Method for reducing background fluorescence of DNA chip by CRISPR system based on transition-state molecular switch
By introducing transition state molecular switches and CRISPR systems in DNA chip detection, combined with Taq polymerase, the problem of excessive fluorescence background signals in DNA chip detection is solved, and higher detection specificity and accuracy are achieved.
Patent Information
- Application Number
- CN202410796097.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-19
- Publication Date
- 2025-01-24
AI Technical Summary
There is a problem of excessive fluorescent background signal during the detection process of existing DNA chips, which leads to nonspecific adsorption and nonspecific recognition of signal molecules, affecting the accuracy of detection.
The CRISPR system based on the transitional molecular switch is used to perform the first screening of nucleic acid through the transitional molecular switch, and the second screening of CRISPR technology is used to combine the TAQ polymerase as the basis for the signal to reduce the fluorescent background signal.
Effectively reduce the fluorescent background signals on the DNA chip, improve the specificity and accuracy of the detection, and simplify the steps of nucleic acid design and operation.
Smart Images
Figure FT_1
Abstract
Description
Technical Field
[0001] The present invention relates to the field of nucleic acid detection technology, and more specifically to a method for reducing the fluorescence background on a DNA chip by combining microfluidics technology, transition state molecular structure preparation and CRISPR / Cas12a technology. Background Art
[0002] Microfluidic chips have the characteristics of high throughput, portability, and low reagent consumption, and can be integrated into independent "chip laboratory" systems. They have several advantages over traditional macroscopic systems, including reduced sample and reagent volume, faster analysis time, and the ability to integrate multiple functions into a single chip. For general DNA chips, capture probes are tools for fixing signal molecules on the chip. One challenge is the nonspecific adsorption of signal probes, which is usually caused by the condensation of a small amount of 3' terminal hydroxyl groups of DNA with aldehyde groups on the chip surface. In addition, nonspecific recognition of DNA can also lead to strong background signals.
[0003] The CRISPR-Cas system uses crRNA to precisely guide the Cas protein to recognize and cut the target nucleic acid, thereby triggering indiscriminate cutting of single-stranded DNA. Using this mechanism, researchers are able to distinguish between single-stranded and double-stranded DNA molecules and generate different signals for nucleic acid quantification by activating the Cas12a trans-cutting activity. However, in more complex samples, nonspecific binding and recognition of crRNA and DNA may produce false positive signals. There have been many studies using Cas12a trans-cutting to obtain lower background, but they may have more complex nucleic acid designs, more cumbersome procedures, and longer reaction times, which complicates the analysis. Therefore, it is hoped that the developed method can reduce nonspecific adsorption on the chip and have a simple and programmable nucleic acid design to reduce nonspecific recognition of crRNA.
[0004] In the present invention, we developed a CRISPR-mediated DNA chip fluorescence detection based on a transition state molecular switch to reduce the background fluorescence on the chip. The introduction of the transition state molecular switch provides a simple programmable nucleic acid design for the first screening of nucleic acids, and the dissociated Taq polymerase serves as the basis for signal retention on the chip, while the introduction of CRISPR technology performs the second screening of nucleic acids and serves as the basis for on-chip fluorescence signal generation and fluorescence background reduction. Each chip can detect 11 samples simultaneously, achieving high-throughput detection. Summary of the invention
[0005] The technical problem to be solved by the present invention is to reduce the fluorescence background of nucleic acid detection on microfluidics.
[0006] A method for reducing background fluorescence of a DNA chip using a CRISPR system based on a transition state molecular switch, characterized by comprising the following steps:
[0007] (1) Treatment of glass substrate
[0008] Use 25×75mm 2 The glass slide was used as the glass substrate. Cyclohexane, anhydrous ethanol, and ultrapure water were used to ultrasonically clean the surface impurities. After cleaning, it was immersed in a 1M NaOH solution and heated at 60°C for 2h for hydroxylation treatment, and then ultrasonically cleaned with deionized water for 10min. Then it was immersed in a mixed solution with a volume ratio of anhydrous ethanol: deionized water: APTES = 95:3:2 for 2h to complete the silanization treatment. Rinse with anhydrous ethanol and a large amount of deionized water, dry with nitrogen, and vacuum dry at 60°C for 1h. Finally, the silanized glass substrate was immersed in a 2.5% glutaraldehyde solution (diluted with 1×PBS solution) for 2h. Rinse with a large amount of deionized water, dry with nitrogen, and vacuum dry at 60°C for 30min. The treated glass substrate was placed in a nitrogen-filled box and stored at 4°C.
