TW202216984A - Bioactive composition test piece structure and detection method thereof capable of simplifying biomedical detection under the surface imaging device and avoiding the test piece structure from occupying the surface imaging device - Google Patents

Abstract

The present invention relates to a bioactive composition test piece structure and a detection method thereof. At least one bioactive composition is disposed on at least one analysis area of a substrate for capturing at least one specimen body. The substrate is provided with at least one marking pattern in at least one analysis area. After a surface imaging device scans the at least one analysis area and obtains a plurality of analysis images in batches, the at least one marking pattern is positioned to make the scanned images overlapped, so as to perform the performance analysis on the at least one bioactive composition. Accordingly, the biomedical detection under the surface imaging device can be simplified, thereby avoiding the test piece structure from occupying the surface imaging device.

Description

Bioactive composition test piece structure and its detection method

The invention relates to a test piece structure and a detection method thereof, in particular to a test piece structure of a biologically active composition and a detection method thereof.

Generally speaking, the performance of protein immobilization on a wafer has several main factors, including stability, uniformity and orientation. Among them, factors such as stability and uniformity can be solved by using Self-Assembled Monolayer (SAM) technology to fix monolayer proteins on the wafer surface by covalent bonding. However, there is still no data to check and follow on the above-mentioned orientation factors. If the Active Binding Site of the protein immobilized on the wafer is buried below, although the above SAM technology can be used to obtain its stability and uniformity, the downward orientation must not be able to match the The antigen (or ligand) above is effectively bound, so the overall effect of the protein on the chip will be very inefficient. Therefore, ordinary users cannot meet the needs of users in actual use.

Therefore, it was developed to screen related drugs by quantitative differences measured by surface imaging devices, and at the same time, by applying an electric field to change the uniform direction of protein immobilization on the wafer, thereby changing the efficiency of protein wafer usage. Not only can it be measured multiple times, but as long as the activity of the protein does not disappear, the experiment system can be reused on the same chip; if combined with microarray technology, multiple drugs can be added to different areas, and the measurement can be obtained on the same chip. The effect of multiple drugs is not only rapid and low-cost, but also a potential protein chip analysis and application technology, which can produce more efficient protein chips, and can reduce costs and improve their quality at the same time.

However, the surface imaging device detection technology developed above requires a long time for the wafer to be combined with the surface imaging device. During the detection process, the experimenter cannot peel off or move the biological wafer for biological culture, or clean the biological wafer. The scanning results of the imaging device are less accurate, that is, the target sample to be analyzed is not effectively attached, or after the detection wafer is peeled off, it takes extra time to reposition, and it is impossible to directly perform the biological culture steps and the scanning steps of the surface imaging device. , and the target positioning of biological analysis is very difficult. In addition, the current improvement of biochip detection lies in planning the flow channels on the biochip, or setting up more micro flow channels, but there is no further improvement in batch processing, so as to greatly improve the efficiency of biodetection

Based on the above-mentioned problems, the present invention provides a biologically active composition test piece structure and a detection method thereof. By non-destructively disposing at least one marking pattern on at least one analysis area on a substrate of the test piece structure, the surface of the test piece structure is After the imaging device completes the analysis and imaging for the at least one analysis area, it can be positioned directly according to the at least one marking pattern and can be superimposed on a plurality of analysis images of the surface imaging device to analyze the biological activity composition on the biochip. In addition, The test piece structure of the present invention is an independent structure, thereby improving the biological detection efficiency.

The main purpose of the present invention is to provide a bioactive composition test piece structure and a detection method thereof, which can accurately locate and superimpose the characteristic analysis images of the multiple surface imaging device by using the marking pattern on the substrate, so as to facilitate the bioactive composition The biological culture analysis can make the test piece structure independent of the surface imaging device for biological culture and cleaning, thereby improving the efficiency of biological detection.

