CN112798141A - Measuring method for surface temperature of gold nanorod, heat transmission device constructed by using measuring method and application - Google Patents

Measuring method for surface temperature of gold nanorod, heat transmission device constructed by using measuring method and application Download PDF

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CN112798141A
CN112798141A CN202011584032.2A CN202011584032A CN112798141A CN 112798141 A CN112798141 A CN 112798141A CN 202011584032 A CN202011584032 A CN 202011584032A CN 112798141 A CN112798141 A CN 112798141A
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游民黎
徐峰
佟昊阳
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Suzhou Dianan Biotechnology Co ltd
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Abstract

The invention provides a measuring method of surface temperature of a gold nanorod, a heat transmission device constructed by using the measuring method and application of the heat transmission device. The measuring method comprises the following steps: connecting a DNA probe to the surface of a gold nanorod through a gold-sulfur bond, dispersing the DNA probe in an alkaline buffer solution, and heating by using laser; the DNA probe is released from the surface of the gold nanorod and emits fluorescence, the fluorescence intensity of the DNA probe is detected, and the surface temperature of the gold nanorod is calculated according to the fluorescence intensity. The invention measures the nanometer local temperature around the gold nanometer particles by utilizing the instability of gold-sulfur bonds, constructs a heat transmission device of the surface temperature field of the gold nanometer rods, and can design the length of a spacer region and a reaction region of a DNA probe and perform nucleic acid amplification by the device; the method can obviously reduce the volume of a PCR reaction system, greatly improve the temperature rise and fall rate in the nucleic acid amplification process and reduce the time required by the amplification reaction.

Description

Measuring method for surface temperature of gold nanorod, heat transmission device constructed by using measuring method and application
Technical Field
The invention belongs to the field of nucleic acid amplification and detection, and particularly relates to a gold nanorod surface temperature measuring method, a heat transfer device constructed by using the same and application of the heat transfer device, in particular to a gold nanorod surface temperature measuring method, a heat transfer device of a gold nanorod surface temperature field and a nucleic acid amplification method based on nano local heating.
Background
Nucleic-acid amplification tests (NAATs) have been widely used in the biomedical field, and have become an indispensable test method for diagnosing cancer, viral and bacterial infections, food safety testing, and environmental monitoring. In the field of nucleic acid detection, Polymerase Chain Reaction (PCR) has become the gold standard for NAATs due to its high sensitivity and specificity, and convenient operation.
The PCR technology needs thermal cycling between different temperatures, so if the PCR reaction is instrumented, a heating part of the instrument needs to have a faster temperature rise and fall rate, and a traditional PCR instrument usually utilizes a metal block for heating and cooling, and the temperature rise and fall rate is relatively low, so that the amplification time is longer. The temperature rise and fall rate of the existing commercial PCR instrument is less than 5 ℃/s; the reported ultra-fast PCR generally uses photothermal nanomaterials as energy converters to heat the entire PCR solution, but has limited improvement (typically < 10-fold) in heating rate compared to commercial PCR devices, while the increase in cooling rate is negligible.
As a gold standard for nucleic acid detection, the traditional PCR technology cannot meet the requirement of increasingly amplified nucleic acid detection due to the long amplification time caused by the low temperature rise and fall rate. Therefore, it is highly desirable to develop a rapid nucleic acid detection method with a rapid temperature increase and decrease rate.
At present, common rapid temperature rise and drop technologies include rotating disc type connection heating, microwave heating, light-driven heating and the like, wherein the rotating disc type heating can enable a PCR system to rapidly enter an environment with a set temperature, but a time-consuming heat transfer process of surface heating is still needed; microwave heating can rapidly heat a PCR system, but temperature control is difficult and a microwave instrument is relied on; while the light-driven heating can directly carry out bulk heating on the PCR system, the dependence on high-concentration photo-thermal nano materials and high-intensity irradiation light also limits the application of the PCR system. Nano-localized heating by plasma photothermal effect has been used to control chemical reactions or release DNA bound on the surface of gold nanoparticles, etc.
