CN103439506B - Rapid detection device and method for interaction of biomacromolecules - Google Patents
Rapid detection device and method for interaction of biomacromolecules Download PDFInfo
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Abstract
The invention relates to the field of bioengineering and aims to provide a rapid detection device and method for interaction of biomacromolecules. The device comprises a bottom plate, wherein the surface of the bottom plate is provided with n detection units, and n is an integral number greater than or equal to 1; each detection unit comprises a reaction hole, a detection hole and a micropipe for connecting the reaction hole and the detection hole; each detection hole is connected to a negative pressure device interface through the corresponding micropipe; the diameter of each micropipe is 0.1 to 2 mm; each reaction hole adopts an open structure and is used for dropping in a reactant; each detection hole adopts a closed structure and a magnetic metal wire is arranged in each detection hole; the direction of each metal wire is consistent with that of the micropipe in the corresponding detection unit. According to the invention, the interaction of antigen-antibodies or two biomacromolecules can be directly observed and the reaction condition can be judged by naked eyes or an instrument only in 1 to 2 minutes. The device is suitable for all biomolecules detected by adopting the ELISA (enzyme-linked immuno sorbent assay) principle and has the characteristics of high efficiency, simplicity, rapidness, convenience, wide application and the like.
Description
Technical field
The invention belongs to bioengineering field, being specifically related to a kind of device and method for detecting interaction of biomacromolecules.
Background technology
Enzyme linked immunosorbent assay (Enzyme-Linked Immunosorbent Assays, ELISA), was widely used in immunology and biochemical field by since Engvall and Perlman report from 1971.Along with the continuous renewal of the updating of method, microplate reader, in the context of detection of antigen and antibody, ELISA be still current test in laboratory analyze in one of the most frequently used technology.The ultimate principle of ELISA method, after antigen and antibody molecule covalent bond, does not affect respective immunologic competence.By antigen coated, close, add primary antibodie, add ELIAS secondary antibody, the step such as substrate colour developing, judge the specificity of antigen and antibody response, by the depth determination antibody of color reaction or the concentration of antigen.This kind of chromogenic reaction carries out quantitative measurement by ELISA detector (microplate reader), makes the detection method that ELISA method becomes a kind of not only special but also responsive.But ELISA reaction, from antigen coated to substrate colour developing, need multiple step, needs within 4-6 hour, even spend the night in time.Current business-like ELISA detection kit, the general ELISA Plate adopted containing pre-coated antigen or antibody, but still need on detection time 2-4 hour.
Therefore, current detection technique needs to solve quick detection reaction problem from the time.For this reason, immune colloidal gold chromatography technology starts to come out.Chromatography detection technique comes across 1988 the earliest, is that Unipath company utilizes the pregnant inspection chromatograph test strip of the one of nano-colloid gold grain Development and Production, uses within prefabricated test strips 3-5 minute, can go out testing result.After this, immune colloid gold is widely used and develops in quick detection reagent.But colloidal gold technique needs the equipment such as collaurum making, metal spraying, point sample of different nano particle, and chromatography reaction often with ELISA reactions vary cause.The antigen that can occur in ELISA and antibody response, but do not occur in colloid gold chromatographic test paper strip.
Therefore, in order to solve the shortcoming of ELISA reaction time length, the low sensitivity issues usually occurred in immunochromatography can be solved again simultaneously.The invention provides a solution, that is: a kind of device of detectable antigens and antibody response (or interaction of biomacromolecules) fast.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, provides a kind of device for fast detecting and method of interaction of biomacromolecules.
For technical solution problem, solution of the present invention is:
There is provided a kind of device for fast detecting of interaction of biomacromolecules, comprise base plate, the surface of base plate is provided with n detecting unit, n is the integer of >=1; Each detecting unit comprises a reacting hole, detect aperture and microtubule both being connected; Each detect aperture is all connected to negative pressure device interface by microtubule; The diameter of described microtubule is 0.1 ~ 2 millimeter; Described reacting hole is opened type, for instilling reactant; Described detect aperture is closed type, and there is a magnetic metal wire of tool its inside, and metal wire is consistent with the direction of microtubule in this group detecting unit.
