Method for determining affinity using microfluidic chip, microfluidic chip and aptamer

Abstract

The application discloses a kind of microfluidic chip determination affinity method and microfluidic chip and nucleic acid aptamer, method includes: gradient concentration's quenching chain solution and nucleic acid aptamer solution reaction, determine the dissociation constant when two chain hybridization equilibrium;Gradient concentration's maydanshin solution and nucleic acid aptamer-quenching chain complex solution are reacted, determine the dissociation constant when maydanshin and nucleic acid aptamer-quenching chain complex hybridization equilibrium;Calculate the equilibrium constant of nucleic acid aptamer and maydanshin binding.The microfluidic chip includes chip ontology, digital microfluidic drive platform, fluorescence detection module.The application determines affinity by microfluidic chip, significantly improves nucleic acid aptamer affinity determination efficiency, with the advantages of low consumption, time-saving and automation.The nucleic acid aptamer screened by the above affinity screening method has high specificity and high affinity binding with maydanshin, is easy to synthesize, low in cost and stable in property.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and microfluidics, and particularly relates to a method for determining affinity using a microfluidic chip, as well as the microfluidic chip and nucleic acid aptamers. Background Technology

[0002] Bioinformatics analysis (phylogenetic trees, homology comparison, molecular docking, etc.) can narrow down 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 rejection of high-affinity nucleic acid sequences. Furthermore, commonly used methods for determining the affinity of nucleic acid aptamers, including isothermal titration calorimetry, flow cytometry, surface plasmon resonance, and chain displacement fluorescence, also suffer from slow testing speeds and high reagent consumption. Therefore, developing high-throughput, automated assay platforms is crucial. Digital microfluidics technology can precisely manipulate microdroplets, offering advantages such as small size, high throughput, and low reagent consumption. Through programmed control, it can achieve automated continuous operation, 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 due to problems such as poor water solubility, low chemical stability, and a narrow therapeutic window. Nucleic acid aptamers, as a class of nucleic acid chains that can specifically recognize target molecules, have many advantages such as stability, small molecular weight, small batch-to-batch variability, and ease of synthesis and modification, and have been widely used in drug delivery. Therefore, using nucleic acid aptamers as carriers to solve the problems related to maytansine may be a feasible solution. However, obtaining nucleic acid aptamers often requires multiple rounds of optimization and iteration, including library design, high-affinity library enrichment, sequencing, and affinity determination of candidate sequences, which are cumbersome and costly. Affinity determination of a large number of candidate sequences is particularly costly in terms of manpower, resources, and time. There is a need to find a method that can rapidly and efficiently screen and identify maytansine nucleic acid aptamers. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a method for determining affinity using a microfluidic chip, as well as the microfluidic chip and nucleic acid aptamers. This method transforms the manual processes of reagent dilution, mixing, and transfer into processes involving the movement, mixing, and splitting of reagent droplets on the microfluidic chip. Compared to repetitive, cumbersome, and manual methods, microfluidic chips significantly improve the efficiency of nucleic acid aptamer affinity determination, reduce sample consumption, and offer advantages such as low cost, time-saving, and automation. Nucleic acid aptamers screened using the above affinity screening method exhibit high specificity and high affinity binding to maytansine, are easy to synthesize, low in cost, and stable in properties.

