Magnetic hydrogel fluorescence sensor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of intracellular sensors, and discloses a magnetic hydrogel fluorescence sensor as well as a preparation method and application thereof. According to the invention, the hydrogel microspheres are used as a carrier of the sensor, on one hand, the fluorescent probe is wrapped in the hydrogel microspheres, so that the problem that the fluorescent probe is easy to lose is controlled, the stability of the sensor is improved, and meanwhile, cell death caused by the fact that the fluorescent probe is free in cells is avoided; on the other hand, the sensor is injected into the cells, so that the concentration change of the intracellular environment calcium ions can be directly obtained. Besides, the sensor and the array type cell carrier are used in a matched mode, the array type cell carrier can limit cell movement and achieve continuity of detection of the same cell, and the sensor can achieve synchronism of multi-cell detection through magnetic control.

Description

Technical Field

[0001] The present invention relates to the technical field of intracellular sensors, and in particular to a magnetic hydrogel fluorescent sensor and a preparation method and application thereof. Background Art

[0002] In recent years, intracytoplasmic sperm injection (ICSI) has become the main assisted reproductive method for treating severe oligospermia, asthenospermia, teratospermia and azoospermia in men. Since its first successful application in 1992, ICSI technology has helped many infertile patients realize their desire to have children. However, despite the increasing application of ICSI, oocyte activation deficiency (OAD) is still one of the important reasons for ICSI failure. OAD is usually caused by the inability of sperm to produce enough phospholipase C-ζ (PLCζ) after entering the oocyte, and the failure to trigger the required calcium ions (Ca 2 +) concentration increases, thereby forming Ca 2 + oscillation to activate the oocyte. The absence of this key step leads to the failure of oocyte activation, thus affecting the ultimate success rate of ICSI.

[0003] To overcome this challenge, artificial oocyte activation (AOA) technology has received widespread attention in recent years. AOA technology simulates Ca2+ by introducing natural or artificial activators (such as PLCζ, A23187, ionomycin, strontium chloride), or even electric pulses. 2 + oscillation, thereby activating the oocyte. However, how to accurately evaluate and control the effect of AOA is a key problem. In this process, the change in calcium ion concentration is particularly important. It is not only a landmark signal for oocyte activation, but also directly affects the subsequent development quality of the embryo.

[0004] Ca 2 + oscillations play a crucial role in early mammalian embryonic development. During fertilization, the mature oocyte undergoes repeated Ca oscillations triggered by sperm factors. 2 + oscillations, with a specific amplitude and duration, transmit activation signals, allowing the oocyte to enter the cell cycle, complete division and form an embryo. During the ICSI process, if the oocyte fails to produce this natural Ca 2 + oscillation, it will lead to activation failure. 2+Real-time monitoring of concentration has become an important means of evaluating the activation status of oocytes. This monitoring can not only help laboratories evaluate the effects of ICSI and AOA in real time, but also serve as a key indicator of oocyte quality, guiding embryologists to more accurately select oocytes with developmental potential, thereby improving the success rate of embryo transplantation.

[0005] In summary, Ca 2 The monitoring of changes in oocyte Ca concentration has important application value in ICSI and AOA processes. 2 + Dynamic, it can not only effectively evaluate the success or failure of ICSI, but also provide key support for optimizing ICSI operations and improving the success rate. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a magnetic hydrogel fluorescent sensor and a preparation method thereof, so that the sensor has low cytotoxicity and avoids the loss of fluorescent probes, thereby realizing long-term real-time monitoring of changes in intracellular calcium ion concentration;

[0007] Another object of the present invention is to provide an application of the above sensor in detecting intracellular calcium ion concentration or in preparing related detection products, especially in the field of detecting intracellular calcium ion concentration of fertilized egg cells.

[0008] In order to solve the above technical problems or at least partially solve the above technical problems, the present invention provides a method for solving the above technical problems or at least partially solving the above technical problems. As a first aspect of the present invention, a magnetic hydrogel fluorescent sensor is provided, comprising hydrogel microspheres, magnetic nanoparticles and calcium ion fluorescent probes; the magnetic nanoparticles and the calcium ion fluorescent probes are filled in a three-dimensional network inside the hydrogel microspheres.

