CN1779461B - A method for encoding multicolor quantum dot microspheres - Google Patents

Abstract

The invention discloses a multi-color quantum dot microsphere coding method, which selects suitable quantum dots for multi-color coding according to the specific needs of biological research. The encoded microspheres with different fluorescence intensity ratios were obtained by controlling the molar concentration ratio of multicolor QDs in the loading solution. For the determined quantum dots, first draw the working curve about the different signal intensity ratios in the microspheres and the QDs concentration ratio in the loading liquid. Through this curve, the effective coding library capacity of the quantum dots is initially evaluated and used to guide the microspheres. Optical encoding. The invention has good accuracy and repeatability, and is simple and easy to operate, and the coded microsphere can be quickly and accurately identified by using a spectrometer, which is beneficial to promoting the development and application of the quantum dot coded microsphere.

Description

A method for encoding multicolor quantum dot microspheres

technical field

The invention belongs to the technical fields of nano-biotechnology and biological analysis, and in particular relates to a method for performing multi-color quantum dot spectral coding on microspheres. Coded microspheres have broad application prospects in gene expression, protein-protein interaction, high-throughput screening, multi-channel biological assays, medical diagnostics, and combinatorial chemistry after biological modification.

Background technique

In recent years, the rapid growth of interest in the analysis of active biomolecules in various complex systems requires and stimulates the invention and development of new analytical detection technologies. Compared with traditional analytical detection technology such as multi-well plate technology, the recently invented microsphere-based analytical detection technology, that is, suspension array technology, has better transport fluidity in the instrumentation device and can provide more molecules per unit volume. The advantages of identifying sites (which helps to improve its detection limit) have good application prospects in biological multivariate analysis, gene expression, medical diagnosis, drug high-throughput screening and combinatorial chemistry, and have become increasingly popular in recent years. The center of attention. However, the bottleneck of this technology lies in the effective encoding of the microspheres. In recent years, many methods of encoding microspheres have emerged. Among them, the multi-color coding technology of microspheres using quantum dot fluorescence signals has attracted much attention due to its advantages of better stability, repeatability, and easy realization of high-throughput detection. However, the multi-color coding based on quantum dots that has been reported so far does not take into account the special requirements of the fluorescence characteristics of biological probes in biological applications, and there are still shortcomings in practicality (such as the overlap between the coding signal and the probe signal, Not conducive to detection, etc.), to be further improved and improved. Therefore, according to the specific needs of biological research, it is necessary and practical to select QDs with suitable emission wavelengths to quantitatively control the multi-color coding of microspheres, and to allow specific wavelength bands to be used as the detection window of marker signals. .

Contents of the invention

The purpose of the present invention is to provide a method for encoding multicolor quantum dot microspheres, which has high accuracy and repeatability, and is easy to operate.

A kind of multi-color quantum dot microsphere encoding method provided by the present invention, its steps are:

(1) According to the emission wavelength of the fluorescently labeled probe to be used, select N kinds of quantum dots whose emission wavelength does not overlap with the fluorescently labeled probe, 2≤N≤10; among the N kinds of quantum dots, randomly select two of them a quantum dot;

(2) prepare the chloroform solution or toluene solution of two kinds of quantum dots selected, measure its molar concentration respectively; Get two kinds of quantum dot solutions selected, join in diluent to obtain quantum dot mixed solution, as loading liquid , the diluent is composed of chloroform and propanol or n-butanol, wherein the volume ratio of chloroform is 40-80%, and the molar concentration and molar concentration ratio of each quantum dot in the mixed solution are calculated;

(3) Add the microspheres to be coded into the loading solution, take them out after fully and evenly adsorbed, and clean the microspheres;

(4) the fluorescence spectrum of the coded microsphere that detection step (3) obtains;

(5) Calculate the fluorescence intensity ratio of the fluorescence spectra of several coded microspheres at the peak positions of the selected two quantum dots, and use the average value of the ratio as the coded signal of the coded microspheres obtained by the concentration ratio loading liquid The average value, the maximum value of the deviation between the intensity ratio of the two peaks of the single-sphere spectrum and the average value is used as the deviation value; Or its logarithmic value as the abscissa and ordinate, draw the coordinate points, and get the deviation value information;

(6) Change the molar concentration ratio of the two selected quantum dots, configure loading solutions with different concentration ratios, repeat steps (3)-(5), obtain the corresponding coded microspheres and corresponding signals and deviations, and draw the coordinate points , get the working curve;

(7) Evaluate the encoding capacity according to the working curve obtained in step (6), and use it for quantitative encoding of the microspheres.

