CN100575929C - A method for detecting gene mutations in cells using a one-dimensional microfluidic biochip - Google Patents

Abstract

The invention discloses a method for detecting gene mutations in cells using a one-dimensional microfluidic biochip, which is characterized in that a functionalized mutation detection chip based on a one-dimensional microfluidic bead array chip is constructed, and total RNA or target DNA The sample is diluted in the hybridization buffer containing fluorescent probes, and flows through the microchannel into the functionalized microbead array under pressure drive. Observe and photograph the surface fluorescence of microbeads with an inverted microscope, and use software to estimate the surface fluorescence intensity of the particles, and judge the mutation type of the detection site according to the difference in fluorescence intensity. The method of the invention solves the defect that the characteristics of micro-detection, high sensitivity and high-throughput analysis are difficult to coexist in the gene mutation analysis method, and is expected to realize the gene mutation analysis at the single-cell level.

Description

A method for detecting gene mutations in cells using a one-dimensional microfluidic biochip

technical field

The invention relates to the biological application field of a micro-total analysis system, in particular to the application and development of a one-dimensional microfluidic biochip in cell gene mutation analysis.

Background technique

Seven countries including the United States, Britain, and China announced in February 2001 that they had completed the first draft of the analysis of the human genome sequence, which transformed the field of human biomedical research from the genome era to the post-genome era, that is, the functional genome era. Geneticists generally believe that an important direction of efforts in the post-genome era is to analyze the variation of human gene sequences, because the study of mutations in human DNA sequences will help to better understand the occurrence, development, and response to drug treatments of human diseases. Therefore, the analysis and detection method of the mutation is very important. Before 1985, mutations such as deletion, insertion and frameshift recombination of genes could be screened out by using Southern blotting. For mutations that cannot be detected by this method, complex and time-consuming DNA sequencing analysis methods can only be applied. Polymerase chain reaction (polymerase chain reaction, PCR) technology is the most significant progress in mutation research, which has made great progress in gene mutation detection technology, but PCR technology is time-consuming, prone to false positives, and cannot detect multiple targets at the same time Points and other shortcomings limit the application of PCR method in mutation detection. The emergence of microarray chip (Microarray) provides a very efficient detection platform for mutation detection, which directly realizes the high throughput and information integration of mutation analysis. However, the analytical instruments of this method are very expensive and the detection sensitivity is low. Microfluidic technology has made it possible to achieve miniaturization, rapidity, and high sensitivity of mutation detection, but it still needs further development in terms of high-throughput performance.

Contents of the invention

The present invention aims to provide a novel detection method that can be applied to the identification and analysis of cell gene mutations, so as to solve the defects that the characteristics of micro detection, high sensitivity and high-throughput analysis are difficult to coexist in the current gene mutation analysis methods, and To promote further development in this field of technology.

The present invention realizes the object of the invention through the following technical solutions:

A method for detecting gene mutations in cells using a one-dimensional microfluidic biochip, including activation of silica microbeads on a one-dimensional microfluidic biochip with alkali, pretreatment with biotin and avidin, and then adding biological The mutation detection capture probe modified by the protein, the silica microbeads modified with the mutation detection capture probe are moved into the small chamber of the microchannel of the biochip one by one to form a functionalized microbead array; the total RNA or target DNA sample is contained in the Diluted in the hybridization buffer with fluorescent probes, and flow through the microchannel into the functionalized microbead array under pressure drive. After hybridization, the highly stringent eluent containing formamide is injected into the channel for elution, and finally observed by a fluorescent inverted microscope And photograph the surface fluorescence of microbeads, and use software to estimate the surface fluorescence intensity of the particles, and judge the mutation type of the detection site according to the difference in fluorescence intensity.

The ratio of the total RNA to the fluorescent probe is 1.5×10 ⁷ grams: 1 mole, and the ratio of the DNA to the fluorescent probe is greater than or equal to 56 grams: 1 mole; the flow rate of the hybridization buffer under the pressure drive is 0.1 μl/ min; the fluorescent probe is a signal probe modified by tetramethylrhodamine; the high stringency eluent contains 60% formamide by volume.

Description of drawings

Figure 1 is a flow chart of a one-dimensional microfluidic biochip used to detect cell gene mutations;

Fig. 2 is the sandwich hybridization model figure that adopts among the present invention;

1-Silica microbeads, 2-Coated biotinylated bovine serum albumin,

3-streptavidin, 4-biotin-modified capture probe,

5-Nucleic acid fragments of mutation detection genes, 6-tetramethylrhodamine-modified signal probes.

