CN111410951A - A kind of composite quantum dot encoded microsphere based on natural prickly pollen and preparation method thereof - Google Patents

Abstract

The invention discloses a composite quantum dot encoding microsphere based on natural prickly pollen and a preparation method thereof. The preparation method uses the natural prickly pollen as an encoding substrate, and the surface is modified layer by layer with quantum dots with different characteristic emission peak positions and intensities. Point; after the pollen substrate is carbonized to eliminate autofluorescence, the porous and thorn-like surface structure provides high porosity and large specific surface area, which can increase the adsorption of target molecules and significantly enhance the detection fluorescence signal and improve detection sensitivity; polyelectrolyte is used The layer-by-layer deposition technology enables the outer surface of the pollen substrate to adsorb different layers, types or sizes of quantum dots layer by layer. The combination of layers of quantum dots can realize the coding form of multiple characteristic emission peaks and intensities, expand the coding amount, and at the same time can precisely control the fluorescent coding information of the coded microspheres to meet the needs of multi-index and high-throughput biological detection.

Description

A composite quantum dot-encoded microsphere based on natural prickly pollen and its preparation method

technical field

The invention relates to the technical field of biological materials, relates to a quantum dot encoded microsphere using natural prickly pollen as a coding substrate and a preparation method thereof, in particular to a composite quantum dot encoded microsphere based on natural prickly pollen and the same Preparation.

Background technique

In order to study a large number of intermolecular interactions, such as the interaction between proteins and nucleic acids, between proteins and proteins, and between proteins and drugs, high-throughput test platforms have emerged one after another, and high-throughput test platforms require high-throughput The molecular carrier, the so-called coding carrier, is used to identify different molecules and the interactions that occur between them, and then analyze the results by reading the corresponding coding information. Among the common encoding carriers, the array biochip uses the fixed positions of the molecules, that is, the coordinates, as codes to distinguish different biomolecules. However, because the coordinates of the array biochip can only be fixed in a two-dimensional space, the recognition reaction is limited. speed and its applications. As a new type of solid-phase encoding carrier, microspheres have become a hot spot in the fields of high-throughput screening and combinatorial chemistry. Compared with other forms of solid support, microspheres have significant advantages: first, the specific surface area of microspheres is large, which enables surface chemical reactions to be carried out in a smaller volume; second, the use of microspheres as a carrier can combine other Auxiliary means such as stirring and liquid scouring can realize a reaction system between solid-liquid reaction and liquid-liquid reaction, thereby speeding up the reaction speed of the system; third, the molecules bound on the surface of the microspheres can be easily It can be separated from the solution; fourthly, the modification of diverse functional groups on the surface of the microspheres can greatly expand the use of the microspheres. These advantages make encoded microspheres play a role in anti-counterfeiting, biological analysis, environmental monitoring and other fields.

At present, the fabrication methods of encoded microspheres mainly include chemical synthesis, template method, photolithography and microfluidic technology. The microspheres obtained by these methods tend to exhibit relatively simple spherical and smooth surface structures. The absence of complex surface topography greatly reduces the efficiency of chemical reactions or optoelectronic interactions at structure-derived active sites, and also hinders the integration of multiple physicochemical properties and functions into encoding vectors. In addition, most of these fabrication methods involve precision machining techniques, complex equipment, time-consuming, labor-intensive, and high-cost, thus limiting the large-scale production and widespread use of encoded microspheres.

Nature provides human beings with abundant materials. These materials have been endowed with delicate and complex microstructures and extraordinary functions after billions of years of evolutionary transformation. The direct use of natural materials saves the tedious process of artificial preparation. Pollen is the germ cell in the stamens of flowering plants, powdery in appearance, pollen grains are micron-sized particles with a highly monodisperse particle size distribution, and their exquisite spiny surface structure is durable even under harsh natural conditions sturdiness. In addition, the natural thorny pollen is very abundant and easy to obtain, so the use of pollen as the base material of the encoding carrier can also significantly reduce the production cost.

Compared with traditional organic fluorescent molecules, quantum dots have obvious advantages: wide excitation spectrum, symmetrical distribution of emission spectrum and narrow width, adjustable color, high photochemical stability, and not easy to photolysis. Quantum dots have a continuous excitation spectrum, and the size of the crystal particles can be adjusted to obtain a continuous emission spectrum. The emission spectrum of quantum dots is very narrow, and only one wavelength of light can simultaneously excite quantum dots of various sizes, so multiple colors of emitted light can be obtained with the smallest optical band gap. Based on the coding advantages of quantum dots, combining semiconductor nanocrystal luminescent quantum dots with different fluorescence colors and fluorescence intensities with natural prickly pollen substrates can establish ideal coding microcarriers for multiplex biological detection.

Therefore, in the present invention, we use natural prickly pollen as an encoding carrier, and modify quantum dots on it layer by layer, and design and invent a composite quantum dot encoded microsphere that can simultaneously measure multiple targets, which can be used for biological detection and analysis. .

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to aim at the deficiencies of the above-mentioned prior art, and to provide a composite quantum dot encoded microsphere based on natural prickly pollen and a preparation method thereof. The method adopts the natural prickly pollen as an encoding carrier, and The quantum dots are modified layer by layer on it to prepare microspheres with unique morphology and multiple coding combinations, which solves the shortcomings of the traditional coding microspheres with simple structure, complicated preparation and insufficient coding quantity.

In order to achieve the above-mentioned technical purpose, the technical solution adopted in the present invention is: a preparation method of composite quantum dot encoded microspheres based on natural prickly pollen, comprising the following steps:

(1) Disperse and screen the thorny pollen, and carbonize the screened pollen grains to obtain the pollen base after eliminating autofluorescence;

(2) The pollen substrate is soaked in the negative polyelectrolyte solution and the positive polyelectrolyte solution in turn to obtain a microsphere substrate with a positively charged surface. Using the polyelectrolyte layer-by-layer deposition technology, the outer surface of the positively charged microsphere substrate is layered layer by layer. Quantum dots with different layers, different types or sizes are adsorbed to obtain composite quantum dot-encoded microspheres based on natural prickly pollen.

