Affinity assay method based on chemotaxis of phospholipid membranes driven by ligand concentration gradients

Abstract

The application discloses an affinity analysis method based on ligand concentration gradient driving phospholipid membrane chemical tropism. The affinity analysis method can analyze the affinity on the phospholipid membrane interface without labeling target molecules. The ligand gradient above the phospholipid membrane interface is established through a microfluidic system, and the phospholipid molecules will be driven to form a complex with the ligand as a receptor, and the phospholipid membrane guides the micron-level tropotaxis of the phospholipid molecules through charge and chemical specificity interaction. In this case, the tropotaxis of the phospholipid receptor will be slowly carried out until the entropy and other intermolecular forces are completely balanced. Through dye labeling of the receptor phospholipid and observation of the final state of the receptor molecule aggregation in the membrane, combined with mathematical statistics, the subtle and transient interaction between the ligand and the receptor can be quantitatively characterized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemical analysis, and particularly relates to an affinity analysis method based on ligand concentration gradient driving phospholipid membrane chemical taxis. BACKGROUND

[0002] Cell membranes and intracellular membranes are collectively referred to as biological membranes. Biological membranes are semi-permeable membranes with special functions, which have important functions such as energy transfer, material transfer and information recognition. These functions play an important role in maintaining cell stability, signal amplification and conversion, and synthesis of biological macromolecules. Although phospholipid membranes are generally involved in biochemical processes, little is known about the physical properties of membrane organization. Phospholipid membranes are difficult to control and manipulate, and conventional methods are not suitable for characterizing systems with such high fluidity and disorder. However, the emergence of supported phospholipid bilayers provides a series of opportunities for precise control and quantitative study of phospholipid membranes and the mechanisms of their influence on biochemical processes. Supported phospholipid bilayers are generally defined as single, continuous phospholipid bilayers on a solid or polymer substrate. Bilayer membranes can be assembled by spontaneously adsorbing and fusing single-layer phospholipid liposomes with appropriate substrates. The supported bilayer is usually separated from the solid substrate by a thin water layer and retains many properties of biological membranes, such as lateral fluidity.

[0003] There are two most commonly used methods for preparing supported lipid bilayers, Langmuir-Blodgett (LB) pulling method and rupture fusion method. The LB pulling method is due to the amphiphilic nature of phospholipids, when dissolved in a non-polar organic solvent, the hydrophilic head group will be located in the water phase, and the hydrophobic tail region will be exposed to the air as the organic solvent evaporates. At this time, the hydrophilic substrate is pulled upwards, and the membrane at the air / water interface will be adsorbed on the substrate to form a single-layer phospholipid membrane. The rupture fusion method is based on the self-assembly of phospholipids, and a liposome solution is prepared in advance, in which a large number of liposomes will automatically adsorb on the solid substrate and then rupture and connect to form a large piece of intact phospholipid bilayer membrane. The hydrophobic effect drives the process of liposomes on the solid support.

[0004] Ligand-receptor binding is ubiquitous in chemistry and biological sciences, and monitoring this interaction is usually done by fluorescently labeling the protein, nucleotide or other object of interest. In fact, fluorescent labeling has become a standard tool for detecting biomolecules. However, protein labeling can interfere with the detection process and is inconvenient to use, which has been the main reason for researchers to urgently develop detection techniques capable of detecting biological analytes in a label-free manner. So far, detection techniques include liquid crystal phase transition, colloidal particle phase transition, quartz crystal microbalance measurement and surface plasmon resonance spectroscopy / imaging, etc. However, despite the great progress in label-free detection, no technique can provide the sensitivity and flexibility based on fluorescence signals. In fact, fluorescence signal measurement can usually be reduced to the single molecule level without the need for subsequent signal amplification steps. In addition, fluorescence-based devices provide fast readout. Finally, in addition to the protein labeling step itself, fluorescence spectroscopy and microscopy are relatively easy to perform. The above advantages give us some enlightenment as to whether this technology can be used for interface analyte detection without the need to label the target molecules. Instead, fluorescent dyes will be embedded on the surface of the detection platform and used as a universal sensing element for ligand-receptor binding. While the biological membrane acts as a cell barrier, it has many natural ligands, and the phospholipid molecules will act as receptors that will be driven by ligand binding. The phospholipid membrane guides the micrometer-scale directed movement of phospholipid molecules through charge and chemical specificity interactions. In this case, the directed movement of the phospholipid receptor will slowly proceed until the entropy and other intermolecular forces are completely balanced. Subsequently, the final state of the receptor molecule aggregation in the membrane will provide a quantitative characterization of the subtle and transient interaction between the ligand and the receptor. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides an affinity analysis method based on ligand concentration gradient driving phospholipid membrane chemical tendency.

