WO2022161460A1 - 聚合酶链反应的方法 - Google Patents

聚合酶链反应的方法 Download PDF

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WO2022161460A1
WO2022161460A1 PCT/CN2022/074585 CN2022074585W WO2022161460A1 WO 2022161460 A1 WO2022161460 A1 WO 2022161460A1 CN 2022074585 W CN2022074585 W CN 2022074585W WO 2022161460 A1 WO2022161460 A1 WO 2022161460A1
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tungsten oxide
oxide nanoparticles
polymerase chain
chain reaction
nucleic acid
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PCT/CN2022/074585
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French (fr)
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黄志嘉
谢达斌
杨理行
李健玮
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黄志嘉
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a method of polymerase chain reaction, and in particular, to a method of polymerase chain reaction using tungsten oxide nanoparticles.
  • PCR polymerase chain reaction
  • template DNA a nucleic acid fragment
  • primer pair ie, forward primer and reverse primer
  • the nucleic acid sequence in the fragment that is complementary to the sequence of the individual primer ie, the binding step
  • the polymerase is based on the nucleic acid sequence of the nucleic acid fragment through the primer-guided extension to copy a new nucleic acid product (commonly referred to as an amplicon) [ie extension (extension) step].
  • the denaturation step is performed at 95° C. for 15 seconds to 30 seconds first, and then the temperature is lowered to an appropriate bonding temperature for 30 seconds to 60 seconds for the bonding step.
  • the extension step is performed at 70°C to 75°C, and the time of this step is determined according to the length of the nucleic acid fragment to be amplified, and is usually 30 seconds to 60 seconds.
  • the denaturation of the aforementioned denaturation step can be completed in an instant, and the extension of the extension step can also improve the polymerization efficiency of the polymerase by precisely controlling the temperature of the reaction solution.
  • the traditional polymerase chain reaction equipment uses metal heating components or water to achieve thermal equilibrium, and its heating rate cannot cope with the temperature changes in the denaturation and extension steps, resulting in long heating time and energy consumption.
  • one aspect of the present invention is to provide a method for polymerase chain reaction.
  • This method uses electromagnetic waves of a specific wavelength to irradiate tungsten oxide nanoparticles with a specific transmittance in the infrared region, so that the low-concentration tungsten oxide nanoparticles can rapidly heat the reaction solution, thereby shortening the heating time.
  • a method for polymerase chain reaction is provided.
  • a plurality of tungsten oxide nanoparticles, nucleic acid fragments and reaction reagents are mixed to form a reaction solution, wherein the concentration of these tungsten oxide nanoparticles after mixing is 50 ppm to 1000 ppm, and the tungsten oxide nanoparticles are larger than 780 nm.
  • the transmittance in the wavelength range of not more than 2000 nm is less than 98%.
  • the reaction solution is subjected to polymerase chain reaction to amplify a part of the sequence of the nucleic acid fragment, wherein the polymerase chain reaction uses electromagnetic waves to irradiate the reaction solution to perform the denaturation step and the extension step, and the wavelength of the electromagnetic waves is 400nm to 2000nm.
  • the transmittance of the tungsten oxide nanoparticles in the wavelength range of 380 nm to 780 nm is greater than 40%.
  • the transmittance of the tungsten oxide nanoparticles in the wavelength range of 400 nm to 600 nm is not less than 50%.
  • the transmittance of the tungsten oxide nanoparticles in the wavelength range of greater than 780 nm and not greater than 1100 nm is not greater than 95%.
  • the tungsten oxide nanoparticles include a plurality of tungsten oxide nanorods, the tungsten oxide nanorods have an average length and an average diameter, and the ratio of the average length to the average diameter is 5-25.
  • these tungsten oxide nanoparticles have the general formula (I) W a O b , in the general formula (I), W represents tungsten, O represents oxygen, and the ratio of b to a (b /a) is 1 to 3.
  • these tungsten oxide nanoparticles have the general formula (II) W a O b M c , in the general formula (II), W represents tungsten, O represents oxygen, M represents nickel, palladium, A metal of platinum or a combination thereof, and the ratio of b to the sum of a and c [b/(a+c)] is 1 to 3.
  • the average heating rate of the denaturation step and the extension step is 2°C/sec to 23°C/sec.
  • the heating time of the denaturation step and the extension step is not more than 15 seconds.
  • the power of the electromagnetic wave is 0.1W to 2W.
  • the method of applying the polymerase chain reaction of the present invention wherein by irradiating the tungsten oxide nanoparticles with a specific transmittance in the infrared region with electromagnetic waves of a specific wavelength, so that the low-concentration tungsten oxide nanoparticles can rapidly heat the reaction solution,
  • the heating time is shortened, and the efficiency of the polymerase chain reaction is improved.
  • FIG. 1 is a flow chart illustrating a method for polymerase chain reaction according to an embodiment of the present invention.
  • FIG. 2 is a graph showing temperature change curves when the reaction reagents are heated by using the nanoparticles of Example 1 and Comparative Example 1 of the present invention, respectively.
  • FIG. 3 is a graph showing the temperature change curve when the reaction reagent is heated by using the nanoparticles of Example 1 to Example 3 of the present invention, respectively.
  • Example 4 is an electrophoresis film image showing the interference comparison test of conventional polymerase chain reaction (polymerase chain reaction without electromagnetic waves) and polymerase chain reaction using electromagnetic waves using the nanoparticles of Example 1 of the present invention, respectively.
  • FIG. 5 is a graph showing the results of a comparative test of background interference using the nanoparticles of Example 1 and Comparative Example 1 of the present invention, respectively.
  • FIG. 6 is a graph showing the amplification of the polymerase chain reaction method according to Application Example 1 of the present invention.
  • the polymerase chain reaction method of the present invention utilizes electromagnetic waves of a specific wavelength to irradiate the tungsten oxide nanoparticles with a specific transmittance in the infrared region, so that the low-concentration tungsten oxide nanoparticles can rapidly heat the reaction solution, thereby shortening the temperature rise. time, and improve the efficiency of polymerase chain reaction.
  • these tungsten oxide nanoparticles have high transmittance in the visible light region, so as to reduce the absorption of excitation light and emission light of the fluorescent agent in the reaction reagent, thereby reducing the background interference of polymerase chain reaction.
  • the method 100 of the polymerase chain reaction is to first mix a plurality of tungsten oxide nanoparticles, nucleic acid fragments and reaction reagents to form a reaction solution, as shown in operation 110 .
  • These tungsten oxide nanoparticles have the ability of photothermal conversion, so they can convert the absorbed electromagnetic waves into thermal energy to heat the reaction solution.