However, no nucleic acid amplification method by nano-localized heating around gold Nanoparticles (nanoparticules) has been reported so far. The first is that nucleic acid amplification requires precise measurement and control of the nano-localized temperature around the gold nanoparticles, and the second is that nucleic acid amplification is difficult to confine in a nano-localized environment around the gold nanoparticles.
Therefore, how to measure and control the nano-local temperature around the gold nanoparticles while limiting nucleic acid amplification to the nano-local environment around the gold nanoparticles is a problem that needs to be solved in the art.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a gold nanorod surface temperature measuring method, a heat transfer model constructed by using the same and application of the heat transfer model. The measuring method can accurately measure the nano local temperature around the gold nanoparticles, and solves the problem of how to measure and control the nano local temperature around the gold nanoparticles. Meanwhile, the constructed heat transmission model is utilized to design a DNA probe with a spacer region and a reaction region, so that the problem of how to limit nucleic acid amplification to a nano local environment around the gold nanoparticles is solved.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for measuring a surface temperature of gold nanorods (AuNRs), the method comprising the steps of:
connecting a DNA probe to the surface of a gold nanorod through a gold-sulfur bond (Au-S), dispersing the DNA probe in an alkaline buffer solution, and heating by using laser; the DNA probe is released from the surface of the gold nanorod and emits fluorescence, the fluorescence intensity of the DNA probe is detected, and the surface temperature of the gold nanorod is calculated according to the fluorescence intensity.
When irradiated with near infrared light (NIR), AuNRs generate heat due to photothermal properties, thereby creating a gradient temperature field around their surface. The temperature is highest at the AuNRs surface and gradually decays to the solution temperature, and measuring the temperature field is critical to designing DNA probes for use in nucleic acid amplification. The Au-S bond between the AuNRs and the DNA probe is a stable and strong chemical bond with a homolytic strength of about 40kcal/mol, but is less stable when exposed to an alkaline buffer, and is gradually dissociated with an increase in temperature. In the present invention, the surface temperature of AuNRs was measured using the instability of Au-S bond.
Wherein, there is certain linear relation between said fluorescence intensity and gold nanorod surface temperature, its linear equation can change according to gold nanorod concentration, its surface modification DNA probe quantity, environmental factor, etc., its linear relation is expressed by equation (1):
y＝a×x-b (1)
wherein y represents log10(fluorescence intensity of released DNA fragments), x represents the surface temperature of gold nanorods; a is any number between 0 and 1, and b is any number greater than 0.
When the concentration of the gold nanorod is 40-60 pM, the concentration of the surface modification DNA probe is (0.1-11) × 1012molecules/cm2And in Tris buffer with pH 8, taking a as 0.0153 and b as 1.7636, the linear relationship can represent the relationship between the surface temperature of the gold nanorods and the fluorescence intensity of the released DNA fragments under specific conditions at 40-90 ℃.
In a preferred embodiment of the present invention, the DNA probe is a thiol-modified DNA probe.
Preferably, the DNA probe is a fluorophore-modified thiolated DNA probe.
Preferably, the pH of the alkaline buffer is 7.8 to 8.5, for example, 7.9, 8.0, 8.1, 8.2, 8.3, or 8.4, and the pH is a main factor affecting the stability of Au — S bond.
Preferably, the alkaline buffer comprises PBS buffer or Tris buffer, preferably Tris buffer. Alkaline Tris-buffers are more likely to cause destabilization of Au-S bonds than alkaline PBS buffers.
In a second aspect, a heat transport device of the surface temperature field of the gold nanorods constructed by the measurement method according to the first aspect is characterized in that the output variable of the heat transport device is the surface temperature field of the gold nanorods;
the heat transport device is expressed by equation (2):
Figure BDA0002866529040000041
where ρ represents density, cpDenotes specific heat, λ denotes thermal conductivity, T denotes temperature, T denotes time, x denotes abscissa, and y denotes ordinate.
