Microfluidic optical observation system and method for chemical tropism of small molecules

Abstract

The present invention discloses a microfluidic optical observation system and method for chemical tropism behavior of small molecules. The microfluidic optical observation system includes an optical platform and a microfluidic chip. The photochemiluminescent substance is used as the research object. By constructing a simulated chemical gradient field of a receptor-ligand pair in a low Reynolds number fluid, the luminescence characteristics of the photochemiluminescent substance are used for image acquisition, and the spontaneous directional migration performance of molecules of different scales and their ordered collections is observed. The motion phase parameters of the receptor are associated with the reversible association reaction information of the ligand, and combined with mathematical statistics, the molecular binding-induced chemotaxis phenomenon is observed.

Description

Technical Field

[0001] The invention belongs to the technical field of simulated chemical analysis, and relates to a microfluidic optical observation system and an observation method for small molecule chemical trend behavior. Background Art

[0002] For a long time, textbooks have been accustomed to using the empirical "collision theory" of classical thermodynamics to phenomenologically explain the molecular reaction process and its approximate dependence on macroscopic thermodynamic conditions, while physical quantities such as the reaction rate constant introduced in dynamics are only suitable for describing the apparent process in general. But the fact is that a general reaction process, even if it has reached an equilibrium state or a non-equilibrium steady state, is inevitably accompanied by the evolution of a series of related states such as the speed, position, and orientation of a large number of molecules (ions). Typical examples include the 1977 Nobel Prize in Chemistry theme "Belousov-Zhabotinsky oscillation reaction", the mechanical cycle of the natural structure "sodium / potassium pump" (1997 Nobel Prize theme), and even the 2016 "molecular machine" and "molecular motor" system. The discovery of these strange phenomena shows that traditional theories are difficult to fully reflect the effects of molecular kinematics and dynamics during energy exchange in reactions, resulting in the real distribution of various types of ensembles in time and space and their fluctuation laws being obscured for a period of time during the reaction, which may hide the chemical tendency of molecules. This characteristic originally refers to the "advantage-avoidance" physiological behavior of single-cell organisms or tissues actively approaching or moving away from higher-intensity special chemical stimuli. When the chemical gradient disappears, bacteria and microorganisms turn to "random walk" like Brownian motion. Given that homogeneous molecules also experience a similar "diffusion-reaction" mode when interacting, and the concentration and position information of the attractant / repellent that triggers cell chemotaxis is sensed by transmembrane protein receptors, the essence of this biological recognition is chemical binding, which initiates complex intracellular signal transduction. It is expected that the establishment of spontaneous chemotactic behavior of molecules in the dispersed phase and the revelation of its nature will help promote those diffusion mass transfer processes that are currently still limited to passive, non-directional and entropic diffusion, such as drug delivery, nanopore-based third-generation gene sequencing, catalytic bed substrate pre-enrichment, membrane filtration / adsorption, water permeation and other important links in the fields of health industry, biochemical engineering, environmental engineering and geochemistry.

