TW202101003A - Composite microbeads and applications thereof capable of identifying the existence of a specific protein - Google Patents

Abstract

Disclosed are composite microbeads and applications thereof. The composite microsbeads are made of magnetic beads and plastic beads for detecting specific proteins, which can be applied to detections in various fields such as biological chemistry, medical science, food safety, etc. A bipolar plate is employed by the present invention to measure the electrical impedance of the composite microbeads, and to draw the electrochemical impedance spectroscopy (EIS) of the composite microbeads, therefore, the present invention not only can identify the existence of a specific protein, but also can identify the type of the specific protein, thereby achieving a goal of conducting a speedy and accurate inspection and classification of specimens.

Description

Composite microbeads and their applications

The present invention is related to medical detection technology, and particularly refers to a composite microbead that can be used to detect the protein to be tested and its application.

Traditional biochemical testing is limited by equipment and technology, and has problems such as high cost, poor efficiency, and human operation pollution. Nowadays, testing instruments can be miniaturized on a chip through MEMS technology, which not only reduces the cost of testing operations , Time cost, also has a considerable contribution to the popularization of advanced detection technology. The microfluidic device introduces a small amount of liquid into a chip element with microchannels for reaction (detection). By combining MEMS technology and microfluidic devices, the functions of sampling, mixing, transmission, pre-processing, separation, reaction, and detection required for biochemical testing can be combined on the microfluidic chip (microelectromechanical chip) to achieve automated analysis. Purpose, and has the advantages of small size, low cost, high detection efficiency, high sensitivity and specificity, low sample and reagent consumption, low energy consumption, and avoiding human operation pollution.

For example, for suspected cases of dengue fever, the most commonly used method is rapid screening with test strips, which has the advantages of low cost, speed and freedom from site restrictions. However, the test strips cannot identify the type of infection, and blood must be drawn and sent to a routine laboratory for testing. The type can be determined, but sending to the laboratory for inspection is time-consuming and requires professionals and Equipment is easy to form loopholes in epidemic prevention. In order to prevent them from happening, the rapid identification of infection types is necessary for the future prevention and control of diseases in Taiwan. Biochip products are more flexible and can provide more information than test strips. However, there are few related products. It is worth mentioning that the biochip system "VereTropTM" produced by the Singapore company "veredus laboratories" was discontinued in 2003. , The existing similar products are only the portable precision diagnostic system "BluBox" launched by the Danish startup "BluSense Diagnostics" in 2017. However, the biochip stage of this product needs to be pre-loaded with biomaterials, and the storage time is short. The manufacturing cost is high, and the detection time is long. The current rapid diagnosis method of dengue fever is rapid screening with test strips, and the cost is about NT$300 each time. The type of infection cannot be identified. It is necessary to draw blood and send it to a routine laboratory test to determine the type, but it takes a long time to send it to the laboratory for testing. When it is wasteful and requires professionals and equipment, it is easy to form a loophole in epidemic prevention. In order to prevent it from happening, the rapid identification of infection type inspection is necessary for future disease prevention and control in Taiwan.

The Republic of China I470224 patent discloses a microfluidic detection system in which the compound to be tested is fixed in the microfluidic channel with a magnetic element, and then the sample is injected into the microfluidic channel to react with the compound for detection. The patent is in During detection, there may be excess specimens or compounds that are not involved in the reaction remaining in the microfluidic channel, causing signal deviation and reducing detection accuracy.

In order to solve the shortcomings of the prior art, the present invention provides a composite microbead and its application, which uses a composite microbead composed of magnetic beads and glue beads for detection Specific protein can be used for detection requirements in many fields such as biochemistry, medicine, food safety, etc. The present invention uses a double electrode plate to measure the electrical impedance of the composite microbeads, and draws the electrochemical impedance spectrum of the composite microbeads, except that the specific In addition to the presence or absence of protein, specific protein types can also be classified to achieve the goal of rapid and accurate inspection and classification of samples.

The present invention provides a composite microbead body, the composite microbead system includes: one or more magnetic beads, the surface of which is planted with a first antibody, the first antibody system is used to capture a protein to be tested; and one or more gel beads, A second antibody is planted on the surface of the gel bead, and the second antibody system can be combined with the first antibody, so that the magnetic bead and the gel bead are combined to form a composite microbead with the protein to be tested.