[0009] (2) Preparation of channel printing chip
[0010] The channel printing chip is prepared on a silicon wafer mold with a channel. The material used is polydimethylsiloxane (PDMS) with a size of 25×75mm. 2 . Mix PDMS and coagulant in a ratio of 10:1, stir evenly and place in a vacuum environment for 20 minutes to remove bubbles. Then pour it onto the silicon plate mold and heat it at 80°C for 2 hours to completely cure the channel printing chip. Finally, peel off the cured PDMS and add a 1mm diameter injection hole and an outlet hole at both ends of the channel.
[0011] (3) Preparation of sample loading chip
[0012] The sample loading chip size is 25×75mm 2 The sample loading chip is prepared by laser etching. The insulating rubber is photoetched to form a designed sample loading pool. Each sample loading chip has 11 sample loading pools, which can independently perform 11 different reactions. The sample loading pool is perpendicular to the flow channel printed on the glass substrate, and the signal is observed at the intersection of the two after the reaction is completed.
[0013] (4) Modification of capture probe
[0014] The channel printing chip is tightly fitted to the glass substrate to ensure that the capture probe is modified on the glass substrate according to the shape of the channel. 1.5 μL of capture probe solution (10 μM) is injected into each injection hole, and a pump is used to suck at the outlet hole to fill the entire channel. When all channels are filled with capture probe solution, seal the injection hole and outlet hole to prevent the capture probe solution from evaporating. Place it in a wet box at 37°C overnight. After peeling off the channel printing chip, place the glass substrate in a vacuum drying oven at 60°C for 1 hour, wash it three times with 0.2% SDS solution and deionized water respectively, and blow it dry with nitrogen. Use 2% BSA / PBS solution to block excess glutaraldehyde for 1 hour. Then wash it three times with PBS solution and deionized water respectively, blow it dry with nitrogen for subsequent experiments. (5) Synthesis of transition state molecular switch and preparation of premixed solution
[0015] Mix 100μM S1 and S2 in TE reaction buffer (pH=8.5). Incubate at 95℃ for 10min, slowly cool to 25℃ at 0.1℃ / s and let stand for 30min. Take 1μL and then add 19μL Taq polymerase and 10× buffer to complete the molecular switch. After incubation at room temperature for 3h, add 5μL dNTPs and dilute to 50mL with TE buffer for use as premix A. Use a solution containing 1μM Cas12a and 1.2μM crRNA as premix B.
[0016] (5) Sample processing and testing
[0017] Take 2 μL of premixed solution A, add 2 μL of the sample to be tested, inject into the reaction pool, react at 60°C for 20 min, add 3 μL of premixed solution B, react at 37°C for 1 h, rinse with SDS solution and a large amount of water, dry, and place in a fluorescence array scanner to read fluorescence.
[0018] Beneficial effects of the present invention
[0019] (1) A method based on transition state molecular switches and CRISPR technology was constructed to reduce the fluorescence background on microfluidic chips.
[0020] (2) The use of transition state molecular switches for pre-screening of nucleic acids using CRISPR technology provides better specificity for the detection method.
[0021] (3) The combination of transition state molecular switches and CRISPR technology can reduce the fluorescence background while generating signal molecules, simplifying the operation steps and nucleic acid design. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 A schematic diagram of the device described in this article. DETAILED DESCRIPTION
[0023] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with embodiments, but the content of the present invention is not limited to the following implementation.
