Fluorescence sensor for temperature detection and preparation method and application thereof

Abstract

The invention discloses a fluorescence sensor for temperature detection and a preparation method and application thereof. The fluorescence sensor comprises a DNA molecule with a stem-loop structure and a fluorescence molecule, wherein the fluorescent molecule is modified at the 3'end or the 5 'end of the DNA molecule. The stem-loop structure of the DNA molecule generates configuration change along with temperature change, and the luminous property of the fluorescent molecule is influenced. According to the fluorescence sensor provided by the invention, the temperature measurement sensitivity of fluorescent molecules is improved by utilizing a DNA stem-loop structure, and the temperature measurement interval can be adjusted by changing the stem-loop structure. Compared with an existing DNA fluorescence temperature sensor, a quenching group does not need to be introduced, the structure is simple, and the cost is lower. The sensor is good in biocompatibility, can be modified on other materials, and has a relatively great application prospect in the fields of biological detection and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and more specifically to a fluorescence sensor for temperature detection, a preparation method thereof and an application thereof. Background Art

[0002] Cells are the basic units of human life activities. Many chemical reactions, such as enzymatic reactions and gene expression, occur in cells. These chemical reactions absorb or release heat, causing changes in cell temperature. Therefore, temperature is an important parameter to indicate the state of cells. Detecting subtle temperature changes in cells is of great significance for studying cell life activities and exploring the mechanisms of disease occurrence.

[0003] Fluorescent molecules are widely used to measure intracellular temperature because their fluorescence intensity changes with temperature. Generally, temperature increases cause the vibration or rotation of fluorescent molecules to intensify, which leads to stronger non-radiative transitions within the molecules and weakens their fluorescence intensity. However, the temperature measurement sensitivity of traditional fluorescent molecules is difficult to exceed 5% / ℃. Fluorescent molecules with sensitive temperature measurement require complex synthesis, are expensive and difficult to commercialize. Stem-loop DNA has good biocompatibility and its structure can change with temperature. It is widely used to improve the temperature measurement sensitivity of fluorescent molecules. Usually, a fluorescent molecule is modified at one end of the stem-loop DNA, and a quenching group or another fluorescent molecule that can undergo fluorescence resonance energy transfer (FRET) is modified at the other end. The temperature is used to cause changes in the DNA configuration, thereby changing the distance between the molecules at both ends of the stem-loop DNA, and realizing sensitive detection of temperature. However, modifying two fluorescent molecules, or one fluorescent molecule and one fluorescent quenching group on the stem-loop DNA has a complex structure and high cost.

[0004] Therefore, the present invention aims to develop a temperature fluorescence sensor which has high relative sensitivity in temperature measurement, low cost and wide application. Summary of the invention

[0005] One object of the present invention is to provide a fluorescence sensor for temperature detection, which solves the problems of low temperature measurement sensitivity using fluorescent molecules directly, complex structure and high cost of existing DNA fluorescence temperature sensors, and provides a new idea for sensor design.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned fluorescence sensor.

[0007] The third object of the present invention is to provide an application of the above-mentioned fluorescence sensor in temperature measurement and a method for detecting temperature.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a fluorescent sensor for temperature detection, wherein the fluorescent sensor comprises a DNA molecule with a stem-loop structure and a fluorescent molecule; wherein the fluorescent molecule is modified at the 3' end or the 5' end of the DNA molecule.

[0010] In the present invention, as the temperature rises, the vibration and rotation of the fluorescent molecule intensify, and the non-radiative transition is enhanced, resulting in a decrease in fluorescence intensity; the stem-loop structure of the DNA molecule undergoes a configuration change as the temperature changes, which affects the luminescence properties of the fluorescent molecule; that is, as the temperature rises, the stem-loop structure of the DNA molecule opens, which can cause the fluorescence intensity of the fluorescent molecule to drop rapidly, and this change is more obvious than the change in fluorescence intensity of the same fluorescent molecule not modified on the stem-loop structure with temperature. Therefore, the fluorescent sensor provided by the present invention utilizes the stem-loop structure of the DNA molecule to improve the temperature measurement sensitivity of the fluorescent molecule, and the temperature measurement range can be adjusted by changing the design of the DNA molecule with the stem-loop structure.

