TW200538548A - The method of biological and/or chemical reaction to fast reach thermally-equilibrium - Google Patents

Abstract

This invention relates to the method of biological and/or chemical reaction to fast reach thermal equilibrium. This method is characterized in that gold nanoparticles are added in the reaction solution during reaction proceeding, wherein the nanoparticle uses gold metal as core to covalently bond the weak acid ionic groups and can be dissolved in the aqueous solution. This invention also relates to the preparation method of nanoparticles which are used in the biological and/or chemical reaction solution to increase the rate of thermal equilibrium, wherein the nanoparticle uses gold metal as core to covalently bond the weak acid ionic groups and can be dissolved in the aqueous solution. The method includes the following steps: (a) the gold salt is combined with the weak acid ionic groups to carry out the nucleation; and (b) removed the unreacted weak acid ionic groups in step (a) to obtain the neutral gold nanoparticles.

Description

200538548 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for biological and / or chemical reactions, in detail °, relates to a method for quickly achieving thermal equilibrium in a biological and / or chemical reaction solution. [Prior art] "Temperature" is a biological reaction or a chemical reaction. It can be used to determine whether a reaction should take place. In biological reactions, temperature is particularly important. Biological reactions involve biological macromolecules (such as nucleic acids and proteins). These biological macromolecules are very sensitive to temperature. Only certain temperature conditions can enable them to maintain a specific molecular configuration, and they can also perform a transposed reaction. . Among various biological and / or chemical reactions, the polymerase chain reaction has particularly strict requirements on temperature and degree. At present, the reaction time of the polymer, enzyme chain reaction and system heat transfer mostly use silicon or aluminum blocks as the heat transfer interface, such as the ABI PRISM® 7000 Sequence manufactured by Applied Biosystems.com.

Detection Systems (its stratospheric reaction is! 0c / sec), which changes the temperature of the reaction solution by controlling the temperature of the silicon or aluminum block and passing through the container containing the reaction solution, but this method takes a long time, and for It is difficult to control the true temperature inside a large volume of reaction solution. Furthermore, for real-time polymerase chain reaction, the external heat transfer effect of the tube wall system of the reaction tube and the equilibrium time reached by convection in the tube are not the same. Poor sensitivity. At present, the above disadvantages are improved in two aspects. One is the reaction system.

O: \ 91 \ 91410.DOC 200538548 improvement, the other is for the improvement of the reaction solution formula. In terms of system improvement, there are the following methods: (1) Changing the volume of the reaction vessel to accelerate heat conduction and convection :: rate, but its effect is limited; ⑺ rapid hot air exchange and the use of Shi Yangguan to improve the heat exchange rate The method attempts to shorten the reaction time, such as the LightCyclerTM instrument developed by Roche, but it still cannot meet the increasingly strict requirements for the temperature equilibrium rate. In terms of improving the reaction solution formula, there are the following methods: (1) The genetic modification method is used to change the nucleic acid synthesis efficiency and reaction sensitivity of the nucleic acid synthetase. The disadvantage of this method is that the process takes two to three years and the cost is high, so this method is not economical. Take Roche ’s Fastst ^ t Taq ™ as an example. After genetic modification, the price has tripled, and its research and development has been conducted for five years. (2) The purity of various drugs is improved, taking nucleic acid drugs as an example. From 95% to 99%, the price is doubled, but the increase in efficiency is not obvious; (3) Add other 'enzymes or proteins that do not perform nucleic acid synthesis to the polymerase chain reaction to increase reaction efficiency and other specificities Uses, such as: Hot-start to reduce other undesired DNA sequences, but the cost of each operation will increase by two to three times, and the development time and the time of genetic improvement are not shortened. (4) Improve the composition of the reaction buffer, increase the stability of the nucleic acid polymerase and reduce the error rate, but its formula is too complicated and the efficiency is not good. The above-mentioned method for improving the heat balance rate is too slow and has the disadvantages of ineffectiveness and / or high cost. [Summary of the Invention] The present invention provides a biological and / or chemical reaction method that achieves thermal equilibrium quickly and efficiently with low cost. O: \ 91 \ 914lO.DOC -6- 200538548 The purpose of the Ben Maoming system is to provide a biological and / or chemical reaction method that quickly reaches thermal equilibrium, which is characterized in that gold nano particles are added to the reaction during the reaction. In the liquid, the gold nanoparticle is centered on gold metal, has a bond ^ weakly acidic ionic group ', and has the characteristics of being soluble in an aqueous solution. Another objective of this month is to provide a polymerase chain reaction method for rapidly reaching thermal equilibrium, which is characterized in that gold nano particles are added to the reaction solution during the reaction, wherein the gold nano particles are made of gold metal. It is a center and covalently bonds a weakly acidic ionic group, and has the characteristics of being soluble in an aqueous solution. Yet another object of the present invention is to provide a reagent for rapid thermal equilibrium rate in biological and / or chemical reaction solution, which comprises a nanometer particle hand, wherein the gold particle is centered on gold metal and covalently bonded. Weakly acidic ionic group 'has the property of being soluble in aqueous solution. Another object of the present invention is to provide a method for preparing gold nano particles for improving the rate of thermal equilibrium in a biological and / or chemical reaction solution, wherein. The helium nanoparticle is centered on gold metal and covalently bonds weakly acidic ionic groups and has the property of dissolving in aqueous solution. The method includes the following steps: (a) A gold salt and a weakly acidic ionic group are subjected to a nucleation reaction. ; And (b) removing unreacted weakly acidic ionic groups in step (a) to obtain neutral gold nanoparticle. [Embodiment] The present invention provides a biological and / or chemical reaction method for quickly reaching thermal equilibrium, characterized in that gold nano particles are added to the reaction solution during the reaction, wherein the gold nano particles are made of gold metal as The center is covalently bonded to a weakly acidic ionic group and has the characteristics of being soluble in an aqueous solution.