Further, in step (1), the thorny pollen is soaked in absolute ethanol and shaken to obtain a pollen dispersion liquid with the pollen grains fully peeled off, and the pollen dispersion liquid is dried to disperse and screen out dry pollen grains with distinct particles. , the dried pollen grains were calcined at a high temperature of 300 °C and nitrogen gas was continuously introduced for 12 hours to completely carbonize the fluorescent organic substances to eliminate autofluorescence.

Further, the spiny pollen is selected from the group consisting of sunflower pollen, ragweed pollen and chrysanthemum pollen.

Further, the positive polyelectrolyte is selected from one of polyacrylamine hydrochloride (PAH) and polyethyleneimine (PEI), and the negative polyelectrolyte is selected from polystyrene sulfonate (PSS), polystyrene One of Acrylic Acid (PAA).

Further, the preparation method of the positive and negative polyelectrolyte solution or the negative polyelectrolyte solution is: dissolving the positive polyelectrolyte or the negative polyelectrolyte solid in a 0.5mol/L NaCl solution to finally obtain a positive polyelectrolyte with a concentration of 1 mg/mL solution or negative polyelectrolyte solution.

Further, in step (2), the positively charged microsphere substrate is immersed in the negatively charged quantum dot solution, the quantum dots are adsorbed on the outer surface of the microsphere substrate, and then the positive-negative-positive polyelectrolyte layer ( The positive-negative-positive polyelectrolyte layer formed by the sequential adsorption of positive polyelectrolyte solution, negative polyelectrolyte solution, and positive polyelectrolyte solution, the positive-negative-positive polyelectrolyte layer separates each layer of quantum dots) and negatively charged quantum dots The electrostatic force between them adsorbs quantum dots of different layers, types or sizes layer by layer.

Further, in step (2), the quantum dots are selected from cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe), and cadmium selenide on zinc sulfide (CdSe@ZnS) quantum dots. A sort of.

Further, in step (2), a layer of positive polyelectrolyte is continuously deposited outside the outermost quantum dots to facilitate the grafting of subsequent detection molecules, wherein the detection molecules are selected from one of DNA, RNA and protein.

The invention also provides a composite quantum dot encoded microsphere based on natural prickly pollen, which is prepared by the above preparation method. Number of layers, different kinds or different sizes of quantum dots.

The present invention also provides a hybrid composite quantum dot encoded microsphere based on natural prickly pollen, the hybrid composite quantum dot encoded microsphere is formed by mixing composite quantum dot encoded microspheres with different specificities, and the The composite quantum dot-encoded microsphere is prepared by the above preparation method, and uses natural prickly pollen as a substrate, and the surface adsorbs quantum dots of different layers, types or sizes layer by layer.

Compared with the prior art, the beneficial effects of the present invention are:

1) The present invention uses natural thorny pollen with good monodispersity as the coding substrate, and its unique and complex porous and thorn-like surface structure provides high porosity and large specific surface area, which can increase the adsorption capacity of target molecules and thus significantly enhance the Detecting fluorescent signals, greatly reducing the detection limit and improving detection sensitivity; at the same time, natural prickly pollen, such as sunflower pollen, comes from a wide variety of sources and is available in large quantities, which can be used for large-scale mass production and greatly reduce costs;

2) The present invention adopts the layer-by-layer adsorption method of polyelectrolyte, so that the outer surface of the pollen substrate can adsorb different layers, different types or different sizes of quantum dots layer by layer. The coding stability of the composite quantum dot coding microspheres is good, the excitation spectrum of the quantum dots is wide, the emission spectrum is symmetrically distributed and the width is narrow, and the coding is always stable during the application process, and the fluorescence characteristic peaks and intensity of the obtained coding microspheres are easily determined. precise control;

3) The present invention adopts the combination of different types and different layers of quantum dots to realize the coding form of multiple characteristic emission peaks and intensities, which greatly expands the coding amount, and the decoding method of the prepared composite quantum dot coding microspheres is simple, and only one Light of one wavelength can simultaneously excite quantum dots with various characteristic emission peaks, and the type and concentration information of the target molecule can be obtained at the same time during decoding. the need for quantitative testing;

4) The composite quantum dot-encoded microspheres prepared by the invention have a wide range of applications, and can realize the immobilization of various functional groups by adsorbing polyelectrolytes, thereby providing conditions for chemical coupling with downstream molecules.

Description of drawings

Fig. 1 is the flow chart of the preparation method of the composite quantum dot-encoded microspheres based on natural prickly pollen of the present invention, wherein, 1 is the natural prickly pollen, 2 is the pollen substrate after eliminating autofluorescence, and 3 is the quantum dots, 4 is the prepared composite quantum dot encoded microsphere;

Figure 2 shows the fluorescence characterization of natural prickly pollen and pollen substrate, in which, Figure a is the image of natural prickly pollen taken in bright field by confocal fluorescence microscope, and Figures b to d are ultraviolet light, blue light and green light in order Autofluorescence images of natural prickly pollen under excitation. Figure e is the image of the pollen substrate taken in bright field by confocal fluorescence microscope. Figures f-h are the pollen substrate after carbonization of natural prickly pollen. Fluorescence images under blue and green excitation;