[0024] Example 1
[0025] A method for reducing the background fluorescence of HPV-16 detected on a DNA chip by using a CRISPR system based on a transition state molecular switch, characterized by comprising the following steps:
[0026] (1) Treatment of glass substrate
[0027] Use 25×75mm 2 The glass slide was used as the glass substrate. Cyclohexane, anhydrous ethanol, and ultrapure water were used to ultrasonically clean the surface impurities. After cleaning, it was immersed in a 1M NaOH solution and heated at 60°C for 2h for hydroxylation treatment, and then ultrasonically cleaned with deionized water for 10min. Then it was immersed in a mixed solution with a volume ratio of anhydrous ethanol: deionized water: APTES = 95:3:2 for 2h to complete the silanization treatment. Rinse with anhydrous ethanol and a large amount of deionized water, dry with nitrogen, and vacuum dry at 60°C for 1h. Finally, the silanized glass substrate was immersed in a 2.5% glutaraldehyde solution (diluted with 1×PBS solution) for 2h. Rinse with a large amount of deionized water, dry with nitrogen, and vacuum dry at 60°C for 30min. The treated glass substrate was placed in a nitrogen-filled box and stored at 4°C.
[0028] (2) Preparation of channel printing chip
[0029] The channel printing chip is prepared on a silicon wafer mold with a channel. The material used is polydimethylsiloxane (PDMS) with a size of 25×75mm. 2 . Mix PDMS and coagulant in a ratio of 10:1, stir evenly and place in a vacuum environment for 20 minutes to remove bubbles. Then pour it onto the silicon plate mold and heat it at 80°C for 2 hours to completely cure the channel printing chip. Finally, peel off the cured PDMS and add a 1mm diameter injection hole and an outlet hole at both ends of the channel.
[0030] (3) Preparation of sample loading chip
[0031] The sample loading chip size is 25×75mm 2 The sample loading chip is prepared by laser etching. The insulating rubber is photoetched to form a designed sample loading pool. Each sample loading chip has 11 sample loading pools, which can independently perform 11 different reactions. The sample loading pool is perpendicular to the flow channel printed on the glass substrate, and the signal is observed at the intersection of the two after the reaction is completed.
[0032] (4) Modification of capture probe
[0033] The channel printing chip is tightly fitted to the glass substrate to ensure that the capture probe is modified on the glass substrate according to the shape of the channel. 1.5 μL of capture probe solution (10 μM) is injected into each injection hole, and a pump is used to suck at the outlet hole to fill the entire channel. When all channels are filled with capture probe solution, seal the injection hole and outlet hole to prevent the capture probe solution from evaporating. Place it in a wet box at 37°C overnight. After peeling off the channel printing chip, place the glass substrate in a vacuum drying oven at 60°C for 1 hour, wash it three times with 0.2% SDS solution and deionized water respectively, and blow it dry with nitrogen. Use 2% BSA / PBS solution to block excess glutaraldehyde for 1 hour. Then wash it three times with PBS solution and deionized water respectively, blow it dry with nitrogen for subsequent experiments. (5) Synthesis of transition state molecular switch and preparation of premixed solution
[0034] Mix 100μM S1 and S2 in TE reaction buffer (pH=8.5). Incubate at 95℃ for 10min, slowly cool to 25℃ at 0.1℃ / s and let stand for 30min. Take 1μL and then add 19μL Taq polymerase and 10× buffer to complete the molecular switch. After incubation at room temperature for 3h, add 5μL dNTPs and dilute to 50mL with TE buffer as premix A. Use a solution containing 1μM Cas12a and 1.2μM crRNA as premix B.
[0035] (5) Sample processing and testing
[0036] Take 2 μL of premixed solution A, add 2 μL of the sample to be tested containing HPV-16, inject into the reaction pool, react at 60°C for 20 minutes, add 3 μL of premixed solution B, react at 37°C for 1 hour, rinse with SDS solution and a large amount of water, dry, and place in a fluorescence array scanner to read fluorescence.