The invention realizes the control of the encoding signal in the microsphere by controlling the molar concentration ratio of QDs with different emission wavelengths in the QDs loading liquid, and can theoretically guide the QDs encoding microsphere according to the experimental results, which has good accuracy and Repeatability, simple and easy operation, and the use of a spectrometer can quickly and accurately identify the encoded microspheres, which is conducive to promoting the development and application of quantum dot encoded microspheres.

Description of drawings

Figure 1 is the ultraviolet absorption and fluorescence spectra of quantum dots, wherein (a) and (b) are the ultraviolet absorption and fluorescence spectra of yellow (575nm) quantum dots, respectively. (c) and (d) are the UV absorption and fluorescence spectra of red (628nm) quantum dots, respectively.

Figure 2 shows the encoding signals of the encoding microspheres obtained from loading solutions with different concentration ratios.

Figure 3 is the relationship curve between the signal intensity ratio of the coded microsphere and the concentration ratio of the two QDs in the corresponding loading solution after taking the logarithm respectively, where the ordinate is lg(I _y /I _r ), and the abscissa is lg(C _y / C _r ).

Detailed ways

The present invention will be further described in detail by taking two kinds of quantum dots as examples below.

(1) According to the luminescence characteristics of specific fluorescent labeled probes used in biological research, such as GFP, FITC, Rh.6G, FAM, HEX, TET, Texas Red, Cascade Blue, Cy3TM, Cy5TM, etc., select two or more suitable emission Quantum dots of wavelengths multi-color code the microspheres. The principle of selection is: the emission wavelength of the quantum dot should avoid the emission wavelength of the labeled probe, so as to avoid the overlapping of the emission peak of the quantum dot and the emission peak of the probe.

(2) Prepare the chloroform solution or toluene solution of the above two kinds of quantum dots, and measure their molar concentrations respectively. Take a small amount of quantum dot solution with a certain volume, add it to a certain volume of diluent composed of chloroform and propanol or chloroform and n-butanol, and dilute to obtain a mixed solution of quantum dots as a loading solution. The volume ratio of methyl chloride is 40-80%. The molar concentration and molar concentration ratio of the above two kinds of quantum dots in the mixed solution are calculated.

(3) Add the microspheres to be encoded into the loading solution at one time, place the loading solution with the microspheres on the shaker and shake fully, after the microspheres are fully and evenly adsorbed to the quantum dots, take them out by centrifugation, and wash off the particles attached to the microspheres with ethanol. Quantum dots on the surface of the spheres, to obtain encoded microspheres obtained from the loading solution at this molar concentration ratio.

(4) Randomly pick some coded microspheres obtained in (3), measure the fluorescence spectrum of the coded microspheres, calculate the fluorescence intensity ratios of these single ball spectra at two peak positions respectively, and obtain the average value of the ratio as this The average value of the coding signals corresponding to the coded microspheres obtained from the loading solution with a concentration ratio, the maximum value of the deviation between the intensity ratio of the two peaks of the single sphere spectrum and the average value is taken as the deviation value of this sample. The molar concentration ratio of the two kinds of quantum dots in the loading solution and the ratio of the fluorescence intensity at the two peaks of the corresponding coded microspheres or their logarithmic values are used as the abscissa and ordinate, respectively, to draw the coordinate points and their deviation value information.

(5) Steps (3) to (4) are repeated to obtain the encoding signals and deviations of the encoding microspheres corresponding to a series of molar concentration ratios of the loading solution. In the coordinate axis, trace the working curve composed of points (lg(I _y /I _r ), lg(C _y /C _r )) (or (I _y /I _r , C _y /C _r ). Among them, I _y and I _r are the fluorescence intensities at two peaks of the microsphere spectrum, respectively, and _Cy and _Cr are the molar concentrations of the two quantum dots in the loading liquid, respectively.

(6) According to the working curve obtained in step (5), if there is no overlap between the deviation values between adjacent points, they can be identified and distinguished; if there is an overlap of deviation values, their identification will be given impact, they should not be used simultaneously in the coding application of the same experiment. In addition, if we want to obtain the coded balls whose coded signal of these two kinds of quantum dots is (I _y /I _x ), we can find the point corresponding to (I _y /I _x ) on the working curve and read out the corresponding loading liquid Concentration ratio value, configure the loading solution of the concentration ratio to encode the pellets, and then obtain the coded pellets with the required coding signal.

