Immobilized fluorescent coded microsphere hydrogel fiber and preparation method and application thereof

Abstract

The application discloses a kind of immobilized fluorescent encoding microspheres hydrogel fiber and its preparation method and application, belong to biomedical technology field, the method is: with water-soluble polymer solution as shell solution, with the water-soluble polymer solution containing fluorescent encoding microspheres as core solution, shell solution and core solution are injected into rotating coagulation bath by coaxial needle, solidified as immobilized fluorescent encoding microspheres hydrogel fiber, or, the water-soluble polymer solution containing fluorescent encoding microspheres is injected into rotating coagulation bath by injection needle, solidified as immobilized fluorescent encoding microspheres hydrogel fiber.The method process is simple, can realize the quick immobilization of fluorescent encoding microspheres, and the hydrogel fiber prepared by the method can be repeatedly observed under fluorescence imaging microscope, can be long-term preserved, has good application prospect in detection field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a kind of immobilized fluorescent encoding microspheres hydrogel fiber and its preparation method and application. BACKGROUND

[0002] The fluorescent encoding microspheres technology is to code the microspheres coated with specific markers with certain fluorescence intensity, so as to realize high-throughput quantitative detection of multiple indicators in biological samples, which has the advantages of rapid detection, simple preparation and sensitive quantitative analysis. The suspended chip using fluorescent encoding microspheres as analysis carrier is widely used in the fields of biomedicine and analysis. This technology uses encoding microspheres as carrier of probe molecules, target detection molecules are combined with microspheres first, and then combined with fluorescent labeled reporter molecules to form an immune complex sandwich. The target molecules can be classified by the encoding information of the microspheres, and the concentration of the target molecules can be determined by the fluorescence intensity of the reporter molecules. However, the current encoding technology has limited capacity and cannot accommodate too many types of fluorescent groups. The short spatial distance also has a great impact on the error of interference signals.

[0003] In the fluorescent encoding microspheres technology, the decoding method is crucial to realize accurate and multiple quantitative detection. At present, the decoding methods mainly include flow decoding and imaging decoding.

[0004] Flow decoding is the current mainstream decoding method, which mainly uses flow cytometry to focus the encoding microspheres into a sheath flow to realize one-way sequential flow, so as to collect the fluorescence signal of the high-speed flowing microspheres. The outstanding advantage of this method is that it has the ability to detect multiple components simultaneously, and the detection accuracy is high. Only the encoding microsphere liquid needs to be placed under the sample needle for self-detection. However, the flow cytometer is composed of a laser, a syringe pump, a PMT, etc. The structure is complex, the cost is high, and the maintenance is difficult. At the same time, the whole system is relatively closed, and it is difficult to directly decode on other reaction platforms (such as microhole array, solid phase carrier, etc.), which limits the application.

[0005] Imaging decoding mainly collects the fluorescence signal of the encoding microspheres coated with immune complexes loaded on the microhole array platform through fluorescence imaging microscope, and decodes by combining image analysis. This method uses simple optical modules, can decode on various platforms, has flexible application scenarios, and has the advantages of low cost and simple maintenance. However, this method must rely on imaging and image analysis for decoding, and in order to ensure the combination of hundreds of thousands of encoding microspheres, a long imaging time is required, the detection speed is slow, and the throughput is low.

[0006] A kind of EGFR mutant gene detection method based on fluorescent encoding microspheres is disclosed in Chinese patent document with publication number CN109097470A, which first fixes a plurality of specific detection probes for EGFR mutant gene on different fluorescent encoding microspheres, then amplifies the fragment containing mutant gene in the sample to be tested by modified ARMS-PCR, and finally hybridizes the amplified fragment with a plurality of encoding microspheres on which specific detection probes are fixed on the surface, and achieves the purpose of judging whether the sample to be tested contains specific EGFR mutant site by flow cytometry or fluorescence imaging technology.

