CN109298188A - One heavy metal species and creatinine combined detection test paper and the preparation method and application thereof - Google Patents
One heavy metal species and creatinine combined detection test paper and the preparation method and application thereof Download PDFInfo
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- CN109298188A CN109298188A CN201811348070.0A CN201811348070A CN109298188A CN 109298188 A CN109298188 A CN 109298188A CN 201811348070 A CN201811348070 A CN 201811348070A CN 109298188 A CN109298188 A CN 109298188A
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- heavy metal
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- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 title claims abstract description 162
- 229940109239 creatinine Drugs 0.000 title claims abstract description 81
- 238000012360 testing method Methods 0.000 title claims abstract description 77
- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 67
- 238000001514 detection method Methods 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 41
- 239000000427 antigen Substances 0.000 claims abstract description 44
- 102000036639 antigens Human genes 0.000 claims abstract description 44
- 108091007433 antigens Proteins 0.000 claims abstract description 44
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000004005 microsphere Substances 0.000 claims abstract description 19
- 229910052737 gold Inorganic materials 0.000 claims abstract description 18
- 239000010931 gold Substances 0.000 claims abstract description 18
- 238000010521 absorption reaction Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000000084 colloidal system Substances 0.000 claims abstract description 11
- 239000002245 particle Substances 0.000 claims abstract description 10
- 239000011248 coating agent Substances 0.000 claims abstract description 7
- 238000000576 coating method Methods 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 31
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 26
- 210000002700 urine Anatomy 0.000 claims description 26
- 238000002156 mixing Methods 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 18
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- 238000010438 heat treatment Methods 0.000 claims description 7
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- 238000005119 centrifugation Methods 0.000 claims description 6
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 5
- 229940038773 trisodium citrate Drugs 0.000 claims description 5
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- 229930006000 Sucrose Natural products 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 150000002978 peroxides Chemical class 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
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- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- MPVDXIMFBOLMNW-ISLYRVAYSA-N 7-hydroxy-8-[(E)-phenyldiazenyl]naphthalene-1,3-disulfonic acid Chemical compound OC1=CC=C2C=C(S(O)(=O)=O)C=C(S(O)(=O)=O)C2=C1\N=N\C1=CC=CC=C1 MPVDXIMFBOLMNW-ISLYRVAYSA-N 0.000 claims description 2
- 150000008065 acid anhydrides Chemical class 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 claims 1
- 238000007689 inspection Methods 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 abstract description 6
- 238000004458 analytical method Methods 0.000 abstract description 4
- 229910052793 cadmium Inorganic materials 0.000 description 20
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 20
- 239000003153 chemical reaction reagent Substances 0.000 description 6
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- 230000000052 comparative effect Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 4
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 4
- 229910052753 mercury Inorganic materials 0.000 description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- 208000005374 Poisoning Diseases 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 208000005882 cadmium poisoning Diseases 0.000 description 3
- 239000011651 chromium Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
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- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 235000005979 Citrus limon Nutrition 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229910052785 arsenic Inorganic materials 0.000 description 2
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- -1 Hydroxyl radical free radical Chemical class 0.000 description 1
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- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
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- 230000001473 noxious effect Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
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- 229950007002 phosphocreatine Drugs 0.000 description 1
- 238000005424 photoluminescence Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229940075930 picrate Drugs 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-M picrate anion Chemical compound [O-]C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-M 0.000 description 1
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- 229910052709 silver Inorganic materials 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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- 238000012546 transfer Methods 0.000 description 1
- SOBHUZYZLFQYFK-UHFFFAOYSA-K trisodium;hydroxy-[[phosphonatomethyl(phosphonomethyl)amino]methyl]phosphinate Chemical compound [Na+].[Na+].[Na+].OP(O)(=O)CN(CP(O)([O-])=O)CP([O-])([O-])=O SOBHUZYZLFQYFK-UHFFFAOYSA-K 0.000 description 1
- 238000004056 waste incineration Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
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
- G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a heavy metal species and creatinine combined detection test paper and the preparation method and application thereof；It is intended to provide a kind of quick, accurate, easy, cheap test strip that can detect heavy metal ion and creatinine simultaneously；Technical points include successively being connected to have sample pad, bonding pad, chromatographic film, creatinine test paper and water absorption pad by bottom plate, and the chromatographic film is equipped with C line and T line, and the heavy metal ion antibody of same colloid gold particle label is sprayed on bonding pad；The heavy metal ion antigen of T location coating fluorescent microsphere label in chromatographic film, it is the BSA of fluorescent microsphere label that C line position is coated；Fluorescence immune chromatography detection and dry analysis field.
Description
Technical field
The present invention relates to fluorescence immune chromatography detection and dry analysis fields, are specifically related to a heavy metal species and creatinine
Combined detection test paper.