[0003] In 2013, an article published in JACS used microchannels to observe dye-labeled urease, laccase, and alkaline phosphatase, and found that these bioactive macromolecules automatically migrated along the direction of increasing substrate concentration gradients; in 2015, Nature reported the use of single-molecule fluorescence correlation spectroscopy, and found that in the intrinsic exothermic catalytic process of urease, laccase, and alkaline phosphatase, their own diffusion coefficients gradually increased, and the diffusion rate formed a linear relationship with the substrate concentration and the cumulative time. The fitted slope was also specifically related to the conversion frequency of each enzyme substrate. Later, when people labeled these proteases with dyes and tried to place their solutions under a fluorescence microscope for observation, they found that these bioactive macromolecules automatically migrated along the direction of increasing substrate concentration gradients, and DNA polymerase also behaved in the same way. The trajectory of the overall cluster movement was comparable to the positive chemotaxis movement of organisms. As a result, the concept of enzyme-catalyzed chemotaxis was first proposed and successfully used for drug release across the blood-brain barrier. Considering that what actually occurs in the cytoplasm and organelles is a chain and cascade of multi-enzyme reactions, in these high-protein-density, high-viscosity metabolic compartments, each enzyme still independently follows its own chemotaxis pattern. In 2017, Science reported that large-scale chaotic turbulent systems such as Kinesin and ATPase can self-organize into a continuous unity of circulating flow, indicating that biomolecular chemotaxis is closely related to life activities. However, the mechanism of enzyme-catalyzed chemotaxis remains unresolved to date. According to the Michaelis-Menten model and its quasi-steady-state approximation, general enzyme catalysis sequentially undergoes two basic stages: reversible substrate binding and irreversible product conversion. Therefore, it is necessary to isolate and examine these two stages from each other in order to clarify the power source of enzyme chemotaxis. In fact, there have been a few reports on the phenomenon of smaller ions / molecules approaching larger objects through non-bonded and non-specific interactions. For example, Paul Braun's team at the University of Illinois at Urbana-Champaign observed the spontaneous convergence of anions to an artificial "enthalpy black hole" composed of localized quaternary ammonium salts at the expense of the freedom of movement of ions in a planar electrostatic field, and developed and applied this phenomenon to accelerate the surface capture of targets. In addition, the low osmotic pressure near the hydrophobic semipermeable membrane can be used to drive the irreversible and firm adsorption of dyes to it. Conversely, when the gradient of monovalent and divalent ions in the Debye layer surrounding the surface of proteins or exosome vesicles is large enough, the mobility of these high molecular weight polymers or micro-nanoparticles will increase hundreds or thousands of times, resulting in so-called diffusion phoretic transport; this mobility is closely related to the size of the colloid and the type of ions, and has been developed for liquid-liquid phase separation of proteins and even crude oil extraction. However, there are currently no literature reports on the observation of small molecule chemical tropism behavior. Summary of the invention

[0004] The purpose of the present invention is to provide a microfluidic optical observation system and method for the chemical tropism behavior of small molecules. The system takes photochemiluminescent molecules as the research object, constructs a simulated chemical gradient field of receptor-ligand pairs in a low Reynolds number fluid, observes the spontaneous directional migration performance of molecules of different scales and their ordered collections, associates the motion phase parameters of the receptor with the reversible association reaction information of the ligand, and combines mathematical statistics to observe the molecular binding-induced chemotaxis phenomenon.

[0005] The technical solution for achieving the purpose of the present invention is as follows:

[0006] Microfluidic optical observation system for small molecule chemical trend behavior, including optical platform and microfluidic chip;

[0007] The microfluidic chip comprises a polydimethylsiloxane (PDMS) chip and a cover glass for microscopic imaging, which are seamlessly bonded; the PDMS chip is provided with more than two microchannel inlets, a reaction chamber and more than one microchannel outlet; each microchannel inlet is respectively injected with a receptor solution or a ligand solution or a buffer solution, the inlet sequence of the receptor solution, the ligand solution and the buffer solution is randomly combined and arranged, each solution converges at the inlet confluence point, enters the reaction chamber, and finally flows out from the microchannel outlet;

[0008] The optical platform comprises an inverted microscope, an external three-channel shutter driver connected to a camera for framing, and a light-emitting diode light source; incident light passes through a fluorescence excitation block and is focused on the bottom of a PDMS chip via an objective lens, and a CCD camera captures the fluorescence imaging of the channel.

[0009] Preferably, the CCD camera is connected to a computer via an image acquisition card, and the acquired fluorescence images are transmitted to the computer for processing.

[0010] In the present invention, the receptor is a photochemiluminescent substance, such as porphyrin substance zinc (II) meso-tetra (4-sulphonato phenyl) porphine (ZnTSPP) and the like.

[0011] In the present invention, the ligand is a substance that can specifically bind to the receptor. For example, the ligand that can specifically bind to ZnTSPP is an organic heterocyclic substance containing N, such as imidazole, pyridine, and the like.

[0012] In the present invention, the buffer solution is PBS buffer or HEPES buffer.

[0013] In a specific embodiment of the present invention, the microfluidic chip is provided with 3 microchannel inlets, 1 inlet confluence point, 1 reaction chamber and 1 microchannel outlet.