[0011] Furthermore, the sensitive temperature measurement interval of the fluorescent sensor is the melting temperature T of the DNA molecule with the stem-loop structure. m ±5°C. It should be noted that, in the present invention, the term "sensitive temperature measurement interval" refers to the range in which the temperature sensor can accurately measure the temperature.

[0012] Furthermore, the term "stem-loop structure" refers to a double-stranded region between paired bases forming a "stem", while the single-stranded region that cannot be paired protrudes to form a "loop", and the preferred number of bases on the stem side of the stem-loop structure is ≥10, and the number of bases in the loop is ≤the number of bases on the stem side.

[0013] Illustratively, the nucleotide sequence of the stem-loop structured DNA molecule includes, but is not limited to, SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3.

[0014] Furthermore, the fluorescent molecule is preferably a temperature-sensitive fluorescent molecule, that is, a substance whose fluorescence intensity decreases sensitively with increasing temperature; illustratively, the fluorescent molecule includes but is not limited to carboxytetramethylrhodamine, rhodamine B or a meso benzene ring-substituted BODIPY derivative.

[0015] In order to expand the application scenarios, the above-mentioned fluorescence sensor of the present invention can also be modified on other materials. According to a specific embodiment of the present invention, the fluorescence sensor also includes a carrier;

[0016] A functional group is modified at one end of the stem-loop structure DNA molecule that is not modified with the fluorescent molecule, so as to couple the DNA molecule modified with the fluorescent molecule to the surface of the carrier;

[0017] Furthermore, the functional group is an aldehyde group, a carboxyl group, an amino group, a thiol group or an azide group.

[0018] Furthermore, the carrier is silicon nanowires, nanoparticles or graphene.

[0019] According to a specific embodiment of the present invention, the 3' end of the stem-loop structured DNA molecule is modified with a fluorescent molecule, and the 5' end is modified with an aldehyde group, and the DNA molecule modified with the fluorescent molecule is coupled to the silicon nanowire via the aldehyde group.

[0020] In a second aspect, the present invention provides a method for preparing a fluorescent sensor for temperature detection, the preparation method comprising:

[0021] Synthesize stem-loop structure DNA molecules according to the temperature measurement interval;

[0022] Modifying the 3' end or 5' end of the DNA molecule with a fluorescent molecule;

[0023] The sensitive temperature range of the fluorescent sensor is the melting temperature T of the stem-loop structure DNA molecule. m ±5℃.

[0024] Furthermore, the preparation method also includes modifying a functional group on the end of the DNA molecule that is not modified with the fluorescent molecule, and then coupling it to the surface of the carrier.

[0025] In a third aspect, the present invention provides application of the above-mentioned fluorescent sensor for temperature detection in detecting temperature.

[0026] Furthermore, the present invention also provides a method for detecting temperature, using the above-mentioned fluorescent sensor for temperature detection to detect temperature, comprising the following steps:

[0027] Determine the excitation wavelength according to the fluorescent molecules modified on the fluorescent sensor, add the fluorescent sensor to the detection system, and adjust the temperature. Each temperature adjustment needs to keep the temperature constant for a period of time, and draw a temperature response standard curve with temperature as the horizontal axis and the fluorescence intensity at the maximum emission wavelength as the vertical axis;

[0028] After incubating the fluorescent sensor with the object to be detected, the fluorescence intensity of the object to be detected at the maximum emission wavelength is obtained;

[0029] The temperature of the object to be measured is calculated based on the temperature response standard curve and the fluorescence intensity at the maximum emission wavelength of the object to be measured.

[0030] Furthermore, the time for maintaining the temperature constant is more than 5 minutes, for example, 5-10 minutes.

