Electrochemical luminescence buffer solution and cleaning fluidTechnical field
The present invention relates to the chemiluminescent buffer and cleaning fluid available for Roche automatic lmunoassays analyzer, belongs to medical treatmentApparatus external diagnosis reagent field.
Background technology
Electrogenerated chemiluminescence (ECL), also referred to as electrochemical luminescence, it is a kind of novel to be that chemiluminescence is combined with electrochemistryResearch method.It is to be electrochemically reacted by applying certain voltage to electrode, certain between product or in product and systemChemically reacted between kind component, electron transition is produced excitation state material to excitation state, excitation state material returns to ground state productionHair tonic light；Either luminescent substance provides energy using electrode and directly carries out redox reaction, generates unstable intermediate stateMaterial, intermediate state material can decompose generation light radiation rapidly.
Roche companies of Switzerland Cobas e411/e601 Electrogenerated chemiluminescent immunoassay systems, it is to apply advanced electrochemistryThe Full-automatic desk type analyzer of luminous (ECL) technology.Thyreoidine, hormone, myocardial damage and tumor-marker, infection are appliedDisease, anaemia, Bone m etabolism etc. widely determine project.Its electrochemiluminescsystem system is tris (bipyridine) ruthenium ([Ru (bpy) 32+])-threePropylamine (TPrA) system, its matching used Procell and Cleancell dosage is larger, and supporting original-pack reagent is particularlyProcell is expensive and toxicity is larger.
Chemiluminescent buffer Procell is tripropyl amine (TPA) cleaning fluid, for safeguarding electrode, transporting reactant mixture, cleaningThe coated particulate of Streptavidin, produce reaction signal；Chemiluminescence cleaning fluid Cleancell, for pipeline after measuring every timeThe cleaning of system and measuring cell and electrodes maintenance.Roche Diagnistics import registration certificate presentation of information Procell is formulated：PhosphateBuffer solution 300mmol/L, tripropyl amine (TPA) 180mmol/L, Qu Gou Ji≤0.1%；Preservative, pH6.8；Cleancell is formulated：KOH 176mmol/L (equivalent to pH value 13.2)；Cleaning agent≤1%.Tris (bipyridine) ruthenium ([Ru (bpy) 32+])-tripropyl amine (TPA)(TPrA) system is a kind of common ECL systems, and wherein tris (bipyridine) ruthenium is electrochemical luminescence agent, and it and electron donor TPrA existElectrode anode surface loses electronics and the oxidation reaction, [Ru (bpy) 3 of divalent occurs simultaneously2+] label is oxidized to the [Ru of trivalent(bpy)33+] label, TPrA is oxidized to radical cation TPrA+*, TPrA+*It is very unstable, spontaneously lose oneProton and form free radical TPrA*, it is strong reductant, by [Ru (bpy) 3 of the electronics to trivalent3+], become and exciteThe Ru (bpy) 3 of state2+, and TPrA itself is oxidized to oxidation product.The Ru (bpy) 3 of excitation state2+Launch a wavelength during decay620nm photon, re-form the Ru (bpy) 3 of ground state2+.The progress that this process is gone round and begun again in electrode surface, produce manyPhoton so that optical signal strengthens.
Because light caused by the irreversibility of TprA autoxidations and interaction between the two is weaker, the system is led toOften the use of large excess of TPrA (is typically TPrA：Ru(bpy)32+=100000 μm of ol/L：1 μm of ol/L), i.e. 100mmol/L,And original-pack reagent adds 180mmol/L TPrA, beyond usual amounts, and TPrA toxicity is identified as high toxicity, rat oral LD50=72mg/kg, its steam or mist have strong impulse to eye, mucous membrane, the upper respiratory tract and skin, for the healthy shadow of docimasterSound is larger.In addition, full automatic biochemical apparatus is after a number of uses, because cleaning performance is bad, from measuring cell to pipeline, easily byThe pollution of reaction solution, long-term protein precipitation, causes blank absorbency to increase, and alarms.
It can be seen that chemiluminescent buffer and cleaning fluid of the further research available for Roche automatic lmunoassays analyzer,The concentration of tripropyl amine (TPA) is especially reduced, and ensures the uniformity of testing result, the buffer solution of stability and accuracy, and haveThe cleaning fluid of more excellent cleaning performance, it is still technical problem urgently to be resolved hurrily, this is also exactly what the present invention was accomplishedLeaned on where basic with power.
