CN111686826A - Micro-fluidic chip with layered structure and application thereof - Google Patents

Abstract

The invention provides a multiple immune analysis system with adjustable detection intervals, the analysis system includes a microfluidic chip with a layered structure and a matching automatic portable instrument, and also provides the application of the immune analysis system. The system's microfluidic immunoassay chip is used for the simultaneous detection of multiple biomarkers with widely varying concentrations. The chip includes a three-dimensional substrate formed by stacking micro-nano fibers and functionalized nanoparticles, so as to simultaneously control the concentration of the patterned capture antibody and the intensity of the chemiluminescence signal, and dynamically adjust the detection range of the immunoassay, so that the present invention can detect the concentration difference up to 10 orders of magnitude biomarkers for combined detection.

Description

Microfluidic chip with layered structure and its application

technical field

The invention belongs to the field of analysis and detection, in particular to a microfluidic chip with a layered structure and its application.

Background technique

Microfluidics is a technology platform for precise manipulation of tiny fluids. The combination of microfluidics and analytical detection enables in vitro diagnostics to develop towards miniaturization, integration and automation. A growing number of microfluidic immunoassay devices enable ultra-fast and precise detection of individual biomarkers. In recent years, there has been an increasing interest and demand for automated and high-throughput detection of multiple analytes in clinical diagnostics. Compared with the detection of a single analyte, parallel detection of multiple analytes has the advantages of shorter analysis time, simpler analysis steps, less sample consumption, higher detection efficiency, and higher cost-effectiveness. Detection of multiple biomarkers in one assay can avoid false positive or false negative results, provide more diagnostic information and increase the diagnostic value of biomarkers. In previous work, many immunoassay devices have the ability to detect multiple biomarkers, including chemiluminescence, fluorescence, colorimetry, etc. However, these methods cannot solve the "single sample multiple indicators" problem well. They still do not meet the needs of clinical use. Most of them still require too much manual work and are too time consuming. Most critically, few of them address the detection of multiple biomarkers with different concentration intervals, which seriously hinders these methods from playing an important role in clinical application. Therefore, it is an urgent problem to develop a multiplex immunoassay system with adjustable detection interval.

SUMMARY OF THE INVENTION

Therefore, the purpose of the present invention is to overcome the defects in the prior art, and to provide a microfluidic chip with a layered structure and its application.

Before describing the content of the present invention, the terms used herein are defined as follows:

The term "POCT" refers to: point-of-care testing.

The term "CRP" refers to: C-reactive protein.

The term "PCT" refers to: Procalcitonin.

The term "IL-6" refers to: "Interleukin 6".

The term "CCD" refers to: Charge Coupled Device.

The term "PMT" refers to: Photomultiplier Tube.

To achieve the above object, a first aspect of the present invention provides a microfluidic chip with a layered structure, the microfluidic chip includes the following three-layer structures: a fluid layer, a substrate layer and an antibody pattern strip.

According to the layered microfluidic chip of the first aspect of the present invention, the antibody pattern strip is a porous film formed by stacking micro-nano fibers;

Preferably, the diameter of the micro-nano fibers is 1000 μm˜100 nm, preferably 20 μm˜500 nm, and most preferably 1 μm;

More preferably, the thickness of the porous film is 1000 μm˜1 μm, preferably 100 μm˜5 μm, and most preferably 10 μm;

Further preferably, the pore size of the pores in the porous film is 100 μm˜500 nm, preferably 10 μm˜1 μm, and most preferably 5 μm.

Preferably, the preparation method of the porous film is selected from one or more of the following: ion etching, sintering, spinning, chemical deposition;

More preferably, the preparation method of the porous film adopts an electrospinning method.

Further preferably, the material of the electrospinning film is selected from one or more of the following: polystyrene, polycarbonate, polyvinylidene fluoride, polycaprolactone, and polylactic acid-glycolic acid copolymer.

According to the microfluidic chip of the layered structure of the first aspect of the present invention, the antibody pattern strip further includes functionalized modified micro-nano particles;

Preferably, the particle size of the micro-nano particles is 10 μm˜50 nm, preferably 1 μm˜100 nm, and most preferably 200 nm.

