LU502571B1 - Microfluidic chip integrating exosome separation and detection and method thereof - Google Patents

Microfluidic chip integrating exosome separation and detection and method thereof Download PDF

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Publication number
LU502571B1
LU502571B1 LU502571A LU502571A LU502571B1 LU 502571 B1 LU502571 B1 LU 502571B1 LU 502571 A LU502571 A LU 502571A LU 502571 A LU502571 A LU 502571A LU 502571 B1 LU502571 B1 LU 502571B1
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Luxembourg
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separation
exosomes
detection
area
antibody
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LU502571A
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German (de)
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Yongyu Liu
Kun Deng
Xuedong Tong
Yong Zhang
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The Third Affiliated Hospital Of Cqmu Gener Hospital
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    • 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
    • B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/4915—Blood using flow cells
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Ecology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
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  • Dispersion Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention provides a microfluidic chip integrating exosome separation and detection and a method thereof, belonging to the field of biomedicine. The microfluidic chip comprises a sample inlet, a separation area, a detection area and a quality control area according to the connection order; the channel of the separation area is coated with antibody for capturing exosomes, the detection area is coated with GPC-1 antibody, and the quality control area is coated with CD63 antibody. According to the invention, a microfluidic chip integrating exosomes separation and detection is constructed, and the microchannel in the separation area is coated with Tim-4, which can be used for capturing tumor-specific exosomes surface markers and realizing the separation of tumor-specific exosomes; the detection area is coated with GPC-1 antibody, and the quality control area is coated with CD63 antibody. CD63 antibody is a universal marker for detecting exosomes, which is used as quality control.