[0031] The beneficial effects of the present invention are as follows:

[0032] (1) The fluorescent sensor of the present invention comprises a stem-loop DNA molecule and a temperature-sensitive fluorescent molecule. The stem-loop structure of the DNA molecule directly affects the microenvironment around the fluorescent molecule as the temperature changes. In addition, the fluorescence intensity of the fluorescent molecule itself changes with temperature, thereby achieving a more sensitive response to temperature. In addition, the other end of the stem-loop DNA molecule does not need to be designed with other fluorescent molecules or quenching groups, so the structure is simple and the cost is lower.

[0033] (2) The present invention is based on a stem-loop structured DNA molecule, which has good biocompatibility and is more conducive to temperature measurement at the biological sample or cell level.

[0034] (3) There is no need to design other fluorescent molecules or quenching groups at one end of the stem-loop DNA molecule to improve the temperature measurement sensitivity. Therefore, additional functional groups can be modified to couple with other molecules or materials, thereby expanding the application range of temperature measurement.

[0035] (4) After the designed stem-loop DNA molecule is coupled to the silicon nanowire, it can realize sensitive measurement of the temperature of different spatial positions of a single cell with high spatial resolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0037] Figure 1 A schematic diagram of the mechanism of temperature detection using the fluorescence sensor provided by the present invention in an embodiment of the present invention is shown.

[0038] Figure 2a The curves showing the change in fluorescence intensity of the fluorescent molecule TAMRA and the fluorescent sensor (No. 1-TAMRA) in Example 1 as a function of temperature are shown.

[0039] Figure 2b The temperature measurement relative sensitivity curve of the fluorescent molecule TAMRA and the fluorescent sensor (No.1-TAMRA) in Example 1 is shown as a function of temperature.

[0040] Figure 3a The curve showing the change in fluorescence intensity of the fluorescence sensor (No. 1-TAMRA) in Example 2 as a function of temperature is shown.

[0041] Figure 3b The curve showing the change in fluorescence intensity of the fluorescence sensor (No. 2-TAMRA) in Example 2 as a function of temperature is shown.

[0042] Figure 3c The curve showing the change in fluorescence intensity of the fluorescence sensor (No. 3-TAMRA) in Example 2 as a function of temperature is shown.

[0043] Figure 4The curves showing the change of fluorescence intensity of the fluorescence sensor (No.1-TAMRA) in Example 3 at different pH values ​​as a function of temperature are shown.

[0044] Figure 5 The curves showing the change of fluorescence intensity of two fluorescent sensors (No.1-TAMRA and CHO-No.1-TAMRA) in Example 4 as a function of temperature are shown.

[0045] Figure 6a A fluorescence image of the SiNW-based fluorescence sensor (SiNW-No. 1) in Example 5 is shown.

[0046] Figure 6b The curve showing the change of fluorescence intensity of the SiNW-based fluorescence sensor (SiNW-No. 1) in Example 5 as a function of temperature is shown.

[0047] Figure 7 The fluorescence image of the SiNW-based fluorescence sensor (SiNW-No. 1) in Example 6 incubated in PC12 cells is shown.

[0048] Figure 8 The curve showing the change in fluorescence intensity of the fluorescence sensor (No. 1-TR) in Example 7 as a function of temperature is shown. DETAILED DESCRIPTION

[0049] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments and drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0050] The present invention provides a fluorescence sensor for temperature detection, which utilizes a stem-loop structured DNA molecule to improve the temperature measurement sensitivity of the fluorescence molecule without introducing a quenching group or a fluorescence resonance energy transfer molecule. Figure 1 As shown, when the temperature decreases, DNA forms a stem-loop structure. In addition to the vibration and rotation of the fluorescent molecules themselves being suppressed by the temperature, the change in the microenvironment of the fluorescent molecules caused by the stem-loop structure will also hinder their vibration and rotation. Compared with the same fluorescent molecules not modified on the DNA molecules, the non-radiative transition is weaker and the fluorescence intensity is greater. When the temperature rises, in addition to the vibration and rotation of the fluorescent molecules themselves being aggravated by the temperature, the stem-loop structure opens, the influence on the microenvironment of the fluorescent molecules gradually decreases, and the hindering effect on the vibration and rotation of the fluorescent molecules decreases. In the temperature range where the stem-loop structure is opened, the influence trend of the stem-loop structure on the vibration and rotation of the fluorescent molecules is the same as the influence trend of the temperature on the vibration and rotation of the fluorescent molecules, thereby improving the temperature measurement sensitivity of the fluorescent molecules in the temperature range where the stem-loop structure is opened.