Claims
1. A method for reducing background fluorescence of a DNA chip using a CRISPR system based on a transition state molecular switch, characterized in that The following steps are involved: (1) Treatment of glass substrate Use 25×75mm 2 The glass slide was used as the glass substrate. Cyclohexane, anhydrous ethanol, and ultrapure water were used to ultrasonically clean the surface impurities. After cleaning, it was immersed in a 1M NaOH solution and heated at 60°C for 2h for hydroxylation treatment, and then ultrasonically cleaned with deionized water for 10min. Then it was immersed in a mixed solution with a volume ratio of anhydrous ethanol: deionized water: APTES = 95:3:2 for 2h to complete the silanization treatment. Rinse with anhydrous ethanol and a large amount of deionized water, dry with nitrogen, and vacuum dry at 60°C for 1h. Finally, the silanized glass substrate was immersed in a 2.5% glutaraldehyde solution (diluted with 1×PBS solution) for 2h. Rinse with a large amount of deionized water, dry with nitrogen, and vacuum dry at 60°C for 30min. The treated glass substrate was placed in a nitrogen-filled box and stored at 4°C. (2) Preparation of channel printing chip The channel printing chip is prepared on a silicon wafer mold with a channel. The material used is polydimethylsiloxane (PDMS) with a size of 25×75mm. 2 . Mix PDMS and coagulant in a ratio of 10:1, stir evenly and place in a vacuum environment for 20 minutes to remove bubbles. Then pour it onto the silicon plate mold and heat it at 80°C for 2 hours to completely cure the channel printing chip. Finally, peel off the cured PDMS and add a 1mm diameter injection hole and an outlet hole at both ends of the channel. (3) Preparation of sample loading chip The sample loading chip size is 25×75mm 2 The sample loading chip is prepared by laser etching. The insulating rubber is photoetched to form a designed sample loading pool. Each sample loading chip has 11 sample loading pools, which can independently perform 11 different reactions. The sample loading pool is perpendicular to the flow channel printed on the glass substrate, and the signal is observed at the intersection of the two after the reaction is completed. (4) Modification of capture probe Fit the channel printing chip tightly to the glass substrate to ensure that the capture probe is modified on the glass substrate according to the shape of the channel. Inject 1.5μL of capture probe solution (10μM) into each injection hole, and use a pump to suck at the outlet hole to fill the entire channel. When all channels are filled with capture probe solution, seal the injection hole and outlet hole to prevent the capture probe solution from evaporating. Put it in a wet box at 37℃ and keep it overnight. After peeling off the channel printing chip, put the glass substrate into a vacuum drying oven at 60℃ for 1h, wash it three times with 0.2% SDS solution and deionized water respectively, and blow it dry with nitrogen. Use 2% BSA / PBS solution to block excess glutaraldehyde for 1h. Then wash it three times with PBS solution and deionized water respectively, and carry out subsequent experiments after drying with nitrogen. (5) Synthesis of transition state molecular switches and preparation of premixed solutions Mix 100μM S1 and S2 in TE reaction buffer (pH=8.5). Incubate at 95℃ for 10min, slowly cool to 25℃ at 0.1℃ / s and let stand for 30min. Take 1μL and then add 19μL Taq polymerase and 10× buffer to complete the molecular switch. After incubation at room temperature for 3h, add 5μL dNTPs and dilute to 50mL with TE buffer for use as premix A. Use a solution containing 1μM Cas12a and 1.2μM crRNA as premix B. (6) Sample processing and testing Take 2 μL of premixed solution A, add 2 μL of the sample to be tested, inject into the reaction pool, react at 60°C for 20 min, add 3 μL of premixed solution B, react at 37°C for 1 h, rinse with SDS solution and a large amount of water, dry, and place in a fluorescence array scanner to read fluorescence.
2. According to claim 1, a method for reducing the background fluorescence of a DNA chip using a CRISPR system based on a transition state molecular switch, characterized in that: A transition state molecular switch is used to pre-screen the nucleic acids recognized by the CRISPR system, generate the target-S1 sequence, and stimulate its trans-cleavage activity after being recognized by Cas12a.
3. According to claim 1, a method for reducing background fluorescence of a DNA chip using a CRISPR system based on a transition state molecular switch, characterized in that: The transition state molecular switch restores the activity of Taq polymerase in the presence of the target, allowing chain extension to occur at 60°C, thereby protecting the signal molecule on the probe.