(7) For the situation of more than two kinds of quantum dots, that is, when N=3, 4, ... or 10, randomly select two kinds of quantum dots, fix the molar concentration of the remaining quantum dots, change the selected two kinds of quantum concentrations, and obtain A series of loading solutions with different concentration ratios. Repeat steps (3)-(5) with the two selected quantum dots to draw a working curve. By changing the selected two kinds of quantum dots and fixing the molar concentration of other quantum dots, another working curve can be obtained. According to this method, multiple working curves can be obtained.

Example

Here, the present invention will be further described in detail by taking the combined use of fluorescent dye FITC (520 nm) as a labeled probe as an example.

(1) Considering that the emission wavelength of FITC (520nm) is in the blue region, we choose yellow (575nm) and red (628nm) CdSe/ZnS quantum dots for two-color coding of commercial polystyrene microspheres. Measure and calculate the original molar concentrations of yellow and red quantum chloroform solutions to be 5.663×10 ^-7 M and 8.59410 ^-8 M respectively.

(2) Change the molar concentration ratio of the two kinds of quantum dots in the loading liquid, we have configured a series of loading liquids with gradient changes in the concentration ratio (the solvent is made up of 60% chloroform and 40% propanol, and the total volume of the loading liquid remains at 0.5ml), the molar concentration ratio of yellow and red quantum dots in the loading solution ranges from 0.3 to 659 (specifically: 0.3, 0.6, 3.3, 6.6, 9.9, 19.8, 39.5, 79.0, 164.7, 329.5, 659). Weigh 11 parts of 0.5 mg polystyrene microspheres, add them to the loading solution of each concentration ratio at one time, place them on a shaker and vibrate for 24 hours, filter to obtain coded microspheres, and wash the microspheres with absolute ethanol 3 times.

(3) Detecting the spectral signal of the coded microspheres. Use a spectrometer to randomly measure 30 single-sphere spectra for each sample, find the average value as the average value of the single-sphere spectrum of the sample, and take the maximum deviation value as the spectral deviation, as shown in Figure 2. It can be seen from Figure 2 that by controlling the gradient change of the concentration ratio of the two quantum dots in the loading liquid, we have obtained a series of coded microspheres with a gradient change in the signal intensity ratio, and the I _y : I _r of the a ~ k spectra are 1: 15.8, 1:9.8, 1:6.2, 1:2.9, 1:2.4, 1:1.7, 1:1.2, 1.3:1, 3.3:1, 4.7:1, 9.1:1. In Figure 3, I _y and I _r are the fluorescence intensities of encoded microspheres at 575 nm and 628 nm, respectively, and _Cy and _Cr are the molar concentrations of yellow and red quantum dots in the loading solution, respectively.

(4) According to the working curve in Fig. 3, it can be seen that the deviation values of most adjacent coding points overlap, and they are not suitable for the same coding application at the same time. We can pick out non-overlapping codes for simultaneous application. In addition, by preparing the (C _y /C _r ) loading solution corresponding to the point on the curve and encoding the microspheres, we can obtain the desired encoded microspheres.

Claims (1)

1. A multicolor quantum dot microsphere encoding method, the steps of which are: (1) According to the emission wavelength of the fluorescently labeled probe to be used, select N kinds of quantum dots whose emission wavelength does not overlap with the fluorescently labeled probe, 2≤N≤10; among the N kinds of quantum dots, randomly select two of them a quantum dot; (2) prepare the chloroform solution or toluene solution of two kinds of quantum dots selected, measure its molar concentration respectively; Get two kinds of quantum dot solutions selected, join in diluent to obtain quantum dot mixed solution, as loading liquid , the diluent is composed of chloroform and propanol or n-butanol, wherein the volume ratio of chloroform is 40-80%, and the molar concentration and molar concentration ratio of each quantum dot in the mixed solution are calculated; (3) Add the microspheres to be coded into the loading solution, take them out after fully and evenly adsorbed, and clean the microspheres; (4) the fluorescence spectrum of the coded microsphere that detection step (3) obtains; (5) Calculate the fluorescence intensity ratio of the fluorescence spectra of several coded microspheres at the peak positions of the selected two quantum dots, and use the average value of the ratio as the coded signal of the coded microspheres obtained by the concentration ratio loading liquid The average value, the maximum value of the deviation between the intensity ratio of the two peaks of the single-sphere spectrum and the average value is used as the deviation value; Or its logarithmic value as the abscissa and ordinate, draw the coordinate points, and get the deviation value information; (6) Change the molar concentration ratio of the two selected quantum dots, configure loading solutions with different concentration ratios, repeat steps (3)-(5), obtain the corresponding coded microspheres and corresponding signals and deviations, and draw the coordinate points , get the working curve; (7) Evaluate the encoding capacity according to the working curve obtained in step (6), and use it for quantitative encoding of the microspheres.

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