CN116449028A - Membrane chromatography detection structure for saliva insulin level and application thereof - Google Patents
Membrane chromatography detection structure for saliva insulin level and application thereof Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a membrane chromatography detection structure of saliva insulin level and application thereof, which are a detachable combined set of a saliva sampling structure and a chromatography detection structure, wherein the saliva sampling structure comprises a sampling head and a sampling connection structure, the chromatography detection structure comprises a detection reagent strip and a sample adding connection structure, a first anti-insulin antibody marked by an indicator is coated on a marker binding pad in the detection reagent strip contained in the chromatography detection structure, a second non-marked anti-insulin antibody is coated on a nitrocellulose membrane, and the membrane is suitable for measuring the insulin level of saliva samples, thereby improving detection efficiency, convenience and accuracy and having important clinical significance.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a membrane chromatography detection structure for saliva insulin level and application thereof.
Background
Insulin is a protein hormone secreted by islet beta cells in the pancreas, consisting of two peptide chains, a and B, regulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, and the like. Insulin is the most important protein hormone for promoting anabolism of human body, and can promote glucose uptake and utilization by tissue cells, promote glycogen synthesis, inhibit gluconeogenesis and reduce blood sugar; the composition can promote fatty acid synthesis and fat storage, and reduce fat decomposition; amino acids can be promoted to enter cells for proteins, and various links of protein synthesis can be promoted to increase protein synthesis.
The immunological detection technology is a saliva insulin level detection method commonly used at present, and comprises an immunonephelometry method, a chemiluminescence method, an enzyme-linked immunosorbent assay method, a chemiluminescence immunoassay method, a time-resolved immunofluorescence method, a quantum dot immunofluorescence chromatography method, a latex immunonephelometry method, a latex enhanced immunonephelometry method, a fluorescence immunochromatography method and the like. High sensitivity, rapidness, convenience, miniaturization, full quantification, automation and household are the development trend of the current clinical immunodetection technology. Point of care testing (POCT) is one of the fastest growing branches of the current immunological testing technology, and chromatographic immunodetection is the most commonly used testing method, wherein colloidal gold chromatography, fluorescence chromatography and latex microsphere chromatography are the most widely used products, and the used testing samples include blood, urine, saliva, sputum, sweat, swab mucus and the like. Blood is most commonly used compared to other samples, but is less accepted than other samples because its sampling pattern is invasive. At present, clinical detection of insulin level adopts blood samples, and interpretation from sampling and detection results is still limited to operation and use of a laboratory and professionals, so that the clinical detection of insulin level cannot be popularized and used to families and medical institutions lacking corresponding detection conditions. However, studies of salivary insulin levels have been largely reported and have a clear correlation with blood insulin levels. A large number of researches prove that the insulin is a protein hormone and important regulation indexes and markers for directly influencing various metabolic related diseases and bad health states including diabetes, and is suitable for detecting various diseases and health states in and out of hospitals, so that the insulin can be developed for detecting and using medical institutions with home self-tests and lack of corresponding detection conditions, has a saliva insulin level rapid detection technology which is simple and convenient to operate, rapid to use and low in cost, is beneficial to improving medical quality and efficiency, and has important clinical significance and application value.
Disclosure of Invention
The invention aims to provide a membrane chromatography detection structure for saliva insulin level and application thereof, which have the advantages of convenient and rapid detection operation, low cost and the like compared with the prior art, and improve the detection quality.
In view of the above, the present invention provides a membrane chromatography detection structure for saliva insulin level, which is characterized in that:
1) The membrane chromatography detection structure is a detachable combined set of a saliva sampling structure and a chromatography detection structure;
2) The chromatographic detection structure comprises a detection reagent strip, a detection shell positioned at the outer side of the detection reagent strip and a sample adding connection structure positioned at the proximal end of the detection shell and corresponding to the proximal end of the detection reagent strip;
3) The detection reagent strip comprises a sample pad, a marker binding pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is positioned at a corresponding position of the sample adding connecting structure and forms liquid phase traffic connection with the sample adding connecting structure, the marker binding pad is coated with a first anti-insulin antibody marked by an indicator, and the nitrocellulose membrane is coated with a second non-marked anti-insulin antibody;
4) The saliva sampling structure comprises a sampling head with liquid adsorption property, a handle-like structure directly connected with the sampling head and a fixed structure for fixing the connection of the sampling head and a non-sampling end of the handle-like structure, and the sampling head and the fixed structure form a sampling connection structure which can be detachably connected with the sampling connection structure together;
5) The sample adding connection structure is of a mutually-communicated double-layer structure and comprises a reagent detection layer for placing the detection reagent strip and a sample collection layer for detachably placing the sampling head, wherein the reagent detection layer is internally provided with a proximal end part of the detection reagent strip, and the sample collection layer is provided with an access port and a sample collection channel of the sampling head and a liquid phase traffic channel which forms a liquid phase traffic flow opening with the reagent detection layer.
The detection reagent strip is a sample pad, a marker binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially stuck on the PVC bottom sheet, and is placed in a detection shell, and the detection shell is of a rigid structure or a flexible structure.
