CN114942332A - Preparation and application of pump-free micro-fluidic chip for thrombin detection - Google Patents
Preparation and application of pump-free micro-fluidic chip for thrombin detection Download PDFInfo
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Abstract
The invention discloses a preparation method of a pump-free micro-fluidic chip, which comprises the following steps of; step S1, surface modification of the gold-plated substrate; step S2, manufacturing the pump-free micro-fluidic chip; step S3, manufacturing a finger-pressure pump for driving liquid to flow in the pump-free micro-fluidic chip; step S4, embedding the gold-plated substrate and sealing the chip. The invention also provides a technical scheme of the application of the pump-free micro-fluidic chip prepared by the pump-free micro-fluidic chip preparation method in thrombin detection. The pump-free micro-fluidic chip designed by the invention has low manufacturing cost, does not need professional operation, and has simple detection operation process. High sensitivity, short detection time and low detection lower limit are realized.
Description
Technical Field
The invention relates to a surface enhanced Raman spectroscopy detection method, in particular to preparation of a SERS-based pump-free micro-fluidic chip and application of the SERS-based pump-free micro-fluidic chip in thrombin detection.
Background
Surface-Enhanced Raman Spectroscopy (SERS) is a powerful vibrational Spectroscopy technique that provides highly sensitive detection of low concentrations of target analytes by amplifying the electromagnetic field generated by local Surface plasmon excitation. Raman spectra of pyridine on coarse silver were first observed in 1974, however, researchers at that time did not recognize that these spectra are caused by any unusual, enhanced or new phenomena. Subsequently, since SERS was formally discovered in 1977, countless researchers have invested great interest and conducted extensive research. SERS enhancers are products of a combination of electromagnetic and chemical enhancement mechanisms. For a highly optimized surface, it may be close to about 1010 and 1011. The advent of SERS has made raman a viable option for biosensing because it allows raman spectroscopic detection of a large number of biomolecules using relatively simple laboratory equipment and even field portable equipment. SERS has high sensitivity, enables the extraction of different spectra from structurally and functionally similar molecules, and eliminates expensive reagents or time-consuming sample preparation steps associated with other techniques such as Polymerase Chain Reaction (PCR).
Thrombin (Thrombin) is a serine protease with multiple functions that can regulate multiple cellular functions including homeostasis, platelet activation, tissue regeneration, inflammation and cancer progression. Thrombin is a major effector protease of the coagulation cascade and, once activated, produces a clot by the process of converting fibrinogen to fibrin. Not only can play a role in procoagulant activity, but also plays a role in coordinating proinflammatory reaction of cells in cells. Overexpression of thrombin promotes inflammatory cytokines, adhesion molecules, angiogenic factors and matrix degrading proteases, thereby inducing metastasis, proliferation and angiogenic processes of tumor cells. In the coagulation process associated with malignant tumors, thrombin concentrations can reach micromolar levels. On the other hand, it has been shown that even patients suffering from diseases associated with abnormal blood coagulation have a high picomolar level of thrombin in their blood. Therefore, in view of the above characteristics, thrombin is likely to be a novel diagnostic factor and also a clinically valuable cancer marker, and its concentration in blood is an indispensable biomarker for tumor diagnosis.
Engvoll and Perlmann two Switzerland scientists developed enzyme-linked immunosorbent assay (ELISA) by modifying Radioimmunoassay (RIA) in 1971, and used this new method to determine IgG levels in rabbit sera. ELISA is a heterogeneous immunoassay technique for detecting soluble antigens and specific antibodies, and various types of ELISA have been developed to improve the specificity of detection based on the specificity of the structure and characteristics of a substance to be detected. One of the ELISA types is the "sandwich" of the double antibody sandwich technique in this modification method, specific antibodies are first adsorbed on a plate, and then reacted with a test sample containing an antigen, and enzyme-labeled specific antibodies are added followed by addition of an enzyme substrate. The "antigen" in the test sample is "captured" and immobilized on the sensitized plate, which can then itself immobilize the antibody labeled by the enzyme. The method has high sensitivity and specificity for detecting high molecular weight antigen.
