CN114910460A - Aptamer-based protein chip and preparation and application thereof - Google Patents
Aptamer-based protein chip and preparation and application thereof Download PDFInfo
- Publication number
- CN114910460A CN114910460A CN202210559206.2A CN202210559206A CN114910460A CN 114910460 A CN114910460 A CN 114910460A CN 202210559206 A CN202210559206 A CN 202210559206A CN 114910460 A CN114910460 A CN 114910460A
- Authority
- CN
- China
- Prior art keywords
- aptamer
- chip
- protein
- substrate
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 134
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 126
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 122
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 46
- 238000000018 DNA microarray Methods 0.000 claims abstract description 23
- 230000000171 quenching effect Effects 0.000 claims abstract description 22
- 238000010791 quenching Methods 0.000 claims abstract description 21
- 238000012123 point-of-care testing Methods 0.000 claims abstract description 13
- 238000004806 packaging method and process Methods 0.000 claims abstract description 3
- 238000001514 detection method Methods 0.000 claims description 39
- 239000012488 sample solution Substances 0.000 claims description 21
- 238000002331 protein detection Methods 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 19
- 230000008569 process Effects 0.000 claims description 16
- 108091034117 Oligonucleotide Proteins 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 12
- 238000004873 anchoring Methods 0.000 claims description 11
- 230000005284 excitation Effects 0.000 claims description 8
- 230000009471 action Effects 0.000 claims description 4
- 239000007790 solid phase Substances 0.000 claims description 4
- 238000000751 protein extraction Methods 0.000 claims description 3
- 230000001360 synchronised effect Effects 0.000 claims description 3
- 238000011897 real-time detection Methods 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000011521 glass Substances 0.000 description 4
- 239000012460 protein solution Substances 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 4
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 239000011888 foil Substances 0.000 description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010011882 Deafness congenital Diseases 0.000 description 1
- 206010013654 Drug abuse Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides an aptamer-based protein chip and preparation and application thereof, and the aptamer-based protein chip comprises a biochip and a chip support, wherein the biochip comprises a substrate and an aptamer fluorescent probe fixed on the substrate, and the aptamer fluorescent probe is combined with a quenching group without a fluorescent signal; when the target protein exists, the target protein promotes the quenching group to be dissociated from the aptamer, and the fluorescent group of the aptamer can be excited to form a fluorescent signal. The biochip and the chip support are assembled into a packaging chip, the structure is simple, the cost is low, the aptamer fluorescent probe can detect a fluorescent signal when meeting target protein, the use and the operation are convenient, and the biochip-based fluorescent probe has obvious economic significance and social benefit in the POCT field.
Description
Technical Field
The invention relates to the field of protein detection, in particular to the technical field of in-vitro diagnosis detection and molecular diagnosis detection, in particular to an aptamer-based protein chip and preparation and application thereof, and more particularly to a protein chip for the instant detection of multiple target proteins
Background
The biochip (biochip) is consistent with the concept of gene chip in early literature, and with the deep research of technology, the biochip is broadly characterized in that the biochemical analysis process is integrated on the surface of the chip according to the principle of specific interaction between biomolecules, so that the high-throughput rapid detection of DNA, RNA, polypeptide, protein and other biological components is realized. Biochips are classified into gene chips, protein chips, tissue chips, cell chips, and the like, according to the type of probes immobilized on the surface of the chips. The research of Chinese biochip starts from 1997-1998, but the development is rapid, 2011, hereditary deafness gene chip developed by Bo and ao organisms is on-market, which is the first self-developed commercial product on the industrialization road of biochip in China. The biochip is especially suitable for multi-factor screening of infectious disease detection, epidemic situation monitoring, tumor marker detection, drug abuse and the like, wherein the number of probes of the medium-low density biochip ranges from dozens to thousands, and is different from the high-density biochip with tens of thousands to millions of probes, higher speciality, higher cost and more complex operation, and the biochip is more suitable for the field detection of sampling sites, ICU monitoring rooms, operating rooms, emergency rooms and the like or the field of point-of-care testing (POCT) of patient beds.