[0014] The microfluidic optical observation method of small molecule chemical tendency behavior includes the following steps:

[0015] Step 1, preparing a receptor solution, a ligand solution and a buffer solution, wherein the receptor solution and the ligand solution are prepared using a buffer solution;

[0016] Step 2, firstly rinse the microfluidic channel with a buffer solution, and then pass the receptor solution, the ligand solution and the buffer solution into the microchannel inlet of the microfluidic chip according to the experimental design;

[0017] Step 3, turn on the light-emitting diode light source to excite the receptor to produce photoluminescence. The fluorescence is transmitted to the CCD camera through the imaging objective to capture the fluorescence imaging of the channel, record the fluorescence images at the inlet confluence point and different sites of the microfluidic channel, and transmit the recorded images to the computer through the image acquisition card for processing and analysis.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The diffusion rate of a substance is generally inversely proportional to its size (Einstein-Stokes equation). The microfluidic optical observation system of the present invention is highly feasible for observing the active migration of low molecular weight receptors to smaller ligands.

[0020] (2) The present invention uses a substance that can generate photoluminescence as a research object, allowing its concentration distribution during affinity analysis in a microfluidic channel to be directly imaged by fluorescence microscopy;

[0021] (3) The Reynolds number of the fluid in the microchannel is small enough, the flow state is laminar, the mass transfer is mainly molecular diffusion, there is no complex flow shape such as turbulence, eddy current, convection, etc., which is close to the real biological viscosity environment;

[0022] (4) By introducing ligands and receptors into different channels respectively, the chemical tropism behavior of small molecule receptors can be observed more intuitively through fluorescence data. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the principle of the microfluidic optical observation system for small molecule chemical attraction behavior.

[0024] Figure 2 Schematic representation of a series of schemes for binding-induced receptor chemotaxis in response to a ligand gradient: (A) blank control; (B) receptor intrareceptor focusing; (C) receptor extrareceptorization; and (D) directed migration.

[0025] Figure 3The internally focused chemotaxis phenomenon of 10 μM ZnTSPP in response to a 100 μM imidazole concentration gradient; (A) Schematic diagram of the Ψ-type 3-inlet microfluidic channel used for the chemotaxis study of the ZnTSPP and imidazole collection system, and the average normalized intensity distribution as a function of the lateral position along the channel width are respectively in the (B) upper (Upper Channel) ROI: confluence point, and (C) lower (Lower Channel) ROI: 4.7 mm down the channel ((a) line: PBS / ZnTSPP / PBS, (b) line: PBS / ZnTSPP+imidazole / PBS); the short dashed line perpendicular to the x-axis in the figure points to the channel axis and the peak position.

[0026] Figure 4 Snapshots of imaging in typical areas for different configurations of stable flow regimes: (A)(B) PBS / ZnTSPP / PBS and (C)(D) PBS / ZnTSPP+imidazole / PBS, collected in pre-specified (A)(C) upper channel and (B)(D) lower channel ROIs (499.4×499.4μm2, 10x objective magnification), respectively. The vertical thin dotted line in the figure indicates the channel axis, which is perpendicular to the thick broken line.

[0027] Figure 5 (A) Ellipsometry visualization of the upper channel microflow ROI (1331.2×499.2μm 2 , 10x objective magnification, pseudocolor rendering) at representative volume flow rates of 1 (a), 5 (b), 10 (c), 50 (d), and 100 (e) μL / h, (B) Dependence of the stability and intensity of the dye signal (white dashed line at the confluence point in (A)) on the flow rate from 1 to 100 μL / h.

[0028] Figure 6 The average normalized PL intensity as a function of the traversal distance for the collective migration of 10 μM ZnTSPP along the channel width toward 100 μM imidazole (line (b)) near the outlet (Lower Channel) is presented in a comparison of the following configurations: (A) imidazole / ZnTSPP / PBS and (B) PBS / ZnTSPP / imidazole, both with PBS / ZnTSPP / PBS (line (a)) as a control. The interpolated broken line perpendicular to the x-axis points to the peak position.