Aptamers are specific DNA or RNA strands selected from a large library of nucleic acids, which are phylogenetically evolved using the "phylogenetic evolution of ligands by exponential enrichment (SELEX)" process and are capable of binding to target molecules with extremely high specificity and affinity. The specific flow of the SELEX scheme is as follows: the random nucleic acid library is incubated with the target molecules and unbound molecules are separated from the bound molecules. The bound nucleic acid is eluted, amplified by PCR, and provides an abundant nucleic acid library for the next cycle. For each target molecule, 6 to 12 consecutive cycles will be performed, and the final enriched pool will be cloned and sequenced. Unlike antibody production methods that rely on induction of the animal's immune system, the SELEX process enables the production of aptamers for non-immunogenic and toxic targets, which are also referred to as "chemical antibodies" because aptamers cannot be obtained by the immune system alone without going through the SELEX process. Compared to antibodies or enzymes that are proteins in nature, aptamers are much smaller in size and complexity, and are highly chemically stable, easy to manufacture, and stable during storage. In view of the above advantages, aptamers are gradually becoming a good alternative to target molecule recognition antibodies.
Disclosure of Invention
The invention aims to solve the problems that the detection is difficult due to low content of thrombin in blood, the operation convenience of an instrument is improved, and the dependence on large-scale precise instruments and professional operation in clinical application is relieved, and develops the preparation of a SERS-based pump-free microfluidic chip with simple operation, low manufacturing cost, short detection time, low detection lower limit and high sensitivity and the application thereof in thrombin detection.
In order to achieve the purpose of the invention, the invention provides the following technical scheme:
a preparation method of a pump-free micro-fluidic chip comprises the following steps;
step S1, surface modification of the gold-plated substrate;
in step S1, the surface modification method of the gold-plated substrate includes:
soaking the gold-plated substrate after hydrophilic treatment in 1mL of a section of DNA aptamer TBA15 solution of 70 nM thrombin for modification of TBA15 aptamer thereon;
then placing the aptamer solution into a vacuum pump to continuously vacuumize for 20 minutes so as to remove gas in the aptamer solution;
after the treatment, the aptamer solution soaked with the gold-plated substrate is put into a honeycomb oscillator to be oscillated for 24 hours;
after the oscillation is finished, taking the treated gold-plated substrates out of the aptamer solution by using tweezers, and putting the gold-plated substrates into a centrifuge tube filled with 1mL of 2 mM 6-MCH solution for soaking for 2 hours;
after soaking, the materials are respectively washed for a plurality of times by deionized water and alcohol, and are used after being naturally dried.
TBA15 DNA aptamer sequence: 5 '-dithiol-TTTTTTTTTTGGTTGGTGTGGTTGG-3';
step S2, manufacturing the pump-free micro-fluidic chip;
the design and manufacturing method of the pump-free micro-fluidic chip comprises the following steps: the design drawing of the pump-free microfluidic chip is carried out on the software Auto CAD2018, the drawn pattern is printed on a transparent film sheet after the design drawing is finished, and the pattern is solidified on a silicon membrane plate by using an ultraviolet photoetching machine. Mixing Polydimethylsiloxane (PDMS) and a curing agent according to the mass ratio of 10:1, pouring the mixture on a silicon template for curing, and obtaining the PDMS chip through mould inversion. The chip length is 45 mm, width is 15 mm, and height is 10 mm.
Step S3, manufacturing a finger-pressure pump for driving liquid to flow in the pump-free micro-fluidic chip;
the manufacturing method of the finger-pressure pump for driving liquid to flow in the chip comprises the following steps: before pouring PDMS on a silicon template with a cured chip pattern, placing a cylindrical copper block with the height of 8 mm and the radius of 5 mm on a specified position on the silicon template as a reserved area of a finger-pressure pump, and then pouring PDMS and a curing agent which are mixed according to the mass ratio of PDMS to the curing agent of 10: 1;
and after the liquid level of the PDMS is stable, placing the culture dish on a heating plate at 75 ℃ for heating, blowing off bubbles generated by heating by using an ear washing ball in time, covering the culture dish with a cover after the bubbles disappear, transferring the culture dish into an oven at 75 ℃ for heating for 2 hours to solidify the PDMS, and obtaining the pump-free microfluidic chip made of the PDMS after heating.