The protein chip can be directly analyzed by crude biological samples (serum, urine and body fluid), can rapidly discover a plurality of biomarkers at the same time, can detect low-abundance proteins, can realize high-flux verification, can realize relatively quantitative analysis and detection, and has wide application prospect in the field of instant POCT detection. However, the protein chip has the following disadvantages: (1) the protein detection chip mainly relies on antibodies and other macromolecules, a rapid, cheap and high-flux protein expression and purification method is established, and a lot of practical problems exist in the high-flux preparation of the antibodies, especially in the large-scale production. (2) The protein chip preparation needs to provide proper temperature and humidity to keep the stability and biological activity of the protein on the chip surface, and the preparation requirement is high. (3) The method needs to solve the problem of a universal high-sensitivity and high-resolution detection method and realize the integration of imaging and data analysis.
Since the concept of the aptamer was proposed in the 90 s of the 20 th century, the research of the aptamer is adjusted and developed, and the aptamer has the advantages of low detection limit, high affinity, strong specificity, easy in-vitro mass synthesis, good repeatability, high stability and easy storage; the aptamer can be widely applied to detection of pesticides, tissues, cells, viruses, proteins, toxins, vitamins, allergens and the like. Because the aptamer is easy to label fluorescence and the activity is not influenced, the aptamer is easy to be combined with various other detection technologies and can be widely applied to various aspects such as cell imaging, new drug development, disease treatment, microorganism detection and the like.
The detection of protein chips based on antibodies usually takes unpackaged bare chips as the main part, generally needs to add a fence and a cover sheet for hybridization during use, and also needs to clean and develop color after hybridization, so that the detection process of the protein chips is complex, the POCT use requirement of convenience and quickness cannot be met, and the application of the protein chips in the POCT field is limited to a certain extent.
Disclosure of Invention
The invention aims to provide a packaged protein chip which has simple structure, simple operation and low cost and is suitable for detecting crude protein extract.
In order to achieve one of the above purposes, the invention provides the following technical scheme:
an aptamer-based protein chip and preparation and application thereof, comprising a substrate and a chip support, wherein the substrate and the chip support form a closed space with only two openings for protein detection; the substrate is a light-transmitting solid-phase sheet structure, one surface of the substrate facing the chip support is anchored with an aptamer of which the end part is provided with a fluorescent group, the aptamer is combined with a quenching group modified by an oligonucleotide chain in a base complementary pairing mode, and the quenching group combined on the aptamer enables the fluorescent group of the aptamer not to emit fluorescence after excitation, so that the fluorescent group is a negative signal of a chip reader; when the target protein is identified and combined with the aptamer, the quenching group is released from the aptamer, and the fluorescent group of the aptamer emits fluorescence after excitation, which is a positive signal of the chip reader; the technical scheme ensures that the target protein can be combined with the aptamer and generate a positive signal on the chip reader as long as the target protein exists in the sample solution to be detected, and relative quantitative analysis can be carried out through the internal standard protein.
According to a preferred embodiment of the invention, the aptamers are arranged in a row and anchored on the substrate and perpendicular to the flow direction of the sample solution to be detected, which is beneficial to capture the target protein and improve the injection flow speed of the sample solution to be detected, thereby accelerating the detection.
According to a preferred embodiment of the present invention, the oligonucleotide chain binding region of the aptamer and the modified oligonucleotide chain binding region of the quencher group are combined together such that the quencher group is released upon binding of the target protein to the aptamer.
According to a preferred embodiment of the invention, the target protein is combined with the aptamer, so that the quenching group can be released and the fluorescent group on the aptamer forms a positive signal in a biochip reader, thereby realizing the instant detection of the target protein and realizing the POCT application requirements.
According to a preferred embodiment of the present invention, the relative quantitative detection of the target protein can be realized by adding the internal standard protein to the sample solution to be detected.
According to a preferred embodiment of the invention, specific aptamers corresponding to a plurality of proteins and combined quenching groups are respectively anchored on different sites of a substrate and assembled with a chip support to form an aptamer-based protein chip, so that synchronous instant detection of the plurality of proteins can be realized.
According to a preferred embodiment of the invention, a sample to be detected is directly input into the detection space formed by assembling the substrate and the chip support, and a POCT detection result is directly formed by using a chip reader.
According to a preferred embodiment of the present invention, only one solution of the protein extract is required for the whole detection process to complete the protein extraction and target protein detection processes in the sample.