In the present invention, described base plate is the base plate of the inert materials such as plastics, pottery or tygon.
In the present invention, described microtubule is the pipeline of tygon, propylene or butylenes plastic material.
In the present invention, the capacity of described reacting hole is 10 ~ 500 microlitres.
In the present invention, described detecting unit has two at least, and each detecting unit mutually side by side and be arranged in parallel, embark on journey by the two ends of its reacting hole and detect aperture apportion base plate and arrangement.
In the present invention, described negative pressure device interface is the interface for connecting peristaltic pump or rubber pipette bulb.
The present invention still further provides a kind of method for quick of the interaction of biomacromolecules based on aforementioned means, comprises the following steps:
(1) according to the product operation instruction of immunomagnetic beads, the first biomacromolecule is coupled on immunomagnetic beads, then adds the second biomacromolecule of material to be detected and at least one mark successively; This process has two kinds of implementations: complete mixing in test tube after, reactant liquid is added reacting hole, or in reacting hole, adds reactant liquid successively;
The first biomacromolecule described is any one in protein, polysaccharide, polypeptide, nucleic acid or chemicals, and described material to be detected and the second biomacromolecule are any one in protein, polypeptide, polysaccharide, toxin, chemicals or nucleic acid.Wherein, the second biomacromolecule can be combined with material to be detected; Label for the second biomacromolecule is any one in collaurum, fluorescent material, chemiluminescent material or upper forwarding luminescent material;
(2) start peristaltic pump, make the reactant liquid flow detection hole in reacting hole; If be coupled to the first biomacromolecule on magnetic bead, material to be detected, mark the second biomacromolecule there occurs interaction, reactant liquid can be adsorbed on detection line when flowing through detect aperture; The color change of magnet-wire can be detected by naked eyes, fluorescence detector, chemiluminescence detector or upper forwarding photodetector.
In the present invention, add in the reactant liquid in reacting hole, the concentration being coupled to the first biomacromolecule on magnetic bead is 0.1 ~ 1 mcg/ml; The concentration of the second biomacromolecule of mark is determined according to the recommendation of its manufacturer.
The characteristic that the present invention utilizes the immunomagnetic beads of activation and biomacromolecule to react, after the first biomacromolecule and immunomagnetic beads coupling, then closes unreacted avtive spot.Then the second biomacromolecule of material to be detected and mark is added.Wherein, with the first biomacromolecule and the second biomacromolecule covalent bond while of thing energy to be detected, and the complex formed with " magnetic ", when reactant flows through detect aperture, by in the magnet wire that can be adsorbed in detect aperture, and can with the naked eye, fluorescence, chemiluminescence or on turn light-emitting appearance and detect.Which kind of instrument is used to detect the label depending on the second biomacromolecule.If label is collaurum, then reactant compound naked eyes are visible.If label is fluorescent material, reaction compound can detect with the fluorescence detector with optical filter.As long as condition possesses, only need 1-2 minute from being reacted to detection.Because reaction carries out in a liquid, therefore maintain the immunologic competence of each reactive material in conventional ELISA, thus achieve the object detected fast.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention directly can observe the interaction between antigen-antibody or two biomacromolecules, only needs 1-2 minute, namely judges response situation by naked eyes or instrument.
This device is applicable to all biomolecule that ELISA principle detects.
Relative to background technology, the present invention has the features such as efficient, simple and direct, quick, convenient, applicable wide.
Accompanying drawing explanation
Fig. 1 is square (2x8 hole) of the present invention reaction unit schematic diagram.
Reference numeral in figure is: reacting hole 1; The metal wire 2 of magnetic; Detect aperture 3; Peristaltic pump interface 4.Wherein, react compound will be adsorbed on " metal wire of magnetic ".
Embodiment
Inventive principle is introduced:
Interaction of biomacromolecules device for fast detecting in the present invention, the principle of foundation is by the specific adsorption between immunomagnetic beads and magnet, and the covalent bond principle of antigen and antibody is made.By one of them biomacromolecule and immunomagnetic beads coupling.Another one biomacromolecule and enzyme, fluorophor, chemiluminescent groups or on turn luminophore coupling.If two biomacromolecules directly or indirectly interact, the magnetic compound of tool can be formed.Thus adsorbed when flowing through magnet wire.