Step S4, embedding the gold-plated substrate and sealing the chip.
The embedding of the gold-plated substrate and the sealing method of the chip comprise the following steps: placing a PDMS chip with one surface of a micro-channel above the micro-channel and placing the PDMS chip and a glass slide into a plasma cleaning machine, firstly vacuumizing the interior of the machine for 70 seconds, and then carrying out oxygen plasma oxidation treatment for 50 seconds by ultraviolet irradiation; after the treatment, taking the two out of the plasma cleaning machine, and quickly placing the gold-plated substrate subjected to hydrophilic treatment and surface modification into a hexagonal cavity arranged in a PDMS chip; after the placement, the slide was sealed to obtain a pump-free microfluidic chip made of PDMS.
The principle that the gold-plated substrate needs to be placed quickly before the PDMS chip is sealed with the glass slide is as follows: the PDMS chip and the glass slide are treated in the plasma cleaning machine in order to open silicon-oxygen bonds on the surfaces of the PDMS chip and the glass slide, but the opened chemical bonds can be closed again at normal temperature and normal pressure, if the gold-plated substrate is not placed in the chip in time, the PDMS chip and the glass slide cannot be tightly combined, and the liquid leakage phenomenon can occur after the reagent is introduced. Therefore, the gold-plated substrate is placed in the PDMS chip within 5 seconds, and then the PDMS chip is sealed with the glass slide, so that the gold-plated substrate and the glass slide can recombine chemical bonds after contacting, thereby forming irreversible sealing and avoiding the liquid leakage phenomenon in the experimental process.
The hydrophilic treatment method of the gold-plated substrate comprises the following steps: placing the gold-plated substrate into a plasma cleaning machine, firstly vacuumizing the interior of the machine for about 70 seconds, then carrying out oxygen plasma oxidation treatment for 5 minutes by ultraviolet irradiation, and taking out the gold-plated substrate to obtain the hydrophilic substrate surface.
Preferably, two flow limiting valves with a height of 50 μm are arranged inside the pump-free microfluidic chip to reduce the flow rate of thrombin and thrombin detection probes in the chip, and the thrombin and thrombin detection probes can sufficiently react with each other before flowing to the gold-plated substrate subjected to hydrophilic treatment and surface modification, so as to increase immune complexes which may be formed on the surface of the gold-plated substrate.
The invention also provides a technical scheme of the application of the pump-free micro-fluidic chip prepared by the pump-free micro-fluidic chip preparation method in thrombin detection.
The technical scheme of the application of the pump-free micro-fluidic chip in thrombin detection specifically adopts a Raman detection method for detection, and the specific steps comprise:
firstly adding thrombin from a liquid inlet by using a pipette, then simultaneously introducing gold nanoparticles serving as a thrombin detection probe and modifying the aptamer TBA29 and the Raman reporter MGITC, and fully reacting thrombin antigen with the detection probe under the action of a snake channel, thereby forming an immune complex with a sandwich structure on a gold-plated substrate incubated with the TBA15 aptamer;
after the reagent is introduced, standing the chip, and after the reaction is carried out for a period of time, introducing PBS buffer solution from the liquid inlet to wash the chip for multiple times for washing off thrombin and thrombin detection probes which are not combined with the TBA15 aptamer on the gold-plated substrate;
finally, the immune complex formed on the gold-plated substrate is subjected to Raman detection according to the principle of controlling variables.
The Raman characteristic peak shift of the thrombin detection probe marked by the Raman reporter molecule MGITC is 1614cm -1 By detecting 1614cm -1 The peak intensity is used for qualitative and quantitative detection of the thrombin. The Raman spectrum is collected by a LabRam HR Evolution system, the power of a helium-neon laser is 10 mW, the selected laser wavelength is 633 nm, and the detection Raman shift range is set to be 700-one 1700cm -1 The integration time was 10 seconds, and the number of integrations was 3 times. The thrombin concentrations to be tested were 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM and 10nM, respectively.