O: \ 9I \ 91410.DOC 200538548 As used herein, the term "biological response" refers to a reaction occurring in a living body or a reaction occurring in vitro from a molecule derived from a living body. Molecules involved in biological reactions include, but are not limited to, macromolecules of nucleic acids, peptides, proteins and sugars. The biological reaction suitable for the present invention preferably includes (but is not limited to) a polymerase chain reaction, an enzyme digestion reaction, a protein hybrid reaction, and a nucleic acid-protein hybrid reaction; more preferably, a polymerase chain reaction; and most preferably, instant polymerization Enzyme chain reaction. The term "chemical reaction" as used herein refers to the reaction of chemical molecules. The chemical reaction suitable for the present invention is preferably a reaction sensitive to temperature changes. \ The term "gold nano particles" used in this article refers to nano-sized particles formed by gold atoms, which are specifically called gold metal as the center and covalently bond weakly acidic ionic groups, and have The characteristics of gold nano particles which are soluble in aqueous solution, wherein the weakly acidic ionic group is preferably citrate or tannin; more preferably citrate. On the other hand, preferably, the particle diameter of the gold nanoparticle is from 1 nanometer to 100 nanometers; more preferably, the particle diameter of the gold nanoparticle is from 1 nanometer to 20 nanometers. In a specific embodiment of the present invention, the gold nano particles are present in a colloidal solution. In order to make the colloid ridge fluid containing gold nano particles can be widely used in various reactions without affecting the acid of the reaction Base number 'The colloidal solution is preferably neutral. The gold nano particles according to the present invention are free gold nano particles, and are suitable for a variety of different reactions. In order to effectively improve the efficiency of the reaction solution to achieve thermal equilibrium, the concentration of gold nanoparticles in the reaction solution according to the present invention is preferably from 1 (). 5_ to 1 () _ 8 blessing; more preferably

