Disc-based multi-staining biochip detection method based on nanoparticle perturbation laser signal

Abstract

The application provides a disc-based multi-staining biochip detection method based on nanoparticle disturbance laser signal, which comprises the following steps: S1, determining staining substances of various to-be-detected organisms according to the concentration detection range of various to-be-detected organism solutions; S2, preparing a staining organism array of various to-be-detected organism solutions on a disc base to obtain a disc-based staining biochip; S3, scanning the disc-based staining biochip based on a blue light drive; and S4, analyzing the BIS error number at each logical position in the scanning result, and determining the concentration of the to-be-detected organism solution according to the BIS error number size. The application can realize the simultaneous detection of the concentrations of various organism solutions, and improves the detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochip detection, and further relates to a high-throughput biochip preparation and detection method. More specifically, the present application relates to a disc-based multi-dye biochip detection method based on nanoparticle disturbance of laser signals, and more particularly to a high-throughput bioarray prepared on a disc surface, scanning by a standard Blu-ray drive, and realizing simultaneous high-throughput quantitative detection of multiple molecules according to different light intensity responses of different color signals. BACKGROUND

[0002] Biochip technology is a micro-biochemical analysis system on a silicon chip or a glass chip surface, which integrates discontinuous analysis processes in the field of life science according to the principle of specific interaction between molecules, so as to realize accurate, rapid and large information detection of cells, proteins, genes and other biotic components.

[0003] The biochip reaction result is displayed by isotope method, chemical fluorescence method, chemiluminescence method or enzyme labeling method, and then detected by a precise scanner or CCD camera technology, and the quantity and sequence information of sample molecules are obtained by computer software analysis. This solid biochip is first developed by the American Bio Company Affymetrix, and has the advantages of high throughput, parallel detection and rapid interpretation. The biggest disadvantage of this method is that the detection instrument is expensive, which limits the popularization and application of biochip, especially in small and medium-sized medical institutions.

[0004] In order to overcome the above-mentioned shortcomings, a series of biochip analyzers have been developed by a research group using commercial electronic equipment. Since 2000, a large number of biochemical detection sensors based on optical drives and their optical heads have appeared, and most of the researches rely on optical drive sensor mechanisms or modify the sensing mechanism. For example, the Maquieira research group in Spain (Morais, S., Carrascosa, J., Mira, D., Puchades, R., & Maquieira, A.. (2007). Microimmunoanalysis on standard compact discs to determine low abundant compounds. Analytical Chemistry, 2007, 79(20), 7628-7635) added a photosensitive trigger and a planar photodiode receiver above the optical drive to extract the transmission light signal of the reflected area on the disc in real time for analysis.

[0005] Compared with traditional biochip detection technology, the bio-sensor based on optical drive / optical head shows obvious advantages in portability and instrument cost, but it needs to prepare special structure optical disc and additional installation of optical elements or photoelectric detector and other hardware devices, which reduces the portability and increases the complexity of operation. In contrast, the error number detection technology of disc quality diagnosis software based on commercialized standard optical drive (Zhang, L., Wang, H., Zhang, X., Li, X., & Yu, H. Z., Indirect competitive immunoassay on a Blu-ray disc for digitized quantitation of food toxins. ACS sensors, 2020, 5(4), 1239-1245) and the optical drive scanning imaging technology based on extraction of internal light intensity signal of optical head (ZL201710656932.5) have the advantages of no modification, simple operation, high sensitivity and on-site rapid detection.

[0006] In addition, in the quantitative detection of biomolecules, sensitivity is an important consideration. In order to improve the sensitivity of the detection system, signal amplification technology is proposed. At present, the traditional signal amplification method includes signal amplification of nanomaterials, enzyme catalytic signal amplification, biotin-avidin signal amplification and hybridization chain reaction signal amplification. Among them, gold nanoparticle (AuNPs) labeling and silver staining are the most commonly used signal amplification method, which can produce obvious and stable gray deposition in a short time and is suitable for solid-state biochip signal amplification. Recently, a new type of enzyme catalytic chromogenic substrate has been used in solid-state biochip signal amplification, which produces insoluble precipitate by reacting with the substrate after enzyme catalytic hydrolysis. Therefore, considering that single staining often appears inconsistent staining degree and oversaturation in multi-index detection, multi-staining biochip is more suitable for multi-index detection system. SUMMARY

[0007] The present application overcomes the deficiencies of the prior art and solves the technical problem of providing a disc-based multi-staining biochip detection method based on nanoparticle disturbance of laser signal to detect multiple biological concentrations.

