A multi-fluorescence coded microsphere decoded by intelligent devices and its preparation method and application

Abstract

The present invention belongs to the field of biological detection technology and relates to a multi-fluorescence-encoded microsphere decoded by an intelligent device, as well as its preparation method and application. The fluorescent-encoded microsphere comprises a rare earth long-afterglow bulk material and a polymer for encapsulating the rare earth long-afterglow bulk material; the rare earth long-afterglow bulk material comprises a rare earth long-afterglow material that emits blue, green, and red monochromatic, dual-color, or tri-color light; and the polymer is formed by cross-linking and curing mercapto-polydimethylsiloxane and vinyl-polydimethylsiloxane. Compared with traditional fluorescent coding, the fluorescent-encoded microspheres of the present invention do not require large-scale preparation and detection equipment, are easy to commercialize, and are expected to achieve rapid and accurate multiple diagnoses in areas with underdeveloped medical equipment.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a multi-fluorescence coded microsphere decoded by an intelligent device, and a preparation method and application thereof. Background Art

[0002] Multiplexed detection technology based on fluorescence coding enables parallel testing of several or even dozens of analytes in a single sample, making it crucial for the early diagnosis of rapidly developing and multi-subtype diseases. Fluorescence coding using materials such as inorganic dyes and quantum dots presents challenges such as strong background fluorescence, photobleaching, and sample damage from the excitation light source. Furthermore, decoding requires specialized instrumentation, significantly limiting its application in rapid biological testing.

[0003] Long-afterglow materials, which have attracted widespread attention in recent years, are a type of rare earth material that can continue to emit light even after the excitation light has ceased. Based on the synthesis method and particle size, long-afterglow materials can be mainly divided into two categories: nanomaterials and bulk materials. Nanomaterials have uniform particle size, but the synthesis process is complex, the yield is low, and the afterglow intensity is low and the duration is short. Currently, their use is limited to biomedical research fields such as cell and in vivo imaging, biological detection and sensing, and drug treatment. Bulk materials are mainly synthesized by high-temperature roasting methods. The particle size is uneven, but the afterglow intensity is visible to the naked eye and the duration is long. Currently, this type of material has been commercialized and is widely used in daily life in decoration, safety instructions, instrumentation displays and other fields.

[0004] Long afterglow occurs when traps within the material matrix capture photons or energy generated by ultraviolet light in a non-radiative manner, a process known as charging. During this luminescence process, excitation and emission are completely separated. Therefore, multicolor long afterglow luminescence can be used as a coding and imaging method, completely avoiding interference from the excitation light and achieving a very high signal-to-noise ratio. This holds promise for achieving background-free, visually discernible fluorescence coding technology. However, there are few reports on the application of long afterglow bulk materials in biological detection. Summary of the Invention

[0005] In order to improve the above technical problems, the present invention provides a fluorescent coded microsphere, comprising a rare earth long afterglow bulk material and a polymer for encapsulating the rare earth long afterglow bulk material;

[0006] The rare earth long afterglow bulk material includes rare earth long afterglow materials that emit blue, green, or red monochromatic light, dual-color light, or tri-color light;

[0007] The polymer is formed by cross-linking and curing mercapto-polydimethylsiloxane and vinyl-polydimethylsiloxane.

[0008] According to an embodiment of the present invention, the rare earth long afterglow bulk material is selected from (Ca4Sr)S:Eu2+ 、Y2O2S:Ti 4 + ,Mg 2+ 、CdSiO3:Sm 3+ 、CaAl2O4:Eu 2+ ,Nd 3+ 、Sr2MgSi2O7:Eu 2+ ,Nd 3+ 、Ba3MgSiO8:Eu 2+ ,Nd 3+ 、SrAl2O4:Eu 2+ ,Dy 3+ 、Ba 13 Al 22 Si 10 O 65 :Eu 2+ 、CaMgSi2O7:Eu 2+ ,Dy 3+ 、CaAl4O7:Ce 3+ ,Tb 3+ By selecting and adjusting the color and ratio of the rare earth long-afterglow bulk material, single-color and mixed-color fluorescent coded microspheres can be produced. Under ultraviolet or fluorescent light, the rare earth long-afterglow bulk material absorbs energy and stores it in traps within the material. When the excitation light is turned off, the energy in the traps is slowly released as afterglow, emitting blue, green, and red light. Each color represents a code. By varying the ratio of the three different colors of long-afterglow materials, a wider range of mixed materials with different luminescent colors can be obtained, thus achieving a richer range of codes.