^ I ^ UIO.DOC 200538548 Ground, the concentration of the gold nanoparticle in the reaction solution is from 10.6 to 10.7 butterfly. -Although metal nano particles have good heat conduction function, but because metal particles are not soluble in water and they will polymerize, they are usually added to lubricating oil and cannot be used in biological and / or chemical reactions. . Conventionally, chemically synthesized knee-coated metal particles with a protective agent are soluble in water and can avoid polymerization, but in the experiments of iron colloidal particles and silver colloidal particles, they have not been successful in promoting heat transfer. The effect of the effect, and for biological reactions that require an enzyme reaction, such as a polymerase chain reaction, in addition to the pure heat transfer effect, factors of enzyme kinetics need to be considered, so only the nanoparticle according to the present invention can not only It is soluble in aqueous solution and does not produce ▲ poly ^ phenomenon, it has been confirmed that when added to the reaction solution, it can promote heat transfer. Because of the inherent heat transfer efficiency and physical properties of energy polymerization, and since the nano-particles are soluble in aqueous solution, they are not prone to polymerization, so they are biological and / or chemical reactions. In this way, the thermal equilibrium efficiency of the reaction liquid can be greatly increased. Taking polymerase chain reaction as an example, the reaction efficiency and sensitivity have been greatly improved, so that the use of general polymerases such as ㈣DNA polymerase can replace the high-performance and high-cost bandit polymerase and allow immediate polymerase chain reaction. . With its high surface area and good heat transfer effect, gold nano particles can make the reaction solution reach thermal equilibrium in tens of femtoseconds (10.100ps). It was found that the inertia was 10-8mM Gold particles with a diameter of 10 to 15 nanometers can increase the sensitivity of the polymerase chain reaction by up to 10,000 times, and the replication reaction is almost a thousand times earlier. This concentration is much larger than the volume used in general literature on nanometer heat transfer research. 3 to 5% M (; Zmfast d electron dynamics in semiconductor and metal colloidal nanoparticlesreffects

O: \ 91 \ 9I410.DOC 200538548 of size and surface, ', Acc.Chem.Res ”V〇1.30, pp.423-429, 1997 and S.Link3C.Burda5Z.L.Wang3and MAEl-Sayed, " Electron dynamics in gold and gold-silver alloy nanoparticles: the influence of a nonequilibrium electron distribution and the size dependence of the electron-phonon relaxation, ff J. ChemJPhys, Vol.lll, ρ · 1255-1264, 1999 ·), in addition, Adding nano-particles according to the present invention can cause an instant polymerase chain reaction of ultra-low concentration DNA templates of 1 replica per unit. The same formula and the same parameters without adding nano-particles require a template of 106-101G replicas. A reaction occurs, so the present invention has the advantages of reducing the cost of reagents, shortening the reaction time, improving yield and detection sensitivity, etc. 1 The present invention also provides a polymerase chain reaction method for quickly reaching thermal equilibrium, which is characterized in that Gold nano particles are in the reaction solution, wherein the gold nano particles are centered on the genus Aurus and are covalently bonded to weakly acidic and ionic groups, and are soluble in water. The invention further provides a reagent for rapidly increasing the thermal equilibrium rate in biological and / or chemical reaction solution, which comprises gold nano particles, wherein the gold nano particles are centered on gold metal and covalently bond weakly acidic. The ionic group has the characteristics of dissolving in an aqueous solution. The present invention also provides a method for preparing gold nano particles for improving the thermal equilibrium rate in a biological and / or chemical reaction solution, wherein the gold nano particles are made of gold metal. It is a center and covalently bonds a weakly acidic ionic group and has the characteristics of dissolving in an aqueous solution. The method includes the following steps: (a) a nucleation reaction with a weakly acidic ionic group with a gold salt; and (b) a removal step ( a) Unreacted weakly acidic ionic groups to prepare neutral gold O: \ 91 \ 91410.DOC -10- 200538548 nano particles. According to the present invention, wherein step 0) uses a gold salt and a weakly acidic ionic group The method for the nucleation reaction of the cluster is well known to those skilled in the art. Gold has the ability to synthesize colloids, and uses the redox method to produce water-soluble nano particles. Among them, the weakly acidic The ionic group is citrate or dansate, more preferably citrate. In a specific embodiment of the present invention, step (a) is to synthesize acidic hydrophilic gold nanoparticle with gold salt and citrate. This law was published by Hayat (M · A · Hayat, " c〇11〇idal

