CN119076071A - Liquid crystal optofluidic chip, optical imaging device and application - Google Patents
Liquid crystal optofluidic chip, optical imaging device and application Download PDFInfo
- Publication number
- CN119076071A CN119076071A CN202411370845.XA CN202411370845A CN119076071A CN 119076071 A CN119076071 A CN 119076071A CN 202411370845 A CN202411370845 A CN 202411370845A CN 119076071 A CN119076071 A CN 119076071A
- Authority
- CN
- China
- Prior art keywords
- liquid crystal
- optical
- channel
- sample
- silicon wafer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Liquid Crystal (AREA)
Abstract
The invention provides a liquid crystal optofluidic chip, an optical imaging device and application, wherein the liquid crystal optofluidic chip comprises a substrate, a closed microstructure is arranged on the substrate, the microstructure comprises a plurality of sample cells and a plurality of optical cells which are the same in number and correspond to each other one by one, each sample cell is communicated with one optical cell corresponding to the sample cell through a micro-mixing channel, each sample cell is provided with a first inlet and outlet pipeline, each optical cell is provided with a second inlet and outlet pipeline, and the micro-mixing channel comprises a first channel and a second channel which are communicated with each other, wherein the first channel is provided with a sample introduction pipeline. The invention combines the liquid crystal birefringence performance based on the aptamer conformational change principle, further develops the liquid crystal optofluidic chip and the optical imaging device, observes different liquid crystal optical appearances of the sample on the liquid crystal optofluidic chip through a polarizing microscope, further realizes the purpose of detecting exosomes in the sample, and has the advantages of high flux, rapid detection and high sensitivity.
Description
Technical Field
The invention relates to the technical field of biological chips and cell detection, in particular to a liquid crystal optofluidic chip, an optical imaging device and application.
Background
The determination of the exosome content has wide application in early diagnosis and treatment of cancer of patients, and has the application fields of early diagnosis of cancer and eclampsia, parkinsonism, platelet function, coagulation and tumor vaccine in the research of clinical test, and the application fields of early diagnosis of cancer, tumor prognosis monitoring, immunotherapy, diagnosis of polycystic ovary syndrome diseases, tissue repair and the like in the recruited clinical test. Therefore, research and detection of exosomes have been receiving extensive attention.
Methods reported for detecting exosomes include Transmission Electron Microscopy (TEM), flow Cytometry (FCM), western Blot detection, nanoparticle-spiking techniques (NTA), and the like. By comprehensively analyzing the above methods, we find that the detection methods have certain defects in the detection of exosomes in biological samples, such as poor detection limit range of exosomes in Flow Cytometry (FCM), can detect exosomes after enrichment and concentration improvement, and show general sensitivity. In addition, some methods such as Transmission Electron Microscopy (TEM), chemical Nanoparticle Tracing (NTA) show desirable analytical performance, but often require precision instrumentation and more stringent human handling. In addition, the existing methods have long testing period, labor intensity, complex instruments and are not suitable for rapid, sensitive and selective detection of exosomes.
The liquid crystal biosensor uses liquid crystal molecules as a signal conversion element of the biosensor, and converts specific recognition binding events among the biomolecules into amplified optical signals for output. The detection principle is that the arrangement orientation of liquid crystal molecules on a functional sensing interface can be changed along with the addition of specific target molecules, so that the change of the liquid crystal to the light refraction direction is caused, and the change of the appearance of the optical image of the liquid crystal under a polarizing microscope is further amplified. The method has the advantages of simple operation, sensitive reaction and the like, and has certain application in the control of small molecular medicines and the detection of proteins. The present invention seeks to explore liquid crystal biosensors that can be used for detection of organisms such as exosomes.
Disclosure of Invention
The invention provides a liquid crystal optofluidic chip, an optical imaging device and application thereof, and aims to develop a novel liquid crystal optofluidic chip and an optical imaging device and explore the possibility of detecting the liquid crystal optofluidic chip and the optical imaging device in an exosome.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
The invention provides a liquid crystal optofluidic chip, which comprises a substrate, wherein the substrate is provided with a closed microstructure, the microstructure comprises a plurality of sample cells and a plurality of optical cells, the sample cells and the optical cells are identical in number and correspond to each other one by one, each sample cell is communicated with one optical cell corresponding to the sample cell through a micro-mixing channel, each sample cell is provided with a first inlet and outlet pipeline, each optical cell is provided with a second inlet and outlet pipeline, and each micro-mixing channel comprises a first channel and a second channel which are communicated with each other, wherein the first channel is provided with a sample introduction pipeline.
Preferably, a plurality of small molecule filtering holes are arranged in the sample cell, the small molecule filtering holes are positioned at one side of the sample cell connected with the micro-mixing channel, and/or a plurality of hole grooves for loading optical components are arranged in the optical cell, preferably, the cross section of each hole groove is rectangular, regular hexagon, regular triangle and circular structure, and more preferably, a plurality of hole grooves are arranged to form a square matrix.
Preferably, the first channel is a hierarchical tree-shaped channel, and/or the second channel comprises a plurality of third channels, and each third channel is respectively communicated with one of the sample cells and one of the optical cells corresponding to the sample cell.
Preferably, the third channel is communicated with the sample cell and the optical cell through pipelines respectively, and more preferably, the third channel is a curve channel.
Preferably, the substrate is a PDMS flexible substrate, and further preferably, the chip further comprises a housing, and more preferably, the housing is a transparent structure.
