Method for determining affinity by using micro-fluidic chip, micro-fluidic chip and nucleic acid aptamer

Abstract

The invention discloses a method for determining affinity by a micro-fluidic chip, the micro-fluidic chip and a nucleic acid aptamer, and the method comprises the following steps: reacting a quenching chain solution with gradient concentration with a nucleic acid aptamer solution, and determining a dissociation constant when the two chains are hybridized and balanced; the method comprises the following steps: reacting a maytansine solution with gradient concentration with a nucleic acid aptamer-quenching chain compound solution, and measuring a dissociation constant when maytansine and a nucleic acid aptamer-quenching chain compound are subjected to hybridization equilibrium; and calculating the balance constant of the combination of the aptamer and the maytansine. The micro-fluidic chip comprises a chip body, a digital micro-fluidic driving platform and a fluorescence detection module. The affinity is determined through the micro-fluidic chip, the affinity determination efficiency of the nucleic acid aptamer is remarkably improved, and the method has the advantages of low consumption, time saving and automation. The nucleic acid aptamer screened by the affinity screening method has high specificity and high affinity combination with maytansine, and is easy to synthesize, low in cost and stable in property.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and microfluidics, and in particular relates to a method for measuring affinity of a microfluidics chip, a microfluidics chip and a nucleic acid aptamer. Background Art

[0002] Bioinformatics analysis (evolutionary trees, homology comparisons, molecular docking, etc.) can narrow the candidate sequence range based on sequence conservation or structural prediction, but existing algorithms are still imperfect in analyzing three-dimensional structure-activity relationships, which may lead to the false screening and elimination of high-affinity nucleic acid sequences. In addition, commonly used methods for determining the affinity of nucleic acid aptamers, including isothermal titration calorimetry, flow cytometry, surface plasmon resonance, and strand displacement fluorescence, also have problems such as slow testing speed and high reagent consumption. Therefore, the development of a high-throughput, automated measurement platform is crucial. Digital microfluidics technology can precisely manipulate microdroplets and has advantages such as small size, high throughput, and low reagent consumption. It can also achieve automated and continuous operation through programmed control, thereby significantly improving experimental efficiency and reducing human error, providing a more efficient and reliable solution for the research and development of nucleic acid aptamers.

[0003] Maytansine is an excellent anti-tumor drug, but its clinical application is limited by poor water solubility, low chemical stability, and a narrow therapeutic window. Aptamers, a class of nucleic acid chains that can specifically recognize target molecules, offer numerous advantages, including stability, small molecular weight, minimal batch-to-batch variability, and ease of synthesis and modification. They have been widely used in drug delivery. Therefore, using aptamers as delivery vehicles may be a viable solution to address the challenges associated with maytansine. However, obtaining aptamers often requires multiple rounds of optimization and iteration, including library design, high-affinity library enrichment, sequencing, and affinity determination of candidate sequences. This process is cumbersome and costly. Affinity determination of a large number of candidate sequences is particularly labor-intensive, time-consuming, and resource-intensive. Therefore, a method for rapidly and efficiently screening and identifying maytansine aptamers is needed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method for affinity determination using a microfluidic chip, a microfluidic chip, and a nucleic acid aptamer. The method for affinity determination using a microfluidic chip transforms processes such as reagent dilution, mixing, and transfer that require manual operation into processes such as movement, mixing, and splitting of reagent droplets on a microfluidic chip. Compared with the repetitive, cumbersome, and manual operation-dependent method, the microfluidic chip significantly improves the efficiency of nucleic acid aptamer affinity determination, reduces sample consumption, and has the advantages of low consumption, time saving, and automation. The nucleic acid aptamer screened out by the above-mentioned affinity screening method has high specificity and high affinity binding to maytansine, is easy to synthesize, has low cost, and is stable in nature.

[0005] In order to solve the above technical problems, the present invention provides a method for measuring affinity using a microfluidic chip, wherein the microfluidic chip includes a chip body 1, a digital microfluidic driving platform 2, and a fluorescence detection module 3; the chip body 1 and the digital microfluidic driving platform 2 are connected in series via a wire; the chip body 1 is provided with a contact electrode 13 connected to the digital microfluidic driving platform 2, a reservoir electrode 14 for storing liquid reagents, a driving electrode 16 for driving the movement, mixing, and splitting of droplets, and a liquid separation electrode 15 for connecting the liquid reservoir electrode 14 and the driving electrode 16; the liquid reservoir electrode 14, the liquid separation electrode 15, and the driving electrode 16 are connected to the contact electrode 13 via electrode leads; The method comprises: S1. By manipulating the digital microfluidic driving platform 2, the buffer solution and the quenching chain in the reservoir electrode 14 are mixed in the chip body 1 to obtain a gradient concentration quenching chain solution; the digital microfluidic driving platform 2 manipulates the gradient concentration quenching chain solution and the nucleic acid aptamer solution to react in the chip body 1 to obtain a nucleic acid aptamer-quenching chain complex solution. The fluorescence detection module 3 obtains an electronic signal by photographing the chip body 1, and draws a fluorescence quenching saturation curve of the hybridization of the nucleic acid aptamer and the quenching chain, thereby determining the dissociation constant K when the nucleic acid aptamer and the quenching chain are in equilibrium. d1 ; S2. By manipulating the digital microfluidic driving platform 2, the buffer solution in the reservoir electrode 14 and the target are mixed in the chip body 1 to obtain a target solution with a gradient concentration; the digital microfluidic driving platform 2 manipulates the target solution with a gradient concentration to mix with the nucleic acid aptamer-quencher chain complex solution in the chip body 1 to react, and the fluorescence detection module 3 obtains an electronic signal by photographing the chip body 1, and draws a fluorescence recovery saturation curve of the target and the nucleic acid aptamer-quencher chain complex, thereby determining the dissociation constant K when the target and the nucleic acid aptamer-quencher chain complex are in hybridization equilibrium. d2 ; S3, according to Kd =K d1 / K d2 Calculate the equilibrium constant for the binding of aptamer to target.