Background technique
Heavy metal original meaning refers to the metal that specific gravity is greater than 5, such as mercury, gold, silver, copper, iron, zinc, lead.Although some heavy metals
It is the microelement of individual need, but most of heavy metal is not, and all heavy metals are accumulated reach in vivo needed for body
To a certain degree, slow poisoning can all be caused.Some heavy metals enter can destroy human normal physiological function in vivo, referred to as toxic
Heavy metal, such as chromium (Cr), cadmium (Cd), mercury (Hg), lead (Pb), arsenic (As).
It is heavy metal-polluted to have a finger in every pie the environmental pollution as caused by heavy metal and its compound.Heavy metal pollution include: chemical pollution,
Motor vehicle exhaust emission, waste incineration etc..Wherein mercury, lead and arsenic etc. influence central nervous system, and copper, cadmium, lead and mercury will affect kidney
Dirty and liver, nickel, copper, cadmium and chromium will affect skin, bone and tooth.Strange illness (minamata disease, pain pain gently then occur for heavy metal poisoning
Disease etc.), severe one will be dead.China's heavy metal pollution is very serious, according to Chinese Ministry of Environmental Protection and Ministry of Land and Resources April 17 in 2014
" report of Chinese soil Survey of contaminating status " of day publication, about 13% heavy metals exceeding standard of national soil, heavy metal pollution range is wide,
It is more to expose crowd to the open air, seriously endanger people's health, but the method for existing clinical detection heavy metal need large-scale instrument, it is complicated for operation,
It is costly, the time is long, need high-throughput Fast Detection Technique (POCT).
China's heavy metal pollution of soil is again the most serious with cadmium pollution, and cadmium is exceeded up to 7%.Cadmium can be accumulated for a long time in vivo,
Can cause acute and chronic poisoning, there is very big damage to the immune system of human body, liver, kidney etc., in addition can cause miscarriage, it is abnormal
Shape, cancer etc..Cadmium is determining carcinogenic substance and is classified as the 6th noxious material being detrimental to health, China Hunan, Guangxi, Guangdong
Deng the crowd it has been found that large quantities of cadmium pollutions are poisoned, but because lacking the reasons such as fast and convenient cheap detection technique, do not carry out so far
Mass survey.
Urinating cadmium is one of most important diagnosis index of chronic cadium poisoning.Since urine cadmium level is related with urine volume, clinic is examined
It is disconnected that correction parameter is used as using urine creatinine.Because creatinine is the end product of metabolism of phosphocreatine in human muscle, can be arranged with urine
Out.The daily output of creatinine is quite stable in normal human urine, and normal range (NR) is 10mg/dL to 300mg/dL, substantially not by food
The influence of protein content and urine volume, therefore it is often used as the correction parameter of other excreta concentration in urine.It is corrected with urine creatinine,
The influence of sampling mode and urine volume to urine cadmium excretion can be eliminated, the one-sidedness that individually observation urine cadmium level generates is avoided.
Most of cadmium normal value is urinated in 1 μ g/g creatinine (or 1 μ g/L) hereinafter, the upper limit is 5 μ g/g creatinines (or 5 μ g/L).It is marked according to country
Quasi- GBZ 17-2015 " diagnosis of occupational cadmium poisoning ", urinate cadmium continuous 2 times is higher than 5 μm of ol/mol creatinines (i.e. 5 μ g/g creatinines)
Diagnosing chronic cadmium poisoning necessity index.
There are many detection method of cadmium, such as: atomic absorption method, spectrophotometry, atomic fluorescence spectrometry, inductive coupling from
Daughter mass spectrography, high performance liquid chromatography etc., when they need expensive instruments, complicated operation and longer detection mostly
Between, and special technical staff is needed to complete.
The detection method of creatinine is also more, such as: alkaline picrate method, enzyme process, capillary electrophoresis, liquid chromatography,
They also need related equipment mostly, complicated for operation, detection speed is slow, testing cost is expensive, clinical use is inconvenient, and
It can not detect simultaneously and be corrected with cadmium.
Therefore, existing Cd test technology is difficult to meet the needs of quickly detecting in vitro, it is necessary to which inventing one kind can be same
When detect Cd2+With the quick diagnosis test paper of creatinine, quick, accurate, easy, cheap practical promotional technique is provided.
The existing cadmium detection method for meeting requirements above has lateral chromatography test strips, and creatinine detection method has dry chemistry test
Paper, the former is fluorescence immune chromatography technology, and the latter is dry analysis technology, but the colloidal gold in fluorescence immune chromatography technology
Color may will affect the result observation of drying chemical reagent paper, and the coloring matter of drying chemical reagent paper may will affect fluorescence immune chromatography
The reaction of technology, therefore the method for also needing research that can detect urine cadmium and creatinine simultaneously.
