CN101000339B - Method for reusing microflow chip - Google Patents
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- CN101000339B CN101000339B CN2007100024523A CN200710002452A CN101000339B CN 101000339 B CN101000339 B CN 101000339B CN 2007100024523 A CN2007100024523 A CN 2007100024523A CN 200710002452 A CN200710002452 A CN 200710002452A CN 101000339 B CN101000339 B CN 101000339B
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Abstract
A method for reutilizing microflow chip repeatedly washes disposable micro flow chip to make washed micro flow chip be at the same quality and the same property as that of original micro flow chip. The kit used for realizing said method is also disclosed.
Description
Technical field
The invention belongs to the biochip technology field, particularly, the present invention relates to the repeated use technology of micro flow chip, comprise method of cleaning micro flow chip and the method for reusing micro flow chip accordingly.The invention still further relates to be used to clean micro flow chip makes it can reusable kit.
Background technology
Micro flow chip (Micro Fluid Chip or Micro Fluidics Lab-Chip, also abbreviate MFLC herein as) be the another new biochip technology of biochip detecting and analysing system that behind the dot matrix chip, grows up, be widely used in biological sample (as, comprise the sample of DNA, RNA and protein) test in.Micro flow chip is 0.5 micron device of supplying with and/or mix multiple liquid and liquid is reacted in passage to the microfluidic channel of 1 mm depth and width on chip, and this device can be with thereon integrated and detect the result by chip analyzer to the test function of test from sample preparation.Theory according to micro-total analysis system, micro flow chip can will comprise sampling, dilute, adds the function of carrying out in the whole laboratory of needs such as reagent, reaction, separation, detection and be incorporated on the microchip, thereby realize microminiaturization, integrated and portability, thereby have extensive applicability the overall process of biological sample processing, detection.By using micro flow chip, not only can make the consumption of costliness, rare Biosample and reagent be reduced to microlitre greatly even receive the upgrading level, and can improve analysis speed, the processing of corresponding reduction biological sample, testing cost greatly.Micro flow chip comprise the sample feeding member (as, sample holes) and microfluidic channel, and can choose members such as comprising micropump, groove wantonly, its structure has had disclosure widely, example in recent years has Chinese patent application CN1730142A, CN1353309A, CN1267089A, U.S. Patent application US2006060811A, US2005167820A, US2005079098A, PCT International Application No. WO 03071262A, WO02086162A, WO0218827A or the like, and has had and manyly become commercialized and sell.
Yet,, make it difficult to clean because micro flow chip inner structure complexity especially is subjected to the influence of the small fluid passage degree of depth and width and test liquid viscosity.If clean with fierce condition, destroy the fine structure that the chip internal micro etch goes out easily, can't reuse; If the cleaning condition gentleness then is difficult to clean residual liquid in the use, produce dirty when causing chip to reuse.Some micro flow chip just is beneficial to clean by design inner structure and purging system, the micro flow chip system design of producing the LC-90 that 5100ALP or Caliper LS produced as Agilent Technologies has proprietary convenient washing, the automatic function of regeneration, so not only increased design difficulty, and versatility is relatively poor.After being to use, another kind of settling mode promptly abandons micro flow chip and no longer repeated use, promptly use disposable micro flow chip (Disposable Micro Fluidics Lab-Chip, also can abbreviate DMFLC as herein), foursquare Lab-on-a-Chip (the disposable micro flow chip of Caliper1000) as the production of U.S. Caliper LS company, the DMFLC of the rhombus that is mated with the Experion system that Agilent Lab Chip DMFLC that Agilent Technology company produces and Bio-RedLaboratories company sell or the Canon DMFLC that CanonUSALS company sells have so just increased use cost.
In view of this, in order to reuse micro flow chip, especially reuse disposable micro flow chip, the inventor is through studying for a long period of time, a kind of cleaning method and kit thereof have been developed, be used to clean the hand micro flow chip, in particular for cleaning disposable micro flow chip, and developed a kind of method of reusing micro flow chip thus.The present invention not only can effectively remove the pollution that chip uses the back to produce, and mild condition, does not destroy chip, make it and can use repeatedly, and performance and quality all can reach the original standard of chip, greatly reduces the use cost of micro flow chip system.In addition, the present invention is simple and easy to do, can be widely used in the clinical trial of mechanisms such as scientific research field and hospital, blood station and disease prevention and control center.
Summary of the invention
The present invention relates to reuse the technology of micro flow chip, provide and cleaned the method and the kit thereof of micro flow chip, and the method for the repeated use micro flow chip that comprises this cleaning method step is provided.
Particularly, aspect first, the invention provides a kind of method of cleaning micro flow chip, it in turn includes the following steps:
(a) clean micro flow chip with solution A, wherein solution A contains sequestrant and detergent;
(b) clean micro flow chip with solution B, wherein solution B contains sequestrant; And
(c) clean micro flow chip with solution C, wherein solution C contains sequestrant.
Wherein, micro flow chip refers to the chip apparatus that is used to detect, analyze the biological or chemical sample, it is by technology such as micro etchs, on chip, form 0.5 micron to the microfluidic channel of 1 mm depth and width supplying with and/or to mix several samples liquid and/or reagent, thereby these liquid can be reacted in passage.Usually, micro flow chip also comprises members such as micropump, groove.According to detected object classification, micro flow chip can be divided into DNA chip (DNA Lab-Chip), RNA chip (RNALab-Chip) and protein chip (Protein Lab-Chip) etc., is respectively applied for DNA, RNA in detection, the analytic sample and protein etc.; According to the access times classification, micro flow chip can be divided into micro flow chip and the disposable micro flow chip that repeatability is used.The method of first aspect of the present invention can be used for cleaning the members such as microfluidic channel of micro flow chip, under the condition of not damaging original chip structure, washes liquid residual in the use, thereby makes chip to reuse.Method applicability of the present invention is strong, can be used for detecting, analyzing the micro flow chip (comprising disposable micro flow chip) of all kinds of samples, and preferably micro flow chip is DNA chip, RNA chip or protein-chip in this method.Protein-chip can detect each proteinoid, for example, can detect the protein in serum or the urine clinically, as lipoprotein, urine protein etc.Nucleic acid chip (it comprises DNA chip and RNA chip) can be used for various dependence electrophoretic techniques separated DNA or RNA, make to detect target position as the dna molecular of pathogen amplified production commonly used in clinical, with DNA chip detection, analysis infectious disease, hereditary disease, variant etc.; And for example, because the RNA molecule is degraded easily, therefore available RNA chip is done quality check control to prepared RNA molecule in clinical or the research.
The method of first aspect of the present invention can replace supporting original cleaning way in the repeated micro flow chip product that uses, but this method is preferred for cleaning disposable micro flow chip, promptly the micro flow chip in this method is disposable micro flow chip, especially this method is preferred for cleaning cited CaliperLS herein, the DMFLC that company produced and sold such as Agilent Technology, Bio-Rad Laboratories and Canon USA LS.
Term among the present invention " detergent " is known to the one of ordinary skill in the art, and it is that a class had not only had hydrophilic group but also had the material of hydrophobic group, can divide polytypes such as negative ion, kation and non-ionic detergent usually.Be familiar with as one of ordinary skill in the art, common anionic detergent has lauryl sodium sulfate and dodecyl semi-annular jade pendant acid sodium; Cationic detergent has geramine, bromogeramine, CTAB, CPC, ZEPH, dequaline chloride, myristylpicolinum bromide (TMPB), domiphen etc.; And non-ionic detergent claims neutral detergent to have: polyethylene glycols, as PEG200; Polyol surfactant is as sorbierite, spans (Span) and Tweens (Tween); Polyoxyethylene aliphatic alcohol ether is as brejs, peregal class; Polyethenoxy alkylphenols is as Igepal CO, polyoxyethylene nonylphenol ether, triton (Triton), Pluronic, bubble enemy; Or the like.How these detergents commercialization, can easily buy.Detergent in the method for first aspect of the present invention in the solution A can be a kind of detergent or the composition that contains multiple detergent, non-ionic detergent preferably, be more preferably the neutral detergent of polyethenoxy alkylphenols class, being more preferably Triton, most preferably is Triton X-100.The content of detergent can be renderd a service to determine according to the decontamination of detergent, accounts for the 0.01-1% (weight) of solution usually, preferably accounts for the 0.05-0.5% (weight) of solution.In a specific embodiment of the present invention, the detergent in the solution A is Triton X-100, and its concentration in solution A is 0.1% (weight).
Term among the present invention " sequestrant " also is known to the one of ordinary skill in the art, it be can chelating ion material, usually can be divided into the amido carboxylic acids (as, EDTA, DTPA, NTA), the polymer phosphate salt (as, sodium hexametaphosphate, tripolyphosphate), polyamine based phosphates class and polymerization of carboxylic acid salt etc.How these sequestrants commercialization, can easily buy.Solution A of the present invention, B and C all contain sequestrant, preferably be selected from one or more sequestrants among EDTA (ethylenediamine tetraacetic acid), DTPA (diethylene-triamine pentaacetic acid), NTA (triacetic acid base ammonia), DCTA (1,2-diaminocyclohexane tetraacetic acid), EDTP (ethylenediamine tetrapropionic acid) and the EGTA (ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA)), being more preferably EDTA, most preferably is EDTA-Na
2The content of sequestrant can determine that the concentration in the solution is 0.05-5mM usually, is preferably 0.2-2mM according to the ability of its chelated metal ions.If contain the sequestrant of identical type among solution A, B and the C, then the sequestrant concentration of the preferred solution C sequestrant concentration that is not more than the sequestrant concentration of solution B and solution B is not more than the sequestrant concentration of solution C.In a specific embodiment of the present invention, the sequestrant among solution A, B and the C is EDTA-Na
2, its concentration in solution A, B and C is respectively 1mM, 0.5mM and 0.5mM.
It is weakly acidic that solution A of the present invention preferably is, and preferably its pH is between 5.0 to 7.0, and more preferably its pH is between 5.5 to 6.5, and more preferably its pH is between 5.7 to 6.3.The adjusting of pH can use acid, alkali, the salt known to the one of ordinary skill in the art to regulate, and preferably uses the pH damping fluid, as phosphate buffered saline(PBS) (PBS), Tris-HCl (Tris-Cl) etc.Usually, the concentration of damping fluid is 1-200mM in the solution, is preferably 5-100mM.In a specific embodiment of the present invention, solution A contains the Tris-HCl of 50mM pH6.0.Solution A of the present invention is further alkali metal containing salt also preferably, and as KCl, NaCl etc., its concentration is generally 1-100mM.
It is neutral that solution B of the present invention and C preferably are basically, and preferably its pH is between 6.0 to 8.0, and more preferably its pH is between 6.5 to 7.5, and more preferably its pH is between 6.7 to 7.3.The adjusting of pH can use acid, alkali, the salt known to the one of ordinary skill in the art to regulate, and preferably uses the pH damping fluid, as phosphate buffered saline(PBS) (PBS), Tris-HCl (Tris-Cl) etc.The concentration of damping fluid is successively decreased among solution A, B and the C.Usually, the content of damping fluid is 1-200mM among solution B and the C, is preferably 5-100mM.In a specific embodiment of the present invention, solution B contains the Tris-HCl of 50mM pH7.0, and solution C contains the Tris-HCl of 10mM pH7.0.Solution B of the present invention is further alkali metal containing salt also preferably, and as KCl, NaCl etc., its concentration is generally 1-100mM less than the concentration of alkali metal salt in the solution A.
Term among the present invention " antiseptic " also is known to the one of ordinary skill in the art, and it is the material that can prevent bacterium and/or fungus growth, and sorbic acid, sorbate (as sorbate potassium), phenmethylol, formaldehyde, phenol, NaN are arranged usually
3(Sodium azide), chloroform, thimerosal (as, ethyl thimerosal), lysozyme or antibiotic or the like.Can further add one or more antiseptics in the solution C of the inventive method.The content of antiseptic can be determined according to its antibacterial efficacy, accounts for the 0.001-1% (weight) of solution usually, preferably accounts for the 0.01-0.1% (weight) of solution.
In sum, detergent in the method for preferred first aspect of the present invention in the solution A is that the pH of non-ionic detergent and solution A is between 5.0 to 7.0, the also further alkali metal containing salt of preferred solution A, more preferably solution A contain EDTA, KCl and Triton X-100 and solution A pH between 5.5 to 6.5, most preferably solution A contains Tris-HCl, the 1mM EDTA-Na of 50mM pH6.0
2, 10mM KCl and 0.1% (weight) Triton X-100.
The pH of solution B is between 6.0 to 8.0 in the method for preferred first aspect of the present invention, the also further alkali metal containing salt of preferred solution B, more preferably solution B contain EDTA and KCl and solution B pH between 6.5 to 7.5, most preferably solution B contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 5mM KCl.
The pH of solution C is between 6.0 to 8.0 in the method for preferred first aspect of the present invention, and preferred solution C also further contains antiseptic, and more preferably solution C contains EDTA and NaN
3And the pH of solution C is between 6.5 to 7.5, and most preferably solution C contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 0.05% (weight) NaN
3
In a specific embodiment of the present invention, select solution A, B and the C of the prescription shown in the table 1-3 for use.
Table 1 solution A is preferably filled a prescription (comprising the Chinese name and the abbreviation of each component)
| 50mM?Tris-HClpH6.0 | 50 mMs trishydroxymethylaminomethane-hydrochloride buffer pH value 6.0 |
| 1mM?EDTA-Na 2 | 1.0 mM disodium ethylene diamine tetraacetate |
| 10mM? |
10 mM potassium chloride |
| 0.1％Triton?X-100 | 0.1% song draws ketone X-100 |
Table 2 solution B is preferably filled a prescription (comprising the Chinese name and the abbreviation of each component)
| 10mM?Tris-HClpH7.0 | 10 mMs trishydroxymethylaminomethane-hydrochloride buffer pH value 7.0 |
| 0.5mM?EDTA-Na 2 | 0.5 mM disodium ethylene diamine tetraacetate |
| 5mM? |
5 mM potassium chloride |
Table 3 solution C is preferably filled a prescription (comprising the Chinese name and the abbreviation of each component)
| 10mM?Tris-HClpH7.0 | 10 mMs trishydroxymethylaminomethane-hydrochloride buffer pH value 7.0 |
| 0.5mM?EDTA-Na 2 | 0.5 mM disodium ethylene diamine tetraacetate |
| 0.05％NaN 3 | 0.05% Sodium azide |
In the method for first aspect of the present invention, cleaning micro flow chip is meant in discarding this chip behind original liquid, add corresponding solution (that is, solution A, B or C) and after discard this solution, add and discard between these two steps and preferably can also place this chip.Particularly, in each step of the inventive method (promptly, step (a) and (b) or (c)) add corresponding solution (promptly to micro flow chip in, solution A, B or C), make corresponding flow of solution cross chip, especially flow through members such as microfluidic channel, thereby wash liquid residual in the use.For fear of mechanical injuries, the inviolent cleaning way of preferred employing can comprise corresponding solution adding micro flow chip sample holes as cleaning micro flow chip in the method for the present invention, places the back and discard this solution from this chip.Wherein, preferably after corresponding solution adds sample holes, can be placed into vortex mixer, with the 2000-2500rpm mixing, more preferably with the 2200rpm mixing; Also can when placing, put back to side by side with the corresponding solution in the pipettor suction sample holes, thus mixing.The adding volume of corresponding solution determines according to the internal volume of concrete chip, and its volume is usually above 1/5th of the internal volume of chip, and can not overflow corresponding solution when chip is placed, and especially is not advisable not overflow corresponding solution at blending process.In a specific embodiment of the present invention, for foursquare Caliper-1000 type DMFLC, every hole adds the corresponding solution of 10 microlitre volumes.In each step, above-mentioned adding and the process that discards corresponding solution can repeat repeatedly, as 2-5 time, preferably repeat 3 times.The time of placing is generally 0.5-60 minute, is preferably 1-10 minute.The temperature of placing is generally 15-35 ℃, is preferably room temperature (that is, 23-27 ℃), is preferably 25 ℃.
More preferably, in the method for first aspect of the present invention, step (a) comprises, solution A is added the micro flow chip sample holes, and room temperature is placed after 1 minute and discard solution A from this chip; Step (b) comprises, solution B is added the micro flow chip sample holes, and room temperature is placed after 1 minute and discard solution B from this chip; And step (c) comprises, solution C is added the micro flow chip sample holes, and room temperature is placed after 1 minute and discard solution C from this chip.After step (c) is finished, can further micro flow chip be placed 5 minutes in room temperature.
After the cleaning of each step is all finished, can use the chip after such processing to carry out the next round sample analysis immediately; Reuse if be not eager, the chip after the cleaning of each step all can being finished is sealed with sealing film, be stored in 4 ℃-8 ℃ standby down, store 1～7 day usually after again repeated use can not influence result of use.Seal film during storage and should seal tightly, otherwise leave 20 ℃ of room temperatures in, can make that entire chip is dewatered, drying in air, causing at last can't reusable consequence.Therefore, if do not use for a long time the chip that cleaned, as reusing after after the cleaning 1～7 day, method of the present invention preferably also comprises again, after step (c) is finished, micro flow chip sealed and 4 ℃-8 ℃ steps of descending storage.In addition,, the liquid that discards as far as possible among the used MFLC to wherein adding solution A, can be placed the back and sealed each hole with sealing film, put into 4 ℃～8 ℃ preservations, carry out cleaning method of the present invention when waiting to have ready conditions again if having little time to clean.
Aspect second, the invention provides a kind of kit that is used to clean micro flow chip, it comprises three containers, it is characterized in that,
Container A comprises solution A, and wherein solution A contains sequestrant and detergent;
Container B comprises solution B, and wherein solution B contains sequestrant; And
Container C comprises solution C, and wherein solution C contains sequestrant.
Corresponding with the method for first aspect of the present invention, the kit of second aspect of the present invention can be used for cleaning micro flow chip, as DNA chip, RNA chip or protein-chip, especially can be used for cleaning disposable micro flow chip.Wherein, solution A, B and C are respectively solution A, B and the C described in the method for first aspect of the present invention.Usually preferred, detergent in the solution A is that the pH of non-ionic detergent and solution A is between 5.0 to 7.0, the also further alkali metal containing salt of preferred solution A, more preferably solution A contain EDTA, KCl and Triton X-100 and solution A pH between 5.5 to 6.5, most preferably solution A contains Tris-HCl, the 1mM EDTA-Na of 50mM pH6.0
2, 10mM KCl and 0.1% (weight) Triton X-100; The pH of solution B is between 6.0 to 8.0, and preferred solution B is further alkali metal containing salt also, more preferably solution B contain EDTA and KCl and solution B pH between 6.5 to 7.5, most preferably solution B contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 5mM KCl; And/or the pH of solution C is between 6.0 to 8.0, and preferred solution C also further contains antiseptic, and more preferably solution C contains EDTA and NaN
3And the pH of solution C is between 6.5 to 7.5, and most preferably solution C contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 0.05% (weight) NaN
3In a specific embodiment of the present invention, solution A, B and C select the prescription shown in the table 1-3 respectively for use, and ie in solution A contains Tris-HCl, the 1mM EDTA-Na of 50mM pH6.0
2, 10mM KCl and 0.1% (weight) Triton X-100; Solution B contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 5mM KCl; And solution C contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 0.05% (weight) NaN
3Also preferred, solution A, B and C contain sequestrant, detergent and/or the salt of higher concentration, perhaps are the water-soluble solid powder, to reduce volume, the convenient storage or transportation can be diluted to above-mentioned suitable working concentration with deionized water when using the solution of these high concentrations.
Solution A, B and C are loaded on respectively among container A, B and the C, and when the concentration of these solution was working concentration, the liquor capacity of being adorned is not less than finished the volume that cleans the required solution of micro flow chip, normally cleans the multiple of a required liquor capacity of micro flow chip.Container A, B and C be respectively can filling solution A, B and C do not destroy the container of corresponding solution composition, as glass container, plastic containers etc.Preferred airtight container A, B and C are to prevent solution evaporation wherein.Container A, B and C are contained in the parcel usually.
Aspect the 3rd, the invention provides a kind of method of reusing disposable micro flow chip, it is characterized in that disposable micro flow chip cleans this micro flow chip with the method for first aspect of the present invention, and then uses this micro flow chip after using.Repeated using method of the present invention can be widely used in each kind disposable micro flow chip, as disposable micro flow chips such as DNA chip, RNA chip, protein chips, treated chip quality is stable and reliable for performance, all can reach consistent result with original chip to the detection sensitivity of sample to be tested, specificity, repeatability.
Below will carry out exemplary description to the method for cleaning micro flow chip of the present invention and the method for kit and repeated use micro flow chip thereof by the drawings and specific embodiments.Yet, should be understood that the instantiation that provides only is to illustrate for example, obviously those of ordinary skill in the art can make various corrections and change to the present invention within the scope of the invention according to the explanation of instructions.
Description of drawings
Fig. 1 has shown the DMFLC that uses the first detection sensitivity to the DNA sample, wherein A to D successively concentration be 10
5, 10
4, 10
3With 10
2The test result of the pcr amplification product of the HBV of individual copy number/ml.
Fig. 2 has shown that the reusable again DMFLC of the same chip piece of no background is to the detection sensitivity of DNA sample after cleaning, and wherein A to D concentration is followed successively by and is respectively 10
5, 10
4, 10
3With 10
2The HBV of individual copy number/ml is through the test result of pcr amplification product.
Fig. 3 has shown the detection sensitivity of the DMFLC of use first to the RNA sample, and wherein A to C RNA concentration successively is respectively the test result of the Cy-5 cRNA sample of 1.0 μ g, 0.5 μ g and the every microlitre of 0.1 μ g.
Fig. 4 has shown that reusable DMFLC is to the detection sensitivity of RNA sample after cleaning, and wherein A to C concentration successively is respectively the test result of 1.0 μ g, 0.5 μ g and 0.1 μ g Cy-5cRNA sample.
Fig. 5 has shown the DMFLC that uses the first detection sensitivity to protein example, and wherein A to C human serum albumins concentration successively is respectively 50,20 and the test result of the every microlitre human serum albumins of 5.0ng sample.
Fig. 6 has shown after cleaning reusable DMFLC to the detection sensitivity of protein example, and wherein A to C human serum albumins concentration successively is respectively 50,20 and the test result of the every microlitre human serum albumins of 5.0ng sample.
Embodiment
1.DMFLC use
Getting a Caliper-1000 type blue DMFLC of foursquare DNA500 (available from U.S. Caliper LS company) upward tests dna sample at Caliper1000 micro flow chip analyser (available from U.S. Caliper LS company).The following summary of test process is finished in regular turn, and the step that does not specialize, reagent are all according to manufacturer's product description operation.
● add 6 μ l dyeing glue in the hole that two indicate G on chip respectively, be labeled as
The hole in add 4 μ l dyeing glue.
● get DNA500 quantitative mark (available from U.S. Agilent Technology), mixing.12 sample wells to Caliper-1000 type DMFLC add 5 μ l DNA500 quantitative mark respectively.Get DNA500 telltale mark (available from U.S. Agilent Technology), mixing.In chip telltale mark hole, add 2 μ lDNA500 telltale marks respectively.
● the pcr amplification product (originating from the HBV PCR micro flow chip kit of Shanghai Haoyuan Biotechnology Co., Ltd.) of getting HBV is respectively with 10
5, 10
4, 10
3With 10
2The sample of individual copy number/ml is in sample wells after the amplification that the 1 μ l amplified production adding of variable concentrations is different.
● the chip that application of sample is finished is put on the oscillator, with 2200-2300g vibration one minute (vibration velocity is as the criterion not allow in the hole liquid spill).After this in 5 minutes chip is placed on the Caliper1000 microflow analysis instrument and carries out interpretation of result.
● use the supporting analysis software of Caliper1000 micro flow chip analyser to analyze, the analysis result that successively decreases according to sample concentration is shown in Figure 1A to Fig. 1 D.
2.DMFLC cleaning reuse to measure
After analysis is finished, DMFLC is taken out from analyser, exhaust raffinate in each sample well and the telltale mark hole, clean DMFLC successively according to the following steps with pipettor.
● add the solution A of 10 μ L prescription as shown in table 1 respectively with pipettor in each hole of this used DMFLC, room temperature exhausts solution A with pipettor after placing 1 minute mixing.Triplicate like this.
● add the solution B of 10 μ L prescription as shown in table 2 respectively with pipettor in each hole of this used DMFLC, room temperature exhausts solution B with pipettor after placing 1 minute mixing.Triplicate like this.
● add the solution C of 10 μ L prescription as shown in table 3 respectively with pipettor in each hole of this used DMFLC, room temperature exhausts solution C with pipettor after placing 1 minute mixing.Triplicate like this.
The DMFLC that the process above-mentioned steps is handled has not had background and can reuse.
3.DMFLC reuse
Reuse DMFLC and analyze with the step identical with the present embodiment part 1, the analysis result that successively decreases according to sample concentration is shown in Fig. 2 A to Fig. 2 D.First the result of Shi Yonging (Figure 1A to Fig. 1 D) and after cleaning reusable result (Fig. 2 A to Fig. 2 D), the relatively demonstration of the picture of Fig. 1 and Fig. 2 same letter numbering, twice sensitivity for analysis and specific assay are the same, show that the result of use of reusable DMFLC after cleaning is identical with the testing result of the DMFLC that uses first, through cleaning reusable DMFLC.
The DMFLC (available from U.S. Caliper LS company) that gets a Caliper-1000 type RNA green goes up at Caliper1000 micro flow chip analyser (available from U.S. Caliper LS company) the RNA sample is tested.Operate with the step identical with embodiment 1, but wherein DNA500 quantitative mark and the specifically labelled step of DNA500 are used in cancellation, and replace the pcr amplification product of HBV with Cy-5cRNA sample (originating from AgilentTechnology company), wherein Cy-5 cRNA sample concentration be respectively 1.0ug, 0.5ug and the every microlitre of 0.1ug.The analysis result that successively decreases according to sample concentration after using first is shown in Fig. 3 A to Fig. 3 C; The reusable analysis result that successively decreases according to sample concentration is shown in Fig. 4 A to Fig. 4 C after cleaning.
First the result of Shi Yonging (Fig. 3) and after cleaning reusable result (Fig. 4), the relatively demonstration of the picture of Fig. 3 and Fig. 4 same letter numbering, twice sensitivity for analysis and specific assay are the same, show that the result of use of reusable DMFLC after cleaning is identical with the testing result of the DMFLC that uses first, through cleaning reusable DMFLC.
Getting a Caliper-1000 type DMFLC (available from U.S. Caliper LS company) upward tests the human serum albumins sample at Caliper1000 micro flow chip analyser (available from U.S. Caliper LS company).Operate with the step identical with embodiment 1, but wherein use standard protein molecular weight (available from AgilentTechnology company) to replace DNA500 quantitative mark and DNA500 telltale mark, and replace the pcr amplification product of HBV with human serum albumins (HSA) (originating from Shanghai Haoyuan Biotechnology Co., Ltd.), wherein the human serum albumins sample concentration be respectively 50,20 and the every microlitre of 5.0ng.The analysis result that successively decreases according to sample concentration after using first is shown in Fig. 5 A to Fig. 5 C; The reusable analysis result that successively decreases according to sample concentration is shown in Fig. 6 A to Fig. 6 C after cleaning.
First the result of Shi Yonging (Fig. 5) and after cleaning reusable result (Fig. 6), the relatively demonstration of the picture of Fig. 5 and Fig. 6 same letter numbering, twice sensitivity for analysis and specific assay are the same, show that the result of use of reusable DMFLC after cleaning is identical with the testing result of the DMFLC that uses first, through cleaning reusable DMFLC.
Claims (18)
1. method of cleaning micro flow chip, it in turn includes the following steps:
(a) clean micro flow chip with solution A, wherein solution A contains sequestrant and detergent, and wherein solution A contains the pH of EDTA, KCl and Triton X-100 and solution A between 5.5 to 6.5;
(b) clean micro flow chip with solution B, wherein solution B contains sequestrant, and wherein solution B contains the pH of EDTA and KCl and solution B between 6.5 to 7.5; And
(c) clean micro flow chip with solution C, wherein solution C contains sequestrant, and wherein solution C contains EDTA and NaN
3And the pH of solution C is between 6.5 to 7.5.
2. method according to claim 1, wherein micro flow chip is DNA chip, RNA chip or protein-chip.
3. method according to claim 1, wherein micro flow chip is disposable micro flow chip.
4. method of cleaning micro flow chip, it in turn includes the following steps:
(a) clean micro flow chip with solution A, wherein solution A contains sequestrant and detergent, and wherein solution A contains Tris-HCl, the 1mM EDTA-Na of 50mM pH6.0
2, 10mM KCl and 0.1% (weight) Triton X-100;
(b) clean micro flow chip with solution B, wherein solution B contains sequestrant, and wherein solution B contains the pH of EDTA and KCl and solution B between 6.5 to 7.5; And
(c) clean micro flow chip with solution C, wherein solution C contains sequestrant, and wherein solution C contains EDTA and NaN
3And the pH of solution C is between 6.5 to 7.5.
5. method of cleaning micro flow chip, it in turn includes the following steps:
(a) clean micro flow chip with solution A, wherein solution A contains sequestrant and detergent, and wherein solution A contains the pH of EDTA, KCl and Triton X-100 and solution A between 5.5 to 6.5;
(b) clean micro flow chip with solution B, wherein solution B contains sequestrant, and wherein solution B contains Tris-HCl, the 0.5mMEDTA-Na of 10mM pH7.0
2With 5mM KCl; And
(c) clean micro flow chip with solution C, wherein solution C contains sequestrant, and wherein solution C contains EDTA and NaN
3And the pH of solution C is between 6.5 to 7.5.
6. method of cleaning micro flow chip, it in turn includes the following steps:
(a) clean micro flow chip with solution A, wherein solution A contains sequestrant and detergent, and wherein solution A contains the pH of EDTA, KCl and Triton X-100 and solution A between 5.5 to 6.5;
(b) clean micro flow chip with solution B, wherein solution B contains sequestrant, and wherein solution B contains the pH of EDTA and KCl and solution B between 6.5 to 7.5; And
(c) clean micro flow chip with solution C, wherein solution C contains sequestrant, and wherein solution C contains Tris-HCl, the 0.5mMEDTA-Na of 10mM pH7.0
2With 0.05% (weight) NaN
3
7. method according to claim 1 is wherein cleaned micro flow chip and is comprised, corresponding solution is added the micro flow chip sample holes, places the back and discard this solution from this chip.
8. method according to claim 7, wherein be 0.5-60 minute standing time.
9. method according to claim 8, wherein be 1-10 minute standing time.
10. method according to claim 7, wherein laying temperature is 15-35 ℃.
11. method according to claim 10, wherein laying temperature is 23-27 ℃.
12. method according to claim 1, wherein,
Step (a) comprises, solution A is added the micro flow chip sample holes, and room temperature is placed after 1 minute and discard solution A from this chip;
Step (b) comprises, solution B is added the micro flow chip sample holes, and room temperature is placed after 1 minute and discard solution B from this chip; And
Step (c) comprises, solution C is added the micro flow chip sample holes, and room temperature is placed after 1 minute and discard solution C from this chip.
13. a kit that is used to clean micro flow chip, it comprises three containers, it is characterized in that,
Container A comprises solution A, and wherein solution A contains sequestrant and detergent, and wherein solution A contains the pH of EDTA, KCl and Triton X-100 and solution A between 5.5 to 6.5;
Container B comprises solution B, and wherein solution B contains sequestrant, and wherein solution B contains the pH of EDTA and KCl and solution B between 6.5 to 7.5; And
Container C comprises solution C, and wherein solution C contains sequestrant, and wherein solution C contains EDTA and NaN
3And the pH of solution C is between 6.5 to 7.5.
14. kit according to claim 13, wherein solution A contains Tris-HCl, the 1mM EDTA-Na of 50mMpH6.0
2, 10mM KCl and 0.1% (weight) Triton X-100.
15. kit according to claim 13, wherein solution B contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 5mM KCl.
16. kit according to claim 13, wherein solution C contains Tris-HCl, the 0.5mMEDTA-Na of 10mM pH7.0
2With 0.05% (weight) NaN
3
17. kit according to claim 13, wherein,
Solution A contains Tris-HCl, the 1mM EDTA-Na of 50mM pH6.0
2, 10mM KCl and 0.1% (weight) Triton X-100;
Solution B contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 5mM KCl; And solution C contains Tris-HCl, the 0.5mM EDTA-Na of 10mM pH7.0
2With 0.05% (weight) NaN
3
18. a method of reusing disposable micro flow chip is characterized in that disposable micro flow chip cleans this micro flow chip with each described method of claim 1 to 12, and then uses this micro flow chip after using.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100024523A CN101000339B (en) | 2007-01-22 | 2007-01-22 | Method for reusing microflow chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100024523A CN101000339B (en) | 2007-01-22 | 2007-01-22 | Method for reusing microflow chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101000339A CN101000339A (en) | 2007-07-18 |
| CN101000339B true CN101000339B (en) | 2011-05-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100024523A Expired - Fee Related CN101000339B (en) | 2007-01-22 | 2007-01-22 | Method for reusing microflow chip |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112048401B (en) * | 2020-08-28 | 2022-05-17 | 上海符贝基因科技有限公司 | Micro-fluidic chip cleaning agent and method thereof |
| CN115044417A (en) * | 2022-05-11 | 2022-09-13 | 苏州莱博睿思生物科技有限公司 | Cleaning fluid, preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1052138A (en) * | 1989-12-02 | 1991-06-12 | 山东大学 | A kind of clean-out system that is used for electronic industry and preparation method thereof |
| US5350458A (en) * | 1989-09-29 | 1994-09-27 | Boehringer Mannheim Gmbh | Method for cleaning a diagnostic analyzer |
| CN1335382A (en) * | 2001-07-23 | 2002-02-13 | 山东大学 | Scavenger composition for superlarge scale IC chip and its prepn |
| CN1680626A (en) * | 2004-04-09 | 2005-10-12 | 上海月旭半导体科技有限公司 | Cleaning liquid of semiconductor chip after chemical mechanical grind |
| CN1872434A (en) * | 2006-06-27 | 2006-12-06 | 济南市第四人民医院 | Method for cleaning out full automatic blood coagulation instrument in STAGO series |
-
2007
- 2007-01-22 CN CN2007100024523A patent/CN101000339B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5350458A (en) * | 1989-09-29 | 1994-09-27 | Boehringer Mannheim Gmbh | Method for cleaning a diagnostic analyzer |
| CN1052138A (en) * | 1989-12-02 | 1991-06-12 | 山东大学 | A kind of clean-out system that is used for electronic industry and preparation method thereof |
| CN1335382A (en) * | 2001-07-23 | 2002-02-13 | 山东大学 | Scavenger composition for superlarge scale IC chip and its prepn |
| CN1680626A (en) * | 2004-04-09 | 2005-10-12 | 上海月旭半导体科技有限公司 | Cleaning liquid of semiconductor chip after chemical mechanical grind |
| CN1872434A (en) * | 2006-06-27 | 2006-12-06 | 济南市第四人民医院 | Method for cleaning out full automatic blood coagulation instrument in STAGO series |
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|---|---|
| CN101000339A (en) | 2007-07-18 |
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