According to a preferred embodiment of the present invention, the substrate is a transparent solid-phase sheet structure, which not only can ensure that the excitation light can irradiate the fluorescent group modified by the aptamer anchored on the substrate through the substrate, but also can ensure that the fluorescent group forms a fluorescent signal after excitation, and can be directly identified through the transparent substrate.
According to the embodiment of the invention, the application of the aptamer-based protein chip in the field of protein detection is protected.
A method for preparing an aptamer-based protein chip, comprising the following steps:
1) respectively carrying out fluorescent group modification and anchoring group modification on two tail ends of the aptamer specifically combined with the target protein, and combining a quenching group modified by oligonucleotide chain onto the aptamer to ensure that the fluorescent group of the aptamer cannot form a positive signal by exciting light;
2) anchoring the aptamer on the surface of the substrate according to a specific position layout by using an anchoring group on the aptamer;
3) the surface of the substrate with the anchoring adapter faces the chip support to complete the assembly of the substrate and the chip support, the substrate and the chip support are tightly connected to form a protein detection space, and the protein detection space only has two holes on the chip support to complete the inlet and outlet of a sample solution to be detected;
4) and forming a protein detection space to finish the preparation of the aptamer-based protein chip.
Use of an aptamer-based protein chip: when the kit is used, a sample is processed by using a protein extracting solution, an obtained extraction solution (a sample solution to be detected, wherein the sample solution to be detected contains target protein) is continuously input into a boxed chip through an input hole of a chip bracket and continuously flows out of an exhaust hole, after the sample solution to be detected enters a packaging chip through the input hole on the chip bracket, the target protein is identified and paired with a corresponding aptamer and a quenching group combined on the aptamer is released, so that a fluorescent group on the aptamer combined with the target protein can emit fluorescence under the action of exciting light of a chip reader, and a positive signal on the chip reader is formed; and the chip reader outputs and determines the type of the target protein in the sample solution to be detected according to the layout and positioning information of various aptamers on the chip. The chip reader can also perform relative quantitative analysis of the target protein according to the fluorescence value of the internal standard protein.
The invention has the beneficial effects that:
the aptamer-based protein chip mainly comprises a substrate and a chip support, wherein the surface of the substrate, on which the aptamer is fixed, faces downwards and is arranged on the chip support to form a closed protein detection space, so that the aptamer-based protein chip is manufactured.
The detection process of the aptamer-based protein chip is simple and convenient, only one solution of protein extract is involved in the detection process, a sample solution (containing target protein) to be detected, which is obtained by mixing the protein extract with a sample, is directly input into the protein detection space of the aptamer-based protein chip, a chip reader is directly used for detection, and a detection result is formed, so that the detection process is simple and quick, and the detection method is suitable for the requirements of the POCT field.
According to the aptamer-based protein chip, relative quantitative analysis of target proteins can be carried out according to fluorescence values of internal standard proteins.
The aptamer-based protein chip is a packaged biochip, so that the operation simplicity of biochip detection is improved, the automation can be realized, the requirement of the biochip in the POCT field can be met, the protein chip can be promoted to approach users and enter thousands of households, the service is effectively provided for common people, and the application range of the biochip technology in the health industry is expanded.
The substrate of the aptamer-based protein chip is of a light-transmitting solid-phase sheet structure, so that excitation light can be ensured to irradiate fluorescent groups modified by the aptamer anchored on the substrate through the substrate, fluorescent signals can be ensured to be formed after the fluorescent groups are excited, the fluorescent signals can be directly identified through the light-transmitting substrate, the process that the traditional naked biochip needs to interpret the fluorescent signals after a fence and a cover plate are removed is omitted, and the operation is simple and easy.
Drawings
FIG. 1 is a cross-sectional view of an aptamer-based protein chip according to the present invention;
FIG. 2 is a top view of an aptamer-based protein chip of the present invention;
FIG. 3 shows the results of example 1 of the aptamer-based protein chip of the present invention;
FIG. 4 shows the results of example 2 of the aptamer-based protein chip of the present invention;
in the figure, 1, a substrate; 2. a chip holder; 3. an aptamer; 4. an anchoring group; 5. a fluorophore group; 6. a quencher group; 7. a modified oligonucleotide chain of a quencher group; 8. the modified oligonucleotide chain of the quenching group is in base pairing hydrogen bond with the aptamer; 9. an input aperture; 10. a discharge hole; 11. a target protein; 12. exciting light emitted by the chip reader; 13. an aptamer fluorophore that emits fluorescence; 14. and (5) sample solution to be tested.
