillomavirus (HPV) 16 and 18 DNAs with sensitivities of 10 and 100 copies within less than one hour. Multiplex detection of HPV16/18 in clinical human swab samples were successfully achieved in the DAMR system using 3D-printed microfluidic device. Furthermore, we demonstrated that target DNA in real human plasma samples can be directly amplified and detected in the DAMR system without complicated sample pre-treatment. As demonstrated, the DAMR system has shown great potential for development of next-generation point-of-care molecular diagnostics.

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Dynamic Aqueous Multiphase Reaction System for One-Pot CRISPR-Cas12a-Based Ultrasensitive and Quantitative Molecular Diagnosis

Analytical Chemistry ◽

10.1021/acs.analchem.0c01459 ◽

2020 ◽

Vol 92 (12) ◽

pp. 8561-8568 ◽

Cited By ~ 3

Author(s):

Kun Yin ◽

Xiong Ding ◽

Ziyue Li ◽

Hui Zhao ◽

Kumarasen Cooper ◽

...

Keyword(s):

Molecular Diagnosis ◽

Reaction System ◽

One Pot ◽

Multiphase Reaction

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A glucose meter interface for point-of-care gene circuit-based diagnostics

Nature Communications ◽

10.1038/s41467-020-20639-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Evan Amalfitano ◽

Margot Karlikow ◽

Masoud Norouzi ◽

Katariina Jaenes ◽

Seray Cicek ◽

...

Keyword(s):

Nucleic Acid ◽

Synthetic Biology ◽

Molecular Diagnostics ◽

Point Of Care ◽

Low Cost ◽

Gene Circuit ◽

Reporter Systems ◽

Glucose Meter ◽

Pandemic Response ◽

Glucose Output

AbstractRecent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

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Nanomaterials for molecular signal amplification in electrochemical nucleic acid biosensing: recent advances and future prospects for point-of-care diagnostics

Molecular Systems Design & Engineering ◽

10.1039/c9me00135b ◽

2020 ◽

Vol 5 (1) ◽

pp. 49-66 ◽

Cited By ~ 11

Author(s):

Léonard Bezinge ◽

Akkapol Suea-Ngam ◽

Andrew J. deMello ◽

Chih-Jen Shih

Keyword(s):

Nucleic Acid ◽

Molecular Diagnostics ◽

Signal Amplification ◽

Point Of Care ◽

Future Prospects ◽

Electrochemical Biosensing ◽

Recent Advances ◽

Point Of Care Diagnostics ◽

Molecular Signal

This account reviews the major amplification strategies utilizing nanomaterials in electrochemical biosensing for robust and sensitive molecular diagnostics.

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Dual-Functional Peroxidase-Copper Phosphate Hybrid Nanoflowers for Sensitive Detection of Biological Thiols

International Journal of Molecular Sciences ◽

10.3390/ijms23010366 ◽

2021 ◽

Vol 23 (1) ◽

pp. 366

Author(s):

Xuan Ai Le ◽

Thao Nguyen Le ◽

Moon Il Kim

Keyword(s):

Room Temperature ◽

Reaction System ◽

Clinical Diagnostics ◽

Fluorescent Detection ◽

One Pot ◽

Catalytic Activities ◽

Cellular Processes ◽

Dual Functional ◽

Copper Phosphate ◽

Reliable Quantification

An effective strategy to detect biological thiols (biothiols), including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), holds significant incentive since they play vital roles in many cellular processes and are closely related to many diseases. Here, we demonstrated that hybrid nanoflowers composed of crystalline copper phosphate and horseradish peroxidase (HRP) served as a functional unit exhibiting dual catalytic activities of biothiol oxidase and HRP, yielding a cascade reaction system for a sensitive one-pot fluorescent detection of biothiols. The nanoflowers were synthesized through the anisotropic growth of copper phosphate petals coordinated with the amine/amide moieties of HRP, by simply incubating HRP and copper(II) sulfate for three days at room temperature. Copper phosphates within the nanoflowers oxidized target biothiols to generate H2O2, which activated the entrapped HRP to oxidize the employed Amplex UltraRed substrate to produce intense fluorescence. Using this strategy, biothiols were selectively and sensitively detected by monitoring the respective fluorescence intensity. This nanoflower-based strategy was also successfully employed for reliable quantification of biothiols present in human serum, demonstrating its great potential for clinical diagnostics.

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Translating Nucleic Acid Amplification Assays to the Microscale: Lab on a Chip for Point-of-Care Molecular Diagnostics

Current Analytical Chemistry ◽

10.2174/1573411012666151020212121 ◽

2016 ◽

Vol 12 (5) ◽

pp. 386-396 ◽

Cited By ~ 2

Author(s):

Michael G. Mauk ◽

Jinzhao Song ◽

Yubing Tong ◽

Haim H. Bau ◽

Changchun Liu

Keyword(s):

Nucleic Acid ◽

Molecular Diagnostics ◽

Point Of Care ◽

Lab On A Chip ◽

Nucleic Acid Amplification

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Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

Journal of Oncology ◽

10.1155/2012/905024 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 16

Author(s):

Anja Gulliksen ◽

Helen Keegan ◽

Cara Martin ◽

John O'Leary ◽

Lars A. Solli ◽

...

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Clinical Sample ◽

Point Of Care ◽

Fluorescent Detection ◽

Clinical Samples ◽

Microfluidic Channels ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Hpv E6

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

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