The content of the invention
As described above, in order to further study available for Roche automatic lmunoassays analyzer chemiluminescent buffer andCleaning fluid, especially reduces the concentration of tripropyl amine (TPA), and ensures the uniformity of testing result, the buffer solution of stability and accuracy,And the cleaning fluid with more excellent cleaning performance, the present inventor conduct in-depth research to this, are paying a large amount of woundsAfter the property made work, so as to complete the present invention.
The present invention is with regard to TPrA and Ru (bpy) 32+Interaction caused by light is weaker sets about, Procell formulas are changedEnter, substantially reduce TPrA dosages, while N is added in prescription, [just] butylaminos of N- bis- ethanol, sodium chloride, PEG 300, thirdThe materials such as diureide, paracresol, enhanced chemiluminescence, reduce the chemiluminescence caused by tripropyl amine (TPA) concentration reduces and weaken, meanwhile,Ensure the uniformity and stability with original-pack reagent.
The present invention adds the compositions such as urea, thiocarbamide Trixton X-100, MES in Cleancell, increases it to condensingThe cleaning performance of the composition such as block and fibrin, and the preservatives of Proclin 300 are added, further safeguard electrode.
Develop and improve the Procell being formulated and Cleancell replacements original-pack Procell and Cleancell, to reduce poisonProperty, cleaning performance is improved, further guard electrode reduces cost, has larger profit margin.
Specifically, the present invention relates to following four aspects, more specifically, one side, the present invention relates to one kindMatching used electrochemical luminescence buffer solution and cleaning fluid, buffer composition include：Sodium dihydrogen phosphate, tripropyl amine (TPA), sodium chloride, grassAcid, Tween 20, Proclin 300；Cleaning fluid component includes：Potassium hydroxide, Trixton X-100, MES, Proclin300。
Preferably, its component of buffer solution therein and content are：250~350mmol/L of potassium dihydrogen phosphate, tripropyl amine (TPA) 60~90mmol/L, 1~10mmol/L of sodium chloride, oxalic acid 3~10mmol/L, Tween 20 0.05~0.2% (w%), Proclin300 0.05~0.2% (w%), pH value 6.6~7.0；Cleaning fluid component and content therein are：Potassium hydroxide 150-200mmol/L, Trixton X-100 0.1~1% (w%), MES 0.1~1% (w%), Proclin 300 0.1~0.5% (w%).
Second aspect, the present invention relates to a kind of matching used electrochemical luminescence buffer solution and cleaning fluid, buffer solution groupDividing includes：Sodium dihydrogen phosphate, tripropyl amine (TPA), sodium chloride, PEG 300, Tween 20, Proclin 300, paracresol；Cleaning fluid groupDividing includes：Potassium hydroxide, Trixton X-100, MES, urea, Proclin 300.
Preferably, its component of buffer solution therein and content are：250~350mmol/L of potassium dihydrogen phosphate, tripropyl amine (TPA) 20~30mmol/L, sodium chloride 1~10mmol/L, PEG300 1~10mmol/L, Tween 20 0.05~0.2% (w%),Proclin 3000.05~0.2% (w%), 0.1~1mmol/L of paracresol, pH value 6.6~7.0；Cleaning fluid component thereinIt is with content：Potassium hydroxide 150-200mmol/L, Trixton X-100 0.1~1% (w%), MES/AES 0.1~1%(w%), urea 0.1~1% (w%), Proclin300 0.1~0.5% (w%).
3rd aspect, the present invention relates to a kind of matching used electrochemical luminescence buffer solution and cleaning fluid, buffer solution groupDividing includes：Sodium dihydrogen phosphate, tripropyl amine (TPA), N, N- bis- [just] butylamino ethanol, sodium chloride, PEG 300, malonyl urea, Tween20, Proclin 300, paracresol, polyvinylpyrrolidone, methylcellulose；Cleaning fluid component includes：Potassium hydroxide,Trixton X-100, MES, thiocarbamide, Proclin 300.