The preparation method of the thrombin detection probe comprises the following steps: first 1 μ L of 0.5M tris (2-carboxyethyl) phosphine (TCEP) was mixed with 50 μ L of a stretch of DNA aptamer TBA29 of 100 μ M thrombin and incubated at room temperature for 6 hours to generate free thiol;
then 100. mu.L of 1. mu.M TBA29 aptamer solution was added to 1mL of gold nanoparticle solution, followed by 2.5M NaCl solution and 0.5M PBS buffer solution to give final concentrations of 0.1M and 10 mM, respectively;
the volumes of the NaCl solution and the PBS buffer solution required to be added are respectively 46.8 mu L and 23.4 mu L by calculation;
incubating the solution in a dry thermostat at 50 ℃ for 48 hours, continuing aging, evenly dividing six times in 48 hours, and adding NaCl solution into the TBA29 aptamer solution, namely adding 7.8 mu L of NaCl solution each time;
two hours before the end of the 48 hour incubation, 4. mu.L of 10 was added -5 M, a Raman reporter MGITC;
finally, the mixed solution is placed in a differential centrifuge to be centrifuged for 15 minutes at the speed of 5500 rpm, a liquid-transfering gun is used for removing the supernatant to remove the free aptamer and MGITC which are not combined with the gold nanoparticles, the precipitate is suspended in PBS buffer solution for standby,
wherein the content of the first and second substances,
TBA29 DNA aptamer sequence: 5 '-dithiol-TTTTTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'.
The synthesis method of the gold nanoparticles comprises the following steps: adding 49 mL of deionized water and 500 mu L of 1% tetrachloroauric acid solution into a clean three-neck flask, placing the mixture into a heating sleeve on a constant-temperature magnetic stirrer, and heating; after boiling, 500 mu L of 1 percent sodium citrate solution is added by a pipette, and after the liquid color is completely changed from transparent to purple, the liquid is heated for 1 hour to cure the synthesized gold nanoparticles, thereby synthesizing the gold nanoparticles with the particle size of about 40 nm.
The invention has the advantages and beneficial effects that:
1. the pump-free micro-fluidic chip designed by the invention has low manufacturing cost, does not need to be operated by professionals, and has simple detection operation process.
And 2, SERS detection realizes high sensitivity, short detection time and low detection lower limit.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of a pump-free microfluidic chip of the present invention;
FIG. 2 is a schematic diagram of the fabrication and assembly of the pump-free microfluidic chip of the present invention;
FIG. 3 is a schematic diagram of the operation of a flow-limiting valve inside the pump-free microfluidic chip of the present invention;
FIG. 4 is a schematic representation of the formation of an immunocomplex on a gold plated substrate;
FIG. 5 is a gradient Raman spectrum of thrombin concentration and its corresponding linear regression plot.
Detailed Description
The technical solution in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.
Fig. 1 is a schematic diagram of a pump-free microfluidic chip of the present invention. It can be seen that the pump-free microfluidic chip mainly comprises three parts, namely a PDMS chip body, a gold-plated substrate and a glass slide.
Fig. 2 is a schematic diagram of the fabrication and assembly of the pump-free microfluidic chip of the present invention. The preparation and assembly method comprises the following steps in sequence:
step S1, surface modification of the gold-plated substrate;
in step S1, the surface modification method of the gold-plated substrate includes:
soaking the gold-plated substrate after hydrophilic treatment in 1mL of a section of DNA aptamer TBA15 solution of 70 nM thrombin for modification of TBA15 aptamer thereon;
then placing the aptamer solution into a vacuum pump to continue vacuumizing for 20 minutes so as to remove gas in the aptamer solution;
after the treatment, the aptamer solution soaked with the gold-plated substrate is put into a honeycomb oscillator to be oscillated for 24 hours;
after the oscillation is finished, taking the treated gold-plated substrates out of the aptamer solution by using tweezers and putting the gold-plated substrates into a centrifuge tube filled with 1mL of 2 mM 6-MCH solution for soaking for 2 hours;
after soaking, the materials are respectively washed for a plurality of times by deionized water and alcohol, and are used after being naturally dried.