In a second aspect, the present invention provides a method for preparing the liquid crystal optical flow control chip according to the first aspect, including the following steps:
step 1, designing a microstructure pattern through a mask plate;
Step 2, transferring the microstructure pattern on the mask plate to a silicon wafer;
and 3, transferring the microstructure pattern on the silicon wafer to a substrate.
Preferably, in the step 2, the microstructure pattern on the mask plate is transferred onto a silicon wafer, and the method includes the following steps:
Step 2-1, the silicon wafer is glued once, preferably a spin coating mode is adopted, the spin coating speed is 500rpm, the acceleration is 500rpm s-1, the time is 5s, and preferably SU8-3000 series photoresist is adopted;
Step 2-2, secondarily gluing the silicon wafer, preferably adopting a spin coating mode, wherein the spin coating speed is 1800-2500rpm, the acceleration is 1800-2500 rpm.s < -1 >, and the time is 60s;
Step 2-3, performing step-by-step pre-baking on the silicon wafer subjected to the secondary gluing, cooling to room temperature, stacking the silicon wafer and the mask plate, performing ultraviolet exposure, and performing step-by-step post-baking on the silicon wafer;
step 2-4, developing the silicon wafer, preferably developing by adopting a PGMEA developer for 4-10min;
Step 2-5, heating the silicon wafer at 120-150 ℃ for 20-30 min, and cooling to room temperature to obtain the silicon wafer with the microstructure;
Further preferably, in the step 2-3, the step-by-step pre-baking includes heating to 50+ -5 ℃ for 10-15 min at room temperature in the first step, heating to 65+ -5 ℃ for 10-15 min in the second step, heating to 95+ -5 ℃ for 10-15 min in the third step, cooling to room temperature in the fourth step, and/or,
The step post-baking includes the first step of heating to 50+ -5 deg.C for 10min at room temperature, the second step of heating to 65+ -5 deg.C for 10min at 50+ -5 deg.C, the third step of heating to 80+ -5 deg.C for 10min at 80+ -5 deg.C, the fourth step of cooling to room temperature at 80+ -5 deg.C, and/or,
The control parameters of the ultraviolet exposure are that the exposure time is 25-30 s, and the ultraviolet intensity is 120-130 mu M cm -2.
Preferably, in the step 3, the microstructure pattern on the silicon wafer is transferred to a substrate, wherein the substrate is a PDMS flexible substrate, and two pieces of PDMS flexible substrates are adopted, wherein one piece of PDMS flexible substrate is provided with a sample cell, an optical cell and a first channel in the microstructure, and the other piece of PDMS flexible substrate is provided with a second channel in the microstructure, and the method specifically comprises the following steps:
Step 3-1, mixing PDMS and a curing agent according to a mass ratio of 10:1, and then eliminating bubbles to obtain liquid PDMS;
Step 3-2, covering the liquid PDMS on the silicon wafer for heat curing, wherein the curing parameters preferably comprise the temperature of 80-90 ℃ and the time of 30-40 min, cooling to room temperature, and stripping the PDMS from the silicon wafer after curing to obtain two PDMS flexible substrates with corresponding microstructures;
Step 3-3, bonding two PDMS flexible substrates, wherein the PDMS flexible substrate with a sample cell, an optical cell and a first channel in the microstructure is positioned at the lower layer, and the PDMS flexible substrate with a second channel in the microstructure is positioned at the upper layer;
further preferably, the preparation method further comprises loading the bonded PDMS flexible substrate into a housing.
In a third aspect, the present invention provides an optical imaging device comprising the liquid crystal optofluidic chip of the first aspect and a polarizing microscope for observing optical phenomena in an optical cell of the liquid crystal optofluidic chip.
In a fourth aspect, the present invention provides a use of the liquid crystal optofluidic chip of the first aspect and/or the optical imaging device of the third aspect for biological detection. Organisms encompass small biological molecules such as cells, viruses, proteins, nucleic acids, and the like.
In a fifth aspect, the present invention provides a method for detecting an exosome, which adopts the optical imaging apparatus according to the third aspect, and includes the following steps:
Mixing liquid crystal with a cationic surfactant solution to obtain a liquid crystal mixed solution, wherein the concentration of the cationic surfactant solution is less than 0.025mM;
Injecting an organism to be detected into a sample pool, introducing the organism to be detected into a micro-mixing channel, and simultaneously injecting an aptamer solution into the micro-mixing channel to be mixed with the organism to be detected, wherein the concentration of the aptamer solution is preferably 45-50 nmol/L;
introducing the mixture further into an optical cell while injecting a liquid crystal mixture into the optical cell to be further mixed with the mixture;
and (3) placing the optical cell under a polarizing microscope for detection, and performing exosome analysis through an optical picture obtained by detection.
Preferably, the cationic surfactant is selected from at least one of cetyltrimethylammonium bromide, dodecyldimethylbenzyl ammonium bromide, octadecyl trimethyl ammonium chloride, methyl ditalloyl ethyl-2-hydroxyethyl methyl ammonium sulfate.
