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Abstract Enzymatic DNA amplification‐based approaches involving intercalating DNA‐binding fluorescent dyes and expensive optical detectors are the gold standard for nucleic acid detection. As components of a simplified and miniaturized system, conventional silicon‐based ion sensitive field effect transistors (ISFETs) that measure a decrease in pH due to the generation of pyrophosphates during DNA amplification have been previously reported. In this article, Bst polymerase in a loop‐mediated isothermal amplification (LAMP) reaction combined with target‐specific primers and crumpled graphene field effect transistors (gFETs) to electrically detect amplification by sensing the reduction in primers is used. Graphene is known to adsorb single‐stranded DNA due to noncovalent π–π bonds, but not double‐stranded DNA. This approach does not require any surface functionalization and allows the detection of primer concentrations at the endpoint of reactions. As recently demonstrated, the crumpled gFET over the conventional flat gFET sensors due to their superior sensitivity is chosen. The endpoint of amplification reaction with starting concentrations down to 8 × 10−21min 90 min including the time of amplification and detection is detected. With its high sensitivity and small footprint, this platform will help bring complex lab‐based diagnostic and genotyping amplification assays to the point‐of‐care.
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Positive feedback drives a secondary nonlinear product burst during a biphasic DNA amplification reaction
Isothermal DNA amplification reactions are used in a broad variety of applications, from diagnostic assays to DNA circuits, with greater speed and less complexity than established PCR technologies. We recently reported a unique, high gain, biphasic isothermal DNA amplification reaction, called the Ultrasensitive DNA Amplification Reaction (UDAR). Here we present a detailed analysis of the UDAR reaction pathways that initiates with a first phase followed by a nonlinear product burst, which is caused by an autocatalytic secondary reaction. The experimental reaction output was reproduced using an ordinary differential equation model based on detailed reaction mechanisms. This model provides insight on the relative importance of each reaction mechanism during both phases, which could aid in the design of product output during DNA amplification reactions.
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- Award ID(s):
- 1847245
- PAR ID:
- 10422060
- Date Published:
- Journal Name:
- The Analyst
- Volume:
- 147
- Issue:
- 20
- ISSN:
- 0003-2654
- Page Range / eLocation ID:
- 4450 to 4461
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D2AN01067D