[0051] The following describes it in detail with reference to specific embodiments.

[0052] Example 1

[0053] 1) Design and prepare the following temperature fluorescence sensor, wherein the nucleotide sequence of the DNA molecule is:

[0054] 5'-TTTTTTTTTTTATCTATCATTATTGTTTTTTTTTTTTTTTACTATTATGTTTAGAT-3'(SEQID No:1)

[0055] According to the reference (Nucleic Acids Research, 2003, Vol. 31, No. 13), the melting temperature of the DNA molecule under 1×PBS is T m The temperature was 36.2° C. The 3' end of the DNA molecule was modified with the fluorescent molecule carboxytetramethylrhodamine (TAMRA), and was named No.1-TAMRA.

[0056] 2) Accurately prepare 1× PBS buffer (1.06 mM potassium dihydrogen phosphate, 3.00 mM disodium hydrogen phosphate heptahydrate, 155 mM sodium chloride, pH=7.20).

[0057] 3) 100 μM of the above No.1-TAMRA dispersed in 1×PBS buffer was annealed at 95°C, slowly cooled to room temperature, and the final concentration dispersed in 1×PBS in a cuvette was 200 nM. The excitation wavelength was fixed at 561 nm, and the temperature was used as the abscissa, and the fluorescence intensity at the normalized emission wavelength of 582.6 nm was used as the ordinate to draw the temperature response curve of the fluorescent sensor modified with TAMRA.

[0058] 4) Weigh TAMRA solid, dissolve it in DMSO, and dilute it with 1×PBS. The final TAMRA concentration in the cuvette is 10 μM and the volume fraction of DMSO is 1%. The excitation wavelength is fixed at 561 nm, and the temperature is used as the horizontal coordinate, and the fluorescence intensity at the normalized emission wavelength of 576.0 nm is used as the vertical coordinate to draw the temperature response curve of the fluorescent molecule TAMRA.

[0059] 5) According to the relative sensitivity formula: The temperature measurement relative sensitivity curves of No.1-TAMRA and TAMRA are plotted as a function of temperature, where S r is the relative sensitivity, F is the relative fluorescence intensity, and T is the temperature.

[0060] The temperature response curve and relative sensitivity curve obtained by the above steps are as follows: Figure 2a and Figure 2b As shown in the figure, in the stem-loop structure DNA molecule, mAt around 37°C, the fluorescence intensity of TAMRA modified on DNA molecules changes more sensitively with temperature than that of TAMRA not on DNA molecules, and its relative sensitivity of temperature measurement can be increased from less than 2.5% / °C to more than 7% / °C.

[0061] Example 2

[0062] 1) Design and prepare three different T m The temperature fluorescence sensor of the DNA molecule is as follows, wherein the nucleotide sequences of the DNA molecule are:

[0063] a)5'-TTTTTTTTTTTATCTAATCATATTGTTTTTTTTTTTTTTTACTATTATGTTTAG AT-3' (SEQ ID No: 1)

[0064] b)5'-TTTTTTTTTTTATCATAATTATTGTTTTTTTTTTTTTTTACTATTTTTTTGAT-3'(SEQ IDNo:2)

[0065] c)5'-TTTTTTTTTTTTATACATTTGTTTTTTTTTTTTACATATGTATAT-3'(SEQ IDNo:3)

[0066] According to the reference (Nucleic Acids Research, 2003, Vol. 31, No. 13), the melting temperatures of the three DNA molecules under 1× PBS are T m They are 36.2°C (SEQ ID No: 1), 22.1°C (SEQ ID No: 2), and 46.7°C (SEQ ID No: 3). The three DNA molecules are all modified with fluorescent molecule TAMRA at the 3' end and named No.1-TAMRA, No.2-TAMRA, and No.3-TAMRA respectively.