Preferably, the overall heat dissipation rate of the temperature field on the surface of the gold nanorod is calculated by a thermal diffusion equation (3):
q＝hA(T-T0)/V (3)
wherein h represents the convection heat transfer coefficient, and A represents the surface area of the gold nanorod; t represents the surface temperature of the gold nanorods, T0Represents the ambient temperature; v represents the total solution amount.
In a third aspect, the use of a heat transport device as described in the second aspect for constructing a nucleic acid amplification method or for preparing a nucleic acid amplification device.
In a fourth aspect, the present invention provides a nucleic acid amplification method based on nano-localized heating and plasma-driven heating, comprising the following steps:
(1) calculating the surface temperature field of the gold nanorods by using the heat transmission device, determining the lengths of a reaction region and a spacer region of the DNA probe, and synthesizing the DNA probe;
(2) connecting the DNA probe to the surface of the gold nanorod, mixing with a nucleic acid template, an enzyme and a reaction buffer solution to prepare a nucleic acid amplification reaction solution, and heating by using laser to amplify the nucleic acid.
According to the nucleic acid amplification method, the temperature rise and fall rate is increased, so that the PCR amplification time is shortened, and the PCR detection efficiency is improved. Nucleic acid amplification by nanoscopic heating around the surface of the gold nanoparticles, confining the nucleic acid amplification to within the nanoscopic environment of the gold nanoparticles (e.g., less than 100nm from the gold nanoparticles), requires only less than 1aL of water to be heated, thereby significantly increasing the heating and cooling rates. Currently, a common method for reducing the PCR volume is to combine photonic PCR with microfluidic technology, so that the PCR volume can be reduced to nL level. Thus, the heating and cooling rates can be increased by tens or even hundreds of times. Also in this case, each gold nanoparticle is only responsible for heating the nucleic acid in its nano-local environment, while the nucleic acid amplification is hardly affected by other gold nanoparticles, and thus the nucleic acid amplification is substantially independent of the concentration of gold nanoparticles.
It should be noted that the nucleic acid amplification method of the present invention is not limited to the photo-thermal nanomaterial gold nanorods, and other photo-thermal nanomaterials capable of being heated by plasmon resonance can be heated by the amplification method of the present invention to achieve ultrafast local heating. The photo-thermal nano material comprises any one or combination of at least two of gold nanorods, ferroferric oxide nano materials, graphene nano materials, carbon nano tube materials, polydopamine nano materials or other photo-thermal nano materials, and the photo-thermal nano materials are preferably the gold nanorods.
Preferably, the incident intensity of the laser is 15-40 mW/mm2For example, it may be 15mW/mm2、20mW/mm2、25mW/mm2、30mW/mm2、35mW/mm2Or 38mW/mm2And the like.
As a preferred embodiment of the present invention, the DNA probe comprises a reaction region including a nucleic acid sequence to be paired with a target nucleic acid, and a spacer region including a nucleic acid sequence consisting of poly-adenine.
The invention also provides a nucleic acid amplification device, which specifically comprises an amplification unit, a laser emission unit and a control unit. In the amplification unit, a photo-thermal nano material connected with a DNA probe, a nucleic acid template, an enzyme and a reaction buffer solution are mixed and subjected to amplification reaction; the laser emission unit emits laser to heat the amplification unit; the control unit amplifies the irradiation intensity, irradiation time, and the like of the laser light according to the amplification program, and completes the nucleic acid amplification program.
In a fifth aspect, the use of the nucleic acid amplification method of the first aspect for nucleic acid detection, gene expression control, or preparation of a nucleic acid amplification device.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the invention provides a method for measuring the nano local temperature around gold nanoparticles by using the instability of Au-S bonds, which can measure the surface temperature of gold nanorods, and meanwhile, the method also proves that the surface temperature of the gold nanorods is mainly related to the intensity of incident laser and unrelated to the concentration of AuNRs, and the temperature of a solution is related to the intensity of the incident laser and the concentration of the AuNRs;
(2) the nucleic acid amplification method provided by the invention has the advantages that the heat transmission device of the surface temperature field of the gold nanorod is utilized to design the DNA probe with the spacer region and the reaction region, the nucleic acid amplification is limited in the nano local environment around the gold nanoparticles, the nucleic acid amplification in the traditional uniform water phase is transferred to the nano local area around the photo-thermal nanoparticles, the PCR volume can be further reduced to the aL level, the volume of the solution to be heated is greatly reduced, the temperature rise and fall rate in the nucleic acid amplification process is greatly improved, the development of the nano local isothermal amplification technology based on the photo-thermal effect is finally realized, and the method can be widely applied to the fields such as clinical medicine development, infectious disease diagnosis, gene expression regulation and the like.