, pick-up unit is square in the present embodiment as shown in Figure 1, and reacting hole is 2x8 hole (in actual use can per sample quantity increase and decrease), is arranged in parallel, does not interfere with each other.Detect aperture 3 and reacting hole 1 one_to_one corresponding, do not interfere with each other between detect aperture 3 and detect aperture 1 yet.The interconnective microtubule in detect aperture 3 side is provided with a peristaltic pump interface 4.Liquid in reacting hole 1 can be sucked in detect aperture 3 by peristaltic pump negative pressure, and finally flow into waste fluid container.In the present invention, also can produce the device replacement peristaltic pump of negative pressure with rubber pipette bulb etc., its fundamental purpose promotes that liquid flows through detect aperture smoothly.
Base plate is the inert materials such as plastics, pottery, tygon, and this material is not combined with biomacromolecules such as protein, polypeptide, polysaccharide, toxin, chemicals, nucleic acid, " magnetic is combined " does not occur with magnetisable material yet and reacts.The capacity of reacting hole 1 is 10 microlitre to 500 microlitres, open.The microtubule material of coupled reaction hole 1 and detect aperture 3 is tygon, propylene or butylenes plastic, and microtubule diameter is 0.1 millimeter to 2 millimeters.Detect aperture 3 is closed type, only comprises the passage that liquid flows through, and capacity depends on microtubule diameter.
Carrying out before detection analyzes, by the immunomagnetic beads coupling of first biomacromolecule and activation, conjugate can be placed on room temperature, 4 according to its character
oc or-20
oc saves backup.Then close unreacted avtive spot on immunomagnetic beads with confining liquid, then magnetic bead conjugate is mixed successively with the second biomacromolecule of material to be checked, mark, add in reacting hole 1.Device is connected with peristaltic pump, starts peristaltic pump, make liquid flow (or with rubber pipette bulb, producing suction).Liquid will flow through detect aperture 3 from reacting hole 1.Detected by naked eyes, fluorescence detection device, chemiluminescence detecting or upper forwarding optical detection device.Positive reaction, should present color in detect aperture 3, and distributes along the metal wire of magnetic.
In the present invention, the product of the usual commodity in use of the second biomacromolecule of mark, concrete reaction density is then determined according to manufacturer's recommendation concentration, and different manufacturer's recommending data slightly changes.
Following embodiment can make the technician of this professional skill field more fully understand the present invention, but does not limit the present invention in any way.
Embodiment 1
1. the device for fast detecting of interaction of biomacromolecules makes
With reference to Fig. 1 custom mold, wherein die size: long x is wide=12x6 centimetre; Material is vinyon.The size of reacting hole 1: the wide x of long x is dark=0.7x0.5x0.3 centimetre, for open, can be used for application of sample.Detect aperture 3 is measure-alike with reacting hole 1, but be closed, only for inlaying the metal wire 2 that is magnetic along microtubule direction, size: the wide x of long x is high=and 0.5x0.1x0.1 centimetre.Reacting hole 1 is arranged in parallel with detect aperture 3, at a distance of 4 centimetres, is connected by microchannel.Microchannel diameter 0.1 centimetre.All pipelines are by after detect aperture, and extension 1 centimetre gathers to peristaltic pump access port.Microchannel is Mold Making, one-shot forming.