[0067] 2) Accurately prepare 1× PBS buffer (1.06 mM potassium dihydrogen phosphate, 3.00 mM sodium dihydrogen phosphate heptahydrate, 155 mM sodium chloride pH = 7.20)

[0068] 3) 100 μM of the above No.1-TAMRA, No.2-TAMRA, and No.3-TAMRA dispersed in 1×PBS were annealed at 95°C and slowly cooled to room temperature. The final concentration dispersed in 1×PBS in a cuvette was 200 nM. The excitation wavelength was fixed at 561 nm, and the temperature was used as the abscissa, and the fluorescence intensity at the normalized emission wavelength of 582.6 nm was used as the ordinate to plot the temperature response curves of the three fluorescent sensors modified with TAMRA.

[0069] The three temperature response curves obtained in the above steps are as follows: Figure 3a , Figure 3b and Figure 3c As shown in the figure, the three stem-loop DNA molecules all have their T m The fluorescence intensity of TAMRA changes with T m The temperature changes nearby are more sensitive, which proves that the method of DNA molecules with stem-loop structure can improve the temperature measurement sensitivity of fluorescent molecules is universal in the design of DNA molecule stem-loop sequences. The DNA molecules with stem-loop structure can be designed according to the possible range of temperature to be measured, and the applicable temperature range is wide.

[0070] Example 3

[0071] 1) Design and prepare the following temperature fluorescence sensor, wherein the nucleotide sequence of the DNA molecule is as shown in SEQ ID No: 1.

[0072] According to the reference (Nucleic Acids Research, 2003, Vol. 31, No. 13), the melting temperature of the DNA molecule under 1×PBS is T m The temperature was 36.2° C. The fluorescent molecule TAMRA was modified at the 3' end of the DNA molecule and named No.1-TAMRA.

[0073] 2) Accurately prepare 1×PBS buffer (1.06 mM potassium dihydrogen phosphate, 3.00 mM disodium hydrogen phosphate heptahydrate, 155 mM sodium chloride, pH=7.20). Use 0.1 M HCl or 0.1 M NaOH to change the pH of the buffer solution to obtain four PBS buffers with different pH values, which are 5.11, 6.17, 7.20 and 8.18, respectively.

[0074] 3) 100 μM of the above No.1-TAMRA dispersed in 1×PBS was annealed at 95°C, slowly cooled to room temperature, and dispersed in a cuvette with PBS of different pH values ​​to a final concentration of 200 nM. The excitation wavelength was fixed at 561 nm, and the temperature was used as the abscissa, and the fluorescence intensity at the normalized emission wavelength of 582.6 nm was used as the ordinate to draw the temperature response curve of the modified No.1-TAMRA at different pH values.

[0075] The temperature response curves at different pH values ​​obtained in the above steps are as follows: Figure 4 As shown. Figure 4 It can be seen that the temperature measurement properties of the designed temperature fluorescence sensor at different pH levels have no obvious differences, so it can be used to measure temperature in environments with different pH levels.

[0076] Example 4

[0077] 1) Design and prepare the following two temperature fluorescence sensors, wherein the nucleotide sequence of the DNA molecule is as shown in SEQ ID No: 1.

[0078] According to the reference (Nucleic Acids Research, 2003, Vol. 31, No. 13), the melting temperature of the DNA molecule under 1×PBS is T m The temperature of the sensor is 36.2℃. The DNA molecule of one sensor is modified with fluorescent molecule TAMRA at the 3' end, named No.1-TAMRA. The DNA molecule of another sensor is modified with fluorescent molecule TAMRA at the 3' end and aldehyde group at the 5' end, named CHO-No.1-TAMRA.