3 is the emission spectrum of the sunflower pollen-based composite quantum dot-encoded microspheres of the present invention, wherein Figure A is the sunflower pollen-based composite quantum dot-encoded microspheres prepared in Example 1 (the microspheres are coded as CdTe 465:CdTe 513:CdTe 626= 1:2:3) and the quantum dots corresponding to the number of layers (one layer of CdTe 465, two layers of CdTe 513, three layers of CdTe626) emission spectra, a1 is prepared in Example 1 based on sunflower pollen The fluorescence emission spectrum of the composite quantum dot encoded microsphere, b1 is the fluorescence emission spectrum of one layer of quantum dots CdTe 465, c1 is the fluorescence emission spectrum of two layers of quantum dots CdTe 513, d1 is the fluorescence emission spectrum of three layers of quantum dots CdTe 626 Fluorescence emission spectrum, Figure B shows the composite quantum dot-encoded microspheres based on ragweed pollen prepared in Example 2 (the microspheres are coded as CdSe 460:CdSe 525:CdSe 620=1:2:1) and their corresponding layers The emission spectrum of the quantum dots (one layer of CdSe 460, two layers of CdSe 525, and one layer of CdSe 620), a2 is the fluorescence emission spectrum of the compound quantum dot-encoded microspheres based on ragweed pollen prepared in Example 2, b2 is The fluorescence emission spectrum of one layer of quantum dots CdSe460, c2 is the fluorescence emission spectrum of two layers of quantum dots CdSe 525, and d2 is the fluorescence emission spectrum of one layer of quantum dots CdSe620;

Figure 4 is the stereoscopic microscope and field emission scanning electron microscope characterization images of dried sunflower pollen grains and sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) in Example 4, wherein, Figure a is a dried sunflower The stereomicroscope images of pollen grains, Figures b and c are SEM images of dried sunflower pollen grains; Figure d is the stereomicroscope images of sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1), Figure d e and f are SEM images of composite quantum dot-encoded microspheres (coded as CdSe 620=1) based on sunflower pollen;

5 is a fluorescence image of different slices of sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) scanned by a laser scanning confocal microscope in Example 4;

Figure 6 shows the composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 460=1), the composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 525=1), and the composite quantum dot-encoded microspheres based on sunflower pollen in Example 4 Fluorescence characterization images of quantum dot-encoded microspheres (coded as CdSe 620=1) under a laser scanning confocal fluorescence microscope, among which, Figure 6a-c shows the composite blue quantum dot-encoded microspheres based on sunflower pollen at different magnifications (coded as CdSe 620=1). are the fluorescence images of CdSe 460=1), Fig. 6d～f are the fluorescence images of the composite green quantum dot-encoded microspheres (coded as CdSe 525=1) based on sunflower pollen at different magnifications, Fig. Fluorescence image of composite red quantum dot-encoded microspheres (coded as CdSe 620=1) of sunflower pollen;

7 is a fluorescence image of the hybrid composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 620=1, CdSe 525=1, CdSe 460=1) under simultaneous excitation by ultraviolet light sources in Example 4, wherein CdSe 620 refers to sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1), CdSe 525 refers to sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 525=1), and CdSe 460 refers to Composite quantum dot-encoded microspheres of sunflower pollen (encoded as CdSe 460=1);

8 is a schematic diagram of the biological detection application process of the composite quantum dot-encoded microspheres prepared in Example 5, wherein 1 is the prepared composite quantum dot-encoded microspheres, 2 is probe DNA, 3 is target DNA, and 4 is modified green Labeled DNA with the fluorescent dye fluorescein carboxylate (FAM, characteristic peak position at 520 nm);

Figure 9 shows the fluorescence intensity of the detected FAM and the standard when the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460=1) and glass beads (Glass Beads), a traditional biological detection product, are used for DNA detection in Example 5. The relationship curve of target DNA concentration, Figures i and ii are sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460=1) and traditional bioassay products with hybrid sandwich structure DNA bound to the surface under modified FAM conditions, respectively. Fluorescence image of glass beads (GlassBeads) in DNA hybridization assay;

Figure 10 shows three kinds of composite quantum dot-encoded microspheres based on sunflower pollen in Example 5 (the codes are CdSe 460:CdSe 620=2:1, CdSe 460:CdSe 620=1:1, CdSe 460:CdSe 620=1:1: 2) Fluorescence spectra obtained for multiplexed and simultaneous DNA detection, among which, Figure a is a composite quantum dot-encoded microsphere based on sunflower pollen (encoded as CdSe 460:CdSe 620= 2:1) The emission fluorescence spectrum of the signal, Figures b~d are three kinds of sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460:CdSe 620= 2:1, CdSe 460:CdSe 620=1:1, CdSe 460:CdSe 620=1:2) emission fluorescence spectrum;

Figure 11 is the fluorescence emission spectrum of natural sunflower pollen and sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe@ZnS 565=1) in Example 6;

12 is a graph showing the change of fluorescence intensity with time of the sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe460=1) after DNA binding in Example 5.

Detailed ways

In order to make those skilled in the art better understand the technical solutions of the present invention, the embodiments of the present invention are described in further detail below with reference to the accompanying drawings.

The experimental methods used in the following examples, unless otherwise specified, are conventional methods, and the used reagents, methods and equipment, unless otherwise specified, are conventional reagents, methods and equipment in the technical field.

The present invention provides a method for preparing composite quantum dot-encoded microspheres based on natural prickly pollen. The process flow is shown in Figure 1, and includes the following steps:

(1) Soak the thorny pollen in absolute ethanol and shake to obtain a pollen dispersion with fully peeled pollen grains. The pollen dispersion is dried to disperse and screen out dry pollen grains with distinct particles. Calcined at a high temperature of 300 °C and continuously passed nitrogen for 12 hours to completely carbonize the fluorescent organic substances to eliminate autofluorescence;

(2) The pollen substrate is soaked in the negative polyelectrolyte solution and the positive polyelectrolyte solution in turn to obtain a microsphere substrate with a positively charged surface. Using the polyelectrolyte layer-by-layer deposition technology, the outer surface of the positively charged microsphere substrate is layered layer by layer. Quantum dots with different layers, different types or sizes are adsorbed to obtain composite quantum dot-encoded microspheres based on natural prickly pollen.

Wherein, the spiny pollen is selected from the group consisting of sunflower pollen, ragweed pollen and chrysanthemum pollen.

Wherein, the positive polyelectrolyte is selected from one of polyacrylamine hydrochloride (PAH) and polyethyleneimine (PEI).

