CN1441061A - Efficient nucleic acid hybridization device and method - Google Patents

Abstract

The invention discloses a device for hybridization reaction between target molecules and probes in liquid, which comprises a trace liquid pipeline containing a first part and a second part which is connected with the first part; wherein the first portion has an irregular cross-section, the second portion has a probe, and a liquid driving member connecting the end of the conduit with the cuvette, wherein the liquid driving member can repeatedly move the target molecule back and forth through the second portion.

Description

Efficient nucleic acid hybridization device and method

technical field

The invention relates to a device for a hybridization reaction between a target molecule and a probe in a liquid, and a method for the hybridization reaction.

Background technique

Molecular biology includes many techniques for analyzing nucleic acids and proteins. Many of these techniques and methods form the basis of clinical diagnostic tests. These techniques include nucleic acid hybridization analysis, restriction enzyme analysis, gene sequence analysis, and isolation and purification of nucleic acids and proteins. For example, nucleic acid hybridization is currently commonly used in genetic research, biomedical research, and clinical diagnosis. However, these techniques involve many complex and time-consuming steps. Their use is often limited by lack of sensitivity, specificity, or reproducibility.

There are many devices and methods that improve the efficiency of hybridization reactions by changing hybridization conditions. For example, US Patent 5,639,423 relates to a device for in situ chemical reactions within a microstructured environment. The device is particularly useful in biochemical reactions that require highly accurate temperature cycling, especially DNA-based controls such as PCR, since the small size of the microdevice facilitates rapid cycle times. US Patent No. 6,238,910 provides a DNA hybridization device capable of accurately controlling temperature and liquid.

In addition, there are techniques to improve the components used in hybridization assay devices. US Patent 5,849,486 discloses a system for molecular biology diagnostics, analysis and multi-step and multiplex reactions utilizing self-moving, self-assembling microelectronic systems to actively control reaction conditions under delicate conditions. US Patent 6,197,565 provides a tiny integrated liquid system for preparation and analysis of various operations, and methods of operating and using these systems. US Patent No. 6,255,050 uses a force, such as centrifugal force, electrophoretic force, gravity, vacuum attraction or pressure, to drive separation and aggregation of nucleic acid base-containing sequences in a hybridization reaction. US Patent No. 6,287,850 discloses a vibrating device for reversibly mediating the back and forth flow of a liquid sample along a nucleic acid array, thereby promoting the hybridization of target sequences of the liquid sample and probes in the nucleic acid array. In addition, Liu et al. improved the micro-liquid biochemical array, and used Motorola (Motorola) glass-based microarray biochip to integrate a large number of parallel micro-liquid channels (the 14th IEEE International Symposium on Micro-Electro-Mechanical Systems 2001, 439-442, 21-25 January 2001).

However, the above-mentioned known techniques cannot provide satisfactory hybridization efficiency, nor can they effectively reduce the time required for hybridization. Therefore, there is a need for an apparatus and method for improving hybridization assays.

SUMMARY OF THE INVENTION An object of the present invention is to provide a device for a hybridization reaction between a target molecule and a probe in a liquid, comprising:

a microfluidic conduit comprising a first section and a second section following said first section, wherein said first section has an irregular cross-section, said second section has a probe, and

A liquid drive element connected to the end of the tube and the test tube, wherein the liquid element can repeatedly move the target molecule back and forth through the second portion.

Another object of the present invention is to provide a method for increasing the hybridization reaction between a target molecule and a probe, comprising the steps of:

(a) providing a trace liquid conduit comprising a first portion and a second portion following said first portion, wherein said first portion has an irregular cross-section, said second portion has a first and second or more probes, wherein said first probe specifically binds said target molecule;

(b) introducing the liquid containing the target molecule into the trace liquid pipeline in the hybridization reaction device of the present invention;

(c) driving said fluid back and forth so that said target molecule can repeatedly pass through said second portion, thereby removing said target molecule non-specifically bound to a second or more probes, remaining in contact with The target molecule to which the first probe binds.

Description of drawings

Fig. 1 shows an example of the irregular cross-sectional shape of the trace liquid channel used in the hybridization reaction device.

Figure 2 illustrates the inventive device.

Figure 3 shows microfluidic channels of different shapes (device I: circle, device II: straight line) with irregular cross-sections.

Figure 4 shows that regardless of the shape of the irregular cross section, the hybridization efficiency of the target DNA driven by the micropump is better than that of the incubated target DNA (control).