[0006] In the above method, further, the microfluidic chip uses a driving voltage as a power to drive the movement, mixing and splitting of the liquid in the chip body 1, and the driving voltage is 140V to 150V.

[0007] The above method further comprises adding 2 mg / mL to 4 mg / mL of Pluronic L64 to the buffer solution.

[0008] Based on a general technical concept, the present invention provides a microfluidic chip, which includes a chip body 1, a digital microfluidic driving platform 2, and a fluorescence detection module 3; the chip body 1 and the digital microfluidic driving platform 2 are connected in series via a wire; The chip body 1 is provided with a contact electrode 13 connected to the digital microfluidic driving platform 2, a liquid reservoir electrode 14 for storing liquid reagents, a driving electrode 16 for driving the movement, mixing and splitting of droplets, and a liquid separation electrode 15 for connecting the liquid reservoir electrode 14 and the driving electrode 16; the liquid reservoir electrode 14, the liquid separation electrode 15, and the driving electrode 16 are connected to the contact electrode 13 through electrode leads.

[0009] The microfluidic chip described above, further, the chip body 1 is assembled by the chip upper plate 4, the chip lower plate 5 and the polymethyl methacrylate gasket to form a sandwich structure; The chip bottom plate 5 is a printed circuit board, on which a contact electrode 13, a liquid reservoir electrode 14, a liquid separation electrode 15 and a driving electrode 16 are provided; The upper electrode plate 4 of the chip is made of indium tin oxide glass and is provided with a sample addition hole 11 and an oil filling hole 12; The gap between the chip upper plate 4 and the chip lower plate 5 is 400 μm.

[0010] The microfluidic chip mentioned above, further, the chip bottom plate 5 is coated with Parylene C and Teflon AF in sequence.

[0011] The above-mentioned microfluidic chip, further, the microfluidic driving platform 2 includes a chip holder 6, a digital microfluidic driving device 7 and a control software 8, and by controlling the control software 8, the digital microfluidic driving device 7 drives the chip body 1 connected to the chip holder 6 through droplets.

[0012] In the above-mentioned microfluidic chip, further, the fluorescence detection module 3 includes a fluorescence microscope 10 and imaging software 9, and the fluorescence microscope 10 is connected to the imaging software 9 via a wire.

[0013] Based on a general technical concept, the present invention provides a nucleic acid aptamer that recognizes maytansine, wherein the nucleic acid aptamer is Seq19-X, and the nucleotide sequence of Seq19-X is shown in SEQ ID NO.1.

[0014] The above-mentioned nucleic acid aptamer, further, one or both ends of the DNA chain of Seq19-X are radioactively labeled, fluorescently labeled, enzyme-labeled, digoxigenin-labeled, or biotin-labeled; and / or a certain position on the nucleotide sequence of Seq19-X is phosphorylated, methylated, amino-labeled, sulfhydryl-labeled, or isotopically labeled.

[0015] Based on a general technical concept, the present invention provides a use of the aforementioned nucleic acid aptamer in the preparation of an anti-tumor drug. The anti-tumor drug is maytansine and Seq19-X. Seq19-X is a drug carrier.

[0016] Compared with the prior art, the advantages of the present invention are: (1) The present invention provides a method for determining affinity using a microfluidic chip. This method can analyze and obtain nucleic acid aptamers from a large number of candidate sequences, and transforms the processes of reagent dilution, mixing, and transfer that require manual operation into the processes of moving, mixing, and splitting reagent droplets on the chip body 1. Compared with the repetitive, tedious, and manual operation-dependent methods, the chip body 1 significantly improves the efficiency of nucleic acid aptamer affinity determination, reduces sample consumption, and has the advantages of low consumption, time saving, and automation. Through programmed control, automated continuous operation can be achieved, thereby significantly improving experimental efficiency and reducing human errors, while avoiding the misscreening and elimination of high-affinity nucleic acid sequences, providing a more efficient and reliable solution for the research and development of nucleic acid aptamers.

