CN103278652B - Total bilirubin detection reagent - Google Patents
Total bilirubin detection reagent Download PDFInfo
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- CN103278652B CN103278652B CN201310200516.6A CN201310200516A CN103278652B CN 103278652 B CN103278652 B CN 103278652B CN 201310200516 A CN201310200516 A CN 201310200516A CN 103278652 B CN103278652 B CN 103278652B
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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 technical field of medical examination, be specifically related to a kind of total bilirubin determination reagent for POCT analyser.
Background technology
Total bilirubin (Totalbilirubin, TBil) be the bilirubinic general name of binding or non-binding produced by heme catabolism in 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 the class medicals diagnosis on disease such as liver and gall, occupy very important status.Current TBil generally adopts various large automatic Biochemical Analyzer to detect, but because large automatic Biochemical Analyzer equipment price is high, and complicated operation, operating personnel need have relevant professional knowledge and accept corresponding training, use complementary conditions to require high, need be equipped with stabilized voltage supply, water purification machine etc., and floor area is large, maintenance cost is high, needs professional's time-based maintenance, and therefore basic medical unit or household person all do not have condition to buy and use.In addition, large hospital patient is many, detects loaded down with trivial details, the long flow path of formality, stand-by period long, and this also brings the huge time to patient and bears, and the more important thing is that patient is affected adversely treatment because not diagnosing in time, even can lose one's life.
Real-time test (point-of-care testing, POCT) is an emerging technical field, and principal feature is rapid results, easy and simple to handle, easily uses and miniaturization.Along with diagnosis and the progress of ancillary technique, and people are to the requirement of the understanding of disease and treatment level raising, and POCT receives publicity gradually.In fact POCT becomes the important research project of external diagnosis reagent and instrument field gradually at European & American Market, existing many products put goods on the market, but this kind of POCT instrument has a major defect at present, be exactly that analyser and reagent consumptive material are expensive especially, for China, there is huge primary care system like this, and using amount of reagent country greatly, instrument and the matched reagent consumptive material of American-European exploitation at present obviously all face the challenge.
Microfluidic 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 sample preparation involved in the fields such as biological and chemical, reaction, separation, detection and cell chulture, sorting, cracking on the chip of a piece tens square centimeters (even less), network is formed by microchannel, running through whole system with controlled fluid, is a kind of technology of the various functions replacing standard biologic or chemical laboratory.Microfluidic chip technology is incorporated into POCT equipment, start the new situation of POCT development, can make laboratory was complicated in the past whole blood quantitatively, the step such as blood cell serum is separated, serum-dilution and Simultaneous Determination, complete in the on-line automatic equalization of chip, reach the synchronous testing goal of multiple mark.POCT analyser in conjunction with microfluidic chip technology have concurrently pin-point accuracy, low need blood volume, simple to operate, detect few, the low cost and other advantages of reagent dosage, the POCT instrument of Sheng Yi company limited as international in Bao Sheng, the Piccolo of Abaxis company, the Afinion etc. of Axis-Shield company, this type of POCT analyser all can realize a small amount of sample can analyze, easy and simple to handle, without cross pollution, can automated job, be highly suitable for China and there is huge primary care system like this and using amount of reagent is national greatly.
Along with Eleventh Five-Year Plan plan purchasing and reinforcement for three grades and second-grade hospital infrastructure device, current middle rank possesses good software and hardware facilities to go to the hospital, but particularly its full-automatic biochemical testing instruments facility of the unit such as commune hospital, clinic is generally not enough for basic medical unit, also do not have the inspection professional of enough numbers, therefore foundation operation POCT analytic system that is simple and easy, cheap, real-time report has important meaning to the effect of the vast basic hospital of performance 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 microfluidic 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, a kind of total bilirubin detection reagent that can be used for the POCT analyser introducing microfluidic chip technology is provided.
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, and wherein dilution consists of the following composition:
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 consists of the following composition:
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, glycine-NaOH buffer, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid buffer, N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, N-tri-(methylol) methyl-2-amino ethanesulfonic acid buffer, piperazine-N, two (2-hydroxyethanesulfonic acid) damping fluid of N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morpholine) ethyl sulfonic acid sodium damping fluid, 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonate buffer, two (2-hydroxyethyl) amino-2-hydroxy-propanesulfonic acid damping fluid of 3-, 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, one in N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid.
Wherein said damping fluid 2 can be the one in 2-(N-morpholine) ethanesulfonic acid buffer, acetate buffer, sodium citrate buffer solution, phosphate buffer.