In the membrane chromatography detection structure, the sampling head consists of a fixed arm and a water absorption pad arranged on the outer side of the fixed arm, and the water absorption pad is of a water absorption material structure.
The water absorbing material is selected from sponge products and polyvinyl alcohol products, and comprises natural and modified high-molecular high-water-absorbing resins and artificially synthesized water-absorbing resins such as starch series, cellulose series, other natural product series, polyvinyl acid salt series, polyvinyl alcohol series, polyoxyethylene series and the like.
In the above-mentioned membrane chromatography detection structure, the reagent testing layer and the sample collection layer of the connection structure of adding a sample are bilayer structure from top to bottom, and the sample collection layer is last, and the reagent testing layer is under, is equipped with between sample collection layer and the reagent testing layer the liquid phase traffic channel uses in the sample collection channel of sample collection layer will the sample collection head inserts during the use, wherein the volume of sample collection channel is less than the volume of sample collection head, and the sample collection channel forms the extrusion to the sample collection head in-process of inserting, adsorb in the liquid phase of sample collection head is passed through the liquid phase traffic channel flows in and is located on the detection reagent strip of reagent testing layer.
In the above-mentioned membrane chromatography detection structure, the reagent testing layer and the sample collection layer of application of sample connection structure are left and right bilayer structure, and the sample collection layer is in one side, and the reagent testing layer is in the opposite side, is equipped with between sample collection layer and the reagent testing layer liquid phase traffic channel uses in the sample collection channel of sample collection layer will the sample collection head inserts during the use, wherein the volume of sample collection channel is less than the volume of sample collection head, and the sample collection channel forms the extrusion to the sample collection head in-process of inserting, adsorb in the liquid phase of sample collection head is through liquid phase traffic channel inflow is located on the detect reagent strip of reagent testing layer.
In the above-mentioned membrane chromatography detection structure, the liquid phase communication channel is a liquid flow opening of the sample pad and/or the label binding pad, which is opened towards the proximal end of the detection reagent strip, so as to form an opening structure for the mutual liquid flow traffic of the sample collection channel and the detection reagent strip in the reagent detection layer.
In the above-mentioned membrane chromatography detection structure, removable combination suit connected mode selects grooved press type, plug-in type or post push type, wherein:
1) The sampling head of the grooved sampling connecting structure is provided with a block-shaped water absorbing structure, the sampling connecting structure is of an upper-lower double-layer structure, a sample collecting channel of the sampling connecting structure is of a grooved structure, the block-shaped sampling head is placed into the grooved sample collecting channel during use, the sampling head is extruded, a liquid phase sample to be detected flows into a detection reagent strip through a liquid phase alternating current channel, and detection is started;
2) The sampling head of the inserted sampling connection structure is wrapped by a sheet-shaped water absorption structure, the sample adding connection structure is of an upper-lower double-layer structure, a sample collecting channel of the sampling connection structure is of a pipeline structure, when the sampling head wrapped by the water absorption structure is inserted into the pipeline sample collecting channel in use, the sampling head is extruded, a liquid phase sample to be detected flows into a detection reagent strip through a liquid phase communication channel, and detection is started;
3) The column pushing type sampling head of the sampling connection structure is wrapped by a sheet-shaped water absorption structure, the sample adding connection structure is of a left-right double-layer structure, a sample collecting channel of the column pushing type sampling head is of a pipeline structure, the sampling head wrapped by the water absorption structure is inserted into the pipeline sample collecting channel during use, the column pushing type sampling head is extruded, a liquid phase sample to be detected flows into a detection reagent strip through a liquid phase communication channel, and detection is started.
In the above membrane chromatography detection structure, the detection reagent strip is at least one of a colloidal gold immunoassay reagent strip, a fluorescent immunoassay reagent strip and a latex microsphere immunoassay reagent strip, and the indicator is at least one of a color microsphere and a fluorescent substance.
In the above-mentioned membrane chromatography detection structure, the membrane chromatography detection structure is configured with a semi-quantitative color chart with the same color as the indicator, and color strips with 3 or more different color depths are printed on the color chart.
In the above-mentioned membrane chromatography detection structure, the membrane chromatography detection structure is configured with a portable quantitative detector including at least one of a colorimetric quantitative analyzer and a fluorescent quantitative analyzer.
In the above membrane chromatography detection structure, the preparation of the detection reagent strip further comprises a biotin/avidin reaction system, the label binding pad is coated with a first anti-insulin antibody and a biotin-labeled second anti-insulin antibody labeled by the indicator, and the nitrocellulose membrane is coated with non-labeled avidin and analogues thereof. At this time, insulin in the sample to be detected is combined with the first insulin-resisting antibody marked by the indicator and the second insulin-resisting antibody marked by the biotin at the beginning of the marker combination pad to form a first insulin-resisting antibody-insulin-biotin marked second insulin-resisting antibody complex marked by the indicator, then flows through a nitrocellulose membrane coated with avidin to be captured to form the first insulin-resisting antibody-insulin-biotin marked second insulin-avidin complex marked by the indicator, and is gathered on a coating strip and visible color or fluorescence is displayed as a basis of quantification or semi-quantification. Avidin and its analogs include avidin, streptavidin and other substances that have specific binding to biotin.