Fig. 5 shows the hybridization efficiency of circular irregular sections.

Figure 6 shows microfluidic channels of different size cross-sections.

Figure 7 shows that the hybridization efficiency of the target DNA driven by the micropump is better than that of the incubated target DNA (control) no matter in the device III or the device IV.

Figure 8 shows that the hybridization signal after 30 minutes in the slow region (large cross-section) of device III is 1.5 times that of the control after 4 hours.

Figure 9 shows that the hybridization signal in the slow region (large cross-section) of device IV after 30 minutes is 2.7 times that of the control after 4 hours, such as 6.1 times that of the control after 30 minutes.

Detailed ways

The present invention utilizes a micro liquid pipeline capable of generating shearing force to shake the liquid back and forth, thereby increasing the hybridization efficiency of target molecules and probes in the liquid and reducing the time required for hybridization.

An object of the present invention is to provide a device for hybridization reaction between a target molecule and a probe in a liquid, comprising:

a microfluidic conduit comprising a first section and a second section following said first section, wherein said first section has an irregular cross-section, said second section has a probe, and

A liquid drive element connected to the end of the tube and the test tube, wherein the liquid element can repeatedly move the target molecule back and forth through the second part.

According to the invention, a probe is a surface-immobilized molecule recognized by a particular target sequence, sometimes referred to as a ligand. Examples of probes that can be studied using this invention include, but are not limited to, agonists and antagonists of cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g. opiate peptides, steroids, etc.), hormone receptors, peptides, Enzymes, substrates, cofactors, drugs, lectins, carbohydrates, oligonucleotides or nucleic acids, oligosaccharides, proteins and monoclonal antibodies.

According to the invention, a target molecule is a molecule with which a given probe has an affinity, sometimes referred to as a receptor. The target molecule can be a naturally occurring molecule or an artificially produced molecule. Plus, they can be used in their unchanged state or in combination with other kinds. Target molecules can be covalently or non-covalently bound to the binding membrane directly or through specific binding media. Examples of target molecules used in the present invention include, but are not limited to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as viruses, cells or other substances), drugs, oligonucleotides or Nucleic acids, peptides, cofactors, lectins, carbohydrates, polysaccharides, cells, cell membranes, and organelles. Preferred target molecules of the invention are nucleic acids, peptides or peptide ribonucleic acids. More preferred target molecules of the present invention are DNA or RNA. More preferred target molecules of the present invention are single-stranded nucleic acids or double-stranded nucleic acids.

According to the invention, the microfluidic conduit of the device comprises a first part and a second part following said first part. According to the invention, the first part has an irregular cross-section. The irregular cross-section is produced by irregularly varying the size of the cross-section of the first portion of the duct. An example of the shape of the first part is shown in Figure 1. Most single-stranded nucleic acid molecules can form a disc-like configuration due to the formation of intramolecular hydrogen bonds. Considering this structure, the region where the hybridization reaction takes place is located within the molecular structure, and therefore, the hybridization reaction is not complete. In the past, only about 8 percent of nucleic acid molecules could react completely. According to the present invention, the first part can generate a shear stress capable of pulling the nucleic acid molecules into a linear configuration, and the formation of the linear nucleic acid molecules facilitates the hybridization reaction. In addition, double-stranded nucleic acids can also be used in the device of the present invention. The shear stress generated by the first part of the present invention can denature double-stranded nucleic acid to generate single-stranded nucleic acid. Similarly, the reactive regions of protein molecules may also be located within the three-dimensional structure. The shear force can damage the three-dimensional structure of the protein, so that the reaction zone is exposed, and the hybridization reaction can be easily performed. According to the invention, the inner surface of the microfluidic conduit is roughened or notched.

According to the invention, the device comprises a fluid drive element connecting the end of said conduit to the test tube. Preferred liquid-driven elements are microgas-driven pumps, micromechanical pumps or microelectrokinetic pumps. More preferably, the micro-mechanical pump is selected from micro-electrostatic pumps, micro-magnetic drive pumps, and micro-diffusion pumps. More preferably, the microelectrokinetic pump is selected from the group consisting of microhydroelectric pumps and microelectrophoretic pumps and microelectroosmotic pumps.

According to the invention, the device further comprises means for energizing said target molecule. A preferred method is a heater such as a thermocycler. Energy can increase the number of collisions between the target molecule and the probe. Therefore, hybridization efficiency can be improved.

A preferred example of the present invention is to illustrate that the device is used in a hybridization reaction between a target molecule and a probe (see FIG. 2 ) in a liquid. Micropump 1 drives fluid flow to valve 2. The liquid flows into the trace liquid pipeline 3 through the test tube 4, and the hybridization reaction is carried out in the pipeline 3. The liquid obtained flows out of line 3 through test tube 5 .

According to the present invention, any known techniques (such as microcasting, etching and bonding methods) can be used to manufacture the device of the hybridization reaction of the present invention, as described in "The 14th IEEE International Symposium on Microelectromechanical Systems 2001, 439- 442 pages, January 21-25, 2001" described in the method. Preferred devices of the invention can be used to remove target molecules that bind non-specifically to the probe.

Another object of the present invention is to provide a method for removing non-specifically bound target molecules between the target molecule and the probe in the hybridization reaction, which comprises the following steps:

(a) providing a trace liquid conduit comprising a first part and a second part following said first part; wherein said first part has an irregular cross-section and said second part has first and second or more probes, wherein said first probe specifically binds to said target molecule;

(b) introducing the liquid containing the target molecule into the micropipe in the hybridization reaction device of the present invention;

(c) driving the liquid to flow back and forth, so that the target molecule can repeatedly pass through the second part, remove the target molecule non-specifically combined with the second or more probes, and retain the target molecule combined with the first probe; The target molecule to which the probe binds.

According to the present invention, the microfluidic device for removing non-specifically bound target molecules used in the method comprises a first part having an irregular cross-section and a second part having first and second or more probes, wherein the The first probe specifically binds to the target molecule. Target molecules that bind non-specifically to other probes can be removed by repeatedly driving the fluid through said second portion.

According to the present invention, the device and method of the present invention can reduce the time required for the hybridization reaction and increase the hybridization efficiency. The present invention provides a commercially viable device for performing hybridization reactions. It is to be understood that the foregoing description is intended to be illustrative and not limiting. From reading the above description, many examples will be apparent to those of skill in the art.

Example

Embodiment 1 hybridization reaction

This example compares the hybridization efficiency of microfluidic channels with different shapes (FIG. 3, device I: circle, device II: straight line).

The four probes, Sp5 (0.5 μM), Alo3 (5 μM), Alo1 (5 μM) and P3 (5 μM) were spotted onto the thin slices with a pipette (fixative solution: 2 × SSC; thin slices: manufactured by Industrial Technology Research Institute sol-gel; the volume of each point: 200nl), reacted at 37°C for 4 hours. The flakes were then sonicated in 0.5% SDS, washed twice with deionized water for one minute each, and air-dried in a DNA hybridization oven (Hybrid Inc.).

There is a specially cast mold comprising a circular or linear first portion of the microfluidic channel tightly combined with the above-mentioned sheet containing specific nucleic acid probes, which is fixed with strong pogo pins. The microfluidic conduits on the casting mold should properly cover the nucleic acid probes immobilized on the sheet so that the nucleic acid probes are exposed to the microfluidic conduits.

Target DNA, Cy5O3 (1 μM; 25-base single-stranded sequence complementary to Alo3, whose 5' end is fluorescently labeled with Cy5), was denatured. A liquid containing 10 μl of target DNA, 20 μl of deionized water, and 30 μl of 2× hybridization buffer enters the microfluidics line. Additional energy is provided to increase hybridization efficiency. The target DNA was driven back and forth with a micropump to perform hybridization (40°C; 1 hour). The target DNA was respectively introduced into another microfluidic tube, and incubated at 40°C for 1 hour for hybridization (as a control). After hybridization, slices were probed for fluorescence using a scanner (ScanArray 4000, General Scan Corporation).

The results are shown in Figure 4 and Figure 5. Figure 4 shows that regardless of the shape of the first part, the hybridization efficiency of the target DNA driven by the micropump is better than that of the incubated target DNA (control). Figure 5 shows that the hybridization efficiency of the linear first part (signal about 3.8 times that of the control) is better than that of the circle (signal about 2.1 times that of the control).

Embodiment 2 hybridization reaction

This example compares the hybridization efficiency of microfluidic channels with different sizes of cross-sections (FIG. 6).

Five probes, Sp5 (0.5 μM), No. 2 probe (5 μM), No. 3 probe (5 μM), No. 4 probe (5 μM) and No. 5 probe (5 μM) were spotted onto the thin slice with a pipette (fixative solution: 2×SSC; sheet: sol-gel manufactured by Industrial Technology Research Institute; volume per spot: 200 nl), and reacted at 37° C. for 4 hours. The flakes were then sonicated in 0.5% SDS, washed twice with deionized water for one minute each, and air-dried in a DNA hybridization oven (Hybrid Inc.).

Two rectilinear microfluidic channels were generated as described in Example 1 with cross-sections 2:1 (Setup III) and 5:1 (Setup IV) and used in the following hybridization experiments.

The target DNA (1K base single-stranded sequence complementary to No. 5 probe, whose 5' end is fluorescently labeled with Cy5 detection) is denatured. A liquid containing 10 μl of target DNA, 20 μl of deionized water, and 30 μl of 2× hybridization buffer enters the microfluidics line. The target DNA was driven back and forth with a micropump to perform hybridization (40°C; 30 minutes). The target DNA was respectively introduced into another microfluidic tube, and incubated at 40°C for 30 minutes for hybridization (as a control). After hybridization, slices were probed for fluorescence using a scanner (ScanArray 4000, General Scan Corporation).

The results are shown in Figures 7 to 9. Figure 7 shows that the hybridization efficiency of the target DNA driven by the micropump is better than that of the incubated target DNA (control) no matter in the device III or the device IV. Figure 8 shows that the hybridization signal after 30 minutes in the slow region (large cross-section) of device III is 1.5 times that of the control after 4 hours. Figure 9 shows that the hybridization signal in the slow region (large cross-section) of device IV after 30 minutes is 2.7 times that of the control after 4 hours, such as 6.1 times that of the control after 30 minutes. These results suggest that the amount of hybridization signal is influenced not only by the kinetic energy used to drive the target DNA, but also by the shape of the microfluidic channel.

While the present invention has been particularly shown and described in preferred reference embodiments, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the invention.

Claims (15)

1. the device of hybridization between target molecule that is used for liquid and the probe is characterized in that comprising:

A micro liquid pipeline, it comprises first part and the first part's second section afterwards that continues, and there is an irregular cross section in wherein said first part, and described second section has a kind of probe, and

A liquid driven element that connects described pipe end and test tube, wherein said liquid cell can repeatedly move around described target molecule by described second section.

2. device according to claim 1 is characterized in that described irregular cross section is that the size of the described first part cross section by the described pipeline of irregular change produces.

3. device according to claim 1 is characterized in that described micro liquid side opposite is coarse or recessed joint is arranged.

4. device according to claim 1 is characterized in that described probe is nucleic acid, peptide or peptide nucleic acid(PNA).

5. device according to claim 4 is characterized in that described nucleic acid is DNA or RNA.

6. device according to claim 4 is characterized in that described nucleic acid is single-chain nucleic acid or double-strandednucleic acid.

7. device according to claim 1 is characterized in that further comprising the device that energy is provided to described target molecule.

8. device according to claim 1, it is characterized in that can be with the target molecule that removes with the non-specific combination of described probe.

9. method that increases hybridization between target molecule and the probe is characterized in that may further comprise the steps:

(a) provide a micro liquid pipeline that contains first part and continue described first part second section afterwards, there is an irregular cross section in wherein said first part, described second section has first and second or more probe, wherein said first probe and described target molecule specific combination;

(b) liquid that will contain described target molecule imports the micro liquid pipeline in the hybridization device of the present invention;

(c) drive described liquid and flow back and forth, described target molecule can be repeated by described second section, therefore remove and second or the described target molecule of the non-specific combination of multiprobe more, keep and the first probe bonded target molecule.

10. method according to claim 9 is characterized in that described probe is a nucleic acid, peptide or peptide nucleic acid(PNA).

11. method according to claim 10 is characterized in that described nucleic acid is DNA or RNA.

12. method according to claim 10 is characterized in that described nucleic acid is single-chain nucleic acid or double-strandednucleic acid.

13. method according to claim 9, the surface that it is characterized in that described pipeline is coarse.

14. method according to claim 9 is characterized in that described irregular cross section is that the size of the described first part cross section by the described pipeline of irregular change produces.

15. method according to claim 9 is characterized in that further comprising the step that energy is provided to described target molecule.

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