[0017] (2) The present invention provides a microfluidic chip for the above method, which controls the movement, mixing, splitting and other functions of discrete droplets based on the principle of electrowetting. That is, the contact angle of the droplets is changed by the action of the electric field (the on and off of the electrodes), so that the droplets are deformed and spread on the surface of the chip, which can more flexibly manipulate the fluid. Compared with the commonly used microfluidic chips based on square drive electrodes 16, the drive electrodes 16 of this chip are cross-shaped, which is conducive to the contact of the droplet edges with adjacent electrodes and facilitates the drive of the droplets. The electrode area of ​​the microfluidic chip is functionally divided into a retention area, an incubation area, and an imaging area, which can meet the characteristics of the chain displacement fluorescence method to achieve the above-mentioned determination of the affinity of the nucleic acid aptamer.

[0018] (3) The present invention provides a nucleic acid aptamer for identifying maytansine screened using the above method. The nucleic acid aptamer can bind to the target substance maytansine with high affinity and high specificity, and the equilibrium dissociation constant is in the nanomolar range. Compared with protein antibodies, the nucleic acid aptamer has better affinity and specificity, low immunogenicity, can be chemically synthesized in vitro, has a small molecular weight, can be modified and substituted at different sites, and has stable chemical properties, is easy to store, and is convenient for labeling. By utilizing the specific interaction between the nucleic acid aptamer and maytansine, it is expected to solve the problems of poor water solubility, low chemical stability, and narrow therapeutic window of maytansine drugs, thereby improving the limitations of maytansine's clinical application (increasing solubility, improving stability, and widening the therapeutic window). Corresponding labeling, modification or substitution of the nucleic acid aptamer is conducive to the tracing of maytansine in vivo, promoting the effectiveness and safety of drug treatment.

[0019] In addition to the above-described objects, features and advantages, the present invention has other objects, features and advantages. The present invention will be further described in detail below with reference to the accompanying drawings and attached tables. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions 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.

[0021] Figure 1 This is a control flow chart of the microfluidic chip in Example 1 of the present invention.

[0022] Figure 2 This is a schematic structural diagram of the chip body 1 in Example 1 of the present invention.

[0023] Figure 3 Dimensions of the chip upper plate 4 in Example 1 of the present invention (unit: mm).

[0024] Figure 4 Dimensions of the chip bottom plate 5 in Example 1 of the present invention (unit: mm).

[0025] Figure 5 This is an investigation of the effect of the droplet driving voltage on the droplet breakup time in Example 2 of the present invention.

[0026] Figure 6 This is an investigation into the effects of the distance between the upper and lower plates of the chip and the surfactant concentration on the ability of droplets to split evenly in Example 2 of the present invention.

[0027] Figure 7 Schematic diagram of the digital microfluidic platform for measuring aptamer affinity in Example 3 of the present invention. Figure A shows the Kd1 determination process; Figure B shows the Kd2 determination process; and Figure C shows the equilibrium constant detection principle.

[0028] Figure 8 This is a schematic diagram of the structure obtained by performing secondary structure simulation on the nucleic acid aptamer seq15-X and seq19-X sequences using the Mfold service in Example 5 of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific preferred embodiments, but the scope of protection of the present invention is not limited thereby.

[0030] The materials, reagents, and instruments used in the following examples can all be purchased from commercial sources. The experimental methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0031] Example 1 A microfluidic chip, see Figure 1 : It includes a chip body 1, a digital microfluidic driving platform 2, and a fluorescence detection module 3; the chip body 1 and the digital microfluidic driving platform 2 are connected in series through wires; the chip body 1 is placed on the stage in the fluorescence detection module 3 and a camera is used to take pictures to obtain fluorescence images for subsequent data processing.

[0032] See also Figure 2 and Figure 3 : The chip body 1 includes a chip upper plate 4, a chip lower plate 5 and a polymethyl methacrylate gasket; the chip upper plate 4, the chip lower plate 5 and the polymethyl methacrylate gasket are assembled to form a sandwich structure.

[0033] The upper electrode plate 4 of the chip is made of indium tin oxide (ITO) glass and is provided with eight sample addition holes 11 with a diameter of 2 mm and one oil addition hole 12 with a diameter of 3.5 mm.

[0034] See also Figure 4The chip's lower electrode plate 5 is a printed circuit board (PCB), with a dielectric layer of Parylene C and a hydrophobic layer of Teflon AF. The dielectric layer is first deposited via chemical vapor deposition, followed by spin coating and high-temperature curing at 170°C. It is equipped with 48 square contact electrodes 13 with a side length of 1.5 mm; 8 rectangular recessed reservoir electrodes 14 measuring 5.5 mm x 4.8 mm for storing small amounts of liquid reagents; and 16 cross-shaped liquid separator electrodes 15 measuring 2.2 mm x 1.83 mm for connecting the reservoir electrodes 14 to the drive electrodes 16 and assisting the reservoir electrodes 14 in splitting small droplets. Twenty-four cross-shaped drive electrodes 16 measuring 2.2 mm x 2.2 mm are used for moving, mixing, and splitting droplets. Forty-eight electrode leads with a line width of 100 μm are used to connect the contact electrodes 13 to the reservoir electrodes 14, the liquid separator electrodes 15, and the drive electrodes 16, respectively. The edge of the droplet on the cross-shaped driving electrode 16 contacts the adjacent electrode, which helps the droplet to move continuously in the chip.