In the above membrane chromatography detection structure, the operation of the membrane chromatography detection structure includes the following steps:
1) Taking out the chromatographic detection structure and the saliva sampling structure, and exposing a sampling head of the saliva sampling structure;
2) The handle structure of the saliva sampling structure is held by hand, the sampling head of the saliva sampling structure is placed in the oral cavity, and saliva samples are naturally absorbed until the sampling head is completely infiltrated by saliva;
3) Taking out and placing a sampling head of the saliva sampling structure into a sample collecting channel of the chromatographic detection structure, extruding, and enabling saliva samples to flow into a sample pad of a detection reagent strip of the reagent detection layer through a liquid phase traffic channel;
4) The saliva sample passes through the sample pad and forwards flows through the marker binding pad, the nitrocellulose membrane and the water absorption pad;
5) And reading the detection result from the observation window to finish detection.
The membrane chromatography detection structure for the salivary insulin level is applied to development of products for rapidly detecting the salivary insulin level.
Due to the adoption of the technical scheme, the invention has the following advantages:
1. the invention relates to a membrane chromatography detection structure of saliva insulin level, which comprises a detachable combination set of a saliva sampling structure and a chromatography detection structure, can detect human insulin level by saliva without interventional collection of blood samples, and has the advantages of convenient detection and high acceptability, thus remarkably improving timeliness of clinical detection and feasibility of popularization and promotion and improving clinical diagnosis and treatment effects.
2. The sampling head of the saliva sampling structure is a detection sampling structure provided with the water absorbing material structure, the water absorbing materials have the quantitative effect of absorbing liquid, the collection capacity of saliva samples is controlled by setting the size of the absorption material structure, and meanwhile, the collection process is natural siphon, and special quantitative equipment is not needed, so that the quantitative operation is feasible and convenient, and the convenience and the quick operability of technical products are realized.
3. The invention adopts a double-layer structure with mutually-communicated sample adding connection structure, and is provided with a reagent detection layer for placing detection reagent strips and a sample collection layer for detachably placing a sampling head, wherein a liquid phase traffic channel with a liquid phase traffic flow opening is arranged between the double layers, the volume of the sample collection layer is smaller than that of the sampling head, and when the sampling head is inserted, the volume of the sample collection layer is smaller than that of the sampling head, so that saliva adsorbed on the sampling head flows into the proximal end part of the detection reagent strips placed in the reagent detection layer, and the detection reaction of the reagent strips is started, thereby not only needing to be provided with a sampling container, but also simplifying the operation process and being more convenient to detect.
4. The colorimetric card for semi-quantitatively detecting the color intensity of the detection strip is arranged, the semi-quantitatively judging of the detection result can be carried out through the comparison of the colorimetric card, the method is suitable for clinical application scenes without accurate quantification and only by observing the variation trend of the saliva insulin level, and the clinical application value is improved.
5. The invention has simple operation steps, is easy to realize household use or self detection, is convenient to use, reduces the waste of raw materials, simultaneously obviously improves the working efficiency, and can be applied to various fields of professional and amateur detection.
Drawings
FIG. 1 is a schematic diagram of the overall structure of the present invention;
FIG. 2 is a schematic longitudinal section of the detachable combined structure of the present invention;
FIG. 3 is a schematic diagram of a test strip according to the present invention;
FIG. 4 is a schematic diagram of a slot press type assembly structure according to the present invention;
FIG. 5 is a schematic longitudinal section of a slot-pressing type composite structure according to the present invention;
FIG. 6 is a schematic diagram of a plug-in combination structure according to the present invention;
FIG. 7 is a schematic longitudinal section of the plug-in combination structure of the present invention;
FIG. 8 is a schematic diagram of a push-push type assembly structure according to the present invention;
FIG. 9 is a schematic longitudinal section of the column pushing type combined structure of the present invention.