[0009] Optionally, the hydrogel microspheres are formed by cross-linking and curing of ultraviolet light-curing monomers. Further optionally, the ultraviolet light-curing monomers include acrylate monomers, acrylamide monomers and acrylic acid monomers.

[0010] Optionally, the magnetic nanoparticles include ferrosoferric oxide nanoparticles, and the calcium ion fluorescent probe includes a Fluo fluorescent probe.

[0011] As a second aspect of the present invention, a method for preparing the sensor is provided, comprising:

[0012] The calcium ion fluorescent probe, the organic solvent and the buffer solution are mixed to form a first aqueous phase, and the ultraviolet light curing monomer, the photoinitiator, the magnetic nanoparticles and the buffer solution are mixed to form a second aqueous phase;

[0013] The first aqueous phase and the second aqueous phase are mixed to form an aqueous solution of a UV-curable monomer precursor;

[0014] The aqueous phase solution of the ultraviolet-curable monomer precursor and the oil phase solution are used to obtain hydrogel droplets through a microfluidic emulsion method, and the sensor is obtained after being cured by ultraviolet light.

[0015] Optionally, the organic solvent includes one or more of dimethyl sulfoxide, acetonitrile, ethanol, methanol, acetone, isopropanol, dichloromethane, chloroform, toluene, dimethylformamide, ethylene glycol, and tetrahydrofuran; the buffer includes one or more of HEPES buffer, PBS buffer, Tris buffer, and MOPS buffer; and the oil phase includes fluorinated oil.

[0016] As a third aspect of the present invention, provided is the use of the sensor in detecting intracellular calcium ion concentration in vitro or in preparing a product for detecting intracellular calcium ion concentration in vitro.

[0017] As a fourth aspect of the present invention, a product for detecting intracellular calcium ion concentration in vitro is provided, comprising a fluorescence detection device, a microinjection device, a cell carrier, a magnet, and the sensor of the present invention.

[0018] Optionally, the cell carrier has one or more independent units, each of which includes one or more grooves for accommodating cells.

[0019] As a fifth aspect of the present invention, a method for detecting intracellular calcium ion concentration in vitro is provided, comprising:

[0020] A calcium ion solution with a gradient concentration is prepared, and the fluorescence intensity at different calcium ion concentrations is measured using the sensor of the present invention, the fluorescence intensity at a certain calcium ion concentration is selected as a benchmark, and the ratios of the fluorescence intensities at other calcium ion concentrations to the benchmark are calculated to obtain a series of relative fluorescence intensity values, and the calcium ion concentrations and the corresponding relative fluorescence intensity values ​​are fitted to obtain a fitting curve;

[0021] The sensor of the present invention is injected into the cell by microinjection technology, and then moved into the cell carrier. At the same time, a magnet is used to exert influence on the cell carrier to control the sensors in the cells to be tested to be in the same plane. Then, a fluorescence detection device is used to obtain the fluorescence intensity in different time periods, and the calcium ion concentration in the cell is detected according to the benchmark and the fitting curve.

[0022] The present invention uses hydrogel microspheres as carriers of sensors. On the one hand, the fluorescent probe is wrapped inside the hydrogel microspheres, thereby controlling the problem of easy loss of the fluorescent probe, improving the stability of the sensor, and preventing the fluorescent probe from being free inside the cell and causing cell death; on the other hand, the sensor is injected into the cell to directly obtain the change in calcium ion concentration in the intracellular environment. In addition, the present invention uses the sensor in combination with an array cell carrier, which can limit cell movement and achieve continuity in detecting the same cell, and the sensor can achieve synchronization of multi-cell detection through magnetic control. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0024] Figure 1 Shown is a schematic structural diagram of the magnetic hydrogel fluorescence sensor of the present invention;

[0025] Figure 2 Shown is a schematic diagram of the preparation of the magnetic hydrogel fluorescent sensor of the present invention by using a microfluidic emulsion method;

[0026] Figure 3 Shown is a schematic diagram of the flow channel structure of a microfluidic chip;

[0027] Figure 4 Shown is the particle size distribution of magnetic hydrogel fluorescent sensor;

[0028] Figure 5 Shown are a physical picture of the cell carrier (a) and a schematic diagram of the array groove structure in each unit on the carrier (b a photo taken under a microscope, c a schematic diagram of the structure);

[0029] Figure 6 Shown is the fluorescence spectrum of the magnetic hydrogel fluorescent sensor;