2. apply
The application of this device can be understood as a kind of immunomagnetic beads adsorption technology based on ELISA principle.Current commercial immunomagnetic beads surface is containing amino, carboxyl, silica-based, Streptavidin, aldehyde radical or sulfydryl isoreactivity group.According to shop instruction by biomolecule and immunomagnetic beads coupling.The coupling of the magnetic bead (Pierce company, article No. 88826 can combine with containing amino biomolecule) that following program activates with Much's bacillus outer membrane protein OmpA and NHS is example, sets forth coupling process and the detection method of immunomagnetic beads and OmpA albumen:
Get 300 microliters of magnetic beads and be placed in 1.5 milliliters of centrifuge tubes, be vertically positioned on the base containing magnet, draw supernatant and abandon.Add 1 mM hydrochloric acid of 1 milliliter of precooling, shake mixing gently, be vertically positioned on the base containing magnet, draw supernatant and abandon.(note: protein concentration is 0.1-2 mg/ml, dissolves with the 50 mM borates of pH 8.5 to add 300 micro liter purified OmpA albumen immediately; Or with not containing the buffer solution of amino pH 7-9; Can not Tris or glycocoll be contained in damping fluid), shake 30 seconds gently.Be placed in and rotate on vortex mixer, room temperature or 4
oc reacts 1 hour.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.Add 1 milliliter of 0.1M glycocoll (pH 2.0), concussion mixing 15 seconds.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.With 0.1M glycocoll (pH 2.0) cyclic washing magnetic bead 2 times.Add 1 milliliter of ultrapure water, concussion mixing 15 seconds.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.Add 1 milliliter of quenching buffers (3M monoethanolamine, pH 9.0) and close the reactive group of magnetic bead surfaces, concussion mixing room temperature reaction 2 hours after 30 seconds.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.Add 1 milliliter of ultrapure water, mixing.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.Add 1 milliliter and store liquid (50 mM borates, pH 8.5,0.05% Sodium azide), concussion mixing 15 seconds.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.2 times are washed with storage liquid.Finally store liquid suspension magnetic bead protein conjugate, in 4 with 300 microlitres
oc saves backup (now OmpA albumen-magnetic bead conjugate final concentration is 10 mg/ml).
By OmpA albumen-magnetic bead conjugate phosphate buffer (10mM, pH 7.4) be diluted to 1 mcg/ml, get 50 microlitres and be placed in 1.5 milliliters of centrifuge tubes, then add the mycobacterium tuberculosis antibody that 50 microlitres are to be detected, then add Protein A/G(that 50 microlitre FITC mark also can with HRP or collaurum or on turn the marks such as light-emitting particles).Shake mixing gently, room temperature reaction 5 minutes.100 microlitre response samples are dripped in the reacting hole 1 in the device for fast detecting of interaction of biomacromolecules.Connect peristaltic pump or rubber pipette bulb, make liquid from reacting hole 1 flow detection hole 3, end reaction liquid all flows out in waste liquid pool.
With the naked eye (be applicable to the Protein A/G of collaurum or HRP mark; If HRP mark, also need to add o-phenylenediamine), fluorescence detection device (being applicable to the fluorescein-labeled Protein A/G such as FITC), upper forwarding optical detection device (being applicable to the Protein A/G of forwarding light particle marker) detect detect aperture and whether occur response line.
In response sample in instillation reacting hole, the concentration of OmpA albumen-magnetic bead conjugate is 1 mcg/ml, the Monitoring lower-cut line concentration of mycobacterium tuberculosis antibody to be detected is 0.001 mcg/ml (all can detect higher than this concentration), the concentration of the Protein A/G of commercial FITC mark is 0.01 mcg/ml (this concentration is recommended by manufacturer, and the working concentration that different manufacturer recommends slightly changes).
Embodiment 2
1. the device for fast detecting of interaction of biomacromolecules makes, identical with embodiment 1.
2. apply
The embodiment of the present embodiment is consistent with the principle of embodiment 1, following program is for nucleic acid, set forth magnetic bead (the Pierce company that itself and Streptavidin activate, article No. 88816, can combine with the biomolecule containing biotin) coupling, set forth coupling process and the detection method of immunomagnetic beads and nucleic acid:
Get 50 microlitres (about 0.5 milligram) magnetic bead and be placed in 1.5 milliliters of centrifuge tubes, be vertically positioned on the base containing magnet, draw supernatant and abandon.Add 1 milliliter of lavation buffer solution (10mM Tris-HCl, pH 7.0, containing 0.1% Tween-20) and shake mixing gently, be vertically positioned on the base containing magnet, draw supernatant and abandon.Add the biotin labeled nucleic acid fragment of 300 microlitre (note: nucleic acid concentration is 2 mcg/ml, dissolves with above-mentioned lavation buffer solution) immediately, shake 30 seconds gently.Be placed in and rotate on vortex mixer, room temperature or 4
oc reacts 1 hour or spends the night.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.Add 300 milliliters of lavation buffer solutions, draw supernatant and abandon, collect magnetic bead.Cyclic washing magnetic bead 2 times, finally uses 300 milliliters of lavation buffer solution suspension magnetic beads.In 4
oc saves backup (now nucleic acid-magnetic bead conjugate final concentration is about 1-2 mcg/ml).