Compared with the prior art, the invention has the beneficial effects that:
The invention combines the traditional microfluidic technology with the liquid crystal optical sensing technology, develops a brand-new liquid crystal optofluidic chip and an optical imaging device which can realize high-flux detection based on the aptamer conformational change principle and the liquid crystal birefringence performance, and further realizes the purpose of detecting exosomes in the sample by observing different liquid crystal optical morphologies of the sample on the liquid crystal optofluidic chip through a polarizing microscope. The liquid crystal optical flow control chip and the optical imaging device are used for detecting exosomes, have the advantages of high flux, rapid detection and high sensitivity, have simple whole operation process, less reagent consumption and short sample processing time, solve the technical problems of complex existing detection methods, high cost and the like, and widen the application range of the liquid crystal optical flow control chip.
Drawings
Fig. 1 is a schematic structural diagram of a liquid crystal optical flow control chip in embodiment 1 of the present invention.
FIG. 2 is a schematic diagram of the structure of a sample cell in example 1 of the present invention.
FIG. 3 is a schematic diagram of the structure of an optical cell in embodiment 1 of the present invention.
Fig. 4 is a diagram illustrating a design structure of a mask in embodiment 2 of the present invention.
In fig. 1 to 4, a square matrix formed by a 1-substrate, a 2-sample cell, a 3-optical cell, a 4-inlet and outlet pipeline I, a 5-inlet and outlet pipeline II, a 6-first channel, a 7-second channel, an 8-sample introduction pipeline, a 9-third channel, a 10-pipeline connecting the sample cell and a micro-mixing channel, a 11-pipeline connecting the micro-mixing channel and the optical cell, a 12-split port I, a 13-split port II, a 14-split port III and a 15-hole groove, a 16-macromolecule filter hole and a 17-square column are shown.
Fig. 5 is a diagram showing different cross-sectional structures (rectangular arrays are formed) of the hole grooves in embodiment 2 of the present invention, wherein the hole grooves are regular hexagons in a diagram a, square in a diagram B, regular triangles in a diagram C, and circular in a diagram D.
FIG. 6 shows the polarization microscope photograph of the liquid crystal optical flow control chip with different concentrations of cetyltrimethylammonium bromide added to the liquid crystal optical flow control chip and the gray scale gradient chart thereof, wherein the concentration of cetyltrimethylammonium bromide solution is 0.02mM, 0.0225mM, 0.025mM, 0.03mM and 0.04mM, wherein the polarization microscope photograph of the liquid crystal modified by the cetyltrimethylammonium bromide solution of the embodiment 3 is shown in the A chart, the polarization microscope photograph of the liquid crystal modified by the cetyltrimethylammonium bromide solution of the B chart, the polarization microscope photograph of the liquid crystal modified by the cetyltrimethylammonium bromide solution of the C chart, the polarization microscope photograph of the liquid crystal modified by the cetyltrimethylammonium bromide solution of the D chart, the polarization microscope photograph of the liquid crystal modified by the cetyltrimethylammonium bromide solution of the E chart, and the gray scale gradient chart.
FIG. 7 is a schematic diagram of a mechanism for detecting an exosome by using an optical imaging device in example 3 of the present invention, wherein the diagram A is a liquid crystal optical topography of an optical cell in which the exosome and the aptamer are not present, the diagram B is a liquid crystal optical topography of an optical cell in which the aptamer is present, and the diagram C is a liquid crystal optical topography of an optical cell in which the exosome and the aptamer are present.
FIG. 8 is a photograph of a gray scale of a polarized light microscope of an optical cell in which a nucleic acid aptamer solution and a 0.02mM cetyltrimethylammonium bromide solution of different concentrations in example 4 of the present invention were added to a liquid crystal optofluidic chip, wherein the concentrations of the nucleic acid aptamer solution were 50nmol/L, 40nmol/L, 20nmol/L, and 10nmol/L, respectively, a photograph of a polarized light microscope of a liquid crystal of a reaction solution of the nucleic acid aptamer solution 10nmol/L and a reaction solution of the 0.02mM cetyltrimethylammonium bromide solution, a photograph of a polarized light microscope of a liquid crystal of a reaction solution of the nucleic acid aptamer solution 20nmol/L and a reaction solution of the 0.02mM cetyltrimethylammonium bromide solution, a photograph of a liquid crystal microscope of a reaction solution of the nucleic acid aptamer solution 40nmol/L and a reaction solution of the 0.02mM cetyltrimethylammonium bromide solution, and a photograph of a gray scale of the nucleic acid aptamer solution 40nmol/L, respectively.
FIG. 9 is a liquid crystal polarization microscope photograph of an optical cell in which an exosome standard solution, a 0.02mM cetyltrimethylammonium bromide solution and a 100nmol/L aptamer solution of different concentrations were added to a liquid crystal optofluidic chip in example 5 of the present invention, wherein FIG. A is a liquid crystal polarization microscope photograph of the exosome standard solution having a concentration of 9.8X10A/L, FIG. B is a liquid crystal polarization microscope photograph of the exosome standard solution having a concentration of 1.95X10A/L, FIG. C is a liquid crystal polarization microscope photograph of the exosome standard solution having a concentration of 3.9X10A/L, and FIG. D is a linear curve of the gray-scale value vs. exosome concentration of the liquid crystal photograph under the polarization microscope.
FIG. 10 is a polarization diagram of an experiment for detecting exosomes by the optical imaging device in example 6 of the present invention, wherein FIG. A is a diagram of introducing liquid crystal, FIG. B is a diagram of introducing an aptamer, and FIG. C is a diagram of introducing a mixed solution of exosomes and aptamer.
