阅读说明：本技术 一种纳米柱阵列微流控芯片及其检测方法 (Nanogolumn array microfluidic chip and detection method thereof ) 是由 靳欣 李歧强 韩琳 于 2019-09-12 设计创作，主要内容包括：本发明公开了一种纳米柱阵列微流控芯片及其检测方法,该芯片包括底层芯片以及分别与其组合使用的探针铺设层芯片和进样层芯片,所述探针铺设层芯片上开设探针铺设流道,所述进样层芯片上开设进样流道,在使用时,所述探针铺设流道和进样流道垂直布置；所述底层芯片上设有m×n个检测区域,其中m为探针铺设流道的个数,n为进样流道的个数,所述检测区域位于探针铺设流道和进样流道的交叉处,且包含大量纳米柱组成的阵列,本发明所公开的芯片及其检测方法样品用量小,检测灵敏度高、检测精度高,捕获效率增加,特别适合用于DNA、RNA、蛋白质的荧光检测。(The invention discloses a nano-column array micro-fluidic chip and a detection method thereof, wherein the chip comprises a bottom chip, a probe laying layer chip and a sample injection layer chip which are respectively combined with the bottom chip, wherein a probe laying flow passage is formed on the probe laying layer chip, a sample injection flow passage is formed on the sample injection layer chip, and when the nano-column array micro-fluidic chip is used, the probe laying flow passage and the sample injection flow passage are vertically arranged; the chip and the detection method thereof have the advantages of small sample consumption, high detection sensitivity, high detection precision and increased capture efficiency, and are particularly suitable for fluorescence detection of DNA, RNA and protein.)

1. A nano-column array micro-fluidic chip is characterized by comprising a bottom chip, a probe laying layer chip and a sample injection layer chip, wherein the probe laying layer chip and the sample injection layer chip are respectively combined with the bottom chip for use; the bottom chip is provided with m multiplied by n detection areas, wherein m is the number of probe laying runners, n is the number of sample injection runners, and the detection areas are positioned at the intersection of the probe laying runners and the sample injection runners and comprise arrays formed by a large number of nano-columns.

2. The nanopillar array microfluidic chip according to claim 1, wherein the probe-laying flow channels are parallel flow channels having injection ports at both ends, the number of the probe-laying flow channels is equal to the number of the probe types, the width of the probe-laying flow channels is greater than the width of the detection region, and the depth of the probe-laying flow channels is greater than the height of the nanopillars.

3. The nanopillar array microfluidic chip according to claim 1, wherein the sample injection flow channels are parallel flow channels with injection ports at both ends, the number of the sample injection flow channels is equal to the number of samples to be detected, the direction of the sample injection flow channels is perpendicular to the direction of the probe laying flow channels, the width of the sample injection flow channels is larger than the lateral width of the detection area, and the depth of the sample injection flow channels is larger than the height of the nanopillars.

4. The nanopillar array microfluidic chip according to claim 1, wherein the array of nanopillars is obtained by nanoimprint or by nanomaterial growth.

5. A detection method of a nano-column array micro-fluidic chip adopts the nano-column array micro-fluidic chip as claimed in claim 1, and is characterized in that a probe laying layer chip is firstly used for being combined with a bottom chip, and different types of probes are injected into a probe laying flow channel, so that specific probe molecules are laid on nano-columns; then uncovering the probe laying layer chip, combining the sample introduction layer chip with the bottom layer chip to ensure that the sample introduction flow channel is vertical to the probe laying flow channel, injecting different samples into the sample introduction flow channel, wherein the samples flow through the detection areas respectively provided with the respective specific probes; and finally, detecting by a fluorescence microscope or a fluorescence scanner, forming fluorescent light spots with different brightness under the excitation of laser with specific wavelength, and further obtaining the type and the content of the specific molecules by fluorescence intensity analysis.

Technical Field

The invention relates to a microfluidic technology, in particular to a nano-column array microfluidic chip and a detection method thereof.