The invention discloses a method for detecting a biologically active composition test piece structure, which is applied to a surface imaging device to detect at least one body carried on a test piece structure, the test piece structure has at least one analysis area, and the test piece structure has at least one analysis area. At least one marking pattern is provided, and the detection method firstly uses at least one linking molecule on the at least one analysis area to immobilize at least one biologically active composition on the analysis area, and then uses the surface imaging device to detect the at least one biologically active composition. Analyzing and imaging is performed to obtain a biological characteristic image, and then the test piece structure is immersed in a sample solution having a plurality of sample bodies, so that the at least one biologically active component captures the at least one body, and then a cleaning device is used to clean the body at least one analysis area for peeling off the sample body that has not been captured by the at least one biologically active component; then, using the surface imaging device to analyze and image the at least one biologically active component and the sample body to obtain a surface characteristic analysis image, and locate and superimpose at least the biological analysis image and the characteristic analysis image according to the at least one marking pattern, so as to obtain a superimposed image of the at least one analysis area, and provide a biological creature in at least a specific position of the analysis area information, thereby analyzing the situation of the at least one biologically active component grasping the at least one entity. As can be seen from the above, in the present invention, at least one marking pattern can be set on the analysis area on the test strip structure to locate and mark the situation of grabbing the at least one body by the at least one biologically active component, and avoid damaging the surface during biological culture or cleaning. Probe of the imaging device.

The present invention provides an embodiment, wherein the at least one entity is at least one cell, at least one bacterium, at least one virus, at least one deoxyribonucleic acid (DNA), at least one ribonucleic acid (RNA) or at least one protein.

The present invention provides an embodiment, wherein before the step of immobilizing at least one biologically active composition on the analysis region with at least one linking molecule on the analysis region, a surface in the analysis region is scanned by the surface imaging device, In order to obtain a blank analysis image, the analysis area is further cleaned by the cleaning device.

The present invention provides an embodiment wherein the analysis area is further cleaned by the cleaning device before the step of scanning the at least one biologically active component is performed by the surface imaging device.

The present invention provides an embodiment, wherein the at least one biologically active component is a globulin or an antibody, and the functional group before the covalent group of the at least one biologically active component is selected from hydroxyl, alkyl, amine, carboxylic acid, and ester group, thioester group, aldehyde group, epoxy group, ethoxy group, ethane group, oxirane group, hydrazine group or thiol group, the functional group before the non-covalent group composed of at least one biological activity is Selected from biotin, avidin, streptavidin, protein, DNA, RNA, ligand or receptor.

The present invention provides an embodiment, wherein in the step of immobilizing at least one biologically active component on the analysis region, further use of hydroxyl group, alkyl group, amine group, carboxylic acid, ester group, thioester group, aldehyde group, epoxy group , ethoxy, ethane, oxirane, hydrazine or thiol functional group compounds link and fix the at least one biologically active composition on the substrate.

The present invention provides an embodiment wherein the surface imaging device is further operated at a temperature of 77K to 400K.

The present invention further discloses a bioactive composition test piece structure, which comprises a substrate, at least one label pattern and a plurality of linking molecules, the substrate has at least one analysis area, and the at least one label pattern is non-destructively disposed on the analysis area , and the plurality of linking molecules are arranged in the at least one analysis area and some of the linking molecules are arranged on the marking pattern. The above-mentioned test piece structure provides a setting environment for biologically active components, and can be analyzed by a surface imaging device into a plurality of analysis images, and the biologically active components linked by these linking molecules can be located and marked by the marking pattern, which can be simultaneously utilized. It is convenient to superimpose the scanned images of the analysis area by the marking pattern.

The present invention provides another embodiment, wherein the setting area of the at least one marking pattern of the substrate is selected from a first surface material, and the remaining areas of the substrate are selected from a second surface material, the first surface material and the The second surface material is selected from metal, plastic, glass, silicon wafer, semiconductor, dielectric layer material, organic material, hydroxylated poly(methyl methacrylate) (PMMA) or a combination thereof, and the first surface material The same material or different material as the second surface material.

The present invention provides another embodiment, wherein the functional groups of the linking molecules are hydroxyl, alkyl, amine, carboxylic acid, ester, thioester, aldehyde, epoxy, ethoxy, ethane , ethylene oxide group, hydrazine group or thiol group.

