CN111686826A - Micro-fluidic chip with layered structure and application thereof - Google Patents
Micro-fluidic chip with layered structure and application thereof Download PDFInfo
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Abstract
The invention provides a multiple immunoassay system with an adjustable detection zone, which comprises: the micro-fluidic chip with a layered structure and a matched automatic portable instrument also provide the application of the immunoassay system. The microfluidic immunoassay chip of the system is used for simultaneously detecting multiple biomarkers under the condition of large concentration difference. The chip comprises a three-dimensional substrate formed by stacking micro-nano fibers and functionalized nano particles, so that the concentration of a patterned capture antibody and the intensity of a chemiluminescent signal are controlled simultaneously, the detection interval of immunoassay is dynamically adjusted, and the chip can be used for carrying out combined detection on biomarkers with concentration differences of 10 orders of magnitude.
Description
Technical Field
The invention belongs to the field of analysis and detection, and particularly relates to a micro-fluidic chip with a layered structure and application thereof.
Background
Microfluidics is a technological platform for precisely manipulating tiny fluids. The combination of microfluidics and analytical testing has led to the development of in vitro diagnostics towards miniaturization, integration and automation. More and more microfluidic immunodetection devices are capable of performing ultra-fast and accurate detection of individual biomarkers. In recent years, there has been an increasing interest and demand for automated and high-throughput detection of a variety of analytes in clinical diagnostics. Compared with the detection of a single analyte, the parallel detection of a plurality of analytes has the advantages of shorter analysis time, simpler analysis steps, less sample consumption, higher detection efficiency and higher cost effectiveness. Detecting multiple biomarkers in one assay can avoid false positive or false negative results, provide more diagnostic information and increase the diagnostic value of the biomarkers. In previous work, many immunoassay devices have the ability to detect multiple biomarkers, including chemiluminescence, fluorescence, colorimetry, and the like. However, these methods do not solve the "single sample multiple index" problem well. They still do not meet the requirements for clinical use. Most of them still require excessive manual operations and are also too time consuming. Most critical is that few of them address the detection of multiple biomarkers with different concentration intervals, which severely hampers the important role of these methods in clinical applications. Therefore, the development of a multiplex immunoassay system with adjustable detection intervals is a problem to be solved urgently.
Disclosure of Invention
Therefore, the present invention is directed to overcome the drawbacks of the prior art, and to provide a microfluidic chip with a layered structure and an application thereof.
Before setting forth the context 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: a charge coupled device.
The term "PMT" means: a photomultiplier tube.
In order to achieve the above object, a first aspect of the present invention provides a microfluidic chip with a layered structure, where the microfluidic chip includes the following three layers: a fluid layer, a base layer and antibody pattern strips.
According to the microfluidic chip with the layered structure, the antibody pattern strip is a porous film formed by stacking micro-nanofibers;
preferably, the diameter of the micro-nano fiber is 1000 μm to 100nm, preferably 20 μm to 500nm, and most preferably 1 μm;
more preferably, the porous membrane has a thickness of 1000 μm to 1 μm, preferably 100 μm to 5 μm, most preferably 10 μm;
further preferably, the pore size of the pores in the porous film is from 100 μm to 500nm, preferably from 10 μm to 1 μm, and most preferably 5 μm.
Preferably, the porous membrane is prepared by a method selected from one or more of the following: ion etching, sintering, spinning and chemical deposition;
more preferably, the preparation method of the porous film is prepared by an electrostatic spinning method.
Further preferably, the material of the electrospun 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 with the layered structure in the first aspect of the invention, the antibody pattern strip further comprises functionalized modified micro-nano particles;
preferably, the particle size of the micro-nano particles is 10 μm to 50nm, preferably 1 μm to 100nm, and most preferably 200 nm.
Preferably, the micro-nano particles are selected from one or more of the following: gold nanoparticles, silica nanoparticles, ferroferric oxide nanoparticles, polystyrene microspheres;
more preferably, the micro-nano particles are gold nano particles.
A second aspect of the invention provides a multiplex immunoassay system having an adjustable detection zone, the assay system comprising:
the layered microfluidic chip of the first aspect, and
a matched automatic portable instrument.
According to a second aspect of the invention, the multiple immunoassay system, the automated portable instrument comprises:
a valve controller;
a liquid driver;
an optical path; and
a CCD camera or a photomultiplier tube.
A third aspect of the invention provides a method of using the immunoassay system of the second aspect, the method comprising the steps of:
(1) selectively coating the antibody pattern strip with an antibody;
(2) cutting the antibody pattern strip, and assembling a chip;
(3) and injecting the biological sample and the reagent into the chip, and automatically detecting by using a portable device.
