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Nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification.

Liu C, Sadik MM, Mauk MG, Edelstein PH, Bushman FD, Gross R, Bau HH - Sci Rep (2014)

Bottom Line: Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings.The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer.The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

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The nuclemeter.(a) A schematic depiction of the cross-section of the nuclemeter, consisting of a sample chamber and a reaction-diffusion conduit. (b) An illustration of the nuclemeter's operation. Initially, only the sample chamber contains the nucleic acid template (top). The template amplifies and diffuses into the conduit, where it continues to amplify at the appropriate amplification temperature (middle). XF indicates the position of the reaction front that propagates with a constant velocity (v0) (bottom). (c) A photograph of a plastic chip housing four nuclemeters.
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f1: The nuclemeter.(a) A schematic depiction of the cross-section of the nuclemeter, consisting of a sample chamber and a reaction-diffusion conduit. (b) An illustration of the nuclemeter's operation. Initially, only the sample chamber contains the nucleic acid template (top). The template amplifies and diffuses into the conduit, where it continues to amplify at the appropriate amplification temperature (middle). XF indicates the position of the reaction front that propagates with a constant velocity (v0) (bottom). (c) A photograph of a plastic chip housing four nuclemeters.

Mentions: Herein, we propose a new paradigm for quantifying the number of target nucleic acid molecules in a sample. We develop a simple, low-cost, reaction-diffusion device, dubbed the “nuclemeter”, affording endpoint, quantitative detection of target nucleic acids based on the position of a reaction front. The nuclemeter is comprised of a sample chamber and a reaction-diffusion conduit, containing all the reagents needed for enzymatic amplification, as well as intercalating dye reporter (Fig. 1a–c). A sample laden with target nucleic acids is introduced into the sample chamber and the amplification reaction is triggered thermally. As time progresses, amplicons diffuse into the reaction-diffusion conduit, where they continue to react and amplify. After a certain time threshold, the conduit consists of two distinct regions (Fig. 1b): the bright, left segment (0 < x < XF), where the amplification reaction has already generated a sufficient number of amplicons to emit detectable fluorescence emission and the dark, right section (x > XF) into which amplicons have not yet diffused. As time proceeds, the reaction front (XF), separating between the bright and dark regions, propagates to the right with a constant velocity (v0). We hypothesize that the position of the reaction front indicates target analyte concentration. Many nuclemeters can be housed on a single chip and imaged simultaneously for concurrent monitoring of multiple amplification processes, calibration standards, and controls. Fig. 1c features a plastic chip with four nuclemeters, many more can be housed on a single substrate. Additionally, many reaction-diffusion conduits (not shown) can branch from a single sample chamber.


Nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification.

Liu C, Sadik MM, Mauk MG, Edelstein PH, Bushman FD, Gross R, Bau HH - Sci Rep (2014)

The nuclemeter.(a) A schematic depiction of the cross-section of the nuclemeter, consisting of a sample chamber and a reaction-diffusion conduit. (b) An illustration of the nuclemeter's operation. Initially, only the sample chamber contains the nucleic acid template (top). The template amplifies and diffuses into the conduit, where it continues to amplify at the appropriate amplification temperature (middle). XF indicates the position of the reaction front that propagates with a constant velocity (v0) (bottom). (c) A photograph of a plastic chip housing four nuclemeters.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256561&req=5

f1: The nuclemeter.(a) A schematic depiction of the cross-section of the nuclemeter, consisting of a sample chamber and a reaction-diffusion conduit. (b) An illustration of the nuclemeter's operation. Initially, only the sample chamber contains the nucleic acid template (top). The template amplifies and diffuses into the conduit, where it continues to amplify at the appropriate amplification temperature (middle). XF indicates the position of the reaction front that propagates with a constant velocity (v0) (bottom). (c) A photograph of a plastic chip housing four nuclemeters.
Mentions: Herein, we propose a new paradigm for quantifying the number of target nucleic acid molecules in a sample. We develop a simple, low-cost, reaction-diffusion device, dubbed the “nuclemeter”, affording endpoint, quantitative detection of target nucleic acids based on the position of a reaction front. The nuclemeter is comprised of a sample chamber and a reaction-diffusion conduit, containing all the reagents needed for enzymatic amplification, as well as intercalating dye reporter (Fig. 1a–c). A sample laden with target nucleic acids is introduced into the sample chamber and the amplification reaction is triggered thermally. As time progresses, amplicons diffuse into the reaction-diffusion conduit, where they continue to react and amplify. After a certain time threshold, the conduit consists of two distinct regions (Fig. 1b): the bright, left segment (0 < x < XF), where the amplification reaction has already generated a sufficient number of amplicons to emit detectable fluorescence emission and the dark, right section (x > XF) into which amplicons have not yet diffused. As time proceeds, the reaction front (XF), separating between the bright and dark regions, propagates to the right with a constant velocity (v0). We hypothesize that the position of the reaction front indicates target analyte concentration. Many nuclemeters can be housed on a single chip and imaged simultaneously for concurrent monitoring of multiple amplification processes, calibration standards, and controls. Fig. 1c features a plastic chip with four nuclemeters, many more can be housed on a single substrate. Additionally, many reaction-diffusion conduits (not shown) can branch from a single sample chamber.

Bottom Line: Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings.The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer.The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

Show MeSH
Related in: MedlinePlus