CN103278652A - Total bilirubin detection reagent - Google Patents
Total bilirubin detection reagent Download PDFInfo
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- CN103278652A CN103278652A CN2013102005166A CN201310200516A CN103278652A CN 103278652 A CN103278652 A CN 103278652A CN 2013102005166 A CN2013102005166 A CN 2013102005166A CN 201310200516 A CN201310200516 A CN 201310200516A CN 103278652 A CN103278652 A CN 103278652A
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- damping fluid
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Abstract
The invention discloses a total bilirubin detection reagent which comprises a diluent and a reaction reagent, wherein the diluent is composed of buffer 1, surfactant, preservative and vitamin C oxidase; and the reaction reagent is buffer 2, sodium chloride, disodium edetate, Tween 80, sodium lauryl sulfate, sodium persulfate, sulfuric acid, preservative and freeze-drying protective agent. The detection reagent disclosed by the invention has favorable sensitivity, accuracy, precision and linearity, and can completely satisfy the clinical examination requirements.
Description
Technical field
The present invention relates to the medical test technical field, be specifically related to a kind of total bilirubin determination reagent for the POCT analyser.
Background technology
Total bilirubin (Totalbilirubin, TBil) be combination or the bilirubinic general name of non-binding type that is produced by the protoheme metabolism in the human body, be mainly used in diagnosing hepatic diseases and obstruction of biliary tract clinically, be one of the most conventional project of medical test, in class medicals diagnosis on disease such as liver and gall, occupy important status.TBil generally adopts various large automatic Biochemical Analyzers to detect at present, but because large automatic Biochemical Analyzer equipment price height, and complicated operation, operating personnel need have relevant professional knowledge and accept corresponding training, and it is high to use complementary conditions to require, and need to be equipped with stabilized voltage supply, water purification machine etc., and floor area is big, the maintenance cost height needs professional's time-based maintenance, so basic medical unit or household person all do not have condition to buy and use.In addition, the large hospital patient is many, detects that formality is loaded down with trivial details, long flow path, stand-by period be long, and this has also brought the huge time to bear to the patient, the more important thing is that patient is affected adversely treatment because can not in time diagnosing, even can lose one's life.
(point-of-care testing is an emerging technical field POCT) to real-time test, and principal feature is to obtain a result fast, and is easy and simple to handle, uses easily and miniaturization.Along with diagnosis and the progress of ancillary technique, and people are to the understanding of disease and the requirement for the treatment of level raising, and POCT receives publicity gradually.In fact POCT becomes the important research project of external diagnosis reagent and instrument field gradually in American-European market, existing many products put goods on the market, but this class POCT instrument has a major defect at present, be exactly that analyser and reagent consumptive material are expensive especially, has huge primary care system like this for China, and the reagent use amount is country greatly, and instrument and the matched reagent consumptive material of American-European exploitation obviously all face the challenge at present.
The micro-fluidic chip technology is one of of paramount importance cutting edge technology in the 21 century world, it is integrated into basic operation units such as specimen preparation related in the fields such as biological and chemical, reaction, separation, detection and cell cultivation, sorting, cracking on the chip of one tens square centimeters (even littler), form network by the microchannel, running through total system with controlled fluid, is a kind of technology in order to the various functions that replace conventional biological or chemical laboratory.The micro-fluidic chip technology is incorporated into POCT equipment, started the new situation of POCT development, can make the complicated whole blood in laboratory in the past quantitatively, blood cell serum separates, serum dilution and step such as mensuration synchronously, finishes in the on-line automatic equalization of chip, reaches the synchronous testing goal of multiple mark.POCT analyser in conjunction with the micro-fluidic chip technology has pin-point accuracy, low blood volume, simple to operate, few, the low cost and other advantages of detection reagent dosage of needing concurrently, as protect and give birth to the international POCT instrument of curing company limited of giving birth to, the Piccolo of Abaxis company, the Afinion of Axis-Shield company etc., this type of POCT analyser can realize that all but small amount of sample can be analyzed, easy and simple to handle, no cross pollution automated job, is highly suitable for China and has huge primary care system and reagent use amount country greatly like this.
