Photothermal sensor based on wettable paper chip and its application in biological detection

Abstract

The present invention provides a photothermal sensing method based on a wettable paper chip, and uses miRNA as an example to verify the biological detection application of the method, which belongs to the field of biosensor technology. The present invention constructs a super-hydrophilic micro-well on a hydrophobic paper substrate, and then modifies a capture probe in the super-hydrophilic micro-well to prepare a miRNA photothermal sensor. In the photothermal sensor for miRNA detection, when miRNA is present, the capture probe, miRNA and reporter probe undergo hybridization in a sandwich structure, and the reporter probe further triggers a hybridization chain reaction of H1 and H2 probes, and the C-rich sequences at the tails of H1 and H2 can adsorb a large amount of Ag. + Under the action of Na2S, Ag2S nanoparticles are generated; the photothermal properties of Ag2S nanoparticles are used to convert the target concentration into temperature output, and a portable infrared thermal imager is used for quantitative analysis, thereby achieving instant quantitative detection of miRNA. The photothermal sensing method based on wettable paper chips proposed in the present invention has the advantages of small sample volume, high sensitivity, low cost, and high throughput.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and in particular relates to a photothermal sensor based on an infiltrative paper chip and a photothermal sensing method thereof, as well as an application in biological detection. Background Art

[0002] Point-of-care testing is a low-cost, simple, fast, portable testing technology that does not require professional technicians to operate. It not only meets the special requirements of time and space for testing, but also enables instant reporting of test results. However, in practical applications, due to the small amount of samples, low concentration of markers, and many interfering factors, the sensitivity and accuracy of currently developed point-of-care testing methods need to be further improved. In addition, most point-of-care testing methods determine the content of the target by color changes, and can only achieve qualitative or semi-quantitative detection. Therefore, there is an urgent need to develop highly sensitive, highly accurate, and quantifiable point-of-care testing technologies.

[0003] In order to achieve the goals of low cost, portability and simple operation, it is often not appropriate to introduce high-end instruments and equipment in the field of instant detection. Photothermal sensing, as an emerging sensing technology, can convert target signals into temperature signals and can achieve quantitative detection using only a portable thermometer. It has the advantages of simplicity, portability and sensitivity, and provides a new idea for instant quantitative detection of markers. However, the currently developed photothermal sensing technology is generally carried out in a solution system, which requires a large amount of samples and there is uneven heat conduction in the solution, making it difficult to ensure the accuracy and reproducibility of the detection, and it is difficult to achieve high-throughput detection of the target object, which limits the application of the photothermal sensing method to a certain extent. Therefore, there is an urgent need to develop a photothermal sensing method with low sample volume, high sensitivity and high throughput. Summary of the invention

[0004] In view of the problems that existing photothermal sensing technology requires a large number of samples, complex labeling, uneven heat transfer and cannot achieve high throughput, the present invention proposes a photothermal sensor and method based on a wettable paper chip. Taking miRNA as an example, the temperature signal is used as the output signal in biological detection. The temperature signal is read by a portable infrared temperature imager, thereby realizing portable quantitative detection of the target object.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is:

[0006] The photothermal sensor based on the wettability paper chip is constructed by hydrophobically modifying the surface of the hydrophilic paper substrate to construct a super-hydrophilic micro-well; then, the hydroxyl groups in the super-hydrophilic micro-well are oxidized to aldehyde groups using NaIO4, and finally, the capture probe is modified in the super-hydrophilic micro-well to obtain the photothermal sensor based on the wettability paper chip.

[0007] The preparation method of the above-mentioned photothermal sensor based on the wettable paper chip mainly comprises the following steps:

[0008] 1) constructing super-hydrophilic micro-wells after hydrophobic modification on the paper substrate surface;

[0009] 2) NaIO4 solution is added dropwise to the super-hydrophilic microwells, reacted in the dark for 30 min-1 h, rinsed with HEPES buffer, and dried with nitrogen; then a phosphate buffer containing a capture probe is added, reacted for more than 2 h, and finally rinsed with HEPES buffer and dried with nitrogen to obtain a photothermal sensor based on a wettable paper chip.

[0010] According to the above scheme, the paper base is a hydrophilic paper base selected from printing paper, cellulose filter paper, nitrocellulose membrane, etc.