[0079] 2) 100 μM of the above No.1-TAMRA and CHO-No.1-TAMRA dispersed in 1×PBS was annealed at 95°C and slowly cooled to room temperature. The final concentration dispersed in 1×PBS in a cuvette was 200 nM. The excitation wavelength was fixed at 561 nm, and the temperature response curves of the two fluorescent sensors were plotted with the temperature as the abscissa and the fluorescence intensity at the normalized emission wavelength of 582.6 nm as the ordinate.

[0080] The temperature response curves of the two fluorescent sensors obtained in the above steps are as follows: Figure 5 As shown in the figure, the aldehyde group at the 5' end of the DNA molecule does not affect the property of the stem-loop structure to improve the temperature measurement sensitivity of the fluorescent molecule. Therefore, the designed fluorescent sensor can be modified on other materials using functional groups to expand its application scenarios.

[0081] Example 5

[0082] 1) Designing and preparing the following temperature fluorescence sensor, wherein the nucleotide sequence of the DNA molecule is shown in SEQ ID No: 1.

[0083] According to the reference (Nucleic Acids Research, 2003, Vol. 31, No. 13), the melting temperature of the DNA molecule under 1×PBS is T m The temperature of the DNA molecule was 36.2° C. The 3' end of the DNA molecule was modified with a fluorescent molecule TAMRA and the 5' end was modified with an aldehyde group, and the DNA molecule was named CHO-No.1-TAMRA.

[0084] 2) Accurately prepare 1× PBS buffer (1.06 mM potassium dihydrogen phosphate, 3.00 mM sodium dihydrogen phosphate heptahydrate, 155 mM sodium chloride pH = 7.20)

[0085] 3) A silicon nanowire array is prepared by metal-assisted chemical etching, and a silicon nanowire array with a silicon dioxide layer on the surface is obtained by thermal oxidation in a tube furnace.

[0086] 4) After the silicon nanowire array was heated with piranha solution (concentrated sulfuric acid and hydrogen peroxide volume ratio of 3:1) for 1 h for surface hydroxylation, it was amino-modified with aminopropyltriethoxysilane to obtain a surface amino-modified silicon nanowire array. The silicon nanowires were scraped off with a scalpel and dispersed in 1×PBS to obtain a silicon nanowire solution with a concentration of 0.2 mg / mL.

[0087] 5) Weigh sodium cyanoborohydride solid and dissolve it in 1×PBS to obtain a 0.3 M sodium cyanoborohydride solution.

[0088] 6) 100 μM of the above CHO-No.1-TAMRA dispersed in 1×PBS was annealed at 95°C and slowly cooled to room temperature. 33 μL of DNA solution was taken and mixed with 114 μL of silicon nanowire solution and 100 μL of sodium cyanoborohydride solution, and shaken at 1000 rpm overnight to obtain a silicon nanowire-based fluorescent sensor named SiNW-No.1.

[0089] 7) SiNW-No.1 was centrifuged at 13000 rpm for 10 min with 1×PBS, centrifuged three times, and finally dispersed in 1 mL of 1×PBS.

[0090] 8) Excited by 561 nm laser, the fluorescence signals of multiple SiNW-No.1 at different temperatures from 570 nm to 620 nm were collected under a confocal microscope, and the fluorescence intensity versus temperature curve was plotted after normalization.

[0091] The fluorescence image of SiNW-No.1 obtained in the above steps under 561nm excitation is as follows Figure 6a The curve of fluorescence intensity changing with temperature is shown in Figure 6b shown. Figure 6a It shows that DNA molecules modified with fluorescent molecules are successfully modified on silicon nanowires; Figure 6b This shows that the temperature differences between different SiNW-No.1 are small and all are within the T of DNA molecules. m The temperature measurement nearby is sensitive.

[0092] Example 6

[0093] 1) Designing and preparing the following temperature fluorescence sensor, wherein the nucleotide sequence of the DNA molecule is shown in SEQ ID No: 1.