The figures are labeled as follows:
saliva sampling structure 1; a chromatographic detection structure 2; a detection reagent strip 3; a handle-like structure 4; a fixed structure 5; a sampling head 6; a reagent detection layer 7; a sample collection channel 8; a sample pad 9; a label binding pad 10; a nitrocellulose membrane pad 11; a water absorbing pad 12; a viewing window 13; a detection housing 14; a test strip holder 15; a loading well 16; a liquid phase traffic channel 17; a PVC support backsheet 18; a detection line 19; a quality control line 20; a slot-press handle-like structure 24; a slot-pressing type fixing structure 25; a slot pressure sampling head 26; a groove-pressure type reagent detection layer 27; a slot press sample acquisition channel 28; a grooved viewing window 33; a slot-pressure type detection housing 34; a slot-press test strip holder 35; a slot-pressure type sample inlet 36; a tank pressure type liquid phase traffic channel 37; a slot-pressure sampling head fixing arm 38; a slot-pressure sampling head absorbent pad 39; a male handle-like structure 44; a male fixation structure 45; an insertion sampling head 46; an insertion reagent detection layer 47; inserting a sample acquisition channel 48; a male sample collection channel proximal end 49; a plug-in viewing window 53; a plug-in detection housing 54; insert test strip holder 55; insertion type sample addition port 56; a plug-in liquid phase traffic channel 57; an insertion sampling head fixing arm 58; an insert-type sampling head suction pad 59; a post-pushing handle-like structure 64; a post-pushing type fixing structure 65; a column pushing sampling head 66; a push-push type reagent detection layer 67; the column pushing type sample collection channel 68; a column-pushing type observation window 73; a push-on detection housing 74; column pushing type test strip bracket 75; a push-on sample addition port 76; column pushing type liquid phase traffic channel 77; a column pushing type sampling head fixing arm 78; a column pushing type sampling head water absorbing pad 79; a column pushing type sampling head fixing arm 80; column pushing type sampling head water absorbing pad 81.
Description of the embodiments
In order to further illustrate the technical means and effects adopted by the present invention to achieve the preset purpose, the following embodiments are used for further illustrating the present invention with reference to the accompanying drawings, but the present invention is not limited to the following description.
As shown in fig. 1 and 2, the integral structure of the invention comprises a saliva sampling structure 1; the chromatographic detection structure 2 and the detection reagent strip 3, wherein the saliva sampling structure 1 comprises a handle-like structure 4, a fixed structure 5 directly connected with the handle-like structure, a sampling head 6 directly connected with the fixed structure 5, and a water absorption pad arranged outside the sampling head, which together form a sampling connection structure capable of being detachably connected with a sample adding connection structure positioned on the chromatographic detection structure 2; the chromatography detection structure 2 is provided with a sample adding connection structure comprising a reagent detection layer 7, a sample collecting channel 8, a sample adding hole 16 and a liquid phase traffic channel 17, a detection shell 14, an observation window 13 and a detection reagent strip bracket 15 positioned in the detection shell 14.
As shown in fig. 3, the test reagent strip of the present invention includes a sample pad 9; a label binding pad 10; the nitrocellulose membrane pad 11 and the water absorbing pad 12 are sequentially arranged on the PVC support negative film 18, the first anti-insulin antibody marked by an indicator is sprayed on the marker binding pad 10 to serve as a detection indicator, the indicator is usually colloidal gold particles, color latex microspheres and fluorescent microspheres, the non-marked second anti-insulin antibody serving as a detection line 19 is coated on the nitrocellulose membrane pad 11, meanwhile, the non-marked non-insulin specific binding antibody such as sheep anti-mouse IgG is coated on the nitrocellulose membrane pad 11, the saliva sample is loaded on the marker binding pad 10 of the detection reagent strip during use, insulin in the sample is combined with the first anti-insulin antibody marked by the indicator to form a first compound marked by the indicator, the first anti-insulin antibody and the insulin are captured through the nitrocellulose membrane pad 11, the non-marked second anti-insulin antibody coated on the nitrocellulose membrane pad 11 is formed, the second compound marked by the indicator, the insulin and the second anti-insulin antibody is simultaneously coated on the nitrocellulose membrane pad 11, the non-marked second anti-insulin antibody is accumulated on the nitrocellulose membrane pad, the insulin is formed, the color development level or the color development level of the insulin is proportional to the color development level of the insulin in the saliva sample, and the color development level is further completed.
As shown in fig. 4 and 5, the detachable combined set of the invention is a slot-pressure type connection structure, wherein the saliva sampling structure 1 is also composed of a handle-like structure 24, a fixed structure 25 directly connected with the handle-like structure, a sampling head 26 directly connected with the fixed structure 25, and a water absorbing pad 29 arranged outside the sampling head, the water absorbing pad 29 arranged on the sampling head 26 is a water absorbing material structure, is arranged on one side of a fixed arm 30, is a block-shaped structure, and is characterized in that a structure with a lever squeezing-like function is formed by the fixed structure 26 and the handle-like structure 24, and is a sampling connection structure capable of being detachably connected with a sample adding connection structure positioned on a chromatography detection structure 2; the chromatographic detection structure 2 is also provided with a sample adding connecting structure comprising a reagent detection layer 27, a sample collecting channel 28, a sample adding hole 36 and a liquid phase traffic channel 37, a detection shell 34, an observation window 33 and a detection reagent strip bracket 35 positioned in the detection shell 34; the sample collection channel 28 is of a groove-shaped structure with an upward opening, the inlet of the groove-shaped structure is a sample adding hole 36, and a liquid flow opening which is positioned at the bottom of the groove-shaped structure and communicated with the reagent detection layer 27 is a liquid phase traffic channel 37; the water absorbing pad 29 of the sampling head 26 is detachably pressed downwards into the groove-shaped sample collecting channel 28 of the sample adding connecting structure, the water absorbing pad 29 is extruded, the sample to be detected is transferred to the detection reagent strip 1 positioned in the reagent detection layer 27 through the open pore structure at the bottom, namely the liquid phase traffic channel 37, and the detection reaction is started.