Described surfactant is non-ionic surfactant, as being selected from TWEEN series (as tween (TWEEN) 20, polysorbate40, polysorbate60, Tween 80), SPAN series is (as class of department (SPAN) 20, class 40 of department, class 60 of department, class 80 of department), TRITON series is (as TritonX-100, TritonX-114, TritonX-405) in concrete material one or more (namely can be TWEEN series, SPAN series or TRITON serial in the concrete material of one, or more than one concrete material in often kind of series, or the concrete material of one in two kinds or more series).
Described antiseptic (comprising the antiseptic in dilution and the antiseptic in reaction reagent) is selected from the one in potassium sorbate, Sodium Benzoate, sodium nitrite, proclin series antiseptic (as Proclin300) or the one in nipagin esters (as methyl p-hydroxybenzoate, ethyl-para-hydroxybenzoate, propylparaben, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isopropyl ester, p-Hydroxybenzoic acid isobutyl ester).
Described freeze drying protectant select in trehalose, sucrose, bovine serum albumin(BSA), Tween 80, triton x-100, AEO (Brij-35) one or several.
It is liquid condition that described TBIL measures dilution in reagent, and reaction reagent is dry powder.
The preparation method that described TBIL measures the dilution of reagent is as follows: mix after described agent formulations is added distilled water and stir evenly.
The preparation method that described TBIL measures the reaction reagent of reagent is as follows: mix after described agent formulations is added distilled water and stir evenly, 0.5-10 μ l reaction reagent is joined reaction detection groove, volatilize 24-72h through freeze-drying (industry routine techniques) or 2-8 DEG C.
The test condition that described TBIL measures reagent is as follows: temperature: 37 DEG C; Detect predominant wavelength 450nm, commplementary wave length 750nm.
Microfluidic chip technology of the present invention is integrated into by basic operation unit on the chip of a piece tens square centimeters (even less), forms network by microchannel, runs through a kind of technology of whole system with controlled fluid.It is two-layer up and down that its feature is that chip is generally divided into, there is the through hole for application of sample on upper strata, the difform fluid channel etc. that lower floor comprises sample cell, dilution liquid bath, sample amounts groove, dilution quantitative slot, reservoir, multiple reaction detection groove, one group of self-inspection groove for system compensation, one group of overflow groove, many groups being preinstalled with reaction reagent control fluid flowing.Its detection method generally comprises following steps: sample solution and dilution are injected in described sample cell and dilution liquid bath through respective through hole by (1); (2) chip described in starter motor rotation; (3) sample solution realizes Separation of Solid and Liquid with quantitative under centrifugal action, and dilution enters dilution quantitative slot simultaneously; (4) quantitative sample and dilution flow into mixing channel and mix; (5) mixed liquid enters reaction detection groove and reaction reagent reacts; (6) in reaction detection groove, in situ detection is carried out by the checkout equipment supporting with chip.
The assay method that described TBIL measures reagent is as follows: 5-20 μ l sample is joined sample cell, 20-100 μ l dilution is joined dilution liquid bath, starter motor, records absorbance A 1, record absorbance A 2 after continuing reaction 5-9min after 37 DEG C of reaction 1min.
The reaction principle that described TBIL measures reagent is: sample first carries out mixing hatching with dilution, to remove the interfering material such as vitamin C and blood fat in sample, after mixed liquor enters reaction detection groove, under sodium chloride, disodium ethylene diamine tetraacetate, Tween 80, lauryl sodium sulfate and sulfuric acid exist, the oxidized dose of sodium peroxydisulfate of the bilirubin direct in sample is oxidized to dehydrobilirubin.Bilirubinic yellow specificity absorbance declines, and by measuring the change of absorbance before and after sodium peroxydisulfate oxidation, calculates the content of TBIL in sample.
Advantage of the present invention and beneficial effect: reagent of the present invention has good sensitivity, accuracy, precision and linear, can meet clinical examination requirement completely.The POCT analyser that reagent of the present invention can be used for introducing microfluidic chip technology detects total bilirubin, thus realizes the foundation of the POCT analytic system of simple and easy, cheap, the real-time report of operation.
Accompanying drawing explanation
The linear result figure of Fig. 1 TBIL.
Fig. 2 TBIL methodology comparison result figure.
Embodiment
To further illustrate 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 amendments to the present invention, such amendment also falls into scope of the present invention.
Following experimental technique if no special instructions, is conventional method, and the experiment material used if no special instructions, all can easily obtain from commercial company.
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.