[0005] To address the aforementioned technical problems, this invention provides a method for determining affinity using a microfluidic chip. 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 wires. 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 separating electrode 15 for connecting the reservoir electrode 14 and the driving electrode 16. The reservoir electrode 14, the separating electrode 15, and the driving electrode 16 are connected to the contact electrode 13 via electrode leads. The method includes: S1. By manipulating the digital microfluidic driving platform 2, the buffer solution and quenching chain in the reservoir electrode 14 are mixed in the chip body 1 to obtain a quenching chain solution of gradient concentration; the digital microfluidic driving platform 2 manipulates the quenching chain solution of gradient concentration to react with the nucleic acid aptamer solution in the chip body 1 to obtain a nucleic acid aptamer-quenching chain complex solution; the fluorescence detection module 3 obtains the electronic signal by photographing the chip body 1, plots the fluorescence quenching saturation curve of the hybridization of nucleic acid aptamer and quenching chain, and thus determines the dissociation constant K at the hybridization equilibrium of nucleic acid aptamer and quenching chain. d1 ; S2. By manipulating the digital microfluidic driving platform 2, the buffer solution and the target substance in the reservoir electrode 14 are mixed in the chip body 1 to obtain a target substance solution of gradient concentration; the digital microfluidic driving platform 2 manipulates the target substance solution of gradient concentration to mix with the nucleic acid aptamer-quencher complex solution in the chip body 1 to carry out the reaction; the fluorescence detection module 3 obtains the electronic signal by photographing the chip body 1, plots the fluorescence recovery saturation curve of the target substance and the nucleic acid aptamer-quencher complex, and thus determines the dissociation constant K at the hybridization equilibrium of the target substance and the nucleic acid aptamer-quencher complex. d2 ; S3, according to Kd =K d1 / K d2 Calculate the equilibrium constant for the binding of the nucleic acid aptamer to the target analyte.

[0006] Furthermore, in the above method, the microfluidic chip drives the liquid in the chip body 1 to move, mix, and split using a driving voltage of 140V to 150V.

[0007] Further, in the above method, 2 mg / mL to 4 mg / mL of Pluronic L64 is added 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 by wires. 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] Furthermore, in the aforementioned microfluidic chip, the chip body 1 is assembled from an upper chip electrode 4, a lower chip electrode 5, and a polymethyl methacrylate gasket to form a sandwich structure. The lower electrode plate 5 of the chip is a printed circuit board, on which a contact electrode 13, a liquid storage 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 loading hole 11 and an oil loading hole 12. The gap between the upper electrode plate 4 and the lower electrode plate 5 of the chip is 400µm.

[0010] Furthermore, in the aforementioned microfluidic chip, the lower electrode plate 5 of the chip is sequentially coated with Parylene C and Teflon AF.

[0011] Furthermore, the microfluidic chip described above includes a microfluidic driving platform 2 comprising 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.

[0012] Furthermore, in the aforementioned microfluidic chip, the fluorescence detection module 3 includes a fluorescence microscope 10 and imaging software 9, wherein the fluorescence microscope 10 is connected to the imaging software 9 via wires.

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

[0014] Furthermore, in the aforementioned nucleic acid aptamer, one or both ends of the Seq19-X DNA strand are radiolabeled, fluorescently labeled, enzyme-labeled, digoxigenin-labeled, or biotin-labeled; and / or, a certain position on the nucleotide sequence of the Seq19-X is phosphorylated, methylated, aminoized, thiolated, or isotopized.

[0015] Based on a general technical concept, this invention provides the application of the above-mentioned nucleic acid aptamer in the preparation of an antitumor drug. The antitumor drug is maytansine and Seq19-X. Seq19-X is the drug carrier.

[0016] Compared with the prior art, the advantages of the present invention are as follows: (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, transforming the manual processes of reagent dilution, mixing, and transfer into processes such as the movement, mixing, and splitting of reagent droplets on the chip body 1. Compared with repetitive, cumbersome, and manual 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 error, while avoiding the misscreening and rejection of high-affinity nucleic acid sequences, providing a more efficient and reliable solution for the research and development of nucleic acid aptamers.

[0017] (2) This invention provides a microfluidic chip for the above-mentioned method, which controls the movement, mixing, and splitting of discrete droplets based on the principle of electrowetting. That is, it uses the effect of an electric field (the switching on and off of the electrodes) to change the contact angle of the droplets, causing the droplets to deform and spread on the chip surface, thus enabling more flexible manipulation of the fluid. Compared with commonly used microfluidic chips based on square driving electrodes 16, the driving electrodes 16 of this chip are cross-shaped, which is beneficial for the droplet edges to contact adjacent electrodes and facilitates droplet driving. The electrode area of ​​this microfluidic chip is functionally divided into a retention area, an incubation area, and an imaging area, which can match the characteristics of the strand displacement fluorescence method to realize the above-mentioned determination of nucleic acid aptamer affinity.