Detailed Description
Exemplary embodiments of the present invention will hereinafter be described in detail with reference to the accompanying drawings, wherein like or similar reference numerals denote like or similar elements. Furthermore, in the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the disclosure. It may be evident, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are shown in schematic form in order to simplify the drawing.
FIG. 1 is a sectional view showing an aptamer-based protein chip according to the present invention, and FIG. 2 is a top view of the aptamer-based protein chip according to the present invention. According to the present general inventive concept, there is provided an aptamer-based protein chip, including a substrate 1 and a chip holder 2, wherein an aptamer 3 having an anchor group 4 and a fluorophore group 5 is immobilized on the substrate 1 via the anchor group 4; the quenching group 6 of the modified oligonucleotide chain 7 with the quenching group realizes the fluorescence quenching effect of the quenching group 6 on the fluorescent group 5 through the base pairing hydrogen bond 8 of the modified oligonucleotide chain of the quenching group and the aptamer, and a chip reader cannot obtain a positive signal; the sample solution 14 to be detected enters the protein detection space through the input hole 9, the target protein 11 and the corresponding aptamer 3 are subjected to specific recognition and combination, the quenching group 6 on the aptamer 3 is promoted to be dissociated from the aptamer 3, the fluorescent group 5 on the aptamer after the quenching group is dissociated can be converted into the aptamer fluorescent group 13 emitting fluorescence under the action of the exciting light 12 emitted by the chip reader, and then a positive signal is formed on the chip reader.
The sample solution 14 to be detected directly enters the protein detection space through the input hole 9 and flows out of the discharge hole 10, the target protein 11 in the sample solution 14 to be detected is respectively combined with different corresponding aptamers and enables the fluorescent group 5 on the corresponding aptamer to be converted into the aptamer fluorescent group 13 emitting fluorescence under the action of the exciting light 12 emitted by the chip reader, and then fluorescent positive signals at different positions are formed in the chip reader, so that synchronous instant detection of multiple proteins is realized.
By placing aptamers to the internal standard proteins on the substrate, relative quantitation of various proteins can be achieved by the internal protein standards. The whole detection process can finish the processes of protein extraction and target protein detection in a sample only by one solution of protein extracting solution, and after the protein extracting solution is directly input into a protein chip, qualitative information and relative quantitative information of related target protein in the sample can be directly obtained on a chip reader; the aptamer-based protein chip is simple to operate and low in cost, can meet the requirements of the POCT detection field, and has remarkable economic significance and social benefit.
Example 1: an aptamer-based protein chip and preparation and application thereof are disclosed, wherein the specific process comprises the following steps:
1) selection of aptamer 3 for BSA: the fluorescent labeling aptamer design was performed according to the BSA aptamer sequence in the literature (Microchim Aacta, 2018, 185(4):241-251.) by designing 6-FAM fluorophore 5 at the 5 'end of the aptamer, spacing 3 adenylates with the aptamer, and designing amino anchor 4 at the 3' end of continuous adenylates of the aptamer. BHQ1 was chosen as quencher 6, whose modified oligonucleotide 7 was 7 nucleotides in length and paired antiparallel to aptamer 3.
2) Committed synthesis of aptamer 3 of BSA: the synthesis was entrusted to Wuhan Kingkurui bioengineering Co., Ltd.
3) Anchoring of the aptamer to the substrate 1: the aldehyde glass substrate is ordered by Biotechnology Limited company of Beijing Boo Kao Crystal dictionary (manufacturer: Boo Crystal dictionary, type: glass substrate, specification: 75mm long, 25mm wide, 1mm thick), and the spotting instrument (import equipment: GESIM Nano-Plotter NP2.1/E) completes the positioning and anchoring of the aptamer 3 on the substrate 1 according to the position and preset position. The array is: 130 rows by 40 columns, nine points are (10,10), (20,20), (30,30), (40,10), (50,20), (60,30), (70,10), (80,20), (90, 10).