Summary of the invention
The first purpose of the invention is to provide a kind of highly sensitive, easy to operate, quick, low at and can detecting simultaneously
Heavy metal ion and creatinine, mutual glitch-free test strip.
One heavy metal species and creatinine combined detection test paper, successively by bottom plate, sample pad, bonding pad, NC film, water absorption pad structure
At the NC film is equipped with C line and T line, and the heavy metal ion that same colloid gold particle label is sprayed on bonding pad is anti-
Body；The heavy metal ion antigen of T location coating fluorescent microsphere label on NC film, coated C line position is fluorescent microsphere label
BSA.
Preferably, above-mentioned a heavy metal species and creatinine combined detection test paper, the heavy metal ion antibody are Cd2+
Antibody or Cr3+Antibody or Hg2+Antibody or Pb2+Antibody or As3+Antibody.
Preferably, above-mentioned a heavy metal species and creatinine combined detection test paper, the heavy metal ion antigen are Cd2+
Antigen or Cr3+Antigen or Hg2+Antigen or Pb2+Antigen or As3+Antigen or other heavy metal ion antigens.
Second technical solution provided by the invention is the system of above-mentioned a heavy metal species and creatinine combined detection test paper
Preparation Method successively includes the following steps:
By colloid gold particle label metallic antibody with metal spraying draw film instrument spray on bonding pad, after drying according to from
Bonding pad is to the direction of water absorption pad, successively the fluorescent marker comlete antigen by 100-140 times of volume dilution and by volume dilution
100-140 times of fluorescent marker BSA is drawn in film instrument coating to NC film with metal spraying, is dried respectively as C line and T line, and again；
Finally sequentially overlap joint pastes sample pad, bonding pad, chromatographic film, creatinine test paper and water absorption pad on bottom plate.
Further, the preparation method of an above-mentioned heavy metal species and creatinine combined detection test paper, the colloidal gold
The method of the metallic antibody of particle marker are as follows:
A, the preparation of colloidal gold:
50mL ultrapure water is housed in 100mL conical flask, places it in magnetic heating stirrer and is stirred and heated to boiling
It rises；The trisodium citrate of 1.8mL 1% is added after the accurate gold chloride that 1mL 1% is added, heating stirring stops adding after ten minutes
Heat continues to stir to prepared colloidal gold solution cooling, and 4 DEG C save backup；
B, the preparation of colloidal gold labeled monoclonal antibody:
The colloidal gold solution 5mL for taking step A to prepare is adjusted the pH value of colloidal gold solution with 0.25M solution of potassium carbonate
It is 8.5, the preventing from heavy metal-iEDTA antibody of 50 μ L 0.85mg/mL is added in side, stirs 30 minutes；The poly- second two of 500 μ L 1% is added
Alcohol 20000 is centrifuged after mixing reaction 30 minutes, abandons supernatant, and sediment is answered lysate with 500mL and is redissolved, 4 DEG C of preservations are standby
With.
Further, the preparation method of an above-mentioned heavy metal species and creatinine combined detection test paper, the redissolution solution
Liquid is to contain 5% trehalose, 2% sucrose, 1%BSA, 0.5%Tween-20 and 0.1%PEG4000 in 0.02M PB solution.
Further, the preparation method of an above-mentioned heavy metal species and creatinine combined detection test paper, the fluorescence mark
Remember comlete antigen the preparation method comprises the following steps:
200 μ L 130nm fluorescent microspheres are added in centrifuge tube, 800 μ L ultrapure waters is added, is protected from light mixing；Then plus
Enter the n-hydroxysuccinimide of 20 μ L 1mg/mL and the carbodiimide of 40 μ L 1mg/mL, room temperature priming reaction 30 divides after mixing
Clock；Fluorescent microsphere after taking activation adds the comlete antigen heavy metal-of the 2mg/mL of corresponding volume in another centrifuge tube
IEDTA-BSA, room temperature is protected from light 2 hours after mixing；10% bovine serum albumin(BSA), which is added, makes BSA finally be 1%, mixes rear chamber
Temperature reaction is centrifugated for 1 hour, and sediment is collected after centrifugation and is redissolved with multiple lysate to twice of original volume, obtains fluorescence
Comlete antigen is marked, 4 DEG C are kept in dark place.