[0008] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a disc-based multi-staining biochip detection method based on nanoparticle disturbance of laser signal, comprising the following steps:

[0009] S1, according to the concentration detection range of various biological solutions to be tested, determine the staining material of various biological to be tested;

[0010] S2, preparing a dyeing bioarray of various biological solutions to be tested on the optical disc base to obtain a dyeing biochip on the optical disc base;

[0011] S3, scanning the dyeing biochip on the optical disc base based on a blue-ray drive;

[0012] S4, analyzing the BIS error number at each logical position in the scanning result, and determining the concentration of the biological solution to be tested according to the BIS error number.

[0013] The laser wavelength of the blue-ray drive is 405 nm.

[0014] The disc-based multi-dyeing biochip detection method based on nanoparticle disturbance laser signal further comprises the following steps:

[0015] Preparing a microfluidic channel on the optical disc base;

[0016] Preparing a dyeing bioarray of various standard concentrations of biological solutions to be tested in the microfluidic channel to obtain a standard dyeing biochip on the optical disc base;

[0017] Scanning the standard dyeing biochip on the optical disc base based on a blue-ray drive;

[0018] Analyzing the BIS error number at each logical position in the scanning result to obtain a BIS error number-concentration curve of various biological solutions to be tested.

[0019] In the step S4, the concentration of the biological solution to be tested is determined according to the BIS error number-concentration curve of various biological solutions to be tested and the corresponding BIS error number.

[0020] In the step S1, the specific method for determining the dyeing color of the biological solution to be tested is:

[0021] The dyeing substances are selected in the order of black, green, yellow, blue, red, cyan, and magenta. For the biological solution to be tested with low concentration, the dyeing substance corresponding to the color at the front is selected, and for the biological solution to be tested with high concentration, the dyeing substance corresponding to the color at the back is selected.

[0022] The specific method of the step S2 is:

[0023] Preparing a microfluidic channel;

[0024] Fixing the microfluidic channel on the surface of the optical disc base after pretreatment of the optical disc base;

[0025] Injecting the biological solution to be tested into the corresponding microfluidic channel, and then injecting the dyeing substance determined in the step S2 into the corresponding microfluidic channel to perform color development reaction, thereby forming a dyeing biochip on the optical disc base including different dyeing colors.

[0026] The microfluidic channel is composed of two vertically placed PDMSs, one of which is used to set the staining substance, and the other is used to set the biological solution to be detected.

[0027] The preparation method of the microfluidic channel is as follows: a microfluidic channel negative mold is prepared by using a 3D light curing technology, then PDMS is poured, and the microfluidic channel is formed after solidification.

[0028] The detection method of the disc-based staining biochip based on nanoparticle disturbance laser signal is used to measure the concentration of different biological solutions.

[0029] The staining substance is 3,3',5,5'-tetramethylbenzidine (TMB), FirePhos phosphatase substrate or BCIP / NBT phosphatase substrate.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] 1. The present application uses a standard optical drive as a detector, a disc-based biochip base, and different colors of biological solutions are dyed by using a blue light drive with different sensitivities to different colors. The concentration of a variety of biological solutions can be detected simultaneously, improving the detection efficiency. Moreover, the color of the dye is selected according to the concentration range of the biological solution, which can avoid saturation caused by high concentration or high sensitivity, and improve the detection range.

[0032] 2. The present application uses 3D printing technology or biological dot sampling to prepare a high-throughput multi-dyeing biochip. Compared with the existing biochip analyzer, the instrument portability is improved while the detection cost is reduced. Compared with the traditional biochip preparation method, the multi-dyeing biochip based on 3D printing has greater flexibility and is suitable for quantitative detection of multi-index high-throughput biological analysis.

[0033] 3. The present application is suitable for micro-detection of harmful ingredients in food, pesticide residues, heavy metals in water, antibiotics, drugs, etc., and can be widely used in clinical examination, diagnosis and individual medical treatment fields, and has very broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is the disc of different color ink dots in the experimental example 1 of the present application;

[0035] Figure 2 is the error number response graph of the different color ink dots obtained in the embodiment 1 of the present application;

[0036] Figure 3 is the scanning graph of the disc surface biotin-streptavidin reaction array dyed by two dyeing methods in the experimental example 2 of the present application

[0037] Figure 4 is the biotin-streptavidin system detection result figure of the TMB color developing method in the experimental example 2 of the present application;

[0038] Figure 5 is the biotin-streptavidin system detection result figure of the TMB color developing and silver staining in the experimental example 2 of the present application. DETAILED DESCRIPTION

[0039] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0040] The embodiments of the present application provide a disc-based multi-staining biochip detection method based on nanoparticle disturbance laser signal, which comprises the following steps:

[0041] S1, determining the staining substances of various to-be-tested organisms according to the concentration detection range of various to-be-tested biological solutions.