4) Preparation of chip holder 2: the entrusted processing was carried out by Beijing Sawahn science and technology Co., Ltd, and the gap between the chip holder 2 and the substrate 1 after the assembly was 0.1 mm, and the chip holder was provided with an input hole 9 and an output hole 10.
5) And (3) finishing the bonding assembly of the substrate 1 anchored with the aptamer and the chip support 2 by using UV shadowless glue to prepare and obtain the encapsulated aptamer-based protein chip.
6) And (3) dissolving BSA in the protein extracting solution to obtain a final concentration of 0.1%, thus obtaining the protein solution 14 to be detected. Inputting a protein solution 14 to be detected into the protein detection space through the input hole 9 by using a syringe pump, flowing out from the discharge hole 10, flushing the protein detection space by using 1 ml of extracting solution, and wrapping the protein chip by using an aluminum foil in the operation process.
7) And (3) placing the protein chip subjected to sample treatment in a chip reader, measuring the detection result of the protein chip, and detecting fluorescent signals at 9 aptamer positions. The results are shown in FIG. 3.
Example 2: an aptamer-based protein chip and preparation and application thereof are disclosed, wherein the specific process comprises the following steps:
1) selection of aptamer 3 for BSA: the design of the fluorescent-labeled aptamer was performed according to the BSA aptamer sequence in the literature (Microchim Aaacta, 2018, 185(4):241-251.), a 6-FAM fluorophore 5 was designed at the 5 'end of the aptamer, 3 adenylate spacers were used between the aptamer and the 6-FAM fluorophore, and an amino anchor group 4 was designed at the 3' end of the aptamer and the end of the continuous adenylate. BHQ1 was chosen as quencher 6, whose modified oligonucleotide 7 was 7 nucleotides in length and paired antiparallel to aptamer 3.
2) Delegated synthesis of aptamer 3 for BSA: the synthesis was entrusted to Securidaceae, Han.
3) Anchoring of the aptamer to the substrate 1: the aldehyde glass substrate is ordered by Biotechnology Limited company of Beijing Boo Kao Crystal dictionary (manufacturer: Boo Crystal dictionary, type: glass substrate, specification: 75mm long, 25mm wide, 1mm thick), and the spotting instrument (import equipment: GESIM Nano-Plotter NP2.1/E) completes the positioning and anchoring of the aptamer 3 on the substrate 1 according to the position and preset position. The array is: 130 rows by 40 columns, and nine points are (10,10), (20,20), (30,30), (40,10), (50,20), (60,30), (70,10), (80,20), (90, 10).
4) Preparation of chip holder 2: the entrusted processing was carried out by Beijing Sawahn science and technology Co., Ltd, and the gap between the chip holder 2 and the substrate 1 after the assembly was 0.1 mm, and the chip holder was provided with an input hole 9 and an output hole 10.
5) And (3) finishing the bonding assembly of the substrate 1 anchored with the aptamer and the chip support 2 by using UV shadowless glue to prepare the protein chip based on the aptamer.
6) And (3) dissolving BSA in the protein extracting solution to obtain a final concentration of 0.001%, namely the protein solution 14 to be detected. Inputting a protein solution 14 to be detected into the protein detection space through the input hole 9 by using a syringe pump, flowing out from the discharge hole 10, flushing the protein detection space by using 1 ml of extracting solution, and wrapping the protein chip by using an aluminum foil in the operation process.
7) And (3) placing the protein chip subjected to sample treatment in a chip reader, measuring the detection result of the protein chip, and detecting fluorescent signals at 9 aptamer positions. The results are shown in FIG. 4.