Further, in a kind of above-mentioned urine heavy metal and acid anhydride combined detection test paper preparation method, the fluorescence
Label BSA's the preparation method comprises the following steps: 200 μ L 130nm fluorescent microspheres are added in 1.5mL centrifuge tube, it is ultrapure to add 800 μ L
Water is protected from light mixing；It is subsequently added into the EDC (carbon two of the NHS (n-hydroxysuccinimide) and 40 μ L 1mg/mL of 20 μ L 1mg/mL
Imines), room temperature priming reaction 30 minutes after mixing；10%BSA (bovine serum albumin(BSA)), which is added, makes BSA final mass volume ratio
1%, it is reacted at room temperature 1 hour after mixing；15000 × g is centrifuged 30 minutes, sediment is collected after centrifugation, and multiple with multiple lysate
Molten twice to original volume obtains fluorescent marker comlete antigen, and 4 DEG C are kept in dark place
Further, the preparation method of an above-mentioned heavy metal species and creatinine combined detection test paper, the redissolution solution
Liquid is that the PBS:10%BSA:Tween-20 of 0.015M pH7.4 is mixed according to volume ratio for 90:10:1.
Further, the preparation method of an above-mentioned heavy metal species and creatinine combined detection test paper, the creatinine examination
Paper the preparation method comprises the following steps:
It is taken out after filter paper is sufficiently submerged in A liquid, in 60 DEG C of dry 1h；It is again that the filter paper after being soaked with A liquid and drying is abundant again
It is taken out after immersing B liquid, in 60 DEG C of dry 20min；Wherein, the A liquid includes 0.4g copper sulphate, 1g trisodium citrate, 0.1g orange
Yellow G and 1L 2mol/LpH6 Tris buffer solution；Wherein, the B liquid includes 10g surfactant, 0.5g reproducibility color
Former, 0.5g peroxide and 1L chloroformic solution.
Final object of the present invention is to provide above-mentioned heavy metal and creatinine combined detection test paper as in urine
The application of heavy metal and/or creatinine level qualitative detection card.
Technical solution provided by the present application has following technological merit:
1, technical solution provided by the present application can detect the heavy metal and creatinine in urine specimen simultaneously, and with tradition
Method has good correlation, and does not as a result interfere with each other, and is the easy detection method to the detection of the heavy metals exceeding standards such as cadmium.
2, technical solution high sensitivity provided by the invention, easy to operate, quick, at low cost, it is easy to process.
3, technical solution provided by the present application has a wide range of application, and can detecte in human or animal heavy metal and creatinine in urine
Level finds heavy metals exceeding standard, intoxication conditions in time, is convenient for clinical diagnosis and prevention.
Figure of description
Fig. 1 is heavy metal and creatinine combined detection test paper structural schematic diagram；
Fig. 2 is the detection reagent card structure schematic diagram with shell；
Fig. 3 is Test paper provided by the invention and graphite oven atomic absorption testing result comparative test testing result；
Fig. 4 is Test paper provided by the invention and conventional methods testing result comparative test testing result；
Fig. 5 is Test paper Cd provided by the invention and creatine concentration ratio comparative test testing result.
Symbology element and its similar component are as follows in figure:
Bottom plate 1, sample pad 2, bonding pad 3, chromatographic film 4, creatinine test paper 5, water absorption pad 6, C line, T line get stuck 7, well
71, observation area 72
Specific embodiment
In order to be more clear the purpose of the present invention, technical solution and advantageous effects, with reference to embodiments and say
Bright book attached drawing, the present invention will be described in further detail.It should be noted that raw material of the present invention, removes special theory
It is bright outer, it is prepared by conventional means or is bought by commercial channel.
Embodiment 1
A heavy metal species and creatinine combined detection test paper provided by the invention, refering to fig. 1, including by PVC bottom plate 1, institute
The bottom plate 1 stated, which is successively connected, sample pad 2, bonding pad 3, chromatographic film 4, creatinine test paper 5 and water absorption pad 6, in the chromatographic film
Equipped with C line and T line, the Cd of same colloid gold particle label is sprayed on bonding pad 32+Antibody (AuNPs-mAb, hereinafter referred to as
Gold labeling antibody)；The Cd of T location coating fluorescent microsphere label in chromatographic film2+Antigen (FNPs-Cd Ag, hereinafter referred to as antigen), C
It is the BSA of fluorescent microsphere label that line position is coated.
The preparation method of above-mentioned heavy metal species and creatinine combined detection test paper is specific as follows:
1, the preparation method of sample pad
The size sample pad sanction that specification is 20cm × 30cm is immersed in sample pad buffer, in drying at room temperature 16-
18h is cut into the sample pad of 2cm × 30cm specification then at 37 DEG C of oven drying 1h after dry.
The buffer formulation of the sample pad is as follows: 0.5%BSA, 2.5% sucrose sugar, 1.5%Tween 20 are dissolved in
In 0.015M pH9 PB buffer.
