US-12624386-B2 - Ultraviolet quantitative label-free detection of DNA amplification

US12624386B2US 12624386 B2US12624386 B2US 12624386B2US-12624386-B2

Abstract

The present document describes methods and systems for amplifying and quantifying amplification of a nucleic acid molecule, with a polymerase chain reaction (PCR) or a loop-mediated isothermal amplification (LAMP), by irradiating, with a heating activation light beam from a continuous wave laser a biological enzymatic reaction mixture in solution comprising a nucleic acid template, a polymerase enzyme, and chemically modified nanoparticles. Quantification of the amplification is achieved by irradiating the biological enzymatic reaction mixture during an annealing and/or elongation steps with an ultraviolet (UV) light source and measuring with a photodetector a transmission change in UV light transmission.

Inventors

Mark Trifiro
Ngoc Anh Minh TRAN
Miltiadis Paliouras
Andrew Kirk
Seung Soo Lee
Padideh MOHAMMADYOUSEF

Assignees

THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY

Dates

Publication Date: 20260512
Application Date: 20191213

Claims (15)

1 . A method of amplifying a nucleic acid molecule and quantifying amplification thereof, of a nucleic acid template with a polymerase chain reaction (PCR) or a loop-mediated isothermal amplification (LAMP), the method comprising: a) performing said PCR or said LAMP and during said PCR or said LAMP, irradiating a biological enzymatic reaction mixture in solution, for a period of time sufficient, with a heating activation light beam from a continuous wave laser, said biological enzymatic reaction mixture comprising said nucleic acid template, a polymerase enzyme and chemically modified nanoparticles comprising nanorods of metal, said heating activation light beam having a wavelength resonant with a surface plasmon of said chemically modified nanoparticles, which activates a plasmon resonance localized at a surface of said chemically modified nanoparticles, which causes a release of heat sufficient to heat a whole of said biological enzymatic reaction mixture and promote said PCR or said LAMP, b) irradiating said biological enzymatic reaction mixture with a monitoring ultraviolet (UV) light from an UV light source during an annealing step, an elongation step, or both, of said PCR or said LAMP, and c) measuring with a photodetector a transmission change in an UV light transmission from said biological enzymatic reaction mixture to quantify amplification of said nucleic acid template, wherein irradiating with said monitoring UV light from an UV light source is for a duration and at an UV light intensity sufficient for measuring said transmission change to measure a free nucleotide concentration to quantify amplification of said nucleic acid template in said biological enzymatic reaction mixture, while avoiding inhibition of amplification of said nucleic acid template.
2 . The method of claim 1 , wherein said PCR is a reverse transcriptase (RT) PCR, or said LAMP is a RT LAMP.
3 . The method of claim 1 , wherein said nanorods of metal are selected from the group consisting of nanorods of Au, Ag, Pd, Pt, Fe, Cu, Al, and Zn.
4 . The method of claim 1 , wherein said chemically modified nanoparticles comprise a surface modification with a chemical compound that prevents association with an active site of said polymerase enzyme and inhibits the polymerase activity of said polymerase enzyme.
5 . The method of claim 4 , wherein said chemical compound is polyethylene glycol.
6 . The method claim 1 , wherein in step a) said irradiating with a heating activation light beam from a continuous wave laser comprises adjusting a power of said activation light beam to regulate a temperature of said biological enzymatic reaction mixture by controlling said release of heat from said chemically modified nanoparticles.
7 . The method of claim 1 , wherein said continuous wave laser is a fibre-coupled laser, a vertical-cavity surface-emitting laser (VCSEL), or a combination of said fibre-coupled laser and said VCSEL.
8 . The method of claim 1 , wherein said activation light beam from said continuous wave laser is provided from a distance of ≤5 mm from said biological enzymatic reaction mixture.
9 . The method of claim 1 , further comprising a step of optically monitoring a temperature change of said biological enzymatic reaction mixture.
10 . The method of claim 9 , wherein said optically monitoring is performed with an infrared thermometer.
11 . The method of claim 9 , wherein said optically monitoring is during said annealing step or during said elongation step.
12 . The method of claim 1 , wherein said duration is from 10 ms to 1000 ms.
13 . The method of claim 1 , wherein said UV light source emits light having a wavelength of from 175 nm to 350 nm.
14 . The method of claim 1 , wherein UV light from said UV light source is focused through an optical lens, a light pipe, or a combination of said optical lens and said light pipe.