Drawings
FIG. 1 is a graph showing the amount of DNA fragments released as a function of AuNRs surface temperature.
FIG. 2 is log10(fluorescence intensity of released DNA fragments) is plotted linearly against AuNRs surface temperature.
FIG. 3 is a graph showing the relationship between the temperature of the solution and the irradiation time under irradiation with different laser intensities.
FIG. 4 is a graph of the different laser intensities as a function of the number of released DNA fragments after 40min irradiation.
Fig. 5 is a bar graph of solution temperature, surface temperature and temperature difference under irradiation of different laser intensities.
FIG. 6 is a graph showing the temperature of the solution as a function of irradiation time at different concentrations of the Au-DNA probe.
FIG. 7 is a graph of the different Au-DNA probe concentrations as a function of the amount of released DNA fragments after 40min of irradiation.
FIG. 8 is a bar graph of solution temperature, surface temperature and temperature difference at different Au-DNA probe concentrations.
FIG. 9 is a scattergram of the local temperature variation of a single AuNR-DNA nano.
Fig. 10 is a temperature profile of a single AuNR-DNA within a longitudinal distance of 100nm from the AuNR center under irradiation with different laser intensities.
FIG. 11 is a schematic diagram showing the temperature ranges of the spacer region and the reaction region on the DNA probe under irradiation with different laser power intensities.
FIG. 12 is a scattergram of the heating rate of the reaction region in the DNA probe under irradiation with different laser intensities.
Figure 13 is a scatter plot of the DNA intensity changes released by DSN enzyme cleavage of miRNA-DNA duplexes at different incubation temperatures.
FIG. 14 is a scatter plot of DNA intensity released by DSN enzyme cleavage of miRNA-DNA duplexes at different laser intensities and corresponding reaction zone mean temperatures.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
Example 1
The instability of gold-sulfur bonds is utilized in this example to provide a method for indirectly measuring the surface temperature of AuNRs.
To clarify the AuNRs surfaceRelationship between temperature and Au-S bond dissociation, AuNRs-DNA was dispersed in Tris buffer at pH 8, with gold nanorods at a concentration of 50pM and surface modification probes at a concentration of 0.1X 1012molecules/cm2(molecule/cm)2) And the entire solution was heated to the design temperature using a dry bath. When the entire solution reached a constant temperature, the AuNRs surface temperature was assumed to be the same as the solution temperature, which could be measured by a thermal infrared imager.
Since the ends of the DNA probes have been modified with organic fluorescent dyes, a recovery of fluorescence can be observed when the DNA probes are released from the AuNRs surface. By quantifying the fluorescence recovery as a function of the solution temperature, the relationship between the released DNA fragments and the surface temperature was obtained after 40 minutes.
As shown in fig. 1, gold-sulfur bonds are gradually dissociated with increasing temperature, resulting in an increase in the number of released DNA fragments with increasing AuNRs surface temperature. When the surface temperature is higher than 40 ℃, the proportion of released DNA increases, which also indicates that the Au — S bond is more unstable at higher temperatures.
This example obtained surface temperature and log in a temperature range of 40 ℃ to 90 ℃ by taking logarithm of fraction of DNA released10(fluorescence intensity of released DNA fragment).