As shown in fig. 6 and 7, in the detachable combined set plug-in connection structure of the invention, the saliva sampling structure 1 is also composed of a handle-like structure 44, a fixed structure 45 directly connected with the handle-like structure, a sampling head 46 directly connected with the fixed structure 45, and a water absorbing pad 59 arranged outside the sampling head, the water absorbing pad 59 arranged on the sampling head 46 is a water absorbing material structure, is wrapped outside the fixed arm 58, is a detection swab-like sampling structure, and can be detachably connected with a sample adding connection structure positioned on the chromatography detection structure 2 to finish sample adding; the chromatographic detection structure 2 is also provided with an upper and lower double-layer sample adding connecting structure comprising a reagent detection layer 47, a sample collecting channel 48, a sample adding hole 56 and a liquid phase traffic channel 57, a detection shell 54, an observation window 53 and a detection reagent strip bracket 55 positioned in the detection shell 54; the sample collection channel 48 is a pipe-shaped structure with an opening at the proximal end, the inlet of the sample collection channel 48 is a sample adding hole 56, the sample enters the sample collection channel 48 through the extension section 49, and a liquid flow opening communicated with the reagent detection layer 47 is arranged at the proximal end of the bottom and is a liquid phase traffic channel 57. In use, the test swab structure of the sampling head 46 is inserted into the sample collection channel 48 through the sample application hole 56, the liquid phase sample to be tested is extruded, and the liquid phase sample to be tested is transferred to the test reagent strip 1 positioned in the reagent test layer 47 through the liquid phase traffic channel 57, and the test reaction is started.
As shown in fig. 8 and 9, the detachable combined set of the column pushing type connection structure of the present invention, wherein the saliva sampling structure 1 is also composed of a handle-like structure 64, a fixing structure 65 directly connected with the handle-like structure, a sampling head 66 directly connected with the fixing structure 65, and a water absorbing pad 79 arranged outside the sampling head, the water absorbing pad 79 arranged on the sampling head 66 is of a water absorbing material structure, is wrapped and arranged on the outer side of the fixed arm 78, is a sample sampling structure for detecting a wiper, and can be detachably connected with a sample adding connecting structure positioned on the chromatographic detection structure 2 to finish sample adding; the chromatographic detection structure 2 is also provided with a left-right double-layer sample adding connecting structure comprising a reagent detection layer 67, a sample collecting channel 68, a sample adding hole 76 and a liquid phase traffic channel 77, a cylindrical detection shell 74, an observation window 73 and a detection reagent strip bracket 75 positioned in the detection shell 74; the sample collection channel 68 is a pipeline-shaped structure with an opening at the proximal end, the inlet of the sample collection channel 68 is a sample adding hole 76, and a liquid flow opening communicated with the reagent detection layer 67 is a liquid phase traffic channel 77 arranged at the bottom of the double-layer sample adding connection structure when the sample collection channel is vertically placed; the outside of the middle section of the fixed structure 65 is provided with an elastic blocking structure 81 which forms a sealing connection with the inlet extension 80 of the sample collection channel 68 when inserted into the inlet extension 80 of the sample collection channel 68. When in use, the detection swab sample structure of the sampling head 66 is inserted into the sample collection channel 8 through the sample adding hole 76 and the inlet extension section 80 of the sample collection channel 68, and then the liquid phase sample to be detected flows back from the sampling head 66 into the inlet extension section 80 of the sample collection channel 68, is connected in a sealed mode and pushed to the liquid phase transportation channel 77, so that the liquid phase sample to be detected is transferred to the detection reagent strip 3 positioned in the reagent detection layer 67, and the detection reaction is started.
In practical operation, when the membrane chromatography detection structure is a colloidal gold immune detection structure, the detection reagent strip 3 is prepared by a colloidal gold method, and a sample pad, a colloidal gold binding pad coated with a colloidal gold-labeled first anti-insulin antibody, a nitrocellulose membrane pad coated with a non-labeled second anti-insulin antibody and a water absorption pad are sequentially adhered on a PVC negative film; when the membrane chromatography detection structure is a color latex microsphere immune detection structure, the detection reagent strip 3 is prepared by a color latex microsphere immune method, and a sample pad, a latex color latex microsphere combination pad coated with a color latex microsphere marker, a nitrocellulose membrane pad coated with a non-marked capture reagent and a water absorption pad are sequentially stuck on a PVC negative film; when the membrane chromatography detection structure is a fluorescence immunoassay detection structure, the detection reagent strip 3 is prepared by a fluorescence immunoassay method by taking fluorescent microspheres or fluorescein as an indicator, and a sample pad, a fluorescent marker binding pad coated with a fluorescent marker, a nitrocellulose membrane pad coated with a non-marked capture reagent and a water absorption pad are sequentially stuck on a PVC negative film.
