CN110951587A - Chip and method for cell filtration, fixation and in-situ fluorescence nucleic acid hybridization experiment - Google Patents

Abstract

The invention provides a chip and a method for cell filtration, fixation and in-situ fluorescence nucleic acid hybridization experiment, comprising a sample inlet, a sample outlet and a reaction chamber; the reaction chamber is internally provided with a micro-column array and micro channels, and the space between the micro-columns is smaller than the size of the cells. The micro-channels comprise a plurality of first micro-channels communicated with the inlet of the reaction chamber and the micro-column array, a plurality of second micro-channels communicated with the outlet of the reaction chamber and the micro-column array, and third micro-channels formed by gaps among the micro-columns and communicated with the first micro-channels and the second micro-channels. The invention integrates cell enrichment, cleaning and nucleic acid hybridization experiments into a chip device, so as to solve the pollution problems caused by long time, complex operation, multi-step operation and the like required by the traditional fluorescence hybridization experiment. After the hybridization experiment is finished, the in-situ fluorescence observation characterization can be directly carried out by using a microscope.

Description

Chip and method for cell filtration, fixation and in-situ fluorescence nucleic acid hybridization experiment

Technical Field

The invention belongs to the technical field of cell analysis chips, and particularly relates to a micro-fluidic chip and a method for carrying out fluorescence nucleic acid hybridization experiments in situ by applying rare cell fixation and filtration.

Background

Single cell genetic analysis is a technique for isolating cells in a population and detecting and analyzing information such as nucleic acids in individual cells. This technique avoids missing small but critical information on cellular molecular biology from analysis of the entire population. It can reflect the difference between cells of single individuals, greatly enhances the understanding of the heterogeneity of cells, and is considered as a novel and potential technical means applied to the analysis of cancer and other fields.

Nucleic acid molecule hybridization based techniques, whereby nucleic acid molecule fragments of known sequence are labeled with a detectable fluorescent signal, can be used to detect unknown nucleic acid sequences in a cell sample, thereby identifying cells with heterogeneity from a population of cells to facilitate downstream analysis.

On the other hand, the clinical cell sample processed by the screening technique has a low volume and a small number of cells in a unit volume of solution, and the cells are usually enriched for subsequent experiments, which also requires additional equipment and operation time.

The traditional fluorescence nucleic acid in situ hybridization experiment relates to a plurality of steps such as early-stage cell processing, middle-stage sample preparation, late-stage result characterization and the like. In the series of operation processes of the step, the transfer of the sample is easy to cause impurity pollution, so that the integrated experimental device is important for improving the success rate of the experiment.

At present, no simple and feasible method can realize filtration, enrichment and in-situ fluorescence hybridization experimental characterization of cells.

Disclosure of Invention

Aiming at the problems, the invention provides a chip, which integrates cell enrichment, cleaning and nucleic acid hybridization experiments into a chip device so as to solve the pollution problems caused by long time, complex operation, multi-step operation and the like of the traditional fluorescence hybridization experiments. After the hybridization experiment is finished, the in-situ fluorescence observation characterization can be directly carried out by using a microscope.

In order to achieve the purpose, the invention adopts the following technical scheme:

the chip is used for filtering and fixing cells and carrying out fluorescence nucleic acid hybridization experiments in situ and comprises a sample inlet, a sample outlet and a reaction chamber; the device is characterized in that a micro-column array is arranged in the reaction chamber, and the space between micro-columns is smaller than the size of cells.

Preferably, the reaction chamber further comprises a microchannel, wherein the microchannel comprises a plurality of first microchannels communicated with the inlet of the reaction chamber and the micropillar array, a plurality of second microchannels communicated with the outlet of the reaction chamber and the micropillar array, and a third microchannel communicated with the first microchannels and the second microchannels and formed by gaps among the micropillars. The channels in the transverse direction and the longitudinal direction between the micro-columns are the same in size. The third channel serves to immobilize the cells and facilitate droplet flow. The first channel and the second channel function to facilitate uniform flow of fluid from the sample inlet to the array and out of the array and to the sample outlet. This ensures that the liquid flows uniformly within the array. So that the liquid environment in which the cells are ultimately immobilized within the array is consistent.