In order to make you examine the characteristics of the present invention and the effect achieved to have a further understanding and understanding, I would like to assist with the embodiment and the description of the cooperation, and the description is as follows:

In view of the problem of detection quality that the existing atomic force microscope biological detection mechanism cannot reliably provide better quality, accordingly, the present invention proposes a biologically active composition test piece structure and a detection method to solve the problem caused by the prior art. The problem of the best detection quality.

Hereinafter, the present invention will further describe the characteristics and matching structures of a biologically active composition test piece structure and its detection method:

First, please refer to the first figure, which is a flowchart of an embodiment of the present invention. As shown in the figure, the biological activity of the present invention constitutes the detection method of the test piece structure, and its steps comprise:

Step S10: provide a test piece structure;

Step S15: cleaning the test piece structure;

Step S20: using the surface imaging device to scan a blank surface in the analysis area to obtain a blank analysis image;

Step S30: using at least one linking molecule of the analysis region to immobilize at least one biologically active composition on the analysis region;

Step S35: cleaning the test piece structure;

Step S40: using the surface imaging device to perform a second scan on the at least one biologically active composition to obtain a biological analysis image;

Step S50: using the test piece structure to immerse in a sample solution having a plurality of sample bodies, so that the at least one biologically active component captures the at least one body;

Step S55: cleaning the test piece structure;

Step S60: using the surface imaging device to scan the at least one biologically active composition and the sample to obtain a characteristic analysis image; and

Step S70: Position and superimpose at least the biological analysis image and the characteristic analysis image according to the marking pattern to obtain a superimposed image of the analysis area and provide biological information on at least a specific position of the analysis area.

Please refer to the second figure A to the second figure G, which are a schematic diagram of providing a test piece structure, a schematic diagram of cleaning a blank test piece, a schematic diagram of connecting biologically active components, a schematic diagram of cleaning biological components, and a immersion sample according to an embodiment of the present invention. Schematic of the solution, schematic of the cleaned sample, and schematic of the superimposed image. As shown in the figure, the bioactive composition test piece structure 1 of the present invention includes a substrate 10, at least one marking pattern 20 and a plurality of linking molecules C, the substrate 10 has an analysis area 12, and the marking pattern 20 is arranged in the analysis area 12, the present embodiment takes a single mark pattern 20 as an example.

In step S10, as shown in Figure 2 A, the marking pattern 20 is provided on the substrate 10 providing a test strip structure 1, and the analysis region 12 is provided on the substrate 10, so the marking pattern 20 is also arranged on the substrate 10. In the analysis area 12, and formed together with the substrate 10, the substrate 10 and the marking pattern 20 are made of materials selected from metal materials, plastics, glass, silicon wafers, semiconductor materials, insulating materials, organic materials, hydroxylated materials Poly(methyl methacrylate) (PMMA) or a combination of the above combination, especially the substrate 10 and the marking pattern 20 are formed of the same or different materials, and the marking pattern 20 is non-destructively disposed on the substrate 10 , that is, deposited on the substrate 10 by chemical vapor deposition or evaporation. Continuing in step S15, as shown in Fig. 2 B, for the detection with more severe conditions, further at the beginning, when the substrate 10 does not carry any substance, use a cleaning device CL to clean the substrate 10, for example: by The substrate 10 is cleaned by spraying the fluid F from the nozzle N to prevent the substrate 10 from sticking to other substances and occupying the analysis area 12 at the beginning, wherein the fluid F is water, physiological buffer solution, alcohol, organic solution, hydrochloric acid, Nitric acid, sulfuric acid or active interface agent. The detection in step S15 in this embodiment under conditions closer to the atmospheric environment can be omitted.

Continuing in step S20, as shown in the second figure C, a cantilever CA of the surface imaging device 40 is connected to a probe P to scan the analysis area 12, and a laser source L generates a projection beam R1 at one end of the cantilever CA , to reflect a reflected beam R2 to a photo sensor M, thereby analyzing and imaging the analysis area 12 through the probe P in a contact or non-contact manner. This embodiment is an atomic force microscope, so the probe P P varies with the fluctuation of an upper surface 102 of the substrate 10 in the analysis region 12 , thereby causing the reflected light beam R2 to generate vibration, frequency or wavelength changes, so that the optical sensor M correspondingly generates a first sensing The test signal SL1 is sent to a processing unit 30, so that the processing unit 30 correspondingly generates a blank analysis image IMG1. The cleaning and blank scanning described above are verifications after mass production of the test piece structure 1 of the present invention. The following are the necessary procedures in the general experimental process of the present invention.