Detailed Description
4-Cyano-4' -pentylbiphenyl (5 CB) in the examples of the present invention was purchased from Tokyo Ke Chemicals. In addition, the specific conditions are not noted in the examples, and are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.
The nucleic acid aptamer sequence used hereinafter is CD63 aptamer (shown in SEQ ID NO. 1): 5'-CACCCCACCTCGCTCCCGTGACACTAATGCTATTTTTTTTTTTTTTT-3' -FAM.
Among the nucleic acid aptamers that may also be used are CD63-2 aptamer (shown as SEQ ID NO. 2): 5'-TAACCACCCCACCTCGCTCCCGTGACACTAATGCTAATTCCAA-3';
CD9-26aptamer (shown in SEQ ID NO. 3):
5′-ATAGTCCCTTGGCGTGCTTCACAACCTTGAACTTGACGCAGGATCGTTCAGTGCGCA CTAGAGCAGGTACGGTGTCA-3′。
The PBS buffer in the examples had the composition 137mol/L NaCl,2.7mmol/L KCl,4.3mmmol/LNa 2HPO4,1.4mmol/L KH2PO4, water as solvent and dilute hydrochloric acid to adjust the pH to 7.4.
Example 1
The embodiment provides a liquid crystal optofluidic chip, the structure of which is shown in fig. 1-5, comprising a PDMS flexible substrate 1, wherein the substrate 1 is provided with a closed microstructure, the microstructure comprises a plurality of sample cells 2 and a plurality of optical cells 3 which are the same in number and in one-to-one correspondence, and each sample cell 2 is communicated with one optical cell 3 corresponding to the sample cell 2 through a micro-mixing channel. Each sample cell 2 is provided with a first inlet and outlet pipeline 4, each optical cell 3 is provided with a second inlet and outlet pipeline 5, and each micro-mixing channel comprises a first channel 6 and a second channel 7 which are communicated with each other, wherein the first channel 6 is provided with a sample inlet pipeline 8. The number of the sample cells 2 and the optical cells 3 is not particularly required, and can be specified according to flux requirements, such as 8, 9,10, 20, 30, 50, 100 and the like, so that the liquid crystal optofluidic chip can realize high-flux biological sample measurement with different concentrations. In all embodiments of the invention, a microstructure of 8 sample cells 2, 8 optical cells 3 is used.
Further, as shown in fig. 2, a plurality of small molecule filtering holes 16 are provided in the sample cell, and the small molecule filtering holes 16 are located at one side of the sample cell 2 connected with the micro-mixing channel. In this embodiment, the small molecular filter hole 6 is formed by disposing a plurality of square columns 17 in the sample cell, and the small molecular filter hole 16 is formed between the square columns 17. In this embodiment, the size of the small molecule filter hole 16 is set below 1um, the small molecule detection (in fig. 2, the spherical particle represents the small molecule detection, the plate-like shape and the central spherical shape represent the cell culture) is usually in the nanometer scale, for example, the secretion such as exosomes is in the scale of 100-150 nm, the small molecule filter hole allows small molecules to pass through, and the large molecule cells are reserved, wherein the large molecule cells refer to cells with the size above 10 um. The size of the small molecule filter hole 16 can be flexibly adjusted according to the size of the component to be detected in the sample. In addition, the dimensions of the sample cell are not particularly limited, and in this example, the sample cell 2 is 1.7cm long, 3mm wide and 100um high.
As shown in fig. 3 and 5, the optical cell 3 is provided with a plurality of holes 15 for loading optical components, the cross section of each hole can be designed into a rectangular (square), a regular hexagon (regular hexagonal), a regular triangle (regular triangular), and a circular structure, and correspondingly, different holes are respectively formed into a square, a regular hexagonal, a regular triangular, and a cylindrical structure, and the optical images formed under the polarizing microscope are respectively rectangular, regular hexagonal, regular triangle, and circular. In this embodiment, a square hole groove structure is adopted, and the optical image formed by each hole groove in fig. 3 to 6 is square. In addition, the optical images formed by arranging the holes and the grooves into regular shapes are uniform and convenient to observe, so that the holes and the grooves in the embodiment are arranged to form a square matrix. In addition, the dimensions of the optical cell are not particularly limited, and in this embodiment, the optical cell 3 is 1.6cm long, 3mm wide, and 100um high. The side of the square hole groove is 80 mu m long and 25 mu m high.
Further, as shown in fig. 4, the first channel 6 is a hierarchical tree-shaped channel, the number of the hierarchical layers is equal to or greater than 2, the number of the hierarchical layers is preferably controlled between 2 and 5, and the uniformity and the efficiency of sample injection can be controlled. In all embodiments of the present invention, a 3-level tree-shaped channel is adopted, the first level is provided with a sample inlet pipe 8 and 2 splitting ports one 12, the second level is provided with 4 splitting ports two 13, the third level is provided with 8 splitting ports three 14, and the splitting ports three 14 are communicated with the third channel 9 of the second channel 7. The components to be introduced enter the second level through the first split port 12 after entering the sample injection pipeline 8, are split into the third level through the second split port 13, and enter the second channel 7 through the third split port 14.