TBA15 DNA aptamer sequence: 5 '-dithiol-TTTTTTTTTTGGTTGGTGTGGTTGG-3';
step S2, manufacturing the pump-free micro-fluidic chip;
and (3) designing the picture on the Auto CAD2018 software, printing the drawn pattern on a transparent film after the drawing is finished, and curing the pattern on a silicon membrane by using an ultraviolet photoetching machine. Before pouring PDMS on a silicon template with a cured chip pattern, a cylindrical copper block with the height of 8 mm and the radius of 5 mm is placed at a specified position on the silicon template as a reserved area of a finger-pressure pump, and then the PDMS and the curing agent which are well mixed according to the mass ratio of the PDMS to the curing agent of 10:1 are poured. After the PDMS liquid level is stable, the culture dish is placed on a heating plate at 75 ℃ for heating, and air bubbles generated by heating are blown off by an ear washing ball in time. After all the bubbles disappeared, the petri dish was covered with a lid and transferred to an oven at 75 ℃ for 2 hours to cure the PDMS. After the heating, the pump-free chip made of PDMS was obtained.
Wherein, the hydrophilic treatment of the gold-plated substrate: the gold-plated substrate was placed in a plasma cleaner, and first, the interior of the plasma cleaner was evacuated for about 70 seconds, and then, oxygen plasma oxidation treatment was performed for 5 minutes by ultraviolet irradiation. After removal, a hydrophilic substrate surface is obtained.
Step S2, manufacturing the pump-free micro-fluidic chip;
the design and manufacturing method of the pump-free micro-fluidic chip comprises the following steps: the design drawing of the pump-free microfluidic chip is carried out on the software Auto CAD2018, the drawn pattern is printed on a transparent film sheet after the design drawing is finished, and the pattern is solidified on a silicon membrane plate by using an ultraviolet photoetching machine. And pouring the mixture on a silicon template for curing after the mixture is mixed according to the mass ratio of Polydimethylsiloxane (PDMS) to a curing agent of 10:1, and obtaining the PDMS chip through mould pouring. The chip length is 45 mm, width is 15 mm, and height is 10 mm.
Step S3, manufacturing a finger-pressure pump for driving liquid to flow in the pump-free micro-fluidic chip;
the manufacturing method of the finger-pressure pump for driving liquid to flow in the chip comprises the following steps: before pouring PDMS on a silicon template with a cured chip pattern, placing a cylindrical copper block with the height of 8 mm and the radius of 5 mm at a specified position on the cylindrical copper block as a reserved area of a finger-pressure pump, and then pouring PDMS and a curing agent which are mixed according to the mass ratio of 10:1 of PDMS to the curing agent;
and after the liquid level of the PDMS is stable, placing the culture dish on a heating plate at 75 ℃ for heating, blowing off bubbles generated by heating by using an ear washing ball in time, covering the culture dish with a cover after the bubbles disappear, transferring the culture dish into an oven at 75 ℃ for heating for 2 hours to solidify the PDMS, and obtaining the pump-free microfluidic chip made of the PDMS after heating.
Step S4, embedding the gold-plated substrate and sealing the chip.
The embedding of the gold-plated substrate and the sealing method of the chip comprise the following steps: placing a PDMS chip with one surface of a micro-channel above the micro-channel and placing the PDMS chip and a glass slide into a plasma cleaning machine, firstly vacuumizing the interior of the machine for 70 seconds, and then carrying out oxygen plasma oxidation treatment for 50 seconds by ultraviolet irradiation; after the treatment, taking the two out of the plasma cleaning machine, and quickly placing the gold-plated substrate subjected to hydrophilic treatment and surface modification into a hexagonal cavity arranged in a PDMS chip; after the placement, the slide was sealed to obtain a pump-free microfluidic chip made of PDMS.