2, the preparation method of coupled antigen, coupled antibody and bonding pad
1) preparation of fluorescent marker comlete antigen: 200 μ L 130nm fluorescent microspheres are added in 1.5mL centrifuge tube, then
800 μ L ultrapure waters are added, are protected from light mixing；It is subsequently added into the NHS (n-hydroxysuccinimide) and 40 μ L of 20 μ L 1mg/mL
The EDC (carbodiimide) of 1mg/mL, room temperature priming reaction 30 minutes after mixing；Fluorescent microsphere after taking activation is in another 1.5mL
Centrifuge tube adds the comlete antigen Cd-iEDTA-BSA of the 2mg/mL of corresponding volume, and room temperature is protected from light 2 hours after mixing；
10%BSA (bovine serum albumin(BSA)), which is added, makes BSA finally be 1%, reacts at room temperature 1 hour after mixing；15000 × g is centrifuged 30 points
Sediment is collected after centrifugation and with the multiple lysate (volume of the PBS:10wt%BSA:Tween-20 of 0.015M pH7.4 in clock
Than redissolving for 90:10:1) to twice of original volume, fluorescent marker comlete antigen is obtained, 4 DEG C are kept in dark place.
2) preparation of fluorescent marker BSA: 200 μ L 130nm fluorescent microspheres are added in 1.5mL centrifuge tube, are added
800 μ L ultrapure waters, are protected from light mixing；It is subsequently added into the NHS (n-hydroxysuccinimide) and 40 μ L 1mg/mL of 20 μ L 1mg/mL
EDC (carbodiimide), room temperature priming reaction 30 minutes after mixing；10%BSA (bovine serum albumin(BSA)), which is added, makes the final matter of BSA
Measuring volume ratio is 1%, is reacted at room temperature 1 hour after mixing；15000 × g is centrifuged 30 minutes, sediment is collected after centrifugation, and use
Multiple lysate (volume ratio of the PBS:10%BSA:Tween-20 of 0.015M pH7.4 is 90:10:1) is redissolved to the two of original volume
Times, fluorescent marker comlete antigen is obtained, 4 DEG C are kept in dark place.
3) preparation of the anti-Cd-iEDTA antibody of colloid gold label:
A, the preparation of colloidal gold:
50mL ultrapure water is housed in 100mL conical flask, places it in magnetic heating stirrer and is stirred and heated to boiling
It rises；The trisodium citrate of 1.8mL 1% is added after the accurate gold chloride that 1mL 1% is added, heating stirring stops adding after ten minutes
Heat continues to stir to prepared colloidal gold solution cooling, and 4 DEG C save backup.
B, the preparation of colloidal gold labeled monoclonal antibody:
The colloidal gold solution 5mL for taking step A to prepare is adjusted the pH value of colloidal gold solution with 0.25M solution of potassium carbonate
It is 8.5.The anti-Cd-iEDTA antibody of 50 μ L 0.85mg/mL is added in side, stirs 30 minutes；500 μ L 1%PEG (poly- second two are added
Alcohol) 20000, mix reaction 30 minutes；4500 × g is centrifuged 15 minutes, abandons supernatant, sediment 500mL is answered lysate
(5% trehalose, 2% sucrose, 1%BSA, 0.5%Tween-20 and 0.1%PEG4000) redissolves, and 4 DEG C save backup.
3, the preparation method of creatinine test paper is as follows
It is taken out after filter paper is sufficiently submerged in A liquid, in 60 DEG C of dry 1h；Wherein, the A liquid includes 0.4g copper sulphate, 1g lemon
Lemon acid trisodium, 0.1g orange G and 1L 2mol/LpH6 Tris buffer solution；Again again by the filter paper after being soaked with A liquid and drying
It is taken out after being sufficiently submerged in B liquid, in 60 DEG C of dry 20min；Wherein, the B liquid includes 10g surfactant, 0.5g reproducibility color
Former, 0.5g peroxide and 1L chloroformic solution.
4、Cd2+It is as follows with the assemble method of creatinine combined detection test paper
The antibody of colloid gold label is drawn film instrument with metal spraying to spray on the bonding pad 3 of polyester film with the amount of 3 μ L/cm,
37 DEG C baking oven 30 minutes, after placed 2 days in 27 DEG C of insulating boxs；According to from bonding pad 2 to the direction of water absorption pad 6, successively pressing body
Product dilutes 120 times of fluorescent marker comlete antigen and draws film instrument by 120 times of volume dilution of fluorescent marker BSA metal spraying with 0.8 μ
In the amount coating to chromatographic film 4 (nitrocellulose filter) of L/cm, respectively as detection line (T line) and lubber-line (C line), and
37 DEG C baking oven 10 hours, after placed 2 days in 27 DEG C of insulating boxs；Finally sequentially overlap joint pastes sample pad 2, bonding pad on bottom plate 1
3, chromatographic film 4, creatinine test paper 5 and water absorption pad 6, by the Cd of preparation2+Detection zone and creatinine detection zone are cut into 3.5mm's as required
Test strips, the Cd of composition2+With creatinine combined detection test paper.