Preferably, its component of buffer solution therein and content are：250~350mmol/L of potassium dihydrogen phosphate, tripropyl amine (TPA) 1~10mmol/L, N, N- bis- [just] butylamino 1~3mmol/L of ethanol, sodium chloride 1~10mmol/L, PEG 300 1~10mmol/L, malonyl urea 1~5mmol/L, Tween 20 0.05~0.2% (w%), Proclin 300 0.05~0.2%(w%), 0.1~1mmol/L of paracresol, 0.1~1mmol/L of polyvinylpyrrolidone, methylcellulose 0.1~1mmol/L, pHValue 6.6~7.0；Cleaning fluid component and content therein are：Potassium hydroxide 150-200mmol/L, Trixton X-100 0.1~1% (w%), MES0.1~1% (w%), thiocarbamide 0.1~1% (w%), Proclin 300 0.1~0.5% (w%).
4th aspect is as follows the present invention relates to the compound method of above-mentioned buffer solution and cleaning fluid：
First, the preparation of chemiluminescent buffer：Composition in addition to tripropyl amine (TPA) is weighed to add in a small amount of water respectively and stirredMixing makes dissolving, add tripropyl amine (TPA) stirring standing after it is complete it is molten after be transferred in volumetric flask, constant volume, shake up, adjust pH value, produce.
2nd, the preparation of cleaning fluid：Appropriate amount is weighed according to the composition in formula, be dissolved in water mixing, lets cool, is transferred toVolumetric flask, constant volume, shake up, produce.
Embodiment
Below by specific embodiment, the present invention is described in detail, but the purposes of these exemplary embodiments andPurpose is only used for enumerating the present invention, not forms any type of any restriction to the real protection scope of the present invention, more non-to incite somebody to actionProtection scope of the present invention is confined to this.
Embodiment 1
A, the component of chemiluminescent buffer (Procell) and preparation：
Potassium dihydrogen phosphate 270mmol/L
Tripropyl amine (TPA) 60mmol/L
Sodium chloride 2mmol/L
Oxalic acid 5mmol/L
Tween 20 0.05% (w%)
Proclin 300 0.05% (w%)
PH value is adjusted to 6.6 with NaOH
Other compositions in addition to tripropyl amine (TPA) are weighed to add in a small amount of water respectively and are stirred to dissolve, tripropyl amine (TPA) is added and stirsMix standing after it is complete it is molten after move in volumetric flask, constant volume, shake up and produce.
B, the component of cleaning fluid (Cleancell) and preparation：
Potassium hydroxide 150mmol/L
Trixton X-100 0.5%
MES 0.5%
Proclin 300 0.3%
Appropriate amount is weighed according to the composition in formula, be dissolved in water mixing, lets cool, and is transferred to volumetric flask, constant volume, shakes upProduce.
Embodiment 2
A, the component of buffer solution (Procell) and preparation：
Potassium dihydrogen phosphate 350mmol/L
Tripropyl amine (TPA) 25mmol/L
Sodium chloride 5mmol/L
PEG 300 5mmol/L
Tween 20 0.1% (w%)
Proclin 300 0.1% (w%)
Paracresol 0.2mmol/L
PH value is adjusted to 7.0 with NaOH
Other compositions in addition to tripropyl amine (TPA) are weighed to add in a small amount of water respectively and are stirred to dissolve, tripropyl amine (TPA) is added and stirsMix standing after it is complete it is molten after move in volumetric flask, constant volume, shake up and produce.
B, the component of cleaning fluid (Cleancell) and preparation：
Potassium hydroxide 200mmol/L
Trixton X-100 0.2%
AES 0.8%
Urea 0.1%
Proclin 300 0.5%
Appropriate amount is weighed according to the composition in formula, be dissolved in water mixing, lets cool, and is transferred to volumetric flask, constant volume, shakes upProduce.
Embodiment 3
A, the component of buffer solution (Procell) and preparation：
Potassium dihydrogen phosphate 300mmol/L
Tripropyl amine (TPA) 5mmol/L
N, N- bis- [just] butylamino ethanol 3mmol/L
Sodium chloride 5mmol/L
PEG 300 3mmol/L
Malonyl urea 5mmol/L
Tween 20 0.1% (w%)
Proclin 300 0.05% (w%)
Paracresol 0.1mmol/L
Polyvinylpyrrolidone 0.1mmol/L
Methylcellulose 0.1mmol/L
PH value is adjusted to 6.8 with NaOH
Other compositions in addition to tripropyl amine (TPA) are weighed to add in a small amount of water respectively and are stirred to dissolve, tripropyl amine (TPA) is added and stirsMix standing after it is complete it is molten after move in volumetric flask, constant volume, shake up and produce.