A fourth aspect of the invention provides the use of a microfluidic chip of the layered structure of the first aspect or an immunoassay system of the second aspect for the preparation of a product for the simultaneous detection of multiple biomarkers in an undiluted sample, preferably a human serum sample, preferably in a range approaching ten orders of magnitude.
A fifth aspect of the invention provides an immunoassay device, the device comprising;
the microfluidic chip of the layered structure of the first aspect, or
The immunoassay system of the second aspect;
preferably, the device can dynamically adjust the detection interval according to different concentrations of the detection object.
The traditional detection is carried out in hospital clinical laboratory or third-party laboratory, and with the continuous improvement of technology, the demand of POCT is gradually increased. In vitro diagnostics have also been slowly expanded beyond clinical laboratories, such as emergency rooms, mobile nursing stations, operating rooms, heavy-town care units, and even patient homes. The POCT immunoassay platform based on microfluidics can quickly and accurately detect the biomarkers. Few have been able to address the challenge of simultaneously detecting multiple biomarkers, particularly those with different concentration intervals, which is normal in many clinical settings, such as early sepsis diagnosis. The system has the advantage of inexplicable performance that the system can simultaneously detect a plurality of biomarkers in undiluted human serum samples, and the detection concentration range of the system is close to ten orders of magnitude. The system can dynamically adjust the detection interval according to different concentrations of the detection object, so that the detection interval meets the requirements of clinical examination. In terms of detection performance, the one-touch operation mode of the present invention not only avoids human operation errors, but also facilitates its application to POCT. Meanwhile, the system of the invention can detect a plurality of markers from mu g/mL to pg/mL. The unique and uneconomical advantage of our system is that our system can simultaneously detect multiple biomarkers in undiluted human serum samples by nearly ten orders of magnitude. In terms of detection performance, the one-touch 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 a variety of markers ranging from μ g/mL to pg/mL.
The multiplex immunoassay system with adjustable detection interval of the present invention may have the following beneficial effects, but is not limited to:
the rapid quantification of the concentration of multiple biomarkers is of great significance for disease diagnosis and for improving the quality of life of people. However, the large difference in biomarker concentration makes it difficult to rapidly detect multiple indicators in a single assay. The microfluidic immunoassay chip of the system is used for simultaneously detecting multiple biomarkers under the condition of large concentration difference. The chip comprises a three-dimensional substrate formed by stacking micro-nano fibers and functionalized nano particles, so that the concentration of a patterned capture antibody and the intensity of a chemiluminescent signal are controlled simultaneously, the detection interval of immunoassay is dynamically adjusted, and the chip can be used for carrying out combined detection on biomarkers with concentration differences of 10 orders of magnitude. By matching with the portable instrument, the invention can complete the whole experiment by quickly and automatically using a drop of serum sample, which has important significance for POCT. The system has the capability of detecting multiple biomarkers and can be used in a range of applications, such as community health centers, ambulatory clinics and even in patients' homes.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, 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.
FIG. 3 shows signal amplification for the multiplex immunoassay system of the present invention.
FIG. 4 shows the principle of the combined detection of multiple biomarkers by the multiplex immunoassay system of the present invention.
FIG. 5 shows that the multiple immunoassay system of the present invention realizes the combined detection of biomarkers in different concentration ranges in one chip by different signal amplification methods.
FIG. 6 shows the results of detection of C-reactive protein in test example 1.
FIG. 7 shows the results of detection of interleukin 6 in test example 2.
FIG. 8 shows the results of the combined detection of C-reactive protein, procalcitonin and interleukin 6 in test example 3.
Reference numerals:
1. a Charge Coupled Device (CCD) camera; 2. a circuit board; 3. a reflector; 4. a valve controller; 5. a chip; 6. a negative pressure connector; 7. a liquid driver; 8. a peristaltic pump; 9. and (5) a lens.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
The reagents and instrumentation used in the following examples are as follows:
reagent: sodium citrate, chloroauric acid and K2CO3From Sigma-Arlatin, horseradish peroxidase and antibodies from Shanghai Yellowshi Bio Inc., bovine serum albumin from doctor Derma bioengineering, Inc.
The instrument comprises the following steps:
CCD cameras are available from photospeed Vision, Inc. (Beijing, China). Lenses and reflectors for reflecting chemiluminescent signals from the microfluidic chip to the CCD camera were purchased from seiko electronics limited (beijing, china). Peristaltic pumps are available from baodingsinuo fluid technology ltd (hebei baoding). Custom Printed Circuit Boards (PCBs) for hardware module automation are manufactured by the gigapeak warqiang electronics technology ltd (shenzhen, china). Other machined parts were machined by the science and technology development ltd, hohengda, china, beijing. Stepper motors are available from Haydon Kerk Motion Solutions, inc. (chan, j). The chip is processed by Shenzhen Senri organosilicon materials GmbH.
Example 1
This example illustrates the structure of the multiplex immunoassay system of the present invention.
The analysis system of the invention mainly comprises two parts: a micro-fluidic chip with a layered structure and a matched automatic portable instrument.
The automatic instrument can mainly complete the whole immune detection and signal extraction process in a full-automatic manner. The device has two main functions: automatically driving the liquid to complete the whole detection; signals were captured to calculate the concentration of each biomarker. The valve controller and liquid driver may drive the fluid according to a programmed setting. The chemiluminescent signal is guided by an optical path and recorded by a CCD camera or PMT. As shown in fig. 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, a peristaltic pump 8, and a lens 9.
As shown in fig. 2, the chip has a multi-layer structure. The most obvious of these are three layers: a fluidic layer with fluidic pathways, a substrate layer with waste recovery and antibody pattern strips for immunoassay. The antibody pattern strip as the immunoreaction substrate is a porous film formed by stacking micro-nano fibers. The porous film can be prepared by using ion etching, sintering, spinning and chemical deposition methods. More preferably, the preparation method of the porous film is prepared by an electrostatic spinning method. Further preferably, the material of the electrospun film is selected from one or more of the following: polystyrene, polycarbonate, polyvinylidene fluoride, polycaprolactone and polylactic acid-glycolic acid copolymer. The aim is primarily to provide a porous three-dimensional substrate on which the immune reaction can be carried out more efficiently. After the antibody is selectively coated, the antibody strips are cut into proper sizes for chip assembly. After the biological sample and the reagent are injected into the chip, the full-automatic portable equipment can complete all steps required by immunoassay and signal analysis, and the requirements of POCT are completely met. As shown in fig. 3, the signal has nine points. Each biomarker had triplicate signal points to yield reliable results. The signal of each spot 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. So as to generate porous three-dimensional reaction substrate and enzyme/antibody modified micro-nano particles to reduce the detection limit (LoD) thereof.
The dynamic adjustment of the detection interval depends on two technologies: the immunoreaction control function of the fiber substrate and the signal amplification function of the functionalized micro-nano particles. The micro-nano particles are selected from one or more of the following: gold nanoparticles, silica nanoparticles, ferroferric oxide nanoparticles, polystyrene microspheres; more preferably, the micro-nano particles are gold nano particles. The gold nanoparticles are prepared into a colloidal gold solution by adopting a sodium citrate reduction chloroauric acid method: adding 1000mL of triple distilled water into a beaker, then adding 10mL of 1 mass percent sodium citrate solution, heating to boil, then adding 20mL of 1 mass percent chloroauric acid solution, heating to a reflux state for 15min, and cooling to obtain the colloidal gold solution; the gold nanoparticles are modified by adopting a physical electrostatic adsorption method: taking 20mL of prepared glueThe gold solution was placed in a beaker and 150. mu. L0.1M K was added2CO3The pH value of the solution is adjusted to 7.6, the solution is stirred uniformly, then the antibody and horseradish peroxidase are added, the stirring is carried out for 30min, then 2mL of 3 mass percent bovine serum albumin solution is added, the centrifugation is carried out, the centrifugation time is 20min, the rotating speed is 13000rpm, the supernatant is discarded, then 0.1 mass percent bovine serum albumin solution is added for redissolving, and then the mixture is placed in a 4-degree refrigerator for storage. For biomarkers in different concentration intervals, the inventor uses a micro-nano fiber stacked film and functionalized modified micro-nano particles to simultaneously control the concentration of a patterned capture antibody and the intensity of a chemiluminescent signal and dynamically adjust the detection interval of immunoassay, so as to detect multiple biomarkers (from mu g/mL to pg/mL). As shown in fig. 4, the inventors were able to use different signal amplification methods to achieve detection for different concentration ranges of biomarkers. The detection limit is decreased from left to right in turn, and dynamic selection can be performed according to the concentration of the biomarker to be detected.
Furthermore, the present inventors have also been able to perform a combined assay of multiple biomarkers using the chip, even if their concentration ranges are very different. As shown in fig. 5, the biomarkers in different concentration ranges can be jointly detected in one chip by different signal amplification modes.
Test example 1
This test example is intended 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: and sequentially coating the anti-human CRP antibody on the surface of the reaction substrate by adopting a protein membrane scribing instrument, a micro-fluidic chip and a spotting instrument, and then cutting to obtain a CRP capture antibody pattern strip. The CRP capture antibody pattern strip comprises three CRP capture antibody patterns.
(2) Chip assembly: and (3) carrying out surface activation on the chip fluid layer and the substrate layer for 1min by using an oxygen-oxygen particle surface cleaning machine, placing a CRP capture antibody pattern strip, and then attaching and packaging the fluid layer and the substrate layer.
(3) Signal detection: and (3) sequentially injecting CRP protein detection reagents, and then putting the CRP protein detection reagents into full-automatic portable equipment until the machine finishes immunoassay and subsequent signal processing.
The detection results are shown in fig. 6, and the values on the left are coating concentrations: 2.5, 5.0, 10. mu.g/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 and 6.25 mu g/mL.
Test example 2
This test example is intended to illustrate the detection of interleukin 6 by the multiplex immunoassay system of the present invention.
(1) Preparing an electrospun porous film: a 10 mass% polystyrene solution was prepared and added to the syringe. The positive electrode and the negative electrode of the electrostatic spinning high-voltage power supply are respectively connected with the syringe needle and the receiving device, and electrospinning is carried out by using high-voltage electricity of 15 kilovolts, and electrospinning is carried out by using voltage with negative voltage of-1 kilovolt. The electrospinning time was 10 minutes, and the resulting electrospun film had a thickness of 10 microns and a diameter of 1 micron.
(2) Preparing and modifying gold nanoparticles: adding 1000mL of triple distilled water into a beaker, then adding 10mL of 1 mass percent sodium citrate solution, heating to boil, then adding 20mL of 1 mass percent chloroauric acid solution, heating to a reflux state for 15min, and cooling to obtain the colloidal gold solution; the gold nanoparticles are modified by adopting a physical electrostatic adsorption method: 20mL of the prepared gold colloid solution was placed in a beaker, and 150. mu. L0.1M K was added2CO3The pH value of the solution is adjusted to 7.6, the solution is stirred uniformly, then an IL-6 detection antibody and horseradish peroxidase are added, the solution is stirred for 30min, then 2mL of 3 mass percent bovine serum albumin solution is added, centrifugation is carried out, the centrifugation time is 20min, the rotating speed is 13000rpm, supernatant is discarded, then 0.1 mass percent bovine serum albumin solution is added for redissolving, and then the mixture is placed in a 4-degree refrigerator for storage.
(3) Coating modification of IL-6 capture antibody: the anti-human IL-6 antibody is coated on the surface of a reaction substrate in sequence by adopting a protein membrane scribing instrument, a micro-fluidic chip and a sample application instrument, and then the IL-6 capture antibody pattern strip is cut. The IL-6 capture antibody pattern strip comprises three IL-6 capture antibody patterns.
(4) Chip assembly: and (3) carrying out surface activation on the chip fluid layer and the substrate layer for 1min by using an oxygen-oxygen particle surface cleaning machine, placing an IL-6 capture antibody pattern strip, and then attaching and packaging the fluid layer and the substrate layer.
(5) Signal detection: after IL-6 protein detection reagent is injected, the sample is put into a full-automatic portable device in sequence until the machine finishes immunodetection and subsequent signal processing.
The detection result is shown in FIG. 7, and the detection interval is 2000-62.5 pg/mL.
Test example 3
This test example is intended to illustrate the results of the combined detection of C-reactive protein, procalcitonin and interleukin 6 by the multiplex immunoassay system of the present invention.
(1) Preparing an electrospun porous film: a 10 mass% polystyrene solution was prepared and added to the syringe. The positive electrode and the negative electrode of the electrostatic spinning high-voltage power supply are respectively connected with the syringe needle and the receiving device, and electrospinning is carried out by using high-voltage electricity of 15 kilovolts, and electrospinning is carried out by using voltage with negative voltage of-1 kilovolt. The electrospinning time was 10 minutes, and the resulting electrospun film had a thickness of 10 microns and a diameter of 1 micron.
(2) Preparing and modifying gold nanoparticles: adding 1000mL of triple distilled water into a beaker, then adding 10mL of 1 mass percent sodium citrate solution, heating to boil, then adding 20mL of 1 mass percent chloroauric acid solution, heating to a reflux state for 15min, and cooling to obtain the colloidal gold solution; the gold nanoparticles are modified by adopting a physical electrostatic adsorption method: 20mL of the prepared gold colloid solution was placed in a beaker, and 150. mu. L0.1M K was added2CO3The pH value of the solution is adjusted to 7.6, the solution is stirred uniformly, then an IL-6 detection antibody and horseradish peroxidase are added, the solution is stirred for 30min, then 2mL of 3 mass percent bovine serum albumin solution is added, centrifugation is carried out, the centrifugation time is 20min, the rotating speed is 13000rpm, supernatant is discarded, then 0.1 mass percent bovine serum albumin solution is added for redissolving, and then the mixture is placed in a 4-degree refrigerator for storage.
(3) Coating modification of CRP, PCT, IL-6 capture antibody: the anti-human CRP, PCT and IL-6 antibodies are coated on the surface of a reaction substrate in sequence by a protein membrane scribing instrument, a micro-fluidic chip and a spotting instrument respectively, and then are cut to obtain CRP, PCT and IL-6 capture antibody pattern strips. The CRP, PCT and IL-6 capture antibody pattern strips respectively comprise 1 CRP capture antibody pattern, 1 PCT capture antibody pattern and 1 IL-6 capture antibody pattern.
(4) Chip assembly: and (3) carrying out surface activation on the chip fluid layer and the substrate layer for 1min by using an oxygen-oxygen particle surface cleaning machine, placing CRP, PCT and IL-6 capture antibody pattern strips, and then attaching and packaging the fluid layer and the substrate layer.
(5) Signal detection: after CRP, PCT and IL-6 protein detection reagents are injected in sequence, the reagent is put into a full-automatic portable device until the machine finishes immunoassay and subsequent signal processing. As shown in FIG. 8, the detection interval for CRP is 100-3.13. mu.g/mL, for PCT is 75-0.31ng/mL, and for IL-6 is 2000-62.5 pg/mL. The concentration range detected is up to ten orders of magnitude.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Claims (10)
1. The microfluidic chip with the layered structure is characterized by comprising the following three layers: a fluid layer, a base layer and antibody pattern strips.
2. The microfluidic chip with a layered structure according to claim 1, wherein the antibody pattern strip is a porous film formed by stacking micro-nanofibers;
preferably, the diameter of the micro-nano fiber is 1000 μm to 100nm, preferably 20 μm to 500nm, and most preferably 1 μm;
more preferably, the porous membrane has a thickness of 1000 μm to 1 μm, preferably 100 μm to 5 μm, most preferably 10 μm;
further preferably, the pore size of the pores in the porous film is from 100 μm to 500nm, preferably from 10 μm to 1 μm, and most preferably 5 μm.
3. The microfluidic chip with a layered structure according to claim 2, wherein the porous membrane is prepared by one or more methods selected from the group consisting of: ion etching, sintering, spinning and chemical deposition.
4. The microfluidic chip of 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 to 50nm, preferably 1 μm to 100nm, and most preferably 200 nm.
5. The microfluidic chip with the layered structure according to claim 4, wherein the micro-nano particles are selected from one or more of the following: gold nanoparticles, silica nanoparticles, ferroferric oxide nanoparticles, polystyrene microspheres;
preferably, the micro-nano particles are gold nano particles.
6. A multiplex immunoassay system having an adjustable detection zone, the assay system comprising:
the layered microfluidic chip according to any one of claims 1 to 5, and
a matched automatic portable instrument.
7. The multiple immunoassay system of claim 6, wherein the automated portable instrument comprises:
a valve controller;
a liquid driver;
an optical path; and
a CCD camera or a photomultiplier tube.
8. Method for using the immunoassay system according to claim 6 or 7, characterized in that it comprises the following steps:
(1) selectively coating the antibody pattern strip with an antibody;
(2) cutting the antibody pattern strip, and assembling a chip;
(3) and injecting the biological sample and the reagent into the chip, and automatically detecting by using a portable device.
9. Use of a microfluidic chip of a layered structure according to any one of claims 1 to 5 or an immunoassay system according to any one of claims 6 to 7 for the preparation of a product for the simultaneous detection of multiple biomarkers, preferably human serum samples, in undiluted samples, preferably in the range of approximately ten orders of magnitude.
10. An immunoassay device, the device comprising;
the layered microfluidic chip of 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 the detection interval according to different concentrations of the detection object.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113720818A (en) * | 2021-08-27 | 2021-11-30 | 广东省大湾区华南理工大学聚集诱导发光高等研究院 | Fluorescence immunoassay system |
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