Background

Fluorescence labeling detection has wide application in marine science, biology and medical research. The use of fluorescence labeling detection enables quantitative detection of organic substances such as DNA, RNA, and proteins. However, in fluorescence detection, the intensity of fluorescence directly affects the sensitivity and accuracy of fluorescence detection, the biomarker and specific probe with fluorescence are very expensive, and the extraction process of biological substances such as DNA, RNA, and protein is complicated, time-consuming, and the reagent cost is high. Therefore, if the usage amount of the sample and the reagent can be reduced, the detection cost is greatly reduced. In addition, the fluorescence detection of non-biochemical substances also has the problem of low fluorescence intensity, which affects the detection sensitivity and accuracy.

Disclosure of Invention

In order to solve the technical problems, the invention provides a nano-column array micro-fluidic chip and a detection method thereof, so as to achieve the purposes of small sample consumption, high detection sensitivity, high detection precision and increased capture efficiency.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a nano-column array micro-fluidic chip comprises a bottom chip, a probe laying layer chip and a sample injection layer chip, wherein the probe laying layer chip and the sample injection layer chip are respectively combined with the bottom chip for use; the bottom chip is provided with m multiplied by n detection areas, wherein m is the number of probe laying runners, n is the number of sample injection runners, and the detection areas are positioned at the intersection of the probe laying runners and the sample injection runners and comprise arrays formed by a large number of nano-columns.

In the above scheme, the probe laying flow channel is a parallel flow channel with injection ports at two ends, the number of the probe laying flow channels is equal to the type of the probes, the width of the probe laying flow channel is larger than that of the detection area, and the depth of the probe laying flow channel is larger than that of the nano-column.

In the above scheme, the sample injection flow channel is a parallel flow channel with injection ports at two ends, the number of the sample injection flow channels is equal to the number of samples to be detected, the direction of the sample injection flow channel is vertical to the direction of the probe laying flow channel, the width of the sample injection flow channel is greater than the lateral width of the detection area, and the depth of the sample injection flow channel is greater than the height of the nano-column.

In the above scheme, the array composed of the nano-pillars is obtained by nano-imprinting or by growth of nano-materials.

A detection method of a nano-column array micro-fluidic chip adopts the nano-column array micro-fluidic chip, firstly, a probe laying layer chip is combined with a bottom layer chip, different types of probes are injected into a probe laying flow channel, and specific probe molecules are laid on a nano-column; then uncovering the probe laying layer chip, combining the sample introduction layer chip with the bottom layer chip to ensure that the sample introduction flow channel is vertical to the probe laying flow channel, injecting different samples into the sample introduction flow channel, wherein the samples flow through the detection areas respectively provided with the respective specific probes; and finally, detecting by a fluorescence microscope or a fluorescence scanner, forming fluorescent light spots with different brightness under the excitation of laser with specific wavelength, and further obtaining the type and the content of the specific molecules by fluorescence intensity analysis.

Through the technical scheme, the nano-column array micro-fluidic chip provided by the invention is a highly integrated biochip prepared by a micro-nano processing technology, and is very suitable for fluorescence detection of DNA, RNA and protein. Compared with the prior art, the invention has the following beneficial effects:

1. the micro-fluidic chip designed by the invention can greatly reduce the use amount of a detection sample and a detection reagent, thereby greatly reducing the sample processing cost and the detection reagent cost.

2. The detection areas of the microfluidic chip designed by the invention are arranged in a dotted and regular manner, the fluorescent detection bright spots of different probes of the same sample can be arranged in a line, and the detection effect is clear at a glance.

3. A large number of nano-column arrays are arranged in the detection area, and the nano-column arrays arranged in order have a fluorescence enhancement effect.

4. Compared with a plane detection area, the nano-column has a large number of specific probes in the lateral direction, and during fluorescence scanning, fluorescence signals are superposed in the longitudinal direction, so that the fluorescence effect is enhanced.

5. Compared with a plane detection region, the three-dimensional structure of the nano-column not only increases the number of probes, but also enhances the contact area between the sample and the detection region, so that more specific molecules with fluorescent labels are combined with the probes, and the detection precision and sensitivity are improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic plan view of a bottom chip according to an embodiment of the present invention;

FIG. 2 is a schematic perspective view of a detection area according to an embodiment of the present invention;

FIG. 3 is a schematic perspective view of a probe-layer chip according to an embodiment of the present invention;

FIG. 4 is a schematic perspective view of a sample injection layer chip according to an embodiment of the present invention;

FIG. 5 is a schematic plan view of a bottom chip and a probe card layer chip according to an embodiment of the present invention;

fig. 6 is a schematic plan view of the bottom chip and the sample injection layer chip according to the embodiment of the present invention.