The present invention provides a method for preparing composite microbeads. The steps include: providing a certain amount of protein to be tested; providing one or more magnetic beads on the surface of which is planted with a first antibody capable of capturing the protein to be tested; The protein to be tested and the magnetic beads are added to the first buffer solution to make the magnetic beads capture the protein to be tested; the magnetic beads are separated from the protein to be tested that has not been captured by magnetic force to remove excess protein to be tested; provide more than one gel The surface of the gel bead is planted with a second antibody that can bind to the first antibody; the magnetic beads and the gel bead are added to the second buffer to bind the first antibody and the second antibody, and then the magnetic The beads are combined with the glue beads to form composite microbeads; the composite microbeads and the unbound glue beads are magnetically separated to remove excess glue beads.

The present invention provides a method for detecting composite microbeads, the steps of which include: providing a certain amount of composite microbeads, and adding the composite microbeads to a third In the buffer, a composite microbead fluid is formed; the composite bead fluid is introduced between a set of double electrode plates, and a signal processing unit measures the difference between the composite bead fluid and the two electrode plates when the composite bead fluid passes between the double electrode plates. Impedance change, the signal processing unit draws the electrochemical impedance spectrum of the composite microbead according to the impedance change, and compares it to determine whether the composite microbead contains a protein to be tested.

In the embodiment of the composite microbead detection method of the present invention, it further includes the following steps: pre-establishing electrochemical impedance spectrum data when the composite microbead does not contain the protein to be tested, as a comparison basis for judging whether the protein to be tested exists .

In an embodiment of the present invention, the method for removing excess magnetic beads and glue beads is washing with phosphate aqueous solution or pure water.

In an embodiment of the present invention, the first, second and third buffers are aqueous phosphate solutions or other types of buffer solutions.

In an embodiment of the present invention, the particle size of the magnetic beads is not smaller than that of the gel beads, and the particle size of the magnetic beads and the gel beads is 0.1-100 μm.

In an embodiment of the present invention, the rubber beads are made of latex, and other kinds of artificial or natural materials can also be used.

In an embodiment of the present invention, the double-electrode plate refers to a double-electrode system structure in an electrochemical system, which can be a parallel-plate double-electrode plate, a coplanar double-electrode plate, or other types of electrochemical double-electrode systems.

In an embodiment of the present invention, the two-electrode plate can be a four-electrode system derived from an electrochemical two-electrode system, and the two-electrode plate can be a four-electrode system. In the electrode end measurement technology, the two points connecting the end points of the voltmeter and the current meter lead.

The above summary and the following detailed description and drawings are all for further illustrating the methods, means and effects of the present invention to achieve the intended purpose. Other objectives and advantages of the present invention will be described in the following description and drawings.

S01~S07‧‧‧Composite microbead preparation method steps

10‧‧‧Composite beads

11‧‧‧Magnetic beads

12‧‧‧Glue beads

13A, 13B‧‧‧Double electrode plate

A1‧‧‧The first antibody

P‧‧‧Protein to be tested

A2‧‧‧Secondary antibody

S101~S111‧‧‧Composite microbead detection method another embodiment step

Figure 1 is a flow chart of the steps of the composite microbead fluid detection method of the present invention.

Figure 2 is the structure of the first embodiment of the composite microbeads and detection system of the present invention

Fig. 3 is a flowchart of another embodiment of the composite microbead detection method of the present invention.

Fig. 4 is a sample diagram of test impedance according to an embodiment of the present invention.

Fig. 5 is a comparison diagram of the discrimination rate of impedance measurement results according to the embodiment of the present invention.

The following is a specific example to illustrate the implementation of the present invention. Those familiar with the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification.

The present invention uses magnetic beads and latex beads as carriers to capture and amplify the signal of the protein to be tested. This patent is to plant the first antibody on the surface of the magnetic beads to capture different types of The protein to be tested is planted with a specific second antibody on the surface of the gel bead, and then the magnetic beads, the protein to be tested and the gel bead are bonded to form a sandwich measurement structure, which can amplify the protein to be tested Electrical signal to determine the presence of the protein to be tested None, feature type, and quantitative detection (that is, detection of the content and ratio of the protein to be tested) can be achieved through the number of bonds.

The "a protein to be tested" in the description of the present invention refers to the target of the present invention to be tested: a specific protein whose quantity is not fixed and depends on the number of samples that can be provided in the actual test occasion, not the quantity of the protein. Limited to one, he first stated.

Figure 1 is a flow chart of the preparation method of composite microbeads of the present invention. The steps include: providing a certain amount of protein S01 to be tested; providing one or more magnetic beads on the surface of which can capture the protein to be tested. An antibody S02; add the protein to be tested and the magnetic beads to the first buffer, so that the magnetic beads capture the protein to be tested S03; magnetically separate the magnetic beads and the protein to be tested that have not been captured, and remove excess Protein S04 to be tested; provide one or more gel beads, the surface of which is planted with a second antibody S05 that can bind to the first antibody; add the magnetic beads and the gel beads to the second buffer to make the first antibody Combined with the second antibody, the magnetic beads and the gel beads are combined to form composite microbeads S06; the composite microbeads and unbound gel beads are separated by a magnetic force to remove excess gel beads S07.