As shown in fig. 2, log10(fluorescence intensity of released DNA fragment) and AuNRs surface temperature have a linear relationship as shown below:
y is 0.0153 x-1.7636; r of which2＝0.9758：
To verify the applicability of this equation, this example also verifies that the concentration of the probe is 0.1 × 10 at different gold nanorod concentrations, i.e. 40pM, 45pM, 55pM, and 60pM and different surface modification probe concentrations12molecules/cm2、0.5×1012molecules/cm2、2×1012molecules/cm2、4×1012molecules/cm2、10×1012molecules/cm2The relationship between fraction of released DNA and AuNRs surface temperature in different cases was found to be equally applicable.
Example 2
This example was used to determine the effect of the power intensity of the incident laser light on the temperature gradient around the AuNRs. Wherein the power intensity of the incident laser light directly affects the input energy and the heat generated by the AuNRs, resulting in a change in the surface temperature.
Dispersing a 46pM AuNRs-DNA probe in a Tris buffer solution with the pH value of 8, wherein the DNA probe is a thiolated DNA probe consisting of a reaction region and a spacer region, the reaction region is responsible for pairing with a target NA and starting NA amplification, and a poly-adenine sequence in the spacer region can adjust the distance from the reaction region to the surface of the AuNRs; five different intensities (7.17 mW/mm) were used together2、9.55mW/mm2、14.51mW/mm2、16.99mW/mm2And 38.22mW/mm2) Is excited by 808nm laser.
As shown in fig. 3, the solution temperature sharply rises at the beginning and then reaches a constant temperature as the irradiation time increases. At the same time, the final constant temperature of the solution is different for different incident laser intensities, and higher intensities will have higher constant temperatures. The highest temperature was obtained in less than 90s for all five different incident laser intensities.
As shown in FIG. 4, after 40 minutes of irradiation, the fluorescence recovery of all groups was recorded and converted to fraction of DNA released, based on surface temperature and log10(fluorescence intensity of released DNA fragments), the surface temperature of AuNRs was calculated.
By comparing the solution temperature and the surface temperature, the temperature difference at different laser intensities can be derived.
As shown in fig. 5, the temperature difference increases as the intensity of the incident laser light decreases. When the intensity of the incident laser is reduced to 9.55mW/mm2The maximum temperature difference reached 20 ℃.
Example 3
This example was used to determine the effect of solution concentration of AuNRs on the temperature gradient around the AuNRs. Among other things, the solution concentration of the AuNRs affects the absorption efficiency and scattering between incident light and the AuNRs.
To understand the temperature field versus AuNRs-DNA concentrationAs a relation, AuNRs-DNA probes of different concentrations were dispersed in Tris buffer at pH 8 with 38.22mW/mm2Is excited by 808nm laser.
As shown in fig. 6, the trend of the solution temperature rise is similar to that at different laser intensities, with the solution temperature reaching a maximum at 90s and then remaining nearly constant, except that the two highest concentrations (i.e., 92pM and 46pM) are slightly reduced.
As shown in fig. 7, after 40 minutes of incubation, the fraction of DNA released at different concentrations of AuNR-DNA probe was obtained and the corresponding AuNRs surface temperature was also calculated.
As shown in fig. 8, by comparing the surface temperature and the solution temperature, the temperature difference decreased with the increase in the concentration of AuNRs under the fixed excitation condition. This can be attributed to the fact that an increase in the concentration of the AuNRs reduces the distance between two adjacent AuNRs, making the solution temperature higher.
Contrary to the apparent change in solution temperature, at constant incident laser intensity, the AuNRs surface temperature only changed slightly with the change in AuNRs concentration.
As can be seen from examples 2 and 3, the surface temperature of AuNRs is mainly related to the incident laser intensity and is independent of the AuNRs concentration, while the solution temperature is related to the incident laser intensity and the AuNRs concentration.
Example 4
To determine the temperature distribution around the AuNR-DNA, this example sets up a two-dimensional heat transport model to simulate the heat transfer process from the AuNRs surface to the surrounding aqueous solution. And during this transfer the heat needs to pass through two models with different thermal conductivity, namely the conjugated DNA layer and the surrounding aqueous solution.