Wherein, the negative polyelectrolyte is selected from one of polystyrene sulfonate (PSS) and polyacrylic acid (PAA).

Further, the preparation method of the positive and negative polyelectrolyte solution or the negative polyelectrolyte solution is: dissolving the positive polyelectrolyte or the negative polyelectrolyte solid in a 0.5mol/L NaCl solution to finally obtain a positive polyelectrolyte solution with a concentration of 1 mg/mL or Negative polyelectrolyte solution.

Further, in step (2), the positively charged microsphere substrate is immersed in the negatively charged quantum dot solution, the quantum dots are adsorbed on the outer surface of the microsphere substrate, and then the positive-negative-positive polyelectrolyte layer ( The polyelectrolyte layer formed by the sequential adsorption of positive polyelectrolyte solution, negative polyelectrolyte solution and positive polyelectrolyte solution, the polyelectrolyte layer separates each layer of quantum dots) and the electrostatic force between the negatively charged quantum dots are different layer by layer adsorption Number of layers, different kinds or different sizes of quantum dots.

Further, the quantum dots are selected from one of cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe), and cadmium selenide in zinc sulfide (CdSe@ZnS) quantum dots.

Further, in step (2), a layer of positive polyelectrolyte is continued to be deposited outside the outermost quantum dots, so as to facilitate the grafting of subsequent detection molecules, and the subsequent detection molecules are selected from one of DNA, RNA, and protein. .

The preparation method of composite quantum dot encoded microspheres based on natural prickly pollen provided by the present invention uses natural prickly pollen as an encoding substrate, has a wide range of material sources, can be used for large-scale mass production and greatly reduces costs, and adopts polyelectrolyte layer by layer. The adsorption method enables the outer surface of the pollen substrate to adsorb quantum dots of different layers, types or sizes layer by layer, which is simple and easy to implement, and can evenly combine various types of quantum dots. It has good performance, and adopts the combination of different types and different layers of quantum points to realize the encoding form of multiple characteristic emission peaks and intensities, which greatly expands the amount of encoding.

Using the above preparation method, composite quantum dot encoded microspheres based on natural prickly pollen can be prepared. The encoded microspheres use natural prickly pollen as a base, and the surface adsorbs quantum dots of different layers, types or sizes layer by layer. .

The above preparation method can also be used to prepare hybrid composite quantum dot-encoded microspheres based on natural prickly pollen, which are formed by mixing composite encoded microspheres with different specificities, and the composite encoded microspheres use natural prickly pollen as a substrate , the surface adsorbs quantum dots of different layers, types or sizes layer by layer.

The above-mentioned composite quantum dot-encoded microspheres or hybrid composite quantum dot-encoded microspheres based on natural prickly pollen use prickly pollen as an encoding substrate, and its porous and prickly surface structure has the benefit of fluorescence enhancement, improving detection sensitivity, and the pollen substrate The quantum dots adsorbed layer by layer on the surface use a combination of different types and layers of quantum dots, which greatly expands the amount of coding, and the coding stability used is good. The decoding method of composite quantum dot-coded microspheres is simple, and only one wavelength of light is used. The quantum dots with a variety of characteristic emission peaks can be excited at the same time, and the type and concentration information of the target molecule can be obtained at the same time when decoding. .

The following are examples:

Example 1

A composite quantum dot-encoded microsphere based on sunflower pollen is prepared by the following steps:

(1) Preparation of sunflower pollen substrate after eliminating autofluorescence by carbonization

Soak the sunflower pollen in absolute ethanol and keep shaking for 20 minutes to obtain a dispersion with fully peeled pollen grains. The dispersion is placed on a hot table and heated at 100 ° C until the ethanol is completely volatilized, and the pollen is in a dry state. Sunflower pollen grains; the dried sunflower pollen grains are placed in a muffle furnace, calcined at a high temperature of 300°C and continuously fed with nitrogen for 12 hours, to obtain a positively charged sunflower pollen substrate after complete carbonization of fluorescent organic substances, as shown in Figure 1;

The natural sunflower pollen and the carbonized sunflower pollen substrate were characterized by laser scanning confocal fluorescence microscopy. Figure 2 shows the fluorescence characterization of the natural sunflower pollen and the sunflower pollen substrate. Figure a is the bright field of the laser confocal fluorescence microscope. The images of natural spiny pollen taken under the following conditions, Figures b-d are the autofluorescence images of natural spiny pollen under the excitation of ultraviolet light, blue light and green light in turn, and Figure e is the pollen substrate taken under the bright field of confocal fluorescence microscope. Images, Figures f-h are the fluorescence images of the pollen substrate after carbonization treatment of natural thorny pollen under the excitation of ultraviolet light, blue light and green light in turn. Comparing the fluorescence images of natural sunflower pollen and sunflower pollen substrate, it can be seen that after carbonization treatment The thorn-like structure of the sunflower pollen substrate was well preserved, and the autofluorescence had been completely removed; the Zeta potential of the sunflower pollen substrate was measured by dynamic light scattering, and the results showed that the surface of the prepared sunflower pollen substrate was positively charged, with an average potential of +27.1mV. ;

(2) Preparation of composite quantum dot-encoded microspheres based on sunflower pollen by layer-by-layer adsorption of polyelectrolytes