Although the embodiment of the present invention is illustrated and described as an encapsulated gene chip in the detailed description, it can be easily understood by those skilled in the art of biochip that the biochip of the present invention can be applied to the field of protein chip. Changes may be made in the described embodiments without departing from the principles and this disclosure, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. An aptamer-based protein chip, and preparation and application thereof, characterized by comprising:
s1, preparing a protein chip by using an aptamer, wherein two tail ends of the aptamer are respectively provided with a fluorescent group and an anchoring group, the anchoring group of the aptamer fixes the aptamer on a substrate, and a quenching group is bonded on the aptamer chain through an oligonucleotide chain connected with the quenching group and enables the fluorescent group of the aptamer not to emit fluorescence after excitation;
s2, assembling the protein chip and the chip support into a packaged chip, wherein the chip support is provided with a sample input hole and a discharge hole, and the sample solution to be detected is continuously input into the packaged chip through the input hole of the chip support and continuously flows out of the discharge hole;
s3 after the sample solution to be tested enters the packaging chip through the input hole on the chip support, the target protein and the aptamer are identified and matched and the quenching group combined on the aptamer is released, the fluorescent group on the aptamer combined with the target protein can emit fluorescence under the action of excitation light, and a positive signal on the chip reader is formed;
and the S4 chip reader outputs and determines the target protein type in the sample solution to be detected according to the layout and positioning information of various aptamers on the chip.
2. The aptamer-based protein chip as claimed in claim 1, wherein the aptamers are arranged in a row and anchored on the substrate perpendicular to the flow direction of the sample solution to be detected, thereby facilitating capture of the target protein and increase of the flow rate of the sample solution to be detected.
3. The aptamer-based protein chip as claimed in claim 1, wherein the target protein binding region of the aptamer is coincident with the modified oligonucleotide chain binding region of the quencher, such that the target protein is bound to the aptamer to facilitate the release of the quencher.
4. The aptamer-based protein chip as claimed in claim 1, wherein the quencher group can be released and the fluorescent group on the aptamer can form a positive signal in a biochip reader when the target protein is combined with the aptamer, thereby realizing the instant detection of the target protein and the POCT application requirements.
5. The aptamer-based protein chip as claimed in claim 1, wherein the relative quantitative detection of the target protein can be realized by adding the internal standard protein into the sample solution to be detected.
6. The aptamer-based protein chip as claimed in claim 1, wherein specific aptamers corresponding to a plurality of proteins and quenching groups bound to the specific aptamers are anchored to different sites of a substrate, respectively, and the specific aptamers and the quenching groups are assembled with a chip support to form the aptamer-based protein chip, so that synchronous real-time detection of the plurality of proteins can be realized.
7. The aptamer-based protein chip as claimed in claim 1, wherein the sample solution to be tested is directly input into the protein detection space formed by assembling the protein chip and the chip holder, and the POCT detection result is directly formed by using a chip reader.
8. The aptamer-based protein chip as claimed in claim 1, wherein the whole detection process requires only one solution of protein extract to complete the protein extraction and target protein detection processes.
9. The aptamer-based protein chip as claimed in claim 1, wherein the substrate is a light-transmissive solid-phase sheet structure, which can ensure that excitation light can irradiate the aptamer-modified fluorophore anchored on the substrate through the substrate, and can ensure that the fluorophore excited to form a fluorescence signal can be directly recognized through the light-transmissive substrate.
10. Protects an aptamer-based protein chip and application of preparation thereof in the field of protein detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210559206.2A CN114910460B (en) | 2022-05-22 | 2022-05-22 | Protein chip based on aptamer and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210559206.2A CN114910460B (en) | 2022-05-22 | 2022-05-22 | Protein chip based on aptamer and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114910460A true CN114910460A (en) | 2022-08-16 |
| CN114910460B CN114910460B (en) | 2024-06-28 |
Family
ID=82769561