The structure diagram of the first embodiment of the composite microbeads of the present invention and the detection system thereof is shown in Figure 2, which is used to briefly explain the structure of the composite microbeads of the present invention and the electric field generated by the double electrode plate to detect the composite microbeads. The principle of electrical impedance. As shown in the figure, the composite microbead body 10 includes one or more magnetic beads 11, and a first antibody A1 is implanted on the surface of the magnetic beads. The first antibody A1 is used to capture a protein P to be tested; and one or more gel beads 12 , The second antibody A2 is planted on the surface of the glue bead 12, and the second The antibody A2 can be biochemically linked and combined with the first antibody A1, so that the magnetic beads 11 and the gel beads 12 are combined to form a composite microbead 10 with the protein P to be tested. The purpose of the present invention using magnetic beads and glue beads to form a composite microbead is to increase the impedance of the object to be measured, so that when it passes between the double electrode plates 13A, 13B, the electric field E flowing between the double electrode plates 13A, 13B Generate a more powerful electrical impedance signal, and then use a signal processing unit (not shown) to draw the electrochemical impedance spectrum of the composite microbead according to the impedance change between the two electrode plates, so as to compare and judge the composite microbead Whether the body contains the protein P to be tested.

In an embodiment of the present invention, the first antibody system is used to capture the protein to be tested, attach it to the magnetic beads, and then combine the biochemical reaction between the second antibody and the first antibody to make the particle size The smaller beads are attached to the surface of the larger magnetic beads. The magnetic beads and gel beads can amplify the electrical signal of the protein to be tested, and by analyzing the electrochemical impedance spectrum pattern of the composite microbeads, the secondary type of the protein to be tested can be further distinguished, for example, for In the detection of multiple types of disease proteins, the inspector can distinguish whether the sample sample has a certain type of disease protein, and distinguish whether it belongs to the disease type A, B, C, or D, providing follow-up drug treatment process more Reference information.

In an embodiment of the present invention, before performing the detection of the composite microbeads, the electrochemical impedance spectrum data when the composite microbeads do not contain the protein to be tested can be established for comparison during actual detection. The double electrode plate for detection can be arranged on a microfluidic sensor chip, and the signal processing unit can be arranged on the microfluidic sensor chip together or independently of the chip. Type of double electrode plate It can be a parallel-plate double-electrode plate, a co-planar double-electrode plate or other types of electrochemical double-electrode system architecture, or a four-electrode system derived from a double-electrode system. Among them, the parallel-plate double-electrode plate and the co-planar double-electrode plate are more Suitable for mass production process of printed circuit board, suitable for mass production to reduce cost. In one embodiment of the present invention, the composite microbead preparation process (magnetic bead, glue bead combination process) and detection program can also be combined and fabricated on a single microfluidic chip or other sensing device, which can be completed more quickly. Test for measuring protein.

The flow chart of another embodiment of the composite microbead detection method of the present invention is shown in Figure 3. By analyzing electrochemical impedance spectroscopy (Electrochemical Impedance Spectroscopy, EIS), the number of microbeads contained is judged, and the antigen (to be tested) Protein) quantity. The steps of this embodiment include: injecting a first sample reagent (pure buffer) into a microfluidic sensor chip, and giving a test signal, and judging whether the microfluidic sensor chip is stable according to the data of electrochemical impedance spectrum (S101); Then inject a second sample reagent into the same microfluidic sensor chip. This sample reagent has similar electrochemical impedance spectroscopy characteristics to the first sample reagent, and contains magnetic particles (magnetic beads) S102 bound to the first antibody on the surface; Obtain the electrochemical impedance spectrum of a test signal, and record it as the first test data S103; then inject the target sample (containing the protein to be tested) into the same microfluidic sensor chip to fully mix it with the second sample reagent S104; Apply a magnetic field to the same microfluid sensor chip to fix the magnetic particles S105; inject the first sample reagent (pure buffer) to make substances other than the magnetic particles leave the microfluid sensor chip S106; then add the third sample reagent On the same microfluidic sensor chip, this sample The reagent contains latex particles (gel beads) S107 with the second antibody bound to the surface; mix the liquid in the microfluidic chip fully, and then fix the magnetic particles to S108; inject the first sample reagent (pure buffer) to make the magnetic particles Other substances leave the microfluid sensor chip S109; give a test signal to obtain its electrochemical impedance spectrum, and record it as the second test data S110; compare the first and second test data, if the influence of the noise factor is excluded There is still a significant difference, indicating that the microfluidic sensor chip contains latex particles, and it can be seen that the target sample contains the target antigen (protein to be tested) S111.