[0006] The purpose of the present application is achieved by the following technical solution: an affinity analysis method based on ligand concentration gradient driving phospholipid membrane chemical tendency, which is realized based on a microfluidic chip used as a reaction chamber, comprising a PDMS device and a cover glass, which are seamlessly bonded; the PDMS device is provided with 3 liquid inlets, 1 liquid outlet and 1 liquid channel; comprising the following steps:

[0007] (1) preparing a unilamellar liposome solution and a buffer solution containing a specific ligand;

[0008] The unilamellar liposome solution is prepared by freeze-thaw extrusion method, specifically: the phospholipid containing NBD dye is dissolved in a buffer solution, and after a plurality of freeze-thaw processes, large unilamellar liposomes are formed, and then a plurality of liposome extruders are extruded, and finally a unilamellar liposome solution with uniform particle size is formed;

[0009] dissolving a specific ligand in a buffer solution to form a buffer solution containing a specific ligand; the specific ligand is a calcium ion, a magnesium ion or a Tim-3 protein;

[0010] (2) The prepared liposome solution in step (1) is introduced from the liquid outlet of the microfluidic chip to fill the microfluidic chip, incubated for 30-60 min, and the phospholipid membrane is formed by rupture fusion;

[0011] (3) The buffer solution is introduced from the three liquid inlets of the microfluidic chip at the same flow rate, and the remaining liposomes above the phospholipid membrane are washed for 20-40 min;

[0012] (4) The buffer solution introduced from the middle liquid inlet is replaced with a buffer solution containing a specific ligand, and the buffer solution containing a specific ligand with gradually increasing concentration is replaced multiple times, the flow rate of the buffer solution on both sides remains unchanged, and the flow rate of the buffer solution containing a specific ligand is consistent with that of the buffer solution on both sides, so as to form a ligand concentration gradient in the microfluidic channel; the specific phospholipid is excited by an LED light source to produce fluorescence, which is observed by a microscope and transmitted to an sCMOS camera through an objective lens, and the fluorescence distribution in the microfluidic channel is photographed at a fixed time, and finally analyzed and numerically fitted by NIS Elements software.

[0013] Further, the concentration of the single-layer liposome solution is 0.5-2 mg / mL.

[0014] Further, in step (4), the buffer solution containing a specific ligand with gradually increasing concentration is replaced multiple times, and the gradually replaced concentration is 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM and 50 mM.

[0015] Further, the composition and mass ratio of the single-layer liposome are glycerophospholipid: cholesterol = 7:3.

[0016] Further, the glycerophospholipid includes phosphatidylcholine and phosphatidylserine.

[0017] Further, the buffer solution includes TrisHCl solution, HEPES solution and MES solution.

[0018] The beneficial effects of the present application are as follows:

[0019] (1) The dye-labeled phospholipid membrane is taken as the research object, and the ligand concentration gradient is established above the phospholipid membrane through the characteristics of the microfluidic system, so as to cause the two-dimensional chemical tendency of the phospholipid molecules in the gradient field direction.

[0020] (2) By observing the interaction between different ligands and phospholipid molecules, and combining mathematical statistics, the affinity between ligands and phospholipids can be estimated, and the fluorescence characteristics of the dye can be visually presented.

[0021] (3) Unlike static analysis in traditional homogeneous systems, the multiphase microfluidic system can more accurately reflect the interaction between micro molecules. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The size of the microfluidic chip and the reaction chamber.

[0023] Figure 2 The experimental schematic diagram.

[0024] Figure 3 The process of making a microfluidic chip.

[0025] Figure 4 The change in channel fluorescence image when the ligand is calcium ion.

[0026] Figure 5 The kinetic curve under different concentrations of calcium ions.

[0027] Figure 6 The Langmuir model fitting result when the ligand is calcium ion (regression coefficient R 2 = 0.984).

[0028] Figure 7 The kinetic curve under different concentrations of magnesium ions.

[0029] Figure 8 The Langmuir model fitting result when the ligand is magnesium ion (regression coefficient R 2 = 0.977)

[0030] Figure 9 The change in channel fluorescence image when the ligand is Tim-3 protein, and the excitation wavelength is 473 nm.

[0031] Figure 10 The channel fluorescence image when the ligand is Tim-3 protein, and the excitation wavelength is 535 nm. DETAILED DESCRIPTION

[0032] The exemplary embodiments will be described in detail herein with reference to the attached drawings. The following description is only exemplary and is not intended to represent all aspects of the present application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the present application as detailed in the appended claims.