Gold ’Principle, Methods and Applications · 1). Among them, the gold salt is preferably HAuCU; on the other hand, the citrate is trisodium citrate (Na ^ I ^ O7 · 2H2〇). This method uses a nucleation reaction (Nucleation) to reduce gold ions to gold atoms as seed crystals, followed by a growth reaction. When the trivalent and v gold ions are used up, the gold particles will stop dysprosium. This method can change the ratio of gold salt to lemon and citrate to formulate gold nano particles with different particle sizes. For example, gold salt The ratio of gold salt to citrate (gold salt · citrate) is 5 to prepare gold nano particles with a particle size of about 10 to 5 nanometers. According to the step 'b' of the present invention, the unreacted weakly acidic ion groups in step (a) are removed to obtain neutral gold nanoparticle. Most of the conventional methods for preparing nano-particles show acidic reactions due to the use of surfactants, and the gold preparation environment is also acidic. Because there is no step to remove weakly acidic ion groups, it is not suitable for neutral or The reaction in alkaline environment, and the reagent for synthesizing gold nano particles will also interfere with the progress of the reaction. For example, the citrate system is a strong clamping agent. For metal ions in the reaction solution, such as magnesium, a clamping reaction occurs. In the method of the present invention, step (b) is further processing the gold nano particles to make O: \ 91 \ 91410.DOC -11-200538548 applicable to biological and / or chemical reactions. In one embodiment of the present invention, high-speed centrifugation is used to make the gold nanoparticles in step (a) into neutral gold nanoparticles. The conditions for high-speed centrifugation are well known to those skilled in the art, such as centrifugation at 10,000 G for 7 minutes. Since gold nano particles with a particle size of less than about 15 nanometers cannot be completely recovered by high-speed centrifugation, the method of the present invention may further include a vacuum extraction step after high-speed centrifugation to concentrate the gold nano particles to obtain the desired Concentration and pH. The following examples illustrate the invention in detail, but it does not mean that the invention is limited only to what is disclosed in these examples. Example 1 Preparation of Nanoparticles1 HAuC14 and Na3C6H507 · 2H20 were prepared by HAuCl4 according to the method of Hayat:

Na3C6Hs〇7 · 2H2O was formulated at a ratio of 5: 1 to prepare hydrophilic nano particles. 4 The particle size of the nano-particles is based on the tunneling electron microscope. The results are shown in Figure i. It can be seen that the diameter of the nano-particles is ΐ3 · 7 ± 0 · 8 nanometers. UV-vis spectrum) detection of nano-particles has a significant absorption peak at a wavelength of 52 nanometers (see Figure 2), and it can also be confirmed that the particle size of the nano-particles is about 13 nanometers. The hydrophilic gold nanoparticle was centrifuged at 10,000 G for 7 minutes, and then concentrated under vacuum at a low temperature to a concentration of 10-4, 10.5, 10-6 41〇-7mM. Example 2: Real-time polymerase chain reaction with nano-particles This example uses the Real-Tmie PCR system (LightCydor) produced by Roche to evaluate the efficiency of nano-particles in promoting thermal equilibrium. The capillary volume of a single experiment is about 20 to 25㈤ 丨. A total of 32 groups of experiments can be used at one time. The composition and ratio of the formula is 2 ml of 〇χO: \ 9I \ 91410.DOC -12- 200538548 PCR buffer , Two primers, 200 M dNTPs (dATP, dCTP, dGTP, and dTTP), 0.5 g / LBSA, 1: 30000 SYBR® Green I (Roche®, Germany), one unit of Supertherm Taq DNA polymerase, 2 L DNA template With 2 mL of the nanogold solution prepared from Example 1. The fragments to be amplified are fluorescein gene (pEGFP-Nl, CLONTECH®) and cDNA of bladder cancer cell gene fragments (Bcl-2 and Nineteen Kda Interacting Protein-3, referred to as BNIP3 cDNA). The two primers used for the fluorescent protein gene are EGFP-NS: 5 ^ TGC AGT GCT TCA GCC GCT AC-3 '(sequence identification number 1) and EGFP-N-AS: 5, -CAG CTC GAT GCG GTT CAC CA -3 * (Sequence Identification Editor 2), which can copy a 173-bp long fragment of the fluorescent protein gene (pEGFP-Nl, CLONTECH®). The GC value of this gene fragment is as high as 60%. In the experiment, Template doses per unit, 102 to 109 copies; for BNIP3 cD, NA use primer pair 5, -CGC AGC TGA AGC ACA TCC-3f (sequence 歹 J identification number 3) and 5, -AAC GAA CCA AGT TAG ACT CCA-3 '(sequence identification number 3), which can amplify a 238bp fragment length, and the GC value is 50.48%. In this example, the fluorescent dye SYBR® Green I is used as the quantitative detection component. At the same time, the F1 mode in the instrument detection mode is selected to detect the absorption wavelength of the light source at 520 nm. The temperature layer setting part is first heated and separated. (denaturation) 95 ° C time is 15 seconds, introduction recombination (annealation) 58 ° C time is 15 seconds and primer extension effect (extention) 72 ° C time is 30 seconds, and the fluorescence quantitative observation temperature layer is 84 ° C The time is 1 second, which is the better experimental parameter used in the general real-time polymerase chain reaction experiment of the experimental formula used. O: \ 91 \ 91410.DOC -13- 200538548 Reduce the reaction time one by one to discuss this experiment. Based on this data as the baseline correlation response results. In order to understand the phenomenon of Chennai particles in the polymerase chain reaction, the particle size of Chennai greens was analyzed by a time light meter and an electron microscope in this embodiment. The results are shown in Figs. 3 and 4. It was found in the analysis that the particle size of the nanometer particles did not change or aggregate. In this example, the mono-temporal layer is also analyzed. As a result, it is found that in addition to the effect of the priming effect of the thermo-layer in the enzyme reaction, the two thermo-layers that are purely related to energy balance (plus # Refining) also has a strengthening effect, which proves that the heat transfer effect affects the polymerase chain reaction. The experimental results of real-time polymerase chain reaction with different concentrations of gold nanoparticles are shown in Fig. 5. In this example, the concentration of gold nano particles reaches 1 (). 8 capsules (that is, 10 mM gold nano particles are mixed in a volume ratio of 1:10) to promote the reaction. The volume of 0.3 to 5% used by Mishao's research literature is low, that is, without adding nano-particles, the template squares per unit of the 106 to ιιο replica should be reacted. After adding the nano-particles, Only one copy of the DNA template per unit is required for the reaction. In this example, the effect of the nanoparticle on the efficiency of the instant polymerase chain reaction was also observed. The cycle time of the nanoparticle with 7.52 × 10-7 mM in different concentrations of the template was 60 seconds, 40 seconds, and 2 seconds. The response in seconds is shown in FIG. 6. It can be seen from FIG. 6 that in the reaction with a standard reaction time of 60 seconds, the unit 1) 1 ^ octa-core version has a concentration of 106 to 109 copies without the addition of gold nanoparticle particles. The product can be detected. Output, and the formulation of adding nano particles can detect the product output in the experiment of the unit DNA template concentration of 102 to 109 copies;