[0011] According to the above scheme, step 1) is specifically as follows: immersing the hydrophilic paper substrate in a n-hexane solution containing octadecyltrichlorosilane to achieve hydrophobic modification of the paper substrate; then, fixing the photomask on the hydrophobically modified paper substrate, and using plasma etching to construct a super-hydrophilic micro-well or a super-hydrophilic micro-well array on the hydrophobically modified paper substrate. The plasma etching time is generally 3-30s, and the super-hydrophilic micro-well is formed in the area through the hole on the photomask, and the other areas are still hydrophobic areas, and the contact angle of the super-hydrophilic micro-well is 0°.

[0012] According to the above scheme, in step 1), after the hydrophilic paper substrate surface is hydrophobically modified, the contact angle of the hydrophobic surface is above 135°, generally within the range of 135°-150°.

[0013] According to the above scheme, in step 1), the area of ​​the super-hydrophilic micro-well is 0.5–7 mm 2 Preferably, the shape of the super-hydrophilic micro-well is generally circular, with a diameter of 0.5-3 mm.

[0014] According to the above scheme, in step 2), the concentration range of NaIO4 solution is 0.5–1M; the amount of NaIO4 solution added is 2.5–7.5 μL / mm 2 .

[0015] According to the above scheme, in step 2), the capture probe is a sequence used to identify and capture the target, and is a biological sequence that can specifically bind to the target detected by the photothermal sensor, thereby identifying and capturing the target.

[0016] According to the above scheme, in step 2), the concentration of the capture probe is 1–10 μM, and the amount of the capture probe added is 2.5–7.5 μL / mm 2 .

[0017] According to the above scheme, in step 2), NaBH3CN is also added to the phosphate buffer of the capture probe at a concentration of 0-0.2%.

[0018] According to the above scheme, in step 2), the HEPES buffer concentration is 10 mM, and the pH value is 7-8; the phosphate buffer concentration is 10 mM, and the pH value is 7-8.

[0019] The present invention also provides an application of the above-mentioned photothermal sensor based on the wettable paper chip to detect biomolecules by photothermal sensing method for real-time quantitative detection of biomolecules. The present invention takes miRNA-182 as an example, and the specific detection steps are as follows:

[0020] a) adding a mixed solution containing a reporter probe, H1, H2, AgNO3 and different concentrations of miRNA to the super-hydrophilic micro-well of the photothermal sensor based on the wettable paper chip of the present invention, reacting at 37° C. for more than 2 h, then washing with HEPES buffer and drying with nitrogen gas;

[0021] b) adding Na2S solution to the super-hydrophilic micro-wells obtained in step a), reacting for a period of time, and then absorbing the solution in the micro-wells;

[0022] c) irradiating the super-hydrophilic micro-well obtained in step b) with a near-infrared laser, and recording the temperature change with an infrared thermal imager, and then drawing a standard curve with the logarithm of the miRNA concentration as the horizontal axis and the temperature change value as the vertical axis, so as to calculate the concentration of the miRNA in the sample to be tested according to the standard curve.

[0023] According to the above scheme, in step a), the amount of the mixed solution added to the super-hydrophilic microwell is generally 2.5–7.5 μL / mm 2 ; In the mixed solution in the super-hydrophilic microwell, the concentration of AgNO3 is 2–4mM, and the concentration range of the reporter probe, H1, and H2 is 0.25–2μM, where the molar ratio of the reporter probe to H1 and H2 is between 1:(1–10):(1–10); the concentration range of MiRNA is above 50pM, generally 50pM–100nM.

[0024] According to the above scheme, in step b), the concentration range of Na2S solution is 5–20 mM, and the addition amount is 2.5–7.5 μL / mm 2 ; The reaction time is generally within 5 minutes.

[0025] According to the above scheme, the miRNA-182 sequence is 5'-UUU GGC AAU GGU AGA ACU CAC ACU-3'.

[0026] According to the above scheme, the reporter probe sequence is 5'-ACC ATT GCC AAA AAC GCT AAT CAC CTG-3'; the capture probe sequences are 5'-NH2-(CH2)6-TTT TAG TGT GAG TTC T-3'.

[0027] According to the above scheme, the H1 and H2 sequences are 5'-CCC CCC CCC CCC ATC ACC TGC TTT TTTCAG GTG ATT AGC GTT-3' and 5'-CCC CCC CCC CCC AAA AAA GCA GGT GAT AAC GCT AATCAC CTG-3', respectively.

