TWI221856B - High performance nucleic acid hybridization device and process - Google Patents

Abstract

The invention discloses a device for hybridization reaction between a target molecule in a fluid and a probe, which comprises a microfluidic channel comprising a first portion and a second portion following said first portion; wherein said first portion have an irregular cross section and said second portion has a probe, and a fluid driving element connected the ends of said channel with tubes, wherein said fluid element can move said target molecules back-and-forth for repeatedly passing through said second portion.

Description

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BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a device for hybridization between a target molecule and a probe in a liquid, and a method for a hybridization reaction. f King Technology Molecular biology includes many technologies for analyzing nucleic acids and proteins. Many of these technologies and methods form the basis of clinical diagnostic analysis and testing. Such 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 genetics research, biomedical research, and clinical diagnostics. However, these technologies involve many complex and time-consuming steps. Their lack of sensitivity, specificity, or reproducibility often limits their applications. There are many devices and methods that improve the efficiency of hybridization reactions by changing hybridization conditions. For example, U.S. Patent No. 5,639,423 relates to a device for in situ chemical reactions in a microstructured environment. The device is particularly advantageous in biochemical reactions that require highly accurate temperature cycling, especially DNA-based controls such as PCR. This is Because the small space typical of microdevices facilitates fast cycle times. U.S. Patent No. 6,23 8,910 provides a DNA hybridization device capable of accurately controlling temperature and liquid. In addition, some technologies have been developed to improve components for hybridization assay devices. U.S. Patent No. 5,849,486 discloses a system for molecular biology diagnosis, analysis, and multi-step and multiplex reactions using a self-moving, self-assembled microelectronic system for actively controlled reactions under delicate conditions. U.S. Patent No. 6,197,565 provides a micro-integrating solution. -4- This paper is sized for China National Standard (CNS) A4 (210X 297 mm).

Body system for the advancement of customers_ Order multiple preparation and analysis operations, as well as methods for operating and using these systems β Wu Guo Patent No. 6,255,050 uses a force such as centrifugal force, electrophoretic force, tongue ^ ^ gravity , Vacuum attraction, or pressure to drive nucleic acid base-containing sequences in hybrid fs that separate aggregations. U.S. Patent No. 6,287,850 discloses a vibration device for reversibly flowing a liquid sample back and forth across a nucleic acid array, thereby promoting the hybridization of the target in the liquid sample and the bismuth needle on the nucleic acid array. In addition, — et al. Developed a microfluidic biochemical array, using Motorola ’s glass microarray biochips to integrate a large number of parallel microfluidic channels (the 14th IEEE International Microelectronic Mechanical Systems Symposium 2001, 439- 442 pages, 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, it is still necessary to invent a device and method for improving the hybridization assay. SUMMARY OF THE INVENTION An object of the present invention is to provide a device for hybridization reaction between a target molecule and a probe in a liquid, including: a trace liquid channel including a first part and a second part following the first part, wherein the first One part has an irregular cross section, the second part has a probe, and a liquid drive element connected to the end of the channel by a pipeline, wherein the liquid drive element can repeatedly move the target molecule back and forth through the second part. Another object of the present invention is to provide an increased target molecule and probe. The paper size is applicable to China National Standard (CNS) A4 (210X 297 mm).

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k -5- __ 3 V. Description of the invention (Method of heterogeneous father reaction, including the following steps: (a) providing a trace liquid channel containing a first part and a second part following the first part, wherein the first part has irregularities The second part has a first probe and a second or more probes, wherein the first probe specifically binds to the target molecule; (b) introducing a liquid containing the target molecule In the micro liquid channel of the hybridization reaction device of the present invention; (c) driving the liquid to flow back and forth, so that the target molecule can pass through the second part repeatedly, thereby removing the non-specific binding with the second or more probes The target molecule is retained, but the target molecule bound to the first probe is retained. Brief Description of the Drawings Figure 1 shows an example of the irregular cross-sectional shape of a trace liquid channel of a hybridization reaction device. Figure 2 illustrates the device of the present invention. (1: Micropump '2: valve, 3: channel, 4: pipeline, 5: pipeline] Figure 3 shows a trace liquid channel with an irregular cross-section with different shapes (device I: circular, device Π: straight). Figure 4 shows Not at Considering the shape of the irregular cross section, the hybridization efficiency of the target DNA driven by the micropump is better than the static target DNA (control group). Figure 5 shows the hybridization efficiency of the circular irregular cross section. Figure 6 shows the Trace liquid channels of different cross sections. Figure 7 shows that the hybridization efficiency of the target DNA driven by the micropump is better than the static target DNA (control group) in either the device or the device. Figure 8 shows the device in the device. After 30 minutes in the slow area of the melon (large cross section) -6- this standard is Tongchuan Chinese National Standard (cNS) A4 (2ίο X '297 public goods) 1221856 A7 Γ ______ B7_ 5. Description of the invention (4 The hybrid signal is 5 times that of the control group after 4 hours. Figure 9 shows that the hybrid signal after 30 minutes in the slow region (large cross section) of the device! V is hybridized after 4 hours in the control group. 2.7 times the signal, which is 6.1 times the hybridization signal after 30 minutes in the control group. DETAILED DESCRIPTION OF THE INVENTION The present invention uses a micro liquid channel that can generate shear force and the liquid to move back and forth to increase the target molecules and probes in the liquid. Hybridization efficiency and reduce 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 trace liquid channel containing a first part and a second part following the first part , Wherein the first part has an irregular cross section, the second part has a probe, and a liquid driving element connected to the end of the channel by a pipeline, wherein the liquid driving element can repeatedly move the target molecule back and forth through the first Part 2. According to the present invention, a 'probe is a molecule immobilized on a surface that can be recognized by a specific target, which is sometimes referred to as a ligand. Examples of probes that can be used in the present invention include, but are not limited to, cell membrane receptors Agonists and antagonists, toxins and mother liquors, viral epitopes, hormones (such as opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, exogenous agglutination Hormones, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins and monoclonal antibodies. According to the present invention, the target molecule has an affinity for a specific probe, which sometimes _______- 7- applies the Chinese National Standard (CNS) A4 specification (static 297 public love) to the paper standard ----____ 1221856 A7