The principle that the gold-plated substrate needs to be placed before the PDMS chip is sealed with the glass slide is as follows: the PDMS chip and the glass slide are treated in the plasma cleaning machine in order to open silicon-oxygen bonds on the surfaces of the PDMS chip and the glass slide, but the opened chemical bonds can be closed again at normal temperature and normal pressure, if the gold-plated substrate is not placed in the chip in time, the PDMS chip and the glass slide cannot be tightly combined, and the liquid leakage phenomenon can occur after the reagent is introduced. Therefore, the gold-plated substrate is placed in the PDMS chip within 5 seconds, and then the PDMS chip is sealed with the glass slide, so that the gold-plated substrate and the glass slide can recombine chemical bonds after contacting, thereby forming irreversible sealing and avoiding the liquid leakage phenomenon in the experimental process.
FIG. 3 is a schematic diagram showing the operation of the flow restriction valve inside the pump-free microfluidic chip of the present invention, wherein two flow restriction valves with a height of 50 μm are disposed inside the pump-free microfluidic chip to reduce the flowing speed of thrombin and thrombin detection probes inside the chip, and the thrombin and thrombin detection probes can react with each other sufficiently before flowing to the gold-plated substrate subjected to hydrophilic treatment and surface modification, so as to increase the immune complex that may be formed on the surface of the gold-plated substrate.
The application method of the pump-free micro-fluidic chip comprises the following steps: the reagent adding step is carried out by firstly pressing the finger-pressing pump with the forefinger to form a pressure difference inside the chip, adding the reagent at the liquid inlet, and loosening the pressure exerted on the finger-pressing pump by the forefinger after the addition is finished to drive the liquid to flow in the micro-channel.
FIG. 4 is a schematic diagram of the formation of immune complexes on a gold-plated substrate, and the preparation process of thrombin detection probes is performed in the following order:
mu.L of 0.5M tris (2-carboxyethyl) phosphine (TCEP) was mixed with 50. mu.L of a stretch of 100. mu.M thrombin of the DNA aptamer TBA29 and incubated at room temperature for 6 hours to generate free thiol groups. Then, 100. mu.L of a 1. mu.M TBA29 aptamer solution was added to 1mL of the gold nanoparticle solution, followed by 2.5M NaCl solution and 0.5M PBS buffer solution to give final concentrations of 0.1M and 10 mM, respectively. The volumes of NaCl solution and PBS buffer required to be added were calculated to be 46.8. mu.L and 23.4. mu.L, respectively. The solution was incubated in a dry thermostat at 50 ℃ for 48 hours to continue aging. Since the aging of DNA aptamer into NaCl solution is a stepwise process, the addition of NaCl solution to TBA29 aptamer solution was divided equally six times in 48 hours, i.e., 7.8. mu.L each time. Two hours before the end of the 48 hour incubation, 4. mu.L of 10 was added -5 M, a Raman reporter MGITC. Finally, the mixed solution was placed in a differential centrifuge and centrifuged at 5500 rpm for 15 minutes, and the supernatant was removed with a pipette gun to remove the free aptamers and MGITC that were not bound to the gold nanoparticles, and the precipitate was suspended in PBS buffer for use.
The experiments performed in the pump-free microfluidic chip were performed in sequence as follows:
thrombin is firstly added from a liquid inlet by a pipette gun, then gold nanoparticles which serve as thrombin detection probes and are modified with the aptamer TBA29 and the Raman reporter molecule MGITC are simultaneously introduced, and a thrombin antigen fully reacts with the detection probes under the action of a snake-like channel, so that an immune complex with a sandwich structure is formed on a gold-plated substrate incubated with the TBA15 aptamer. And (3) standing the chip after the reagent is introduced, and introducing PBS buffer solution from the liquid inlet to wash the chip for multiple times after the reaction is carried out for a period of time so as to wash away thrombin which is not combined with the TBA15 aptamer on the gold-plated substrate and the thrombin detection probe. This formed a sandwich-like immune complex on the gold-plated substrate.
FIG. 5 is a Raman spectrum of a thrombin concentration gradient and its corresponding linear regression plot.