[0042] In the step S1, the specific method for determining the staining colors of the to-be-tested biological solutions is as follows:

[0043] The staining substances are selected in the order of black, green, yellow, blue, red, cyan and magenta. For the to-be-tested biological solutions with low concentration, the staining substances corresponding to the colors at the front are selected, and for the to-be-tested biological solutions with high concentration, the staining substances corresponding to the colors at the back are selected. In the present embodiment, the existing mature solid substrates, 3,3',5,5'-diaminobenzidine (TMB), FirePhos phosphatase substrate, BCIP / NBT phosphatase substrate and the horseradish peroxidase (HRP) / alkaline phosphatase (AP) labeled on the biomolecules are selected to have a color developing reaction, so that the biological array presents different colors.

[0044] S2, preparing the staining biological array of various to-be-tested biological solutions on the optical disc base to obtain the optical disc base staining biochip.

[0045] The specific method of the step S2 is as follows:

[0046] S201, preparing a microfluidic channel; the preparation method of the microfluidic channel is: using 3D light curing technology to prepare a microfluidic channel negative mold, then pouring PDMS, and forming a microfluidic channel after curing. A 3D modeling software SOLIDWORKS is used to design a microfluidic channel negative mold; then a light solid 3D printing technology is used to print a microchannel model, and the printing material is photosensitive resin; then a PDMS main agent and a cross-linking curing agent with a proportion of 10:1 are heated and cured at 70°C for 3h; finally, the prepared PDMS microchannel is cut and demolded for standby.

[0047] S202, fixing the microfluidic channel on the surface of the Blu-ray disc base after pretreatment.

[0048] The surface of the Blu-ray disc is coated with a "Hard CoatTM" film (a unique substrate material, lactone composite material), so the surface film of the Blu-ray disc is completely immersed in a glass culture dish containing a NaOH (1.0M, 55°C) solution, hydrolyzed for 1.5h, then taken out, washed with deionized water, and blown dry with N2; then the prepared microfluidic channel is firmly fixed on the surface of the Blu-ray disc, a mixed solution of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) with a concentration of 100mM and NHS (N-hydroxysuccinimide) with a concentration of 45mM is injected into the microchannel, and activated at room temperature for 2.5-3h; Amine-PEG2-biotin biomolecules are injected, and fixed at room temperature for 4-5h; a 4% BSA blocking solution is injected and acts for 1.5-2h.

[0049] S203, injecting the to-be-tested biological solution into the corresponding microfluidic channel, then injecting the staining substance determined in step S2 into the corresponding microfluidic channel to perform a color development reaction, and forming a Blu-ray disc base stained biological chip including different staining colors.

[0050] Further, in this embodiment, the prepared microfluidic channel is composed of two vertically placed PDMSs, one of which is used to set the staining substance, and the other is used to set the to-be-tested biological solution, and the two PDMSs are vertically placed, and only the channel overlap part occurs specific reaction, in this embodiment, a plurality of microfluidic channels are arranged on the surface of the Blu-ray disc base, and the preparation of a high-throughput biological array can be realized.

[0051] S3, scanning the Blu-ray disc base stained biological chip based on the Blu-ray drive.

[0052] S4, analyzing the BIS error number at each logical position in the scanning result, and determining the concentration of the to-be-tested biological solution according to the BIS error number.

[0053] Specifically, the laser wavelength of the blue light drive is 405 nm. According to the different sensitivities of 405 nm laser to different colors, the dye color suitable for the reaction system is selected to realize the simultaneous detection of multiple biomolecules.

[0054] Further, the detection method of the embodiment further includes the step of obtaining the BIS error number-concentration curve of each to-be-detected biological solution, and the specific method is:

[0055] Preparation of a microfluidic channel on a disc base;

[0056] Preparation of a dyeing biological array of various standard concentrations of to-be-detected biological solutions in the microfluidic channel to obtain a standard disc base dyeing biological chip;

[0057] Scanning of the standard disc base dyeing biological chip based on a blue light drive;

[0058] Analysis of the BIS error number at each logical position in the scanning result to obtain the BIS error number-concentration curve of each to-be-detected biological solution.