[0094] According to the reference (Nucleic Acids Research, 2003, Vol. 31, No. 13), the melting temperature of the DNA molecule under 1×PBS is Tm The temperature of the DNA molecule was 36.2° C. The 3' end of the DNA molecule was modified with a fluorescent molecule TAMRA and the 5' end was modified with an aldehyde group, and the DNA molecule was named SiNW-No.1.

[0095] 2) Accurately prepare 1× PBS buffer (1.06 mM potassium dihydrogen phosphate, 3.00 mM sodium dihydrogen phosphate heptahydrate, 155 mM sodium chloride pH = 7.20)

[0096] Accurately prepare 200 mL of complete cell culture medium: 176 mL of DMEM, 20 mL of fetal bovine serum, 2 mL of penicillin-streptomycin solution (100×), and 2 mL of MEM non-essential amino acid solution (100×).

[0097] 3) A silicon nanowire array is prepared by metal-assisted chemical etching, and a silicon nanowire array with a silicon dioxide layer on the surface is obtained by thermal oxidation in a tube furnace.

[0098] 4) After the silicon nanowire array was heated with piranha solution (concentrated sulfuric acid and hydrogen peroxide volume ratio of 3:1) for 1 h for surface hydroxylation, it was amino-modified with aminopropyltriethoxysilane to obtain a surface amino-modified silicon nanowire array. The silicon nanowires were scraped off with a scalpel and dispersed in 1×PBS to obtain a silicon nanowire solution with a concentration of 0.2 mg / mL.

[0099] 5) Weigh sodium cyanoborohydride solid and dissolve it in 1×PBS to obtain a 0.3 M sodium cyanoborohydride solution.

[0100] 6) 100 μM of the above CHO-No.1-TAMRA dispersed in 1×PBS was annealed at 95°C and slowly cooled to room temperature. 33 μL of DNA solution was taken and mixed with 114 μL of silicon nanowire solution and 100 μL of sodium cyanoborohydride solution, and shaken at 1000 rpm overnight to obtain a silicon nanowire-based fluorescent sensor named SiNW-No.1.

[0101] 7) SiNW-No.1 was centrifuged at 13000 rpm for 10 min with 1×PBS, centrifuged three times, and finally dispersed in 600 μL of complete cell culture medium.

[0102] 8) PC12 cells were detached with pancreatic enzyme, and the cell solution was fully mixed with SiNW-No.1 solution, incubated in a cell culture incubator (37°C, 5% CO2 volume fraction) for 48 hours, and then stained with CMAC dye. CMAC dye was excited with a 405nm laser, and blue fluorescence signals in the 430nm to 490nm band were collected; TAMRA molecules on SiNW-No.1 were excited with 561nm, and orange fluorescence signals in the 570nm to 620nm band were collected.

[0103] Fluorescence images showing the relationship between SiNW-No.1 and cells Figure 7 As shown. Figure 7 It can be seen that the fluorescence of CMAC and SiNW-No.1 in the cell appear on the same focal plane, and the two overlap on the plane, indicating that SiNW-No.1 successfully enters the cell and can perform temperature measurement with high spatial resolution in a single cell.

[0104] Example 7

[0105] 1) Design and prepare the following temperature fluorescence sensor, the nucleotide sequence of the DNA molecule is shown in SEQ ID No: 1.

[0106] According to the reference (Nucleic Acids Research, 2003, Vol. 31, No. 13), the melting temperature of the DNA molecule under 1×PBS is T m The temperature of the DNA molecule was 36.2° C. The fluorescent molecule Texas Red was modified at the 3' end and named No.1-TR.

[0107] 2) Accurately prepare 1× PBS buffer (1.06 mM potassium dihydrogen phosphate, 3.00 mM sodium dihydrogen phosphate heptahydrate, 155 mM sodium chloride pH = 7.20)

[0108] 3) 100 μM of the above fluorescent sensor dispersed in 1×PBS was annealed at 95°C and slowly cooled to room temperature. The final concentration of the fluorescent sensor dispersed in 1×PBS in a cuvette was 200 nM. The excitation wavelength was fixed at 561 nm, and the temperature response curve of DNA was plotted with temperature as the abscissa and the fluorescence intensity at the normalized emission wavelength of 611.0 nm as the ordinate.