Preferably, when the chip device is horizontally placed, the heights of the sample outlet, the second micro-channel and the third micro-channel are sequentially increased, and the heights of the third micro-channel and the first micro-channel are equal.

Preferably, the spacing between the microcolumns is 5-14 um.

Preferably, the microcolumn is characterized by comprising an upper part and a lower part, wherein the upper part is a circular truncated cone, the lower part is a cube, the radius of the upper surface of the circular truncated cone is 4-15um, the radius of the lower surface of the circular truncated cone is 8-18um, the length of the cube is 15-30um, and the height of the cube is 4-30 um.

Preferably, slide rails are arranged on two sides of the chip device, and a sealing cover is embedded in each slide rail and can seal the chip.

The method for carrying out the cell fluorescent nucleic acid hybridization experiment in situ based on the chip comprises the following steps:

step 1: cleaning the chip;

step 2: dripping cell dispersion liquid drop by drop above the micro-column array at time intervals of 1-10 s;

and step 3: the sample outlet is connected with a syringe pump to continuously suck out the inflowing liquid;

and 4, step 4: adding a cell cleaning solution from the sample inlet, and cleaning for 5-10 minutes;

and 5: adding hybridization reaction liquid into a sample inlet;

step 6: closing the sample outlet injection pump;

and 7: the whole chip is closed, and nucleic acid hybridization experiments and fluorescence characterization are carried out.

Has the advantages that:

(1) traditional nucleic acid fluorescence hybridization experiments require multiple procedures for cell processing, hybridization experiments, and characterization, involving multiple reagents. The invention integrates multiple operations, avoids impurity pollution caused by multi-step operation, and has small reagent consumption and low cost.

(2) The chip device is small in size, convenient to store and carry, closed by the sealing cover, and prevented from volatilizing and being polluted by impurities in the hybridization experiment. Through the external equipment, the chip can be convenient carry out the control of temperature and humidity.

(3) Simple operation and no need of complex professional background.

(4) Can cooperate upstream and downstream analysis and detection equipment, chip array structure is from taking the filter effect, can filter the bulky solution that contains a small amount of cells, can be with cell enrichment, avoids still must carrying out the complicated operation of cell enrichment before the hybridization. After the hybridization experiment is finished, the chip sample wafer can be directly moved, downstream fluorescence characterization can be conveniently and directly carried out, and other operations are not needed.

(5) The array is suitable for cells with various uniform sizes, and the size of the array can be designed and modified according to the size of the cells in an actual sample, so that the array has broad-spectrum applicability.

(6) The invention integrates multiple operations, reduces the processing time and steps of the whole process, shortens the detection period and has strong controllability.

Drawings

FIG. 1 is an overview of a chip device, wherein A is a sample outlet; b is a micro-column array; c, a sample inlet; d, micro-channel; and E, closing the cover.

Fig. 2 is a side view of a chip arrangement.

Fig. 3 is a front perspective view of the chip. A is a sample outlet; c is a sample inlet; d1 is a first micro channel; d2 is a second micro channel; d3 is a third microchannel.

FIG. 4 is a view showing the structure of a micropillar array.

Fig. 5 is a schematic view of a single microcolumn in the microcolumn array.

Detailed Description

The invention will be further described with reference to the accompanying drawings.

As shown in fig. 1, the present invention designs a microfluidic chip based on transparent plastic or glass material, the chip includes: sample inlet, water conservancy diversion channel, cell array room, play appearance mouth and closing cap.

1) The sample inlet and the sample outlet mainly perform inflow and outflow of liquid samples (including cell washing solution and reaction reagent).

2) The flow guide channel is designed into multiple channels and is responsible for communicating fluid entering from the sample inlet to the array chamber and guiding liquid flowing out from the array chamber to the sample outlet. The micro-channels comprise a plurality of first micro-channels communicated with the inlet of the reaction chamber and the micro-column array, a plurality of second micro-channels communicated with the outlet of the reaction chamber and the micro-column array, and third micro-channels formed by gaps among the micro-columns and communicated with the first micro-channels and the second micro-channels.

3) The cell array chamber consists of a micro-column array and a micro-channel, wherein the micro-array is used for fixing and filtering cells, and the micro-channel is used for circulating liquid, so that the cells can be conveniently cleaned and reaction reagents can be conveniently provided.

4) The closing cap is used for closing the whole reaction system, and avoids the evaporation of a large amount of liquid during the high-temperature nucleic acid hybridization reaction.

As shown in fig. 2, the closing cover E can slide, and the sliding cover can be opened when performing cell manipulation experiments; and closing the sliding cover when the fluorescence hybridization experiment is carried out, so as to seal the whole reaction chamber.

As shown in fig. 3, the bottom heights of the sample outlet a, the sample inlet C, the first microchannel D1, the second microchannel D2, and the third microchannel D3 are in the following relationship: the design of A < D2< D3 ═ D1, the left low and the right high can facilitate the fluid to flow under the condition of natural gravity, and the backflow is avoided.

As shown in FIG. 4, the third microchannel D3 has a width of 5-14 um. The distance is designed in relation to the particular cell to be manipulated and may be modified according to the size of the cell of the test sample.

As shown in FIG. 5, radius R1 is 4-15um, radius R2 is 8-18um, length L is 15-30um, and height H is 4-30 um.

Taking the chip made of polydimethylsiloxane as an example, the cell experiment method comprises the following steps:

1) taking out the clean chip and taking down the sealing cover;

2) putting the chip into a plasma cleaning instrument for treatment for 2 minutes;

3) dripping the treated chip into cell dispersion liquid drop by drop above the micro-column array at a time interval of 1-10 s;

4) the sample outlet is connected with a syringe pump to continuously suck out the inflowing liquid;

5) after all the cell sap is dripped, adding a cell cleaning solution from the sample inlet, and cleaning for 5-10 minutes;

6) adding hybridization reaction liquid into a sample inlet;

7) after 5 minutes, the injection pump at the sample outlet is closed;

8) the cover seals the whole chip, and the nucleic acid hybridization experiment and the fluorescence characterization are carried out.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims (7)

1. The chip is used for filtering and fixing cells and carrying out fluorescence nucleic acid hybridization experiments in situ and comprises a sample inlet, a sample outlet and a reaction chamber; the device is characterized in that a micro-column array is arranged in the reaction chamber, and the space between micro-columns is smaller than the size of cells.

2. The chip of claim 1, wherein the reaction chamber further comprises microchannels, the microchannels comprise a plurality of first microchannels communicating the inlet of the reaction chamber with the micropillar array, a plurality of second microchannels communicating the outlet of the reaction chamber with the micropillar array, and a third microchannels communicating the first microchannels with the second microchannels formed by the gaps between the micropillars.

3. The chip of claim 2, wherein the height of the sample outlet, the second microchannel, and the third microchannel increases sequentially when the chip device is horizontally placed, and the height of the third microchannel is equal to the height of the first microchannel.

4. The chip for cell filtration, immobilization and in situ fluorescence nucleic acid hybridization assay according to claim 1, wherein the spacing between the micro-pillars is 5-14 um.

5. The chip of claim 1, wherein the micro-column comprises an upper part and a lower part, the upper part is a circular truncated cone, the lower part is a cube, the radius of the upper surface of the circular truncated cone is 4-15um, the radius of the lower surface of the circular truncated cone is 8-18um, the length of the cube is 15-30um, and the height of the cube is 4-30 um.

6. The chip for cell filtration, immobilization and in situ fluorescence nucleic acid hybridization assay according to claim 1, wherein slide rails are disposed on both sides of the chip device, and a sealing cap is embedded in the slide rails and can seal the chip.

7. The method for performing cell fluorescent nucleic acid hybridization experiments in situ based on the chip of claim 1, comprising the following steps:

step 1: cleaning the chip;

step 2: dripping cell dispersion liquid drop by drop above the micro-column array at time intervals of 1-10 s;

and step 3: the sample outlet is connected with a syringe pump to continuously suck out the inflowing liquid;

and 4, step 4: adding a cell cleaning solution from the sample inlet, and cleaning for 5-10 minutes;

and 5: adding hybridization reaction liquid into a sample inlet;

step 6: closing the sample outlet injection pump;

and 7: the whole chip is closed, and nucleic acid hybridization experiments and fluorescence characterization are carried out.

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