O: \ 91 \ 9U10.DOC -14- 200538548 In the reaction in which the quasi-reaction time is shortened to 2/3 (40 seconds), the formulation of adding nano-particles to the unit DNA template concentration is between 103 and 109 copies. The product output can be detected in the experiment. In the reaction with a standard reaction time shortened to 1/3 (40 seconds), the unit DNA template concentration can be detected in the 109 replica without the addition of gold nanoparticle formula. There is product output, and the formula added with nano particles can detect the product output in the experiment of the unit DNA branch concentration of 107 to 109 copies, which is sufficient to prove that the nano particles according to the present invention are added. The particles can elevate the polymerase chain reaction formula containing Supertherm Taq DNA poiymerase to the range of use of instant polymerase chain reaction. The effect of the reaction of the 7.52x1 0-7 mM gold nanoparticle particles on the 1.06 complex I template on the ampHfication curve was further observed. The results are shown in FIG. 7 and FIG. 8. Since the addition of nano particles can make the threshold cycle (CT) earlier if cycle, according to the generalized pCR efficiency defined by the LightCycler ™ instrument and instrument operating manual (instrument pr0t〇c〇1) The formula is as follows: 'Slope = -l / logE E is the efficiency of the polymerase chain reaction. According to the comparison of the above formula, it is found that the slope value has decreased from -8 · 566 to -4.818, in other words, the efficiency has doubled (0 · 9〇7 increased to ι · 59), and the ideal value is 2. It can be seen that the addition of gold nanoparticles can significantly improve the reaction efficiency of the polymerase chain reaction. In this example, the effect of different concentration of gold nanoparticle particles on the cycle number (CT value) is also explored. The experiment is performed with a template with a number of 106 copies, and the ct value is measured separately. The results are shown in FIG. 9. 9 It can be seen that when the agronomic degree of the nanoparticle in the reaction solution is 1CT6 mM to 10 · 7 mM, the CT value is greatly reduced. It can be seen that the reaction efficiency is good and the reaction occurs early. Therefore, the concentrated