[0030] Figure 7 The magnetic hydrogel fluorescent sensor is shown to detect Ca 2+ Sensing curve of concentration; wherein, A = 2.91395 ± 0.03687, t = 361.57068 ± 29.20517, B = 2.91395 ± 0.03687;

[0031] Figure 8 Shown is a schematic diagram of the injection of magnetic hydrogel fluorescent sensor into fertilized egg cells;

[0032] Fig. 9 Shown is the detection of intracellular Ca 2+ Schematic diagram of the overall method implementation of concentration;

[0033] Figure Number:

[0034] 1 is a water phase injection needle, 2 is an oil phase injection needle, 3 is a microfluidic chip, 4 is an ultraviolet lamp, 5 is a hydrogel collection device, 6 is a suction needle, 7 is an injection needle, 8 is a magnetic hydrogel fluorescent sensor, and 9 is a fertilized egg cell. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0037] In the first aspect of the present invention, a sensor for detecting intracellular calcium ion concentration is provided, comprising hydrogel microspheres, magnetic nanoparticles and calcium ion fluorescent probes; the magnetic nanoparticles and calcium ion fluorescent probes are filled in a three-dimensional network inside the hydrogel microspheres, and the structural schematic diagram is shown in FIG. Figure 1 .

[0038] In certain embodiments of the present invention, the hydrogel microspheres are formed by cross-linking and curing of ultraviolet light-cured monomers, and an intricate three-dimensional network is formed inside the hydrogel microspheres, thereby confining the magnetic nanoparticles and the calcium ion fluorescent probe in the three-dimensional grid.

[0039] In certain embodiments of the present invention, the UV-curable monomers include acrylate monomers, acrylamide monomers and acrylic acid monomers. The acrylate monomers include but are not limited to polyethylene glycol diacrylate (PEG-DA), polyethylene glycol acrylate (PEG-Acrylate), and polyethylene glycol methacrylate (PEG-MA).

[0040] In certain embodiments of the present invention, the magnetic nanoparticles include ferrosoferric oxide nanoparticles, and the calcium ion fluorescent probe includes a Fluo fluorescent probe, such as a Fluo-8 fluorescent probe.

[0041] In a second aspect of the present invention, a method for preparing the sensor is provided, comprising:

[0042] The calcium ion fluorescent probe, the organic solvent and the buffer solution are mixed to form a first aqueous phase, and the ultraviolet light curing monomer, the photoinitiator, the magnetic nanoparticles and the buffer solution are mixed to form a second aqueous phase;

[0043] The first aqueous phase and the second aqueous phase are mixed to form an aqueous solution of a UV-curable monomer precursor;

[0044] The aqueous phase solution of the ultraviolet-curable monomer precursor and the oil phase solution are used to obtain hydrogel droplets through a microfluidic emulsion method, and the sensor is obtained after being cured by ultraviolet light.

[0045] In certain embodiments of the present invention, the organic solvent includes one or more of dimethyl sulfoxide, acetonitrile, ethanol, methanol, acetone, isopropanol, dichloromethane, chloroform, toluene, dimethylamide, ethylene glycol, and tetrahydrofuran. In some other embodiments of the present invention, the calcium ion fluorescent probe is dissolved in dimethyl sulfoxide, and the concentration can be adjusted accordingly according to the demand for fluorescence brightness. If the brightness is not enough, the concentration can be appropriately increased. If it is too bright, the concentration can be appropriately reduced. The present invention usually selects within the range of 0.8mM-1.5mM, for example, 1mM; in the first aqueous phase, the volume ratio of the dimethyl sulfoxide solution containing the calcium ion fluorescent probe to the buffer solution is 1:4-1:6, for example, 1:5.

[0046] In certain embodiments of the present invention, the magnetic nanoparticles can be first dissolved in a buffer solution, and then mixed with a UV-curable monomer and a photoinitiator, wherein the mass ratio of the UV-curable monomer, the photoinitiator and the buffer solution containing the magnetic nanoparticles is 10:(0.1-1):9, for example, the mass percentages are 50%, 5% and 45% respectively; wherein the photoinitiator is determined according to the type of UV-curable monomer, for example, when monomers such as PEG-DA, PEG-Acrylate and PEG-MA are selected, 2-hydroxy-2-methylpropiophenone can be selected as the photoinitiator. In other embodiments of the present invention, the mass percentage of the magnetic nanoparticles in the buffer solution is 0.01-0.05%.