[0007] Since the above two decoding methods have defects, there is currently a need for a fluorescent encoding microsphere technology that can achieve high loading rate of microspheres and simple decoding system, suitable for wide application. SUMMARY

[0008] The present application provides a preparation method of hydrogel fiber immobilized with fluorescent encoding microspheres, which is simple in process and can realize rapid immobilization of fluorescent encoding microspheres. The hydrogel fiber prepared by the method can be repeatedly observed under a fluorescence imaging system, can be stored for a long time, and improves the decoding convenience of fluorescent encoding microspheres.

[0009] The specific technical solutions adopted are as follows:

[0010] A preparation method of hydrogel fiber immobilized with fluorescent encoding microspheres, using one of method 1 or method 2:

[0011] Method 1: using water-soluble polymer solution as shell layer solution, using water-soluble polymer solution containing fluorescent encoding microspheres as core layer solution, uniformly injecting the shell layer solution and the core layer solution into the rotating coagulation bath through coaxial needle at a constant speed, and solidifying into hydrogel fiber immobilized with fluorescent encoding microspheres;

[0012] Method 2: uniformly injecting water-soluble polymer solution containing fluorescent encoding microspheres into the rotating coagulation bath through injection needle at a constant speed, and solidifying into hydrogel fiber immobilized with fluorescent encoding microspheres.

[0013] The fluorescent encoding microspheres include fluorescent microspheres or microspheres carrying fluorescently labeled immune complex sandwich.

[0014] The present application uses hydrogel fiber to realize the packaging of fluorescent encoding microspheres, and the method is fast and simple. In the hydrogel fiber prepared by the method of the present application, the fluorescent encoding microspheres are aggregated into linear shape, which is conducive to subsequent in situ labeling and imaging observation.

[0015] Preferably, the concentration of water-soluble polymer in the shell solution of Method 1 is 10-30 mg / mL, the concentration of water-soluble polymer in the core solution of Method 1 is 0.5-20 mg / mL, and the concentration of fluorescently encoded microspheres is 0.1-5 mg / mL.

[0016] Preferably, in Method 2, the concentration of water-soluble polymer in the water-soluble polymer solution containing fluorescently encoded microspheres is 2-10 mg / mL, and the concentration of fluorescently encoded microspheres is 0.1-5 mg / mL.

[0017] A too high concentration of fluorescently encoded microspheres can cause needle blockage, and too many fluorescent microspheres are difficult to arrange in the middle of the hydrogel fiber, which can cause stacking and is not conducive to subsequent observation. A too low concentration of microspheres can result in too few observable microspheres in the hydrogel fiber, which can affect the observation effect. The above concentration range is conducive to the formation of hydrogel fibers and can avoid fiber breakage and the like.

[0018] Preferably, in Method 1 or Method 2, the water-soluble polymer solution is a sodium alginate solution or a polyvinyl alcohol solution; when the water-soluble polymer solution is a sodium alginate solution, the coagulation bath is selected from a calcium chloride solution, a barium chloride solution, an ammonium sulfate solution, or an aluminum sulfate solution; and when the water-soluble polymer solution is a polyvinyl alcohol solution, the coagulation bath is selected from a sodium sulfate solution, a zinc sulfate solution, or a boric acid solution.

[0019] Further preferably, in Method 1 or Method 2, the water-soluble polymer solution is a sodium alginate solution, and the coagulation bath is a calcium chloride solution with a concentration of 10-70 mg / mL.

[0020] Preferably, in Method 1, the particle size of the fluorescently encoded microspheres is 20-50 μm, and the coaxial needle is of a 30G inner shaft and a 21G outer shaft; and in Method 2, the particle size of the fluorescently encoded microspheres is 10-30 μm, and the injection needle is of a 32G type.

[0021] Preferably, in Method 1, the core solution is pushed through the coaxial needle at a speed v1, and the shell solution is pushed through the coaxial needle at a speed v2; the coagulation bath is placed on a rotating disc and rotated at a speed of 0.5-5 rad / s; wherein 100 μL / min≤v1≤1000 μL / min, and v2 is calculated according to the following formula:

[0022]

[0023] Preferably, in Method 2, the water-soluble polymer solution containing fluorescently encoded microspheres is pushed through the injection needle at a speed of 100-1000 μL / min; and the coagulation bath is placed on a rotating disc and rotated at a speed of 0.5-5 rad / s.