[0018] (3) This invention provides a nucleic acid aptamer for recognizing maytansine, screened using the above method. This nucleic acid aptamer can bind to the target drug maytansine with high affinity and high specificity, and the equilibrium dissociation constant is in the nanomolar range. Compared with protein antibodies, this nucleic acid aptamer has better affinity and specificity, lower immunogenicity, can be chemically synthesized in vitro, has a small molecular weight, can be modified and substituted at different sites, and is chemically stable, easy to store, and easy to label. Utilizing the specific interaction between this 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, thereby improving the limitations of maytansine's clinical application (improving solubility, improving stability, and broadening the therapeutic window). Appropriate labeling, modification, or substitution of this nucleic acid aptamer is beneficial for the in vivo tracking of maytansine, promoting the effectiveness and safety of drug treatment.

[0019] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the accompanying drawings and tables. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0021] Figure 1 This is a flowchart of the microfluidic chip control process in Embodiment 1 of the present invention.

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

[0023] Figure 3 This is a dimensional diagram (unit: mm) of the upper electrode plate 4 of the chip in Embodiment 1 of the present invention.

[0024] Figure 4 This is a dimensional diagram (unit: mm) of the lower electrode plate 5 of the chip in Embodiment 1 of the present invention.

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

[0026] Figure 6 This study examines the influence of the distance between the upper and lower electrode plates of the chip and the surfactant concentration on the droplet splitting ability in Embodiment 2 of the present invention.

[0027] Figure 7 This is a schematic diagram of the determination of nucleic acid aptamer affinity using a digital microfluidic platform in Embodiment 3 of the present invention. In the diagram, A represents the determination process for Kd1; B represents the determination process for Kd2; and C represents the principle of equilibrium constant detection.

[0028] Figure 8 This is a schematic diagram of the structure obtained by using the Mfold service to simulate the secondary structure of nucleic acid aptamer seq15-X and seq19-X sequences in Embodiment 5 of the present invention. Detailed Implementation

[0029] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0030] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

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

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

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

[0034] See also Figure 4The lower electrode 5 of the chip is a printed circuit board with a Parylene C dielectric layer and a Teflon AF hydrophobic layer. Its fabrication process involves first creating the dielectric layer using chemical vapor deposition, followed by spin coating and high-temperature curing at 170°C to create the hydrophobic layer on the printed circuit board. It has 48 square contact electrodes 13 with a side length of 1.5 mm; 8 rectangular recessed liquid reservoir electrodes 14 (5.5 mm × 4.8 mm) for storing small amounts of liquid reagents; 16 cross-shaped dispensing electrodes 15 (2.2 mm × 1.83 mm) for connecting the liquid reservoir electrodes 14 and the driving electrodes 16, assisting the liquid reservoir electrodes 14 in splitting small droplets. There are 24 cross-shaped driving electrodes 16 (2.2 mm × 2.2 mm) for droplet movement, mixing, and splitting. 48 electrode leads with a line width of 100 μm are used to connect the contact electrodes 13 to the liquid reservoir electrodes 14, dispensing electrodes 15, and driving electrodes 16, respectively. The edge of the droplet on the cross-shaped drive electrode 16 contacts the adjacent electrode, which helps the droplet move continuously in the chip.

[0035] The microfluidic drive platform 2 (purchased from Xiamen Deyun Core Technology Co., Ltd.) includes a chip holder 6, a digital microfluidic drive device 7, and control software 8. The control software 8 is manually operated to enable the digital microfluidic drive 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 signals from the chip body 1 through the imaging software 9.

[0037] The contact electrode 13 of the lower electrode plate 5 in the chip body 1 receives electrical signals from the driving device 7 by contacting the spring pin on the chip support 6 in the microfluidic drive platform 2. The computer controls the digital microfluidic drive device 7 via control software 8, thereby controlling the droplets on the chip body 1 to perform operations such as movement, mixing, and splitting. Specifically: Droplet movement is achieved by de-energizing the electrode where the droplet is located and energizing the adjacent electrodes that the droplet's edge contacts; the droplet moves to the adjacent electrode. Droplet mixing is achieved by de-energizing the electrode where the droplet is located, then energizing the adjacent electrodes that the droplet's edge contacts, and then sequentially switching the energizer on and off along a square path in a cycle. Droplet splitting is achieved by de-energizing the electrode where the droplet is located and then energizing the electrodes on both sides of the droplet's edge.

[0038] After the droplet reaction is complete, the fluorescence microscope 10 is used to take pictures by the imaging software 9 to obtain fluorescence images, which are then analyzed using ImageJ.

[0039] Example 2 A method for fabricating a microfluidic chip according to Embodiment 1 includes the following steps: (1) Preparation of chip body 1.

[0040] 1.1. Surface pretreatment is performed on the lower electrode plate 5 of the chip; after pretreatment, the surface dielectric layer is fabricated by chemical vapor deposition, and then a hydrophobic layer is fabricated on the printed circuit board by spin coating and high temperature curing at 170℃. Surface pretreatment and preparation of the surface hydrophobic layer are performed on the upper electrode plate 4 of the chip.

[0041] 1.2, according to Figure 2 The method involves placing the lower electrode 5 of the chip on the chip support 6, aligning the contact electrode 13 on the lower electrode 5 with the top electrode on the chip support 6, placing polymethyl methacrylate (PMMA) pads on both sides of the lower electrode 5, and placing the upper electrode 4 on top of the pads with the conductive surface of the upper electrode 4 facing down (the entire conductive surface of the upper electrode 4 is conductive; during use, the edges of the conductive surface of the upper electrode 4 are protected with tape, then a hydrophobic layer is spin-coated onto the entire conductive surface (the area with the hydrophobic layer is non-conductive), and finally the tape is removed (the area protected by the tape is conductive; this part can be connected to the ground electrode), and aligned with the lower electrode 5 to form a sandwich structure. The height between the upper and lower electrodes can be controlled by adjusting the number of tape layers on the pads (each layer is approximately 50µm). 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 electrode 13. Specifically, it includes: 2.1 Inject silicone oil into the gap between the upper and lower plates of the chip through the oil filling hole 12 on the upper plate 4 of the chip to make the droplet drive smooth and prevent the droplet from evaporating.

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

[0044] 2.3 The accompanying control software 8 controls the digital microfluidic drive device 7 to output the required electrical signals, which are transmitted to the contact electrodes 13 of the chip via spring pins. The contact electrodes 13 control the flow of liquid in the reservoir electrode 14 and the dispensing electrode 15, respectively. The control software 8 controls the on / off state of the electrodes in real time. For example, in real-time control mode, it can control the on / off state of a small number of electrodes for chip driveability testing; in step-by-step control mode, it can load a preset program to control the on / off state of a series of electrodes for automated droplet control on the chip. By setting parameters such as electrode driving voltage, frequency, and energizing time, the droplets can be driven.

[0045] (3) In the retention area, droplets are generated by equal splitting to form quenched chain concentration gradient droplets with a gradient interval of 2. The quenched chain gradient droplets and nucleic acid aptamer droplets are mixed and reacted in the incubation area for 10 min. Then, the droplets are driven to the imaging area to collect fluorescence signals. The chip body 1 is placed on the stage of a fluorescence microscope, and fluorescence images of the droplets are collected. A 10× objective lens is selected to perform microscopic imaging of the droplets. The appropriate focal length is adjusted and kept fixed, and the exposure time is set. The gray values ​​of the fluorescence images are extracted using ImageJ. Based on the difference in gray values ​​(the difference between the gray values ​​of the fluorescent droplets on the chip and the gray values ​​of the buffer), a fluorescence quenching saturation curve is plotted.

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

[0047] The distance between the upper and lower plates 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 electrodes was changed (50V, 80V, 110V, 130V, 140V, 150V), and the droplet generation effect was observed by taking pictures.

[0048] Figure 5 The effect of different driving voltages on droplet actuation is shown in the figure. It can be seen from the figure that when the driving voltage is greater than or equal to 140V, the droplets can be rapidly split (split time less than 5s). Therefore, the driving voltage was set to 140V for subsequent experiments.

[0049] Experiment 2: Investigating the concentration of the surfactant Pluronic L64 in the droplets on the chip and the distance between the upper and lower electrodes of the chip. The concentrations of the surfactant Pluronic L64 in the buffer solution were adjusted to 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL; the distances between the upper and lower electrodes of the chip were adjusted to 220 µm, 265 µm, 310 µm, 355 µm, and 400 µm; and their effects on the uniform splitting of droplets were investigated.

[0050] Figure 6 This paper illustrates the effect of the concentration of the surfactant Pluronic L64 in the droplets on the chip and the distance between the upper and lower electrodes on the droplet actuation effect. The figure shows that as the distance between the upper and lower electrodes gradually increases, the concentration of surfactant required for uniform droplet splitting also increases. When the height of the upper and lower electrodes is 400 µm, the concentration of surfactant in the droplet must be greater than or equal to 2.0 mg / mL for the droplet to split normally. To facilitate droplet actuation and fluorescence signal acquisition, a chip upper and lower electrode distance of 400 µm and a droplet surfactant concentration of 2 mg / mL were selected for the above experiments.

[0051] Example 3 A method for determining affinity using a microfluidic chip as described in Example 1 includes the following steps: (1) Determine the dissociation constant K between the nucleic acid aptamer and the quenched strand. d1 Specifically, this includes: 1.1 Add 10µL of 100nM nucleic acid aptamer solution, 10µL of buffer solution, and 10µL of 3200nM quencher to each reservoir electrode 14 of the microfluidic chip.

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

[0053] 1.3 After the reaction, the large droplet is split into two smaller droplets, one of which is driven into the reaction zone. The other droplet fuses with a new buffer droplet. Repeating the above operation yields quenched chain droplets with gradient concentrations (0 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM).

[0054] 1.4. Quenching chain droplets of different concentrations were fused with nucleic acid aptamer droplets in the reaction zone and reacted at room temperature for 10 min. The mixture was then driven to the imaging zone, and the fluorescence signal was read using a fluorescence microscope. The grayscale values ​​of the fluorescence images were analyzed using ImageJ, and fluorescence quenching saturation curves of the hybridization of the nucleic acid aptamer and the quenching chain were plotted to determine the dissociation constant K at hybridization equilibrium between the two chains. d1 Dissociation constant K d1 The calculation formula is shown in Equation I.

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

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

[0057] 2.3. After the reaction, the large droplet is split into two smaller droplets. One droplet is driven to the reaction zone, and the other droplet is fused with a new buffer droplet. Repeating the above operation will yield maytansine droplets with gradient concentrations (0µM, 1.56µM, 3.125µM, 6.25µM, 12.5µM, 25µM, 50µM and 100µM).

[0058] 2.4. Small droplets of maytansine at different concentrations were fused with small droplets of the nucleic acid aptamer-quencher complex in the reaction zone. The mixture was reacted at room temperature for 10 min, then driven to the imaging zone, and the fluorescence signal was read using a fluorescence microscope. The grayscale values ​​of the fluorescence images were analyzed using ImageJ, and fluorescence recovery saturation curves of the target binding to the nucleic acid aptamer-quencher complex were plotted to determine the dissociation constant K at hybridization equilibrium. d2 Dissociation constant K d2 The calculation formula is shown in Formula II.

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

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

[0061] Figure 7 This describes the principle behind the microfluidic chip-based assay for the affinity determination of maytansin aptamers. In the figure, A represents K. d1 The measurement process; B in the diagram represents K. d2 The measurement process is as follows; C in the figure represents the principle of equilibrium constant detection. As can be seen from the figure: In this invention, the aptamer labeled with a fluorescent group is complementary to the base of the quenching chain. At this time, the fluorescence is in a quenched state. By plotting the fluorescence quenching saturation curve of the hybridization between the aptamer and the quenching chain, the dissociation constant K at which the hybridization reaches equilibrium is determined. d1 Then, the target compound was added under optimal quenching ratio conditions. Due to the binding of the target compound to the aptamer, the aptamer conformation changed, leading to the dissociation of the quenched chain and the recovery of fluorescence. Fluorescence recovery saturation curves were plotted for the competition between the target compound and the nucleic acid aptamer-quenched chain complex, and the dissociation constant K at which the target compound induces the dissociation of the nucleic acid aptamer-quenched chain complex reached equilibrium was determined. d2 K d1 With K d2 By taking the ratio, the equilibrium dissociation constant K for the binding of the nucleic acid aptamer to the target can be obtained. d .

[0062] The nucleic acid aptamers were obtained by screening using SELEX technology and labeled with FAM fluorescent groups. The quenching strands were labeled with BHQ-1 quenching groups. 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 Nucleic acid aptamer affinity was determined using a combination of traditional manual sample loading and ELISA reader analysis. The specific detection steps are as follows: (1) Dissociation constant K between nucleic acid aptamers and quenched strands d1 The calculation was performed by hybridizing a nucleic acid aptamer to a final concentration of 50 nM with a series of quenched strands at different concentrations (0 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM) at 25°C for 1 h. Afterward, the samples were added to multi-well plates, and the fluorescence signal of the solution was acquired using a multi-mode microplate reader. Fluorescence quenching saturation curves of the hybridization of the nucleic acid aptamer and the quenched strand were plotted to determine the dissociation constant K at which complementary hybridization reaches equilibrium. d1 .

[0064] (2) The dissociation constant K between the target substance, medanin, and the nucleic acid aptamer-quencher hybridization complex. d2 The calculation is as follows. Based on the fluorescence quenching curve obtained in the previous step, the optimal quenching ratio can be determined. K d2 The determination was performed by adding different concentrations of the target analyte (0 µM, 1.56 µM, 3.125 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, and 100 µM) to the complex under optimal quenching ratio conditions, and incubating at 25 °C for 1 h. Afterwards, the samples were added to multi-well plates, and the fluorescence signal was measured using a multi-mode microplate reader. Fluorescence recovery curves of maytansin binding to the nucleic acid aptamer-quencher complex were plotted, and the dissociation constant K at which maytansin induces the nucleic acid aptamer-quencher complex to reach equilibrium was calculated. d2 .

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

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

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

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

[0074] Table 2: Results of nucleic acid aptamer affinity determination using different methods.

[0075]

[0076] The affinity assay method based on a microfluidic chip in Embodiment 3 of this invention largely integrates this process. Users only need to add a small amount of sample, and then, through the platform's automated control, they can quickly obtain the detection results. This automated assay method reduces the single assay time from 105 min to 45 min, only about half the time required by the manual sample addition combined with an ELISA reader, significantly improving the assay efficiency. As can be seen from Table 2, there are slight differences between the assay results and the results obtained manually combined with an ELISA reader, but the affinity K... d The values ​​are all on the same order of magnitude (see the comparison in columns 2 and 5 of Table 2). The results indicate that this microfluidic platform can be used to determine the affinity of nucleic acid aptamers.

[0077] (3) The amount of reagent consumed by manual sample addition combined with the enzyme-linked immunosorbent assay (ELISA) in Example 3 and Comparative Example 1 were investigated respectively. The results of the amount of reagent consumed by manual sample addition combined with the ELISA in Comparative Example 1 are listed in Table 3, and the results of the amount of reagent consumed in Example 3 are listed in Table 4.

[0078] Table 3: Reagent consumption of manual sample addition combined with ELISA reader in Comparative Example 1.

[0079]

[0080] Note: The above results are based on a single K measurement. d2 The reagent consumption required for the value.

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

[0082]

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

[0084] The results in Tables 3 and 4 show that, in the comparative examples, there are significant differences in reagent consumption between the two methods for determining nucleic acid aptamer affinity. Here, we take the K value of Seq2-X as an example. d2 The determination of [specific substance] is described in detail below. When using an ELISA reader, eight 80µL solutions of aptamer-quencher complex and eight 80µL solutions of maytansin (concentrations of 0µM, 1.56µM, 3.13µM, 6.25µM, 12.5µM, 25µM, 50µM, and 100µM, diluted manually) are incubated. The fluorescence value of the mixed solution is then measured using an ELISA reader, as shown in Table 3. The total sample consumption is 1280µL. The digital microfluidic platform also required incubation of eight 1.92 µL nucleic acid aptamer-quencher chain complex solutions with eight 1.92 µL maytansine solutions (concentrations of 0 µM, 1.56 µM, 3.13 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, and 100 µM, with automated droplet dilution on the chip). The fluorescence values ​​of the mixed solutions were then measured using a fluorescence microscope, as shown in Table 4. The total sample consumption of the digital microfluidic platform was 30.72 µL. By comparison, we found that the sample consumption of the digital microfluidic platform was only 1 / 42 of that of the microplate reader, significantly reducing reagent consumption.

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

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

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

[0088]

[0089] Affinity was determined for the seven candidate nucleic acid aptamers in Table 5, and the results are listed in Table 6.

[0090] Table 6: Determination results of affinity of 7 candidate nucleic acid aptamers.

[0091]

[0092]

[0093] As shown in Table 6, the nucleic acid aptamers screened according to the method of this invention have the following equilibrium dissociation constant (K) for Seq19-X. d (Within 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, as follows: 5'-CTCTCGGGACGACAAATTGTGTGTAGAGGGAGTGGGTCATTTTGTCGTCCC-3'.

[0095] Figure 8 This is a schematic diagram of the secondary structure obtained by using the Mfold service to simulate the nucleic acid aptamer seq15-X and seq19-X sequences.

[0096] Furthermore, radioactive labeling, fluorescent labeling, enzyme labeling, digoxigenin labeling, and biotin labeling of one or both ends of the Seq19-X DNA strand can achieve the same or similar technical effects as in Example 1. In this example, the fluorescent substance is the FAM fluorescent group. FAM is labeled at the 5' end of Seq19-X, denoted as the nucleic acid aptamer FAM-Seq19-X. However, other fluorescent groups can also produce the same technical effects, such as the Cy5 fluorescent group.

[0097] In this invention, while keeping the overall structure of the nucleic acid aptamer unchanged, a certain position on the nucleotide sequence of the nucleic acid aptamer Seq19-X is phosphorylated, methylated, aminated, thiolated, or isotopized.

[0098] Further, in the aforementioned nucleic acid aptamer, the amination is performed by labeling an amino group -NH2 at the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq19-X; and the thiolation is performed by labeling a thiol group -SH at the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq19-X.

[0099] In this invention, the DNA sequence of the nucleic acid aptamer is any one of the following four sequences: (1) The nucleotide sequence of the nucleic acid aptamer Seq19-X has more than 60% homology; (2) A sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer Seq19-X; (3) The RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer Seq19-X; (4) is a derivative of Seq19-X, wherein the derivative is a thiophosphate 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 by the nucleic acid aptamer.

[0100] The aforementioned nucleic acid aptamers can bind to maytansine with high affinity and high specificity, with equilibrium dissociation constants in the nanomolar range. Compared to protein antibodies, nucleic acid aptamers have lower immunogenicity, can be synthesized in large quantities in vitro, have small molecular weights, can be modified and substituted at different sites, and are chemically stable, easy to store, and easy to label. Simultaneously, nucleic acid aptamers have good water solubility; after binding to the lipid-soluble drug maytansine, they can improve the drug's solubility, avoiding the toxic side effects caused by the addition of organic solvents (such as DMSO) or surfactants (such as Tween). Utilizing the specific interaction between nucleic acid aptamers and maytansine can improve the stability of maytansine, enhance its cytotoxicity, and thus broaden its therapeutic window. Using the nucleic acid aptamers of this invention as drug delivery carriers for maytansine can overcome the limitations of maytansine's clinical application (improving solubility, stability, and broadening the therapeutic window), which is of great significance for the design and development of maytansine in clinical applications. Meanwhile, labeling, modifying or substituting the nucleic acid aptamers of the present invention is beneficial for the tracing of maytansine in vivo, helps to reveal the mechanisms of drug absorption, distribution, metabolism and excretion, and promotes the effectiveness and safety of drug therapy.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for determining affinity using a microfluidic chip, characterized in that, 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 by wires; 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 separating electrode (15) for connecting the reservoir electrode (14) and the driving electrode (16); the reservoir electrode (14), the separating electrode (15), and the driving electrode (16) are connected to the contact electrode (13) through electrode leads; The method includes: S1. By manipulating the digital microfluidic driving platform (2), the buffer solution and quenching chain in the reservoir electrode (14) are mixed in the chip body (1) to obtain a quenching chain solution with gradient concentrations; the digital microfluidic driving platform (2) manipulates the quenching chain solution with gradient concentrations to react with the nucleic acid aptamer solution in the chip body (1) to obtain a nucleic acid aptamer-quenching chain complex solution; the fluorescence detection module (3) obtains the electronic signal by taking a picture of the chip body (1), plots the fluorescence quenching saturation curve of the hybridization of nucleic acid aptamer and quenching chain, and thus determines the dissociation constant K when the hybridization of nucleic acid aptamer and quenching chain reaches equilibrium. d1 ; S2. By manipulating the digital microfluidic driving platform (2), the buffer solution and the target substance in the reservoir electrode (14) are mixed in the chip body (1) to obtain a target substance solution with gradient concentration; the digital microfluidic driving platform (2) manipulates the target substance solution with gradient concentration and the nucleic acid aptamer-quencher complex solution to mix and react in the chip body (1); the fluorescence detection module (3) obtains the electronic signal by taking a picture of the chip body (1), plots the fluorescence recovery saturation curve of the target substance and the nucleic acid aptamer-quencher complex binding, and thus determines the dissociation constant K of the target substance and the nucleic acid aptamer-quencher complex at hybridization equilibrium. d2 ; S3, according to K d =K d1 / K d2 Calculate the equilibrium constant for the binding of the nucleic acid aptamer to the target analyte.

2. The method according to claim 1, characterized in that, The microfluidic chip uses a driving voltage 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, characterized in that, Add 2 mg / mL to 4 mg / mL of Pluronic L64 to the buffer solution.

4. A microfluidic chip according to any one of claims 1 to 3, characterized in that, 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 by wires; 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 separating electrode (15) for connecting the reservoir electrode (14) and the driving electrode (16); the reservoir electrode (14), the separating 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 the upper electrode plate (4), the lower electrode plate (5) and the polymethyl methacrylate gasket to form a sandwich structure; The lower electrode plate (5) of the chip is a printed circuit board, on which a contact electrode (13), a liquid storage 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 has a sample loading hole (11) and an oil filling hole (12) on it. The gap between the upper electrode plate (4) and the lower electrode plate (5) of the chip is 400µm.

6. The microfluidic chip according to claim 5, characterized in that, The lower electrode plate (5) of the chip 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) includes 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 droplet driving.

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 wires.

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