[0028] The principle of the present invention is to construct a photothermal sensing platform based on a wettable paper chip to achieve sensitive, quantitative, instant, and high-throughput detection of miRNA. Figure 1 As shown, the present invention first constructs a super-hydrophilic micro-well on the surface of a hydrophobic paper substrate, and modifies the capture probe for subsequent detection. Due to the difference in wettability between the super-hydrophilic micro-well and the substrate, the present invention can limit the reaction droplets to the super-hydrophilic micro-well area, enrich the detection signal, and improve the detection sensitivity. When a target (such as miRNA) is present, the capture probe, miRNA, and reporter probe hybridize in a sandwich structure and are fixed in the super-hydrophilic micro-well. Subsequently, the reporter probe further triggers a hybridization chain reaction of the H1 and H2 probes. The C-rich sequences at the tails of H1 and H2 can adsorb a large amount of Ag. + , under the action of Na2S, Ag2S nanoparticles are generated. The photothermal properties of Ag2S nanoparticles are used to generate heat under near-infrared light excitation, and the concentration of the target is converted into a temperature signal output, and quantitative analysis and detection can be achieved using a portable thermal imager.

[0029] In addition, the detection target of the present invention is not limited to miRNA. Other targets such as proteins, small molecules, etc. are combined using a sandwich structure and induce HCR reaction. The method of further generating photothermal materials on the HCR product should also be considered as the protection scope of the present invention.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The photothermal sensor of the wettable paper chip of the present invention uses a temperature signal as an output signal, reads the temperature signal by a portable infrared temperature imager, and realizes portable quantitative detection of the target object by a photothermal sensing method, avoiding the use of large instruments and equipment and the need for professional operators.

[0032] 2. The present invention utilizes the signal enrichment capability of super-hydrophilic micro-wells and the HCR dual strategy to improve the detection sensitivity. At the same time, since the size of the micro-wells is 1-3 mm, only a few microliters of sample are required, which greatly reduces the required sample volume. After the Ag2S nanoparticles are generated, the solution in the micro-wells is removed before photothermal detection, which not only shortens the laser irradiation time, but also avoids the problem of uneven heat conduction in the detection system, thereby improving the accuracy of detection. A large number of super-hydrophilic micro-wells can be constructed on the same hydrophobic paper substrate, thereby realizing high-throughput detection of the target object.

[0033] 3. The present invention can not only realize high-throughput detection of the target by constructing a superhydrophilic-hydrophobic pattern on a paper substrate, but also limit the reaction to the superhydrophilic microwell area through the difference in wettability between the superhydrophilic microwell and the hydrophobic substrate, so that the detection signal can be enriched and the sensitivity of analysis and detection can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of miRNA photothermal sensing based on wettable paper chip.

[0035] Figure 2 This is a characterization diagram of the contact angle of the hydrophobic surface of the wettable paper chip.

[0036] Figure 3 Characterization diagram of contact angle of super-hydrophilic micro-wells in wettable paper chip

[0037] Figure 4 Actual picture of red aqueous droplets fixed in superhydrophilic micro-wells.

[0038] Figure 5 Thermal images of miRNA responses at different concentrations taken by a portable infrared temperature imager and the corresponding standard curve.

[0039] Figure 6 This is a selective experiment diagram.

[0040] Figure 7 This is a diagram of a reproducible experiment.

[0041] Figure 8 This is a diagram of the miRNA detection experiment in serum. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0043] In the following examples, Ag2S nanoparticles were synthesized in situ in microwells using the C-rich fragments in the hairpin probes H1 and H2 as templates, and had a spherical morphology and a particle size of about 7 nm.

[0044] In the following embodiments, the power of the near-infrared laser is 1.75 W and the wavelength is 808 nm.

[0045] Example

[0046] 1. Preparation of hydrophobic paper substrate: Cut cellulose filter paper into 2.5 cm × 2.5 cm size, soak in n-hexane solution containing 3% octadecyltrichlorosilane, and react for 2 hours. Then rinse the paper substrate with ethanol and dry it in an oven at 45°C to obtain a hydrophobic paper substrate ( Figure 2 ), and the hydrophobic surface contact angle is 146.6±2.3°.

[0047] 2. Preparation of super-hydrophilic microwell array: A 2.5 cm × 2.5 cm, 1 mm thick steel mask was made, with 4 × 4 circular holes of the same size and 1 mm in diameter on the mask. The mask was placed on a hydrophobic paper substrate and fixed with a binder clip. The paper substrate covered with the mask was placed in a plasma cleaner for 5 seconds. The exposed portion of the circular holes on the mask was converted back into a super-hydrophilic ( Figure 3 ), while the covered part remains hydrophobic, thus obtaining a wettable paper base. Figure 4 As shown, the red droplets will be confined in the superhydrophilic microwells and will not diffuse outward, and each droplet is an independent reaction system, without cross contamination between droplets.