Be described below in conjunction with the performance of form to the embodiment of the present invention 1 gained reagent.
1, precision
Table 1, precision assessment result
2, linear
The analysis thing adding variable concentrations with standard serum samples outward detects, and TBIL linearly the results are shown in Fig. 1.
3, methodology Comparability test
Compare with the TBIL end value that automatic clinical chemistry analyzer measures, result is as Fig. 2, wherein X-axis represents determination data in automatic clinical chemistry analyzer Hitachi 7180, POCT analyser (the Taiwan Bao Sheng world of micro flow chip technology is introduced in Y-axis representative, Amishield, TMO-100) upper 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, calculate gained with statistical software EPEvaluatorrelease6.Experimental result shows reagent of the present invention good sensitivity, can meet the improvement of U.S. clinical laboratory completely and amend legislation.
Table 2 reagent detection sensitivity
From above-mentioned testing result, reagent of the present invention has good sensitivity, accuracy, precision and linear, can meet clinical examination requirement completely.
Claims (7)
1. a total bilirubin detection reagent, is characterized in that: this reagent comprises dilution and reaction reagent, and wherein dilution consists of the following composition:
Damping fluid 1 pH 6.5-7.5:0.01-1.0 mol/L;
Surfactant 0.1-10.0% (mass percent);
Antiseptic 0.1-10.0% (mass percent);
Ascorbic acid oxidase 1-100 KU/L;
Wherein said reaction reagent consists of the following composition:
Damping fluid 2 pH 3.0-5.0:0.1-1.0 mol/L;
Sodium chloride 45 g/L;
Disodium ethylene diamine tetraacetate 2-10 g/L;
Tween 80 5-20 ml/L;
Lauryl sodium sulfate 10-100 g/L;
Sodium peroxydisulfate 100-200 g/L;
Sulfuric acid 0.1-2 ml/L;
Antiseptic 0.1-1 g/L;
Freeze drying protectant 10-30 g/L;
Described surfactant be selected from TWEEN series, SPAN series, TRITON serial in one or more of concrete material;
Described freeze drying protectant is one or several in trehalose, sucrose, bovine serum albumin(BSA), Tween 80, triton x-100, AEO.
2. total bilirubin detection reagent according to claim 1, is characterized in that: described damping fluid 1 is the amino damping fluid of trihydroxy methyl, glycine-NaOH buffer, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid buffer, N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, N-tri-(methylol) methyl-2-amino ethanesulfonic acid buffer, two (2-hydroxyethanesulfonic acid) damping fluid of piperazine-N, N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morpholine) ethyl sulfonic acid sodium damping fluid, 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonate buffer, two (2-hydroxyethyl) amino-2-hydroxy-propanesulfonic acid damping fluid of 3-, 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, one in N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid.
3. total bilirubin detection reagent according to claim 1, is characterized in that: described damping fluid 2 is the one in 2-(N-morpholine) ethanesulfonic acid buffer, acetate buffer, sodium citrate buffer solution, phosphate buffer.
4. total bilirubin detection reagent according to claim 1, is characterized in that: described antiseptic is the one in a kind of or nipagin esters in potassium sorbate, Sodium Benzoate, sodium nitrite, proclin series antiseptic.
5. total bilirubin detection reagent according to claim 4, 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.
6. total bilirubin detection reagent according to claim 4, is characterized in that: described proclin series antiseptic is Proclin300.
7. total bilirubin detection reagent according to claim 1, is characterized in that: in described total bilirubin detection reagent agent, dilution is liquid condition, and reaction reagent is dry powder.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106814037B (en) * | 2015-11-30 | 2018-12-11 | 山东博科生物产业有限公司 | A kind of sodium nitrite method total bilirubin (oxidizing process) detection kit |
| CN106896101A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of TBA detection reagent |
| CN106896102A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of total bilirubin 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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|---|---|---|---|---|
| EP0795753A1 (en) * | 1994-12-02 | 1997-09-17 | Nitto Boseki Co., Ltd. | Method for determining bilirubin |
| JP4469435B2 (en) * | 1999-03-10 | 2010-05-26 | 三菱化学メディエンス株式会社 | Method for stabilizing bilirubin oxidase |
| CN1687786A (en) * | 2005-04-29 | 2005-10-26 | 张抗 | Testing kit of full-liquid enzyme method combined with bilirubin |
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| CN102944683A (en) * | 2012-11-16 | 2013-02-27 | 李立和 | Double reagent method for detecting indirect bilirubin kit and preparation method |
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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. |