In step S30, as shown in the second diagram D, in this embodiment, a first biologically active composition Y1 and a second biologically active composition Y2 are used as an example, and a first linking molecule C1 and a second linking molecule C2 are disposed on the On the substrate 10, since the material of the marking pattern 20 and the substrate 10 are the same or different, the first linking molecule C1 and the second linking molecule C2 may be different. The first bioactive composition Y1 in this embodiment It is the same composition as the second biologically active composition Y2, such as: antibody, antigen, DNA probe, RNA probe, enzyme, protein or globulin, which binds the first linking molecule C1 and the first linking molecule C1 by covalent bond. Two linking molecules C2, the functional groups of the first biologically active component Y1 and the second biologically active component Y2 in the covalent bond are hydroxyl, alkyl, amine, carboxylic acid, ester, thioester, aldehyde, ring oxy, ethoxy, ethane, oxirane, hydrazine or thiol group, so the first linking molecule C1 and the second linking molecule C2 have hydroxyl, alkyl, amine, carboxylic acid , ester, thioester, aldehyde, epoxy, ethoxy, ethane, oxirane, hydrazine or thiol functional group compounds. In step S35, as shown in the second diagram E, the fluid F is sprayed on the substrate 10 by the nozzle N of the cleaning device CL, so that other substances not linked by the linking molecules C1 and C2, whether they are Proteins or other material particles will be washed away by the fluid F and removed. The detection of step S35 in this embodiment under conditions closer to the atmospheric environment can be omitted.

In step S40, as shown in the second figure F, the surface imaging device 40 analyzes and images the analysis area 12 carrying the first biologically active composition Y1 and the second biologically active composition Y2. This embodiment is the surface imaging. The cantilever CA of the device 40 suspends the probe P to scan the first biologically active composition Y1 and the second biologically active composition Y2 on the analysis area 12 , thereby allowing the projection beam R1 generated by the laser source L The reflected light beam R2 is reflected and generated, so that the light sensor M generates a corresponding second sensing signal SL2 according to the reflected light beam R2, and transmits it to the processing unit 30 to correspondingly generate a biological analysis image IMG2.

In step S50, as shown in the second figure G, the substrate 10 is immersed in a sample solution LQ, and in the solution LQ, the first bioactive composition Y1 and the second bioactive composition Y2 of this embodiment are bonded The sample body SP in the sample solution LQ, that is, the functional group of the non-covalent bond of the first biologically active composition Y1 and the second biologically active composition Y2 is bonded to the sample bodies SP, wherein the functional group is are selected from biotin, avidin, streptavidin, protein, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), ligand or receptor, so these sample SPs are cells, Bacteria, DNA molecules, RNA molecules, viruses or proteins. In step S55, as shown in the second figure H, the fluid F is sprayed on the substrate 10 by the nozzle N of the cleaning device CL, so that the first biological activity is not composed of Y1 and the second biological activity. The other substances captured by the composition Y2, whether they are proteins or other substance particles, will be washed away by the fluid F and removed.