[0035] The microfluidic driving platform 2 (purchased from Xiamen Deyunxinzhun Technology Co., Ltd.) includes a chip holder 6, a digital microfluidic driving device 7 and control software 8. The control software 8 is manually controlled to enable the digital microfluidic driving device 7 to drive the chip body 1 through droplets.

[0036] The fluorescence detection module 3 includes a fluorescence microscope 10 and imaging software 9. The fluorescence microscope 10 acquires the signal on the chip body 1 through the imaging software 9.

[0037] The contact electrode 13 of the chip bottom plate 5 in the chip body 1 receives the electrical signal sent by the drive device 7 by contacting the spring pin on the chip holder 6 in the microfluidic drive platform 2. The digital microfluidic drive device 7 is controlled by the computer through the control software 8, and then the droplets on the chip body 1 are controlled to move, mix, split and other operations. Droplet movement: by cutting off the power to the electrode where the droplet is located and energizing the adjacent electrode that the edge of the droplet touches, the droplet is moved, that is, moved to the adjacent electrode. Droplet mixing: by cutting off the power to the electrode where the droplet is located, and then energizing the adjacent electrode that the edge of the droplet touches, and then turning the power on and off the adjacent electrodes in a square route and circulating, the droplet mixing is achieved. Droplet splitting: by cutting off the power to the electrode where the droplet is located, and then energizing the electrodes on both sides of the droplet edge, the droplet splitting is achieved.

[0038] After the droplet reaction is completed, the fluorescence microscope 10 is controlled by the imaging software 9 to take pictures to obtain fluorescence images, which are then analyzed using Image J.

[0039] Example 2 A method for preparing the microfluidic chip of Example 1 comprises the following steps: (1) Prepare the chip body 1.

[0040] 1.1. Pre-treat the surface of the chip's lower plate 5. After pre-treating, form a surface dielectric layer using chemical vapor deposition, and then form a hydrophobic layer on the printed circuit board by spin coating and high-temperature curing at 170°C. Pre-treat the surface of the chip's upper plate 4 and prepare the surface hydrophobic layer.

[0041] 1.2, according to Figure 2 The chip bottom plate 5 is placed on the chip holder 6, so that the contact electrode 13 on the chip bottom plate 5 is aligned with the top pin on the chip holder 6. Polymethyl methacrylate (PMMA) gaskets are placed on both sides of the chip bottom plate 5. The chip top plate 4 is placed on the gaskets, with the conductive surface facing down (the entire conductive surface of the top plate is conductive. During use, the conductive surface of the top plate is protected by tape. The entire conductive surface is then spin-coated with a hydrophobic layer (the hydrophobic area is non-conductive). Finally, the tape is removed (the area protected by the tape is conductive and can be connected to the ground electrode). The chip bottom plate 5 is aligned to form a sandwich structure. By adjusting the number of tape layers on the gasket (each layer is approximately 50µm), the height between the upper and lower plates can be controlled. The chip body 1 is successfully assembled.

[0042] (2) The control software 8 is connected to the chip holder 6 via the digital microfluidic drive device 7, and the chip holder 6 controls the chip body 1 via the contact electrodes 13. Specifically including: 2.1. Inject silicone oil into the gap between the upper and lower plates of the chip through the filling hole 12 of the upper plate 4 of the chip to ensure smooth droplet driving and prevent droplet volatilization.

[0043] 2.2. The nucleic acid aptamer solution, quenching chain solution and screening buffer are loaded into the reservoir electrode 14 through the sample loading hole 11 respectively.

[0044] 2.3. The digital microfluidic drive device 7 is controlled by the supporting control software 8 to output the required electrical signal, which is transmitted to the contact electrode 13 of the chip via the spring pin. The contact electrode 13 controls the flow of liquid in the liquid reservoir electrode 14 and the liquid separation electrode 15 respectively. The control software 8 controls the power on and off of the electrodes in real time. For example, the real-time control mode can be used to control the power on and off of a small number of electrodes in real time for chip driveability testing. The preset program can be loaded through the step-by-step control mode to power on and off a series of electrodes for automatic control of droplets on the chip. After setting parameters such as the electrode drive voltage, frequency, and power-on time, the droplets can be driven.

[0045] (3) In the retention area, droplets were evenly split to generate quenching chain concentration gradient droplets with a gradient interval of 2 times; the quenching chain gradient droplets were mixed with nucleic acid aptamer droplets in the incubation area for 10 minutes; the droplets were then driven to the imaging area to collect fluorescence signals. The chip body 1 was placed on the stage of a fluorescence microscope to collect fluorescence images of the droplets. A 10× objective lens was selected to perform microscopic imaging of the droplets. The appropriate focal length was adjusted and kept fixed, and the exposure time was set. The grayscale value of the fluorescence image was extracted using Image J. Based on the difference in grayscale values ​​(the difference in grayscale value between the fluorescent droplets on the chip and the grayscale value of the buffer solution), a fluorescence quenching saturation curve was plotted.

[0046] Experiment 1: Optimize the effect of electrode driving voltage on droplet formation during the preparation process.

[0047] The distance between the upper and lower electrodes of the chip was set to 400µm. Droplets containing 2mg / mL surfactant (Pluronic L64) were added to the buffer solution. The driving voltage of the electrode was changed (50V, 80V, 110V, 130V, 140V, 150V), and photos were taken to observe the droplet generation effect.