[0059] Further, in the step S4, the concentration of the to-be-detected biological solution is determined according to the BIS error number-concentration curve of each to-be-detected biological solution and the corresponding BIS error number. In the embodiment,

[0060] Experimental Example 1: Preparation and detection of biological chips with different color ink dots

[0061] (1) Seven ink dots with the same size and different colors are printed on the surface of a blue light disc by using a biological dot sampling technology, as shown in FIG. 1. The colors along the radius of the disc from inside to outside are black, red, green, yellow, blue, magenta, and cyan, respectively. Then, the disc is placed in a drying oven at 56°C for 15 min. Figure 1

[0062] (2) The prepared biological disc is placed in a standard light drive, and a conventional light drive (PLEXTOR PX-LB950UE external blue light) and a free disc quality diagnosis software (plextutilities 1.3.3.1) are used to read the multi-dyeing biological chip, and the reading speed is set to 8x to scan the entire disc to collect and determine the error BIS (Burst Indicating Subcode) and LDC (Long Distance Code) distribution. During the data reading process, LDC represents the number of LDC check code bytes used when the data needs to be corrected, and BIS represents the number of BIS check code bytes used when the data needs to be corrected.

[0063] ​(3) During the scanning and reading process of the biochip, the reading laser wavelength is 405nm. When the laser shines on the biochip array area, it will cause interference such as absorption and scattering, which will cause changes in the laser signal received by the photodetector in the optical head. The sensitivity of ink dots of different colors at 405nm is different. The order of sensitivity from high to low is black, green, yellow, blue, red, cyan, and magenta.

[0064] (4) BIS error count results are as follows Figure 2 As shown, the higher the sensitivity, the higher the number of errors generated. Therefore, the number of errors represents the strength of the biological array signal, indicating that the biochip in this embodiment can realize the quantitative detection of biomolecules by utilizing the number of errors. Figure (a) is a distribution diagram of the error number peaks of different colored ink dots and the radial distance of the optical disc, and Figure (b) is a distribution diagram of the error number density of different colored ink dots.

[0065] Experiment Example 2: Preparation and Detection of Biochips Using Silver Staining and TMB Color Development

[0066] Step 1: Preparation of multi-stain biochips:

[0067] (1) Preparation of microfluidic channels: The negative mold of the microfluidic channel (5 channel grooves, each channel is 0.5×0.5×30mm) was designed using the 3D modeling software SOLIDWORKS; then the microchannel model was printed using photopolymerization 3D printing technology, and the printing material was photosensitive resin; then the PDMS main agent and crosslinking curing agent were heated and cured at 70℃ for 3h in a ratio of 10:1; finally, the prepared PDMS microchannel was cut and demolded for later use.

[0068] (2) Preparation of multi-staining biochips: The "Hard Coat™" film (a unique substrate material, lactone composite material) on the surface of Blu-ray discs was used to completely immerse the Blu-ray disc surface film in a solution of NaOH (1.0M, 55°C).

[0069] C) In the solution of the glass Petri dish, hydrolysis 1.5h after taking out, washed with deionized water, and N2 blow dry Blu-ray disc; after that, the prepared PDMS template is firmly fixed on the Blu-ray disc surface, the mixed solution of 100mM EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 45mM NHS (N-hydroxysuccinimide) is injected into the microchannel, and activated at room temperature for 2.5-3h; inject Amine-PEG2-biotin biomolecules, and fix at room temperature for 4-5h; inject 4% BSA blocking solution for 1.5-2h; inject HRP-streptavidin and Nanogold-streptavidin conjugate solution into the corresponding microfluidic channel according to different concentration changes, and react for 45min-1h; finally, use silver staining solution and TMB substrate for color development reaction for 1-5min, and form different color bioarrays, and use PBS buffer solution to wash and dry for analysis.

[0070] As shown in Figure 3 The figure after staining is shown, and the upper array is the silver staining result, and the lower array is the TMS color development result.

[0071] Step two: reading and analyzing the multi-staining biochip

[0072] (1) Put the prepared bio-disc into the standard optical drive, and use the conventional optical drive (PLEXTOR PX-LB950UE external Blu-ray) and free disc quality diagnosis software (plextutilities1.3.3.1) to read the multi-staining biochip, and set the reading speed to 8x to scan the entire disc, so as to collect and determine the error BIS (Burst Indicating Subcode, Burst Indicating Subcode) and LDC (Long Distance Code, Long Distance Code) distribution. During the data reading process, LDC represents the number of LDC check code bytes used when the data needs to be corrected, and BIS represents the number of BIS check code bytes used when the data needs to be corrected.