The second channels 7 comprise a plurality of third channels 9, and each third channel 9 is respectively communicated with one of the sample cells 2 and one of the optical cells 3 corresponding to the sample cell 2, i.e. the number of third channels 9 is the same as the number of sample cells 2 and the number of optical cells 3. In all embodiments of the invention, 8 third channels 9 are used. The third channel 9 is connected to the sample cell 2 and the optical cell 3 via pipes 10,11, respectively. In this embodiment, each third channel 9 is configured such that a "mouth-shaped" channel is connected to a spiral channel, where the "mouth-shaped" channel is in one-to-one correspondence with and connected to the third split-port 14, and the mouth-shaped "channel is located on the side of the sample cell 2, and the spiral channel is located on the side of the optical cell 3.
In order to better protect the liquid crystal optofluidic chip, a shell can be arranged, and the shell and the chip can be of an integrated structure or a detachable structure. When the case and the chip are integrally formed, the case is preferably provided in a transparent structure. The housing is used to prevent the liquid crystal optofluidic chip from being contaminated or damaged, and the structure without the housing is shown in fig. 1.
The liquid crystal optical flow control chip of the embodiment can realize uniform mixing of different components in the micro-mixing channel, divide a multi-step complex experiment into a similar pipeline structure, accurately control the volume of injected solution, rapidly complete mixing, and solve the problems of volume error, complex operation and the like caused by manual mixing. The mixture is then collected into an optical zone, which may be simultaneously filled with optical components such as liquid crystals, thereby utilizing different optical phenomena to detect, identify and distinguish the substances in the components. The chip can simultaneously carry out multiple groups of experiments, simultaneously observe multiple groups of experimental phenomena, and is convenient, fast and efficient.
Example 2
The preparation method of the liquid crystal optofluidic chip of the embodiment 1 and the assembly of the optical imaging device are as follows:
(1) Geometric patterns with different sizes and shapes are drawn by adopting AutoCAD software, a mask plate with the size of 5 inches multiplied by 2.3mm is customized, a mask plate design diagram of a microstructure is shown in fig. 4, black represents an opaque part, and white represents a transparent part.
(2) The photoresist is coated on a silicon wafer by adopting SU8-3000 series photoresist, the spin coating is divided into two steps, the first step is that the speed is 500rpm, the acceleration is 500 rpm.s -1, the time is 5s, the second step is that the speed is 1800-2500rpm, the acceleration is 1800-2500 rpm.s -1, the time is 60s, the spin coating thickness is about 20-60 mu M, the step pre-baking is carried out, the room temperature is naturally cooled, the mask plate and the coated silicon wafer are aligned in a photoetching machine for ultraviolet exposure, the ultraviolet exposure parameters are that the exposure time is 25s, the ultraviolet intensity is 126 mu M.cm -2, the step post baking is carried out, the developing is carried out by adopting a PGMEA developer, the isopropanol is used for cleaning after the developing is completed, the photoetching condition is observed by a microscope after the nitrogen silicon wafer, and the film is hardened, the photoetching is kept on a 120 ℃ hot plate for 30min, and then the room temperature is naturally cooled.
The step-by-step pre-baking comprises the steps of heating to 50+/-5 ℃ for 10-15 min at room temperature in the first step, heating to 65+/-5 ℃ for 10-15 min in the second step, heating to 95+/-5 ℃ for 10-15 min in the third step, and cooling to the room temperature in the fourth step at 95 ℃.
The step post-baking comprises the steps of heating to 50+/-5 ℃ at room temperature for 10min in the first step, heating to 65+/-5 ℃ at 50+/-5 ℃ at 65+/-5 ℃ for 10min in the second step, heating to 80+/-5 ℃ at 80+/-5 ℃ for 10min in the third step, and cooling to room temperature in the fourth step at 80+/-5 ℃.
(3) The method comprises the steps of pouring PDMS into a mold, namely fully stirring and mixing PDMS and a curing agent according to a mass ratio of 10:1, then eliminating bubbles in vacuum to obtain liquid PDMS, uniformly pouring the liquid PDMS onto a silicon wafer, performing thermal curing, wherein curing parameters comprise a temperature of 90 ℃ and a time of 30min, naturally cooling to room temperature, peeling the PDMS from the silicon wafer after curing, cutting the PDMS into two pieces of PDMS, bonding, particularly bonding, wherein a sample pool, an optical pool and a PDMS flexible substrate of a first channel in a microstructure are positioned at a lower layer, a PDMS flexible substrate of a second channel in the microstructure is positioned at an upper layer, and cleaning a non-micro channel part on the PDMS flexible substrate, and a plasma cleaning machine is adopted for the instrument, wherein plasma gas is air, and the time is 6min. The microstructure was masked with 3M tape during the plasma cleaning process. The bonding process requires removal of masking material of the upper and lower substrates.
After bonding, the openings of the inlet and outlet pipelines I, the inlet and outlet pipeline II and the sample feeding pipeline and the openings of the connecting pipelines of the sample cell, the micro-mixing channel and the optical cell are arranged by using a puncher with the outer diameter of 1.25 mm. And obtaining the liquid crystal optical flow control chip for standby. The present embodiment does not load the housing to the liquid crystal optofluidic chip.
The optical imaging device is assembled by adopting a silica gel hose with the inner diameter of 0.5mm and the outer diameter of 1.5mm as an inlet and outlet pipeline I, an inlet and outlet pipeline II and a sample injection pipeline II, and the inlet and outlet pipeline I and the inlet and outlet pipeline II are also connected with an injection device which is a fluid conveying device consisting of an injection pump and an injection needle. And then adding a polarizing microscope, placing an optical area of the liquid crystal optofluidic chip under the polarizing microscope, enabling two linear polarizers of the polarizing microscope to vertically cross, receiving detection light beams by one side of an optical pool of the liquid crystal optofluidic chip, realizing detection according to the light beam change of the other side passing through the optical pool, and receiving an optical image of the liquid crystal optofluidic chip by using a CCD electronic element.