Figure 3 shows the sensitivity results of one-dimensional microfluidic biochip detection of gene mutations;

Fig. 4 shows the results of one-dimensional microfluidic biochip detecting the mutation of codon 175 of p53 gene in three tumor cells, CNE2, A549 and SKBr-3.

The one-dimensional microfluidic biochip uses a sandwich model as a hybridization detection method. The principle of the model is: as shown in Figure 2, biotinylated bovine serum albumin 2 is bound to the solid-phase interface silica microbeads 1 through physical adsorption. On the surface, streptavidin 3 is strongly bound to biotin and immobilized on the surface of silica microbeads, biotin-modified capture probe 4 is specific to avidin 3 on the surface of silica microbeads through modified biotin The biotin-modified capture probe 4 can specifically select and recognize the complementary matching sequence in the target nucleic acid sequence 5, while the other tetramethylrhodamine-modified signal probe 6 It can match and bind with the target nucleic acid sequence 5, and the combination of the three forms a hybrid structure similar to a sandwich, which can realize the specific detection of the target nucleic acid sequence without labeling.

The method of the present invention has the advantages that: the process of the present invention uses mRNA and DNA as detection objects, and does not require the PCR process used in the conventional method, thereby avoiding the situation of false positives caused by contamination in the PCR process; The chip has the dual characteristics of trace detection and high-throughput detection, so the defects that are difficult to coexist in conventional mutation analysis methods such as trace detection, high sensitivity, and high-throughput analysis are better resolved; It can still distinguish the region of single base mutation, and the simultaneous detection of multiple targets can be carried out with one injection; in addition, the method of the present invention can detect and observe the hybridization and elution process of the probe in real time with the assistance of CCD and computer software, And it is expected to use the method of the present invention to realize gene mutation analysis at the single cell level, and provide an efficient analysis method for studying the occurrence and development of human diseases and personalized medicine.

Detailed ways

Example 1, one-dimensional microfluidic mutation detection chip detects p53 gene mutation with DNA sequence as object:

1. Preparation of silica microbeads for mutation detection: Take about 10-20 mg of silica microbeads with a diameter of 40 μm and place them in a 1.5ml Ep tube, add 500 μl of 0.01M NaOH to activate the surface for 20 minutes, wash with TE 3 times, remove Supernatant: Add 80μl 0.1-1.0mg/ml biotinylated bovine serum albumin (Biotin-BSA), place on a low-temperature shaker at 4°C, shake at 350rpm for 12-48h, then wash with TE for 3 times, Biotin- BSA is bound to the particle surface through physical adsorption; add 80 μl 0.1-1.0 mg/ml streptavidin (Streptavidin), place it on a low-temperature shaker at 4°C, shake at 350 rpm for 4-8 hours, and the streptavidin can be combined with the particles The biotin on the surface is strongly bound and fixed on the surface of the particle, washed with TE for 3 times; add 50 μl of 0.1-1.0 μM biotin-modified mutation detection capture probes Capture-C, Capture-G, Capture-T and Capture-A respectively (As shown in Table 1), shake at 350 rpm for 12-48 hours at 4°C, wash with TE and store at 4°C for later use. The capture probe is specifically bound to the avidin on the surface of the particle through the modified biotin and immobilized on the particle, thereby forming a functionalized silica particle with the ability to detect p53 gene mutations.

2. Prepare a one-dimensional microfluidic mutation detection chip: Mix polydimethylsiloxane and curing agent thoroughly, remove air bubbles in a vacuum pump, then spread it on the positive template of the chip, and place it in an oven at 75°C for 20-40 minutes , take it out after curing, and finally peel off the polydimethylsiloxane (PDMS) film base from the positive template, which contains many small chambers for holding microbeads; place the film base in an inverted fluorescent On the microscope, functionalized microbeads with a diameter of about 40 μm modified with mutation detection probes are placed in the small chambers in the substrate microchannels by means of micromanipulation: first, use a set of imported burning needles, pulling needles and The micromanipulation micropipette is prepared by the grinding needle system, and the micropipette is fixed on the micromanipulation system of the inverted fluorescence microscope. Indoors, each chamber corresponds to different functionalized microbeads for multi-target detection of samples; finally, clean glass slides and substrates are tightly combined to complete chip packaging.

3. According to the operation process of Fig. 1, the hybridization buffer (the composition of the hybridization buffer is: 10mM Potassium phosphate buffer (pH7.6), 300mMNaCl, 0.1% Tween 20 by volume and 30% formamide by volume) passed through the one-dimensional biochip microchannel at a flow rate of 0.1 μl/min under pressure drive Put the functionalized microbead array for mutation detection, after hybridization for 20min, wash the channel with 5 μl of TE; then, under the pressure drive, flow into the high stringency elution buffer containing 60% formamide (volume percentage) at a flow rate of 2 μl/min. The composition of the elution buffer is: 10mM potassium phosphate buffer (pH7.6), 150mMNaCl, 0.1% Tween 20 and 60% formamide by volume percentage), using matching (Capture-C and p53-G) and single-base mismatch (Capture-G, Capture-T, Capture-A and p53-G) sequences were identified by elution, and the channel was washed again with TE after 5 min. Place the reacted chip under the CCD imaging system of the fluorescent inverted microscope according to the process shown in Figure 1, observe and photograph the particles, and analyze with the fluorescent image analysis software, the mutation detection sensitivity shown in Figure 3 can be obtained the result of. The results showed that when the concentration of the target sequence was 0.04nM, the signal-to-noise ratio was 4 (the signal-to-noise ratio was defined as the fluorescence signal generated by the perfectly matched hybridization divided by the fluorescent signal generated by the single-base mismatched hybridization, and it was considered that only the perfectly matched The fluorescence signal generated by matching hybridization divided by the fluorescent signal generated by single base mismatch hybridization is greater than 4 (the data is valid), as the concentration of target sequence increases, the fluorescence generated by perfectly matching hybridization also continues to increase. When the target sequence When the concentration reaches 3nM, the fluorescence signal generated by the perfect matching hybridization reaches the maximum, thus it can be seen that the method of the present invention can identify single base mutation at the target sequence concentration of 0.04nM.

Oligonucleotide probes synthesized in table 1 for detecting p53 gene mutations

code describe sequence Capture-C A capture probe whose recognition point is C 5'-GGGCAG<u>C</u>GCCTCACAACCAAAAAAAAAAA-Biotin-3' Capture-G A capture probe whose recognition point is G 5’-GGGCAG<u>G</u>GCCTCACAACCAAAAAAAAAAA-Biotin-3’ Capture-T A capture probe whose recognition point is T 5’-GGGCAG<u>T</u>GCCTCACAACCAAAAAAAAAAA-Biotin-3’ Capture-A The capture probe whose recognition point is A 5’-GGGCAG<u>A</u>GCCTCACAACCAAAAAAAAAAA-Biotin-3’ p53-G p53 gene target sequence 5’-GGTTGTGAGGC<u>G</u>CTGCCCAAGCGAGCACTGCCCAACAACA CCAGC-3’ F-probe signal probe 5'TAMRA-AAAAAAAAAAAGCTGGTGTTGTTGGGCAGTGCTCGCTT-3'

Example 2, one-dimensional microfluidic mutation detection chip detects p53 gene mutation with mRNA sequence as object:

1. Preparation of silica microbeads for mutation detection: Take about 10-20 mg of silica microbeads with a diameter of 40 μm and place them in a 1.5ml Ep tube, add 500 μl of 0.01M NaOH to activate the surface for 20 minutes, wash with TE 3 times, remove Supernatant: Add 80μl 0.1-1.0mg/ml biotinylated bovine serum albumin (Biotin-BSA), place on a low-temperature shaker at 4°C, shake at 350rpm for 12-48h, then wash with TE for 3 times, Biotin- BSA is bound to the particle surface through physical adsorption; add 80 μl 0.1-1.0 mg/ml streptavidin (Streptavidin), place it on a low-temperature shaker at 4°C, shake at 350 rpm for 4-8 hours, and the streptavidin can be combined with the particles The biotin on the surface is strongly bound and fixed on the surface of the particle, washed with TE three times; 50 μl of 0.1-1.0 μM biotin-modified mutation detection capture probes Capture-C, Capture-G, Capture-T and Capture- A (as shown in Table 1), shake at 350 rpm for 12-48 hours at 4°C, wash with TE and store at 4°C for later use. The capture probe is specifically bound to the avidin on the surface of the particle through the modified biotin and immobilized on the particle, thereby forming a functionalized silica particle with the ability to detect p53 gene mutations.

2. Prepare a one-dimensional microfluidic mutation detection chip: Mix polydimethylsiloxane and curing agent thoroughly, remove air bubbles in a vacuum pump, then spread it on the positive template of the chip, and place it in an oven at 75°C for 20-40 minutes , take it out after curing, and finally peel off the PDMS sheet base from the positive template, which contains many small chambers for accommodating microbeads. The one-dimensional microfluidic biochip base casted with polydimethylsiloxane is placed on an inverted fluorescence microscope, and functionalized microbeads with a diameter of about 40 μm modified with mutation detection probes are placed on the inverted fluorescence microscope. Placed in the small chamber in the microchannel: first, use a set of imported burning needle, pulling needle and grinding needle system to prepare the micromanipulation micropipette, fix the micropipette on the micromanipulation system of the inverted fluorescence microscope, and then Under micro-conditions, put different functionalized microbeads into the small chambers in the channel according to the position coding method, and each small chamber corresponds to different functionalized microbeads for multi-target detection of samples; The base is tightly combined to complete the package of the chip.

3. total RNA was extracted respectively from these three kinds of cells of nasopharyngeal carcinoma cell (CNE2), lung cancer cell (A549) and breast cancer cell (SKBr-3), and quantified by ultraviolet absorption method; respectively get 3 μ g (the three Seed cells) total RNA in 20 μl of hybridization buffer (the hybridization buffer composition is: 10 mM potassium phosphate buffer (pH7.6), 300 mM NaCl, 0.1% Tween 20 by volume and 30% by volume Formamide) was diluted, and the signal probe modified by tetramethylrhodamine was added at the same time so that the final concentration in the hybridization buffer was 10nM, and then passed through the one-dimensional biological For the functionalized microbead array in the chip, after the hybridization reaction for 20 min, the channel was washed with TE and eluted with a high stringency elution buffer for 5 min under pressure drive (the composition of the elution buffer is: 10 mM potassium phosphate buffer ( pH7.6), 150mMNaCl, 0.1% Tween 20 by volume and 60% formamide by volume), the flow rate is 2 μl/min; the chip after the reaction is placed under the CCD imaging system in the fluorescent inverted microscope, The particles were observed and photographed, and analyzed with fluorescence image analysis software, and the results of mutation detection sensitivity as shown in Figure 4 can be obtained. The results showed that only SKBr-3 cells had a base conversion (CGC>CAC) among the three tumor cells, while the codon 175 of the p53 gene in the other two cells had no mutation and was still wild type. This result is consistent with the result of traditional sequence determination.

Claims (3)

1. method of utilizing one-dimensional microflow controlled biochip to detect gene mutation in the cell, comprise that the silicon dioxide microballon activates with alkali on the one-dimensional microflow controlled biochip, carry out pre-service with biotin and Avidin again, the sudden change that adds biotin modification then respectively detects capture probe, the silicon dioxide microballon of having modified sudden change detection capture probe is moved into one by one the cell of described biochip microchannel, form the functionalization micropearl array, it is characterized in that total RNA or target DNA sample are diluted in the hybridization buffer that includes fluorescence probe, and under pressure-driven, flow through the microchannel and enter the functionalization micropearl array with the flow velocity of 0.1 μ l/min, wash-out is carried out in the high rigorous eluent injection channel that will contain formamide after the hybridization, the volume fraction of described formamide in the rigorous eluent of height is 60%, observe and take bead surface fluorescence at last by the fluorescence inverted microscope, and with software the surface fluorescence intensity of particle is estimated, judge the mutation type of this detection site according to the difference of fluorescence intensity.

2. the method for utilizing one-dimensional microflow controlled biochip to detect gene mutation in the cell according to claim 1, the ratio that it is characterized in that described total RNA and fluorescence probe is 1.5 * 10 ⁷Gram: 1 mole, the ratio of DNA and fluorescence probe restrains more than or equal to 56: 1 mole.

3. the method for utilizing one-dimensional microflow controlled biochip to detect gene mutation in the cell according to claim 1 and 2 is characterized in that the signal probe that described fluorescence probe is modified for the tetramethyl rhodamine.

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