[0109] The temperature response curve of the fluorescence sensor obtained in the above steps is as follows: Figure 8 As shown. Figure 8 It can be seen that the fluorescence intensity of Texas Red is also affected by the opening of the DNA stem-loop structure as the temperature changes. m The fluorescence intensity nearby is more sensitive to temperature changes, indicating that the improvement of the temperature measurement sensitivity of fluorescent molecules by stem-loop DNA molecules is universal for fluorescent molecules. However, in order to make the sensor have better temperature measurement sensitivity, it is recommended to select fluorescent molecules that are more sensitive to temperature changes.

[0110] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A fluorescence sensor for temperature detection, characterized in that: The fluorescent sensor comprises a DNA molecule with a stem-loop structure and a fluorescent molecule; wherein the fluorescent molecule is modified at the 3' end or the 5' end of the DNA molecule.

2. The fluorescence sensor according to claim 1, characterized in that The sensitive temperature measurement range of the fluorescent sensor is the melting temperature T of the stem-loop structure DNA molecule. m ±5℃.

3. The fluorescence sensor according to claim 1 or 2, characterized in that: In the DNA molecule of the stem-loop structure, the number of bases on the stem side is ≥10, and the number of bases in the loop is ≤ the number of bases on the stem side.

4. The fluorescence sensor according to claim 1 or 2, characterized in that: The nucleotide sequence of the stem-loop structure DNA molecule is shown in SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No:

3.

5. The fluorescence sensor according to claim 1 or 2, characterized in that: The fluorescent molecule is a temperature-sensitive fluorescent molecule; Preferably, the fluorescent molecule is rhodamine B, carboxytetramethylrhodamine or a mesobenzene ring-substituted BODIPY derivative.

6. The fluorescence sensor according to claim 1, characterized in that: The fluorescent sensor further comprises a carrier; A functional group is modified at one end of the stem-loop structure DNA molecule that is not modified with the fluorescent molecule, so as to couple the DNA molecule modified with the fluorescent molecule to the surface of the carrier; Preferably, the functional group is an aldehyde group, a carboxyl group, an amino group, a thiol group or an azide group; Preferably, the carrier is silicon nanowires, nanoparticles or graphene.

7. The method for preparing a fluorescent sensor for temperature detection according to any one of claims 1 to 6, characterized in that: The preparation method comprises: Synthesize stem-loop structure DNA molecules according to the temperature measurement interval; Modifying the 3' end or 5' end of the DNA molecule with a fluorescent molecule; The sensitive temperature range of the fluorescent sensor is the melting temperature T of the stem-loop structure DNA molecule. m ±5℃; Preferably, the preparation method further comprises modifying a functional group on the end of the DNA molecule that is not modified with the fluorescent molecule, and then coupling it to the surface of the carrier.

8. Use of the fluorescent sensor for temperature detection according to any one of claims 1 to 6 in detecting temperature.

9. A method for detecting temperature, characterized in that: Detecting temperature using the fluorescent sensor for temperature detection according to any one of claims 1 to 6 comprises the following steps: Determine the excitation wavelength according to the fluorescent molecules modified on the fluorescent sensor, add the fluorescent sensor to the detection system, and adjust the temperature. Each temperature adjustment needs to keep the temperature constant for a period of time, and draw a temperature response standard curve with temperature as the horizontal axis and the fluorescence intensity at the maximum emission wavelength as the vertical axis; After incubating the fluorescent sensor with the object to be detected, the fluorescence intensity of the object to be detected at the maximum emission wavelength is obtained; The temperature of the object to be measured is calculated based on the temperature response standard curve and the fluorescence intensity at the maximum emission wavelength of the object to be measured.

10. The method according to claim 9, characterized in that The time for maintaining the temperature constant is more than 5 minutes.

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