The Raman spectrum is collected by a LabRam HR Evolution system, a laser spot is firstly moved to a hexagonal detection area bearing a gold-plated substrate, and then a microscope 50 times of objective lens is used and a fine focusing screw is adjusted to focus the laser spot on the gold-plated substrate. The power of the He-Ne laser is 10 mW, the selected laser wavelength is 633 nm, and the detected Raman shift range is set to be 700-one 1700cm -1 The integration time was 10 seconds, and the number of integrations was 3 times. After taking experimental data, they were baseline removed and background noise suppressed using labspec5.0 software. Each line is the average of five hits of antigen at that concentration. Taking Raman shift as abscissa and Raman spectrum signal intensity as ordinate, observing 1614cm -1 Quantitative analysis of thrombin was performed on the change in SERS intensity of the peak. The thrombin detection range is 0.01-10nM, and the lower detection limit is 0.01 nM. Alternatively, thrombin was selected at varying concentrations at 1614cm -1 The Raman intensity is plotted into a linear regression curve, the linear fitting degree reaches 0.97354, and a good linear relation between the thrombin concentration and the SERS peak intensity can be seen.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the pump-free micro-fluidic chip is characterized by comprising the following steps;
step S1, surface modification of the gold-plated substrate;
soaking the gold-plated substrate after hydrophilic treatment in a section of DNA aptamer TBA15 solution of thrombin for modifying TBA15 aptamer thereon;
TBA15 DNA aptamer sequence: 5 '-dithiol-TTTTTTTTTTGGTTGGTGTGGTTGG-3';
step S2, manufacturing the pump-free micro-fluidic chip;
printing the drawn pattern on a transparent film, and curing the pattern on a silicon membrane plate by using an ultraviolet lithography machine;
after being mixed well, Polydimethylsiloxane (PDMS) and a curing agent are poured on a silicon template for curing, and a PDMS chip is obtained through mould pouring;
step S3, manufacturing a finger-pressure pump for driving liquid to flow in the pump-free micro-fluidic chip;
step S4, embedding the gold-plated substrate and sealing the chip.
2. The method for preparing a pump-free microfluidic chip according to claim 1, wherein in step S1, the surface modification method of the gold-plated substrate comprises:
soaking the gold-plated substrate after hydrophilic treatment in 1mL of a section of DNA aptamer TBA15 solution of 70 nM thrombin for modification of TBA15 aptamer thereon;
then placing the aptamer solution into a vacuum pump to continuously vacuumize for 20 minutes so as to remove gas in the aptamer solution;
after the treatment, the aptamer solution soaked with the gold-plated substrate is placed into a honeycomb oscillator to be oscillated for 24 hours;
after the oscillation is finished, taking the treated gold-plated substrates out of the aptamer solution by using tweezers and putting the gold-plated substrates into a centrifuge tube filled with 1mL of 2 mM 6-MCH solution for soaking for 2 hours;
after soaking, the materials are respectively washed for a plurality of times by deionized water and alcohol, and are used after being naturally dried.
3. The method for preparing a pump-free microfluidic chip according to claim 2, wherein the hydrophilic treatment method of the gold-plated substrate comprises: placing the gold-plated substrate into a plasma cleaning machine, firstly vacuumizing the interior of the machine for about 70 seconds, then carrying out oxygen plasma oxidation treatment for 5 minutes by ultraviolet irradiation, and taking out the gold-plated substrate to obtain the hydrophilic substrate surface.
4. The method for preparing a pump-free microfluidic chip according to claim 1, wherein in step S3, the method for manufacturing the finger pump for driving the liquid to flow inside the chip comprises: before pouring PDMS on a silicon template with a cured chip pattern, placing a cylindrical copper block with the height of 8 mm and the radius of 5 mm at a specified position on the cylindrical copper block as a reserved area of a finger-pressure pump, and then pouring the mixed PDMS and a curing agent according to the mass ratio of 10:1 of the PDMS to the curing agent;
and after the liquid level of the PDMS is stable, placing the culture dish on a heating plate at 75 ℃ for heating, blowing off bubbles generated by heating by using an ear washing ball in time, covering the culture dish with a cover after the bubbles disappear, transferring the culture dish into an oven at 75 ℃ for heating for 2 hours to solidify the PDMS, and obtaining the pump-free microfluidic chip made of the PDMS after heating.
5. The method of claim 4, wherein two flow-limiting valves with a height of 50 μm are disposed inside the pump-free microfluidic chip to reduce the flow rate of thrombin and thrombin detection probes inside the chip, and the thrombin and thrombin detection probes can react with each other sufficiently before flowing to the gold-plated substrate subjected to hydrophilic treatment and surface modification, thereby increasing the possibility of immune complexes forming on the surface of the gold-plated substrate.
6. The method for preparing a pump-free microfluidic chip according to claim 1, wherein in step S4, the embedding of the gold-plated substrate and the sealing of the chip are performed by: placing a PDMS chip with one surface of a micro-channel above the micro-channel and placing the PDMS chip and a glass slide into a plasma cleaning machine, firstly vacuumizing the interior of the machine for 70 seconds, and then carrying out oxygen plasma oxidation treatment for 50 seconds by ultraviolet irradiation; after the treatment, taking the two out of the plasma cleaning machine, and quickly putting the gold-plated substrate subjected to hydrophilic treatment and surface modification into a hexagonal cavity arranged in a PDMS chip; after the placement, the slide was sealed to obtain a pump-free microfluidic chip made of PDMS.
7. Use of a pump-free microfluidic chip prepared by the method according to any one of claims 1 to 6 in thrombin detection.
8. The application of the pump-free microfluidic chip in thrombin detection according to claim 7, wherein the detection is performed by a Raman detection method, and the specific steps comprise:
firstly adding thrombin from a liquid inlet by using a pipette, then simultaneously introducing gold nanoparticles serving as a thrombin detection probe and modifying the aptamer TBA29 and the Raman reporter MGITC, and fully reacting thrombin antigen with the detection probe under the action of a snake channel, thereby forming an immune complex with a sandwich structure on a gold-plated substrate incubated with the TBA15 aptamer;
after the reagent is introduced, standing the chip, and after the reaction is carried out for a period of time, introducing PBS buffer solution from the liquid inlet to wash the chip for multiple times for washing off thrombin and thrombin detection probes which are not combined with the TBA15 aptamer on the gold-plated substrate;
finally, Raman detection is carried out on the immune complex formed on the gold-plated substrate according to the principle of controlling variables.
9. The pump-free microfluidic chip of claim 8, wherein the thrombin detection probe is prepared by the following steps: first 1 μ L of 0.5M tris (2-carboxyethyl) phosphine (TCEP) was mixed with 50 μ L of a stretch of DNA aptamer TBA29 of 100 μ M thrombin and incubated at room temperature for 6 hours to generate free thiol;
then 100. mu.L of 1. mu.M TBA29 aptamer solution was added to 1mL of gold nanoparticle solution, followed by 2.5M NaCl solution and 0.5M PBS buffer solution to give final concentrations of 0.1M and 10 mM, respectively;
the volumes of the NaCl solution and the PBS buffer solution required to be added are respectively 46.8 mu L and 23.4 mu L by calculation;
incubating the solution in a dry thermostat at 50 ℃ for 48 hours, continuing aging, and adding NaCl solution into the TBA29 aptamer solution for six times in an average way within 48 hours, namely adding 7.8 mu L of NaCl solution for each time;
two hours before the end of the 48 hour incubation, 4. mu.L of 10 was added -5 M Raman reporter MGITC;
finally, the mixed solution is placed in a differential centrifuge to be centrifuged for 15 minutes at the speed of 5500 rpm, a liquid-transfering gun is used for removing the supernatant to remove the free aptamer and MGITC which are not combined with the gold nanoparticles, the precipitate is suspended in PBS buffer solution for standby,
wherein, the sequence of TBA29 DNA aptamer: 5 '-dithiol-TTTTTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'.
10. The application of the pump-free microfluidic chip in thrombin detection according to claim 8, wherein the synthesis method of the gold nanoparticles comprises the following steps: adding 49 mL of deionized water and 500 mu L of 1% tetrachloroauric acid solution into a clean three-neck flask, placing the mixture into a heating sleeve on a constant-temperature magnetic stirrer, and heating; after boiling, 500 mu L of 1 percent sodium citrate solution with mass fraction is added by a pipette, after the liquid color is completely changed from transparent to purple, the heating is continued for 1 hour to cure the synthesized gold nano-particles, thereby synthesizing the gold nano-particles with the particle size of about 40 nm.
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