The PAH and PSS solid particles were dissolved in 0.5mol/L NaCl solution to prepare PAH solution and PSS solution with a concentration of 1 mg/mL; the sunflower pollen substrate after eliminating autofluorescence was immersed in PSS solution, and allowed to stand for adsorption. 20 minutes, centrifuge, rinse three times with ultrapure water to wash off excess PSS solution, and obtain negatively charged pollen microspheres. The negatively charged pollen microspheres are then immersed in PAH solution, and also after standing for adsorption for 20 minutes, Centrifuge, rinse with ultrapure water three times to obtain a sunflower pollen substrate with a positively charged surface, soak the pollen substrate in an aqueous solution of thioglycolic acid-modified blue quantum dots CdTe 465 (characteristic emission peak at 465 nm), and stand to absorb 60 Centrifuge after 2 minutes, and then wash with ultrapure water three times; referring to the above-mentioned polyelectrolyte adsorption steps, adsorb positive-negative-positive polyelectrolyte layer on the outer surface of blue quantum dot CdTe 465 (sequentially adsorb PAH solution, PSS solution, PAH solution), Then adsorb the first layer of green quantum dots CdTe 513 on the outer surface of the positive-negative-positive polyelectrolyte layer, repeat the above operation, continue to adsorb the second layer of green quantum dots CdTe 513, and then adsorb three layers of red quantum dots through the polyelectrolyte layer-by-layer deposition technology. dot CdTe 626 to prepare composite quantum dot encoded microspheres based on sunflower pollen; the encoded microspheres use quantum dots of different colors (characteristic emission peaks) and different intensities (layers) for composite encoding, and the final code is CdTe 465: CdTe 513: CdTe 626 = 1:2:3;

For the prepared sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdTe 465:CdTe 513:CdTe626 = 1:2:3) and the quantum dots corresponding to the number of layers (one layer of CdTe 465, two layers of CdTe 513, Three layers of CdTe 626) were measured under the same ultraviolet excitation light, and their fluorescence emission spectra were measured using a fiber optic spectrometer, as shown in Figure 3A, where a1 was the sunflower pollen-based composite quantum dots prepared in this example. The fluorescence emission spectrum of the encoded microspheres, b1 is the fluorescence emission spectrum of one-layer quantum dots CdTe 465, c1 is the fluorescence emission spectrum of two-layer quantum dots CdTe 513, d1 is the fluorescence emission spectrum of three-layer quantum dots CdTe 626 , it can be seen from the figure that after the quantum dots are modified on the pollen substrate, the fluorescence intensity does not decrease significantly.

Example 2

A composite quantum dot-encoded microsphere based on ragweed pollen is prepared by the following steps:

(1) Preparation of ragweed pollen substrate after autofluorescence elimination by carbonization

Soak the ragweed pollen in absolute ethanol and keep shaking for 20 minutes to obtain a dispersion with fully peeled pollen grains. The dispersion is placed on a hot table and heated at 100 ° C until the ethanol is completely volatilized, and the pollen is in a dry state. Drying the ragweed pollen grains; placing the dried ragweed pollen grains in a muffle furnace, calcining at a high temperature of 300° C. and continuously feeding nitrogen for 12 hours, to obtain a positively charged ragweed pollen substrate after the fluorescent organic substances are completely carbonized;

(2) Preparation of composite quantum dot-encoded microspheres based on ragweed pollen by polyelectrolyte layer-by-layer adsorption

The PAH and PAA solid particles were dissolved in 0.5mol/L NaCl solution, respectively, to prepare a PAH solution and a PAA solution with a concentration of 1 mg/mL; the ragweed pollen substrate after eliminating autofluorescence was immersed in the PAA solution, and allowed to stand. Adsorbed for 20 minutes, centrifuged, rinsed three times with ultrapure water to wash off excess PAA solution, then immersed the pollen microspheres in PAH solution, also stood for adsorption for 20 minutes, centrifuged, and rinsed three times with ultrapure water to obtain The positively charged ragweed pollen substrate, referring to the polyelectrolyte layer-by-layer adsorption method in Example 1, adsorbed thioglycolic acid-modified blue quantum dots layer by layer through a polyelectrolyte isolation layer (sequentially adsorbing PAH solution, PAA solution, and PAH solution). CdSe 460, the first layer of green quantum dots CdSe 525, the second layer of green quantum dots CdSe 525, and the red quantum dots CdSe 620 were used to prepare composite quantum dot encoded microspheres based on ragweed pollen; the encoded microspheres used different colors ( Characteristic emission peaks) and quantum dots of different intensities (layers) are compositely encoded, and the final encoding is CdSe 460: CdSe 525: CdSe 620 = 1:2:1;

The prepared ragweed pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460:CdSe 525:CdSe 620 = 1:2:1) and their corresponding quantum dots (one layer of CdSe 460, two layers of CdSe 525, a layer of CdSe 620) were measured under the same ultraviolet excitation light, and their fluorescence emission spectra were measured using a fiber optic spectrometer as shown in Figure 3B, where a2 was prepared in this example. The composite quantum based on sunflower pollen The fluorescence emission spectrum of dot-coded microspheres, b2 is the fluorescence emission spectrum of one layer of quantum dots CdSe 460, c2 is the fluorescence emission spectrum of two layers of quantum dots CdSe 525, d2 is the fluorescence emission spectrum of one layer of quantum dots CdSe 620 It can be seen from the figure that after the quantum dots are modified on the pollen substrate, their fluorescence intensity does not decrease significantly.

Example 3

A hybrid composite quantum dot-encoded microsphere based on chrysanthemum pollen is prepared by the following steps:

(1) Soak chrysanthemum pollen in anhydrous ethanol and keep shaking for 20 minutes to obtain a dispersion with fully peeled pollen particles. Heat the dispersion on a hot table at 100°C until the ethanol is completely volatilized and the pollen is in a dry state. Screen out the particles Distinct dry ragweed pollen grains; the dry chrysanthemum pollen grains are placed in a muffle furnace, calcined at a high temperature of 300°C and continuously fed with nitrogen for 12 hours to obtain a positively charged chrysanthemum pollen substrate after the fluorescent organic substances are completely carbonized;

(2) Hybrid composite quantum dot-encoded microspheres based on chrysanthemum pollen were prepared by layer-by-layer adsorption of polyelectrolytes