[0073] (2) During the scanning and reading process of the biochip, the reading laser wavelength is 405nm, and when the laser irradiates the biochip array area, it will cause interference such as absorption and scattering of the laser, so that the laser signal received by the photodetector in the optical head changes; at the same time, the error correction software generates error numbers LDC and BIS; the error number represents the intensity of the bioarray signal, so as to realize the quantitative detection of biomolecules.

[0074] Figure 4 is the error number detection result of Nanogold-streptavidin after TMB color development, which isFigure 4 It can be seen that, with the increase of the concentration of Nanogold-streptavidin (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 ng / mL), the error peak value also increases.

[0075] Figure 5 The error number detection results of TMB color development and silver staining of HRP-streptavidin, 1-6 represent the silver staining results of different concentrations of reactants, and 1'-6' represent the TMB color development results of different concentrations of reactants. It can be seen from the figure that, with the increase of the concentration of Nanogold-streptavidin, the amplitude of the error number generated after TMB color development and silver staining color development appears a clear gradient. When the concentration is 0.05 mg / mL, no obvious error peak appears; when the concentration is 0.8 mg / mL, no error peak appears.

[0076] The results show that: the two staining methods can coexist well on a disc; therefore, according to the color and the corresponding intensity, this double-channel staining system can identify and quantify two analytes in a mixed sample.

[0077] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting multiple staining biochips on optical discs based on nanoparticle-perturbed laser signals, characterized in that, Includes the following steps: S1. Determine the staining substances of various biological samples based on the concentration detection range of the various biological solutions to be tested; S2. Prepare staining biological arrays of various biological solutions to be tested on optical disc substrates to obtain optical disc-based staining biological chips; S3. Scanning optical disc-based staining biochips using a Blu-ray drive; S4. Analyze the number of BIS errors at each logical location in the scan results, and determine the concentration of the biological solution to be tested based on the magnitude of the number of BIS errors; In step S1, the specific method for determining the staining color of the biological solution to be tested is as follows: Select the staining substances in the order of black, green, yellow, blue, red, cyan, and magenta. For biological solutions with low concentrations, select the staining substance corresponding to the earlier color; for biological solutions with high concentrations, select the staining substance corresponding to the later color. The laser wavelength of the Blu-ray drive is 405nm; In step S4, the concentration of the biological solution to be tested is determined based on the BIS error number-concentration curve of various biological solutions to be tested and the corresponding BIS error number. The specific method for step S2 is as follows: Fabrication of microfluidic channels; After pre-treating the optical disc substrate, the microfluidic channel is fixed on the surface of the optical disc substrate; The biological solution to be tested is injected into the corresponding microchannel, and then the staining material determined in step S2 is injected into the corresponding microchannel to carry out the color reaction, forming a disc-based staining biochip with different staining colors. The microfluidic channel is prepared by using 3D photopolymerization technology to prepare a microfluidic channel negative mold, then pouring in PDMS, and curing to form a microfluidic channel.

2. The method for detecting multiple staining biochips on optical discs based on nanoparticle-perturbed laser signals according to claim 1, characterized in that, It also includes the following steps: Microfluidic channels were fabricated on optical disc substrates; A standard optical disc-based staining biochip was obtained by preparing staining bioarrays of various standard concentrations of biological solutions to be tested in a microfluidic channel. Scanning of standard optical disc-based staining biochips using a Blu-ray drive; By analyzing the number of BIS errors at each logical location in the scan results, BIS error-concentration curves for various biological solutions to be tested were obtained.

3. The method for detecting multiple staining biochips on optical discs based on nanoparticle-perturbed laser signals according to claim 1, characterized in that, The microfluidic channel is composed of two vertically placed PDMS channels, one of which is used to set the staining material and the other is used to set the biological solution to be tested.

4. The method for detecting multi-staining biochips based on optical discs using nanoparticle-perturbed laser signals according to claim 1, characterized in that, Used to measure the concentration of different biological solutions.

5. The method for detecting multiple staining biochips on optical discs based on nanoparticle-perturbed laser signals according to claim 1, characterized in that, The staining material is 3,3',5,5'-benzidine (TMB), FirePhos phosphatase substrate, or BCIP / NBT phosphatase substrate.

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