[0048] Figure 5 Figure 2 shows the effect of different drive voltages on droplet actuation. As can be seen from the figure, when the drive voltage is 140V or greater, droplets can be rapidly split (split time is less than 5s). Therefore, the drive voltage was set to 140V for subsequent experiments.

[0049] Experiment 2: Examining the concentration of the surfactant Pluronic L64 in the droplets on the chip and the spacing between the upper and lower plates of the chip The concentration of the surfactant Pluronic L64 in the buffer was adjusted to 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL, respectively. The spacing between the upper and lower plates of the chip was adjusted to 220 µm, 265 µm, 310 µm, 355 µm, and 400 µm, respectively. The effects of these adjustments on the uniform splitting of droplets were investigated.

[0050] Figure 6 The effect of the concentration of the surfactant Pluronic L64 in the droplets on the chip and the spacing between the upper and lower plates on the droplet actuation performance is shown in the figure. As the spacing between the upper and lower plates increases, the surfactant concentration required for uniform droplet splitting increases. When the upper and lower plates are 400µm high, the surfactant concentration in the droplets must be at least 2.0mg / mL for droplet splitting to be successful. To facilitate droplet actuation and fluorescence signal acquisition, a 400µm spacing between the upper and lower plates and a 2mg / mL droplet surfactant concentration were selected for the above experiments.

[0051] Example 3 A method for measuring affinity using the microfluidic chip of Example 1 comprises the following steps: (1) Determination of the dissociation constant K between the aptamer and the quencher chain d1 . Specifically including: 1.1. Add 10 µL of 100 nM aptamer solution, 10 µL of buffer, and 10 µL of 3200 nM quencher strand to each reservoir electrode 14 of the microfluidic chip.

[0052] 1.2. The control software 8 controls the three liquid reservoir electrodes 14 to generate one droplet each, and drives the small droplets of buffer solution and the small droplets of quenching chain to merge into large droplets, and mix them evenly to react.

[0053] 1.3. Split the large droplet after the reaction into two small droplets. Drive one droplet to the reaction zone. The other droplet merges with a new buffer droplet. Repeat the above steps to obtain quenching chain droplets with a gradient concentration (0nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, 400nM, and 800nM).

[0054] 1.4. The quencher chain droplets of different concentrations in the reaction zone were fused with the aptamer droplets and reacted at room temperature for 10 minutes. The droplets were then driven to the imaging zone and the fluorescence signal was read using a fluorescence microscope. The grayscale value of the fluorescence image was analyzed using Image J, and the fluorescence quenching saturation curve of the hybridization of the aptamer and the quencher chain was plotted to determine the dissociation constant K at the hybridization equilibrium of the two chains. d1 Dissociation constant K d1 The calculation formula of is shown in Formula I.

[0055] (Formula I) (2) Dissociation constant K between the target and the aptamer-quencher hybrid complex d2 The determination of . Specifically includes the following steps: 2.1. Add 10 µL of 100 nM aptamer-quencher complex solution, 10 µL of buffer, and 10 µL of 200 µM maytansine to each reservoir electrode 14 of the microfluidic chip.

[0056] 2.2. The control software 8 controls the three liquid reservoir electrodes 14 to generate one droplet each, and drives the small droplets of buffer solution and maytansine to merge into large droplets, and mix them evenly to react.

[0057] 2.3. Split the reacted large droplet into two small droplets, drive one droplet to the reaction zone, and merge the other droplet with a new buffer droplet. Repeat the above operation to obtain small droplets of maytansine with a gradient concentration (0µM, 1.56µM, 3.125µM, 6.25µM, 12.5µM, 25µM, 50µM and 100µM).

[0058] 2.4. The reaction zone was fused with maytansine droplets of varying concentrations and aptamer-quencher complex droplets. The reaction was allowed to proceed for 10 minutes at room temperature before being driven to the imaging zone. Fluorescence signals were read using a fluorescence microscope. The grayscale values ​​of the fluorescence images were analyzed using Image J, and the fluorescence recovery saturation curve of the target and aptamer-quencher complex was plotted to determine the dissociation constant K at hybridization equilibrium. d2 Dissociation constant K d2 The calculation formula of is shown in Formula II.

[0059] (Formula II) (3) According to K d =K d1 / K d2 Calculate the equilibrium constant of aptamer-target binding.

[0060] (4) After the chip is used, remove the upper and lower plates from the chip holder 6 in turn, rinse the upper and lower plates with ultrapure water and ethanol, blow dry with nitrogen, and store them in a chip box.

[0061] Figure 7 This is the principle of affinity determination of maytansine nucleic acid aptamer based on microfluidic chip. d1 The determination process of K d2 The determination process of the equilibrium constant is shown in Figure C. As can be seen from the figure, the present invention pairs the aptamer labeled with a fluorescent group with the quencher chain base complementarily. At this time, the fluorescence is in a quenched state. By plotting the fluorescence quenching saturation curve of the hybridization of the aptamer and the quencher chain, the dissociation constant K when the hybridization reaches equilibrium is determined. d1 Then, the target is added under the optimal quenching ratio. Due to the binding of the target and the aptamer, the conformation of the aptamer is changed, and the quencher chain dissociates, allowing the fluorescence to recover. The fluorescence recovery saturation curve of the competition between the target and the aptamer-quencher chain complex is drawn to determine the dissociation constant K when the target induces the dissociation of the aptamer-quencher chain complex to reach equilibrium. d2 . K d1 With K d2 By making the ratio, we can get the equilibrium dissociation constant K of the binding between the aptamer and the target. d .

[0062] The nucleic acid aptamer was obtained by SELEX technology and labeled with a FAM fluorescent group. The quencher chain was labeled with a BHQ-1 quencher group. The screening buffer was PBS buffer (10 mM, pH 7.4, used for DNA sample dilution and incubation). The target solution was diluted and incubated using a screening buffer containing 2.5% DMSO.

[0063] Comparative Example 1 The affinity of nucleic acid aptamers was determined by traditional manual addition combined with a microplate reader. The specific detection method steps are as follows: (1) Dissociation constant K between aptamer and quencher chain d1 Calculation of the final concentration of 50 nM nucleic acid aptamer was hybridized and incubated with a series of different concentrations (0nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, 400nM and 800nM) of quencher chain at 25°C for 1 hour. Afterwards, the sample was added to a multi-well plate, and the fluorescence signal of the solution was obtained using a multifunctional microplate reader. The fluorescence quenching saturation curve of the hybridization of the nucleic acid aptamer and the quencher chain was drawn to determine the dissociation constant K when the complementary hybridization of the two reached equilibrium. d1 .

[0064] (2) Dissociation constant K between the target compound maytansine and the nucleic acid aptamer-quencher chain hybrid complex d2 According to the fluorescence quenching curve obtained in the previous step, the optimal quenching ratio can be determined. d2 The determination is to add different concentrations of target (0µM, 1.56µM, 3.125µM, 6.25µM, 12.5µM, 25µM, 50µM and 100µM) to the complex under the optimal quenching ratio conditions and incubate at 25°C for 1 hour. Afterwards, the sample is added to a multi-well plate and the fluorescence signal is measured by a multifunctional microplate reader. The fluorescence recovery curve of the binding of maytansine to the nucleic acid aptamer-quencher chain complex is drawn, and the dissociation constant K when the nucleic acid aptamer-quencher chain complex reaches equilibrium induced by maytansine is calculated. d2 .

[0065] (3) Equilibrium dissociation constant K d The acquisition is based on K d1 With K d2 The ratio is calculated, which reflects the binding affinity between the nucleic acid aptamer and maytansine.

[0066] The method of this embodiment can be used not only to detect the affinity of nucleic acid aptamers that recognize maytansine, but also to detect other nucleic acid aptamers, which can be achieved by replacing the corresponding target solution.

[0067] Experiment 3: The affinity of the existing maytansine nucleic acid aptamer was determined according to the method of Example 3.

[0068] (1) Select 10 existing maytansine nucleic acid aptamers: The DNA sequences used in the examples are Seq1-X, Seq2-X, Seq4-X, Seq5-X, Seq6-X, Seq7-X, Seq8-X, Seq9-X, Seq10-X and Seq11-X.

[0069] The DNA sequences of these 10 nucleic acid aptamers are listed in Table 1.

[0070] Table 1: DNA sequences of 10 nucleic acid aptamers.

[0071]

[0072] (2) The affinity of the nucleic acid aptamers was determined according to the method of Example 3.

[0073] The affinity of the 10 nucleic acid aptamers was measured according to the method of Example 3. The affinity measurement results are listed in Table 2.

[0074] Table 2: Aptamer affinity determination results corresponding to different methods.

[0075]

[0076] The affinity determination method based on the microfluidic chip in Example 3 of the present invention integrates this process to a large extent. The user only needs to add a small amount of sample, and then through the automated control of the platform, the test results can be quickly obtained. This automated determination method shortens the single determination time from 105min to 45min, which is only about half the time of the manual addition combined with the enzyme reader method, significantly improving the efficiency of the determination. As can be seen from Table 2: There is a slight difference between the determination results and the results of the manual combined with the enzyme reader determination, but the affinity K d The values ​​are all in the same order of magnitude (see the comparison between columns 2 and 5 in Table 2). The results show that this microfluidic platform can be used to determine the affinity of nucleic acid aptamers.

[0077] (3) The manual addition of samples combined with the consumption of enzyme-labeled reagents in Example 3 and Comparative Example 1 were investigated respectively. The results of the manual addition of samples combined with the consumption of enzyme-labeled reagents in Comparative Example 1 are listed in Table 3, and the results of the reagent consumption in Example 3 are listed in Table 4.

[0078] Table 3: Manual addition combined with enzyme reader reagent consumption in Comparative Example 1.

[0079]

[0080] Note: The above results are for one K measurement. d2 The reagent consumption required.

[0081] Table 4: Reagent consumption in the microfluidic chip in Example 3.

[0082]

[0083] Note: The minimum volume of droplets that can be driven on the chip body 1 used is 1.92 µL.

[0084] From the results in Table 3 and Table 4, we can see that in the comparative example, there are also great differences in reagent consumption between the two nucleic acid aptamer affinity determination methods. Here, the K d2 The determination of aptamer-quencher complexes is described in detail in Table 3. Microplate reader analysis requires incubation of eight 80µL aptamer-quencher complex solutions with eight 80µL maytansine solutions (at concentrations of 0µM, 1.56µM, 3.13µM, 6.25µM, 12.5µM, 25µM, 50µM, and 100µM, respectively, with manual dilutions). The fluorescence of the mixed solutions is then measured using a microplate reader, as shown in Table 3. The total sample consumption is 1280µL. The digital microfluidics platform also requires incubation of eight 1.92µL aptamer-quencher complex solutions with eight 1.92µL maytansine solutions (at concentrations of 0µM, 1.56µM, 3.13µM, 6.25µM, 12.5µM, 25µM, 50µM, and 100µM, with automated on-chip droplet dilution). The fluorescence of the mixed solutions is then measured using a fluorescence microscope. As shown in Table 4, the total sample consumption for the digital microfluidics platform is 30.72µL. By comparison, the digital microfluidics platform consumes only 1 / 42 of the sample consumption for microplate reader analysis, significantly reducing reagent consumption.

[0085] Example 4 The method of Example 3 was used to screen nucleic acid aptamers that recognize maytansine, and nucleic acid aptamers with high affinity and high specificity for maytansine were screened from a large number of nucleic acid aptamers.

[0086] The screened nucleic acid aptamers are listed in Table 5.

[0087] Table 5: DNA sequences of candidate nucleic acid aptamers.

[0088]

[0089] The affinity of the 7 candidate nucleic acid aptamers in Table 5 was measured, and the results are listed in Table 6.

[0090] Table 6: Affinity determination results of 7 candidate nucleic acid aptamers.

[0091]

[0092]

[0093] From the results in Table 6, we can see that the equilibrium dissociation constant (K) of the nucleic acid aptamers screened according to the method of the present invention is d ) are in the nanomolar range.

[0094] Example 5 A nucleic acid aptamer for maytansine: Seq19-X. The nucleotide sequence of Seq19-X is shown in SEQ ID NO. 1, and is as follows: 5'-CTCTCGGGACGACAAATTGTGTGTAGAGGGAGTGGGTCATTTTGTCGTCCC-3'.

[0095] Figure 8 Schematic diagram of the structure obtained by performing secondary structure simulation of nucleic acid aptamer seq15-X and seq19-X sequences using the Mfold service.

[0096] Furthermore, one or both ends of the DNA strand of Seq19-X can be labeled with radioactivity, fluorescence, enzymes, digoxigenin, or biotin, all of which can achieve the same or similar technical effects as in Example 1. In this example, the fluorescent substance is a FAM fluorescent group. Seq19-X is labeled with FAM at the 5' end, resulting in the nucleic acid aptamer FAM-Seq19-X. However, other fluorescent groups, such as the Cy5 fluorescent group, can also produce the same technical effects.

[0097] In the present invention, under the premise that the overall structure of the nucleic acid aptamer remains unchanged, a certain position on the nucleotide sequence of the nucleic acid aptamer Seq19-X is phosphorylated, methylated, aminoated, sulfhydrylated or isotopized.

[0098] The above-mentioned nucleic acid aptamer, further, the amination is to label the 5' end or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq19-X with an amino group -NH2; the thiolation is to label the 5' end or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq19-X with a thiol group -SH.

[0099] In the present invention, the DNA sequence of the nucleic acid aptamer is a nucleic acid aptamer of any one of the following four sequences: (1) The homology with the nucleotide sequence of the nucleic acid aptamer Seq19-X is greater than 60%; (2) a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer Seq19-X; (3) an RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer Seq19-X; (4) A derivative of Seq19-X, wherein the derivative is a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer Seq19-X, or a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer.

[0100] The aforementioned aptamers can bind to maytansine with high affinity and specificity, with an equilibrium dissociation constant in the nanomolar range. Compared to protein antibodies, aptamers are less immunogenic, can be synthesized in batches in vitro, have a low molecular weight, can be modified and substituted at different locations, and are chemically stable, easy to store, and convenient for labeling. Aptamers are also highly water-soluble. When combined with the fat-soluble drug maytansine, they can increase the drug's solubility and avoid the toxic side effects caused by the addition of organic solvents (such as DMSO) or surfactants (such as Tween). The specific interaction between aptamers and maytansine can improve the stability of maytansine, enhance its cytotoxicity, and thus broaden its therapeutic window. Using the aptamers of the present invention as drug delivery vehicles for maytansine can improve the limitations of maytansine's clinical application (increasing solubility, improving stability, and broadening the therapeutic window), which is of great significance for the design and development of maytansine drugs in clinical applications. At the same time, corresponding labeling, modification or substitution of the nucleic acid aptamer of the present invention is beneficial to the tracing of maytansine in the body, helps to reveal the mechanism of drug absorption, distribution, metabolism and excretion, and promotes the effectiveness and safety of drug treatment.

[0101] The above description is only a preferred embodiment of the present invention and does not constitute any formal limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for measuring affinity using a microfluidic chip, characterized in that: The microfluidic chip comprises a chip body (1), a digital microfluidic driving platform (2), and a fluorescence detection module (3); the chip body (1) and the digital microfluidic driving platform (2) are connected in series via a wire; the chip body (1) is provided with a contact electrode (13) connected to the digital microfluidic driving platform (2), a liquid reservoir electrode (14) for storing liquid reagents, a driving electrode (16) for driving the movement, mixing and splitting of droplets, and a liquid separation electrode (15) for connecting the liquid reservoir electrode (14) and the driving electrode (16); the liquid reservoir electrode (14), the liquid separation electrode (15), and the driving electrode (16) are connected to the contact electrode (13) via electrode leads; The method comprises: S1. By manipulating the digital microfluidic driving platform (2), the buffer solution and the quenching chain in the liquid reservoir electrode (14) are mixed in the chip body (1) to obtain a gradient concentration quenching chain solution; the digital microfluidic driving platform (2) manipulates the gradient concentration quenching chain solution and the nucleic acid aptamer solution to react in the chip body (1) to obtain a nucleic acid aptamer-quenching chain complex solution, and the fluorescence detection module (3) obtains an electronic signal by photographing the chip body (1), and draws a fluorescence quenching saturation curve of the hybridization of the nucleic acid aptamer and the quenching chain, thereby determining the dissociation constant K when the nucleic acid aptamer and the quenching chain are in equilibrium. d1 ; S2. The digital microfluidic driving platform (2) is manipulated to mix the buffer solution in the reservoir electrode (14) and the target in the chip body (1) to obtain a target solution with a gradient concentration; the digital microfluidic driving platform (2) manipulates the target solution with a gradient concentration to mix with the nucleic acid aptamer-quencher chain complex solution in the chip body (1) to react, and the fluorescence detection module (3) obtains an electronic signal by photographing the chip body (1), and draws a fluorescence recovery saturation curve of the target and the nucleic acid aptamer-quencher chain complex, thereby determining the dissociation constant K when the target and the nucleic acid aptamer-quencher chain complex are in hybridization equilibrium. d2 ; S3, according to K d =K d1 / K d2 Calculate the equilibrium constant for the binding of aptamer to target.

2. The method according to claim 1, characterized in that The microfluidic chip uses a driving voltage as a driving force to drive the movement, mixing and splitting of the liquid in the chip body (1), and the driving voltage is 140V to 150V.

3. The method according to claim 1, wherein 2 mg / mL to 4 mg / mL of Pluronic L64 was added to the buffer.

4. A microfluidic chip according to any one of claims 1 to 3, characterized in that: The microfluidic chip comprises a chip body (1), a digital microfluidic driving platform (2), and a fluorescence detection module (3); the chip body (1) and the digital microfluidic driving platform (2) are connected in series via a wire; The chip body (1) is provided with a contact electrode (13) connected to the digital microfluidic driving platform (2), a liquid reservoir electrode (14) for storing liquid reagents, a driving electrode (16) for driving the movement, mixing and splitting of droplets, and a liquid separation electrode (15) for connecting the liquid reservoir electrode (14) and the driving electrode (16); the liquid reservoir electrode (14), the liquid separation electrode (15) and the driving electrode (16) are connected to the contact electrode (13) through electrode leads.

5. The microfluidic chip according to claim 4, characterized in that: The chip body (1) is assembled from a chip upper plate (4), a chip lower plate (5) and a polymethyl methacrylate gasket to form a sandwich structure; The chip lower electrode plate (5) is a printed circuit board, on which a contact electrode (13), a liquid reservoir electrode (14), a liquid separation electrode (15) and a driving electrode (16) are provided; The chip upper plate (4) is made of indium tin oxide glass and is provided with a sample addition hole (11) and an oil addition hole (12); The gap between the chip upper plate (4) and the chip lower plate (5) is 400µm.

6. The microfluidic chip according to claim 5, wherein The chip bottom plate (5) is coated with Parylene C and Teflon AF in sequence.

7. The microfluidic chip according to claim 4, characterized in that: The microfluidic driving platform (2) comprises a chip holder (6), a digital microfluidic driving device (7) and control software (8). By controlling the control software (8), the digital microfluidic driving device (7) drives the chip body (1) connected to the chip holder (6) through droplets.

8. The microfluidic chip according to claim 4, characterized in that: The fluorescence detection module (3) includes a fluorescence microscope (10) and imaging software (9), and the fluorescence microscope (10) is connected to the imaging software (9) via a wire.

9. A nucleic acid aptamer screened by the method of claim 1, characterized in that: The nucleic acid aptamer is Seq19-X that recognizes maytansine, and the nucleotide sequence of Seq19-X is shown in SEQ ID NO.

1.

10. The nucleic acid aptamer according to claim 9, characterized in that One or both ends of the DNA chain of Seq19-X are radioactively labeled, fluorescently labeled, enzyme-labeled, digoxigenin-labeled, or biotin-labeled; and / or, a certain position on the nucleotide sequence of Seq19-X is phosphorylated, methylated, amino-labeled, sulfhydryl-labeled, or isotopically labeled.

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