The PEI and PSS solid particles were dissolved in 0.5mol/L NaCl solution, respectively, to prepare a PEI solution and a PSS solution with a concentration of 1 mg/mL; the chrysanthemum pollen substrate after eliminating autofluorescence was immersed in the PSS solution, and allowed to stand for adsorption. 20 minutes, centrifuge, rinse three times with ultrapure water to wash off excess PSS solution, then soak the pollen microspheres in PEI solution, also stand for adsorption for 20 minutes, centrifuge, rinse three times with ultrapure water to obtain the surface The positively charged chrysanthemum pollen substrate, referring to the polyelectrolyte layer-by-layer adsorption method in Example 1, adsorbed the first layer of blue quantum dots CdS 540, The second layer of blue quantum dots CdS 540 and the third layer of blue quantum dots CdS 540 are prepared to obtain a chrysanthemum pollen-based composite quantum dot encoded microsphere encoded as CdS 540=3; with reference to the above-mentioned polyelectrolyte layer-by-layer adsorption method, Quantum dots CdS 540, the first layer of quantum dots CdS 580, the second layer of quantum dots CdS 580, and the third layer of quantum dots CdS 580 were adsorbed layer by layer on the outer surface of the chrysanthemum pollen substrate, and the prepared code was CdS 540:CdS 580= 1: The composite quantum dot coding microsphere based on chrysanthemum pollen according to 3; with reference to the above-mentioned polyelectrolyte layer-by-layer adsorption method, quantum dots CdS 540, quantum dots CdS 580 and quantum dots CdS 620 are adsorbed layer by layer on the outer surface of the chrysanthemum pollen substrate, and the prepared code is CdS 540:CdS580:CdS 620=1:1:1 chrysanthemum pollen-based composite quantum dot-encoded microspheres; mixing the above three encoded microspheres to obtain three different specific chrysanthemum pollen-based hybrid quantum dots Coding microspheres, that is, the combination of quantum dot coding microspheres with CdS 540=3, CdS 540: CdS 580= 1: 3, CdS 540: CdS 580: CdS 620= 1: 1: 1 respectively;

In the embodiment, a layer of PEI solution is deposited outside the outermost quantum dots of each composite quantum dot encoded microsphere, which can be used for the detection of various molecules to be tested, and the molecules are selected from one of DNA, RNA and protein.

Example 4: Characterization experiment of composite quantum dot-encoded microspheres based on natural prickly pollen

4.1) Preparation of dry sunflower pollen grains, sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe620=1), sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe 460=1), and sunflower pollen-based composite Quantum dot encoded microspheres (coded as CdSe 525=1);

With reference to Example 1, sunflower pollen was soaked in absolute ethanol and kept oscillating for 20 minutes to obtain a dispersion with fully peeled pollen grains. Dry sunflower pollen grains with distinct particles; take part of the dried sunflower pollen grains and dry sunflower pollen grains in a muffle furnace, calcine at a high temperature of 300 ° C and continuously pass nitrogen for 12 hours to obtain the sunflower pollen pollen base after eliminating autofluorescence. The sunflower pollen substrate was immersed in PSS solution and PAH solution with a concentration of 1 mg/mL in turn, and then the positively charged sunflower pollen substrate was immersed in quantum dot CdSe 620 to obtain a surface-modified layer of red based on natural sunflower pollen. Composite quantum dot-encoded microspheres of quantum dots CdSe 620, namely sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1); similarly, the surface-positively charged sunflower pollen substrate was immersed in quantum dot CdSe 525 , obtained a composite quantum dot-encoded microsphere with a layer of green quantum dots CdSe 525 based on natural sunflower pollen, that is, a sunflower pollen-based composite quantum dot-encoded microsphere (coded as CdSe 525=1); the surface is positively charged The sunflower pollen substrate was soaked in quantum dots CdSe 460 to obtain composite quantum dot-encoded microspheres based on natural sunflower pollen with a layer of blue quantum dots CdSe 460, that is, sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe 460=1);

4.2) The dried sunflower pollen grains prepared in step 4.1) and the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) were characterized by stereo microscopy and field emission scanning electron microscopy, and the results are shown in Figure 4; It can be seen from Fig. 4d–f that the diameter of the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) is in the range of 20–30 μm, which is about 10 μm smaller than that of natural sunflower pollen (Fig. 4a–c). , this is the result of high temperature calcination at 300 °C. Although the diameter of the composite quantum dot-encoded microspheres becomes smaller, the retention of the pollen-like structure is better. At the same time, it can be clearly observed that the quantum dot CdSe 620 is uniformly distributed on the surface of the pollen substrate, indicating that the natural Spiny pollen is the coding substrate. Its unique and complex porous and thorn-like surface structure provides high porosity and large specific surface area. The layer-by-layer adsorption method of polyelectrolyte can uniformly bind various types of quantum dots;

4.3) Fluorescence images of different slices scanned under a laser scanning confocal microscope on the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) prepared in step 4.1), as shown in Figure 5, red quantum dots CdSe620 (bright white part in the figure) covers the entire pollen surface and is only distributed on the surface, indicating the use of spiny pollen with good monodispersity as the coding substrate, and its unique complex porous and spiny surface structure provides high porosity and relatively Large specific surface area, effective quantum dot coating can be obtained by layer-by-layer adsorption of polyelectrolyte, which can increase the adsorption amount of target molecules and significantly enhance the detection fluorescence signal, greatly reduce the detection limit, and improve the detection sensitivity;

4.4) For the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460=1) prepared in step 4.1), the sunflower pollen-based composite quantum dot-coded microspheres (coded as CdSe 525=1), sunflower pollen-based The composite quantum dot-encoded microspheres (coded as CdSe 620=1) were subjected to fluorescence characterization under a laser scanning confocal fluorescence microscope. Fluorescence image with the code CdSe 460=1), the blue quantum dot CdSe 460 (the bright part in the figure) covers the entire pollen surface and is only distributed on the surface, Figure b, c are Figure a under different magnifications, Figure 6d- f is the fluorescence image of the composite quantum dot-encoded microspheres (coded as CdSe 525=1) based on sunflower pollen. The green quantum dot CdSe 525 (the bright part in the figure) covers the entire pollen surface and is only distributed on the surface. Figures e and f respectively Figure d under different magnifications, Figure 6g-i are the fluorescence images of the composite quantum dot-encoded microspheres (coded as CdSe 620=1) based on sunflower pollen, the red quantum dot CdSe 620 (the bright part in the figure) covers the entire pollen The surface and only distributed on the surface, Figures h, i are Figures g under different magnifications, and it can be observed that there is stronger fluorescence at the top of the spikes on the pollen surface;