[0048] 3. Super-hydrophilic micro-well modified capture probe: 2 μL 0.5M NaIO4 was added to the super-hydrophilic micro-well, reacted in the dark for 0.5 h, then rinsed with HEPES buffer and dried with nitrogen. Then, 4 μL of phosphate buffer containing 0.7 μM capture probe and 0.1% NaBH3CN was added to the micro-well, reacted at room temperature for 2 h. Then, rinsed with HEPES buffer, dried with nitrogen, and stored at 4°C for future use.

[0049] The 5' end of the capture probe is modified with an amino group, and its sequence is: 5'-NH2-(CH2)6-TTT TAG TGT GAG TTCT-3'.

[0050] 4. Add 4μL of a mixture of target miRNA-182 (the concentrations of miRNA-182 are 0nM, 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 50nM, and 100nM, respectively), 0.5μM reporter probe, 1μM H1, 1μM H2, and 3mM AgNO3 to the super-hydrophilic micro-wells, and react at 37°C for 2.5h. After the reaction is completed, rinse with HEPES buffer and blow dry with nitrogen. Next, add 2μL of 10mM Na2S solution to the super-hydrophilic micro-wells, react for 5 minutes, and then remove the solution from the micro-wells. Finally, irradiate the micro-wells with an 808nm laser, and record the temperature with a portable infrared thermal imager. Plot the relationship between the increased temperature value and the logarithm of the concentration of the target miRNA-182 to obtain a standard curve ( Figure 5 ), and the linear regression equation calculated according to the standard curve was △T=8.07lgC+15.32 (△T is the temperature value increased compared with the initial temperature, C is the concentration of the target miRNA), and the linear correlation coefficient R=0.995.

[0051] The miRNA-182 sequence is: 5'-UUU GGC AAU GGU AGA ACU CAC ACU-3'; the reporter probe sequence is: 5'-ACC ATT GCC AAA AAC GCT AAT CAC CTG-3'; the H1 sequence is: 5'-CCC CCC CCC CCCATC ACC TGC TTT TTT CAG GTG ATT AGC GTT-3'; the H2 sequence is 5'-CCC CCC CCC CCC AAA AAAGCA GGT GAT AAC GCT AAT CAC CTG-3'.

[0052] 5. Selectivity experiment: The target miRNA-182, interfering miRNA-375, miRNA-205, miRNA-155 and blank group were used to verify the selectivity of the sensor, and the concentrations were all 100nM. The detection conditions were the same as those for establishing the standard curve. Figure 6As shown in the figure, when miRNA-375, miRNA-205, and miRNA-155 are present, the temperature in the microwells does not change significantly, and the temperature change is similar to that of the blank group. When the target miRNA is present, the temperature of the microwell increases significantly. These conditions indicate that due to the high sequence dependence of nucleic acid hybridization, the photothermal sensor has good selectivity for the detection of the target miRNA-182. The target miRNA-182 sequence is: 5'-UUU GGC AAU GGU AGA ACU CAC ACU-3'; the interfering miRNA-375 sequence is: 5'-UAG CUU AUC AGA CUG AUG UUG A-3'; the interfering miRNA-205 sequence is: 5'-UCC UUC AUU CCACCG GAG UCU G-3'; the interfering miRNA-155 sequence is: 5'-UUA AUG CUA AUC GUG AUA GGG G-3'.

[0053] 6. Reproducibility experiment: The sensor established by the present invention was used to detect different concentrations of target miRNA-182. Each concentration was detected 6 times using 6 independent sensors. The detection conditions were the same as those for establishing the standard curve. Figure 7 As shown, at the same concentration, the temperature rise values ​​are consistent. For 0nM, 0.5nM, 5nM, and 50nM target miRNA, the standard deviations of six repeated measurements are 7.08%, 4.71%, 5.17%, and 2.42%, respectively, indicating that the photothermal sensor constructed in the present invention has good repeatability.