A heavy metal species and creatinine combined detection test paper prepared by embodiment 1, which are refilled into plastics, gets stuck 7；The modeling
Material, which gets stuck, 7 is provided with well 71 and observation area 72, and the position of well 72 is located at 2 position of sample pad；Observation area is located at detection line
The position in (T line), lubber-line (C line) region and creatinine test paper (5) block, forms complete Cd2+With creatinine combined detection test paper,
Refering to Fig. 2.
In order to preferably use heavy metal provided by the present application and creatinine combined detection test paper, technology provided by the present application
Creatinine, Cd are also established in scheme respectively2+Detection curve:
1. the preparation method of creatinine standard curve carries out as follows
(1) the creatinine reference material of 0mg/dL, 10mg/dL, 50mg/dL, 150mg/dL, 300mg/dL are prepared；
(2) the above-mentioned five kinds of reference materials of 100 μ L is taken to be separately added into the big well for the test strips that embodiment 1 prepares,
It develops the color completely after 1min, is detected with instrument；
It (3) is linearly y=1.047x+2.4204 within the scope of 0~300mg/L, R2=0.9938.Cd2+Standard curve
Preparation method carries out as follows.
2.Cd2+The preparation method of standard curve carries out as follows:
(1) it takes healthy human urine to be added to 1mM chelating agent EDTA-2Na, reacts 5min.
(2) Cd is added in the urine sample handled well2+Standard solution is formulated as follows the Cd of concentration respectively2+Reference material:
100ng/mL、50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.13ng/mL、1.56ng/mL、0.73ng/mL。
(3) standard solution of 8 kinds of concentration of the 100 aforementioned preparations of μ L is taken to be separately added into the small of the test strips of the preparation of embodiment 1
In well, by calculating Cd after 15min2+In the detection line of detection zone on fluorescence intensity and lubber-line fluorescence intensity ratio,
Draw standard curve.In the well that the urine sample Jing Guo preceding processing is added to test card in sample detection, detection line is calculated
With the ratio of fluorescence intensity on lubber-line, then Cd in urine sample can be calculated by standard curve2+Concentration.Spirit of the invention
Sensitivity is 0.21ng/mL, is linearly y=0.817x+0.3094, R in the detection range of 0.73ng/mL to 100ng/mL2=
0.9926。
Cd provided by the invention is given below in the technical solution provided in order to better understand the present invention2+It is detected with creatinine
The testing principle of card
Cd2+Testing principle
Cd provided by the invention2+When not being loaded with creatinine detection card detection reagent, coated fluorescence on T, C line on NC film
Microballoon conjugate, in the case where exciting under light action, T, C line can show photoluminescence line.When test sample will be added, sample is in capillary
Under effect, successively pass through sample pad, bonding pad, NC film, to the movement of the direction of water absorption pad.
If being free of Cd in sample2+, the gold labeling antibody on bonding pad flows through NC film under the drive of sample, on NC film T line
Antigen binding, colloid gold particle can make fluorescent microsphere (with antigen be coupled) fluorescence occur energy transfer, fluorescence excitation
Light time, T line do not fluoresce, and thus display is wireless, which is also referred to as fluorescent quenching.
If containing Cd in sample2+, Cd in sample2+It can make gold labeling antibody can not be with NC with the gold labeling antibody on bonding pad
Antigen binding on film T line, under fluorescent exciting, T line shows fluorescence.Cd in sample i.e. to be detected2+On NC film T line
Antigenic competition bonding pad on gold labeling antibody.If Cd2+Concentration is higher, can be with the gold labeling antibody of the antigen binding on NC film T line
Fewer, the degree of T line fluorescent quenching is lower, and fluorescence intensity is stronger.Cd in sample at this time2+Content by T, C fluorescence intensity according to
T/C is calculated.
If there is not fluorescence at C when test, show that this measured value is invalid, should carry out repeating experiment.
Creatinine testing principle:
Cu under acid condition, in test paper2+Citrate is reacted with creatinine in sample, generates Cu2+Creatinine complex compound,
Redox reaction occurs for this complex compound and TMB, generates Cu+Creatinine complex compound and TMB+ recycle peroxide to generate a large amount of
Hydroxyl radical free radical, so that Cu+ is oxidized to Cu2+, Cu2+Reacting above is repeated with TMB again, entire reaction process is recycled,
TMB+ journey blue in above-mentioned reaction, creatine concentration is higher, and blue product is more, and blue is deeper.At this time in sample creatinine content by
Chart scanner reads absorption luminous intensity and is calculated.