In step S60, as shown in the second figure I, the surface imaging device 40 determines the first biological activity composition Y1 and the second biological activity composition Y2 and the first biological activity composition Y1 and the second biological activity composition Y2. The captured sample bodies SP are analyzed and imaged. The cantilever CA of the surface imaging device 40 in this embodiment suspends the probe P to scan the first biologically active composition Y1 and the second biologically active composition Y2 and The sample bodies SP captured by the first bioactive composition Y1 and the second bioactive composition Y2 are thus reflected by the projection beam R1 generated by the laser source L to generate the reflected beam R2, so that the light The sensor M generates a corresponding third sensing signal SL3 according to the reflected light beam R2, and transmits it to the processing unit 30 to generate a characteristic analysis image IMG3 correspondingly. In step S70, the processing unit 30 superimposes the blank analysis image IMG1 to the characteristic analysis image IMG3, and positions the blank analysis image IMG1 to the characteristic analysis image IMG3 according to the marking pattern 20, so as to make the blank The analysis image IMG1, the biological analysis image IMG2, and the characteristic analysis image IMG3 are superimposed at the same position in the analysis area 12, so that a corresponding stack of the blank analysis image IMG1, the biological analysis image IMG2, and the characteristic analysis image IMG3 is obtained. The IMGO image is combined to accurately analyze the culture state of the biologically active composition of the substrate 10 at the same location. The above embodiment takes a single analysis area 12 as an example, but the present invention is not limited to this. The present invention can also present the analysis area 12 in an array and position it with a coordinate system, so as to be applied to different biological studies, for example: individual different organisms The active composition is correspondingly set in different analysis areas to analyze all the properties of the sample body.

The cleaning step mentioned above is for illustration, and the cleaning device CL of the cleaning step of the present invention can not only be cleaned by spraying the fluid F from the nozzle N, but also can be immersed in a cleaning liquid (for example: water, physiological buffer solution, alcohol, organic solution, hydrochloric acid, nitric acid, sulfuric acid or active interface agent) to avoid non-analyzed substances remaining in the analysis area 12 . In addition, since the test strip structure 1 of the present invention is not equipped with the structure of the surface imaging device 40, the cleaning step and the biological culture can be carried out separately, thereby preventing the probe P of the surface imaging device 40 from being affected or damaged, thereby improving the biological performance. detection efficiency. The above-mentioned marking pattern 20 is not limited to the analysis area 12 , and may be disposed outside the analysis area 12 . The above-mentioned surface imaging device 40 is exemplified by the atomic force microscope in the scanning probe microscope, in addition, it can be an optical microscope, an electron microscope, a scanning microscope, a near-field optical microscope, an electrostatic force microscope, or a tunneling microscope. type microscope (STM). The surface imaging device 40 described above is further operated at a temperature of 77K to 400K.

To sum up, the bioactive composition test piece structure and its detection method of the present invention provide a surface imaging device that does not need to build a flow channel combined with a substrate, so that the substrate can be biologically cultured and cleaned, and avoid the need for biological culture or cleaning. Destroy the probe of the surface imaging device, thereby improving the efficiency of biological detection.

Therefore, the present invention is indeed novel, progressive and available for industrial use, and it should meet the requirements of patent application in my country's patent law.

However, the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the scope of implementation of the present invention. All changes and modifications made in accordance with the shape, structure, features and spirit described in the scope of the patent application of the present invention are equivalent. , shall be included in the scope of the patent application of the present invention.

1: Test piece structure 10: Substrate 12: Analysis area 20: Mark Pattern 30: Processing unit 40: Surface imaging device C: linker molecule C1: The first linking molecule C2: Second linker molecule CA: Cantilever CL: Cleaning device F: fluid IMG1: Blank Analysis Image IMG2: Bioanalytical Imaging IMG3: Characterization Image IMGO: Overlay images L: laser source LQ: solution M: light sensor P: Probe SL1: The first sensing signal SL2: The second sensing signal SL3: The third sensing signal SP: sample body Y1: The first biologically active composition Y2: Second bioactive composition S10-S70: Steps

Figure 1: It is a flow chart of an embodiment of the present invention; The second figure A: it is a schematic diagram of providing a test piece structure according to an embodiment of the present invention; Second Figure B: it is a schematic diagram of the cleaning blank test piece of an embodiment of the present invention; Second Figure C: It is a schematic diagram of a blank analysis image according to an embodiment of the present invention; The second figure D: it is a schematic diagram of the linked biologically active composition of an embodiment of the present invention; The second figure E: it is a schematic diagram of the cleaning biological composition according to an embodiment of the present invention; Second Figure F: it is a schematic diagram of a bioanalytical image according to an embodiment of the present invention; Second G figure: it is a schematic diagram of immersion in the sample solution according to an embodiment of the present invention; The second figure H: it is a schematic diagram of a cleaning sample according to an embodiment of the present invention; The second I figure: it is a schematic diagram of a characteristic analysis image according to an embodiment of the present invention; and Second Figure J: It is a schematic diagram of a superimposed image according to an embodiment of the present invention.