[0029] Figure 7The average normalized intensity distribution across the channel width for 10 μM ZnTSPP in the absence and presence of 100 μM imidazole: (A) imidazole / ZnTSPP / PBS and (B) PBS / ZnTSPP / imidazole, both with PBS / ZnTSPP / PBS as blank control. The right panel shows snapshots of the channel under different conditions, with the upper channel corresponding to the profiles shown in (A) and (B). DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0031] The coexistence of ZnTSPP and organic amines will cause dsp 2 The axial coordination between the hybridized zinc and the lone pair of electrons on the ligand N occurs in a strict 1:1 stoichiometry, resulting in a five-coordinate complex. However, the association is weak and quite reversible due to the Jahn-Teller distortion of the macrocycle and its competitive hydration in the aqueous phase. Such unstable binding is critical for chemical targeting, because strong and irreversible bonding will offset its continuous tracking of the reactants (i.e., substrate depletion effect). In addition, the fluorescent activity of zinc porphyrin, combined with its optical stability and resistance to self-aggregation in the above interaction, makes it an ideal probe for mass transport imaging.

[0032] Figure 1 The schematic diagram of the principle of the microfluidic optical observation system for the chemical tropism behavior of small molecules. In the following embodiments, the microfluidic optical observation system for the chemical tropism behavior of small molecules is used, with ZnTSPP as the receptor, to observe the receptor chemical tropism phenomenon induced by the binding of ligands in the presence of various organic amine ligands. The chemical tropism of the binding-induced receptor in response to the ligand gradient includes receptor intra-focusing, receptor extracellular expansion and directional migration. In the following embodiments, the observation is carried out by the following steps:

[0033] Step 1: Prepare a 10 mM PBS solution at pH 9.0, and use the solution to prepare receptor solution A: 10 μM ZnTSPP solution and ligand solution B: 100 μM imidazole solution.

[0034] Step 2: Rinse the microfluidic channel thoroughly with buffer solution.

[0035] Step 3: If Figure 2 As shown in A, the following solutions were introduced into the three inlets of the channel from left to right. This configuration was recorded as PBS / ZnTSPP / PBS. The fluorescence data of the inlet confluence point of the microfluidic channel and the sampling point 4.7 mm below the inlet were recorded as blank controls.

[0036] Step 4: Figure 2As shown in B, PBS / ZnTSPP+imidazole / PBS were introduced into the three channels respectively, and the fluorescence data of the inlet confluence point of the microfluidic channel and the sampling point 4.7 mm below the inlet were recorded as the intrareceptor focusing phenomenon observation system.

[0037] Step 5: Figure 2 As shown in C, imidazole / ZnTSPP / imidazole were introduced into the three channels respectively, and the fluorescence data of the inlet confluence point of the microfluidic channel and the sampling point 4.7 mm below the inlet were recorded as the receptor epitaxial phenomenon observation system.

[0038] Step 6: Figure 2 As shown in D, imidazole / ZnTSPP / PBS were introduced into the three channels respectively, and the fluorescence data of the inlet confluence point of the microfluidic channel and the sampling point 4.7 mm below the inlet were recorded as the receptor directional migration phenomenon observation system.

[0039] Figure 2 Schematic representation of a series of schemes for binding-induced receptor chemotaxis in response to a ligand gradient: (A) blank control; (B) receptor intrareceptor focusing; (C) receptor extrareceptorization; and (D) directed migration.

[0040] In the following embodiments, the microfluidic chip is prepared by conventional methods in the art. The production process of the PDMS chip is as follows: the required volume of monomer (Sylgard 184Silicone elastomer kit, Dow Corning) is stirred and mixed with a high elastic crosslinker in a mass ratio of 10:1, and then degassed and defoamed in a vacuum dryer for 2 hours. Subsequently, the solution is poured on the microchannel template of the silicon wafer and aged in a 70°C oven for 12 hours. The solidified PDMS block is peeled off from the template, and a stainless steel needle is used as a puncher to perforate the entrances and exits of each channel. Then, PDMS and a clean cover glass are placed in a PDC-32G plasma cleaner / disinfector and high pressure is applied for 45 seconds. The PDMS is gently pressed on the pretreated cover glass with a little force of the fingers to make the two seamlessly bonded, and annealed at 100°C for 5 minutes. The coverslips used for microscopic imaging were pretreated as follows: the coverslips were placed on a ceramic holder (22 width × 40 length × 0.15 mm thickness, Fisherbrand, Fisher Scientific) and immersed in a mixture of near-boiling 7X detergent (MP Biomedicals LLC.) and ultrapure water (≥18 MΩ) for 2 h. Then, the glass slides were rinsed thoroughly with ultrapure water and the surface was dried with N2, and then annealed in a muffle furnace at 530 °C for 6 h.

[0041] In the following examples, the optical platform used includes an inverted microscope (Nikon Eclipse Ti2-U), an external UNIBLITZ VMM-D3 three-channel shutter driver connected to a camera viewfinder, and a light-emitting diode (LED) light source (Lumen Dynamics X-Cite Series 120Q, 100W). The incident light passes through the green fluorescence excitation block (λ ex =538~580nm), and focused on the bottom of the PDMS chip through a 10x objective lens (numerical aperture: 0.25, Ph1 DL∞ / -, focal distance: 10.5). The fluorescence imaging of the channel was captured by a MicroMax 1024CCD camera (Princeton Instruments), and real-time shooting was performed using the (Timelapse) module of Universal Imaging MetaMorph software. Specific parameters: exposure interval 30s, cycle 20min, exposure time: 250ms. The recorded images were transferred to the computer for processing and analysis through an image acquisition card. The specific analysis method is as follows: the fluorescence intensity of the region of interest (ROI) was selected, the background was subtracted and the average was taken; then the channel width was used as the function independent variable, and normalization was performed using Origin 8.0 software. In order to draw the manifold in the microchannel, all images were rendered in 16-bit RGB "pseudo-color", and the lower limit of the contrast was calibrated to 2000 and the upper limit was 13000, replacing the grayscale image.

[0042] Example 1

[0043] This embodiment provides a microfluidic chip system for observing the cohesive chemical tropism of ZnTSPP under the influence of imidazole ligands, and the specific steps are as follows:

[0044] (1) Prepare 10 mM PBS buffer solution at pH 9.0, and use it to prepare receptor solution A: 10 μM ZnTSPP solution and ligand solution B: 100 μM imidazole solution;

[0045] (2) fully rinsing the microfluidic channel with a buffer solution;

[0046] (3) The microchannel was configured with three inlets from left to right, and PBS / ZnTSPP / PBS were introduced respectively. The fluorescence data of the inlet confluence point of the microfluidic channel and the sampling point 4.7 mm below the inlet were recorded (e.g. Figure 3 B and (a) line in 3C);

[0047] (4) The microchannel was configured with three inlets from left to right, and PBS / ZnTSPP+imidazole / PBS were introduced respectively. The fluorescence data of the inlet confluence point of the microfluidic channel and the sampling point 4.7 mm below the inlet were recorded (e.g. Figure 3 B and (b) line in 3C).

[0048] Can be Figure 3 In C, the cohesion of line (b) relative to line (a) is observed, which is the chemical tendency of ZnTSPP to focus inward under the influence of imidazole. Figure 4 As shown in the microscopic imaging, by establishing an imidazole concentration gradient in the flow channel where ZnTSPP is located, the outward propagation of ZnTSPP against itself was observed. This behavior is consistent with the traditional definition of chemical taxis, thus confirming the existence of molecular chemotaxis.

[0049] Example 2

[0050] This embodiment provides a flow rate optimization experiment of a microfluidic chip system for observing the chemical tropism behavior of small molecule receptors, and the specific steps are as follows:

[0051] (1) Prepare a 10 mM PBS solution (pH 9.0), and use it to prepare receptor solution A: 10 μM ZnTSPP solution and ligand solution B: 100 μM imidazole solution;

[0052] (2) fully rinsing the microfluidic channel with a buffer solution;

[0053] (3) The microchannel is configured with three inlets from left to right, and PBS / ZnTSPP / PBS are introduced respectively;

[0054] (4) The flow rates were controlled at 1, 5, 10, 50, and 100 μL / h, and the fluorescence intensity distribution of ZnTSPP in the lateral direction of the channel at the confluence point was recorded. Figure 5 A), and its relationship with flow rate (such as Figure 5 B) From Figure 5 It can be seen that the data point at which the flow rate is 50 μL / h is the optimal speed.

[0055] Example 3

[0056] This embodiment provides a microfluidic optical observation system for the chemical tropism behavior of ZnTSPP directional migration under the influence of imidazole ligands, and the specific steps are as follows:

[0057] (1) Prepare a 10 mM PBS solution (pH 9.0), and use it to prepare receptor solution A: 10 μM ZnTSPP solution and ligand solution B: 100 μM imidazole solution;

[0058] (2) fully rinsing the microfluidic channel with a buffer solution;

[0059] (3) The microchannel was configured with three inlets from left to right, and PBS / ZnTSPP / PBS were introduced respectively, and the fluorescence data of the sampling point 4.7 mm below the inlet of the microfluidic channel were recorded (e.g. Figure 6 A and 6B (a) line), as blank control;

[0060] (4) The microchannel was configured with three inlets from left to right, and imidazole / ZnTSPP / PBS were introduced respectively, and the fluorescence data of the sampling point 4.7 mm below the inlet of the microfluidic channel were recorded (e.g. Figure 6 Line (b) in A);

[0061] (5) PBS / ZnTSPP / imidazole were introduced into the microchannel in the order of the three inlets from left to right, and the fluorescence data of the sampling point 4.7 mm below the inlet of the microfluidic channel were recorded (e.g. Figure 6 (b) line in B).

[0062] Depend on Figure 7 It can be seen that the lateral flow of imidazole breaks the mirror symmetry of the normal distribution of ZnTSPP in the lateral direction. It is biased towards the side of the imidazole gradient, and the peak drifts to the left as a whole by 16 μm (peak spacing), which reveals that the ZnTSPP flow as a whole actively migrates into the original imidazole area and migrates out of PBS at the same time, which proves that ZnTSPP can effectively perform chemical chemotaxis and chemotaxis far-across micrometer distances to chase imidazole.

Claims

1. A microfluidic optical observation system for small molecule chemical tropism behavior, characterized in that: Includes optical platform and microfluidic chip; The microfluidic chip comprises a PDMS chip and a cover glass for microscopic imaging, which are seamlessly bonded; the PDMS chip is provided with more than two microchannel inlets, a reaction chamber and more than one microchannel outlet; each microchannel inlet is respectively injected with a receptor solution or a ligand solution or a buffer solution, the inlet sequence of the receptor solution, the ligand solution and the buffer solution is randomly combined and arranged, each solution converges at an inlet confluence point, enters the reaction chamber, and finally flows out from the microchannel outlet, the receptor is ZnTSPP, and the ligand is imidazole; The optical platform comprises an inverted microscope, an external three-channel shutter driver connected to a camera for framing, and a light-emitting diode light source; incident light passes through a fluorescence excitation block and is focused on the bottom of a PDMS chip via an objective lens, and a CCD camera captures the fluorescence imaging of the channel.

2. The microfluidic optical observation system according to claim 1, characterized in that: The CCD camera is connected to a computer via an image acquisition card, and the acquired fluorescence images are transmitted to the computer for processing.

3. The microfluidic optical observation system according to claim 1, characterized in that: The buffer solution is PBS buffer or HEPES buffer.

4. The microfluidic optical observation system according to claim 1, characterized in that: The microfluidic chip is provided with three microchannel inlets, one inlet confluence point, one reaction chamber and one microchannel outlet.

5. A method for observing the chemical tendency behavior of small molecules using the microfluidic optical observation system according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1, preparing a receptor solution, a ligand solution and a buffer solution, wherein the receptor solution and the ligand solution are prepared using a buffer solution; Step 2, firstly rinse the microfluidic channel with a buffer solution, and then pass the receptor solution, the ligand solution and the buffer solution into the microchannel inlet of the microfluidic chip according to the experimental design; Step 3, turn on the light-emitting diode light source to excite the receptor to produce photoluminescence. The fluorescence is transmitted to the CCD camera through the imaging objective to capture the fluorescence imaging of the channel, record the fluorescence images at the inlet confluence point and different sites of the microfluidic channel, and transmit the recorded images to the computer through the image acquisition card for processing and analysis.

Images