The semi-quantitative detection specific operation comprises the following steps: 1) Taking out the saliva sampling structure 1, the chromatographic detection structure 2 and the semi-quantitative colorimetric card 82, and exposing a sampling head of the saliva sampling structure 1; 2) Placing a sampling head of the saliva sampling structure into an oral cavity, and naturally absorbing saliva samples until the sampling head is completely infiltrated by saliva; 3) The sampling head of the sampled saliva sampling structure 1 is inserted into a sample adding connection structure of the chromatographic detection structure 2, the sampled saliva sample is transferred to a sample pad, and forwards flows through a marker binding pad, a nitrocellulose membrane and a water absorption pad; 4) And reading the detection result from the observation window, comparing the color depth of the detection line with the semi-quantitative color comparison card 82, and reading the semi-quantitative range value to finish the detection.
The specific quantitative detection operation comprises the following steps: 1) Taking out the saliva sampling structure 1, the chromatographic detection structure 2 and the semi-quantitative colorimetric card 82, and exposing a sampling head of the saliva sampling structure 1; 2) Placing a sampling head of the saliva sampling structure into an oral cavity, and naturally absorbing saliva samples until the sampling head is completely infiltrated by saliva; 3) The sampling head of the sampled saliva sampling structure 1 is inserted into a sample adding connection structure of the chromatographic detection structure 2, the sampled saliva sample is transferred to a sample pad, and forwards flows through a marker binding pad, a nitrocellulose membrane and a water absorption pad; 4) And (3) placing the chromatographic detection structure 2 in a quantitative detector, reading the detection result, and quantitatively calculating the measurement value to finish detection.
Experimental study of the invention: the following experiments illustrate the detection method and the effect of the present invention, but are not limiting of the present invention. The experimental methods used in the following experiments are conventional methods unless otherwise specified. The materials, reagents and the like used, unless otherwise specified, are all commercially available.
Experiment one: saliva insulin detection experiment by immune colloidal gold method:
1. preparing a detection reagent strip:
preparing a detection reagent strip by adopting a double-antibody sandwich method by adopting a conventional immune colloidal gold detection technology, and adopting the membrane chromatography detection structure to prepare a detection kit for saliva insulin detection experiments, wherein a colloidal gold mark of a detection line T of the detection reagent strip indicates that an antibody is a first anti-insulin monoclonal antibody of 10ug/ml, and the detection reagent strip is coated on a glass cellulose membrane colloidal gold binding pad by adopting colloidal gold particles with the particle size of 50 nm; the capture antibody of the detection line T of the detection reagent strip is 1.2mg/ml paired second anti-insulin monoclonal antibody, and the paired second anti-insulin monoclonal antibody is coated on a nitrocellulose membrane pad; the capture antibody of the quality control line C of the detection reagent strip is a goat anti-mouse IgG polyclonal antibody of 1.0mg/ml, and the goat anti-mouse IgG polyclonal antibody is coated on a nitrocellulose membrane pad and used for capturing the colloidal gold labeled anti-insulin monoclonal antibody which is not specifically captured. The two ends of the nitrocellulose membrane pad printing membrane are respectively stuck with a water absorption pad and a colloidal gold mark bonding pad, and one side of the colloidal gold mark bonding pad is stuck with a sample pad. Placing the stuck detection sheet on a slitter, and cutting into detection reagent strips with the thickness of 3.0 mm.
2. The membrane chromatography detection structure of the invention is prepared by the following steps:
the Solidworks is adopted to design the upper cover, the base and the saliva sampling structure of the detection shell of the push-press type connection detection of the membrane chromatography detection structure, and 3D printing samples are used for preparing the membrane chromatography detection structure samples for experimental detection. Red color cards with different color depths are printed.
3. Experimental method and results:
during the experiment, the prepared detection reagent strip and the membrane chromatography detection structure are assembled into an integrated detection structure, the assembled detection structure is placed into an aluminum foil sealing bag with a drying agent, and the aluminum foil sealing bag is sealed on a sealing machine and labeled. The saliva of a freshly collected healthy person is centrifuged at 10000rpm/min for 10 minutes, and the supernatant is prepared into Changxiu human recombinant insulin glargine injection (100 IU/ml) with the concentration of 1, 5, 10, 15, 20, 25 and 30 uIU/ml. Taking 100ul of insulin detention solution, directly dripping insulin detention solutions with different concentrations to a sample adding window of a chromatographic detection structure, standing for 20 minutes, checking the color depth of a detection line through an observation window, photographing, designing and printing a semi-quantitative color comparison card according to the photographing result, and taking the semi-quantitative color comparison card as a comparison standard for judging the follow-up saliva sample semi-quantitative detection result.
The prepared saliva sampling structure is used for respectively collecting 1-hour saliva samples of fasting and postprandial of 10 healthy donors, the saliva samples are respectively and directly inserted into a sample collecting channel of a chromatographic detection structure, the saliva sampling structure is stood for 20 minutes, the color depth of a detection line is checked through an observation window, the saliva sampling structure is colorimetric with a semi-quantitative colorimetric card, the concentration range of a colorimetric result is judged, the fasting result is about 1-10uU/ml, the postprandial time is about 5-25uU/ml, and the saliva sampling structure accords with the detection range of normal people.