The thermal conductivity and density of the DNA layer were 0.3W/(mK) and 1.7g/cm, respectively3The thermal conductivity coefficient of the water environment is about 0.6W/(m.K), and the density is 1g/cm3The heat transfer model can be simplified to a two-dimensional transient heat transfer problem without a heat source, and a parabolic partial differential equation of thermal diffusion is used as shown in equation (2):
Figure BDA0002866529040000101
in the formula: ρ represents a density; c. CpRepresents specific heat; λ represents the thermal conductivity, T represents the temperature, T represents the time, x represents the abscissa, and y represents the ordinate.
Assuming that the AuNRs are evenly distributed in the water, a single AuNR surrounded by water in the box can be considered by applying periodic boundary conditions on the box surface.
According to experimental data, the boundary condition of the AuNR surface is set to a constant temperature. For the cooling process, heat is dissipated to the surroundings by natural convection at the surface of the PCR tube.
To simulate the surface cooling effect, the computational domain should include the entire AuNRs and aqueous solution in the tube. However, this is quite time consuming to calculate. To simplify the process, we consider the overall heat dissipation rate as a "heat sink" uniformly distributed in the solution, i.e., adding a heat source term to the heat diffusion equation, the resulting model is shown in equation (3):
q＝hA(T-T0)/V (3)
in the formula: h represents a convective heat transfer coefficient and has a value of 10W/(m)2K); a represents the surface area of the gold nanorods, and the value is 5.07X 10-4m2；T0Representing the ambient temperature, at a value of 293.15K; v represents the total solution amount and is 1X 10-6m3。
Example 5
In this embodiment, simulation result simulation and data analysis are performed on the model provided in embodiment 4. Since the thermal resistance along the heat transfer path changes, two stages were used in this example to simulate heat flux.
The simulated temperature field around a single AuNR is shown in fig. 9, and a scatter plot shows the temperature change along the long axis of the AuNR. Simulation results show that the temperature drops rapidly near the AuNRs surface and the decay slows as the distance from the AuNR surface increases.
As shown in fig. 10, AuNRs produced different temperature ranges within 100nm under excitation with lasers of different intensities.
As shown in FIG. 11, the temperature ranges of the spacer region and the reaction region can be obtained from the heat transfer model according to the calculated length of the DNA probe. Simulation results show that the temperature variation in the reaction zone is very narrow, with a maximum variation of only 2 ℃. If we assume that the conversion time from light to heat is negligible, the simulation results indicate that the reaction region is able to reach temperature equilibrium in μ s.
As shown in FIG. 12, an ultra-high heating rate (up to 10) was obtained in the reaction region of the DNA probe5DEG C/s). In these cases, the nano-localized environment under irradiation with greater laser intensity exhibits lower heating and cooling rates due to the longer time required to span a larger temperature range.
In summary, the photothermal effect of AuNRs establishes a temperature field suitable for achieving ultra-fast NA amplification, due to the ultra-fast heating/cooling rates and limited temperature changes, with no significant effect on the activity of the NA polymerase.
Example 6
This example serves as a conceptual proof to demonstrate that isothermal amplification mediated by double-strand specific nucleases (DSN enzymes) can be performed in a nano-local environment.
The DSN enzyme can efficiently recognize and enzyme a DNA strand in a complete complementary paired DNA double strand or a DNA/RNA hybrid double strand, and has little effect on single-stranded DNA and single/double-stranded RNA. Based on this feature, this example performed DSN-mediated isothermal amplification around AuNRs in PBS buffer as proof-of-concept for AuNRs surface plasmon-driven NA amplification.
Isothermal amplification was performed in PBS buffer because the Au-S bond was stable in weakly alkaline PBS buffer even at high temperatures (up to 60 ℃) compared to Tris buffer. The DNA probe modified by the organic dye is combined with the AuNRs surface, and the fluorescence release of the organic dye on the metal surface is inhibited.
In this example, the biomarker miRNA-21 associated with various cancers was selected as a model target. The DNA sequence in the reaction region may specifically pair with the target miRNA-21 to form a duplex structure.