O: \ 91 \ 91410.DOC -15- 200538548 degrees is preferably 10 · 6 mM to 1 (T7 mM. The above examples are only for illustrating the principle of the present invention and its efficacy ', rather than limiting the present invention. Therefore, Xi Modifications and changes made by those skilled in the art to the above-mentioned embodiments still do not violate the spirit of the present invention. The scope of the rights of the present invention should be listed in the scope of patent applications mentioned later. &Quot; [Schematic description] Figure 1 is The particle size of gold nanocrystals synthesized by the redox method observed under an electron microscope. Figure 2 shows the absorption peaks of gold nanometers synthesized by the redox method measured with a spectrometer. Figure 3 shows a spectrometer Measure the absorption peaks of the particle size of gold nanoparticle before and after participating in the polymerase chain reaction. Figure 4 shows the particle size of the golden rice particle before and after participating in the polymerase and chain reaction with an electron microscope. Figure 5 shows the difference The electrophoresis of the product of gold nanoparticle concentration participating in the real-time polymerase chain reaction. Figure 6 shows the results of the polymerase chain reaction of gold nano particles at different reaction times. A, C and E are the results of the reaction without gold nano particles. B, d & f is plus The reaction of gold nanoparticles; A and B are 60 seconds per cycle, (: and! ^ Are 40 seconds per cycle, and E and F are 20 seconds per cycle. Figure 7 shows the initial, Instant polymerase chain reaction efficiency diagram of DNA template with a concentration of 106 copies per unit. Figure 8 shows the instant polymerase chain reaction efficiency of generalized nano-particles on a DNA template with an initial concentration of 100 copies per unit. Illustration.

O: \ 91 \ 91410.DOC -16- 200538548 Figure 9 is the CT value graph of gold nanoparticle with different concentration when the DNA template is 106 copies per unit.

O: \ 91 \ 91410.DOC -17- 200538548

Sequence Listing < 110 > National Cheng Kung University < 120 > Biological and / or Chemical Reaction Method for Rapid Thermal Equilibrium < 130 > None < 160> 4 < 170> Patentln version 3.2 < 210 > 1 < 211 > 20 < 212 > DNA < 213〉 artificial < 400 > 1 tgcagtgctt cagccgctac < 210 > 2 < 211 > 20 < 212 > DNA < 213 > artificial < 400 > 2 cagctcgatg cggttcacca < 210 > < 211 > 18 < 212 > DNA < 213 > artificial < 400 > 3, cgcagctgaa gcacatcc 18

O: \ 91 \ 91410.DOC 200538548 < 210 > < 211 > < 212 > < 213 > 4 21 DNA artificial < 400 > 4 aacgaaccaa gttagactcc a

O: \ 91 \ 91410.DOC

Claims (1)