Experiment II: saliva insulin detection experiment by color latex microsphere immunochromatography:
1. preparing a detection reagent strip:
preparing a detection reagent strip by adopting a double-antibody sandwich method by adopting a conventional latex microsphere immunochromatography technology, preparing a detachable chromatographic detection structure detection kit by adopting the membrane chromatography detection structure of the invention for insulin detection experiments, wherein the color latex microspheres are all biological 300nm red microspheres, and the color latex microsphere mark of a detection line T of the detection reagent strip indicates that an antibody is a first anti-insulin monoclonal antibody of 50ug/ml, and coating the first anti-insulin monoclonal antibody on a glass cellulose membrane binding pad; the capture antibody of the detection line T of the detection reagent strip is 1.2mg/ml paired second anti-insulin monoclonal antibody, and the paired second anti-insulin monoclonal antibody is coated on a nitrocellulose membrane pad; the capture antibody of the quality control line C of the detection reagent strip is a goat anti-mouse IgG polyclonal antibody of 1.0mg/ml, and the goat anti-mouse IgG polyclonal antibody is coated on a nitrocellulose membrane pad and used for capturing the color latex microsphere marked anti-insulin monoclonal antibody which is not specifically captured. The two ends of the nitrocellulose membrane pad printing membrane are respectively stuck with a water absorption pad and a color latex microsphere marking combination pad, and one side of the color latex microsphere marking combination pad is stuck with a sample pad. Placing the stuck detection sheet on a slitter, and cutting into detection reagent strips with the thickness of 3.0 mm.
2. The membrane chromatography detection structure of the invention is prepared by the following steps:
the Solidworks is adopted to design the upper cover, the base and the saliva sampling structure of the detection shell of the plug-in connection detection of the membrane chromatography detection structure, and 3D printing samples are used for preparing the membrane chromatography detection structure samples for experimental detection. Red color cards with different color depths are printed.
3. Experimental method and results:
during the experiment, the prepared detection reagent strip and the membrane chromatography detection structure are assembled into an integrated detection structure, the assembled detection structure is placed into an aluminum foil sealing bag with a drying agent, and the aluminum foil sealing bag is sealed on a sealing machine and labeled. The saliva of a freshly collected healthy person is centrifuged for 10 minutes at 10000RPM/min, and the supernatant is taken to prepare Changxiu human recombinant insulin glargine injection (100 IU/ml) with the concentration of 1, 5, 10, 15, 20, 25 and 30 uIU/ml. Taking 100ul of insulin detention solution, directly dripping insulin detention solutions with different concentrations to a sample adding window of a chromatographic detection structure, standing for 20 minutes, checking the color depth of a detection line through an observation window, photographing, designing and printing a semi-quantitative color comparison card according to the photographing result, and taking the semi-quantitative color comparison card as a comparison standard for judging the follow-up saliva sample semi-quantitative detection result.
The prepared saliva sampling structure is used for respectively collecting 1-hour saliva samples of fasting and postprandial of 10 healthy donors, the saliva samples are respectively and directly inserted into a sample collecting channel of a chromatographic detection structure, the saliva sampling structure is stood for 20 minutes, the color depth of a detection line is checked through an observation window, the saliva sampling structure is colorimetric with a semi-quantitative colorimetric card, the concentration range of a colorimetric result is judged, the fasting result is about 1-10uU/ml, the postprandial time is about 5-30uU/ml, and the saliva sampling structure accords with the detection range of normal people.
Claims (11)
1. A membrane chromatography assay for saliva insulin levels, characterized by:
1) The membrane chromatography detection structure is a detachable combined set of a saliva sampling structure and a chromatography detection structure;
2) The chromatographic detection structure comprises a detection reagent strip, a detection shell positioned at the outer side of the detection reagent strip and a sample adding connection structure positioned at the proximal end of the detection shell and corresponding to the proximal end of the detection reagent strip;
3) The detection reagent strip comprises a sample pad, a marker binding pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is positioned at a corresponding position of the sample adding connecting structure and forms liquid phase traffic connection with the sample adding connecting structure, the marker binding pad is coated with a first anti-insulin antibody marked by an indicator, and the nitrocellulose membrane is coated with a second non-marked anti-insulin antibody;
4) The saliva sampling structure comprises a sampling head with liquid adsorption property, a handle-like structure directly connected with the sampling head and a fixed structure for fixing the connection of the sampling head and a non-sampling end of the handle-like structure, and the sampling head and the fixed structure form a sampling connection structure which can be detachably connected with the sampling connection structure together;
5) The sample adding connection structure is of a mutually-communicated double-layer structure and comprises a reagent detection layer for placing the detection reagent strip and a sample collection layer for detachably placing the sampling head, wherein the reagent detection layer is internally provided with a proximal end part of the detection reagent strip, and the sample collection layer is provided with an access port and a sample collection channel of the sampling head and a liquid phase traffic channel which forms a liquid phase traffic flow opening with the reagent detection layer.