  • the transmittance of the tungsten oxide nanoparticles in the wavelength range of more than 780 nm and not more than 2000 nm is less than 98%. Equivalently, the absorbance of the tungsten oxide nanoparticles in the infrared region is not less than 2%. If the absorbance of the tungsten oxide nanoparticles in the infrared region is less than 2%, the tungsten oxide nanoparticles cannot effectively absorb electromagnetic waves to convert them into heat energy, so the reaction solution cannot be heated effectively.
  • the transmittance of the tungsten oxide nanoparticles in the wavelength range greater than 780 nm and not greater than 1100 nm is not greater than 95%.
  • the reaction solution used in the polymerase chain reaction method 100 contains a fluorescent agent, and the fluorescent agent emits fluorescence after being irradiated by excitation light, and the fluorescence is used to detect the quantity of nucleic acid products, thus serving as a basis for quantifying the original nucleic acid fragment content.
  • the added tungsten oxide nanoparticles do not interfere with the absorption of excitation light and the emission of fluorescence by the fluorescent agent, and the subsequent quantitative accuracy and/or sensitivity of the polymerase chain reaction method can be improved.
  • the wavelengths of excitation light and emission light of the fluorescent agent are usually in the visible light region, so the subsequent quantitative accuracy and/or the PCR method can be improved by controlling the transmittance of the tungsten oxide nanoparticles in the visible light region. sensitivity.
  • the transmittance of the tungsten oxide nanoparticles in the wavelength range of 380 nm to 780 nm (corresponding to the visible light region) is greater than 40%.
  • the transmittance of the tungsten oxide nanoparticles in the wavelength range of 400 nm to 600 nm is not less than 50%.
  • the transmittance of the tungsten oxide nanoparticles is greater than 40% in a part of the wavelength range of the visible light region, such as 450 nm, 500 nm, 550 nm and combinations thereof (eg 450 nm to 550 nm or 500 nm to 550 nm).
  • the tungsten oxide nanoparticles may have absorbance in the visible light region, but not only in the wavelength range of excitation light and fluorescence, or the absorbance is very low and will not cause interference, such as absorption degree is less than 28%, preferably less than 20%, and more preferably less than 10%.
  • the aforementioned tungsten oxide nanoparticles can also convert the absorbed visible light into thermal energy to heat the reaction solution.
  • the polymerase chain reaction method 100 of the present invention can eliminate the step of gel electrophoresis without removing tungsten oxide nanoparticles by gel electrophoresis, thereby simplifying the subsequent step of quantifying the content of nucleic acid fragments.
  • the polymerase chain reaction method can selectively perform the step of gel electrophoresis to remove the tungsten oxide nanoparticles, and then perform the subsequent step of quantifying the content of nucleic acid fragments, so as to further improve the accuracy of quantification and / or sensitivity.
  • Tungsten oxide nanoparticles can have various shapes, such as spherical, rod, wire, or columnar.
  • the tungsten oxide nanoparticles can be, but are not limited to, tungsten oxide nanorods.
  • the tungsten oxide nanorods can have an average length and an average diameter, wherein the average length can be from 250 nm to 550 nm and the average diameter can be from 20 nm to 50 nm.
  • the aforementioned average diameter can also be referred to as the average width when viewed in a cross-section passing through the long axis of the nanorod.
  • the ratio of the average length to the average diameter may be 5 to 25.
  • the aforementioned ratio of the average length to the average diameter is also referred to as the aspect ratio.
  • the aspect ratio may be 6 to 23.
  • the absorption peak of the tungsten oxide nanoparticles shifts to short wavelengths (such as moving to the infrared region), thereby reducing or eliminating the absorption in the visible light region, thus reducing the interference to the fluorescence emitted by the fluorescent agent.
  • the tungsten oxide nanoparticles have the general formula (I) W a O b , where W represents tungsten, O represents oxygen, and the ratio of b to a (b/a) is 1 to 3.
  • W represents tungsten
  • O represents oxygen
  • the ratio of b to a (b/a) is 1 to 3.
  • the tungsten oxide nanoparticles have absorbance in the infrared region, so as to improve the photothermal conversion ability of the tungsten oxide nanoparticles.
  • the ratio of b to a is preferably greater than 2 and less than 3, wherein the oxygen atoms of the tungsten oxide nanoparticles are insufficient and free electrons exist, so the absorption of infrared light by the tungsten oxide nanoparticles is enhanced, thereby further enhancing the photothermal conversion of the tungsten oxide nanoparticles Ability.
  • the constituent elements of the tungsten oxide nanoparticles may selectively include a transition metal element of Group VIII.
  • the material of the tungsten oxide nanoparticles includes undoped and doped tungsten oxide, and these doped transition metal elements can enhance the infrared light absorption of the tungsten oxide nanoparticles, thereby enhancing the properties of these nanoparticles. The ability to convert light to heat.
  • the tungsten oxide nanoparticles have the general formula (II) W a O b M c , where W represents tungsten, O represents oxygen, and M represents nickel, palladium, platinum and combinations thereof, and the ratio of b to the sum of a and c [b/(a+c)] is 1 to 3.
  • W represents tungsten
  • O represents oxygen
  • M represents nickel, palladium, platinum and combinations thereof
  • the ratio of b to the sum of a and c [b/(a+c)] is 1 to 3.
  • the ratio of b to the sum of a and c is in the aforementioned range, the infrared light absorption of the tungsten oxide nanoparticles is further enhanced, thereby further enhancing the photothermal conversion capability of these nanoparticles.
  • nucleic acid fragment serves as template DNA.
  • the nucleic acid fragment of the present invention is not particularly limited, and may be known to those with general knowledge in the technical field of the present invention.
  • nucleic acid fragments may comprise nucleic acid of infectious microorganisms, such as bacteria, viruses, fungi, parasites, and the like.
  • the aforementioned reaction reagents may include polymerases, primer pairs, nucleotide bases, fluorescent agents and buffer solutions, all of which may be known to those with common knowledge in the technical field of the present invention.
  • the design of primer pairs must be based on the sequences of the aforementioned nucleic acid fragments, so that the primers are adhered to the single strands of the nucleic acid fragments, thereby realizing the purpose of polymerase chain reaction.
  • the concentration of the tungsten oxide nanoparticles is 50 ppm to 1000 ppm. If the concentration of the tungsten oxide nanoparticles is less than 50 ppm, the heating effect of too few tungsten oxide nanoparticles is not good for the heating of the reaction solution, and the heating time cannot be shortened. Conversely, if the concentration of tungsten oxide nanoparticles is greater than 1000 ppm, excessive tungsten oxide nanoparticles greatly reduce the transmittance in the visible light region, and interfere with the fluorescence emitted by the fluorescent agent, so the content of nucleic acid fragments cannot be quantified. Further, the concentration of the tungsten oxide nanoparticles is preferably 50 ppm to 500 ppm, more preferably 300 ppm to 350 ppm.
  • the polymerase chain reaction method 100 performs polymerase chain reaction on the reaction solution to amplify the partial sequence of the nucleic acid fragment, as shown in operation 120 .
  • the aforementioned polymerase chain reaction uses electromagnetic waves to irradiate the reaction solution to perform the denaturation step and the extension step.
  • the tungsten oxide nanoparticles in the reaction solution are irradiated by electromagnetic waves, which generate heat energy after absorbing the electromagnetic waves, and the heat energy heats the reaction solution.
  • the wavelength of the electromagnetic wave is 400nm to 2000nm, which is equivalent to the frequency of the electromagnetic wave is 150THz to 750THz.
  • electromagnetic waves include visible light, near infrared rays, and far infrared rays. Since electromagnetic waves are absorbed by tungsten oxide nanoparticles, the absorption characteristics of tungsten oxide nanoparticles determine the wavelength of electromagnetic waves. As previously mentioned, the absorption properties of tungsten oxide nanoparticles can be affected by shape and dopants.
  • the wavelength of the electromagnetic wave may be from 780 nm to 2000 nm, so that the tungsten oxide nanorods absorbing near infrared rays generate thermal energy, thereby heating the reaction solution.
  • tungsten oxide nanoparticles that absorb visible light can also heat the reaction solution under visible light irradiation.
  • the power of the electromagnetic wave will affect the heating rate of the tungsten oxide nanoparticles to the reaction solution, wherein the greater the power, the greater the heating rate, and vice versa.
  • the power of the electromagnetic waves may be 0.1W to 2W.
  • the electromagnetic wave with this power can quickly generate enough heat energy for the tungsten oxide nanoparticles, so that the average heating rate can meet the requirements of the temperature change in the denaturation step and the extension step, thereby shortening the heating time, and By precisely controlling the temperature of the reaction solution, the polymerization efficiency of the polymerase is improved.
  • the average heating rate for the denaturation and extension steps is 2°C/sec to 23°C/sec.
  • the average heating rate is in the aforementioned range, it can correspond to the requirement of rapid heating in the denaturation step, so as to shorten the heating time, and precisely control the heating in the extension step, so as to improve the polymerization efficiency of the polymerase, thus improving the polymerase chain reaction efficiency. efficiency.
  • the heating time of the denaturation step and the extension step is not more than 15 seconds.
  • the heating time of the denaturation step and the extension step may be 5 seconds to 15 seconds and 1 second to 4 seconds, respectively.
  • the heating time is in the aforementioned range, the heating time can be shortened, thereby improving the efficiency of the polymerase chain reaction.
  • the heating time of the denaturation step and the extension step is not more than 10 seconds.
  • the tungsten oxide nanorods of Example 1 are prepared by a hydrothermal method, wherein 1 to 300 mg of tungsten halide, 1 to 20 mL of 0.1 to 20 mL of 0.1 to 1.5 M hydrochloric acid, and 1 to 20 mL of ink [containing polyethylene glycol ( PEG), ethylene glycol (EG), polyvinylpyrrolidone (PVP) and glycerol], and then the mixture was obtained, and then reduced with 0.01 mL to 1 mL of N2H4 to obtain the tungsten oxide nanorods of Example 1. Then, evaluation was performed by the evaluation method described later.
  • Example 2 The doped tungsten oxide nanorods of Examples 2 to 3 and the iron oxide nanoparticles of Comparative Example 1 were produced in a similar manner to Example 1. The difference is that Examples 2-3 reduce tungsten halide, and Example 2 adds 0.1 mg to 80 mg of platinum halide, while Example 3 adds 0.1 mg to 80 mg of palladium halide. In addition, in Comparative Example 1, the halide of iron was used instead of the halide of tungsten. The evaluation results of the foregoing Examples 1 to 3 and Comparative Example 1 are shown in Table 1 below.
  • the transmittance test is to use a spectrometer to measure the spectrum of the tungsten oxide nanoparticles.
  • the spectrum includes the visible light region and the infrared light region, and the test conditions are those commonly used by those with common knowledge.
  • the size test is to measure the size, such as particle size, of the tungsten oxide nanoparticles using an electron microscope, wherein the test conditions are those conventionally used by those with common knowledge.
  • the size includes the average length and the average diameter, and the ratio of the average length to the average diameter (also referred to as the aspect ratio and rounded up) is obtained.
  • the element content test is carried out using a chemical element analyzer, and the composition formula of the tungsten oxide nanoparticles is calculated from the measured element content, wherein the test conditions are those conventionally used by those with common knowledge.
  • the comparison test of the heating rate is to add the nanoparticles prepared in Examples 1 to 3 and Comparative Example 1 to the reaction reagent with a volume of 20 ⁇ L, and then irradiate these reactions containing nanoparticles with an electromagnetic wave with a wavelength of 808 nm and a power of 1 W.
  • Reagents for heating and record the temperature curve of these reaction reagents, as shown in Figure 2 and Figure 3, and divide the temperature difference from 28 ° C to 60 ° C (ie 32 ° C) by the required time.
  • the average heating rate was obtained for the interval, and the results are shown in Table 1.
  • the interference test of the amplification reaction is carried out by the same polymerase chain reaction as the subsequent application example 1, wherein the same one uses tungsten oxide nanorods and irradiates electromagnetic waves, and the difference between the similar ones is that no tungsten oxide nanorods or tungsten oxide nanorods are used. Electromagnetic waves were not irradiated to confirm that the tungsten oxide nanorods could heat the reaction solution by irradiating electromagnetic waves, and the temperature of the heated reaction solution could allow the polymerase to carry out the polymerase chain reaction. In addition, it can also be confirmed that these tungsten oxide nanorods do not affect the nucleic acid amplification reaction of the polymerase.
  • Carrying out the aforementioned three steps in sequence is called a cycle, and the cycle is repeated 40 times to terminate, that is, to complete the amplification of a part of the sequence of the nucleic acid fragment, thereby obtaining a nucleic acid product, and then perform gel electrophoresis on the obtained nucleic acid product, and use the electrophoresis results.
  • the temperature of the reaction solution can be controlled by irradiating tungsten oxide nanorods with electromagnetic waves, so that the polymerase can carry out the polymerase chain reaction, and these tungsten oxide nanorods can not affect the nucleic acid amplification reaction of the polymerase.
  • the results are shown in Figure 4.
  • the comparison test of background interference is to add the nanoparticles prepared in Example 1 and Comparative Example 1 to the reaction reagent with a volume of 20 ⁇ L, irradiate the reaction reagent containing nanoparticles with excitation light with a wavelength of 488 nm, and measure the amount of the nanoparticles.