H++Cu2+Citrate+creatinine → Cu2+Creatinine+citric acid
Cu2+Creatinine+TMB → Cu+Creatinine+TMB+
Cu+Creatinine+ROOH → RO+OH-+Cu2+Creatinine
In order to better illustrate technical solution provided by the present application, Cd provided by the present application is given below2+Combine with creatinine
The specifically used method of test strip
Immunity analysis instrument is opened, chip identical with test strips lot number is inserted into；Reagent strip outer packing is removed when use, is taken out
Reagent strip is horizontally arranged；It is accurate to draw the 100 full urine samples of μ l, 1 percent volume 1mM chelating agent EDTA-2Na are added, instead
Answer 5min.Urine sample is added in the well of detection card, then starts timing；After reacting at room temperature 15min, it will test card and put
Enter into the card slot of instrument；" test " key on instrument is clicked, instrument will start to test, and read the OD value and Cd of creatinine2+
The size of fluorescence signal, and show urine cadmium/creatinine ratio result.
The advantages of in order to prove technical solution provided by the invention, the comparative experiments example of the present invention and other methods:
Doubtful 100, cadmium poisoning Urine in Patients sample are collected, respectively with the present invention and traditional sampling Graphite Furnace Atomic Absorption light
Spectrometry surveys (ASS) Cd2+The comparison of creatinine method is surveyed with biochemical reaction instrument, as a result as shown in FIG. 1 to 3.Wherein, the present invention detects
Cd2+It is linearly related to graphite oven atomic absorption testing result, linear equation y=1.018x+0.9288, R2=
0.9495；To traditional creatinine detection method same linear related, linear equation y=0.9796x-3.068, R2=
0.9519；Cd2+/ creatinine (μm ol/mol) linear equation is y=1.1757x-0.1542, R2=0.9425.
The above result shows that the present invention can detect the Cd in urine specimen simultaneously2+With creatinine, and with conventional method have
There is good correlation, as a result do not interfere with each other, is the easy detection method to Cd poisoning detection.
Cr3+Antibody or Hg2+Antibody or Pb2+Antibody or As3+Antibody
Even if it should be strongly noted that the Cd that scheme provided by the present application not only can detecte2+With creatinine, phase is replaced
Corresponding antibody, such as Hg2+Antibody or Pb2+Antibody or As3+Antibody, and the corresponding corresponding antigen of replacement is such as: Cr3+Antigen
Or Hg2+Antigen or Pb2+Antigen or As3+Antigen equally can detecte corresponding heavy metal.
According to the disclosure and teachings of the above specification, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out change and modification appropriate.Therefore, the invention is not limited to the specific embodiments disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In use some specific terms, these terms are merely for convenience of description, does not limit the present invention in any way.
Claims (10)
1. a heavy metal species and creatinine combined detection test paper, including by bottom plate (1), which is characterized in that the bottom plate (1) according to
Secondary linking has sample pad (2), bonding pad (3), chromatographic film (4), creatinine test paper (5) and water absorption pad (6), sets in the chromatographic film
There are C line and T line, the heavy metal ion antibody of same colloid gold particle label is sprayed on bonding pad (3)；In chromatographic film (4)
The heavy metal ion antigen of upper T location coating fluorescent microsphere label, it is the BSA of fluorescent microsphere label that C line position is coated.
2. according to right want 1 described in a heavy metal species and creatinine combined detection test paper, which is characterized in that the heavy metal
Ion antibody is Cd2+Antibody or Cr3+Antibody or Hg2+Antibody or Pb2+Antibody or As3+Antibody or other heavy metals from
Sub- antibody；The heavy metal ion antigen is Cd2+Antigen or Cr3+Antigen or Hg2+Antigen or Pb2+Antigen or As3 +Antigen or other heavy metal ion antigens.
3. the method for preparing a heavy metal species and creatinine combined detection test paper described in claim 1, which is characterized in that successively
Include the following steps:
The metallic antibody of colloid gold particle label is drawn film instrument with metal spraying to spray on bonding pad, according to from combination after drying
The direction of water absorption pad is padded, successively the fluorescent marker comlete antigen by 100-140 times of volume dilution and by volume dilution 100-
140 times of fluorescent marker BSA is drawn in film instrument coating to chromatographic film with metal spraying, respectively as C line and T line, is dried again, finally,
Successively being connected in bottom plate (1) has sample pad (2), bonding pad (3), chromatographic film (4), creatinine test paper (5) and water absorption pad (6), and
The heavy metal ion antibody of same colloid gold particle label is sprayed on bonding pad (3)；T location is coated with fluorescence in chromatographic film
The heavy metal ion antigen of microballoon label, it is the BSA of fluorescent microsphere label that C line position is coated.