The composite quantum dot-encoded microspheres based on sunflower pollen (coded as CdSe 460=1), the sunflower pollen-based composite quantum dot-coded microspheres (coded as CdSe 525=1), and the sunflower pollen-based composite quantum dot-coded microspheres ( Coded as CdSe 620=1), three hybrid composite quantum dot-encoded microspheres based on sunflower pollen with different specificities were obtained, that is, the quantum dot codes were CdSe 460=1, CdSe 525=1, CdSe 620=1 respectively. Combination of composite quantum dot-encoded microspheres; hybrid composite quantum dot-encoded microspheres based on sunflower pollen (coded as CdSe 460=1, CdSe 525=1, CdSe 620=1) were subjected to fluorescence under a laser scanning confocal fluorescence microscope Characterization, as shown in Figure 7, under the excitation of the same ultraviolet light source, quantum dots with multiple characteristic emission peaks can be excited at the same time, and red (coded as CdSe 620=1) and green (coded as CdSe 525= 1), blue (coded as CdSe 460=1) three-color coded microspheres, indicating that the method for decoding the composite quantum dot coding microspheres or the hybrid composite quantum dot coding microspheres based on natural prickly pollen prepared by the present invention is simple , quantum dots with multiple characteristic emission peaks can be excited simultaneously with only one wavelength of light.

Example 5: DNA Hybridization Assay Experiment

(1) Taking the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460=1) prepared in Example 4 as an example, the probe DNA was immobilized on the composite quantum dot-encoded microspheres. The specific implementation process is as follows: First, the probe DNA is amine functionalized at the 5' end, and labeled with the green fluorescent dye fluorescein carboxylate (FAM, the characteristic peak position is 520 nm) at the 5' end of the labeled DNA; the hybrid sandwich structure is shown in Figure 8. The prepared composite quantum dot-encoded microspheres with carboxyl groups were added to 50 mL of freshly prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC (4.2 mg/mL) solution, meanwhile, the active intermediate was stabilized with 50 mL of N-hydroxysuccinimide NHS (5 mg/mL) solution; then 1 mL of amine-functionalized probe DNA solution was added to the above mixture, and the sample Incubate for 2 hours at 4°C with continuous shaking at room temperature in the dark. Since the carboxyl group of the composite quantum dot-encoded microspheres can form an amide bond with the amino group of the amine-functionalized DNA, the probe DNA can be immobilized. On the composite quantum dot-encoded microspheres, after centrifugation, the unbound excess probe DNA was washed three times with phosphate buffered saline solution; then, the 5' end-labeled FAM-labeled DNA and the target DNA mixture were added in equimolar ratios in turn. , and the samples were continuously shaken at 4°C and incubated for 6 hours under the condition of room temperature and dark; The hybrid sandwich structure was formed on the surface with the code CdSe460=1), and the composite quantum dot-encoded microspheres based on sunflower pollen (coded as CdSe 460=1) with the hybrid sandwich structure DNA bound on the surface were subjected to fluorescence by laser scanning confocal microscope and fiber optic spectrometer. Characterization of images and emission spectra.

Referring to the above steps, using glass beads (Glass Beads), a traditional biological detection product, as a control, and under the same conditions as other experimental parameters, compare the composite quantum dot-encoded microspheres based on sunflower pollen (coded as CdSe460=1) and Glass Beads Performance in DNA hybridization assay experiments. Fluorescence images and emission spectra of Glass Beads with hybridized sandwich DNA under modified FAM conditions were characterized by laser scanning confocal microscopy and fiber optic spectrometer. As shown in Fig. 9i, 9ii, the green fluorescence of the labeled dye FAM can be detected in the fluorescence images, and the fluorescence intensity emitted by the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460=1) is much higher than that of Glass Beads. As shown in Figure 9, the fluorescence curve proves that the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460=1) can detect a wider range of DNA concentrations than Glass Beads, and the detection limit of Glass Beads is further measured as 1090 pM, while the detection limit of the quantum dot-encoded microspheres prepared by the present invention is only 9.7 pM. It can be seen that the prickly pollen is used as the encoding substrate, and its unique and complex porous and thorn-like surface structure provides high porosity and a large ratio. The surface area increases the adsorption amount of the target molecule, which significantly enhances the detection fluorescence signal, greatly reduces the detection limit, and improves the detection sensitivity.

(2) Determination of multiple DNA target sequences

Referring to the preparation method of Example 3, prepare hybrid composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 460:CdSe 620=2:1, 1:1, 1:2), refer to the above steps of this example (1), different probe DNAs (1 nmol/L) were immobilized on three kinds of sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe 460:CdSe 620=2:1, CdSe 460:CdSe 620= 1:1, CdSe 460:CdSe 620=1:2), mix different carrier-probe conjugates at room temperature, and add four target DNA molecules with different sequences in an equimolar ratio, and put the mixture at 37 °C. Incubate with continuous shaking for 6 hours in the dark to obtain hybrid composite quantum dot-encoded microspheres (coded as CdSe 460:CdSe 620=2:1, 1:1, 1:2) with hybrid sandwich DNA bound on the surface; then Spectral signals reflected on different composite quantum dot-encoded microspheres were detected with a spectrometer (encoded microspheres were washed 3 times with buffer before fluorescence measurement). The results are shown in Figure 10. Figure a is the emission fluorescence spectrum of the composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 460:CdSe 620=2:1) without target signal under the condition of labeled DNA unmodified FAM. Figures b to d show three kinds of composite quantum dot-encoded microspheres based on sunflower pollen with hybrid sandwich structure DNA bound on the surface in turn under the condition of modified FAM (the codes are CdSe 460:CdSe 620=2:1, CdSe 460:CdSe 620 =1:1, CdSe 460:CdSe 620=1:2) emission fluorescence spectrum, the characteristic peak at the wavelength of 520 nm is the emission peak of the detected target signal; from the detection results shown in Figure 10, it can be seen that the mixed composite There is no cross-interference in the detection of quantum dot-encoded microspheres, which can clearly reflect the types of target molecules and quantitatively detect the concentration information of the molecules; it shows that the present invention adopts the combination of different types and different layers of quantum dots to achieve multiple characteristic emission peaks and intensities The coding format of TM greatly expands the coding amount, and the type and concentration information of the target molecule can be obtained at the same time during decoding, and there is no cross-interference during detection, which can meet the needs of simultaneous detection of multiple indicators and high-throughput detection.

Example 6: Emission Spectra and Stability Characterization of Quantum Dot Encoding

The reference is the preparation method of the composite quantum dot-encoded microspheres described in Example 1. Using natural sunflower pollen as a substrate, a layer of CdSe@ZnS565 quantum dots is modified on the surface of the sunflower pollen substrate by polyelectrolyte layer-by-layer deposition technology, thereby preparing Sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe@ZnS565=1), natural sunflower pollen and sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe@ZnS565=1) were analyzed by fluorescence spectrophotometer The measurement of excitation spectrum and emission spectrum is shown in Figure 11. It can be observed that the composite quantum dot-encoded microspheres based on sunflower pollen (coded as CdSe@ZnS565=1) (referred to as composite quantum dot-encoded microspheres in the figure) The excitation spectrum is wide, and the emission spectrum is symmetrically distributed and narrow in width. Compared with natural sunflower pollen, the fluorescence of the composite quantum dot-encoded microspheres has a strong optical encoding advantage;

In the DNA hybridization assay experiment in Example 5, the fluorescence intensity of the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460=1) after binding to DNA was detected by a fiber optic spectrometer. The results are shown in Figure 12. It can be seen that the fluorescence intensity of the quantum dots has a small decrease in a long time; it shows that the composite coding microspheres based on the natural spiny pollen prepared by the present invention have better coding stability in the application process.

The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions that belong to the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims (10)

1. a preparation method based on the composite quantum dot coding microsphere of natural prickly pollen, is characterized in that, comprises the following steps: (1) Disperse and screen the thorny pollen, and carbonize the screened pollen grains to obtain the pollen base after eliminating autofluorescence; (2) The pollen substrate is soaked in the negative polyelectrolyte solution and the positive polyelectrolyte solution in turn to obtain a microsphere substrate with a positively charged surface. Using the polyelectrolyte layer-by-layer deposition technology, the outer surface of the positively charged microsphere substrate is layered layer by layer. Quantum dots with different layers, different types or sizes are adsorbed to obtain composite quantum dot-encoded microspheres based on natural prickly pollen. 2. The preparation method according to claim 1, characterized in that: in step (1), the spiny pollen is immersed in absolute ethanol and shaken to obtain a pollen dispersion liquid from which the pollen grains are fully peeled off, and the pollen dispersion liquid is subjected to Drying treatment is used to disperse and screen dry pollen grains with distinct particles. The dry pollen grains are calcined at a high temperature of 300°C and continuously passed in nitrogen for 12 hours to completely carbonize the fluorescent organic substances to eliminate autofluorescence. 3. The preparation method according to claim 2, wherein the spiny pollen is selected from the group consisting of sunflower pollen, ragweed pollen and chrysanthemum pollen. 4. The preparation method according to claim 1, wherein the positive polyelectrolyte is selected from one of polyacrylamine hydrochloride and polyethyleneimine, and the negative polyelectrolyte is selected from polystyrene sulfonate One of sodium and polyacrylic acid. 5. preparation method according to claim 4 is characterized in that: the preparation method of described positive polyelectrolyte solution or negative polyelectrolyte solution is: positive polyelectrolyte or negative polyelectrolyte solid is dissolved in 0.5mol/L NaCl solution , and finally obtain a positive polyelectrolyte solution or a negative polyelectrolyte solution with a concentration of 1 mg/mL. 6 . The preparation method according to claim 5 , wherein in step (2), the positively charged microsphere substrate is immersed in a negatively charged quantum dot solution, and the quantum dots are adsorbed on the outside of the microsphere substrate. 7 . On the surface, quantum dots of different layers, types or sizes are adsorbed layer by layer through the electrostatic force between the positive-negative-positive polyelectrolyte layer and the negatively charged quantum dots. 7 . The preparation method according to claim 6 , wherein in step (2), the quantum dots are selected from the group consisting of cadmium sulfide, cadmium selenide, cadmium telluride, and cadmium selenide on zinc sulfide quantum dots. 8 . kind. 8 . The preparation method according to claim 7 , wherein in step (2), a layer of positive polyelectrolyte is continuously deposited outside the outermost layer of quantum dots, so as to facilitate subsequent detection of the grafting of molecules. The molecule is selected from one of DNA, RNA, and protein. 9. A composite quantum dot-encoded microsphere based on natural thorny pollen, characterized in that, obtained by the preparation method according to any one of claims 1-8, the composite quantum dot-encoded microsphere is made of natural The thorn-like pollen was used as the substrate, and the surface adsorbed different layers, different types or different sizes of quantum dots layer by layer. 10. A hybrid composite quantum dot encoded microsphere based on natural prickly pollen, wherein the hybrid composite quantum dot encoded microsphere is formed by mixing different specificity composite quantum dot encoded microspheres. The composite quantum dot-encoded microspheres are prepared by the preparation method described in any one of claims 1-8, with natural prickly pollen as a substrate, and the surface adsorbs quantum dots of different layers, types or sizes layer by layer.

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