  • the intensity of the fluorescence emitted at a wavelength of 520 nm is shown in Table 1 and Figure 5, where the fluorescence intensity measured using the reactant without nanoparticles added is used as a 100% reference to obtain the concentration of the reactant added with nanoparticles. The fluorescence intensity.
  • Application Example 1 is to use the tungsten oxide nanorods prepared in Example 1 to perform polymerase chain reaction, wherein tungsten oxide nanorods, nucleic acid fragments and reaction reagents are mixed to form a reaction solution with a volume of 20 ⁇ L.
  • the reaction reagents include polymerase, primer pairs, nucleotide bases, fluorescent agents and buffer solutions, wherein the polymerase, nucleotide bases, fluorescent agents and buffer solutions are reagents manufactured by Roche (commercial products). Named KAPA SYBR FAST qPCR Kit), and add the reaction reagents according to the instruction manual of this product.
  • the concentration of the nucleic acid fragment is 0.1 ⁇ g/mL
  • the addition volume is 1 ⁇ L
  • the forward primer of the primer pair is as shown in SEQ ID NO:1.
  • sequence, the reverse primer is the sequence shown in SEQ ID NO:2.
  • the polymerase chain reaction was performed using a real-time detection polymerase chain reaction machine.
  • Use the same reaction temperature and time conditions as the aforementioned amplification reaction interference test repeat to a predetermined number of cycles (as shown in Figure 6) and terminate, that is, to complete the amplification of a part of the sequence of the nucleic acid fragment, thereby obtaining a nucleic acid product.
  • the fluorescent signal of the amplified nucleic acid product is detected, and the increased signal difference ⁇ Rn is calculated.
  • the obtained fluorescence signal amplification curve is shown in FIG. 6 .
  • the cooling step of the PCR in Application Example 1 uses the same fan as the conventional PCR instrument to cool down, and the conditions can be the same as the conventional PCR instrument, so it will not be repeated.
  • Comparative Application Example 1 was carried out in a manner similar to that of Application Example 1. The difference is that in Comparative Application Example 1, iron oxide nanoparticles are used to replace tungsten oxide nanorods, and the amplification curve is shown in FIG. 7 .
  • the transmittance of the tungsten oxide nanomaterials of Examples 1 to 3 at 800 nm to 1100 nm is compared with that of the iron oxide nanoparticles of Comparative Example 1. Therefore, the nanomaterials of Examples 1 to 3 can absorb electromagnetic waves (eg, infrared light) with a wavelength of 800 nm to 1100 nm, and convert them into heat energy to heat the reaction solution, thereby increasing the average heating rate.
  • electromagnetic waves eg, infrared light
  • the nanomaterials of Examples 1 to 3 can heat the reaction solution more quickly, so as to more effectively control the temperature of the reaction solution in the denaturation step and the extension step, such as shortening the heating time, thereby improving the efficiency of the polymerase chain reaction .
  • both the platinum-doped tungsten oxide nanorods of Example 2 and the palladium-doped tungsten oxide nanorods of Example 3 can increase the oxygen vacancy through the doped transition metal, and increase the Free electrons, which can increase the absorption of nanomaterials in the infrared region (that is, reduce the transmittance in the infrared region), thus improving the photothermal conversion rate of nanomaterials.
  • the nanomaterials of Examples 1 to 3 are all rod-shaped, which have vertical and horizontal Compare. With an aspect ratio of 5 to 25, the nanorods can further enhance the photothermal conversion rate of the nanomaterials, so the temperature of the reaction solution in the denaturation step and the extension step can be more effectively controlled.
  • M represents a standard product
  • C represents a nucleic acid product obtained by a polymerase chain reaction method without using nanoparticles
  • A1 represents a tungsten oxide nanorod of 660 ppm and irradiated with an electromagnetic wave of 808 nm.
  • A2 represents the nucleic acid product obtained by the polymerase chain reaction method using 330 ppm tungsten oxide nanorods and irradiating 808 nm electromagnetic waves
  • B1 represents using 660 ppm tungsten oxide nanorods
  • B2 represents the nucleic acid product obtained by the polymerase chain reaction method using 330 ppm of tungsten oxide nanorods and not irradiating electromagnetic waves.
  • the nucleic acid products obtained therefrom are the same as those obtained by the method without nanomaterials, namely two.
  • the base pair length is 100bp.
  • the brightness of the nucleic acid product obtained using 330 ppm of tungsten oxide nanorods was consistent with the brightness of the nucleic acid product obtained using 660 ppm of tungsten oxide nanorods. Accordingly, the added tungsten oxide nanorods will not affect the nucleic acid amplification reaction of the polymerase.
  • the transmittance of the tungsten oxide nanomaterials of Examples 1 to 3 at 450 nm to 550 nm is higher than that of the iron oxide nanoparticles of Comparative Example 1. Therefore, the nanomaterials of Examples 1 to 3 have lower absorption of electromagnetic waves (such as visible light) with a wavelength of 450 nm to 550 nm, so as to reduce the absorption of excitation light and emission light for the fluorescent agent in the reaction reagent, thereby reducing background interference.
  • the background interference generated by the iron oxide nanoparticles of Comparative Example 1 is 7 to 28 times that of the tungsten oxide nanorods of Example 1.
  • ⁇ Rn represents the difference in fluorescence intensity of the reaction solution containing nanomaterials before and after the nucleic acid amplification reaction.
  • PCR reactions can be performed using 82.5 ppm to 660 ppm of the tungsten oxide nanorods of Example 1.
  • 125 ppm of the iron oxide nanoparticles of Comparative Example 1 was necessary to achieve a similar ⁇ Rn fluorescence intensity difference (about 25000) to 660 ppm of tungsten oxide. Accordingly, the tungsten oxide nanorods have better light transmittance, so the interference of the tungsten oxide nanorods on the fluorescence detection of nucleic acid products can be reduced.
  • the concentration of tungsten oxide nanorods was 660 ppm
  • the maximum ⁇ Rn was 25,000
  • the concentration of iron oxide nanoparticles was 125 ppm
  • the polymerase chain reaction method of the present invention utilizes electromagnetic waves of a specific wavelength to irradiate tungsten oxide nanoparticles with a specific transmittance in the infrared region, so that the low-concentration tungsten oxide nanoparticles can rapidly heat and react. solution, thereby shortening the heating time and improving the efficiency of the polymerase chain reaction.