DSNs recognize DNA-RNA duplexes and cleave the DNA sequence into short fragments, thereby releasing the organic dye from the vicinity of the AuNR and restoring fluorescence, while the target miRNA remains unchanged and pairs with the next DNA probe. By repeating the cleavage reaction without interruption, free organic dye, which can be measured, accumulates in the solution. The final recovered fluorescence of the organic dye can be determined by the amount of target miRNA, DSN activity and reaction time.
First, we investigated DSN activity at different temperatures in PBS buffer, where the amount of miRNA added was 10nM and the reaction time was 40 minutes.
As shown in FIG. 13, by changing the temperature of the PBS buffer, released DNA fragments can be obtained, and when the temperature is lower than 50 ℃, the released DNA increases with the increase of the temperature, which confirms that the activity of DSN increases with the increase of the temperature of the solution within this temperature range. However, when the temperature of the PBS buffer reached 60 ℃, the amount of released DNA decreased dramatically, indicating that the DSN began to lose activity at this temperature.
As shown in FIG. 14, different amounts of released DNA were obtained under irradiation with different laser intensities by heating the PBS buffer with laser irradiation instead of a dry bath.
Meanwhile, the temperature range of the reaction region was obtained by the heat transfer model, and the average temperature of the reaction region at the corresponding laser intensity is also shown in fig. 14. When the average temperature of the reaction region was 53.2 ℃, the released DNA was the highest. When the average temperature was higher or lower than 53.2 ℃, a significant reduction in released DNA was found.
The results show that DSN showed higher activity at 53.2 ℃, which is consistent with the results of the DSN activity test. Therefore, proof of concept of isothermal amplification near the AuNRs under laser irradiation was successfully verified.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims (10)

1. A method for measuring the surface temperature of a gold nanorod is characterized by comprising the following steps:
connecting a DNA probe to the surface of a gold nanorod through a gold-sulfur bond, dispersing the DNA probe in an alkaline buffer solution, and heating by using laser;
the DNA probe is released from the surface of the gold nanorod and emits fluorescence, the fluorescence intensity of the DNA probe is detected, and the surface temperature of the gold nanorod is calculated according to the fluorescence intensity.
2. The measurement method according to claim 1, wherein the linear relationship between the fluorescence intensity and the surface temperature of the gold nanorod is as shown in formula (1):
y＝a×x-b (1)；
wherein y represents log10(fluorescence intensity of released DNA fragments), x represents the surface temperature of gold nanorods; a is any number between 0 and 1, and b is any number greater than 0;
preferably, the concentration of the gold nanorods is 40-60 pM, and the concentration of the DNA probe is (0.1-11) × 1012Molecule/cm2When a is 0.0153 and b is 1.7636.
3. The method of measurement according to claim 1 or 2, wherein the DNA probe is a thiolated-modified DNA probe;
preferably, the DNA probe is a thiol-modified DNA probe modified by a fluorescent group;
preferably, the pH value of the alkaline buffer solution is 7.8-8.5;
preferably, the alkaline buffer is a Tris buffer.
4. A heat transport device of the surface temperature field of the gold nanorods constructed by using the measurement method according to any one of claims 1 to 3, wherein the output variable of the heat transport device is the surface temperature field of the gold nanorods;
the calculation formula of the output variable of the heat transport device is expressed by equation (2):
Figure FDA0002866529030000021
where ρ represents density, cpThe specific heat is expressed, lambda represents the thermal conductivity coefficient, T represents the surface temperature of the gold nanorod, T represents time, x represents the abscissa, and y represents the ordinate.
5. The heat transport device according to claim 4, wherein the overall heat dissipation rate of the temperature field of the gold nanorod surface is calculated by a heat diffusion equation (3):
q＝hA(T-T0)/V (3)
wherein h represents the convection heat transfer coefficient, and A represents the surface area of the gold nanorod; t represents the surface temperature of the gold nanorod, T0Represents the ambient temperature; v represents the total solution amount.
6. Use of the heat transport device according to claim 4 or 5 for constructing a nucleic acid amplification method or for preparing a nucleic acid amplification device.