[0047] In certain embodiments of the present invention, the buffer comprises one or more of HEPES buffer, PBS buffer, Tris buffer, and MOPS buffer, wherein the pH value of the buffer is controlled at 7.2±0.2, which is close to the working environment of the sensor.

[0048] In some embodiments of the present invention, the oil phase includes fluorinated oil, such as HFE7500. In other embodiments of the present invention, the oil phase is a fluorinated oil with a surfactant added, and the addition of a surfactant can reduce the interfacial tension to prevent the water phase and the oil phase from being rapidly separated after mixing, and can form a stable interface layer around the droplets to prevent the droplets from contacting and agglomerating; the mass percentage of the surfactant is 2-5%, and the surfactant includes but is not limited to Krytox series surfactants, such as Krytox 157FSH; Zonly series surfactants, such as Zonyl FSN or Zonyl FSO; 3M fluorinated surfactants, such as 3M FC-4430 and 3M FC-4432, and the like.

[0049] In certain embodiments of the present invention, the microfluidic emulsion method is to pass an incompatible liquid through a microfluidic chip to generate droplets at the intersection of water and oil, and then cure the droplets by ultraviolet irradiation to form hydrogel microspheres by cross-linking. The process diagram is shown in Figure 2 and Figure 3 . By adjusting the pressure, the water phase flow rate and the oil phase flow rate are controlled to control the particle size of the microspheres to the required size. The particle size of the microspheres is determined by the diameter of the subsequent injection equipment. For example, the oil phase inlet pressure is always maintained at 1950 mbar, and the particle size of the microspheres is controlled by gradually adjusting the water phase pressure. First, the water phase pressure is set to 1700 mbar, and then decreased by 50 mbar each time, and adjusted to 1650 mbar, 1600 mbar, 1550 mbar, until it finally reaches 1300 mbar. When the water phase pressure reaches 1300 mbar, the particle size of the generated magnetic fluorescent hydrogel microspheres is around 5 microns, and its particle size distribution diagram is shown as follows. Figure 4 shown.

[0050] In certain embodiments of the present invention, the collected magnetic fluorescent hydrogel microspheres are irradiated with a UV curing lamp to complete curing. The cured magnetic hydrogel microspheres are located in the emulsion and float on the upper layer of the fluorinated oil. The lower transparent fluorinated oil is carefully sucked and removed from the upper layer. A demulsifier is then added, and the mixture is centrifuged after slight shaking. After the centrifugation, the liquid shows obvious stratification. Carefully aspirate the lower layer of demulsifier, add the upper layer of liquid to the buffer solution, and centrifuge and wash it several times to finally obtain a magnetic hydrogel fluorescent sensor.

[0051] In the third aspect of the present invention, there is provided the use of the sensor in detecting intracellular calcium ion concentration in vitro or in preparing a product for detecting intracellular calcium ion concentration in vitro.

[0052] In certain embodiments of the present invention, the present invention takes the detection of calcium ions in fertilized egg cells as an example to further illustrate the technical solution of the present invention.

[0053] In a fourth aspect of the present invention, a product for detecting intracellular calcium ion concentration in vitro is provided, comprising a fluorescence detection device, a microinjection device, a cell carrier, a magnet, and the sensor of the present invention.

[0054] Since fertilized egg cells are different from adherent cells during the culture process, they will not adhere to the surface of the culture medium, but move freely in a suspended state. This suspended state will bring certain challenges when detecting calcium ion oscillations, because the movement of fertilized egg cells may have an adverse effect on the stability and accuracy of the detection. Therefore, in certain embodiments of the present invention, the cell carrier has one or more independent units, and each independent unit includes one or more grooves, and the grooves are used to accommodate cells. In other embodiments of the present invention, the cell carrier is made by 3D printing using materials with good biocompatibility, such as BIO resin materials. More specifically, the printed film carrier is 1 cm long and wide, 300 microns thick, and 12 independent areas are divided on its surface. In each area, 9 array micro-circular hole grooves with a diameter of 200 microns are evenly distributed to accommodate cells, such as fertilized egg cells. See the structural schematic diagram. Figure 5 In some other embodiments of the present invention, there is a concave channel between each adjacent micro-circular hole groove to enhance the information exchange between cells, which can make the fertilized egg cell develop better to a certain extent.

[0055] In a fifth aspect of the present invention, a method for detecting intracellular calcium ion concentration in vitro is provided, comprising:

[0056] A calcium ion solution with a gradient concentration is prepared, and the fluorescence intensity at different calcium ion concentrations is measured using the sensor of the present invention, the fluorescence intensity at a certain calcium ion concentration is selected as a benchmark, and the ratios of the fluorescence intensities at other calcium ion concentrations to the benchmark are calculated to obtain a series of relative fluorescence intensity values, and the calcium ion concentrations and the corresponding relative fluorescence intensity values ​​are fitted to obtain a fitting curve;

[0057] The sensor of the present invention is injected into the cell by microinjection technology, and then moved into the cell carrier. At the same time, a magnet is used to exert influence on the cell carrier to control the sensors in the cells to be tested to be in the same plane. Then, a fluorescence detection device is used to obtain the fluorescence intensity in different time periods, and the calcium ion concentration in the cell is detected according to the benchmark and the fitting curve.

[0058] In certain embodiments of the present invention, Ca is first prepared. 2 + mother solution. During the experiment, a pipette was used to take the solution containing the magnetic hydrogel fluorescent sensor of the present invention and add it to the microcontainer at the bottom of the confocal culture dish. Then, a certain volume of Ca 2+ mother liquor is added to the solution to make its calcium ion concentration reach a certain predetermined value. Subsequently, the same volume of Ca 2 + mother liquor, so that the calcium ion concentration in the solution gradually increases, and a series of calcium ion solutions with gradient concentrations are obtained.

[0059] Figure 6 The fluorescence spectrum of the magnetic hydrogel fluorescent sensor is shown in Figure 2. Figure 6 Results and instrument parameters, the present invention sets the fluorescence excitation wavelength to 488nm, the detection wavelength to 525nm, and the present invention takes pictures at different Ca 2 + concentrations, and analyzed the fluorescence intensity changes in each image. Due to the different loading contents of the fluorescent probe, in order to ensure the accuracy of the detection, a certain calcium ion concentration was selected as the benchmark to calculate the fluorescence intensity with different Ca 2 + concentration changes. Finally, we can get the relative fluorescence intensity of Ca 2+ The fitting curve of concentration sensing, such as Figure 7 shown.

[0060] In the process of detecting the intracellular calcium ion concentration of the fertilized egg cell of the present invention, the magnetic hydrogel fluorescent sensor of the present invention is injected into the fertilized egg cell by using the piezoelectric ultrasonic microscope injection technology (usually one sensor is injected into one fertilized egg cell). During the injection, the position of the cell nucleus of the fertilized egg is observed to avoid damaging the cell nucleus and ensure that the sensor is located in the cytoplasm, so as to detect the change of the cytoplasmic calcium ion concentration caused by the release of calcium ions from the endoplasmic reticulum. Figure 8 The injected fertilized oocytes are moved to the culture dish containing the above-mentioned cell carriers to restrain the fertilized oocytes and prevent them from moving and losing the detection target during the detection process, thereby accurately detecting the Ca of a single oocyte. 2+ Concentration parameter. At the same time, the magnetic properties of the sensor are used to control the position of the sensors in all fertilized egg cells to be in the same plane as much as possible through the attraction of neodymium magnets before detection to solve the detection error caused by the sensors not being in the same plane, thereby improving the accuracy of simultaneous detection of multiple fertilized eggs. The overall method implementation diagram is shown in Fig. 9 .

[0061] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified. In the specific embodiments of the present invention, unless otherwise specified, the experimental environment and parameter conditions of each group in the specific embodiment test are consistent with each other except for the differences clearly stated.

[0062] The following is a further description of a magnetic hydrogel fluorescent sensor provided by the present invention and its preparation method and application.

[0063] Embodiment 1:

[0064] Take 50 micrograms of Fluo-8 fluorescent probe freeze-dried powder and dissolve it in 50 microliters of DMSO solution to prepare a fluorescent probe solution with a concentration of about 1 mM, and add 250 microliters of HEPES buffer to obtain the probe solution.

[0065] Prepare a HEPES buffer solution containing magnetic nano-ferroferric oxide: take 0.23g HEPES and 0.04g magnetic nano-ferroferric oxide, mix them evenly into 100ml deionized water, adjust the pH to 7.2, and use an ultrasonic instrument to ultrasonicate the solution at a frequency of 30kHz for 5 minutes to make the nanoparticles in the solution evenly dispersed.

[0066] PEG-DA (3 g) and 5% 2-hydroxy-2-methylpropiophenone (0.3 g) were weighed and mixed in a HEPES buffer (2.7 g) containing magnetic nano-ferrosoferric oxide to form a uniform PEG-DA solution.

[0067] The probe solution and the PEG-DA solution were mixed at a ratio of 1:5 to obtain a PEG-DA precursor aqueous solution.

[0068] A microfluidic chip is used, and the channel diameter at the intersection of water and oil in the microfluidic chip is 30 microns, such as Figure 3 As shown. The aqueous phase is the above-mentioned PEG-DA precursor aqueous solution. The oil phase is a fluorinated oil (HFE7500) containing 2% surfactant. The particle size of the microspheres is adjusted by controlling the flow rate of the aqueous phase. The specific method is to keep the oil phase inlet pressure at 1950 mbar at all times, and control the particle size of the microspheres by gradually adjusting the pressure of the aqueous phase. First, the aqueous phase pressure is set to 1700 mbar, and then decreases by 50 mbar each time, and is adjusted to 1650 mbar, 1600 mbar, 1550 mbar, until it finally reaches 1300 mbar. When the aqueous phase pressure reaches 1300 mbar, the particle size of the generated magnetic fluorescent hydrogel microspheres is around 5 microns, and its particle size distribution diagram is shown in the figure. Figure 4 As shown. The collected magnetic fluorescent hydrogel microspheres are irradiated with a UV curing lamp (10W, 365nm) for 20s to complete the curing. The cured magnetic hydrogel microspheres are located in the emulsion and float on the upper layer of HFE7500. Carefully aspirate and remove the lower transparent HFE7500. Subsequently, a demulsifier is added at a volume ratio of 1:1, and the mixture is centrifuged at 5000rpm for 30 seconds after slight shaking. After the centrifugation, the liquid shows obvious stratification. Carefully aspirate the lower layer of demulsifier, add the upper layer of liquid to HEPES buffer, and centrifuge and wash several times to finally obtain a magnetic hydrogel fluorescent sensor with a size of about 5 microns. The structural diagram is shown in Figure 1 .

[0069] Embodiment 2:

[0070] Laser confocal microscopy was used to measure the fluorescence properties of the magnetic hydrogel fluorescent sensor. First, 1.47 mg of anhydrous CaCl2 was weighed and dissolved in 10 ml of deionized water to obtain a 1 mM calcium ion standard solution, which was then diluted to a concentration of 10 μM Ca 2+ During the experiment, 200 μl of the solution containing the fluorescent hydrogel microsensor was taken with a pipette and added to the microcontainer prepared at the bottom of the confocal culture dish for convenient calibration and detection. Then, 2 μl of Ca 2+ The mother solution was added to the solution to make the calcium ion concentration reach 99nmol / l. Subsequently, 2μl of Ca 2+ mother liquor, so that the calcium ion concentration in the solution gradually increased. After 20 times of gradual addition, the final solution had Ca 2+ The concentration increased to 1667nmol / l.

[0071] The fluorescence excitation wavelength was set to 488 nm and the detection wavelength was set to 525 nm. Due to the different loading contents of the fluorescent probe, in order to ensure the accuracy of the detection, the fluorescence intensity at a calcium ion concentration of 99 nmol / l was used as the benchmark to calculate the fluorescence intensity at different Ca 2 + concentration changes. Finally, we get the relative fluorescence intensity of Ca 2+ The concentration sensor fitting curve is as follows Figure 7 shown.

[0072] Through 3D printing, SMS (Sub-pixel Micro Scanning) micro-nano 3D printer was used, using BIO resin material with good biocompatibility. The printed film carrier is 1 cm long and wide, 300 microns thick, and divided into 12 independent areas on its surface. In each area, 9 array micro-circular holes with a diameter of 200 microns are evenly distributed to accommodate fertilized egg cells. There are concave channels between each adjacent micro-circular hole to enhance the information exchange between cells. See the schematic diagram of the structure. Figure 5 .

[0073] The magnetic hydrogel fluorescent sensor of Example 1 was injected into the fertilized egg cell using piezoelectric ultrasonic microscope injection technology. During the injection, the position of the nucleus of the fertilized egg was observed to avoid damaging the nucleus and ensure that the sensor was located in the cytoplasm, so as to detect the change in cytoplasmic calcium ion concentration caused by the release of calcium ions from the endoplasmic reticulum. The injected oocyte was moved to a culture dish containing the above-mentioned film carrier to restrain the oocyte and prevent it from moving and losing the detection target during the detection process, thereby accurately detecting the Ca concentration of a single fertilized egg cell. 2+Concentration parameters. At the same time, the magnetic properties of the sensor are used to control the position of the sensors in all fertilized egg cells to be in the same plane as much as possible through the attraction of neodymium magnets before detection to solve the detection error caused by the sensors not being in the same plane, thereby improving the accuracy of simultaneous detection of multiple fertilized eggs.

[0074] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0075] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A magnetic hydrogel fluorescent sensor, characterized in that: It comprises hydrogel microspheres, magnetic nanoparticles and calcium ion fluorescent probes; the magnetic nanoparticles and calcium ion fluorescent probes are filled in a three-dimensional network inside the hydrogel microspheres.

2. The sensor according to claim 1, characterized in that The hydrogel microspheres are formed by cross-linking and curing of ultraviolet light-cured monomers.

3. The sensor according to claim 2, characterized in that The ultraviolet light curing monomers include acrylate monomers, acrylamide monomers and acrylic acid monomers.

4. The sensor according to claim 1, characterized in that The magnetic nanoparticles include ferroferric oxide nanoparticles, and the calcium ion fluorescent probe includes a Fluo fluorescent probe.

5. The method for preparing the sensor according to claim 1, characterized in that: include: The calcium ion fluorescent probe, the organic solvent and the buffer solution are mixed to form a first aqueous phase, and the ultraviolet light curing monomer, the photoinitiator, the magnetic nanoparticles and the buffer solution are mixed to form a second aqueous phase; The first aqueous phase and the second aqueous phase are mixed to form an aqueous solution of a UV-curable monomer precursor; The aqueous phase solution of the ultraviolet-curable monomer precursor and the oil phase solution are used to obtain hydrogel droplets through a microfluidic emulsion method, and the sensor is obtained after being cured by ultraviolet light.

6. The preparation method according to claim 5, characterized in that: The organic solvent includes one or more of dimethyl sulfoxide, acetonitrile, ethanol, methanol, acetone, isopropanol, dichloromethane, chloroform, toluene, dimethylformamide, ethylene glycol, and tetrahydrofuran; the buffer includes one or more of HEPES buffer, PBS buffer, Tris buffer, and MOPS buffer; and the oil phase includes fluorinated oil.

7. Use of the sensor according to any one of claims 1 to 4 in detecting intracellular calcium ion concentration in vitro or in preparing a product for detecting intracellular calcium ion concentration in vitro.

8. A product for detecting intracellular calcium ion concentration in vitro, characterized in that: It comprises a fluorescence detection device, a microinjection device, a cell carrier, a magnet, and the sensor according to any one of claims 1 to 4.

9. The product according to claim 8, characterized in that The cell carrier has one or more independent units, each of which includes one or more grooves for accommodating cells.

10. A method for detecting intracellular calcium ion concentration in vitro, characterized in that: include: Prepare calcium ion solutions with gradient concentrations, use the sensor described in any one of claims 1 to 4 to measure the fluorescence intensity at different calcium ion concentrations, select the fluorescence intensity at a certain calcium ion concentration as a benchmark, calculate the ratio of the fluorescence intensity at other calcium ion concentrations to the benchmark, obtain a series of relative fluorescence intensity values, and perform fitting with the calcium ion concentration and the corresponding relative fluorescence intensity value to obtain a fitting curve; The sensor described in any one of claims 1 to 4 is injected into the cell by microinjection technology, and then moved into a cell carrier. At the same time, a magnet is used to exert influence on the cell carrier to control the sensors in the cells to be tested to be in the same plane. Then, a fluorescence detection device is used to obtain the fluorescence intensity in different time periods, and the calcium ion concentration in the cell is detected based on the benchmark and the fitting curve.

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