200538548 The scope of patent application: 1. A biological and / or chemical reaction method for quickly achieving thermal equilibrium, characterized in that gold nano particles are added to the reaction solution during the reaction, wherein the gold particles are made of gold metal It is a center and covalently bonds a weakly acidic ionic group, and has the characteristics of being soluble in an aqueous solution. 2. The method according to item 1 of the patent application range, wherein the reaction is a reaction sensitive to changes in temperature. 3. The method according to item 2 of the scope of patent application, wherein the reaction is a polymerase chain reaction. 4. The method according to item 3 of the scope of patent application, wherein the reaction is a real-time polymerase chain reaction. ‘5. The method according to item 丨 of the application, wherein the weakly acidic ionic group is citrate or tannin. 6. The method according to claim 5 of the patent application, wherein the weakly acidic ionic group is citrate. 7. The method according to item (1) of the patent application range, wherein the particle size of the gold nanoparticle is from 1 nanometer to 100 nanometers. 8. The method according to item 7 of the patent application, wherein the particle size of the gold nanoparticles is from 1 nm to 40 nm. 9. According to the patent application ## ^^^, the cloth 1 method is used, wherein the aqueous solution in which the nano-particles are dissolved is a colloidal solution. 10. The method according to item 7 of the patent application, wherein the colloidal solution is neutral O: \ 91 \ 91410.DOC 200538548. 11. The method according to item 丨 of the patent application scope, wherein the wave length of the gold nanoparticle in the reaction solution is from 10.5 mM to 1CT8 mM. 1 2. The method according to item 丨 丨 in the scope of patent application, wherein the concentration of the gold nanoparticle in the reaction solution is 10-6 mM to 10-7 mM. Π · —A polymerase chain reaction method for rapidly achieving thermal equilibrium, characterized in that gold nano particles are added to the reaction solution during the reaction, wherein the gold nano particles are centered on gold metal and weakly covalently bonded. It is an acidic ionic group and has the characteristics of being soluble in an aqueous solution. 14. The method according to item 13 of the scope of patent application, wherein the reaction is an instant polymerase chain reaction. -≪ 15. The method according to item 13 of the patent application, wherein the weakly acidic ionic group is citrate or tannin. V Shishen W's method of patent scope item 15, wherein the weakly acidic ionic group is citrate. 17. The method as described in the patent scope, wherein the particle of Chennai seeking particles is from 1 nm to 100 nm. 18. The method according to item 17 of the scope of patent application, wherein the particle diameter of the gold nanoparticle is from 1 nanometer to 40 nanometers. 19. The method according to the scope of the patent application, wherein the aqueous solution in which the gold nanoparticle is dissolved is a colloidal solution. O: \ 9l \ 91410.DOC 200538548 2 0. The method of claim 19 in the scope of patent application, wherein the colloidal solution is neutral. 21. The method according to item 13 of the patent application, wherein the concentration of the gold nanoparticle in the reaction solution is from 1CT5 mM to 10.8 mM. 22. The method according to item 21 of the patent application scope, wherein the concentration of the nano-particles in the reaction solution is 1 (T6 mM to 10.7 mM. 23.-Rapid in biological and / or chemical reaction solutions A reagent for improving the rate of thermal equilibrium 'includes gold nano particles, wherein the gold nano particles are centered on gold metal and covalently bond weakly acidic ion groups, and have the characteristics of dissolving in a permanent solution. 24. Such as applying for a patent The reagent of the scope item 23, wherein the reaction is a reaction sensitive to changes in temperature. 25 · 'As the reagent of the scope of application for the scope item 23, wherein the reaction is a polymerase chain reaction. 26. If the scope of the patent application is 25 Reagent, in which the reaction is an instant polymerase chain reaction. 27. The reagent as described in item 23 of the patent application, wherein the weakly acidic ionic group is a rod nucleic acid or tannin. 28. If applying for a patent The 27th item is a lacking reagent, wherein the weakly acidic ionic group is citrate. 29. The reagent according to the 23rd item of the patent application, wherein the particle of the gold nanoparticle O: \ 91 \ 91410.DOC 200538548 is From 1 nm to 100 nm 30. The reagent according to item 29 of the patent application, wherein the particle size of the gold nanoparticle is controlled from 1 nanometer to 40 nanometers. 3 1. The reagent according to item 23 of the patent application, wherein the dissolved The aqueous solution of gold nanoparticles is in a colloidal solution. 32. The reagent according to item 31 of the patent application, wherein the colloidal solution is neutral. 33. The reagent of item 19 in the patent application, wherein the gold nanoparticles The concentration in the reaction solution is from 1 (T5 mM to 10-8 mM., 34. The reagent of the range 27, wherein the degree of the gold nanoparticle in the reaction solution is 1 (Γ6 mM to 10.7 mM 35. A method for improving the thermal equilibrium rate of nano particles in biological and / or chemical reaction liquids', wherein the gold particles are covalently bonded to weakly acidic ions with gold metal as the center Group, and has the characteristics of being soluble in aqueous solution, the method includes the following steps: (a) ionic group undergoes a nucleation reaction; and (b) unreacted weakly acidic ionic group to obtain neutral gold nanoparticle. 36. The method of item 35, wherein the gold salt is HAuCl4. 37. Method of item 35 Method, wherein the weakly acidic ion group is citrate or tanninate. 3 8. The method according to item 37, wherein the weakly acidic ion group is lemon O: \ 91 \ 91410.DOC 200538548 acid group 39. The method according to item 38, wherein the citrate is trisodium citrate (Na3C6H507 · 2H2〇). 40. The method according to item 29, wherein step (b) is performed by high-speed centrifugation to remove step (a). Unreacted weakly acidic ionic group. 41. The method of scope item 40, wherein step (b) further comprises a vacuum extraction step to concentrate the gold nanoparticle. O: \ 91 \ 91410.DOC