Description

DESCRIPTION LU502571
MICROFLUIDIC CHIP INTEGRATING EXOSOME SEPARATION AND DETECTION AND METHOD THEREOF
TECHNICAL FIELD The invention relates to the field of biomedicine, in particular to a microfluidic chip integrating exosome separation and detection and a method thereof.
BACKGROUND The incidence and mortality of malignant tumors are high, and its prevention and treatment is a big problem faced by all countries in the world. At present, the clinical diagnosis of tumor mainly depends on tissue biopsy, serological screening and imaging examination, but these methods have poor sensitivity and specificity, and the results are lagging behind. Most tumor patients have metastasis or are at an advanced stage when they are diagnosed. At present, it is urgent to find better diagnostic targets and establish better detection methods to realize the early and concomitant diagnosis of tumors. In recent years, it has been found that exosomes not only play an important role in cell-to-cell information exchange and signal transduction, but also tumor-derived exosomes are involved in regulating the growth, development, invasion and metastasis of tumors. Because exosomes are formed by the invagination of cellular multivesicular membranes, their membrane proteins can more directly reflect the origin, physiological and pathological state of living tumor cells than tumor markers isolated from body fluids, and at the same time, they are more stable. Therefore, exosomes protein markers have the potential to become better markers for early and concomitant diagnosis of tumors.
Most of the traditional microfluidic chips for exosomes are single chips for separation or detection, but there is no report on the integrated microfluidic chip for exosomes separation and detection.
SUMMARY The purpose of the present invention is to provide an integrated microfluidic chip for exosomes separation and detection and a method thereof, so as to solve the problems existing in the prior art. The invention utilizes the antibody group of tumor-specific exosome membrane protein, and provides a more accurate method for early and concomitant diagnosis of pancreatic cancer by constructing an integrated microfluidic chip for exosome separation and detection.
To achieve the above purpose, the present invention provides the following solutions: LU502571 À microfluidic chip integrating exosomes separation and detection comprises a sample inlet, a separation area, a detection area and a quality control area according to a connection order; the channel of the separation area 1s coated with antibody for capturing exosomes, the detection area is coated with GPC-1 antibody, and the quality control area 1s coated with CD63 antibody.
Further, the separation area comprises array units composed of fishbone microchannels, each array unit is eight individual fishbone microchannels, and the eight fishbone microchannels are connected with the sample inlet through tube connectors.
Further, the antibody coated by the channel is Tim-4.
A method for separating exosomes by chip comprises: adding a body fluid sample into a sample inlet, capturing the exosomes in the body fluid sample by a capture antibody coated in the channel of the chip, and then eluting with eluent to obtain the exosomes.
Further, the injection flow rate of the body fluid sample is 20 uL/min.
Further, the eluent is glycine-HCI buffer with a pH of 2.8-8.5.
Further, the flow rate of the eluent is 80 uL/min.
The invention discloses the following technical effects: According to the invention, a microfluidic chip integrating exosomes separation and detection is constructed, and the microchannel in the separation area is coated with Tim-4, which can be used for capturing tumor-specific exosomes surface markers and realizing the separation of tumor-specific exosomes. The detection area is coated with GPC-1 antibody, and the quality control area is coated with CD63 antibody. CD63 antibody is a universal marker for detecting exosomes, which is used as quality control. GPC-1 is a specific marker of pancreatic cancer, which is used for the diagnosis of pancreatic cancer.
The microfluidic chip integrated with exosome separation and detection has the advantages of high throughput, small volume and simple operation, has great potential in specific separation and detection of exosome subgroups, and can contribute to the diagnosis and detection of pancreatic cancer based on exosome.
BRIEF DESCRIPTION OF THE FIGURES In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the embodiments. Obviously, the drawings in the following description are only som&J502571 embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without any creative effort.
Fig. 1 is a schematic diagram of the structure of a microfluidic chip integrating exosomes separation and detection; Fig. 2 shows the microchannel structure of the array unit in the separation area.
In Figure, 1: sample inlet; 2: separation area, 3: detection area, 4: quality control area.
DESCRIPTION OF THE INVENTION Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be taken as a limitation of the present invention, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present invention.
It should be understood that the terms mentioned in the present invention are only used to describe specific embodiments, and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Any stated value or intermediate value within the stated range, and any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
Unless otherwise stated, all technical and scientific terms used herein have the same meanings commonly understood by those of ordinary skill in the field to which this invention relates. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.
Without departing from the scope or spirit of the present invention, it is obvious to those skilled in the art that many modifications and changes can be made to the specific embodiments of the present specification. Other embodiments obtained from the description of the present invention will be obvious to the skilled person. The description and embodiment of thlat/502571 invention are only exemplary.
As used in this paper, the terms "comprising", "including", "having" and "containing" are all open terms, meaning including but not limited to.
Antibodies Tim-4, GPC-1 and CD63 are purchased from Shanghai Enzyme-linked Organisms.
Embodiment 1 The microfluidic chip with exosome separation and detection integration has the structure as shown in Figure 1. The chip includes a sample inlet, a separation area, a detection area and a quality control area. The sample inlet, the separation area, the detection area and the quality control area are connected in turn, and the body fluid sample can enter the chip from the sample inlet and reach the detection area and the quality control area after passing through the separation area.
The separation area includes a plurality of array units composed of fishbone microchannels, as shown in Figure 2, each array unit is eight individual fishbone microchannels, and the eight fishbone microchannels are connected with the sample inlet through tube connectors. The total height (h) of the micro-channel is 50 um, the ratio of the height of the groove to the height (a) of the channel is set to 0.8, and the channel structure is etched on the silicon wafer by standard photolithography; the angle (0) between the V-shape and the channel axis is 45, the main wave vector Q = 2 x/100 um (as shown in Figure 2), the length of each array unit is 2500 pm, the interval of fishbone microchannels is 300 um, and the channel area is 39000 um long and 22115 um wide.
After the sample enters from the sample inlet, it is divided into eight separate fishbone microchannels to ensure the mechanical integrity and uniform flow distribution of the whole chip.
The micro-channel of the separation region is coated with Tim-4, which can be used to capture the surface markers of tumor-specific exosomes and realize the separation of tumor-specific exosomes.
The detection area is coated with GPC-1 antibody, and the quality control area is coated with CD63 antibody. CD63 antibody is a universal marker for detecting exosomes, which is used as quality control. GPC-1 is a specific marker of pancreatic cancer, which is used for th&/502571 diagnosis of pancreatic cancer. Interpretation criteria: Fluorescence signals were detected in both the detection area and the quality control area, and it was interpreted as suspected pancreatic cancer; The detection area is negative, the quality control area is positive, and it is suspected to exclude pancreatic cancer. The test area is negative, and the quality control area is negative. It is judged that the test is invalid, and no exosomes have been isolated, so it needs to be reexamined.
Embodiment 2 Sample separation and detection: Take the serum samples of pancreatic cancer patients and normal people out of the refrigerator at 80°C, and perform the following operations respectively: Thaw slowly on ice, and take 200 uL of serum to flow through fishbone microfluidic chip at a flow rate of 20 uL/min; then, use 2.5 mM CaCl; flush the channel in the chip at a flow rate of uL/min for 10 min, and introduce the air by a pressure pump to discharge the liquid in the chip; then, at a flow rate of 80 uL/min, please add 500 ul glycine -HCI buffer with pH 4.0 (pH
2.8-8.5 can achieve the same effect) for elution; finally, use the pressure pump to introduce air into the channel to ensure that all the remaining liquid in the channel flows out; at the other end of the chip, use a 1.5 mL centrifuge tube to collect eluent. The concentration of exosome particles in pancreatic cancer patients is 1.8x10"° particles/mL, and the detection area and quality control area were double positive. The concentration of exosome particles in normal people is
7.5x10® particles/mL, and the detection area and quality control area are double negative.
The above-mentioned embodiments only describe the preferred mode of the invention, but do not limit the scope of the invention. On the premise of not departing from the design spirit of the invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the scope of protection determined by the claims of the invention.

Claims (7)

CLAIMS LU502571
1. A microfluidic chip integrating exosomes separation and detection, characterized by comprising a sample inlet, a separation area, a detection area and a quality control area according to the connection order; the channel of the separation area is coated with an antibody for capturing exosomes, the detection area is coated with a GPC-1 antibody, and the quality control area is coated with CD63 antibody.
2. The microfluidic chip integrating exosomes separation and detection according to claim 1, characterized in that the separation area comprises array units composed of fishbone microchannels, each array unit composes of eight individual fishbone microchannels, and the eight fishbone microchannels are connected with the sample inlet through tube connectors.
3. The microfluidic chip integrating exosomes separation and detection according to claim 1, characterized in that the antibody coated by the channel is Tim-4.
4. A method for separating exosomes by chip according to claim 1, characterized by comprising: adding a body fluid sample into a sample inlet, capturing the exosomes in the body fluid sample by a capture antibody coated in the channel of the chip, and then eluting with eluent to obtain the exosomes.
5. The method according to claim 4, characterized in that the injection flow rate of the body fluid sample is 20 uL/min.
6. The method according to claim 4, characterized in that the eluent is a glycine-HCI buffer with a pH of 2.8-8.5.
7. The method according to claim 6, characterized in that the flow rate of the eluent is 80 uL/min.
LU502571A 2022-07-27 2022-07-27 Microfluidic chip integrating exosome separation and detection and method thereof LU502571B1 (en)

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