By nucleic acid-magnetic bead conjugate phosphate buffer (10mM, pH 7.4) be diluted to 0.1 mcg/ml, get 50 microlitres and be placed in 1.5 milliliters of centrifuge tubes, then 50 microlitre the second biomolecule to be detected (small molecule toxins is added, concentration is 1 nanogram-1 mcg/ml), add 50 microlitre toxin specific antibodies (monoclonal antibody, extension rate is 1:10000), add two anti-(sheep anti-mouse antibodies of FITC mark, extension rate is 1:5000) of 50 microlitre marks again.Shake mixing gently, room temperature reaction 5 minutes.100 microlitre response samples are dripped in the reacting hole 1 in the device for fast detecting of interaction of biomacromolecules.Connect peristaltic pump or rubber pipette bulb, make liquid from reacting hole 1 flow detection hole 3, end reaction liquid all flows out in waste liquid pool.Detecting step is with embodiment one.
In response sample in instillation reacting hole, the concentration of nucleic acid-magnetic bead conjugate is 0.1 mcg/ml, the Monitoring lower-cut concentration of small molecule toxins to be detected is 1 nanograms/milliliter (all can detect higher than this concentration), the concentration of toxin specific antibody is 1 nanograms/milliliter, the concentration of the sheep anti-mouse antibody of commercial FITC mark is 0.01 mcg/ml (this concentration is recommended by manufacturer, and the working concentration that different manufacturer recommends slightly changes).
Embodiment 3
1. the device for fast detecting of interaction of biomacromolecules makes, identical with embodiment 1.
2. apply
The embodiment of the present embodiment is consistent with embodiment 1 principle, following program, for antibody, sets forth magnetic bead (Pierce company, article No. 88802 that itself and ProteinA/G activate, can combine with antibody molecule) coupling, set forth coupling process and the detection method of immunomagnetic beads and antibody:
Get 50 microlitres (about 0.5 milligram) magnetic bead and be placed in 1.5 milliliters of centrifuge tubes, be vertically positioned on the base containing magnet, draw supernatant and abandon.Add 150 microlitre lavation buffer solutions (10mM Tris-HCl, pH 7.0, containing 0.1% Tween-20) and shake mixing gently, be vertically positioned on the base containing magnet, draw supernatant and abandon.Add 1 milliliter of lavation buffer solution, centrifuge tube is vertically positioned on the base containing magnet, draw supernatant and abandon.Add 500 microliters of sample (as: serum, cell culture, ascites etc. containing antibody) immediately, shake 30 seconds gently.Be placed in and rotate on vortex mixer, room temperature reaction 1 hour.Centrifuge tube is vertically positioned on the base containing magnet, draws supernatant and abandon, collect magnetic bead.Add 300 milliliters of lavation buffer solutions, draw supernatant and abandon, collect magnetic bead.Cyclic washing magnetic bead 2 times, finally uses 300 milliliters of lavation buffer solution suspension magnetic beads.In 4
oc saves backup.
Get 50 gel of antibody-magnetic bead conjugate and be placed in 1.5 milliliters of centrifuge tubes, then add 50 microlitres mark, can with the small molecule toxins of antibody response on magnetic bead, add the aptamer that the FITC that can react with toxin marks again, shake mixing gently, room temperature reaction 5 minutes.100 microlitre response samples are dripped in the reacting hole 1 in the device for fast detecting of interaction of biomacromolecules.Connect peristaltic pump or rubber pipette bulb, make liquid from reacting hole 1 flow detection hole 3, end reaction liquid all flows out in waste liquid pool.Detecting step is with embodiment one.
In response sample in instillation reacting hole, the concentration of antibody-magnetic bead conjugate is 0.5 mcg/ml, the Monitoring lower-cut concentration of toxin to be detected is 1 nanograms/milliliter (all can detect higher than this concentration), and the aptamer concentration of FITC mark is 0.1 mcg/ml.