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Abstract

一种聚合酶链反应的方法，此方法使用特定波长的电磁波照射于红外光区具有特定的透过率的氧化钨纳米粒子，以使低浓度的氧化钨纳米粒子可快速加热反应溶液，从而缩短升温时间，且提升聚合酶链反应的效率。

Description

聚合酶链反应的方法 技术领域
本发明涉及一种聚合酶链反应的方法，且特别涉及一种使用氧化钨纳米粒子的聚合酶链反应的方法。
背景技术
习知的聚合酶链反应(PCR)是利用加热将核酸片段(通称为模板DNA)互补的双股分开(即变性步骤)，再以引子对(即正向引子及反向引子)标的至核酸片段中的与个别引子的序列互补的核酸序列(即黏合步骤)，然后聚合酶依据核酸片段的核酸序列通过引子引导延伸复制出新的核酸产物(通称为扩增子)[即延展(extension)步骤]。依序经历前述的三个步骤(变性步骤、黏合步骤及延长步骤)称为一个循环。进行一个循环后，接续回到前述的变性步骤，以进行下一个循环，直至核酸产物的数量达到预定的目标为止。
举例而言，在PCR中，先于95℃持续15秒至30秒，以进行变性步骤，再降温至适当的黏合温度，持续30秒至60秒，以进行黏合步骤。然后，于70℃至75℃进行延展步骤，此步骤的时间依据待增幅的核酸片段的长度决定，通常为30秒至60秒。前述的变性步骤的变性可在瞬间就完成，且延展步骤的延展也能够通过精准地控制反应溶液的升温，而提高聚合酶的聚合效率。然而，传统的聚合酶连锁反应仪器是使用金属加温组件或水达成热平衡，其加热速率无法应对变性步骤及延展步骤的温度变化的需求，进而导致加热时间冗长且耗能。
有鉴于此，亟需发展一种新的聚合酶链反应的方法，以改善习知的聚合酶链反应的上述缺点。
发明内容
有鉴于上述的问题，本发明的一态样是在于提供一种聚合酶链反应的方法。此方法使用特定波长的电磁波照射于红外光区具有特定的透过率的氧化钨纳米粒子，以使低浓度的氧化钨纳米粒子能快速加热反应溶液，从而缩短升温时间。
根据本发明的一态样，提出一种聚合酶链反应的方法。于此方法中，混合多个氧化钨纳米粒子、核酸片段及反应试剂，以形成反应溶液，其中混合后此些氧化钨纳米粒子的浓度为50ppm至1000ppm，且此些氧化钨纳米粒子在大于780nm且不大于2000nm的波长范围的透过率为小于98％。然后，对反应溶液进行聚合酶链反应，以增幅核酸片段的一部分序列，其中聚合酶链反应使用电磁波照射反应溶液，以进行变性步骤及延展步骤，且电磁波的波长为400nm至2000nm。
依据本发明的一实施例，此些氧化钨纳米粒子于380nm至780nm的波长范围的透过率为大于40％。
依据本发明的另一实施例，此些氧化钨纳米粒子于400nm至600nm的波长范围的透过率为不小于50％。
依据本发明的另一实施例，此些氧化钨纳米粒子在大于780nm且不大于1100nm的波长范围的透过率为不大于95％。
依据本发明的又一实施例，此些氧化钨纳米粒子包含多个氧化钨纳米棒，此些氧化钨纳米棒具有平均长度及平均直径，且平均长度与平均直径的比值为5至25。
依据本发明的又一实施例，此些氧化钨纳米粒子具有通式(I)W aO b，于通式(I)中，W代表钨，O代表氧，且b与a的比值(b/a)为1至3。
依据本发明的又一实施例，此些氧化钨纳米粒子具有通式(II)W aO bM c，于通式(II)中，W代表钨，O代表氧，M代表镍、钯、铂或其组合的金属，且b与a及c的总合的比值[b/(a+c)]为1至3。
依据本发明的又一实施例，变性步骤及延展步骤的平均加热速率为2℃/sec至23℃/sec。
依据本发明的又一实施例，变性步骤及延展步骤的升温时间不大于15秒。
依据本发明的又一实施例，电磁波的功率为0.1W至2W。
应用本发明的聚合酶链反应的方法，其中通过以特定波长的电磁波照射于红外光区具有特定的透过率的氧化钨纳米粒子，以使低浓度的氧化钨纳米粒子可快速加热反应溶液，从而缩短升温时间，且提升聚合酶链反应的效率。
附图说明
为了对本发明的实施例及其优点有更完整的理解，现请参照以下的说明并配合相应的附图。必须强调的是，各种特征并非依比例描绘且仅是为了图解目的。相关附图内容说明如下：
图1为绘示根据本发明的一实施例的聚合酶链反应的方法的流程图。
图2为绘示分别使用本发明的实施例1及比较例1的纳米粒子进行加热反应试剂时的温度变化曲线。
图3为绘示分别使用本发明的实施例1至实施例3的纳米粒子进行加热反应试剂时的温度变化曲线。
图4为绘示分别使用本发明的实施例1的纳米粒子进行传统的聚合酶链反应(未使用电磁波的聚合酶链反应)与使用电磁波的聚合酶链反应的干扰比较试验的电泳胶片影像。
图5为绘示分别使用本发明的实施例1及比较例1的纳米粒子进行背景干扰的比较试验的结果图。
图6为绘示根据本发明的应用例1的聚合酶链反应的方法的增幅曲线。
图7为绘示根据本发明的比较应用例1的聚合酶链反应的方法的增幅曲线。
其中，附图标记：
100：方法
110，120：操作
具体实施方式
以下仔细讨论本发明实施例的制造和使用。然而，可以理解的是，实施例提供许多可应用的发明概念，其可实施于各式各样的特定内容中。所讨论的特定实施例仅供说明，并非用以限定本发明的范围。
本发明的聚合酶链反应的方法是利用特定波长的电磁波照射于红外光区具有特定的透过率的氧化钨纳米粒子，以使低浓度的氧化钨纳米粒子能够快速加热反应溶液，从而缩短升温时间，且提升聚合酶链反应的效率。此外，此些氧化钨纳米粒子于可见光区具有高的透过率，以降低对于反应试剂中的荧光剂的激发光及放射光的吸收，从而降低聚合酶链反应的背景干扰。
请参阅图1，聚合酶链反应的方法100是先混合多个氧化钨纳米粒子、核 酸片段及反应试剂，以形成反应溶液，如操作110所示。此些氧化钨纳米粒子具备光热转换的能力，故能将吸收的电磁波转换成热能，以加热反应溶液。
氧化钨纳米粒子在大于780nm且不大于2000nm的波长范围的透过率为小于98％。相当于，氧化钨纳米粒子于红外光区的吸收度为不小于2％。倘若氧化钨纳米粒子于红外光区的吸收度小于2％，氧化钨纳米粒子不能有效地吸收电磁波以转换成热能，故不能有效地加热反应溶液。较佳地，氧化钨纳米粒子在大于780nm且不大于1100nm的波长范围的透过率为不大于95％。