[0024] The liquid in the coagulation bath rotates, the rotating liquid generates shearing action, the water-soluble polymer solution containing fluorescent coded microspheres is gradually elongated and thinned under the action of the shearing force, according to the principle of particle migration in the microchannel, the fluorescent coded microspheres spontaneously stabilize at the same position away from the axis of the pipeline, and the slow injection speed avoids the radial movement of the water-soluble polymer solution containing fluorescent coded microspheres that may occur before forming, so that the fluorescent coded microspheres in the prepared hydrogel fiber are arranged in the same line. Subsequently, only axial movement of the fiber is required to image and observe all the fluorescent coded microspheres, without the need to move the fiber in each direction to capture the microspheres.

[0025] The application further provides a hydrogel fiber prepared by the preparation method of the immobilized fluorescent coded microsphere hydrogel fiber.

[0026] The application further provides application of the hydrogel fiber in the detection field such as circulating tumor cell detection and molecular level marker detection.

[0027] The application further provides a decoding method of the fluorescent coded microsphere, comprising:

[0028] The preparation method of the immobilized fluorescent coded microsphere hydrogel fiber is adopted to immobilize the to-be-detected fluorescent coded microsphere in the hydrogel fiber;

[0029] The fluorescence imaging system is used to detect the fluorescence signal of the to-be-detected fluorescent coded microsphere in the hydrogel fiber, and the to-be-detected fluorescent coded microsphere is decoded according to the relationship between the fluorescence signal and the information of the fluorescent coded microsphere which has been constructed.

[0030] After the microspheres labeled with antibodies and the fluorescent dyes labeled with paired antibodies react with the target detection antigens (to-be-detected markers), the microspheres carrying the fluorescent-labeled immune complex sandwich are formed, that is, the to-be-detected fluorescent coded microspheres.

[0031] The above method can realize repeated observation of the fluorescent coded microspheres, and the decoding has good repeatability and accuracy.

[0032] Compared with the prior art, the application has the following beneficial effects:

[0033] The preparation method of the immobilized fluorescent coded microsphere hydrogel fiber in the application has the advantages of simple process, easy operation, short preparation time, long-term storage of the prepared hydrogel fiber, repeated observation under the fluorescence imaging system, improved decoding convenience of the fluorescent coded microspheres, and application in the fields of circulating tumor cell detection, liquid chip analysis and molecular level marker detection. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Schematic diagram of the device for preparing the hydrogel fiber in Example 1, with reference numerals: 101 - coaxial needle, 102 - first injection pump, 103 - second injection pump, 104 - first syringe, 105 - second syringe, 106 - receiver.

[0035] Figure 2 This is a microscopic morphology of the hydrogel fiber prepared in Example 1 (50x microscope).

[0036] Figure 3 This is a comparison chart of the fluorescence imaging and image processing signals of the hydrogel fiber prepared in Example 1 (100x magnification).

[0037] Figure 4 Fluorescence and bright field images (50x magnification) of the hydrogel fibers in Example 1 stored for 40 days, wherein A is the fluorescence image and B is the bright field image.

[0038] Figure 5 This is a comparison chart of the fluorescence imaging and image processing signals of the hydrogel fiber prepared in Example 2 (100x magnification). DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the examples and accompanying drawings. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The operating methods in the following examples where no specific conditions are specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0040] The schematic diagram of the device for preparing the hydrogel fiber using method 1 is as follows Figure 1 As shown, the device includes a coaxial needle 101, a first injection pump 102 for injecting the core layer solution, a second injection pump 103 for injecting the shell layer solution, a first syringe 104, a second syringe 105 and a receiver 106; the core layer solution and the shell layer solution are injected into the coaxial needle 101 through the first syringe 104 and the second syringe 105 respectively, and solidified in the rotating receiver 106; the first syringe 104, the second syringe 105 and the coaxial needle 101 are connected by a hose and a Luer connector.

