CN102649931A - Preparation method for microarray biochip - Google Patents

Abstract

The invention relates to a method for preparing a microarray biochip. M printing nozzles are used to form a nozzle group, and the nozzles are installed parallel to the X direction and arranged along the Y direction; Control the action of the corresponding nozzle on the nozzle corresponding to each step position, and spray an index biomolecule solution to the predetermined position of one or more microarray biochip substrates arranged along the X direction; after passing through the sequence of M nozzles The action is to spray the biomolecule solutions of M indicators one by one onto the predetermined positions of the biochip matrix to form a spotting array. This method can realize the preparation of microarray biochips for multi-index and multi-person parallel detection; compared with the existing products, it has the advantages of fast speed, high precision, wide applicability, high efficiency, less cross-contamination, easy maintenance and use, The characteristics of low manufacturing cost are very suitable for large-scale production of biochips, and also suitable for scientific research and application.

Description

A kind of preparation method of microarray biochip

technical field

The invention relates to a biochip preparation technology, in particular to a preparation method of a microarray biochip for microarray parallel detection.

Background technique

The preparation method of existing biochip can basically be divided into two classes: a class is in-situ synthesis (that is, the direct in-situ synthesis of oligonucleotide probes on the surface of the support); a class is directly sampled after pre-synthesis, A small amount of DNA solution is directly placed in the form of a microarray and solidified on the biochip substrate. Because the in situ synthesis method is very complicated, most of the DNA microarray chips use the synthetic spotting method. The preparation of the protein chip, due to the limitation of the method, is prepared by the method of first synthesis and then spotting. The synthetic spotting method is to accurately and quickly spot different probe samples quantitatively on positively charged nylon membranes or Silicon wafers and other corresponding positions, after solidification, DNA microarrays or protein chips can be obtained.

There are two traditional methods of spotting, one is contact spotting, that is, the spotting needle directly contacts the surface of the biochip substrate, and the sample is left on the matrix; the other is non-contact spotting, which uses the principle of piezoelectricity or The special micro-spray spotting nozzle made by the principle of micro-solenoid valve on-off, sprays the sample to the surface of the biochip substrate through the capillary.

In contact spotting, the spotting needle needs to be dipped into the source plate first to pick up the sample, and then a certain volume of liquid droplet is formed on the tip of the needle after being lifted, and then moved to the corresponding position of the glass slide, and the sample is transferred by contacting the substrate to form a sample. point, and repeat this process until the chip preparation is complete. The disadvantages of contact spotting are obvious: the solid sampling needle carries a small amount of sample, and each sampling can only be sampled once, and the production process is very slow; the distance between each chip and the source plate is different, and the sampling needle is transferred from the microwell plate The time required for the sample to reach the chip varies greatly, and the degree of influence of evaporation on the droplet varies greatly, resulting in poor uniformity of sample diameter: the diameter of the sample near the source plate is larger, and the diameter of the farther away is smaller.

In non-contact spotting, the hollow spotting needle is first dipped into the source plate to absorb a small amount of sample, and then the sample is sprayed point by point onto the chip surface by the pointing needle nozzle. Compared with contact spotting, the non-contact spotting needle can draw a sample multiple times at one time, which can relatively improve the efficiency of spotting, but it still needs to spray samples point by point, and it is impossible to spot multiple people and multiple indicators at the same time.

Research and experiments have shown that the physical precision of industrial inkjet heads can reach 185dpi-360dpi, and the minimum ink droplet volume can reach 14pL-80pL, which can be used for the printing of biochips.

Contents of the invention

The present invention aims at the problems of low efficiency of biological preparation and the inability to print multiple samples at the same time, and proposes a preparation method of a microarray biochip, which uses an industrial inkjet printing nozzle to prepare a "jet-printing" microarray biochip. The rapid production of microarray biochips for multi-indicator and multi-person testing can be realized. the

The technical scheme of the present invention is: a method for preparing a microarray biochip, specifically comprising the following steps:

1) Use M print nozzles to form a nozzle group, the nozzles are installed parallel to the X direction and arranged along the Y direction;

2) The biochip matrix moves relative to the nozzle group along the Y direction, and the action of the corresponding nozzle on the nozzle corresponding to each step position is controlled by the driving signal, and an index of biomolecular solution is sprayed to one or the other nozzles arranged along the X direction. Predetermined positions on a plurality of microarray biochip substrates;

3) Through the sequential actions of M nozzles, the biomolecule solutions of M indicators are sequentially sprayed onto the predetermined positions of the biochip matrix to form a spotting array.

The dot-like density and the spacing of the dot-like array are controlled by the driving signal to control the gating of the nozzles at a certain distance. If the distance between adjacent nozzles is e, the dot spacing in the dot-like array is n·e, that is, two working The nozzle spacing is n·e, n is the number of nozzle intervals between two working nozzles, the spacing control method of the spotting array is similar, and the spacing between two adjacent spotting arrays is m·e, that is, the distance between m nozzles .

The size of the spot is controlled by the amplitude and frequency of the driving signal.

Each nozzle only sprays one kind of index biomolecule solution.

The sample points in the spot pattern array have the same spacing in the X direction and the Y direction, which is an integer multiple of the nozzle spacing.

The nozzles corresponding to the sample points of the same index arranged in the X direction are driven by the same driving signal.

The relative movement of the chip substrate and the nozzle group along the Y direction may be that the nozzle group moves and the chip substrate is fixed; or the nozzle group is fixed and the chip substrate moves.

The working mode of the nozzle used can be piezoelectric or thermal.

The biomolecule solution may be nucleic acid or protein, nucleic acid including DNA probes.

The biochip substrate can be one or more membrane substrates, one or more glass slides, or one or more porous plates.

The beneficial effect of the present invention is that: the preparation method of the microarray biochip of the present invention can realize the preparation of the microarray biochip for multi-indicator and multi-person parallel detection; Wide range of performance, high efficiency, less cross-contamination, easy maintenance and use, low manufacturing cost, etc., are very suitable for large-scale production of biochips, and are also suitable for scientific research and applications.

Description of drawings

Fig. 1 is the schematic diagram of the sprinkler group of the spotting instrument used for preparing the biochip of the present invention;

2 is a schematic diagram of an example of a method for preparing a microarray biochip of the present invention;

Fig. 3 is a structure diagram of the biochip microarray prepared by the microarray biochip preparation method of the present invention.

Detailed ways

As shown in Figure 1 or Figure 2, 1-8 is the nozzle group composed of nozzles, 9 is the biochip substrate, 10-17 is the schematic position of the working nozzle, a and b are two different index sample points in a typical array structure . The biochip substrate 9 moves along the Y direction. When the predetermined position of the chip substrate 9 moves to the position of the first nozzle 1, the driving signal controls the corresponding nozzle actions at 10, 11 and 12, 13 of the nozzle 1, and the biomolecular solution Sample a is sprayed onto the substrate.

When the predetermined position of the chip moves to the position of the second spray head 2, the driving signal controls the corresponding nozzles at 14, 15 and 16, 17 of the spray head to spray the biological solution sample b onto the matrix. This completes the first row of spot samples in the X direction. The preparation methods of other spotting arrays are the same, and are completed by No. 3 to No. 8 nozzles respectively. The final result is shown in FIG. 3 as the structural diagram of the biochip microarray.

The dot density and the spacing of the dot array are realized by controlling the gating of the nozzles at a certain spacing by the driving signal. If the distance between adjacent nozzles 10 and 11 is e, the distance between the sample points in the spotting array is n·e, that is, the distance between two working nozzles is n·e, and n is the number of nozzle intervals between the two working nozzles. The smaller the number of nozzle intervals between the selected working nozzles, the smaller the sample point spacing and the higher the point sample density. The spacing control method of spotting arrays is similar, and the spacing between two adjacent spotting arrays is recorded as m·e, that is, the distance between m nozzles.

The size of the sample point is controlled by the amplitude and frequency of the driving signal. A driving signal with a smaller amplitude produces small droplets, a signal with a larger amplitude can produce medium-sized droplets, and two tight pulses can continuously eject small droplets and Medium-sized droplets that coalesce in the air and land on the substrate to form larger spots.

The method of the invention can realize the preparation of the microarray biochip for multi-indicator and multi-person parallel detection. Compared with the existing products, the speed is faster than the contact mechanical pointing method and the non-contact micro-spray pointing needle pointing speed, especially for mass production; high precision, and the inkjet pointing liquid droplet is small , high density, good precision, accurate quantification, uniform sample points, and good reproducibility; wide applicability, can spot samples on a variety of material substrates, which can be one or more nylon membranes or nitrocellulose membranes and other membranes The substrate can also be one or more rigid substrates such as glass slides, or one or more porous plates; the efficiency is high, and this spotting platform can simultaneously spot samples for multiple people within the inkjet range horizontally, and also multiple nozzles The group can work at the same time; vertically, multiple people can be flow-lined, and samples with multiple indicators can be sampled, which greatly improves the efficiency of sample spotting; cross-contamination is less, and one nozzle unit is only responsible for sampling one of the indicators, saving replacement samples It also effectively reduces the cross-contamination between different samples.

Claims (10)

1. A method for preparing a microarray biochip, characterized in that, specifically comprising the steps of: 1) Use M print nozzles to form a nozzle group, the nozzles are installed parallel to the X direction and arranged along the Y direction; 2) The biochip matrix moves relative to the nozzle group along the Y direction, and the action of the corresponding nozzle on the nozzle corresponding to each step position is controlled by the driving signal, and an index of biomolecular solution is sprayed to one or the other nozzles arranged along the X direction. Predetermined positions on a plurality of microarray biochip substrates; 3) Through the sequential actions of M nozzles, the biomolecule solutions of M indicators are sequentially sprayed onto the predetermined positions of the biochip matrix to form a spotting array. 2. according to the described microarray biochip preparation method of claim 1, it is characterized in that, the spacing of described dot sample density and dot sample array controls the gating of nozzle on certain spacing by driving signal and realizes, if the spacing of adjacent nozzles is e, the distance between the sample points in the spotting array is n e, that is, the distance between two working nozzles is n e, and n is the number of nozzle intervals between the two working nozzles. The spacing control method of the spotting array is similar. The distance between two adjacent dot-like arrays is m·e, that is, the distance between m nozzles. 3. The preparation method of the microarray biochip according to claim 1, characterized in that the size of the spot is controlled by the amplitude and frequency of the driving signal. 4. The method for preparing a microarray biochip according to claim 1, wherein each nozzle only sprays a biomolecular solution of one index. 5 . The method for preparing a microarray biochip according to claim 1 , wherein the spacing between the sample points in the spotting array is the same in the X direction and the Y direction, which is an integer multiple of the nozzle spacing. 6 . 6 . The method for preparing a microarray biochip according to claim 1 , wherein the nozzles corresponding to the sample points of the same index arranged in the X direction are driven by the same driving signal. 7. The microarray biochip preparation method according to claim 1, characterized in that, the relative movement of the chip matrix along the Y direction with the nozzle group can be that the nozzle group moves, and the chip matrix is fixed; it can also be that the nozzle group is fixed, and the chip substrate movement. 8. The preparation method of microarray biochip according to claim 1, characterized in that, the working mode of the nozzle used can be piezoelectric or thermal. 9. The method for preparing a microarray biochip according to claim 1, wherein the biomolecule solution may be nucleic acid or protein, and the nucleic acid includes a DNA probe. 10. microarray biochip preparation method according to claim 1, is characterized in that, described biochip substrate can be one or more membrane substrates, also can be one or more glass slides, can also be one or more Multiple well plates.

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