[0054] 8. Determination of target miRNA-182 in serum: Fetal bovine serum was diluted to 1%, and different concentrations of target miRNA-182 were added, including 5nM, 10nM, 50nM, and 100nM. The detection conditions were the same as those for establishing the standard curve. The temperature change was measured using the photothermal sensor constructed by the present invention. Figure 8 As shown, for serum samples containing different concentrations of miRNA-182, the detection results of the detection method established by the present invention in serum are consistent with the detection results in buffer, indicating that the established sensing method has good anti-interference ability and accuracy.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A photothermal sensing method for biomolecule detection, characterized in that: The main steps are: a) adding a mixed solution containing a reporter probe, hairpin probes H1 and H2, AgNO3 and different concentrations of the target to the super-hydrophilic microwell of the photothermal sensor, reacting at 36-38°C for more than 2 hours, then washing with a buffer solution and drying; b) adding Na2S solution to the super-hydrophilic micro-wells obtained in step a), reacting for a period of time, and then absorbing the solution in the micro-wells; c) irradiating the super-hydrophilic micro-well obtained in step b) with a near-infrared laser, and recording the temperature change with an infrared thermal imager, and then drawing a standard curve with the logarithm of the target concentration as the abscissa and the temperature change value as the ordinate, so as to calculate the concentration of the target in the sample to be tested according to the standard curve; The preparation method of the photothermal sensor is to first construct a super-hydrophilic micro-well after hydrophobic modification on the surface of a hydrophilic paper substrate; Then the hydroxyl groups in the super-hydrophilic micro-wells were oxidized to aldehyde groups, and finally the capture probes were modified in the super-hydrophilic micro-wells to obtain a photothermal sensor based on a wettable paper chip. The target is a biomolecule that can hybridize with the capture probe and the reporter probe in a sandwich structure; the reporter probe can trigger a hybridization chain reaction between the hairpin probes H1 and H2; the tails of the hairpin probes H1 and H2 contain C-rich sequences that can adsorb Ag. + .

2. A photothermal sensing method for biomolecule detection according to claim 1, characterized in that The main steps of the preparation method of the photothermal sensor based on the wettable paper chip are as follows: 1) constructing super-hydrophilic micro-wells after hydrophobic modification on the paper substrate surface; 2) NaIO4 solution is added dropwise to the super-hydrophilic micro-wells, and the hydroxyl groups in the super-hydrophilic micro-wells are oxidized to aldehyde groups after a reaction for a period of time, and then the solution is rinsed with a buffer solution and dried; then a phosphate buffer solution containing a capture probe is added, and the solution is reacted for more than 2 hours, and finally the solution is rinsed with a buffer solution and dried to obtain a photothermal sensor based on a wettable paper chip.

3. A photothermal sensing method for biomolecule detection according to claim 2, characterized in that Step 1) is specifically as follows: immersing the hydrophilic paper substrate in a n-hexane solution containing octadecyltrichlorosilane to achieve hydrophobic modification of the paper substrate; then, fixing a photomask on the hydrophobically modified paper substrate, and using plasma etching to construct a super-hydrophilic micro-well or a super-hydrophilic micro-well array on the hydrophobically modified paper substrate.

4. A photothermal sensing method for biomolecule detection according to claim 2, characterized in that In step 1), the area of ​​the super-hydrophilic micro-well is 0.7-7 mm 2 step 2), the concentration range of NaIO4 solution is 0.5-1M; the amount of addition is 2.5-7.5μL / mm 2 .

5. A photothermal sensing method for biomolecule detection according to claim 2, characterized in that In step 2), the concentration of the capture probe is 1-10 μM, and the amount of the capture probe added is 2.5-7.5 μL / mm 2 .

6. A photothermal sensing method for biomolecule detection according to claim 1, characterized in that In step a), the amount of the mixed solution added to the super-hydrophilic microwell is 2.5-7.5 μL / mm 2 ; In the mixed solution in the super-hydrophilic microwell, the concentration of AgNO3 is 2-4mM, and the concentration range of the reporter probe, H1, and H2 is 0.25-2μM, where the molar ratio of the reporter probe to H1 and H2 is 1:(1-10):(1-10); the concentration range of the target is greater than 50pM.

7. A photothermal sensing method for biomolecule detection according to claim 1, characterized in that In step b), the concentration range of the Na2S solution is 5-20 mM, and the amount of addition is 2.5-7.5 μL / mm 2 ; The reaction time is within 5 minutes.

8. The photothermal sensing method for biomolecule detection according to claim 1, characterized in that The target is miRNA-182; the reporter probe sequence is 5'-ACC ATT GCC AAA AAC GCT AAT CAC CTG-3'; the capture probe sequence is 5'-NH2-(CH2)6-TTT TAG TGT GAG TTC T-3'; the H1 and H2 sequences are 5'-CCC CCC CCC CCC ATC ACC TGC TTT TTT CAG GTG ATT AGC GTT-3' and 5'-CCC CCC CCCCCC AAA AAA GCA GGT GAT AAC GCT AAT CAC CTG-3', respectively.

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