Binding

k A7 B7 6 V. Description of the invention (

The surface is rough or notched. Q According to the invention, the device includes a liquid drive element connected to the end of the channel in a pipeline. The preferred liquid-driven element is a gas-driven micropump, a mechanical micropump, or an electric micropump. More preferably, the mechanical micropump is selected from the group consisting of an electrostatic micropump magnetically driven micropump and a diffusion micropump. Even more preferably, the electric micropump is selected from the group consisting of a hydroelectric micropump, an electrophoretic micropump, and an electroosmotic micropump. According to the present invention, the device further includes a means for supplying energy to the target molecule. The preferred component is a heater such as a temperature cycler. Energy increases the number of collisions between the target molecule and the probe. Therefore, hybridization efficiency can be improved. A preferred embodiment of the present invention is provided here to exemplify a device for hybridization reaction between a liquid bidding molecule and a probe (see FIG. 2). Micropump i drives fluid aa ML to valve 2. The liquid flows into the trace liquid channel 3 through the line 4, and the hybridization reaction proceeds in the channel 3. The obtained liquid flows out of the channel 3 through the line 5. According to the present invention, any known technique (such as micro-casting, etching, and bonding methods) Both can be used to make the device of the hybridization reaction of the present invention, such as the method described in "The 14th IEEE International Symposium on Microelectromechanical Systems, 2001, pages 439-442, January 21-25, 2001. The present invention The device is preferably used to remove a target molecule that is non-specifically bound to a probe. Another object of the present invention is to provide a method for removing a target molecule that is non-specifically bound in a hybridization reaction between a target molecule and a probe, including the following Steps: (a) Provide a trace liquid channel, including the first part and the continuation of the first " 9-this paper size it standard (CNS) A4 specifications (210X297 public love) " '----- 彡(7) The second part of a knife; wherein the first part has an irregular cross section, and the second part has a first probe and a second or more probes, wherein the first Probe and target molecule Heteroconjugate; (b) introducing the liquid containing the target molecule into the microchannel of the hybridization reaction device of the present invention; (0) driving the liquid to flow back and forth, so that the target molecule passes through the first knife repeatedly, thereby removing the second kind One or more probes are non-specifically bound to the target, while retaining the target molecule bound to the first probe. According to the present invention, the trace liquid channel for removing the non-specifically bound target molecule used in the method includes The first part of the irregular cross section, and the second part having the first probe and the second or more probes, wherein the first probe specifically binds to the target molecule. Non-specific to other probes The bound target molecule can be removed by repeatedly driving the liquid through the second part. 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 feasible Device for performing hetero-X reaction. It can be understood that the above description is intended to clarify rather than limit. By reading the above description, many specific examples are familiar with the technology. It is obvious. Example Example 1 Wei Diaoyu This example compares the hybridization efficiency of trace liquid channels of different shapes (Figure 3, device I: circular, device Π: straight line). Needle, Sp5 (0.5 # M), Alo3 (5 / zM), Alol (5 / zM) and this paper size are applicable to China National Standard (CNS) Α4 specification (21G X 297 public director) " 1221856 A7 ______— B7 5 2. Description of the invention (8) P3 (5 # Μ) point to the wafer with a pipette (fixed buffer · 2 X ssc; wafer · s01-gei developed by the Institute of Industrial Technology; the volume of each point: 200 nl) 'for 4 hours at 37 ° C. The wafer was then sonicated in 0.5% SDS, and the strips were washed twice with deionized water for one minute each time 'and air-dried in a DNA hybrid X oven (Hybrid Inc.). With a powerful spring clip, a special mold cover having a trace liquid channel including a circular or linear first portion is tightly coupled to the wafer on which the specific nucleic acid probe is fixed. The microfluidic channel on the mold cover should properly cover the nucleic acid probe fixed on the wafer, so that the nucleic acid probe is exposed to the microfluidic channel. The target DNA Cy503 (1 #M; 25 base pair single-stranded sequence complementary to Alo3, whose 5 ' end is fluorescently labeled with Cy5 probe) was denatured. A 2 X hybridization buffer containing 10 #! Target DNA, 20 // 1 deionized water, and 30 β 1 was introduced into a micro-fluid channel. Provide additional energy to increase crosses. The target DNA was driven back and forth with a micropump for hybridization (40 ° C; 1 hour). The target DNA was introduced into another microfluidic channel and allowed to stand at 40 eC for 1 hour for hybridization (as a control group). After hybridization, the wafer was scanned for fluorescence with a scanner (ScanArray 4000, General Scanning Inc.). The results are shown in Figures 4 and 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 stationary target DNA (control group). Figure 5 shows that the linear first part (the signal is approximately 3.8 times the signal of the control group) has better hybridization efficiency than the circular first part (the signal is approximately 2.1 times the signal of the control group). Example 2 Hybrid Anti-Pang ^ _ _ -11-This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 1221856 A7 _ — ___ B7 V. Description of the invention (9) This example is different Hybridization efficiency of micro liquid channels with large and small cross sections (Figure 6). Five kinds of probes 'Sp5 (0.5 vM), No. 2 probe (5 / ZM), No. 3 probe (5 # Μ)' No. 4 probe (5 to μ) and No. 5 probe (5 / Z Μ) point onto the wafer with a pipette (fixing buffer: 2 x ssc; wafer: sol-gel developed by the Institute of Industrial Technology; volume at each point: 200 nm), react at 37 ° C 4 hour. The wafer was then sonicated in 0.5% SDS, washed twice with deionized water, one minute each time, and air-dried in a DNA hybridization oven (Hybrid Inc.). Two linear trace liquid channels with cross-sectional areas of 2: 1 (device M) and 5: 1 (device IV) were generated according to the description of Example 1 and used in the following hybridization experiments. The target DNA (1K base pair single-stranded sequence complementary to probe 5 whose 5 ' end is fluorescently labeled with Cy5 probe) was denatured. A liquid containing 10 β 1 labeled DNA, 20 ^ 1 deionized water, and 30 // 1 2X hybridization buffer was introduced into the micro liquid channel. The target DNA was driven back and forth with a micropump for hybridization (40 ° C; 30 minutes). The target DNA was introduced into another microfluidic channel and allowed to stand at 40 ° C for 30 minutes to perform hybridization (as a control group). After hybridization, the 'far chip' uses a scanner (ScanArray 4000, General Scanning Inc.) to detect fluorescence. The results are shown in Figures 7 to 9. Fig. 7 shows that the hybridization efficiency of the target DNA driven by the micropump is better than that of the stationary target DNA (control group) in either 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 the device Π is 1.5 times the hybridization signal after 4 hours in the control group. -12- This paper size applies to China National Standard (CNS) A4 (210X 297mm)