B, the component of cleaning fluid (Cleancell) and preparation：
Potassium hydroxide 180mmol/L
Trixton X-100 0.8%
MES 0.2%
Thiocarbamide 0.5%
Proclin 300 0.5%
Appropriate amount is weighed according to the composition in formula, be dissolved in water mixing, lets cool, and is transferred to volumetric flask, constant volume, shakes upProduce.
Embodiment 4
First, Accuracy Verification experiment is determined
1st, the Procell and Cleancell configured embodiment 1 is placed in the Elecsys2010 electrochemical luminescences of RocheImmunoassay system carries out checking test on corresponding position, takes 30 patients serums to be tested, contrast and original-pack supporting examinationThe correlation of agent Procell, Cleancell testing result.
As a result show, Procell, Cleancell of autogamy embodiment 1 and original-pack Procell, Cleancell performance oneCause property is good.
2nd, the Procell and Cleancell configured embodiment 2 is placed in the Elecsys2010 electrochemical luminescences of RocheImmunoassay system carries out checking test on corresponding position, takes 30 patients serums to be tested, contrast and original-pack supporting examinationThe correlation of agent Procell, Cleancell testing result.
As a result show, autogamy embodiment 2Procell, Cleancell is consistent with original-pack Procell, Cleancell performanceProperty is good.
3rd, the Procell and Cleancell configured embodiment 3 is placed in the Elecsys2010 electrochemical luminescences of RocheImmunoassay system carries out checking test on corresponding position, takes 30 patients serums to be tested, contrast and original-pack supporting examinationThe correlation of agent Procell, Cleancell testing result.
As a result show, autogamy embodiment 3Procell, Cleancell is consistent with original-pack Procell, Cleancell performanceProperty is good.
It in summary it can be seen, show Self-made reagent in the case where greatly reducing tripropyl amine (TPA) concentration, while remain to completeImmunoassay on fully-automatic analyzer, meet the goldstandard with original-pack reagent uniformity, and result is accurately and reliably, it is seen that thisProcell, Cleancell of the improvement formula of invention have quite excellent safety and reliability.
Embodiment 5
2nd, Cleancell cleaning performances checking test
The cleaning of pipe-line system and measuring cell and electrodes maintenance after Cleancell effect mainly measures every time, thisInvention improves formula, to improve its cleaning action to pipeline and measuring cell, to therein on the basis of the formula of original-pack reagentReaction solution is mainly that the albumen remained is cleaned, and first reacts a large amount of blank serums and thyroid-stimulating hormone kit, then willAbove reaction solution is sub-packed in blank cuvette, is stood 30min, is outwelled, and is existed using autogamy Cleancell and imported reagentsCuvette cleaning (respectively carrying out 20 groups of parallel tests) is carried out in the case of identical addition, the water of equivalent is added after cleaning, is shakenIt is even, under protein maximum absorption wavelength 280nm, absorbance is tested, observes result.
1. the Cleancell configured in embodiment 1 is tested according to above verification method, as a result see the table below：
2. the Cleancell configured in embodiment 2 is tested according to above verification method, as a result see the table below：
3. the Cleancell configured in embodiment 3 is tested according to above verification method, as a result see the table below：
It in summary it can be seen, autogamy embodiment 1-3, improve the Cleancell of formula compared with imported reagentsCleancell albumen cleaning performance is more excellent, therefore the Cleancell of the improvement formula of the present invention, cleaning action enhancing.So as toSo that the matching used electrochemical luminescence buffer solution of the present invention and cleaning fluid will have more actual industrial application value.
It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to the protection model of the limitation present inventionEnclose.In addition, it should also be understood that, after the technology contents of the present invention have been read, those skilled in the art can make each to the present inventionKind change, modification and/or variation, all these equivalent form of values equally fall within the guarantor that the application appended claims are limitedWithin the scope of shield.