2. The membrane chromatography detection apparatus according to claim 1, wherein said sampling head is composed of a fixed arm and a water-absorbing pad provided outside said fixed arm, said water-absorbing pad being of a water-absorbing material structure.
3. The membrane chromatography detection structure according to claim 1, wherein the reagent detection layer and the sample collection layer of the sample addition connection structure are of an upper-lower double-layer structure, the sample collection layer is arranged on the upper side, the reagent detection layer is arranged on the lower side, the liquid phase traffic channel is arranged between the sample collection layer and the reagent detection layer, the sampling head is inserted into the sample collection channel of the sample collection layer for use, the volume of the sample collection channel is smaller than that of the sampling head in use, the sample collection channel extrudes the sampling head in the insertion process, and liquid phase adsorbed on the sampling head flows into a detection reagent strip positioned on the reagent detection layer through the liquid phase traffic channel.
4. The membrane chromatography detection structure according to claim 1, wherein the reagent detection layer and the sample collection layer of the sample addition connection structure are of a left-right double-layer structure, the sample collection layer is arranged on one side, the reagent detection layer is arranged on the other side, the liquid phase traffic channel is arranged between the sample collection layer and the reagent detection layer, the sampling head is inserted into the sample collection channel of the sample collection layer for use, the volume of the sample collection channel is smaller than that of the sampling head in use, the sample collection channel extrudes the sampling head in the insertion process, and liquid phase adsorbed on the sampling head flows into a detection reagent strip positioned on the reagent detection layer through the liquid phase traffic channel.
5. The membrane chromatography detection apparatus according to claim 1, wherein said liquid phase communication channel is a liquid flow opening of a sample pad and/or a label binding pad opening toward a proximal end of said detection reagent strip, thereby forming an open structure in which the sample collection channel and the detection reagent strip in said reagent detection layer are in liquid flow communication with each other.
6. The membrane chromatography detection apparatus according to claim 1, wherein said removable combination set selects a slot press type, a plug type, or a push type, wherein:
1) The sampling head of the grooved sampling connecting structure is provided with a block-shaped water absorbing structure, the sampling connecting structure is of an upper-lower double-layer structure, a sample collecting channel of the sampling connecting structure is of a grooved structure, the block-shaped sampling head is placed into the grooved sample collecting channel during use, the sampling head is extruded, a liquid phase sample to be detected flows into a detection reagent strip through a liquid phase alternating current channel, and detection is started;
2) The sampling head of the inserted sampling connection structure is wrapped by a sheet-shaped water absorption structure, the sample adding connection structure is of an upper-lower double-layer structure, a sample collecting channel of the sampling connection structure is of a pipeline structure, when the sampling head wrapped by the water absorption structure is inserted into the pipeline sample collecting channel in use, the sampling head is extruded, a liquid phase sample to be detected flows into a detection reagent strip through a liquid phase communication channel, and detection is started;
3) The column pushing type sampling head of the sampling connection structure is wrapped by a sheet-shaped water absorption structure, the sample adding connection structure is of a left-right double-layer structure, a sample collecting channel of the column pushing type sampling head is of a pipeline structure, the sampling head wrapped by the water absorption structure is inserted into the pipeline sample collecting channel during use, the column pushing type sampling head is extruded, a liquid phase sample to be detected flows into a detection reagent strip through a liquid phase communication channel, and detection is started.
7. The membrane chromatography detection structure according to claim 1, wherein the detection reagent strip is at least one of a colloidal gold immunoassay reagent strip, a fluorescent immunoassay reagent strip, and a latex microsphere immunoassay reagent strip, and the indicator is at least one of a color microsphere and a fluorescent substance.
8. The membrane chromatography detection apparatus according to claim 1, wherein said membrane chromatography detection apparatus is provided with a semi-quantitative color chart having 3 or more color strips of different color depths printed thereon in accordance with the color of said indicator.
9. The membrane chromatography detection structure of claim 1, wherein said membrane chromatography detection structure is configured with a portable quantitative detector comprising at least one of a colorimetric quantitative analyzer and a fluorescent quantitative analyzer.
10. The membrane chromatography detection structure of claim 1, wherein the operational use of the membrane chromatography detection structure comprises the steps of:
1) Taking out the chromatographic detection structure and the saliva sampling structure, and exposing a sampling head of the saliva sampling structure;
2) The handle structure of the saliva sampling structure is held by hand, the sampling head of the saliva sampling structure is placed in the oral cavity, and saliva samples are naturally absorbed until the sampling head is completely infiltrated by saliva;
3) Taking out and placing a sampling head of the saliva sampling structure into a sample collecting channel of the chromatographic detection structure, extruding, and enabling saliva samples to flow into a sample pad of a detection reagent strip of the reagent detection layer through a liquid phase traffic channel;
4) The saliva sample passes through the sample pad and forwards flows through the marker binding pad, the nitrocellulose membrane and the water absorption pad;
5) And reading the detection result from the observation window to finish detection.
11. Use of the membrane chromatography detection structure of claim 1 in the development of a rapid detection product for salivary insulin levels.
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