▲ 1221856 A7 B7 V. Description of the invention (10) Figure 9 shows that the hybridization signal after 30 minutes in the slow area (large cross-section) of device IV is 2.7 times the hybridization signal after 4 hours in the control group, which is the control. After the group 30 minutes, the hybridization signal was 6.1 times. These results indicate that the size of the hybridization signal is affected not only by the kinetic energy used to drive the target DNA, but also by the design of trace liquid channels. Although the present invention is specifically shown and described with reference to its preferred specific examples, those skilled in the art will understand that there can be many changes in form and detail without departing from the spirit and scope of the invention.

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-13- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

Claims (1)

1221856 ABCD VI. Scope of patent application The probe is a non-specific binding target molecule. 9. A method for increasing the target molecule and probe 41. The method for restoring needle-to-foot hybridization includes the following steps: (1) providing a trace liquid channel containing a first part and a second part following the first part, wherein the first part Has an irregular cross-section, the second part has a first probe and a second or more probes, wherein the first probe specifically binds to the target molecule; ⑼ introducing a liquid containing the target molecule In the micro liquid channel of the hybridization reaction device of the present invention; (c) driving the liquid to flow back and forth, so that the target molecule is repeatedly passed through the first part, thereby removing the target molecule non-specifically bound to the second or more probes While retaining the target molecule bound to the first probe. 10. The method according to item 9 of the scope of patent application, wherein the probe is a nucleic acid, a peptide or a peptide nucleic acid. 11. A method according to item 10 of the patent application, wherein the nucleic acid is DNA or RNA. 12. The method according to item 10 of the scope of the patent application, wherein the nucleic acid is a single-stranded nucleic acid or a double-stranded nucleic acid. 13. The method according to item 9 of the scope of patent application, wherein the surface of the channel is rough. -15- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1221856 A8 B8 C8 D8, patent application scope 14. According to the method of patent application scope item 9, the irregular cross section is Produced by irregularly changing the size of the cross section of the first portion of the channel. 15. The method according to item 9 of the patent application scope, further comprising the step of supplying energy to the target molecule. Su m -16- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)