15 . The method of claim 1 , wherein said quantifying amplification of said nucleic acid template is performed in real-time.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority of U.S. provisional patent No. 62/779,132 filed on Dec. 13, 2018, the specification of which is hereby incorporated by reference in its entirety. BACKGROUND (a) Field The subject matter disclosed generally relates to methods and systems for amplifying nucleic acid molecules and quantifying amplification thereof. More specifically, the subject matter disclosed relates to methods and systems for amplifying nucleic acid molecules and quantifying amplification thereof with plasmonic heating and quantification of amplification with UV light transmission. (b) Related Prior Art DNA amplification technologies such as the polymerase chain reaction (PCR) and loop mediated amplification (LAMP) are methods widely used in molecular biology to make multiple copies of a specific DNA segment. Using PCR or LAMP, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. For example, PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics. Traditional PCR instruments commonly uses heating elements referred to as the Peltier block or element and relies on the contact of the sample tubes with the heating block to provide heating and cooling to the sample. The advantages of Peltier elements are that the technology is well understood, thoroughly tested, and Peltier elements are readily available of-the-shelf at a low cost. One disadvantage of Peltier modules for use in PCR is that they are relatively slow when working with microliter reaction volumes, as the entire Peltier element, heat spreader and reaction sample's temperature must be changed at each cycle. The use of a water bath in conjunction with a robotic arm can be used to speed up the process. This, however, will increase cost, complexity and is certainly not a portable system. Moving to direct non-contact methods, such as plasmonic PCR, which rely on electromagnetic radiation as the mechanism for heat transfer, can potentially address the shortcomings of contemporary PCR machines. One caveat of these non-contact methods of heating is that the wall-plug efficiency is typically not 100%. As an example, lasers have a wall plug efficiency of about 40-50%. DNA and other nucleic acids are regularly quantitated with PCR. Fluorescent labels can be added directly into the PCR mix to measures its fluorescent signal at each cycle. Fluorescent DNA-intercalating dyes such as ethidium bromide and SYBR™/Pico™ green are a simple and straightforward compound, which can be used in quantitative PCR (qPCR). In fact, the first qPCR assays employed ethidium bromide as a detection mechanism, as it also binds the double stranded DNA produced during amplification. However, concerns over safety and sensitivity of ethidium bromide led to the use of alternative fluorescent intercalating dyes, including SYBR™/Pico™ Green dye, in the qPCR reaction. Like ethidium bromide SYBR™/Pico™ Green preferentially binds to double-stranded DNA, but with a much greater affinity. The use of SYBR™ green and pico green has improved the sensitivity of detection meaning that fewer PCR cycles are required. These dyes are also far more quantitative, as the emission of fluorescence is directly proportional to the amount of PCR product generated. However, these dyes are costly and need to perpetually be added to the reaction mixture. Therefore, doing away with the addition of fluorescent labels would be advantageous. Therefore, there is a need for methods of quantitating the production of DNA during DNA amplification reaction, or mitigating the disadvantages of currently existing methods for quantitating the production of DNA during DNA amplification reaction. SUMMARY According to an embodiment, there is provided a method of amplifying a nucleic acid molecule and quantify amplification thereof, with a polymerase chain reaction (PCR) or a loop-mediated isothermal amplification (LAMP), through bulk heating a biological enzymatic reaction mixture in solution containing a nucleic acid template,a polymerase enzyme, andchemically modified nanoparticles comprising nanorods of metal, having photo-thermal properties, to promote the PCR or the LAMP, comprising:a) irradiating the chemically modified nanoparticles with an activation light beam from a continuous wave laser to provide excitation for a period of time to activate the photo-thermal properties of the chemically modified nanoparticles, such that the chemically modified nanoparticles release heat sufficient to provide the heating to the whole reaction mixture in solution and promote the PCR or the LAMP, andb) irradiating the biological enzymatic reaction mixture, during an annealing step, an elongation step, or both, with an ultraviolet (UV) light source and measuring