Example 3
Functional test of liquid crystal optofluidic chip:
Adopting the optical imaging device of the embodiment 2, firstly injecting liquid crystal into the optical cells of the liquid crystal optical flow control chip through the inlet and outlet pipeline II, wherein the adding amount is about 2 mu L of each optical cell;
The preparation of cetyltrimethylammonium bromide solution, which is prepared by dissolving cetyltrimethylammonium bromide in PBS buffer solution for 30min with ultrasonic dissolution time, and respectively preparing cetyltrimethylammonium bromide solution with concentration of 0.02mM, 0.0225mM, 0.025mM, 0.03mM and 0.04mM, wherein the cetyltrimethylammonium bromide solution with different concentrations is respectively added into an optical cell of a liquid crystal optical flow control chip through a second different inlet and outlet pipeline, the addition volume of each optical cell is 110 mu L, and the liquid crystal shows bright appearance within 5 min when the concentration of the cetyltrimethylammonium bromide solution is less than 0.025mM, and the liquid crystal shows dark appearance within 5 min when the concentration of the cetyltrimethylammonium bromide solution is more than or equal to 0.025 mM. Thus, the concentration of cetyltrimethylammonium bromide solution is controlled to <0.025mM, preferably 0.02mM. Cetyl trimethyl ammonium bromide is a cationic surfactant, and the invention can also select dodecyl dimethyl benzyl ammonium bromide, octadecyl trimethyl ammonium chloride and methyl ditalloyl ethyl-2-hydroxyethyl methyl ammonium sulfate.
In the above-described process, the dropping amount of the liquid crystal is not particularly limited as long as the prepared liquid crystal biosensor is a uniform dark field under a polarizing microscope.
The detection principle of the exosome by utilizing the optical imaging device is shown in figure 7, when the exosome and the aptamer are not present in the optical pool of the liquid crystal optical flow control chip, the cetyltrimethylammonium bromide monolayer film at the interface still maintains the vertical arrangement of liquid crystal molecules, a liquid crystal image shows a dark optical morphology under a polarized light microscope (figure 7A), when the aptamer is added, the concentration of the cetyltrimethylammonium bromide at the interface is reduced to destroy the originally formed cetyltrimethylammonium bromide monolayer film, so that the liquid crystal molecules are converted into parallel/inclined arrangement from the vertical arrangement, the liquid crystal image shows a bright optical morphology under the polarized light microscope (figure 7B), and when the mixed solution of the aptamer and the exosome is added, the cetyltrimethylammonium bromide monolayer film at the interface still maintains the vertical arrangement of the liquid crystal molecules, and the liquid crystal image shows the dark optical morphology under the polarized light microscope (figure 7C). Based on the above principle, the qualitative and quantitative detection process of exosomes can be obtained by combining the gray-concentration standard curve of example 5.
In addition, the sample cell can be used as a cell culture unit according to the requirement, and the hole groove for loading the optical component in the optical cell can be adjusted according to different requirements, so that the grid shape size in the formed liquid crystal image can be changed.
Example 4
Determination of aptamer concentration:
Adopting the optical imaging device of the embodiment 2, firstly injecting liquid crystal into the optical cells of the liquid crystal optical flow control chip through the inlet and outlet pipeline II, wherein the adding amount is about 2 mu L of each optical cell;
The CD63 aptamer lyophilized powder is dissolved in PBS buffer solution, and is uniformly mixed by vortex to prepare aptamer solutions with the concentrations of 50nmol/L, 40nmol/L, 20nmol/L and 10nmol/L respectively, the aptamer solutions with the different concentrations are uniformly mixed with 0.02mM cetyltrimethylammonium bromide solution (the preparation method is the same as that of example 3) by vortex, the mixture is incubated at 37 ℃ for 30min and then added into an array optical cell of a liquid crystal biosensor, the dropping volume of each optical cell is 110 mu L, and the observation is carried out by using a polarized light microscope, so that as shown in figure 8, when the concentration of the aptamer solution is more than or equal to 50nmol/L, the liquid crystal has an all bright morphology, and when the concentration of the aptamer solution is gradually reduced from 50nmol to 10nmol, the morphology of the liquid crystal is gradually converted from all bright to all dark, and the bright area range is gradually reduced. Therefore, the concentration of the nucleic acid aptamer solution is suitably controlled to 45 to 50nmol/L, preferably 50nmol/L.
Example 5
Obtaining an exosome gray-concentration standard curve:
Adopting the optical imaging device of the embodiment 2, firstly injecting liquid crystal into the optical cells of the liquid crystal optical flow control chip through the inlet and outlet pipeline II, wherein the adding amount is about 2 mu L of each optical cell;
Extracting exosomes, namely selecting 3 rd-4 th generation human umbilical cord Mesenchymal Stem Cells (MSC) with good state, replacing fresh serum-free culture medium when the MSC is fused to about 75%, culturing for 48 hours, and collecting supernatant. Placing the supernatant into a centrifuge tube, centrifuging at 4deg.C for 30min at 2000g, removing cell debris, filtering the retained supernatant with a sterile filter membrane (0.45 μm), placing into an overspeed centrifuge tube, and centrifuging at 4deg.C for 70min at 1000g to obtain exosome concentrate. The concentrate was transferred to a new ultracentrifuge tube and centrifuged at 4℃and 100000g for 70min. The pellet was diluted with PBS and centrifuged again at 4℃and 100000g for 70min. The obtained exosome concentrate was filtered through sterile filter (0.22 μm), resuspended in a small volume of PBS and refrigerated at-80℃for subsequent experiments to obtain the laboratory self-made exosome standard.