进一步，聚合酶链反应的方法100使用的反应溶液含有荧光剂，此荧光剂遭受激发光照射后而放射荧光，此荧光用于检测核酸产物的数量，从而作为定量原始的核酸片段含量的依据。添加的氧化钨纳米粒子对于荧光剂吸收激发光及放射荧光愈不干扰，则可提高聚合酶链反应的方法后续的定量的准确性及/或灵敏度。
再者，通常荧光剂的激发光与放射光的波长位于可见光区，故可通过控制氧化钨纳米粒子于可见光区的透过率来提高聚合酶链反应的方法后续的定量的准确性及/或灵敏度。在一些实施例中，氧化钨纳米粒子于380nm至780nm的波长范围(相当于可见光区)的透过率为大于40％。较佳地，氧化钨纳米粒子于400nm至600nm的波长范围的透过率为不小于50％。在一些具体例中，氧化钨纳米粒子于可见光区的波长范围的一部分波长的透过率为大于40％，例如450nm、500nm、550nm及其组合(如450nm至550nm或500nm至550nm)。然而，在另一些实施例中，氧化钨纳米粒子于可见光区可具有吸收度，而仅于激发光及荧光的波长范围不具吸收度，或者其吸收度很低，而不会产生干扰，例如吸收度小于28％，较佳小于20％，且更佳小于10％。再者，前述的氧化钨纳米粒子亦可将所吸收的可见光转换成热能，以加热反应溶液。
据此，在上述的实施例中，本发明的聚合酶链反应的方法100可排除胶电泳步骤，而无需通过胶电泳移除氧化钨纳米粒子，从而简化后续定量核酸片段含量的步骤。然而，在另一些实施例中，聚合酶链反应的方法可选择性进行胶电泳步骤，以移除氧化钨纳米粒子后，再进行后续定量核酸片段含量的步骤，以更提高定量的准确性及/或灵敏度。
氧化钨纳米粒子可具有各种形状，例如球状、棒状、线状或柱状。在一些实施例中，氧化钨纳米粒子可为但不限于氧化钨纳米棒。氧化钨纳米棒可具有 平均长度及平均直径，其中平均长度可为250nm至550nm，且平均直径可为20nm至50nm。若以穿过纳米棒的长轴的剖面观之，前述的平均直径亦可称作平均宽度。
在一些具体例中，平均长度与平均直径的比值可为5至25。前述的平均长度与平均直径的比值亦称为纵横比。较佳地，纵横比可为6至23。当纵横比为前述的范围时，氧化钨纳米粒子的吸收峰往短波长(如移至红外光区)移动，从而降低或消除于可见光区的吸收，故减少对荧光剂放射荧光的干扰。
在一些实施例中，氧化钨纳米粒子具有通式(I)W aO b，于通式(I)中，W代表钨，O代表氧，且b与a的比值(b/a)为1至3。当氧化钨纳米粒子具有通式(I)时，氧化钨纳米粒子于红外光区具有吸收度，以提升氧化钨纳米粒子的光热转换的能力。b与a的比值较佳可为大于2且小于3，其中氧化钨纳米粒子的氧原子不足而存在自由电子，故增强氧化钨纳米粒子吸收红外光，从而更提升氧化钨纳米粒子的光热转换的能力。
除了氧及钨之外，氧化钨纳米粒子的组成元素可选择性包含第Ⅷ族的过渡金属元素。换句话说，氧化钨纳米粒子的材料包含未经掺杂的及经掺杂的氧化钨，此些掺杂的过渡金属元素可增强氧化钨纳米粒子的红外光吸收，从而提升此些纳米粒子的光热转换的能力。
举例而言，在一些实施例中，氧化钨纳米粒子具有通式(II)W aO bM c，于通式(II)中，W代表钨，O代表氧，M代表镍、钯、铂及其组合的金属，且b与a及c的总合的比值[b/(a+c)]为1至3。当b与a及c的总合的比值为前述的范围时，更增强氧化钨纳米粒子的红外光吸收，从而更提升此些纳米粒子的光热转换的能力。
此外，前述的核酸片段作为模板DNA。本发明的核酸片段没有特别限制，且可为本发明技术领域中具有公知常识者所习知者。举例而言，核酸片段可包含感染性微生物的核酸，如细菌、病毒、真菌及寄生虫等。
再者，前述的反应试剂可包含聚合酶、引子对、核苷酸碱基、荧光剂及缓冲溶液，其皆可为本发明技术领域中具有公知常识者所习知者。如具有公知常识者所理解的，引子对的设计必需依据前述的核酸片段的序列，以使引子黏合至核酸片段的单股上，进而实现聚合酶链反应的目的。
在氧化钨纳米粒子、核酸片段及反应试剂形成的反应溶液中，氧化钨纳米 粒子的浓度为50ppm至1000ppm。倘若氧化钨纳米粒子的浓度小于50ppm，过少的氧化钨纳米粒子对于反应溶液的加热效果不佳，而不能达成缩短升温时间。反之，倘若氧化钨纳米粒子的浓度大于1000ppm，过多的氧化钨纳米粒子大幅降低可见光区的透过率，而干扰荧光剂放射荧光，故无法定量核酸片段的含量。进一步，氧化钨纳米粒子的浓度较佳可为50ppm至500ppm，更佳可为300ppm至350ppm。
于操作110后，聚合酶链反应的方法100对反应溶液进行聚合酶链反应，以增幅核酸片段的部分序列，如操作120所示。前述的聚合酶链反应是使用电磁波照射反应溶液，以进行变性步骤及延展步骤。于变性步骤及延展步骤中，电磁波照射反应溶液中的氧化钨纳米粒子，其吸收电磁波后产生热能，此热能加热反应溶液。
电磁波的波长为400nm至2000nm，相当于电磁波的频率为150THz至750THz。换句话说，电磁波包含可见光、近红外线及远红外线。由于电磁波被氧化钨纳米粒子所吸收，所以氧化钨纳米粒子的吸收特性决定电磁波的波长。如前所述，氧化钨纳米粒子的吸收特性可受形状及掺杂物的影响。举例而言，在一些实施例中，电磁波的波长可为780nm至2000nm，以使吸收近红外线的氧化钨纳米棒产生热能，进而加热反应溶液。相同地，吸收可见光的氧化钨纳米粒子亦可于可见光照射下，加热反应溶液。
此外，电磁波的功率会影响氧化钨纳米粒子对于反应溶液的加热速率，其中功率愈大，加热速率愈大，且反之则相反。在一些实施例中，电磁波的功率可为0.1W至2W。当电磁波的功率为前述的范围时，具有此功率的电磁波能够使氧化钨纳米粒子快速产生足够的热能，以使平均加热速率达到变性步骤及延展步骤的温度变化的需求，从而缩短升温时间，且通过精准控制反应溶液的升温，而提高聚合酶的聚合效率。
在一些实施例中，变性步骤及延展步骤的平均加热速率为2℃/sec至23℃/sec。当平均加热速率为前述的范围时，其可对应于变性步骤的快速升温的需求，以缩短升温时间，且精准控制延展步骤的升温，而提高聚合酶的聚合效率，故提升聚合酶链反应的效率。在一些具体例中，变性步骤及延展步骤的升温时间不大于15秒。较佳地，变性步骤及延展步骤的升温时间可分别为5秒至15秒及1秒至4秒。当升温时间为前述的范围时，可更缩短升温时间，从而提升 聚合酶链反应的效率。较佳地，变性步骤及延展步骤的升温时间不大于10秒。