Preferably, the micro-nano particles are selected from one or more of the following: gold nanoparticles, silica nanoparticles, triferromagnetic nanoparticles, polystyrene microspheres;

More preferably, the micro-nano particles are gold nanoparticles.

A second aspect of the present invention provides a multiplex immunoassay system with adjustable detection intervals, the assay system comprising:

The layered microfluidic chip of the first aspect, and

Matching automatic portable instrument.

According to the multiplex immunoassay system of the second aspect of the present invention, the automatic portable instrument comprises:

valve controller;

liquid drive;

the light path; and

CCD camera or photomultiplier tube.

A third aspect of the present invention provides a method for using the immunoassay system described in the second aspect, the method comprising the following steps:

(1) selectively coating the antibody pattern band with an antibody;

(2) cutting the antibody pattern strip, and assembling the chip;

(3) The biological samples and reagents are injected into the chip, and the automatic portable equipment is used for detection.

The fourth aspect of the present invention provides the microfluidic chip of the layered structure described in the first aspect or the immunoassay system of the second aspect in the preparation of a product for simultaneous detection of multiple biomarkers in an undiluted sample For application, the undiluted sample is preferably a human serum sample, and the range of the multiple biomarkers is preferably close to ten orders of magnitude.

A fifth aspect of the present invention provides an immune detection device, the device comprising;

The layered microfluidic chip of the first aspect, or

The immunoassay system of the second aspect;

Preferably, the device can dynamically adjust its detection interval according to different concentrations of the detected substances.

Traditional testing is carried out in the laboratory department of a hospital or a third-party laboratory. With the continuous advancement of technology, people's demand for POCT is gradually increasing. In vitro diagnosis has also gradually expanded beyond laboratory departments, such as emergency rooms, mobile nursing stations, operating rooms, intensive care units and even patients' homes. The microfluidic-based POCT immunoassay platform enables rapid and accurate detection of biomarkers. But few have been able to address the challenge of simultaneously detecting multiple biomarkers, especially those biomarkers with different concentration intervals, which are the norm in many clinical settings such as early sepsis diagnosis. The unparalleled advantage of the present invention is that the system can simultaneously detect multiple biomarkers in undiluted human serum samples with a detection concentration range close to ten orders of magnitude. The system can dynamically adjust its detection range according to the different concentrations of the test substances, so that it can meet the needs of clinical testing. In terms of detection performance, the one-key operation mode of the present invention not only avoids human operation errors, but also facilitates its application to POCT. At the same time, the system of the present invention can detect a variety of markers from μg/mL to pg/mL. A unique and unparalleled advantage of the inventors' system is that the inventors' system can simultaneously detect nearly ten orders of magnitude of multiple biomarkers in undiluted human serum samples. In terms of detection performance, the one-key operation mode of the present invention not only avoids human operation errors, but also facilitates its application to POCT. Meanwhile, the immunoassay system of the present inventors can detect various markers from μg/mL to pg/mL.

The multiplex immunoassay system with adjustable detection interval of the present invention can have but not limited to the following beneficial effects:

Rapid quantification of the concentrations of multiple biomarkers is of great significance for disease diagnosis and improvement of people's quality of life. However, the large variability in biomarker concentrations makes it difficult to rapidly detect multiple indicators in a single assay. The system's microfluidic immunoassay chip is used for the simultaneous detection of multiple biomarkers with widely varying concentrations. The chip includes a three-dimensional substrate formed by stacking micro-nano fibers and functionalized nanoparticles, so as to simultaneously control the concentration of the patterned capture antibody and the intensity of the chemiluminescence signal, and dynamically adjust the detection range of the immunoassay, so that the present invention can detect the concentration difference up to 10 orders of magnitude biomarkers for combined detection. With the portable instrument of the present invention, the present invention can quickly and automatically complete the entire experiment with a drop of serum sample, which is of great significance to POCT. With the ability to detect multiple biomarkers, the system can be used in a range of applications, such as community health centers, mobile clinics, and even patients' homes.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

FIG. 1 shows the structure of the multiplex immunoassay system of the present invention.

FIG. 2 shows the structure of the microfluidic chip of the present invention.

Figure 3 shows the signal amplification of the multiplex immunoassay system of the present invention.

Figure 4 shows the principle of combined detection of multiple biomarkers by the multiplex immunoassay system of the present invention.

FIG. 5 shows that the multiplex immunoassay system of the present invention realizes the combined detection of biomarkers in different concentration ranges in one chip through different signal amplification methods.

FIG. 6 shows the detection results of C-reactive protein in Test Example 1. FIG.

FIG. 7 shows the detection results of interleukin-6 in Test Example 2. FIG.

FIG. 8 shows the combined detection results of C-reactive protein, procalcitonin and interleukin-6 in Test Example 3. FIG.

Reference number:

1. Charge-coupled device (CCD) camera; 2. Circuit board; 3. Reflector; 4. Valve controller; 5. Chip; 6. Negative pressure connector; 7. Liquid driver; 8. Peristaltic pump; 9. Lens .

Detailed ways

The present invention is further described below through specific examples, but it should be understood that these examples are only used for more detailed and specific description, and should not be construed as being used to limit the present invention in any form.

This section provides a general description of the materials and test methods used in the tests of the present invention. While many of the materials and methods of operation used for the purposes of the present invention are known in the art, the present invention is described in as much detail as possible. It is clear to those skilled in the art that, in the context, if not specifically stated, the materials and methods of operation used in the present invention are well known in the art.

The reagents and instruments used in the following examples are as follows:

Reagents: sodium citrate, chloroauric _acid , and K2CO3 _were purchased from Sigma Aladdin, horseradish peroxidase and antibodies were purchased from Shanghai Yemin Biological Co., Ltd., and bovine serum albumin was purchased from Boster Bioengineering Co., Ltd.

instrument:

The CCD camera was purchased from Lightspeed Vision Co., Ltd. (Beijing, China). Lenses and reflectors for reflecting the chemiluminescence signal from the microfluidic chip to the CCD camera were purchased from Qisai Electronics Co., Ltd. (Beijing, China). The peristaltic pump was obtained from Baoding Snow Fluid Technology Co., Ltd. (Baoding, Hebei). Custom printed circuit boards (PCBs) for hardware module automation were manufactured by Jufeng Huaqiang Electronic Technology Co., Ltd. (Shenzhen, China). Other machined parts are processed by Xinghong Shengda Technology Development Co., Ltd. (Beijing, China). Stepper motors were purchased from Haydon Kerk Motion Solutions, Inc., (Changzhou, Jiangsu, China). The chips are processed by Shenzhen Senri Silicone Materials Co., Ltd.

Example 1

This example is used to illustrate the structure of the multiplex immunoassay system of the present invention.

The analysis system of the invention mainly includes two parts: a microfluidic chip with a layered structure and a matching automatic portable instrument.

The automated instrument can mainly complete the entire immune detection and signal extraction process automatically. The device has two main functions: automatically drive the liquid to complete the entire assay; and capture the signal to calculate the concentration of each biomarker. Valve controllers and fluid actuators can be programmed to drive fluids. The chemiluminescence signal is guided by the optical path and recorded by a CCD camera or PMT. As shown in Figure 1, the main components of the instrument include a charge-coupled device (CCD) camera 1, a circuit board 2, a reflector 3, a valve controller 4, a chip 5, a negative pressure connector 6, a liquid driver 7, and a peristaltic pump 8 and lens 9.

As shown in Figure 2, the chip has a multi-layer structure. Three of these are most evident: a fluidic layer with fluid pathways, a substrate layer with waste recovery, and antibody patterned strips for immunoassays. The antibody-patterned strip as the immunoreactive substrate is a porous film formed by stacking micro-nanofibers. Porous films can be prepared using ion etching, sintering, spinning, and chemical deposition methods. More preferably, the preparation method of the porous film adopts an electrospinning method. Further preferably, the material of the electrospinning film is selected from one or more of the following: polystyrene, polycarbonate, polyvinylidene fluoride, polycaprolactone, and polylactic acid-glycolic acid copolymer. The main purpose is to provide a porous three-dimensional substrate on which the immune response can be carried out more efficiently. After selectively coating the antibody, cut the antibody strip to the appropriate size for chip assembly. After the biological samples and reagents are injected into the chip, the fully automatic portable device can complete all the steps required for immunoassay and signal analysis, fully meeting the requirements of POCT. As shown in Figure 3, the signal has nine points. Each biomarker has triplicate signal points to generate reliable results. The signal at each point was analyzed to calculate the concentration of each biomarker. For different biomarkers, different amplification methods were used to adjust the dynamic range to the natural range of their human serum samples. To generate porous three-dimensional reaction substrates and enzyme/antibody-modified micro-nanoparticles to reduce their limit of detection (LoD).

The dynamic regulation of the detection range is fundamentally dependent on two technologies: the control of the immune response of the fiber substrate and the signal amplification of the functionalized micro-nanoparticles. The micro-nano particles are selected from one or more of the following: gold nanoparticles, silica nanoparticles, triferromagnetic nanoparticles, polystyrene microspheres; more preferably, the micro-nano particles are gold nanoparticles particles. Gold nanoparticles are prepared by sodium citrate reduction chloroauric acid method to prepare colloidal gold solution: add 1000 mL of three-distilled water to the beaker, then add 10 mL of 1 mass % sodium citrate solution, heat to boiling, and then add 20 mL of 1 mass Percentage of chloroauric acid solution, heated to reflux for 15 min and then cooled to obtain the colloidal gold solution; the modification of gold nanoparticles adopts the physical electrostatic adsorption method: take 20 mL of the prepared colloidal gold solution into a beaker, add 150 μL of 0.1 M The pH of the solution was adjusted to 7.6 with the K ₂ CO ₃ solution, stirred evenly, then the antibody and horseradish peroxidase were added, stirred for 30 min, and then 2 mL of 3 mass % bovine serum albumin solution was added, and centrifuged for 20 min. The rotation speed was 13000 rpm, the supernatant was discarded, 0.1 mass % bovine serum albumin solution was added for reconstitution, and then the solution was placed in a refrigerator at 4 degrees for storage. For biomarkers in different concentration ranges, the inventors used micro-nanofiber stacked films and functionalized modified micro-nanoparticles to simultaneously control the concentration of patterned capture antibodies and the intensity of chemiluminescence signals, and dynamically adjust the detection range of immunoassays , to detect multiple biomarkers (from μg/mL down to pg/mL). As shown in FIG. 4 , for biomarkers in different concentration ranges, the inventors can use different signal amplification methods to achieve detection. Decreasing the detection limit from left to right can be dynamically selected based on the concentration of the biomarker to be detected.

Not only that, the inventors can also use the chip for combined detection of multiple biomarkers, even though their concentration ranges are very different. As shown in Figure 5, biomarkers in different concentration ranges are combined in one chip through different signal amplification methods.

Test Example 1

This test example is used to illustrate the detection of C-reactive protein by the multiplex immunoassay system of the present invention.

(1) Coating modification of C-reactive protein capture antibody: After the anti-human CRP antibody was sequentially coated on the surface of the reaction substrate using a protein stripper, a microfluidic chip, and a spotter, the pattern strip of the CRP capture antibody was cut out. bring. The CRP capture antibody pattern strip includes three CRP capture antibody patterns.

(2) Chip assembly: Surface activation of the chip fluid layer and substrate layer for 1 min using a particle surface cleaning machine such as oxygen, after placing the CRP capture antibody pattern strips, the fluid layer and the substrate layer are bonded and packaged.

(3) Signal detection: After injecting the CRP protein detection reagent in turn, put it into a fully automatic portable device until the machine completes the immune detection and subsequent signal processing.

The detection results are shown in Figure 6. The values on the left are the coating concentrations: 2.5, 5.0, and 10 μg/mL. The higher the concentration, the stronger the signal, but the narrower the detection interval. The above values are several concentrations tested: 100, 50, 25, 12.5, 6.25 μg/mL.

Test Example 2

This test example is used to illustrate the detection of interleukin-6 by the multiplex immunoassay system of the present invention.

(1) Preparation of electrospun porous film: A 10 mass % polystyrene solution was prepared and added to a syringe. The positive and negative electrodes of the high-voltage power supply for electrospinning are respectively connected to the syringe needle and the receiving device, and electrospinning is performed with a high voltage of 15 kV, and a negative voltage of -1 kV is used for receiving electrospinning. The electrospinning time was 10 minutes, and the resulting electrospun membrane had a thickness of 10 μm and a diameter of 1 μm.

(2) Preparation and modification of gold nanoparticles: add 1000 mL of three-distilled water to the beaker, then add 10 mL of 1 mass% sodium citrate solution, and after heating to boiling, add 20 mL of 1 mass percent chloroauric acid solution , heated to reflux for 15 min, and then cooled to obtain the colloidal gold solution; the modification of gold nanoparticles adopts the physical electrostatic adsorption method: take 20 mL of the prepared colloidal gold solution and place it in a beaker, add 150 μL of 0.1 M K ₂ CO ₃ solution Adjust the pH of the solution to 7.6, stir evenly, then add IL-6 detection antibody and horseradish peroxidase, stir for 30 min, then add 2 mL of 3 mass% bovine serum albumin solution, and centrifuge for 20 min at a rotational speed of 13000 rpm, discard the supernatant, add 0.1 mass % bovine serum albumin solution for reconstitution, and store in a refrigerator at 4 degrees.

(3) Coating modification of IL-6 capture antibody: After the anti-human IL-6 antibody was sequentially coated on the surface of the reaction substrate using a protein stripper, a microfluidic chip, and a spotter, IL-6 was prepared by cutting. Capture antibody patterned bands. The IL-6 capture antibody pattern strip includes three IL-6 capture antibody patterns.

(4) Chip assembly: The fluid layer and the substrate layer of the chip were activated for 1 min using a particle surface cleaning machine such as oxygen.

(5) Signal detection: After injecting the IL-6 protein detection reagent in turn, put it into a fully automatic portable device until the machine completes the immune detection and subsequent signal processing.

The detection results are shown in Figure 7, and the detection interval is 2000-62.5 pg/mL.

Test Example 3

This test example is used to illustrate the combined detection results of C-reactive protein, procalcitonin and interleukin-6 by the multiple immunoassay system of the present invention.

(1) Preparation of electrospun porous film: A 10 mass % polystyrene solution was prepared and added to a syringe. The positive and negative electrodes of the high-voltage power supply for electrospinning are respectively connected to the syringe needle and the receiving device, and electrospinning is performed with a high voltage of 15 kV, and a negative voltage of -1 kV is used for receiving electrospinning. The electrospinning time was 10 minutes, and the resulting electrospun membrane had a thickness of 10 μm and a diameter of 1 μm.

(2) Preparation and modification of gold nanoparticles: add 1000 mL of three-distilled water to the beaker, then add 10 mL of 1 mass% sodium citrate solution, and after heating to boiling, add 20 mL of 1 mass percent chloroauric acid solution , heated to reflux for 15 min, and then cooled to obtain the colloidal gold solution; the modification of gold nanoparticles adopts the physical electrostatic adsorption method: take 20 mL of the prepared colloidal gold solution and place it in a beaker, add 150 μL of 0.1 M K ₂ CO ₃ solution Adjust the pH of the solution to 7.6, stir evenly, then add IL-6 detection antibody and horseradish peroxidase, stir for 30 min, then add 2 mL of 3 mass% bovine serum albumin solution, and centrifuge for 20 min at a rotational speed of 13000 rpm, discard the supernatant, add 0.1 mass % bovine serum albumin solution for reconstitution, and store in a refrigerator at 4 degrees.

(3) Coating modification of CRP, PCT, IL-6 capture antibodies: The anti-human CRP, PCT, IL-6 antibodies were sequentially coated on the reaction substrate by a protein stripper, a microfluidic chip, and a spotter. After the surface, the pattern strips of CRP, PCT and IL-6 capture antibodies were prepared by cutting. The CRP, PCT, and IL-6 capture antibody pattern strips respectively comprise one CRP capture antibody pattern, one PCT capture antibody pattern, and one IL-6 capture antibody pattern.

(4) Chip assembly: use a particle surface cleaning machine such as oxygen to activate the surface of the chip fluid layer and the base layer for 1 min. After the CRP, PCT, and IL-6 capture antibody pattern strips are placed, the fluid layer and the base layer are attached. package.

(5) Signal detection: After injecting CRP, PCT, and IL-6 protein detection reagents in sequence, put them into a fully automatic portable device until the machine completes the immune detection and subsequent signal processing. The detection results are shown in Figure 8. The detection range of CRP is 100-3.13 μg/mL, the detection range of PCT is 75-0.31 ng/mL, and the detection range of IL-6 is 2000-62.5 pg/mL. The range of concentrations detected is up to ten orders of magnitude.

Although this invention has been described to a certain extent, it will be apparent that suitable changes in various conditions may be made without departing from the spirit and scope of the invention. It is to be understood that the invention is not limited to the embodiments described, but is to be included within the scope of the claims, which include equivalents for each of the elements described.

Claims (10)

1. A microfluidic chip with a layered structure, wherein the microfluidic chip comprises the following three-layer structure: a fluid layer, a substrate layer and an antibody pattern strip. 2. The microfluidic chip of a layered structure according to claim 1, wherein the antibody pattern strip is a porous film formed by stacking micro-nano fibers; Preferably, the diameter of the micro-nano fibers is 1000 μm˜100 nm, preferably 20 μm˜500 nm, and most preferably 1 μm; More preferably, the thickness of the porous film is 1000 μm˜1 μm, preferably 100 μm˜5 μm, and most preferably 10 μm; Further preferably, the pore size of the pores in the porous film is 100 μm˜500 nm, preferably 10 μm˜1 μm, and most preferably 5 μm. 3 . The microfluidic chip with a layered structure according to claim 2 , wherein the preparation method of the porous film is selected from one or more of the following: ion etching, sintering, spinning, and chemical deposition. 4 . 4. The microfluidic chip of the layered structure according to any one of claims 1 to 3, wherein the antibody pattern strip further comprises functionalized modified micro-nano particles; Preferably, the particle size of the micro-nano particles is 10 μm˜50 nm, preferably 1 μm˜100 nm, and most preferably 200 nm. 5. The microfluidic chip with a layered structure according to claim 4, wherein the micro-nano particles are selected from one or more of the following: gold nanoparticles, silicon dioxide nanoparticles, triferromagnetic tetroxide Nanoparticles, polystyrene microspheres; Preferably, the micro-nano particles are gold nanoparticles. 6. A multiplex immune analysis system with adjustable detection interval, characterized in that, the analysis system comprises: The layered microfluidic chip according to any one of claims 1 to 5, and Matching automatic portable instrument. 7. The multiplex immunoassay system according to claim 6, wherein the automatic portable instrument comprises: valve controller; liquid drive; the light path; and CCD camera or photomultiplier tube. 8. The method of using the immunoassay system according to claim 6 or 7, wherein the method comprises the following steps: (1) selectively coating the antibody pattern band with an antibody; (2) cutting the antibody pattern strip, and assembling the chip; (3) The biological samples and reagents are injected into the chip, and the automatic portable equipment is used for detection. 9. The layered microfluidic chip of any one of claims 1 to 5 or the immunoassay system of any one of claims 6 to 7 is prepared for simultaneous detection of multiple organisms in undiluted samples For the application of the marker product, the undiluted sample is preferably a human serum sample, and the range of the multiple biomarkers is preferably close to ten orders of magnitude. 10. An immune detection device, characterized in that the device comprises; The layered microfluidic chip according to any one of claims 1 to 5, or The immunoassay system of any one of claims 6 to 7; Preferably, the device can dynamically adjust its detection interval according to different concentrations of the detected substances.

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