[0009] According to an embodiment of the present invention, the mass ratio of the rare earth long-afterglow bulk material, mercapto-PDMS, and vinyl-PDMS is 1:(1.1-1.5):1, for example 1:1.2:1. By controlling the ratio of mercapto-PDMS to vinyl-PDMS, uncrosslinked thiols remain on the surface of the fluorescently encoded microspheres, which can serve as modification sites for nucleic acid probes.

[0010] According to an embodiment of the present invention, the surface of the polymer has thiol groups, and the nucleic acid probe is modified on the surface of the polymer via the thiol groups.

[0011] According to an embodiment of the present invention, the particle size of the fluorescent coded microspheres is not less than 100 μm, for example, 100 μm, 120 μm, 150 μm, 160 μm, 200 μm, 240 μm, 250 μm, 300 μm, 350 μm, or 400 μm, so that the fluorescent coded microspheres can be directly photographed and recognized by a camera of a smart device (e.g., a mobile phone or tablet).

[0012] The present invention also provides a method for preparing the fluorescent coded microspheres, comprising the following steps:

[0013] (1) mixing a rare earth long afterglow bulk material, mercapto-polydimethylsiloxane and vinyl-polydimethylsiloxane, and then mixing with a photoinitiator solution;

[0014] (2) Under stirring conditions, the mixture obtained in step (1) is added dropwise to the surfactant solution, and irradiated with ultraviolet light until the microspheres are solidified.

[0015] According to an embodiment of the present invention, the particle size of the rare earth long afterglow bulk material is 1-5 μm, for example, 1 μm, 2 μm, 3 μm, 4 μm, or 5 μm.

[0016] According to an embodiment of the present invention, the photoinitiator is selected from photoinitiators known in the art, such as one or more of benzoin, benzoin dimethyl ether, benzoin isopropyl ether, and benzoin butyl ether.

[0017] According to an embodiment of the present invention, the photoinitiator solution is an acetone solution of the photoinitiator.

[0018] According to an embodiment of the present invention, the surfactant is selected from sodium lauryl sulfate, styrene-maleic anhydride random copolymer or Tween, preferably sodium lauryl sulfate. In a preferred embodiment, the surfactant is sodium lauryl sulfate, and the sodium lauryl sulfate solution is an aqueous sodium lauryl sulfate solution, preferably at a concentration of 3 to 10%.

[0019] According to an embodiment of the present invention, the preparation method further comprises the following step: (3) reacting the microspheres obtained in step (2) with a maleimide-modified nucleic acid probe, and washing the microspheres after the reaction is completed.

[0020] The present invention also provides the use of the fluorescent coded microspheres in nucleic acid detection.

[0021] The present invention also provides an in vitro nucleic acid detection method, which uses the above-mentioned fluorescent coded microspheres for detection.

[0022] According to an embodiment of the present invention, the detection method includes the following steps: adding the above-mentioned fluorescent coding microspheres to the sample to be tested, incubating, using ultraviolet light-excited nucleic acid dye as a reporter group, and under the irradiation of the excitation light source, photographing the luminescence of the fluorescent coding microspheres before and after incubation by an intelligent device to determine whether there is a target nucleic acid in the sample to be tested; the surface of the polymer of the fluorescent coding microspheres has a thiol group, and the nucleic acid probe is modified on the surface of the polymer by the thiol group. Specifically, when there is a target nucleic acid in the sample to be tested, the target nucleic acid will combine with the nucleic acid probe on the surface of the fluorescent coding microspheres to form a reverse complementary double-stranded pairing, and the nucleic acid dye is embedded in the double-stranded chain. Under the irradiation of the excitation light source (such as ultraviolet lamp), the fluorescent coding microspheres will simultaneously exhibit long afterglow fluorescence and nucleic acid dye fluorescence; if there is no target nucleic acid in the sample to be tested, the nucleic acid probe on the surface of the fluorescent coding microspheres remains in a single-stranded state, the nucleic acid dye cannot be embedded, and under the irradiation of the excitation light source (such as ultraviolet lamp), the fluorescent coding microspheres only exhibit long afterglow fluorescence. In other words, if there is a target nucleic acid, the luminescence color of the fluorescent coding microspheres before and after incubation is different.

[0023] According to an embodiment of the present invention, the excitation light source includes common light source devices such as fluorescent lamps, smartphone flashlights, (portable) ultraviolet lamps, flashlights, etc.

[0024] Beneficial effects

[0025] The coded microspheres of the present invention are composed of a multi-color adjustable fluorescent long-afterglow bulk material and a cross-linked polydimethylsiloxane encapsulation material. The fluorescent coding material is a rare earth long-afterglow material that emits red, green, and blue primary colors. This material can absorb light emitted by the flash of a smart device (such as a mobile phone) and store the energy in a trap within the material matrix. After the excitation light source of the device flash is turned off, the energy in the trap will emit red, green, and blue light in the form of afterglow. The afterglow light can be captured by the camera of the smart device, and the coded signal can be interpreted. The use of polydimethylsiloxane encapsulation can protect the internal long-afterglow material and facilitate the preparation of coded microspheres with uniform particle size and modifiable surface.

[0026] The microspheres prepared by the present invention have a long afterglow time and can still be photographed by a smartphone camera 10 seconds after the excitation light source is turned off.

[0027] The multi-encoded microspheres are made of long-afterglow bulk materials. The afterglow luminescence intensity is high, the duration is long, and there is no background fluorescence signal. No large-scale preparation and detection equipment is required, and it is easy to excite and read, thus realizing the portability of multiple detection. The preparation method is simple and easy to commercialize. It can provide assistance for rapid classification and diagnosis of diseases in the wild and areas with scarce medical resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1Afterglow spectra of three long afterglow bulk materials: blue, green, and red.

[0029] Figure 2 Optical microscope photos of encoded microspheres of different sizes prepared by the stirring method.

[0030] Figure 3 Afterglow photos of color-coded microspheres.

[0031] Figure 4 Photos of blue-coded microspheres bound to the non-reporting group ethidium bromide (EB) with UV light on and off.

[0032] Figure 5 In the figure, (a) Schematic diagram of the process of multiple detection using a smartphone; (b) turning on the UV light to detect whether there are reporter groups bound to the surface of the coded microspheres; turning off the UV light to interpret the code of the microspheres. DETAILED DESCRIPTION

[0033] The fluorescent coding microspheres provided by the present invention have a core part composed of a single-color or multi-color adjustable rare earth long-afterglow bulk material, which is wrapped into a microsphere by a macromolecular polymer and modified with a specific DNA sequence on the outer surface. The rare earth long-afterglow bulk material includes rare earth long-afterglow materials that emit blue, green, and red light. Under the irradiation of ultraviolet light or fluorescent light, the bulk material absorbs energy and stores it in the trap of the material; after the excitation light is turned off, the energy in the trap is slowly released in the form of afterglow, emitting blue, green, and red light respectively. Each color represents a code. By changing the dosage ratio of the three different colors of long-afterglow materials, more mixed materials with different luminescent colors can be obtained, thereby achieving rich codes. These bulk materials are evenly dispersed in a hydrophobic high molecular polymer to prepare micron-sized coding beads with uniform particle size.

[0034] The polymer of the present invention is a macromolecule formed by cross-linking mercapto-polydimethylsiloxane (PDMS) and vinyl-PDMS. Both compounds are colorless and transparent liquids with strong fluidity. After mixing in a certain proportion, they can be further mixed with long-afterglow bulk materials in any proportion. Spherical droplets are obtained by using micro-droplet technology. Under the action of a curing agent, cross-linking occurs between mercapto-PDMS and vinyl-PDMS molecules, and microspheres with smooth and hydrophobic surfaces are formed after curing. The PDMS on the surface of the microspheres prevents the aqueous solution from contacting the long-afterglow bulk material, thereby ensuring the stability of the material structure and luminescent properties; at the same time, PDMS has excellent light transmittance and has almost no effect on the emission of afterglow light. By adjusting the ratio of mercapto-PDMS to vinyl-PDMS, uncross-linked thiols remain on the surface of the microspheres, which can be used as modification sites for nucleic acid probes.

[0035] The surface of the encoded microspheres prepared by the present invention can be modified with nucleic acid probes. Using a UV-excited nucleic acid dye as a reporter group, the presence of the target nucleic acid can be determined. For example, if the target nucleic acid is present in the analyte, it will bind to the probe on the microsphere surface to form a reverse-complementary double-stranded pair. The nucleic acid dye will then embed into the double-stranded pair. Under UV illumination, the encoded microspheres will exhibit both long-lasting fluorescence and fluorescence from the nucleic acid dye. If the target nucleic acid is absent, the probe on the microsphere surface remains single-stranded, preventing the nucleic acid dye from embedding. Under UV illumination, the encoded microspheres will only exhibit long-lasting fluorescence.

[0036] The droplet generation technology used in this invention includes microfluidic chips, stirring methods, and centrifugation methods. It is simple to operate and has high yields, capable of producing several grams of coded microspheres at a time. The generated droplet size is uniform and controllable.

[0037] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0038] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0039] Example 1

[0040] Fluorescently coded microspheres and their preparation:

[0041] (1) The blue (CaAl2O4:Eu) 2+ ,Nd 3+ ), red (Ca4Sr)S:Eu 2+ ), green (SrAl2O4:Eu 2+ ,Dy 3+ ) crushing and sorting the three long afterglow bulk materials to obtain a long afterglow material with a particle size of about 1-5 μm;

[0042] (2) Selecting a single-color long-afterglow material after sorting, or mixing a multi-color long-afterglow material, and repeatedly mixing the prepared mixed material in a mortar; mixing the long-afterglow material with thiol-PDMS and vinyl-PDMS in a mass ratio of 1:1.2:1;

[0043] (3) dissolving the photoinitiator benzoin dimethyl ether in acetone to prepare a solution, adding it to the above-mentioned long afterglow material-PDMS mixture, and mixing thoroughly;

[0044] (4) Dissolve SDS in ultrapure water to prepare a 5% SDS solution. Take about 10 mL each time and add it to a 50 mL EP tube. Place it in a magnetic rotor on a magnetic stirrer and add the mixture prepared in (3) dropwise. Simultaneously, use a portable UV lamp to irradiate the microspheres until they solidify.

[0045] (5) Use stainless steel filters with different pore sizes to sort the solidified microspheres.

[0046] Figure 1 The afterglow spectra of the three long afterglow bulk materials (blue, green and red) in step (1) are shown.

[0047] Figure 2 The optical microscope photographs of coded microspheres of different sizes of 240 μm, 200 μm, 160 μm, 120 μm and 100 μm obtained by sorting in step (5) are shown.

[0048] Figure 3 Afterglow images of coded microspheres of different colors. From left to right, the images show blue, green, red, blue-green, and red-green coded microspheres, with a particle size of 120 μm. Each coded microsphere was placed in a dark location, and a cell phone flash was used as the laser light source. After turning off the flash, the smartphone camera immediately took the image.

[0049] Example 2

[0050] Detection of target nucleic acid:

[0051] (1) Place blue coded microspheres in a 1.5 mL centrifuge tube, add maleimide-modified DNA probe-1, and stir for 24 hours. Remove the supernatant and rinse twice with ultrapure water to remove unbound probes to obtain probe-modified blue coded microspheres, which are then stored in ultrapure water. Under the same conditions, DNA probes-2, -3, and -4 were added to the red, green, and blue-green surfaces, respectively.

[0052] (2) 10 μl (approximately 20) of each of the four probe-modified coding microspheres were mixed and added to the sample to be tested (containing a single-stranded DNA complementary to probe 1) and stirred for incubation. After hybridization, the supernatant was removed and washed twice with ultrapure water. The reporter group hexadidium bromide was added and incubated for 2 minutes. The supernatant was removed and washed twice with ultrapure water. The microspheres were aspirated and placed on a glass slide.

[0053] (3) Place the slide in a dark place, illuminate the microspheres with a portable UV lamp, turn on the iPhone 11 smartphone camera, and take photos when the UV lamp is on, 0 seconds after the UV lamp is turned off, and 10 seconds after the UV lamp is turned off.

[0054] Figure 4The following are photos of blue-coded microspheres with and without reporter groups bound to ethidium bromide, with the UV light on and off. As can be seen from the photos, when the UV light is on, the light emitted by the two coded microspheres differs significantly: the blue-coded microspheres without reporter groups emit a strong blue, while the blue-coded microspheres with reporter groups emit a strong blue-violet. When the UV light is off, both microspheres emit a soft blue.

[0055] Figure 5 In the figure, (a) is a schematic diagram of the multiple detection process using a smartphone. First, turn on the UV light to detect whether there is a reporter group bound to the surface of the coded microspheres; then turn off the UV light and interpret the code of the microspheres (b), obtaining the results of the target DNA detection using the above four coded microspheres. When the UV light is on, the red coded microspheres, green coded microspheres, and cyan coded microspheres emit strong red, green, and cyan-green fluorescence, respectively, and the blue coded microspheres emit strong blue-purple fluorescence; immediately after turning off the UV light, take a picture; wait 10 seconds and take another picture, and you can still see the red coded microspheres, green coded microspheres, cyan coded microspheres, and blue coded microspheres emit red, green, cyan, and blue soft afterglow, respectively. This indicates that the sample to be tested contains only the target nucleic acid that is complementary to probe 1. This result is consistent with the fact that the sample to be tested contains a single-stranded DNA that is complementary to probe 1, indicating that the detection of target nucleic acids using fluorescent coded microspheres is feasible and accurate.

[0056] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A fluorescent coded microsphere, characterized in that: The fluorescent coded microspheres include a rare earth long afterglow bulk material and a polymer for encapsulating the rare earth long afterglow bulk material; The rare earth long afterglow bulk material includes a rare earth long afterglow material that emits blue, green, and red monochromatic light, two-color light, or three-color light, and the particle size of the rare earth long afterglow bulk material is 1-5 μm; The polymer is formed by cross-linking and curing mercapto-polydimethylsiloxane and vinyl-polydimethylsiloxane; The mass ratio of the rare earth long afterglow bulk material, mercapto-polydimethylsiloxane and vinyl-polydimethylsiloxane is 1:(1.1-1.5):1; The particle size of the fluorescent coding microspheres is 100-400 μm.

2. The fluorescent coded microsphere according to claim 1, characterized in that The rare earth long afterglow bulk material is selected from (Ca4Sr)S:Eu 2+ 、Y2O2S:Ti 4+ ,Mg 2+ 、CdSiO3:Sm 3+ 、CaAl2O4:Eu 2+ ,Nd 3+ 、Sr2MgSi2O7:Eu 2+ ,Nd 3+ 、Ba3MgSiO8:Eu 2+ ,Nd 3+ 、SrAl2O4:Eu 2+ ,Dy 3+ 、Ba 13 Al 22 Si 10 O 65 :Eu 2+ 、CaMgSi2O7:Eu 2+ ,Dy 3+ 、CaAl4O7:Ce 3+ ,Tb 3+ One or more of .

3. The fluorescent coded microsphere according to claim 1, characterized in that The surface of the polymer has thiol groups, and the nucleic acid probe is modified on the surface of the polymer via the thiol groups.

4. The fluorescent coded microsphere according to any one of claims 1 to 3, characterized in that: The particle size of the fluorescent coding microspheres is 120 to 350 μm.

5. The method for preparing the fluorescent coded microspheres according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) mixing a rare earth long afterglow bulk material, mercapto-polydimethylsiloxane and vinyl-polydimethylsiloxane, and then mixing with a photoinitiator solution; (2) Under stirring conditions, the mixture obtained in step (1) is added dropwise to the surfactant solution, and irradiated with ultraviolet light until the microspheres are solidified.

6. The preparation method according to claim 5, characterized in that The surfactant is selected from sodium lauryl sulfate, styrene-maleic anhydride random copolymer or Tween, and / or the initiator is selected from one or more of benzoin, benzoin dimethyl ether, benzoin isopropyl ether and benzoin butyl ether.

7. The preparation method according to claim 6, characterized in that The surfactant is sodium lauryl sulfate.

8. The preparation method according to claim 5, characterized in that The preparation method further comprises the following steps: (3) reacting the microspheres obtained in step (2) with a maleimide-modified nucleic acid probe, and washing the microspheres after the reaction is completed.

9. Use of the fluorescently encoded microspheres according to any one of claims 1 to 4 in nucleic acid detection for non-diagnostic and non-therapeutic purposes.

10. An in vitro nucleic acid detection method for non-diagnostic and non-therapeutic purposes, characterized in that: Detection is performed using the fluorescent coded microspheres described in any one of claims 1 to 4.

11. The non-diagnostic and non-therapeutic in vitro nucleic acid detection method according to claim 10, characterized in that: The detection method comprises the following steps: adding the fluorescently encoded microspheres to a sample to be tested, incubating the microspheres, using a nucleic acid dye excited by ultraviolet light as a reporter group, and photographing the luminescence of the fluorescently encoded microspheres before and after incubation under irradiation with an excitation light source by a smart device to determine whether the target nucleic acid is present in the sample to be tested; the surface of the polymer of the fluorescently encoded microspheres has thiol groups, and the nucleic acid probe is modified on the surface of the polymer via the thiol groups; The excitation light source is selected from a fluorescent lamp, a smartphone flashlight, an ultraviolet lamp or a flashlight.

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