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210559206.2A Active CN114910460B (en) | 2022-05-22 | 2022-05-22 | Protein chip based on aptamer and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114910460B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117373564A (en) * | 2023-12-08 | 2024-01-09 | 北京百奥纳芯生物科技有限公司 | Method and device for generating binding ligand of protein target and electronic equipment |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006015810A2 (en) * | 2004-08-06 | 2006-02-16 | Diacdem Chip Technology Gmbh | Fluorescence-based assays for the rapid quantitative analysis of biomolecules (proteins and nucleic acids) by accumulation on cells or beads |
| JP2009209094A (en) * | 2008-03-04 | 2009-09-17 | Sharp Corp | Microchip for protein extraction, protein extraction apparatus, protein measurement apparatus, protein extraction method using them, and air conditioner |
| CN112391448A (en) * | 2020-04-29 | 2021-02-23 | 湖北中医药大学 | DNA nano molecular machine for analyzing exosome and surface protein and application |
| CN113215312A (en) * | 2021-04-28 | 2021-08-06 | 山东莱博生物科技有限公司 | Coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application |
| CN114381550A (en) * | 2021-12-03 | 2022-04-22 | 中国科学院精密测量科学与技术创新研究院 | Multi-target nucleic acid detection kit and detection method for HPV typing |
-
2022
- 2022-05-22 CN CN202210559206.2A patent/CN114910460B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006015810A2 (en) * | 2004-08-06 | 2006-02-16 | Diacdem Chip Technology Gmbh | Fluorescence-based assays for the rapid quantitative analysis of biomolecules (proteins and nucleic acids) by accumulation on cells or beads |
| JP2009209094A (en) * | 2008-03-04 | 2009-09-17 | Sharp Corp | Microchip for protein extraction, protein extraction apparatus, protein measurement apparatus, protein extraction method using them, and air conditioner |
| CN112391448A (en) * | 2020-04-29 | 2021-02-23 | 湖北中医药大学 | DNA nano molecular machine for analyzing exosome and surface protein and application |
| CN113215312A (en) * | 2021-04-28 | 2021-08-06 | 山东莱博生物科技有限公司 | Coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application |
| CN114381550A (en) * | 2021-12-03 | 2022-04-22 | 中国科学院精密测量科学与技术创新研究院 | Multi-target nucleic acid detection kit and detection method for HPV typing |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117373564A (en) * | 2023-12-08 | 2024-01-09 | 北京百奥纳芯生物科技有限公司 | Method and device for generating binding ligand of protein target and electronic equipment |
| CN117373564B (en) * | 2023-12-08 | 2024-03-01 | 北京百奥纳芯生物科技有限公司 | Method and device for generating binding ligand of protein target and electronic equipment |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114910460B (en) | 2024-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105392895B (en) | Improved measuring method | |
| Lal et al. | Antibody arrays: an embryonic but rapidly growing technology | |
| JP2018054629A (en) | Co-binder assisted assay method | |
| KR20170036659A (en) | Improved assay methods | |
| US20040029109A1 (en) | Novel integrated circuit chip for bioassays | |
| CN103149369A (en) | Protein chip for detecting esophageal squamous carcinoma marker and kit box of protein chip | |
| CN101144815A (en) | Preparation method of liquid phase protein chip | |
| CN114910460A (en) | Aptamer-based protein chip and preparation and application thereof | |
| JP2022511168A (en) | Analytical systems with microfluidic devices and related methods | |
| CN112858670B (en) | Multiple digital ELISA detection method and microfluidic chip | |
| ES2421257T3 (en) | Method and apparatus for simultaneous testing of several analytes with an internal control | |
| Xu et al. | Multiplex biomarker analysis biosensor for detection of hepatitis B virus | |
| JP2004527735A (en) | Biochemical methods and devices for detecting protein properties | |
| JP2004527735A5 (en) | ||
| CN104931688B (en) | A kind of microstructured optical fibers biochip and preparation method thereof | |
| CN114966051A (en) | Microfluidic protein chip and manufacturing method and application thereof | |
| US20070141576A1 (en) | Biological chip and use thereof | |
| EP3517955A1 (en) | Multi-unit for performing biochemical test and immune response test, and test method using same | |
| CN115236322A (en) | Capillary tube detection plate and preparation and application thereof | |
| CN114245829A (en) | Optimized nucleic acid probes for detecting analytes | |
| CN1521272B (en) | Novel ligand detecting method | |
| US7544474B2 (en) | Method for detection of multiple test materials in a sample | |
| CN109932513B (en) | Application of RPPA and PWG in preparation of respiratory tract pathogen detection product | |
| EP3779442A1 (en) | Target substance detection method, target substance detection kit, and target substance detection apparatus | |
| Duffy et al. | Multiplex Cytokine Assays |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20221207 Address after: Room 2119, Floor 2, Unit 104, Building 10, No. 25, Jinghai 4th Road, Daxing District, Beijing 100176 Applicant after: Beijing baionaxin Biotechnology Co.,Ltd. Address before: Room B1--5675, Building 3, No. 20 Yongan Road, Shilong Economic Development Zone, Mentougou District, Beijing 102300 Applicant before: BEIJING HUANIU SHIJI BIOTECHNOLOGY Research Institute |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant |