Universal qualitative analysis method for biomolecules

Abstract

The invention provides a universal qualitative analysis method for biomolecules, which comprises the following steps of: firstly, establishing a relationship between key nucleic acid and key protein corresponding to a target disease, then establishing an identification biochip capable of interpreting the target disease, and identifying the target disease through a reaction nucleic acid probe and a reaction protein antibody probe which are fixed on the identification biochip. The target nucleic acid and the target protein of the identification biochip and the specimen are reacted, and then comparison and interpretation are performed according to the color generation image of the color generation reaction of the identification biochip, so that the identification biochip has the characteristics of convenience in operation and high yield.

Description

TECHNICAL FIELD

[0001] The present application relates to a universal biomolecular qualitative analysis method for detecting biomarkers of general diseases in a qualitative manner. BACKGROUND

[0002] With the development of modern medical technology and the progress of medical equipment, the decoding of the corresponding gene sequence and genome sequence of various diseases can be established, and the possible effects or interactive regulation of the tens of genes involved in the above diseases on the organism can be determined, so that the relevant personnel can make more accurate judgments.

[0003] Furthermore, although there is information about the corresponding gene sequence of the related disease, current technical problems are mostly limited, so that each test can only focus on single gene or single protein research, but most diseases involve the interaction of multiple genes or multiple proteins, and the reaction path may be related to the reaction path and mechanism of the disease, so it is necessary to provide a qualitative analysis of the target nucleic acid and the target reaction protein of the protein, so as to reduce the above-mentioned shortcomings. SUMMARY

[0004] The purpose of the present application is to provide a universal biomolecular qualitative analysis method for qualitative analysis and judgment of the test body by identifying the biomicrochip corresponding to the selected target disease.

[0005] To achieve the above object, the present application provides a general biomolecule qualitative analysis method, which mainly includes establishing the relationship between a target disease and its corresponding key nucleic acid and key protein, searching the data of the corresponding key protein and the data of the corresponding key nucleic acid according to the specified target disease; establishing the nucleic acid mutation site and the protein mutation site of the target disease, respectively establishing at least one nucleic acid mutation site or at least one protein mutation site according to the key nucleic acid and the key protein of the target disease obtained by searching the relationship between the target disease and its corresponding key nucleic acid and key protein; checking the nucleic acid mutation site and the protein mutation site of the target disease, comparing whether the characteristic performance of the target disease is related according to the characteristic performance of the nucleic acid mutation site and the characteristic performance of the protein mutation site; establishing a discrimination biological chip for distinguishing the target disease, forming at least one corresponding reaction nucleic acid probe or at least one reaction protein antibody probe according to the nucleic acid mutation site and the protein mutation site of the target disease, and then at least one of the reaction nucleic acid probe combined with the modified nucleic acid or the reaction protein antibody probe is fixed on a carrier platform in a high-energy mode, thereby forming a discrimination biological chip for distinguishing the target disease, wherein the modified nucleic acid improves the specificity of the reaction nucleic acid probe; sample sampling and preservation, performing sample sampling and preserving the sample; extraction, purification and calibration of the sample, extracting the required target nucleic acid or target protein biomolecule material from the sample obtained after the sample sampling and preservation according to the detection purpose, purifying the biomolecule material with the target nucleic acid and the target protein, and completing the calibration by combining the biomolecule material with at least one nucleic acid calibration substance or at least one protein calibration substance; establishing a target nucleic acid reaction, activating the discrimination biological chip corresponding to the target disease and the sample that has completed calibration, then performing hybridization reaction of the target nucleic acid and the reaction nucleic acid probe of the discrimination biological chip; establishing a target protein reaction, adding an enzyme complex to the discrimination biological chip corresponding to the target disease and the sample that has completed calibration to perform enzyme reaction for activation, then performing affinity reaction of primary antibody and secondary antibody, thereby completing the reaction of the reaction protein antibody probe of the discrimination biological chip and the target protein; establishing a color development reaction, after the reaction of the reaction nucleic acid probe or the reaction protein antibody probe of the discrimination biological chip and the corresponding target nucleic acid or target protein is completed, the reaction nucleic acid probe or the reaction protein antibody probe of the discrimination biological chip will produce a color development reaction, and an image capturing module is used to capture a color development image of each discrimination biological chip; and performing comparison and interpretation, the color development image is compared and interpreted with the data of a standard image database by a processing module with an artificial intelligence system; wherein the target protein is identified by a specific primary antibody, and the target protein is identified by a specific secondary antibody and the enzyme complex.

[0006] Further, the recognition biochip is immobilized with the reaction nucleic acid probe and the antibody probe required for the reaction on a porous solid support platform.

[0007] Further, each of the reaction nucleic acid probes is one of a DNA probe or a RNA probe.

[0008] Further, the target protein reaction includes the following steps: target sequence recognition of the antigen of the target protein obtained from the subject using a specific primary antibody; specific primary antibody recognition of the enzyme complex using a specific secondary antibody combined with the primary antibody; and addition of a substrate corresponding to the enzyme complex to allow the substrate to react with the enzyme complex.

[0009] Further, the color development reaction includes the following steps: dephosphorylation of the reaction protein antibody probe of the recognition biochip; reaction of the substrate with a color development chemical; and emission of light of a corresponding wavelength.

[0010] Further, the target nucleic acid reaction includes the following steps: activation of the recognition biochip; hybridization of the reaction nucleic acid probe of the recognition biochip with the target nucleic acid of the subject; and analysis of the hybridization signal and improvement of the accuracy thereof.

[0011] Further, the modified nucleic acid is selected from one of LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), or 2’-OMe RNA.

[0012] Further, the melting temperature of the reaction nucleic acid probe is between 40°C and 60°C.

[0013] Further, the reaction nucleic acid probe and the reaction protein antibody probe are immobilized on the support platform using one of inkjet printing or laser transfer.

[0014] The universal biomolecule qualitative analysis method of the present application is to establish the relationship between the target disease and the corresponding key nucleic acid and key protein, and then establish the recognition biochip with the reaction nucleic acid probe of the nucleic acid mutation site and the reaction protein antibody probe of the protein mutation site of the target disease. The reaction nucleic acid probe is combined with a modified nucleic acid at least one of the nucleic acid mutation sites, and the DNA and RNA corresponding to the target nucleic acid or the protein corresponding to the target protein are extracted, purified and labeled after the sample to be detected is extracted, purified and labeled. The sample and the recognition biochip establish a target nucleic acid reaction or a target protein reaction, the reaction nucleic acid probe of the recognition biochip or the reaction protein antibody probe produces a hybridization reaction with the corresponding target nucleic acid and an antibody recognition reaction with the corresponding reaction protein antibody probe to produce a color reaction, and different wavelengths of color produced by the color reaction are intercepted. The processing module with artificial intelligence system and the data of the standard image database are compared and interpreted. Thus, the present application is not only suitable for any target disease and has universality, but also can be used to detect multiple genes and the expression changes of genes in organisms, and has the characteristics of convenient operation and high yield. BRIEF DESCRIPTION OF DRAWINGS

[0015] Fig. 1 : Flowchart of the universal biomolecule qualitative analysis method of the present application;

[0016] Fig. 2 : Schematic diagram of the recognition biochip of the universal biomolecule qualitative analysis method of the present application after color reaction to obtain color image.

[0017] SYMBOL DESCRIPTION

[0018] 10: Establish the relationship between the target disease and the corresponding key nucleic acid and key protein

[0019] 20: Establish the nucleic acid mutation site and the protein mutation site of the target disease

[0020] 30: Check the nucleic acid mutation site and the protein mutation site of the target disease

[0021] 40: Establish a recognition biochip for the target disease

[0022] 41: Recognition biochip

[0023] 50: Sample collection and preservation

[0024] 60: Extraction, purification and labeling of the sample

[0025] 70: Establish a target nucleic acid reaction

[0026] 80: Establish a target protein reaction

[0027] 90: Establishing a color reaction

[0028] 91: Color image

[0029] 95: Performing comparison and interpretation DETAILED DESCRIPTION

[0030] The technical features and operation of the present application are described below with reference to several preferred embodiments, together with the accompanying drawings, to provide a review for reference. Furthermore, the drawings of the present application are not necessarily drawn to scale for the purpose of illustration, and the proportions in the drawings are not intended to limit the scope of the present application.

[0031] For the technology of the present application, please refer to Figs. 1-2 The present application provides a general type of biomolecule qualitative analysis method, which mainly includes:

[0032] Establishing a relationship between a target disease and its corresponding key nucleic acid and key protein 10, retrieving data of the key protein corresponding to the target disease from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database according to the specified target disease, and retrieving data of the key nucleic acid corresponding to the target disease from the Online Mendelian Inheritance in Man (OMIM) database, but the database for retrieving the key protein corresponding to the target disease is not limited thereto;

[0033] Establishing nucleic acid mutation sites and protein mutation sites of the target disease 20, respectively establishing at least one nucleic acid mutation site or at least one protein mutation site according to the key nucleic acid and key protein of the target disease obtained by the relationship between the target disease and its corresponding key nucleic acid and key protein 10;

[0034] Checking the nucleic acid mutation sites and protein mutation sites of the target disease 30, comparing whether the characteristic manifestations of the target disease are related according to the characteristic manifestations of the nucleic acid mutation sites and the characteristic manifestations of the protein mutation sites, wherein, in this embodiment, the selected nucleic acid mutation sites and protein mutation sites are checked whether they are related by comparing literature data and other biological databases related to the target disease, but not limited thereto;

[0035] The recognition biochip 40 for identifying the target disease is established, and the nucleic acid mutation sites and the protein mutation sites corresponding to the target disease are respectively formed into at least one reaction nucleic acid probe or at least one reaction protein antibody probe. The reaction nucleic acid probe is combined with a modified nucleic acid at at least one of the nucleic acid mutation sites, and at least one of the reaction nucleic acid probe or the reaction protein antibody probe combined with the modified nucleic acid is fixed to a carrier platform by high-energy mode to form a recognition biochip 41 for identifying the target disease. The reaction nucleic acid probe and the corresponding modified nucleic acid can improve the specificity after being combined. In this embodiment, one modified nucleic acid is combined with each of the selected nucleic acid mutation site and the nucleic acid mutation site before and after the selected nucleic acid mutation site, but this is not limited. The reaction nucleic acid probe and the reaction protein antibody probe can be fixed to the carrier platform by one of mechanical spotting, ink-jetting or photolithography. The reaction nucleic acid probe has a melting temperature of 40-80°C when the nucleic acid length is the same. The modified nucleic acid is selected from one of LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid) or 2'-OMeRNA. The carrier platform in this embodiment is a porous solid carrier platform. The reaction nucleic acid probe is one of a DNA probe or a RNA probe. The recognition biochip 41 is formed by fixing the reaction nucleic acid probe and the reaction protein antibody probe to the carrier platform. Further, the melting temperature of the reaction nucleic acid probe in this embodiment is 40-60°C, but this is not limited. That is, the recognition biochip 41 can also fix only one of the reaction nucleic acid probe or the reaction protein antibody probe to the carrier platform.

[0036] Sample sampling and preservation 50, sample sampling is performed on the person to be examined, and the sample is preserved. When the sample is preserved, better sampling equipment or suitable sample preservation liquid can be used to optimize preservation.

[0037] Extraction, purification and calibration of the sample 60, the target nucleic acid or target protein and other biomolecule substances are extracted from the sample obtained by the sample sampling and preservation 50 according to the detection purpose, and the biomolecule substances with the target nucleic acid and the target protein are purified, and the target nucleic acid and the target protein are combined with at least one nucleic acid calibration substance or at least one protein calibration substance to complete calibration.

[0038] The target nucleic acid reaction 70 is established by activating the recognition biochip 41 corresponding to the target disease and the calibrated sample, and then hybridizing the target nucleic acid with the reaction nucleic acid probe of the recognition biochip 41. The target nucleic acid reaction further includes the following steps: activating the recognition biochip 41; hybridizing the reaction nucleic acid probe of the recognition biochip 41 with the target nucleic acid of the sample; and analyzing the signal of the hybridization reaction and improving the accuracy. In this embodiment, the signal is enhanced by using a specific primary antibody and a specific secondary antibody, and the accuracy is improved by using a high-resolution optical detection instrument to enhance the color signal.

[0039] The target protein reaction 80 is established by adding an enzyme complex to the recognition biochip 41 corresponding to the target disease and the calibrated sample to activate the enzyme reaction, and then performing affinity reaction of the primary antibody and the secondary antibody. In this way, the reaction protein antibody probe of the recognition biochip 41 and the target protein complete the reaction. The target protein reaction further includes the following steps: using a specific primary antibody to identify the target sequence of the antigen of the target protein obtained from the sample; using a specific secondary antibody to combine with the primary antibody and perform specific primary antibody identification with the enzyme complex; and adding a substrate corresponding to the enzyme complex to perform catalytic reaction of the substrate and the enzyme complex.

[0040] The color reaction 90 is established by reacting the reaction nucleic acid probe or the reaction protein antibody probe of the recognition biochip 41 with the corresponding target nucleic acid or target protein, respectively. The reaction nucleic acid probe or the reaction protein antibody probe of the recognition biochip 41 produces a color reaction, and an image capturing module captures a color image 91 of each recognition biochip 41. The color reaction further includes the following steps: dephosphorylation of the reaction protein antibody probe of the recognition biochip 41 to produce a reaction of the substrate and a color chemical substance, and emit light of a corresponding wavelength to form the color image 91. The corresponding wavelength of light can be visible light color and corresponding wavelength, or infrared light and corresponding infrared wavelength, but is not limited thereto; and

[0041] The comparison and interpretation 95 is performed by a processing module with an artificial intelligence system to compare and interpret the color image 91 with the data of a standard image database, so as to provide relevant personnel to interpret whether the sample has the characteristics of the target disease.

[0042] Wherein, the recognition biochip 41 is fixed with the reaction nucleic acid probe and the reaction protein antibody probe at the same time, and is reacted with the corresponding target nucleic acid, and the target protein is recognized by the specific primary antibody; and the target protein is recognized by the specific secondary antibody and the enzyme complex.

[0043] Please refer to Fig. 1 With Fig. 2 As shown, after confirming the target disease, the user searches for the key nucleic acid and the key protein of the target disease, and samples the specimen. The specimen can be the oral mucosa, nasal mucosa, saliva, blood, sweat, urine, feces, cerebrospinal fluid, joint fluid, pleural effusion, pericardial effusion, or other cells or tissues that can be sampled for genes or proteins. After sampling the specimen, the sampling equipment or the storage conditions of the specimen are optimized to avoid contamination of the specimen and affect the accuracy of subsequent analysis. After the specimen is sequentially extracted for nucleic acid to obtain the required DNA or RNA of the target nucleic acid, and extracted for protein to obtain the required target protein, the reaction nucleic acid probe and the reaction protein antibody probe fixed on the recognition biochip 41 are set according to the target disease. Therefore, after the specimen is labeled, the specimen and the recognition biochip 41 are placed in the same container, and the target nucleic acid reaction is established first, and then the target protein reaction is established. After the reaction nucleic acid probe and the reaction protein antibody probe of the recognition biochip complete the color reaction with the corresponding target nucleic acid and target protein, the processing module compares the obtained color image 91 with the data of the standard image database through an artificial intelligence system to obtain a qualitative reaction, and determines whether the sampled specimen has the characteristics of the target disease.

[0044] To sum up, the universal biomolecule qualitative analysis method of the present application is to establish the relationship between the target disease and the corresponding key nucleic acid and key protein, and then establish the recognition biochip 41 with the nucleic acid mutation site of the reaction nucleic acid probe and the protein mutation site of the reaction protein antibody probe of the target disease. The reaction nucleic acid probe is combined with a modified nucleic acid at least one of the nucleic acid mutation sites, and the sample to be detected is extracted, purified and labeled with the corresponding target nucleic acid DNA and RNA or the corresponding target protein. After the protein is extracted, purified and labeled, the sample is combined with the recognition biochip 41 to establish the target nucleic acid reaction and the target protein reaction. The reaction nucleic acid probe and the reaction protein antibody probe of the recognition biochip 41 are combined with the corresponding target nucleic acid to produce a hybridization reaction and an antibody recognition reaction with the corresponding reaction protein antibody probe to produce a color reaction. Different wavelengths of color produced by color reaction are intercepted, and the processing module with artificial intelligence system and the data of the standard image database are compared and interpreted. Thus, the present application is not only suitable for any target disease and has universality, but also the recognition biochip 41 is simultaneously fixed with the reaction nucleic acid probe or the reaction protein antibody probe, and can be used to detect multiple genes, multiple antigens and the expression changes of genes in the organism, and has the characteristics of convenient operation and high yield.

Claims

1. A universal qualitative analysis method for biomolecules, characterized in that, It mainly includes: Establish the relationship between target diseases and their corresponding key nucleic acids and key proteins, and retrieve data on the corresponding key proteins and key nucleic acids based on the specified target disease; Establish the nucleic acid mutation sites and protein mutation sites of the target disease. Based on the relationship between the target disease and its corresponding key nucleic acid and key protein, retrieve the key nucleic acid and key protein of the target disease and establish at least one nucleic acid mutation site or at least one protein mutation site respectively. The nucleic acid and protein mutation sites of the target disease are examined, and the correlation between the characteristic features of the nucleic acid mutation sites and the characteristic features of the protein mutation sites is compared with the characteristic features of the target disease. A biochip for identifying a target disease is constructed. At least one reactive nucleic acid probe or at least one reactive protein antibody probe is formed corresponding to the nucleic acid mutation sites and protein mutation sites identified for the target disease. Each reactive nucleic acid probe binds a modified nucleic acid to at least one of the nucleic acid mutation sites. Then, at least one of the reactive nucleic acid probes or the reactive protein antibody probes bound to the modified nucleic acid is immobilized on a carrier platform using a high-energy method, thereby forming a biochip for identifying the target disease. The modified nucleic acid enhances the specificity of the reactive nucleic acid probes. Specimen sampling and preservation: Specimen samples are collected and preserved. The extraction, purification, and labeling of the specimen involve extracting the target nucleic acid or target protein biomolecules from the specimen obtained after sampling and preservation, according to the detection purpose, and purifying the biomolecules containing the target nucleic acid and the target protein. The target nucleic acid is bound to at least one nucleic acid label corresponding to the target protein, or to at least one corresponding protein label to complete the labeling. Establish a target nucleic acid reaction, activate the identification biochip corresponding to the target disease with the labeled sample, and then perform a hybridization reaction between the target nucleic acid and the reaction nucleic acid probe of the identification biochip; To establish a target protein reaction, the identification biochip corresponding to the target disease and the labeled sample are respectively added to an enzyme complex to activate the enzyme reaction. Then, the primary antibody and the secondary antibody undergo an affinity reaction, thereby enabling the reactive protein antibody probe of the identification biochip to react with the target protein. A colorimetric reaction is established. Once the reactive nucleic acid probe or reactive protein antibody probe of the biochip has reacted with the corresponding target nucleic acid or target protein, a colorimetric reaction will occur. An image capture module is then used to capture a colorimetric image of each biochip. The color image is compared and interpreted by a processing module with an artificial intelligence system with data from a standard image database. The target protein is identified by a specific primary antibody. Specifically, the target protein is identified by a specific primary antibody against the enzyme complex using a specific secondary antibody.

2. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, The identification biochip is formed by immobilizing the required reactive nucleic acid probes and antibody probes on a porous solid-state carrier platform.

3. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, Each of these nucleic acid probes is either a DNA probe or an RNA probe.

4. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, Furthermore, the establishment of the target protein reaction includes the following steps: The target sequence is identified by using a specific primary antibody and the antigen of the target protein obtained from the sample. The specific secondary antibody binds to the primary antibody and then specifically recognizes the primary antibody in the enzyme complex. as well as Add a substrate corresponding to the enzyme complex to allow the substrate to catalyze the reaction with the enzyme complex.

5. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, Furthermore, the establishment of this colorimetric reaction includes: Dephosphorylation is performed to dephosphorylate the reactive protein antibody probe of the biochip, causing the receptor to react with a chromogenic chemical substance and emit light of the corresponding wavelength.

6. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, Furthermore, the establishment of the target nucleic acid reaction includes the following steps: An activation reaction is carried out to activate the biochip. Perform a hybridization reaction, causing the reactive nucleic acid probe of the biochip to hybridize with the target nucleic acid of the sample; and Analyze the signals of hybridization reactions and improve their accuracy.

7. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, Furthermore, the modified nucleic acid is composed of one of LNA, PNA, or 2'-OMeRNA.

8. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, Furthermore, the melting temperature of the reactive nucleic acid probe is between 40°C and 80°C.

9. The qualitative analysis method for biomolecules as described in claim 1, characterized in that, Furthermore, the reactive nucleic acid probe and the reactive protein antibody probe are immobilized on the carrier platform using either inkjet printing or laser transfer.

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