[0041] Example 1 Preparation of hydrogel fibers

[0042] To 0.1 g of sodium alginate particles, add 10 ml of deionized water. After adding a magnet, thoroughly oscillate and mix using a vortex mixer at room temperature (25°C) for 2 h to obtain a sodium alginate solution with a concentration of 10 mg / ml, which is used as the shell solution.

[0043] Take 50 μL of 41.7 μm yellow-green fluorescent microspheres solution (catalog number 18242-2, excitation wavelength 441 nm, emission wavelength 485 nm) with a mass fraction of 2.7% purchased from Polysciences, 100 μL of the above prepared sodium alginate solution, and 350 μL of deionized water, mix uniformly for 5 min using an ultrasonic cleaner, and take the mixed solution as the core layer solution; in the core layer solution, the concentration of sodium alginate is 2 mg / ml, and the concentration of yellow-green fluorescent microspheres is 2.7 mg / mL.

[0044] Take 0.6 g of anhydrous calcium chloride particles and add them to 20 ml of deionized water, stir uniformly to obtain a calcium chloride solution with a mass concentration of 30 mg / mL for cross-linking of the sodium alginate hydrogel.

[0045] Place the core layer solution and the shell layer solution in the first syringe 104 and the second syringe 105 respectively, and install the first syringe 104 and the second syringe 105 on the first injection pump 102 and the second injection pump 103 respectively for uniform injection, set the injection speed of the first injection pump 102 to 500 μL / min, the perfusion volume to 0.1 ml, and the injection speed of the second injection pump 103 to 4580 μL / min, the perfusion volume to 0.92 ml. The coaxial needle used is an inner shaft 30G (inner diameter 160 μm) and an outer shaft 21G (inner diameter 510 μm), since the coaxial needle 101 is in a concentric circular structure, the speed ratio of the inner and outer shafts is the same as the cross-sectional ratio of the circular and annular shapes, thereby ensuring that the outlet speeds of the core layer solution and the shell layer solution are consistent.

[0046] Start the first injection pump 102 and the second injection pump 103 at the same time, inject the core layer solution and the shell layer solution into the calcium chloride solution in the receiver 106 using the coaxial needle 101, and place the receiver 106 on the motorized turntable rotating at an angular velocity of 1.25 rad / s, i.e. at a speed of 0.125 m / s in the axial direction at the tip of the coaxial needle, thereby obtaining a uniform hydrogel fiber of immobilized fluorescent coded microspheres. The injection speed is relatively slow compared to the normal injection speed in order to ensure stable and uniform injection, and the rotating culture dish is used to stabilize the diameter of the formed hydrogel fiber, and the obtained hydrogel fiber is stored in a deionized water solution, which can realize long-term stable immobilization of fluorescent microspheres.

[0047] Preparation of a hydrogel fiber immobilizing multiple microspheres

[0048] Add 10 ml of deionized water to 0.3 g of sodium alginate particles, and after adding the magnetic particles, mix uniformly at room temperature (25°C) using a vortex mixer for 2 h to obtain a sodium alginate solution with a concentration of 30 mg / ml;

[0049] Take 2.7 mg of 24 μm red fluorescent microspheres purchased from Thermo Fisher (product number 36-5B, excitation wavelength 542 nm, emission wavelength 612 nm), add 100 μL of deionized water, and mix well at room temperature (25°C) using a vortex mixer to obtain a red fluorescent microsphere solution with a mass fraction of 2.7%;

[0050] Take 25 μL of 26 μm yellow-green fluorescent microsphere solution with a mass fraction of 2.7% purchased from Polysciences (product number 18241-2, excitation wavelength 441 nm, emission wavelength 485 nm), 25 μL of the above-prepared red fluorescent microsphere solution, 250 μL of the above-prepared sodium alginate solution, and 750 μL of deionized water, and mix well using an ultrasonic cleaner for 5 min to form a water-soluble polymer solution containing fluorescently encoded microspheres, wherein the concentration of sodium alginate is 7.14 mg / mL, the concentration of yellow-green fluorescent microspheres is 0.64 mg / mL, and the concentration of red fluorescent microspheres is 0.64 mg / mL.

[0051] Take 1 g of anhydrous calcium chloride particles and add 20 ml of deionized water, stir well to obtain a calcium chloride solution with a mass concentration of 50 mg / mL for cross-linking of the sodium alginate hydrogel.

[0052] Place the water-soluble polymer solution containing fluorescently encoded microspheres in a 2.5 ml syringe, install the syringe on a syringe pump for uniform injection, set the injection speed of the syringe pump to 300 μL / min, the perfusion volume to 0.1 ml, and use a 32G needle (inner diameter 110 μm) bent at a right angle and vertically inserted into the calcium chloride solution.

[0053] Start the syringe pump and inject the water-soluble polymer solution containing fluorescently encoded microspheres into the calcium chloride solution in the receiver at a uniform speed, place the receiver on a motorized turntable rotating at an angular velocity of 1 rad / s, i.e. the injected fiber is stretched at a speed of 0.1 m / s in the axial direction, thereby obtaining a uniform hydrogel fiber immobilized with two types of fluorescently encoded microspheres. The injection speed is relatively slow compared to the normal injection speed to ensure stable and uniform injection, and the rotating culture dish is used to stabilize the diameter of the formed hydrogel fiber, and the obtained hydrogel fiber is stored in a deionized water solution to achieve long-term stable fluorescent microsphere immobilization.

[0054] Example 3 Imaging decoding experiment

[0055] Place the hydrogel fiber obtained in Example 1 on the stage of a fluorescence imaging microscope, use a blue filter with an excitation wavelength of 480 nm to excite the fluorescent signal, and obtain a fluorescence image.

[0056] The bright field image of the hydrogel fiber prepared in Example 1 under 50 times magnification is shown in Figure 2 It can be seen that the fluorescent microspheres are uniformly distributed in the center of the fiber channel formed by the core solution inside the hydrogel fiber.

[0057] The hydrogel fiber prepared in Example 1 was moved in the direction parallel to the fiber at a speed ranging from 0.5 to 2 cm / min by using a stepping motor, and 50 frames of imaging video were recorded by using Andor SOLIS software. A specific size interval (42 μm x 84 μm) was taken for movement in the direction parallel to the fiber in each frame of video (pixels: 512*512), the interval width was the same as the diameter of the fluorescent microspheres, and the average gray value change graph of each frame of different interval was read by using matlab. When the gray value peak value exceeded 80 (this value is related to the size of the selected interval and is not unique), it was considered that a single complete fluorescent microsphere was detected. Some video and data processing results are shown in Figure 3 .

[0058] The hydrogel fiber prepared in Example 1 can be stored for a long time and repeatedly detected. The fluorescence and bright field images of the hydrogel fiber prepared in Example 1 stored for 40 days are shown in Figure 4 It can be observed from B in Figure 4 that although the shell layer and the core layer form a fiber fusion, the fluorescent microspheres are still located at the center position of the hydrogel fiber, and the fluorescence imaging thereof can be observed from A in Figure 4 , which proves the stability of the method.

[0059] The hydrogel fiber prepared in Example 2 was placed on the stage of the fluorescence imaging microscope, and blue filter with an excitation wavelength of 480 nm and green filter with an excitation wavelength of 545 nm were used for fluorescence signal excitation, respectively. The hydrogel fiber prepared in Example 2 was moved in the direction parallel to the fiber at a speed ranging from 0.5 to 2 cm / min by using a stepping motor, and 50 frames of imaging video were recorded by using Andor SOLIS software. A specific size interval (25 μm x 50 μm) was taken for movement in the direction parallel to the fiber in each frame of video (pixels: 512*512), the interval width was the same as the diameter of the fluorescent microspheres, and the average gray value change graph of each frame of different interval was read by using matlab. When the gray value peak value exceeded 80 (this value is related to the size of the selected interval and is not unique), it was considered that a single complete fluorescent microsphere was detected. Some video and data processing results are shown in Figure 5 , and the left and right fluorescence images represent the fluorescence images of red fluorescent microspheres excited by green filter and yellow-green fluorescent microspheres excited by blue filter, respectively.