以下利用实施例以说明本发明的应用，然其并非用以限定本发明，任何熟习此技艺者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰。
纳米粒子的制造
实施例1
实施例1的氧化钨纳米棒是利用水热法制得，其中混合1mg至300mg的钨的卤化物、1mL至20mL的0.1M至1.5M的盐酸及1mL至20mL的油墨[包含聚乙二醇(PEG)、乙二醇(EG)、聚乙烯吡咯烷酮(PVP)及甘油的水溶液]后获得混合物，再以0.01mL至1mL的N2H4进行还原，以获得实施例1的氧化钨纳米棒。然后，以后述的评价方式进行评价。
实施例2至3及比较例1
实施例2至3的经掺杂的氧化钨纳米棒及比较例1的氧化铁纳米粒子均以与实施例1相似的方法制造。不同的是，实施例2至3减少钨的卤化物，且实施例2添加0.1mg至80mg的铂的卤化物，而实施例3添加0.1mg至80mg的钯的卤化物。此外，比较例1使用铁的卤化物取代钨的卤化物。前述实施例1至3及比较例1的评价结果如下表1所示。
评价方式
1.氧化钨纳米粒子的透过率的试验
透过率的试验是使用光谱仪量测氧化钨纳米粒子的光谱，光谱包含可见光区及红外光区，其中试验条件为具有公知常识者所惯用者。
2.氧化钨纳米粒子的尺寸的试验
尺寸的试验是使用电子显微镜量测氧化钨纳米粒子的尺寸，如粒径，其中试验条件为具有公知常识者所惯用者。当氧化钨纳米粒子为氧化钨纳米棒时，尺寸包含平均长度及平均直径，并求得平均长度与平均直径的比值(亦称作纵横比，且以四舍五入计)。
3.氧化钨纳米粒子的元素含量的试验
元素含量的试验是使用化学元素分析仪进行，并通过测得的元素含量计算出氧化钨纳米粒子的组成通式，其中试验条件为具有公知常识者所惯用者。
4.不同纳米粒子的加热速率的比较试验
加热速率的比较试验是分别添加实施例1至3及比较例1所制得的纳米粒 子至体积为20μL的反应试剂，再以波长为808nm且功率为1W的电磁波照射含有纳米粒子的此些反应试剂，以进行加热，并纪录此些反应试剂的温度变化曲线，如图2及图3所示，并以由28℃升温至60℃的温度差值(即32℃)除以所需的时间区间求得平均加热速率，结果显示于表1。
5.干扰的试验
5.1增幅反应的干扰试验
增幅反应的干扰试验是以与后续应用例1相似及相同的聚合酶链反应进行，其中相同者为使用氧化钨纳米棒且照射电磁波，而相似者的差异处在于，未使用氧化钨纳米棒或未照射电磁波，以确认氧化钨纳米棒可通过照射电磁波来加热反应溶液，且经加热的反应溶液的升温情况可使聚合酶进行聚合酶链反应。此外，亦可确认此些氧化钨纳米棒不影响聚合酶进行核酸增幅反应。
详述之，使用浓度分别为660ppm及330ppm的氧化钨纳米棒，并以程控波长为8.08×10 2nm的电磁波照射反应溶液的条件，以对反应溶液进行聚合酶链反应。开启功率为1W的电磁波，从25℃以5℃/sec至10℃/sec的平均加热速率加热反应溶液至95℃(升温时间为7至15秒)；降低电磁波的功率后，持温1秒至30秒，进行变性步骤；关闭电磁波并开启风扇，以降温至56℃，持温1秒至30秒，进行黏合步骤；然后开启功率为1W的电磁波，以5℃/sec至10℃/sec的平均加热速率加热反应溶液至72℃(升温时间为1.6至3.2秒)，持续1秒至30秒，以进行延展步骤。依序进行前述的三个步骤称作一个循环，且重复循环40次后终止，即完成增幅核酸片段的一部分序列，从而获得核酸产物，再对获得的核酸产物进行胶电泳，并以电泳的结果确认通过电磁波照射氧化钨纳米棒可控制反应溶液的升温情况，以使聚合酶进行聚合酶链反应，且此些氧化钨纳米棒可不影响聚合酶进行核酸增幅反应，其结果如图4所示。
5.2不同纳米粒子的背景干扰的比较试验
背景干扰的比较试验是分别添加实施例1及比较例1所制得的纳米粒子至体积为20μL的反应试剂中，以波长为488nm的激发光照射含有纳米粒子的反应试剂，并量测其所放射的波长为520nm的荧光的强度，结果如表1及图5所示，其中使用未添加纳米粒子的反应试剂测得的荧光强度作为100％的基准，以求得添加纳米粒子的反应试剂的荧光强度。
表1
Figure PCTCN2022074585-appb-000001
备注：「N/A」表示未进行此项试验。
聚合酶链反应的方法
应用例1
应用例1是使用前述的实施例1所制得的氧化钨纳米棒进行聚合酶链反应，其中混合氧化钨纳米棒、核酸片段及反应试剂，以形成体积为20μL的反应溶液。详述之，反应试剂包含聚合酶、引子对、核苷酸碱基、荧光剂及缓冲溶液，其中聚合酶、核苷酸碱基、荧光剂及缓冲溶液是使用由罗氏公司制造的试剂(商品名为KAPA SYBR FAST qPCR套件)，且依据此商品使用说明书添加反应试剂。再者，以基因转殖的大肠杆菌的核酸萃取物为例子，核酸片段的浓度为0.1μg/mL，其添加体积为1μL，且引子对的正向引子为如SEQ ID NO:1所示的序列，反向引子为如SEQ ID NO:2所示的序列。
然后，使用实时检测聚合酶链反应机器进行聚合酶链反应。使用与前述的增幅反应的干扰试验相同的反应温度及时间的条件，重复至预定的循环次数 (如图6所示)后终止，即完成增幅核酸片段的一部分序列，从而获得核酸产物。每次循环结束即检测所扩增核酸产物的荧光信号，并计算增加的信号差值ΔRn，所得到的荧光信号增幅曲线如图6所示。此外，需说明的是，应用例1的聚合酶链反应的降温步骤是使用与习知的PCR仪器相同的风扇进行降温，且其条件可与习知的PCR仪器相同，故不再赘述。
比较应用例1
比较应用例1是以与应用例1相似的方法进行。不同的是，比较应用例1使用氧化铁纳米粒子取代氧化钨纳米棒，其中增幅曲线如图7所示。
根据表1的透过率与平均加热速率的结果，以及图2至图3，实施例1至3的氧化钨纳米材料于800nm至1100nm的透过率相较于比较例1的氧化铁纳米粒子更低，故实施例1至3的纳米材料可吸收波长为800nm至1100nm的电磁波(例如红外光)，并转换成热能，以加热反应溶液，从而提高平均加热速率。据此，实施例1至3的纳米材料能够更快速地加热反应溶液，以利于更有效地控制变性步骤及延展步骤的反应溶液的温度，如缩短升温的时间，从而提升聚合酶链反应的效率。