Be more than in conjunction with specific embodiments son the present invention is done further describe.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; under the premise without departing from the principles of the invention; the present invention and application thereof also have various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (8)
1. the device for fast detecting of interaction of biomacromolecules, comprises base plate, it is characterized in that, the surface of base plate is provided with n detecting unit, and n is the integer of >=1; Each detecting unit comprises a reacting hole, detect aperture and microtubule both being connected; Each detect aperture is all connected to negative pressure device interface by microtubule; The diameter of described microtubule is 0.1 ~ 2 millimeter; Described reacting hole is opened type, for instilling reactant; Described detect aperture is closed type, and there is a magnetic metal wire of tool its inside, and metal wire is consistent with the direction of microtubule in affiliated detecting unit.
2. device according to claim 1, it is characterized in that, described base plate is the base plate of plastics or pottery.
3. device according to claim 1, it is characterized in that, described microtubule is the pipeline of tygon, propylene or butylenes plastic material.
4. device according to claim 1, it is characterized in that, the capacity of described reacting hole is 10 ~ 500 microlitres.
5. according to the device described in Claims 1-4 any one, it is characterized in that, described detecting unit has two at least, and each detecting unit mutually side by side and be arranged in parallel, embark on journey by the two ends of its reacting hole and detect aperture apportion base plate and arrangement.
6. according to the device described in Claims 1-4 any one, it is characterized in that, described negative pressure device interface is the interface for connecting peristaltic pump or rubber pipette bulb.
7. based on a method for quick for the interaction of biomacromolecules of device described in claim 1, it is characterized in that, comprise the following steps:
(1) according to the product operation instruction of immunomagnetic beads, the first biomacromolecule is coupled on immunomagnetic beads, then adds the second biomacromolecule of material to be detected and at least one mark successively; This process has two kinds of implementations: complete mixing in test tube after, reactant liquid is added reacting hole, or in reacting hole, adds reactant liquid successively;
The first biomacromolecule described is any one in protein, polysaccharide, polypeptide, nucleic acid or chemicals, described material to be detected and the second biomacromolecule are any one in protein, polypeptide, polysaccharide, toxin, chemicals or nucleic acid, and the second biomacromolecule of described mark can be combined with material to be detected; Mark for the second biomacromolecule is any one in collaurum, fluorescent material, chemiluminescent material or upper forwarding luminescent material;
(2) start peristaltic pump, make the reactant liquid flow detection hole in reacting hole; If be coupled to the first biomacromolecule on magnetic bead, material to be detected, mark the second biomacromolecule there occurs interaction, reactant liquid can be adsorbed on detection line when flowing through detect aperture; The color change of magnet-wire can be detected by naked eyes, fluorescence detector, chemiluminescence detector or upper forwarding photodetector.
8. method according to claim 7, is characterized in that, adds in the reactant liquid in reacting hole, and the concentration being coupled to the first biomacromolecule on magnetic bead is 0.1 ~ 1 mcg/ml; The concentration of the second biomacromolecule of mark is determined according to the recommendation of its manufacturer.
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| CN109613245B (en) * | 2019-01-09 | 2023-03-31 | 新疆医科大学 | Method for detecting targeting of protein-alliinase conjugate |
| CN109856392A (en) * | 2019-02-22 | 2019-06-07 | 武汉巴菲尔生物技术服务有限公司 | The device for fast detecting and method of interaction of biomacromolecules |
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Effective date of registration: 20150608 Address after: 276000 16-1-001, 369 middle Chongqing Road, Licang District, Shandong, Qingdao Patentee after: Qingdao Ruijie Bio-Tech Co., Ltd. Address before: Yuhang District, Hangzhou City, Zhejiang Province, 311121 West No. 1500, 2-825 Patentee before: Hangzhou Golden Biotek Co., Ltd. |
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Effective date of registration: 20210923 Address after: 266000 Room 101, building 5, blue biomedical industrial park, No. 368 Hedong Road, high tech Zone, Qingdao, Shandong Patentee after: Qingdao batfi Technology Development Co.,Ltd. Address before: 16-1-001, No. 369, Chongqing Middle Road, Licang District, Qingdao, Shandong 276000 Patentee before: QINGDAO RUIJIE BIO-TECH Co.,Ltd. |