[0033] The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in the present application and the accompanying claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "and / or," as used herein, refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0034] It is to be understood that, although the terms first, second, third, etc. can be used herein to describe various information, the information should not be limited to these terms. These terms are only used to distinguish one piece of information from another. For example, a first information can be termed a second information, and, similarly, a second information can be termed a first information, without departing from the scope of the present application. Depending on the context, the word "if' as used herein can be interpreted as meaning "when" or "in response to determining."

[0035] The present application will be described in detail below with reference to the attached drawings. The features of the embodiments and implementation described below can be combined with each other as long as there is no conflict.

[0036] The microfluidic chip is used as a reaction chamber, including a PDMS device and a cover glass, which are seamlessly bonded. The PDMS device is provided with 3 liquid inlets, 1 liquid outlet and 1 liquid channel. The geometric size of the reaction chamber ranges from 234 μm in width, 2 cm in length and 100 μm in height. The size of the microfluidic chip and the reaction chamber is shown in Figure 1 .

[0037] In the following examples, in the inverted fluorescence microscopic imaging system equipped with the microfluidic chip, NBD dye-labeled phosphatidylserine (NBD-DOPS) is used as the receptor, Ca 2+ , Mg 2+ and Alexa Fluor@555 dye-labeled Tim-3 protein are used as the ligand, and the phospholipid molecule is observed to be driven by the ligand gradient to flow and aggregate in the phospholipid membrane, Figure 2 is an experimental schematic diagram. (Phospholipids and dye-labeled phospholipids are purchased from Avanti Company)

[0038] In the following examples, the microfluidic chip is prepared by soft lithography. At present, the most commonly used materials for preparing microfluidic chips are inorganic materials such as silicon and glass, and new polymer materials. Compared with inorganic materials, polymer materials such as polydimethylsiloxane (PDMS) have better light transmission and insulation, better performance, simpler preparation technology, higher precision, and lower cost. The PDMS chip production process is as follows: mix the PDMS monomer and the high-elasticity curing agent in a mass ratio of 10:1, place them in a vacuum pump, and repeatedly vacuumize until there are no bubbles. Then, pour the mixture into a customized silicon mold master sheet, and if there are small bubbles during pouring, they must be removed with a dust-free pipette. Heat the mold in an oven at 70°C for 30 minutes to cure the upper and lower surfaces simultaneously. After cooling, remove the mold, carefully cut the individual chip model along the frame with a blade, and then punch it with a matching punch (imported three-hole, one-hole outlet). Use transparent tape to adhere the upper and lower surfaces of the chip to remove impurities and dust. In order to obtain a strong seal between the PDMS and the cover glass, treat the PDMS block and the prepared hydrophilic thin glass sheet with an electric spark vacuum detector for 45s to activate the alkyl hydroxyl group, and then combine the treated channel side with the thin glass sheet. Finally, place the microfluidic chip on a constant temperature heating plate at 90°C for 30 minutes. The preparation process is shown in Figure 3 .

[0039] In the following examples, the freeze-thaw extrusion method used to prepare liposomes includes the following steps: mix the required raw materials with chloroform in a mass ratio of "POPC:NBD-DOPS:Cholesterol=69:1:30", where NBD dye is labeled at the tail of the DOPS fatty chain, and its ultraviolet fluorescence spectrum is shown in Figure 2 . Dry the above mixture with nitrogen and place it in a vacuum drying box for 4 hours to remove any residual solvent. Rehydrate the lipid mixture with a buffer solution, and then perform repeated freeze-thaw treatment. Cool in liquid nitrogen for 5 minutes and then transfer to a 40°C water bath for heating for 5 minutes, which is one cycle. Perform 10 freeze-thaw cycles. Finally, use a liposome extruder to extrude and filter 8 times under a 200nm polycarbonate hydrophilic membrane to form liposomes with uniform pore size, and store them in a 4°C refrigerator until use.

[0040] In the following examples, the microscope used is an inverted fluorescence microscope (Nikon Eclipse Ti2-U) from a light-emitting diode (LED) light source. The incident light passes through a fluorescence excitation module (λ exThe fluorescence images of the channel were captured by a Nikon's CMOS camera and imaged in real time and taken by NIS Elements software. The resulting images were processed and analyzed by selecting the Region of Interest (ROI). The processing procedure included background subtraction, inter-group averaging, normalization, and numerical fitting based on the Langmuir isotherm model.

[0041] In the following examples, a physical quantity η is defined to quantify the tendency of phospholipid in the membrane. The lateral position is defined as x, with the inside of the left wall of the channel as the starting point, and the distance range of x is 0≤x≤L, L being the width of the channel. The solution flows along the y direction through the channel, and the total fluorescence intensity is is a function of x, measured in a differential unit of size dy in the x direction. The experimental condition is defined as α, representing the ligand concentration.

[0042] Definition The minimum value of the measured fluorescence intensity is approximated to be the background noise value:

[0043]

[0044] I α (x) is the fluorescence intensity in the differential unit:

[0045]

[0046] A α is the fluorescence integral area of the middle channel:

[0047]

[0048] is the fluorescence integral area of all channels:

[0049]

[0050] A α and respectively represent the dye fluorescence intensity of the middle channel and the entire channel. At the same time, it is assumed that the ratio of the two is equal to the ratio of the dye concentrations of the middle channel and the entire channel, i.e.

[0051]

[0052] Example 1

[0053] This example provides a method for measuring the binding of phosphatidylserine (PS) to Ca 2+A technique that utilizes concentration gradients to induce two-dimensional chemitropic motion within phospholipid membranes, and is used to analyze phospholipid membranes and Ca2+. 2+ The bonding force between them, the specific steps are as follows:

[0054] (1) Prepare a 10 mM Tris-HCl buffer solution with pH 7.4, and use this solution to prepare a liposome solution with a concentration of 1 mg / mL. Prepare a solution containing Ca... 2+ The buffer solutions had ion concentrations of 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, and 50 mM, respectively.

[0055] (2) Add 20 μL of liposome solution from the outlet of the microfluidic chip to fill the reaction chamber and incubate at 40°C for 40 min.

[0056] (3) Tris-HCl buffer solution was simultaneously introduced into all three channel inlets at a flow rate of 100 μL / h for 20 min to wash away any remaining liposomes above the phospholipid membrane. The solution in the middle channel was then replaced with a solution containing Ca. 2+ The buffer solution remains unchanged on both sides, and the flow rate remains unchanged.

[0057] (4) Use the Timelapse module in NIS Elements software to take timed photos. Specific parameters are: exposure time 1 second, exposure interval 10 minutes, and period 140 minutes. Select a laminar flow stability point near the confluence point as the ROI, such as... Figure 3 As shown by the dashed line, the changes in light intensity distribution of the ROI are recorded, and the channel fluorescence image is as follows. Figure 4 As shown.

[0058] (5) Gradually increase the calcium ion concentration and calculate the η value under different concentration gradients. Record the kinetic trend of its change with time and fit it with the Langmuir isotherm adsorption equation: Where η is the degree of PS aggregation under different ligand gradients, B corresponds to the maximum aggregation degree at high ligand concentrations, and K D It is the equilibrium dissociation constant of the PS-ligand complex, c ligand Refers to ligand concentration.

[0059] (6) From Figure 5 It can be seen that the curve tends to plateau after 80 minutes. Therefore, it can be considered that under these conditions, the binding of calcium ions and PS reaches saturation after 80 minutes. Thus, the η value at T = 100 min is selected to represent the degree of phospholipid aggregation after binding reaches saturation. The η value at this moment is plotted as a function of calcium ion concentration and fitted to a Langmuir curve, as shown below. Figure 6 As shown. The dissociation equilibrium constant K of the obtained calcium ion affinity with PS is... Dis 75 ± 17 μΜ.

[0060] In some embodiments, the single-layer liposome solution can also be selected to have a concentration of 0.5-2 mg / mL;

[0061] In some embodiments, the buffer solution can also be selected to be HEPES solution and MES solution.

[0062] Example 2

[0063] Example 2 is only different from Example 1 in that the ligand is changed to Mg 2+ , and the rest is the same as Example 1, the kinetic curve and the fitting result are shown in Figure 7 and 8 . The dissociation equilibrium constant K D of the obtained magnesium ion and PS affinity is 276 ± 44 μΜ.

[0064] Example 3

[0065] Example 3 is only different from Example 1 in that the ligand is changed to Alexa Fluor@555 dye-labeled Tim-3 protein, and the channel fluorescence image is shown in Figure 9 and 10 . Wherein Figure 9 is the fluorescence image under 473 nm wavelength excitation, and the results show that the protein as a biological macromolecule can also cause the migration of PS in a similar manner. But unlike ions, the protein has a larger molecular weight, and the phospholipid-protein complex formed after binding with PS will affect the diffusion coefficient of PS, so it takes longer to reach a steady state. Figure 10 is the fluorescence image under 535 nm wavelength excitation, and the results show that under this microfluidic system, the fluid is in a laminar flow state, that is, the transverse diffusion of the ligand during the flow process can be ignored, so the ligand concentration gradient can be constructed as needed.

[0066] The above examples are only used to illustrate the design idea and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and the protection scope of the present application is not limited to the above examples. Therefore, any equivalent changes or modifications made in accordance with the principles disclosed by the present application are within the scope of protection of the present application.

[0067] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the application be limited only by the scope of the claims, including any equitable

[0068] It is to be understood that the application is not limited to the precise construction already described above and shown in the drawings, and that various modifications and changes can be made by those skilled in the art without departing from the scope thereof.

Claims

1. An affinity assay method based on chemotaxis of phospholipid membranes driven by a ligand concentration gradient, characterized by, The method is realized based on a microfluidic chip serving as a reaction chamber, comprising a PDMS device and a cover glass which are seamlessly bonded; the PDMS device is provided with three liquid inlets, one liquid outlet and one liquid channel; comprising the following steps: (1) preparing a unilamellar liposome solution and a buffer solution containing a specific ligand; The unilamellar liposome solution is prepared by freeze-thaw extrusion method, specifically: dissolving phospholipid containing NBD dye in a buffer solution, forming large unilamellar liposomes through multiple freeze-thaw processes, and then extruding through a liposome extruder multiple times to finally form a unilamellar liposome solution with uniform particle size; Dissolve a specific ligand in a buffer solution to form a buffer solution containing a specific ligand; the specific ligand is calcium ion, magnesium ion or Tim-3 protein; (2) pass the liposome solution prepared in step (1) from the liquid outlet of the microfluidic chip, so that it fills the microfluidic chip, incubate for 30-60 min, and form a phospholipid membrane through rupture fusion; (3) pass the buffer solution from the three liquid inlets of the microfluidic chip at the same flow rate for 20-40 min to flush the remaining liposomes above the phospholipid membrane; (4) replace the buffer solution passed from the middle liquid inlet with a buffer solution containing a specific ligand, and replace the buffer solution containing a specific ligand with gradually increasing concentrations multiple times, the flow rates of the buffer solutions on both sides remain unchanged, and the flow rate of the buffer solution containing a specific ligand is consistent with that of the buffer solutions on both sides, so as to form a ligand concentration gradient in the microfluidic channel; excite the specific phospholipid to produce fluorescence by an LED light source, observe using a microscope, and transmit to an sCMOS camera through an objective lens, take pictures of the fluorescence distribution in the microfluidic channel at regular time intervals, and finally process and analyze by NIS Elements software and numerical fitting; comprising: Define a physical quantity η to quantify the tendency of phospholipid in the membrane; Definitions The minimum value of the measured fluorescence intensity is approximated to be the background noise value: I α (x) is the fluorescence intensity within the differential unit: A α Fluorescence integrated area for intermediate channel: Fluorescence integrated area for all channels: Wherein, the distance range of x is 0≤x≤L, L is the channel width, and x represents the lateral position; F is the total fluorescence intensity; and based on The dissociation equilibrium constant K D ; B corresponds to the maximum degree of aggregation at high ligand concentration, c ligand denotes the ligand concentration.

2. The affinity assay method based on chemotaxis of phospholipid membranes driven by ligand concentration gradient according to claim 1, characterized in that, The concentration of the unilamellar liposome solution ranges from 0.5 to 2 mg / mL.

3. The affinity assay method based on chemotaxis of phospholipid membranes driven by ligand concentration gradient according to claim 1, characterized in that, In step (4), the buffer solution containing a specific ligand with gradually increasing concentrations is replaced multiple times, and the gradually replaced concentrations are 0.01 mM, 0.1 mM, 0.5 Mm, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM and 50 mM.

4. The affinity assay method based on chemotaxis of phospholipid membranes driven by ligand concentration gradient according to claim 1, characterized in that, The component and mass ratio of the unilamellar liposome are glycerophospholipid: cholesterol = 7:

3.

5. The affinity assay method based on chemotaxis of phospholipid membranes driven by ligand concentration gradient according to claim 4, characterized in that, The glycerophospholipid includes phosphatidylcholine and phosphatidylserine.

6. The affinity assay method based on chemotaxis of phospholipid membranes driven by ligand concentration gradient according to claim 1, characterized in that, The buffer solution includes TrisHCl solution, HEPES solution and MES solution.

Images