4. the preparation method of a heavy metal species and creatinine combined detection test paper according to claim 3, which is characterized in that
The method of the metallic antibody of the colloid gold particle label are as follows:
A, the preparation of colloidal gold:
50mL ultrapure water is housed in 100mL conical flask, places it in magnetic heating stirrer and is stirred and heated to boiling；It is quasi-
The trisodium citrate of 1.8mL 1% is really added after the gold chloride of addition 1mL 1%, heating stirring stops heating after ten minutes and continues
It stirs to prepared colloidal gold solution cooling, 4 DEG C save backup；
B, the preparation of colloidal gold labeled monoclonal antibody:
The pH value of colloidal gold solution is adjusted to by the colloidal gold solution 5mL for taking step A to prepare with 0.25M solution of potassium carbonate
8.5, the preventing from heavy metal-iEDTA antibody of 50 μ L 0.85mg/mL is added in side, stirs 30 minutes；500 μ L, 1% polyethylene glycol is added
20000, it is centrifuged after mixing reaction 30 minutes, abandons supernatant, sediment is answered into lysate with 500mL and is redissolved, 4 DEG C save backup.
5. the preparation method of a heavy metal species and creatinine combined detection test paper according to claim 4, which is characterized in that
The multiple lysate be 0.02M PB solution in containing 5% trehalose, 2% sucrose, 1%BSA, 0.5%Tween-20 and
0.1%PEG4000.
6. the preparation method of a heavy metal species and creatinine combined detection test paper according to claim 3, which is characterized in that
The fluorescent marker comlete antigen the preparation method comprises the following steps:
200 μ L 130nm fluorescent microspheres are added in centrifuge tube, 800 μ L ultrapure waters is added, is protected from light mixing；It is subsequently added into 20
The carbodiimide of the n-hydroxysuccinimide of μ L 1mg/mL and 40 μ L 1mg/mL, room temperature priming reaction 30 minutes after mixing；
Fluorescent microsphere after taking activation adds the comlete antigen heavy metal-iEDTA- of the 2mg/mL of corresponding volume in another centrifuge tube
BSA, room temperature is protected from light 2 hours after mixing；10% bovine serum albumin(BSA), which is added, makes BSA finally be 1%, reacts at room temperature after mixing
It is centrifugated within 1 hour, sediment is collected after centrifugation and is redissolved with multiple lysate to twice of original volume, it is complete to obtain fluorescent marker
Holoantigen, 4 DEG C are kept in dark place.
7. the preparation method of a heavy metal species and acid anhydride combined detection test paper according to claim 3, which is characterized in that institute
The fluorescent marker stated BSA's the preparation method comprises the following steps: 200 μ L 130nm fluorescent microspheres are added in 1.5mL centrifuge tube, adds
800 μ L ultrapure waters, are protected from light mixing；It is subsequently added into the n-hydroxysuccinimide of 20 μ L 1mg/mL and the EDC of 40 μ L 1mg/mL
(carbodiimide), room temperature priming reaction 30 minutes after mixing；10%BSA (bovine serum albumin(BSA)), which is added, makes BSA final mass body
Product reacts at room temperature 1 hour after mixing than being 1%；15000 × g is centrifuged 30 minutes, is collected after centrifugation sediment, and with redissolution
Solution liquid is redissolved to twice of original volume, obtains fluorescent marker comlete antigen, 4 DEG C are kept in dark place.
8. the preparation method of a heavy metal species and creatinine combined detection test paper, feature according to claim 6 or 7
It is, the multiple lysate is that the PBS:10%BSA:Tween-20 of 0.015M pH7.4 is mixed for 90:10:1 according to volume ratio
It closes.
9. the preparation method of a heavy metal species and creatinine combined detection test paper according to claim 4, which is characterized in that
The creatinine test paper the preparation method comprises the following steps:
It is taken out after filter paper is sufficiently submerged in A liquid, in 60 DEG C of dry 1h；The filter paper after being soaked with A liquid and drying is sufficiently submerged in B again again
It is taken out after liquid, in 60 DEG C of dry 20min；Wherein, the A liquid include 0.4g copper sulphate, 1g trisodium citrate, 0.1g orange G with
And 1L 2mol/LpH6Tris buffer solution；Wherein, the B liquid include 10g surfactant, 0.5g reproducibility chromogen,
0.5g peroxide and 1L chloroformic solution.
10. heavy metal described in claim 1 and creatinine combined detection test paper heavy metal and/or creatinine level inspection in urine
The application of survey.
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| CN115718160A (en) * | 2022-11-24 | 2023-02-28 | 健尔圣(珠海)医药科技有限公司 | Method for detecting GMDTC (GMDTC) and metabolite thereof in plasma by LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry) |
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