[0060] In the embodiment, the fluorescent microspheres in the hydrogel fiber can be normally fluorescently imaged and bright field imaged, and then it can be illustrated that the fluorescently encoded microspheres in the hydrogel fiber have normal fluorescent signal and imaging capability, and can be applied to precise detection of disease markers and the like. The microspheres sandwiched by the fluorescently labeled immune complex are decoded subsequently.

[0061] It can be seen that the method can realize preparation of the hydrogel fiber of the immobilized fluorescently encoded microspheres, has simple process, is convenient to operate, has short preparation time, and the obtained hydrogel fiber can be detected by imaging of a fluorescent imaging microscope and simple data processing. Meanwhile, the hydrogel fiber is easy to store, avoids the defect that the flow cytometer can only be observed in one direction, has good repeatability, can be observed for multiple times, and also avoids the problem that the traditional imaging decoding mode is too complex and waste to load the microspheres.

[0062] The above embodiment has described the technical solution of the present application in detail, and it should be understood that the above description is only a specific embodiment of the present application, and is not used to limit the present application. Any modification, supplement or similar replacement within the principle range of the present application should be included in the protection range of the present application.

Claims

1. A method for preparing hydrogel fibers immobilized with fluorescent coded microspheres, characterized in that: Use method 1: Method 1: A water-soluble polymer solution is used as the shell solution, and a water-soluble polymer solution containing fluorescently encoded microspheres is used as the core solution. The shell solution and the core solution are uniformly injected into a rotating coagulation bath through a coaxial needle to solidify into a hydrogel fiber with immobilized fluorescently encoded microspheres; In the shell solution of method 1, the concentration of the water-soluble polymer is 10-30 mg / mL, in the core solution of method 1, the concentration of the water-soluble polymer is 0.5-20 mg / mL, and the concentration of the fluorescently encoded microspheres is 0.1-5 mg / mL; In method 1, the water-soluble polymer solution is a sodium alginate solution, and the coagulation bath includes a calcium chloride solution, a barium chloride solution, an ammonium sulfate solution, or an aluminum sulfate solution; In method 1, the particle size of the fluorescent coded microspheres is 20-50 μm; In method 1, the core solution is v 1 Push through the coaxial needle, the shell solution v 2 through the coaxial needle; the coagulation bath is placed on a turntable and rotated at a speed of 0.5-5 rad / s; wherein, 100 μL / min≤ v 1≤1000 μL / min, v 2 is calculated according to the following formula: 。 2. The method for preparing hydrogel fibers immobilized with fluorescent coded microspheres according to claim 1, characterized in that: In method 1, the coaxial needle has a model of 30G inner shaft and 21G outer shaft.

3. A hydrogel fiber, characterized in that: The hydrogel fiber is prepared by the preparation method of the immobilized fluorescent coding microspheres according to claim 1 or 2.

4. Application of the hydrogel fiber according to claim 3 in the field of detection.

5. A decoding method for fluorescent coded microspheres, characterized in that: include: The method for preparing the hydrogel fiber with immobilized fluorescent coded microspheres according to claim 1 or 2 is used to immobilize the fluorescent coded microspheres to be tested inside the hydrogel fiber; The fluorescence imaging system is used to detect the fluorescence signal of the fluorescent coded microspheres to be tested inside the hydrogel fiber, and the fluorescent coded microspheres to be tested are decoded according to the relationship between the constructed fluorescence signal and the information of the fluorescent coded microspheres.

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