7. A nucleic acid amplification method comprising the steps of:
(1) calculating the surface temperature field of the gold nanorods by using the heat transport device of claim 4 or 5, determining the lengths of the reaction region and the spacer region of the DNA probe, and synthesizing the DNA probe;
(2) connecting the DNA probe to the surface of the gold nanorod, mixing with a nucleic acid template, an enzyme and a reaction buffer solution to prepare a nucleic acid amplification reaction solution, and heating by using laser to amplify the nucleic acid.
8. The method for amplifying a nucleic acid according to claim 7, wherein the laser has an incident intensity of 15 to 40mW/mm2。
9. The method for amplifying a nucleic acid according to claim 7 or 8, wherein the reaction region of the DNA probe comprises a nucleic acid sequence that pairs with a target nucleic acid;
preferably, the spacer of the DNA probe comprises a nucleic acid sequence consisting of poly-adenine.
10. Use of the nucleic acid amplification method of any one of claims 7 to 9 for nucleic acid detection or gene expression regulation.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114136481A (en) * 2021-10-27 2022-03-04 天津大学 In-situ measurement method and device for temperature at microwave-induced microscopic hot spot and application

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030143604A1 (en) * 2001-11-30 2003-07-31 Storhoff James J. Real-time monitoring of PCR amplification using nanoparticle probes
CN103599070A (en) * 2013-11-26 2014-02-26 上海交通大学 Preparation method of temperature and fluorescence probe of lipidosome loaded with gold nanocluster and anti-cancer drug
CN103611946A (en) * 2013-12-08 2014-03-05 福建医科大学 Preparation method of gold nanocluster fluorescent materials protected by methionine
CN104004642A (en) * 2014-04-30 2014-08-27 南京邮电大学 Nucleic acid sensor and preparation method thereof and multi-element detection method based on sensor
CN105022873A (en) * 2015-07-10 2015-11-04 中南大学 On-line reconstruction method of isothermal stamping mold temperature field
CN109135729A (en) * 2018-06-28 2019-01-04 华南理工大学 A kind of temperature response type luminescent gold nano-micelle and preparation method thereof and the application in visualization temperature detection
CN110655796A (en) * 2019-11-15 2020-01-07 江苏科技大学 Nano-gold fibroin composite membrane and preparation method and application thereof
CN111763713A (en) * 2020-03-23 2020-10-13 天津大学 Method and kit for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purpose

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030143604A1 (en) * 2001-11-30 2003-07-31 Storhoff James J. Real-time monitoring of PCR amplification using nanoparticle probes
CN103599070A (en) * 2013-11-26 2014-02-26 上海交通大学 Preparation method of temperature and fluorescence probe of lipidosome loaded with gold nanocluster and anti-cancer drug
CN103611946A (en) * 2013-12-08 2014-03-05 福建医科大学 Preparation method of gold nanocluster fluorescent materials protected by methionine
CN104004642A (en) * 2014-04-30 2014-08-27 南京邮电大学 Nucleic acid sensor and preparation method thereof and multi-element detection method based on sensor
CN105022873A (en) * 2015-07-10 2015-11-04 中南大学 On-line reconstruction method of isothermal stamping mold temperature field
CN109135729A (en) * 2018-06-28 2019-01-04 华南理工大学 A kind of temperature response type luminescent gold nano-micelle and preparation method thereof and the application in visualization temperature detection
CN110655796A (en) * 2019-11-15 2020-01-07 江苏科技大学 Nano-gold fibroin composite membrane and preparation method and application thereof
CN111763713A (en) * 2020-03-23 2020-10-13 天津大学 Method and kit for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purpose

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
袁鸣: "肽-金复合纳米结构的合成及生物医学应用研究", 中国博士学位论文全文数据库, pages 020 - 254 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114136481A (en) * 2021-10-27 2022-03-04 天津大学 In-situ measurement method and device for temperature at microwave-induced microscopic hot spot and application
CN114136481B (en) * 2021-10-27 2023-12-01 天津大学 In-situ measurement method and equipment for temperature at microwave-induced microscopic hot spot and application

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