S10-S70: Steps

Claims (10)

A method for detecting a biologically active composition test piece structure, which is applied to a surface imaging device to detect at least one body carried on a test piece structure, the test piece structure has at least one analysis area, and the test piece structure is provided with at least one Marking pattern, the detection method steps include: The blank analysis image uses at least one linking molecule of the analysis area to immobilize at least one biologically active composition on the analysis area; Using the surface imaging device to analyze and image the at least one biologically active component to obtain a biological analysis image; Utilize the test strip structure to immerse or contact at least one sample solution with a plurality of sample bodies, so that the at least one biologically active composition captures the at least one body; cleaning the substrate with a cleaning device; Using the surface imaging device to analyze and image the at least one biologically active composition and the sample to obtain a characteristic analysis image; and According to the marking pattern, the biological analysis image and the analysis area of the characteristic analysis image of the biological characteristic analysis image are positioned to obtain a superimposed image of the analysis area and provide biological information on at least a specific position of the analysis area. The method for detecting the structure of a biologically active composition test strip according to claim 1, wherein the at least one entity is at least one cell, at least one bacterium, at least one enzyme, at least one deoxyribonucleic acid (DNA), at least one ribonucleic acid ( RNA), at least one virus, or at least one protein. The method for detecting the structure of a biologically active composition test piece as claimed in claim 1, wherein before the step of immobilizing at least one biologically active composition on the analysis region by using at least one linking molecule of the analysis region, further comprising: Using the surface imaging device to analyze and image a blank surface in the at least one analysis region; and The at least one analysis area is cleaned with the cleaning device. The method for detecting the structure of a biologically active composition test piece according to claim 1, wherein before the step of scanning the at least one biologically active composition with the surface imaging device, the cleaning device is used to clean the at least one analysis area. The method for detecting the structure of a biologically active composition test strip according to claim 1, wherein the at least one biologically active composition is an antibody, an antigen, a deoxyribonucleic acid (DNA) probe, a ribonucleic acid (RNA) probe, an enzyme, a protein or globulin, the functional group before the covalent group of the at least one biologically active composition is selected from hydroxyl, alkyl, amine, carboxylic acid, ester, thioester, aldehyde, epoxy, ethoxy group, ethane group, oxirane group, hydrazine group or thiol group, the functional group before the at least one biologically active composition is selected from biotin, avidin, streptavidin protein, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), ligand or receptor. The method for detecting the structure of a biologically active composition test piece according to claim 1, wherein in the step of immobilizing at least one biologically active composition on the analysis region, further use of hydroxyl, alkyl, amine, carboxylic acid, ester, Compounds with functional groups of thioester, aldehyde, epoxy, ethoxy, ethane, oxirane, hydrazine or thiol groups link and immobilize the at least one biologically active composition on the substrate. The method for detecting the structure of a biologically active composition test piece according to claim 1, wherein the surface imaging device is further operated at a temperature of 77K to 400K. A biologically active composition test piece structure, it comprises: a substrate having at least one analysis region; at least one marking pattern non-destructively disposed on the substrate, the at least one marking located inside or outside the analysis area; and A plurality of linking molecules are arranged in the analysis area and some of the linking molecules are arranged on the marking pattern. The bioactive composition test piece structure according to claim 8, wherein the setting area of the at least one marking pattern of the substrate is selected from a first surface material, the rest area of the substrate is selected from a second surface material, the The first surface material and the second surface material are selected from metal materials, plastics, glass, silicon wafers, semiconductor materials, insulating materials, organic materials, hydroxylated poly(methyl methacrylate) (PMMA) or combinations thereof, And the first surface material and the second surface material are the same material or different materials. The bioactive composition test piece structure according to claim 8, wherein the functional groups of the linking molecules are hydroxyl, alkyl, amine, carboxylic acid, ester, thioester, aldehyde, epoxy, ethyl oxy, ethane, oxirane, hydrazine or thiol groups.

Images