Along with Eleventh Five-Year Plan plan purchasing and strengthen at three grades and second-grade hospital infrastructure device, middle rank possesses good software and hardware facilities to go to the hospital at present, but basic medical unit particularly its full-automatic biochemical testing instruments facility of unit such as commune hospital, clinic is generally not enough, the check professional who does not also have enough numbers, therefore setting up operation POCT analytic system simple and easy, cheap, real-time report has important meaning to bringing into play the effect of vast basic hospital in prevention from suffering from the diseases and diagnosis and treatment.And provide a kind of total bilirubin determination reagent that can be used for the POCT analyser of above-mentioned introducing micro-fluidic chip technology to be called problem demanding prompt solution.
Summary of the invention
The present invention is directed to the above-mentioned deficiency of prior art, provide a kind of total bilirubin that can be used for introducing the POCT analyser of micro-fluidic chip technology to detect reagent.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of total bilirubin determination reagent, and this reagent comprises dilution and reaction reagent, wherein dilution is to be grouped into by following one-tenth:
Damping fluid 1(pH6.5-7.5) 0.01-1.0mol/L;
Surfactant 0.1-10.0% (mass percent);
Antiseptic 0.1-10.0% (mass percent);
Ascorbic acid oxidase 1-100KU/L;
Wherein said reaction reagent is to be grouped into by following one-tenth:
Damping fluid 2(pH3.0-5.0) 0.1-1.0mol/L;
Sodium chloride 45g/L;
Disodium ethylene diamine tetraacetate 2-10g/L;
Tween 80 5-20ml/L;
Lauryl sodium sulfate 10-100g/L;
Sodium peroxydisulfate 100-200g/L;
Sulfuric acid 0.1-2ml/L
Antiseptic 0.1-1g/L;
Freeze drying protectant 10-30g/L.
Wherein said damping fluid 1 can be the amino damping fluid of trihydroxy methyl, glycocoll-NaOH damping fluid, N-2-hydroxyethyl piperazine-N'-2-ethyl sulfonic acid damping fluid, N-three (methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, N-three (methylol) methyl-2-tarine damping fluid, piperazine-N, two (2-hydroxyethanesulfonic acid) damping fluids of N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morpholine) ethyl sulfonic acid sodium damping fluid, the 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonic acid damping fluid, two (2-hydroxyethyl) amino of 3--2-hydroxy-propanesulfonic acid damping fluid, 3-(cyclohexylamine)-2-hydroxyl-1-propane sulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid damping fluid, 3-(cyclohexylamine)-1-propane sulfonic acid damping fluid, 3-morpholine propane sulfonic acid damping fluid, a kind of in N-three (methylol) the methyl-3-aminopropanesulfonicacid acid damping fluid.
Wherein said damping fluid 2 can be a kind of in 2-(N-morpholine) ethyl sulfonic acid damping fluid, acetate buffer, sodium citrate buffer solution, the phosphate buffer.
Described surfactant is non-ionic surfactant, as be selected from TWEEN series (as tween (TWEEN) 20, polysorbate40, polysorbate60, Tween 80), SPAN series is (as (SPAN) 20 of class of department, class of department 40, class of department 60, class of department 80), TRITON series is (as TritonX-100, TritonX-114, TritonX-405) one or more of concrete material (namely can be TWEEN series in, a kind of concrete material in SPAN series or the TRITON series, or more than one concrete material in every kind of series, or a kind of concrete material in two kinds or the above series).
Described antiseptic (comprising antiseptic in the dilution and the antiseptic in the reaction reagent) is selected from a kind of in a kind of or nipagin esters (as methyl p-hydroxybenzoate, ethyl-para-hydroxybenzoate, propylparaben, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isopropyl ester, p-Hydroxybenzoic acid isobutyl ester) in potassium sorbate, Sodium Benzoate, sodium nitrite, the proclin series antiseptic (as Proclin300).
Described freeze drying protectant selects one or several in trehalose, sucrose, bovine serum albumin(BSA), Tween 80, triton x-100, the AEO (Brij-35).
Dilution is liquid condition in the described TBIL mensuration reagent, and reaction reagent is dry powder.
The preparation method of the dilution of described TBIL mensuration reagent is as follows: stir evenly mixing behind the described reagent composition adding distilled water.
The preparation method of reaction reagent that described TBIL measures reagent is as follows: described reagent composition is added to mix behind the distilled water stir evenly, 0.5-10 μ l reaction reagent is joined the reaction detection groove, volatilize 24-72h through freeze-drying (industry routine techniques) or 2-8 ℃.
The test condition that described TBIL measures reagent is as follows: temperature: 37 ℃; Detect predominant wavelength 450nm, commplementary wave length 750nm.
Micro-fluidic chip technology of the present invention is that basic operation unit is integrated on the chip of one tens square centimeters (even littler), forms network by the microchannel, runs through a kind of technology of total system with controlled fluid.It is two-layer up and down that its characteristics are that chip generally is divided into, the upper strata is useful on the through hole of application of sample, and lower floor comprises the difform fluid channel that responds sample cell, dilution liquid bath, sample quantitative slot, dilution quantitative slot, reservoir, a plurality of prepackage the reaction detection groove of reagent, one group of self check groove that is used for system compensation, one group of overflow groove, many group control fluids flow etc.Its detection method generally comprises following steps: (1) is injected into sample solution and dilution in described sample cell and the dilution liquid bath through through hole separately; (2) starter motor rotates described chip; (3) sample solution is realized Separation of Solid and Liquid and quantitative under centrifugal action, and dilution enters the dilution quantitative slot simultaneously; (4) quantified sample is mixed with dilution inflow mixing channel; (5) mixed liquid enters the reaction detection groove and reaction reagent reacts; (6) by in the reaction detection groove, carrying out in situ detection with the supporting checkout equipment of chip.
The assay method of described TBIL mensuration reagent is as follows: 5-20 μ l sample joined sample cell, 20-100 μ l dilution joined the dilution liquid bath, and starter motor, record absorbance A 1 behind 37 ℃ of reaction 1min continues to record absorbance A 2 behind the reaction 5-9min.
The reaction principle that described TBIL measures reagent is: sample mixes with dilution earlier hatches, with interfering materials such as the vitamin C in the removal sample and blood fat, after mixed liquor enters the reaction detection groove, in the presence of sodium chloride, disodium ethylene diamine tetraacetate, Tween 80, lauryl sodium sulfate and sulfuric acid, oxidized dose of sodium peroxydisulfate of the bilirubin direct in the sample is oxidized to dehydrobilirubin.Bilirubinic yellow specificity absorbance descends, and by measuring the variation of sodium peroxydisulfate oxidation front and back absorbance, calculates the content of TBIL in the sample.
Advantage of the present invention and beneficial effect: reagent of the present invention has good sensitivity, accuracy, precision and linearity, can satisfy the clinical examination requirement fully.Reagent of the present invention can be used for introducing on the POCT analyser of micro-fluidic chip technology and detects total bilirubin, thereby realizes that operation is simple and easy, cheap, the foundation of the POCT analytic system of real-time report.
Description of drawings
The linear figure as a result of Fig. 1 TBIL.
Fig. 2 TBIL methodology comparison result figure.
Embodiment
To further specify the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many modifications to the present invention, such modification also falls into scope of the present invention.
Following experimental technique is conventional method if no special instructions, and employed experiment material all can easily be obtained from commercial company if no special instructions.
Embodiment 1
Dilution:
Amino damping fluid (pH7.0) 0.1mol/L of trihydroxy methyl
TWEEN80 5%
Proclin300 0.1%
Ascorbic acid oxidase 1KU/L
Reaction reagent:
Phosphate buffer (pH4.0) 0.1mol/L
Disodium ethylene diamine tetraacetate 2g/L
Tween 80 5ml/L
Lauryl sodium sulfate 10g/L
Sodium chloride 45g/L
Sodium peroxydisulfate 100g/L
Sulfuric acid 0.1ml/L
Potassium sorbate 0.1g/L
Bovine serum albumin(BSA) 5g/L.
Embodiment 2
Dilution:
Amino damping fluid (pH7.5) 0.5mol/L of trihydroxy methyl
TRITON X-100 5%
Methyl p-hydroxybenzoate 1%
Ascorbic acid oxidase 2KU/L
Reaction reagent:
2-(N-morpholine) ethyl sulfonic acid sodium damping fluid (pH3.0) 0.5mol/L
Damping fluid 2(pH3.0-5.0) 1.0mol/L
Disodium ethylene diamine tetraacetate 10g/L
Tween 80 20ml/L
Lauryl sodium sulfate 20g/L
Sodium chloride 45g/L
Sodium peroxydisulfate 150g/L
Sulfuric acid 1ml/L
Proclin300 1g/L
Trehalose 10g/L.
Embodiment 3
Dilution:
3-morpholine propane sulfonic acid damping fluid (pH6.5) 1.0mol/L
SPAN20 10%
Sodium Benzoate 1%
Ascorbic acid oxidase 10KU/L
Reaction reagent:
Sodium citrate buffer solution (pH5.0) 1mol/L
Disodium ethylene diamine tetraacetate 5g/L
Tween 80 10ml/L
Lauryl sodium sulfate 20g/L
Sodium chloride 45g/L
Sodium peroxydisulfate 50g/L
Sulfuric acid 1ml/L
Proclin300 1g/L
AEO 10g/L.
Describe below in conjunction with the performance of form to the embodiment of the invention 1 gained reagent.
1, precision
Table 1, precision assessment result
2, linearity
The analyte that adds variable concentrations with the standard serum sample outward detects, and the TBIL linearity the results are shown in Fig. 1.
3, methodology comparison test
Compare with the TBIL end value of measuring on the automatic clinical chemistry analyzer, result such as Fig. 2, wherein X-axis represents determination data in the automatic clinical chemistry analyzer Hitachi 7180, POCT analyser (the living world of Taiwan guarantor of micro flow chip technology is introduced in the Y-axis representative, Amishield TMO-100) goes up determination data.
4, detection sensitivity
The appraisal procedure of the detection sensitivity standard deviation of 10-20 dummy signal strength, and the standard deviation of 3-15 least significant non-zero sample signal strength are calculated gained with the EPEvaluatorrelease6 of statistical software.Experimental result shows that reagent of the present invention has good sensitivity, can meet the improvement of U.S. clinical labororatory fully and amend legislation.
Table 2 reagent detection sensitivity
From above-mentioned testing result as can be known, reagent of the present invention has good sensitivity, accuracy, precision and linearity, can satisfy the clinical examination requirement fully.
Claims (9)
1. a total bilirubin detects reagent, and it is characterized in that: this reagent comprises dilution and reaction reagent, and wherein dilution is to be grouped into by following one-tenth:
Damping fluid 1(pH6.5-7.5) 0.01-1.0mol/L;
Surfactant 0.1-10.0% (mass percent);
Antiseptic 0.1-10.0% (mass percent);
Ascorbic acid oxidase 1-100KU/L;
Wherein said reaction reagent is to be grouped into by following one-tenth:
Damping fluid 2(pH3.0-5.0): 0.1-1.0mol/L;
Sodium chloride 45g/L;
Disodium ethylene diamine tetraacetate 2-10g/L;
Tween 80 5-20ml/L;
Lauryl sodium sulfate 10-100g/L;
Sodium peroxydisulfate 100-200g/L;
Sulfuric acid 0.1-2ml/L;
Antiseptic 0.1-1g/L;
Freeze drying protectant 10-30g/L.
2. total bilirubin according to claim 1 detects reagent, it is characterized in that: described damping fluid 1 is the amino damping fluid of trihydroxy methyl, glycocoll-NaOH damping fluid, N-2-hydroxyethyl piperazine-N'-2-ethyl sulfonic acid damping fluid, N-three (methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, N-three (methylol) methyl-2-tarine damping fluid, piperazine-N, two (2-hydroxyethanesulfonic acid) damping fluids of N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morpholine) ethyl sulfonic acid sodium damping fluid, the 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonic acid damping fluid, two (2-hydroxyethyl) amino of 3--2-hydroxy-propanesulfonic acid damping fluid, 3-(cyclohexylamine)-2-hydroxyl-1-propane sulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid damping fluid, 3-(cyclohexylamine)-1-propane sulfonic acid damping fluid, 3-morpholine propane sulfonic acid damping fluid, a kind of in N-three (methylol) the methyl-3-aminopropanesulfonicacid acid damping fluid.
3. total bilirubin according to claim 1 detects reagent, it is characterized in that: a kind of in 2-(N-morpholine) ethyl sulfonic acid damping fluid, acetate buffer, sodium citrate buffer solution, the phosphate buffer of described damping fluid 2.
4. total bilirubin according to claim 1 detects reagent, it is characterized in that: described surfactant is to be selected from one or more of concrete material in TWEEN series, SPAN series, the TRITON series.
5. total bilirubin according to claim 1 detects reagent, it is characterized in that: described antiseptic is a kind of in a kind of or nipagin esters in potassium sorbate, Sodium Benzoate, sodium nitrite, the proclin series antiseptic.
6. total bilirubin according to claim 5 detects reagent, and it is characterized in that: described nipagin esters is methyl p-hydroxybenzoate, ethyl-para-hydroxybenzoate, propylparaben, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isopropyl ester or p-Hydroxybenzoic acid isobutyl ester.
7. total bilirubin according to claim 5 detects reagent, it is characterized in that: described proclin series antiseptic is Proclin300.
8. total bilirubin according to claim 1 detects reagent, and it is characterized in that: described freeze drying protectant is one or several in trehalose, sucrose, bovine serum albumin(BSA), Tween 80, triton x-100, the AEO.
9. total bilirubin according to claim 1 detects reagent, it is characterized in that: dilution is liquid condition in the agent of described total bilirubin detection reagent, and reaction reagent is dry powder.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586384A (en) * | 2016-03-11 | 2016-05-18 | 威尚生物技术（合肥）有限公司 | Total bilirubin detection kit |
| CN106353512A (en) * | 2016-08-15 | 2017-01-25 | 山东博科生物产业有限公司 | Novel reagent for detecting blood total bilirubin through synthetic chemistry oxidation method |
| CN106814037A (en) * | 2015-11-30 | 2017-06-09 | 山东博科生物产业有限公司 | A kind of sodium nitrite method total bilirubin（Oxidizing process）Detection kit |
| CN106896102A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of total bilirubin detection reagent |
| CN106896101A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of TBA detection reagent |
| CN110274881A (en) * | 2018-03-14 | 2019-09-24 | 济南煊赫生物科技有限公司 | A kind of stabilization, total bilirubin (enzymatic measurement) detection reagent of strong antijamming capability and detection method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106814037A (en) * | 2015-11-30 | 2017-06-09 | 山东博科生物产业有限公司 | A kind of sodium nitrite method total bilirubin（Oxidizing process）Detection kit |
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| CN106896102A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of total bilirubin detection reagent |
| CN106896101A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of TBA detection reagent |
| CN105586384A (en) * | 2016-03-11 | 2016-05-18 | 威尚生物技术（合肥）有限公司 | Total bilirubin detection kit |
| CN106353512A (en) * | 2016-08-15 | 2017-01-25 | 山东博科生物产业有限公司 | Novel reagent for detecting blood total bilirubin through synthetic chemistry oxidation method |
| CN110274881A (en) * | 2018-03-14 | 2019-09-24 | 济南煊赫生物科技有限公司 | A kind of stabilization, total bilirubin (enzymatic measurement) detection reagent of strong antijamming capability and detection method |
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| CN103278652B (en) | 2015-04-15 |
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Inventor after: Zou Bingde Inventor after: Zou Jihua Inventor after: Zhang Guichun Inventor after: Wo Yanbo Inventor before: Zou Bingde Inventor before: Zou Jihua Inventor before: Wo Yanbo Inventor before: Zhang Guichun Inventor before: Zhou Haibin |
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Address after: 315104 Ningbo, Yinzhou, Central District, No. Qiming South Road, No. 299 Patentee after: Meikang biological Polytron Technologies Inc Address before: 315104 Ningbo, Yinzhou, Central District, No. Qiming South Road, No. 299 Patentee before: Ningbo Meikang Biotechnology Co., Ltd. |