The method comprises the steps of dissolving an exosome standard substance in PBS buffer solution in a vortex manner to prepare exosome standard solutions with the concentration of 3.9X10-7/mL, 1.95X10-7/mL and 9.8X10-6/mL, adding CD63 nucleic acid aptamer solution into the exosome standard solutions with different concentrations, uniformly mixing, enabling the volume ratio of the nucleic acid aptamer to the exosome standard solution to be 1:1, enabling the final concentration of the nucleic acid aptamer in the mixed solution to be 100nmol/L, enabling the nucleic acid aptamer to react for 60min at 4 ℃, obtaining a reaction solution ①, enabling the reaction solution ① to be uniformly mixed with cetyltrimethylammonium bromide solution, enabling the volume ratio of the cetyltrimethylammonium bromide solution to be 1:1, enabling the final concentration of the cetyltrimethylammonium bromide in the mixed solution to be 0.02mM, and enabling the reaction solution to react for 30min at 37 ℃, and obtaining a reaction solution 3235. The reaction solution ② was added into the optical cells, the drop volume of each optical cell was 110. Mu.L, and observation was performed using a polarizing microscope, as shown in FIG. 9, when the concentration of the exosome standard solution was not less than 3.9X10A 7/mL, the liquid crystal exhibited a dark morphology, when the concentration of the exosome standard solution was not more than 9.8X10A 6/mL, the liquid crystal exhibited a bright morphology, and when the concentration of the exosome standard solution was gradually decreased from 3.9X10A 7/mL to 9.8X10A 6/mL, the liquid crystal morphology was gradually bright from total darkness. The collected optical images were subjected to pixel analysis by MATLAB (version 9.6.0, r2019 a) software to obtain the average gray values of the liquid crystal images and plotted by originpro9.1 software. The exosome has a good linear relationship between the gray value of the liquid crystal picture and the exosome concentration in the concentration range of 9.8X10-6/mL to 3.9X10-7/mL, and the correlation coefficient is 0.9996 (D diagram in FIG. 9). The detection limit of the liquid crystal biosensor for detecting exosomes is 1.86×10ζ6/ml.
Example 6
Exosome samples were tested using the optical imaging device of example 2:
Firstly, injecting liquid crystal into the optical cells of the liquid crystal optical flow control chip through the inlet and outlet pipelines II, wherein the addition amount is about 2 mu L of each optical cell, forming a liquid crystal film with a certain thickness, and simultaneously anchoring the liquid crystal molecules on the upper interface of the liquid crystal film to be vertically arranged. As shown in fig. 10, panel a, is a POM plot of a liquid crystal vertical anchoring microstructure.
Then, 0.01mM cetyltrimethylammonium bromide solution was added to the first syringe needle, and the mixture was fixed in the first syringe pump, and the injection volume was 30. Mu.L.
Exosomes were obtained by the method of extraction of exosomes according to example 5, and resuspended in PBS to a concentration of 3.9X10A 7/mL, added to injection needle two, fixed in injection pump two and injected at a volume of 30. Mu.L.
The injection needle for preparing the CD63 aptamer into a solution with the concentration of 40nmol/L by using PBS buffer solution is fixed in an injection pump, and the injection volume is 60 mu L.
The flow rates of the 3 liquids respectively injected into the optical cell, the sample cell and the micro-mixing channel are 25 mu L.min-1, 150 mu L.min-1 and 80 mu L.min-1, and finally the liquids are mixed in the optical cell, and the obtained results are shown as a diagram B and a diagram C in FIG. 10 by adopting a polarizing microscope, and the acquired optical images are subjected to pixel analysis by MATLAB (version 9.6.0, R2019 a) software, and the concentration of the exosomes is calculated to be 3.9X10A/mL by combining an exosome gray-concentration standard graph. The exosome is further prepared into a solution with the concentration of 4.5X10-7/mL, 5.0X10-7/mL, 5.5X10-7/mL and 6.0X10-7/mL, the experimental process is repeated, repeated verification of results is carried out, the calculation results are consistent with the target concentration, and the example results prove that the liquid crystal sensor has high detection accuracy when being used for exosome.