其次，参照表1的元素含量的结果，实施例2的掺杂铂的氧化钨纳米棒及实施例3的掺杂钯的氧化钨纳米棒皆可通过掺杂的过渡金属提高氧空缺，而增加自由电子，此自由电子可提升纳米材料于红外光区的吸收度(也就是降低红外光区的透过率)，故提升纳米材的光热转换率。
进一步，根据表1的尺寸及平均加热速率的结果，以及图2至图3，相较于球状的比较例1的氧化铁纳米粒子，实施例1至3的纳米材料均为棒状，其具有纵横比。通过5至25的纵横比，纳米棒可更提升纳米材的光热转换率，故可更有效地控制变性步骤及延展步骤的反应溶液的温度。
此外，根据图4，「M」代表标准品，「C」代表未使用纳米粒子的聚合酶链反应方法所获得的核酸产物，「A1」代表使用660ppm的氧化钨纳米棒且照射808nm的电磁波的聚合酶链反应方法所获得的核酸产物，「A2」代表使用330ppm的氧化钨纳米棒且照射808nm的电磁波的聚合酶链反应方法所获得的核酸产物，「B1」代表使用660ppm的氧化钨纳米棒且未照射电磁波的聚合酶链反应方法所获得的核酸产物，「B2」代表使用330ppm的氧化钨纳米棒且未照射电磁波的聚合酶链反应方法所获得的核酸产物。
详述之，使用浓度为660ppm及330ppm的实施例1的氧化钨纳米棒进行聚合酶链反应方法，由其所获得的核酸产物与未使用纳米材进行的方法所获得的核酸产物相同，即二者的碱基对长度均为100bp。其次，经由电泳胶片影像的分析，使用330ppm的氧化钨纳米棒所获得的核酸产物的亮度与使用660ppm的氧化钨纳米棒所获得的核酸产物的亮度一致。据此，添加的氧化钨纳米棒不会影响聚合酶进行核酸增幅反应。
进一步，根据表1的透过率与背景干扰的结果，以及图5，实施例1至3的氧化钨纳米材料于450nm至550nm的透过率相较于比较例1的氧化铁纳米粒子更高，故实施例1至3的纳米材料对于波长为450nm至550nm的电磁波(例如可见光)的吸收较低，以降低对于反应试剂中的荧光剂的激发光及放射光的吸收，从而降低背景干扰。进一步，根据背景干扰的结果，经计算后(以四舍五入计)，比较例1的氧化铁纳米粒子产生的背景干扰为实施例1的氧化钨纳米棒产生的背景干扰的7至28倍。
接续，根据图6及图7，「ΔRn」代表含有纳米材料的反应溶液于核酸增幅反应前与后的荧光强度的差值。使用82.5ppm至660ppm的实施例1的氧化钨纳米棒均可进行PCR反应。然而，必需低至125ppm的比较例1的氧化铁纳米粒子才能达到与660ppm氧化钨相近的ΔRn荧光强度差值(约25000)。据此，氧化钨纳米棒具有较佳的透光率，故可降低氧化钨纳米棒对检测核酸产物荧光的干扰。进一步，当氧化钨纳米棒的浓度为660ppm时，最大的ΔRn为25000，然而当氧化铁纳米粒子的浓度为125ppm时，其最大的ΔRn为24000。若视二者ΔRn相当，二者的浓度的比值为5.28倍(660/125＝5.68)，此亦可看出，使用实施例1的氧化钨纳米棒的方法具有较低的干扰。
综上所述，本发明的聚合酶链反应的方法是利用特定波长的电磁波照射于红外光区具有特定的透过率的氧化钨纳米粒子，以使低浓度的氧化钨纳米粒子能够快速加热反应溶液，从而缩短升温时间，且提升聚合酶链反应的效率。
虽然本发明已以实施方式揭露如上，然其并非用以限定本发明，在本发明所属技术领域中任何具有公知常识者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视所附的权利要求所界定的范围为准。

Claims (10)

  1. 一种聚合酶链反应的方法，其特征在于，包含：
    混合多个氧化钨纳米粒子、一核酸片段及一反应试剂，以形成一反应溶液，其中混合后该些氧化钨纳米粒子的一浓度为50ppm至1000ppm，且该些氧化钨纳米粒子在大于780nm且不大于2000nm的一第一波长范围的一第一透过率为小于98％；以及
    对该反应溶液进行该聚合酶链反应，以增幅该核酸片段的一部分序列，其中该聚合酶链反应使用一电磁波照射该反应溶液，以进行一变性步骤及一延展步骤，且该电磁波的一波长为400nm至2000nm。
  2. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该些氧化钨纳米粒子于380nm至780nm的一第二波长范围的一第二透过率为大于40％。
  3. 根据权利要求2所述的聚合酶链反应的方法，其特征在于，该些氧化钨纳米粒子于400nm至600nm的一第三波长范围的一第三透过率为不小于50％。
  4. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该些氧化钨纳米粒子在大于780nm且不大于1100nm的一第四波长范围的一第四透过率为不大于95％。
  5. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该些氧化钨纳米粒子包含多个氧化钨纳米棒，该些氧化钨纳米棒具有一平均长度及一平均直径，且该平均长度与该平均直径的一比值为5至25。
  6. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该些氧化钨纳米粒子具有一通式(I)W aO b，于该通式(I)中，W代表钨，O代表氧，且b与a的一比值(b/a)为1至3。
  7. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该些氧化钨纳米粒子具有一通式(II)W aO bM c，于该通式(II)中，W代表钨，O代表氧，M代表镍、钯、铂或其组合的金属，且b与a及c的总合的一比值[b/(a+c)]为1至3。
  8. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该变性步骤及该延展步骤的一平均加热速率为2℃/sec至23℃/sec。
  9. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该变性步骤及该延展步骤的一升温时间不大于15秒。
  10. 根据权利要求1所述的聚合酶链反应的方法，其特征在于，该电磁波的一功率为0.1W至2W。
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