In order to further verify the feasibility of the composite microbeads proposed in the present invention, the detection method of the biochip, and the dual-electrode plate microfluidic chip, the experiments used composite microbeads without the protein to be tested (the surface of the magnetic beads is bonded to BSA). (Bovine Serum Albumin) and primary antibody; BSA and secondary antibody bound to the surface of gel beads), and composite microbeads with protein to be tested (BSA, primary antibody and protein to be tested are bound to the surface of magnetic beads, gel The surface layer of the beads is bound to BSA and secondary antibody) for measurement. The following table is the concentration of the analyte used in this test. The concentration of the protein to be tested on the surface of the beads is 20μg/ml, and the difference between the magnetic beads and the gel beads Concentrations are all prepared by mixing with PBS, and the preparation method is prepared with a volume ratio of microbeads to PBS of 1:1. It should also be noted that the composite microbeads in this experiment are all mixed and bonded outside the microfluidic chip before measurement, and the surface layer of the microbead is bonded to the protein to be tested and the mixing time of the two microbeads is about approximately 20 minutes.

In one embodiment of the present invention, the wafer used for measurement is made by PDMS reversal molding, wherein the electrode of the wafer is composed of a single-core copper wire, and the width and height of the micro channel are about 300 μm and 400 μm, respectively. Use the peristaltic pump as the power source to push the object under test. In addition, the concentration of the magnetic beads used in the present invention is 10 mg/ml, and the first antibody has been planted on the surface of the magnetic beads, and BSA is used to fill the void of the magnetic beads that are not bound to the antibody. The concentration of the gel beads used is 1%, and the surface of the gel beads is also bound to the secondary antibody and BSA.

The present invention uses the surface layer of magnetic beads that simultaneously bind the primary antibody, BSA, and the protein to be tested to mix with the surface layer of gel beads that have been planted with the second antibody and BSA, wherein the concentrations of the magnetic beads and gel beads are 10 mg/ml and 1 %, and the mixing volume ratio of the two liquids is 1:1. As for the magnetic beads with the first antibody bound on the surface, and the magnetic beads with the second antibody bound to the protein (NS1) Glue beads, the value change curve between the two, the test impedance sample diagram of the embodiment of the present invention is shown in Figure 4, the horizontal axis is the number of samples, the left vertical axis is the impedance per unit value, and the right vertical axis is the impedance offset , Compare the impedance offset and the impedance per unit value of the pure magnetic beads and the composite microbeads of the present invention. It can be seen from the figure that the two curves are finally stable, that is, the absolute value of the offset is less than 0.2%. Comparing the values after compensation of the two curves, it can be seen that the impedance standard per unit value of the magnetic beads bound to the first antibody is about It is 0.999 standard, and the impedance standard per unit value after compensation of the magnetic beads with the protein to be tested and the secondary antibody gel bead is about 0.994 standard, the difference between the two is 0.005 standard, and the impedance offset is about 0.47% , Far greater than 0.2%, so through the detection method of the present invention, it can be accurately judged whether the sample contains the protein to be tested.

In addition, in order to explore and analyze the identification rate of the present invention in detecting the protein to be tested, the present invention is carried out twice with mixed magnetic beads (surface-bonded BSA, antibody and protein to be tested) and gel beads (surface-bonded). The measurement results of BSA and antibody) and the comparison of the discrimination rate of the measurement results are shown in Figure 5. The composite microbead detection method of the present invention is used for two tests, and it can be diagnosed that the sample contains the protein to be tested. The rate is 100%.

Therefore, the present invention provides a composite microbead and its application. The composite microbead composed of magnetic beads and gel beads is used to detect specific proteins. The present invention uses a double electrode plate to measure the electrical impedance of the composite microbead. The electrochemical impedance spectrum of the composite microbeads is drawn. In addition to distinguishing the presence or absence of specific proteins, it can also classify specific protein types to achieve the goal of rapid and accurate inspection and classification of samples. Microfluidic sensor chips with dual electrode plates can use printed circuit board technology Because of its high production efficiency and the potential for popularization, the present invention can be used for testing needs in biochemistry, medicine, food safety and other fields. The preparation and detection steps of the composite microbeads of the present invention can be shrink-packed on a single microchip, and have the effects of rapid inspection and typing, and are quite helpful for biochemical inspection technologies such as biotechnology products, disease prevention, medicine, and food safety. beneficial.

The above-mentioned embodiments are only illustrative of the features and effects of the present invention, and are not used to limit the scope of the essential technical content of the present invention. Anyone familiar with this technique can modify and change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be as listed in the scope of patent application described later.

10‧‧‧Composite beads

11‧‧‧Magnetic beads

12‧‧‧Glue beads

13A, 13B‧‧‧Double electrode plate

A1‧‧‧The first antibody

P‧‧‧Protein to be tested

A‧‧‧Secondary antibody

Claims (16)

A composite microbead body includes: one or more magnetic beads on which a first antibody is planted on the surface of the magnetic bead, and the first antibody system is used to capture a protein to be tested; and one or more gel beads on which a second antibody is planted on the surface. Antibodies, the second antibody system can be combined with the first antibody, so that the magnetic beads and the gel beads are combined to form a composite microbead with the protein to be tested. The composite microbead according to claim 1, wherein the particle size of the magnetic beads is larger than that of the gel beads. The composite microbead according to claim 1, wherein the particle size of the magnetic beads and the gel beads is 0.1-100 μm. The composite microbead according to claim 1, wherein the glue bead is made of latex. A method for preparing composite microbeads, the steps include: providing a certain amount of protein to be tested; providing one or more magnetic beads on the surface of which are planted with a first antibody capable of capturing the protein to be tested; Add magnetic beads to the first buffer solution to make the magnetic beads capture the protein to be tested; magnetically separate the magnetic beads from the protein to be tested that has not been captured, and remove excess protein to be tested; provide one or more gel beads, the gel A second antibody that can bind to the first antibody is planted on the surface of the beads; Add the magnetic beads and the glue beads to the second buffer to bind the first antibody and the second antibody, and then combine the magnetic beads and the glue beads to form composite microbeads; separate the composite microbeads by magnetic force With unbound glue beads, remove excess glue beads. The method for preparing composite microbeads according to claim 5, wherein the first buffer is an aqueous phosphate solution or other kinds of buffer solutions. The method for preparing composite microbeads according to claim 5, wherein the second buffer is an aqueous phosphate solution or other kinds of buffer solutions. The method for preparing composite microbeads according to claim 5, wherein the particle size of the magnetic beads is not smaller than that of the colloidal beads. The method for preparing composite microbeads according to claim 5, wherein the particle size range of the magnetic beads and the gel beads is 0.1-100 μm. The method for preparing composite microbeads according to claim 5, wherein the rubber beads are made of latex or other kinds of artificial or natural materials. A method for detecting composite microbeads, the steps include: providing a certain amount of composite microbeads, adding the composite microbeads to a third buffer solution to form a composite microbead fluid; and introducing the composite microbead fluid into a Between the two electrode plates, a signal processing unit measures the impedance change of the double electrode plate when the composite microbead fluid passes between the double electrode plates, and the signal processing unit draws the composite bead body according to the impedance change Electrochemical impedance spectroscopy is used to compare and judge whether the composite microbead contains a protein to be tested; wherein the steps of the preparation method of the composite microbead include: Provide a certain amount of the protein to be tested; provide one or more magnetic beads on the surface of which are planted with a first antibody that can capture the protein to be tested; add the protein to be tested and the magnetic beads to the first buffer to make the magnetic Beads capture the protein to be tested; magnetically separate the magnetic beads from the protein to be tested that have not been captured to remove excess protein to be tested; provide one or more gel beads on the surface of which can bind to the first antibody The second antibody; add the magnetic beads and the glue beads to the second buffer to bind the first antibody to the second antibody, and then combine the magnetic beads and the glue beads to form composite microbeads; separate the magnetic beads Composite microbeads and unbound glue beads to remove excess glue beads. The composite microbead detection method according to claim 11, which further includes the following steps: pre-establishing electrochemical impedance spectrum data when the composite microbead does not contain the protein to be tested, as a ratio for judging whether the protein to be tested is present On the basis. The method for detecting composite microbeads according to claim 11, wherein the third buffer is an aqueous phosphate solution or other types of buffer solutions. The composite microbead detection method according to claim 11, wherein the double electrode plate is a double electrode system structure in an electrochemical system. The composite microbead detection method according to claim 11, wherein the double electrode plate is a parallel plate double electrode plate or a coplanar double electrode plate. The composite microbead detection method according to claim 11, wherein the two-electrode plate is two points connecting the end points of the voltmeter and the current meter wire in the four-terminal measurement technique.

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