In the figure, 1, a bottom chip; 2. laying a layer chip by using the probe; 3. sampling layer chips; 4. detecting a region; 5. a nanopillar; 6. a probe is laid at the inlet of the flow channel; 7. laying a probe at an outlet of the flow passage; 8. a sample injection flow channel inlet; 9. an outlet of the sample injection flow channel; 10. laying a flow channel by the probe; 11. and a sample injection flow channel.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

The invention provides a nano-column array micro-fluidic chip and a detection method thereof.

A nano-column 5 array micro-fluidic chip comprises a bottom chip 1, a probe laying layer chip 2 and a sample injection layer chip 3, wherein the probe laying layer chip 2 and the sample injection layer chip 3 are respectively combined with the bottom chip for use, a probe laying flow channel 10 is formed in the probe laying layer chip 2, a sample injection flow channel 11 is formed in the sample injection layer chip 3, and the probe laying flow channel 10 and the sample injection flow channel 11 are vertically arranged when the micro-fluidic chip is used.

As shown in fig. 1 and fig. 2, m × n detection regions 4 are disposed on the bottom chip 1, where m is the number of probe laying flow channels 10, n is the number of sample injection flow channels 11, and the detection regions 4 are located at the intersection of the probe laying flow channels 10 and the sample injection flow channels 11 and include an array composed of a large number of nano-pillars 5.

As shown in fig. 3, the probe laying flow path 10 is a parallel flow path having injection ports at both ends, and as shown in fig. 3, one end of the flow path is a probe laying flow path inlet 6, and the other end is a probe laying flow path outlet 7; the number of the probe laying flow channels 10 is equal to the type of the probes, the width of the flow channels is larger than that of the detection area 4, and the depth of the flow channels is larger than that of the nano-columns 5.

As shown in fig. 4, the sample injection channel 11 is a parallel channel with injection ports at both ends, as shown in fig. 4, one end of the channel is a sample injection channel inlet 8, and the other end is a sample injection channel outlet 9; the number of the sample introduction runners 11 is equal to that of samples to be detected, the direction of the runners is vertical to the direction of the probe laying runner 10, the width of the runners is larger than the lateral width of the detection area 4, and the depth of the runners is larger than the height of the nano-columns 5.

The array of nano-pillars 5 is obtained by nano-imprinting or by nano-material growth.

The number of probe laying flow channels 10 of the invention determines the maximum number of probe types, the number of sample injection flow channels 11 determines the maximum number of single chip detection samples, the flow channels are mutually vertical, and the number and the topological structure of the detection areas 4 of the bottom chip 1 are determined together. And the bottom chip 1 is provided with a detection area 4 formed by nano-pillars 5. The detection region 4 may be any shape, but the length and width of the detection region 4 are respectively smaller than the width of the probe laying channel 10 and the sample injection channel 11, and are located at the intersection of the two upper flow channels. The functionalized bottom chip 1 is bonded to the probe-placement layer chip 2 in order to place different specific probes in each row of detection regions 4. After the probe is well combined with the nano-column 5 of the detection area 4 of the bottom chip 1, the probe is taken off and the chip 2 of the laying layer is laid, and then the bottom chip 1 is combined with the chip 3 of the sampling layer, so that each row of detection area 4 is positioned in different sampling flow channels, and each sample (pre-fluorescence mark) can flow through each row of detection area 4, and then contacts with different specific probes and generates specific combination with different degrees.

The detection method of the nano-column 5 array microfluidic chip comprises the following steps:

firstly, a probe laying layer chip 2 is combined with a bottom layer chip 1, as shown in fig. 5, probes of different types are injected into a probe laying flow channel 10, so that specific probe molecules are laid on a nano column 5; then uncovering the probe laying layer chip 2, then combining the sample introduction layer chip 3 with the bottom layer chip 1, as shown in figure 6, injecting different samples into the sample introduction flow channel 11, wherein the samples flow through the detection areas 4 respectively provided with respective specific probes; and finally, detecting by a fluorescence microscope or a fluorescence scanner, forming fluorescent light spots with different brightness under the excitation of laser with specific wavelength, and further obtaining the type and the content of the specific molecules by fluorescence intensity analysis.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.