The result shows that the liquid crystal biosensor component can effectively detect exosomes in a liquid sample, and the method can rapidly, sensitively, simply and conveniently detect trace exosomes in an array manner, and the whole detection process takes about 10 minutes.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (12)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411370845.XA CN119076071A (en) | 2024-09-27 | 2024-09-27 | Liquid crystal optofluidic chip, optical imaging device and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411370845.XA CN119076071A (en) | 2024-09-27 | 2024-09-27 | Liquid crystal optofluidic chip, optical imaging device and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN119076071A true CN119076071A (en) | 2024-12-06 |
Family
ID=93692736
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411370845.XA Pending CN119076071A (en) | 2024-09-27 | 2024-09-27 | Liquid crystal optofluidic chip, optical imaging device and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119076071A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120163094A (en) * | 2025-05-16 | 2025-06-17 | 中国人民解放军国防科技大学 | Preparation method and application of liquid colloidal crystal thin film material |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040185551A1 (en) * | 2003-03-20 | 2004-09-23 | Northeastern Ohio Universities College Of Medicine | Self-contained assay device for rapid detection of biohazardous agents |
| CN103278449A (en) * | 2013-06-19 | 2013-09-04 | 中国科学院电子学研究所 | Multi-channel optical detection device |
| US20170131266A1 (en) * | 2014-03-19 | 2017-05-11 | The Regents Of The University Of Colorado, A Body Corporate | Binding Detection Using Liquid Crystal |
| CN114235714A (en) * | 2022-02-28 | 2022-03-25 | 之江实验室 | Method for realizing one-step nucleic acid detection by using liquid crystal sensor |
| CN116087112A (en) * | 2023-01-03 | 2023-05-09 | 南方科技大学 | Liquid crystal-based antimony ion detection sensor, preparation method thereof and detection platform |
| CN117805100A (en) * | 2023-12-29 | 2024-04-02 | 苏州汉朗东越科技有限公司 | Liquid crystal detection platform based on micro-fluidic control and CRISPR/Cas system |
-
2024
- 2024-09-27 CN CN202411370845.XA patent/CN119076071A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040185551A1 (en) * | 2003-03-20 | 2004-09-23 | Northeastern Ohio Universities College Of Medicine | Self-contained assay device for rapid detection of biohazardous agents |
| CN103278449A (en) * | 2013-06-19 | 2013-09-04 | 中国科学院电子学研究所 | Multi-channel optical detection device |
| US20170131266A1 (en) * | 2014-03-19 | 2017-05-11 | The Regents Of The University Of Colorado, A Body Corporate | Binding Detection Using Liquid Crystal |
| CN114235714A (en) * | 2022-02-28 | 2022-03-25 | 之江实验室 | Method for realizing one-step nucleic acid detection by using liquid crystal sensor |
| CN116087112A (en) * | 2023-01-03 | 2023-05-09 | 南方科技大学 | Liquid crystal-based antimony ion detection sensor, preparation method thereof and detection platform |
| CN117805100A (en) * | 2023-12-29 | 2024-04-02 | 苏州汉朗东越科技有限公司 | Liquid crystal detection platform based on micro-fluidic control and CRISPR/Cas system |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120163094A (en) * | 2025-05-16 | 2025-06-17 | 中国人民解放军国防科技大学 | Preparation method and application of liquid colloidal crystal thin film material |
| CN120163094B (en) * | 2025-05-16 | 2025-08-12 | 中国人民解放军国防科技大学 | Preparation method and application of liquid colloid crystal film material |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11045806B2 (en) | Integrated type microfluidic electrochemical biosensor system and method for rapid biochemical analysis | |
| CN109499634B (en) | Microfluidic chip and preparation method and detection method thereof | |
| CN109499633B (en) | Bedside diagnosis micro-fluidic chip and preparation method and detection method thereof | |
| TWI743430B (en) | Self-driven microfluidic chip for rapid influenza a detection | |
| CN202066861U (en) | Special microfluidic chip used for diagnosing lung cancer rapidly | |
| US10981164B2 (en) | Chip and application thereof | |
| US10144911B2 (en) | Method and device for separation of particles and cells using gradient magnetic ratcheting | |
| CN119076071A (en) | Liquid crystal optofluidic chip, optical imaging device and application | |
| CN108344866A (en) | A kind of fluidic chip detecting system and the method based on system progress sample detection | |
| Dhondt et al. | Preparation of multi-omics grade extracellular vesicles by density-based fractionation of urine | |
| CN113447470B (en) | Surface-enhanced Raman scattering substrate, detection chip and preparation method | |
| CN109735439A (en) | Self-driven micro-fluidic detection chip and preparation method and application thereof | |
| Xu et al. | A novel microfluidic chip for fast, sensitive quantification of plasma extracellular vesicles as biomarkers in patients with osteosarcoma | |
| Li et al. | Multiple on-line active valves based centrifugal microfluidics for dynamic solid-phase enrichment and purification of viral nucleic acid | |
| CN113528310B (en) | A bionic microfluidic chip for simulating cervical microenvironment and preparation method thereof | |
| JP3041423B1 (en) | Polymerase chain reaction device using integrated microwell | |
| CN107442186A (en) | Micro-fluidic chip, the analytical equipment based on micro-fluidic chip and analysis method | |
| Qiu et al. | Efficient EVs separation and detection by an alumina-nanochannel-array-membrane integrated microfluidic chip and an antibody barcode biochip | |
| CN115219437B (en) | A flexible array microchannel liquid crystal sensor, preparation method thereof, liquid crystal filling method and application thereof | |
| CN211402400U (en) | Exosome identification device | |
| CN113687061B (en) | Digital Quantitative Detection System of Biological Targets Based on Virtual Segmentation Method | |
| WO2021233186A1 (en) | Biological target digital quantitative detection method and system based on virtual segmentation technology | |
| CN113322156B (en) | Bionic micro-fluidic chip for simulating fallopian tube microenvironment and preparation method thereof | |
| CN201449391U (en) | A syphilis diagnostic device containing gold colloidal antigen-sensitive membrane-coated electrodes | |
| CN116952811B (en) | Method for detecting integrity of exosome membrane based on nano-flow detector and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |