CN102662015B - Biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold - Google Patents
Biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold Download PDFInfo
- Publication number
- CN102662015B CN102662015B CN2012101058284A CN201210105828A CN102662015B CN 102662015 B CN102662015 B CN 102662015B CN 2012101058284 A CN2012101058284 A CN 2012101058284A CN 201210105828 A CN201210105828 A CN 201210105828A CN 102662015 B CN102662015 B CN 102662015B
- Authority
- CN
- China
- Prior art keywords
- biological sample
- gold
- inorganic salts
- purification method
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
A biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold comprises the following steps of: (1) adding a hydrophilic organic solvent into a biological sample according to the volume ratio of the biological sample containing nano-gold to the hydrophilic organic solvent being 1: (0.5-10), and uniformly mixing to obtain a mixed sample; (2) adding inorganic salt into the mixed sample according to the ratio of the volume of the biological sample containing nano-gold to the weight of inorganic salt being 1: (0.05-5), followed by ultrasonic dissolving for 1-3 minutes, and oscillating for 1-3 minutes by the use of a vortex oscillator; and (3) standing at the temperature of minus 20-20 DEG C, or centrifuging and partitioning, and taking an upper layered organic phase clear liquid. The method provided by the invention requires no complex reagent and separation equipment, has advantages of low consumption, environmental protection, strong adaptability and good repeatability, is simple and rapid, and can be used to simultaneously realize separation of nano-gold and sufficient purification of matrix in the biological sample.
Description
Technical field
The present invention relates to a kind of purification method of biological sample, especially relate to a kind of purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts.
Background technology
In recent years, nm of gold was widely used in biomedicine field due to its unique physicochemical characteristics.In biology-nanoanalysis technical field, the homogeneous phase biosensor technique that due to disperseing/gather based on nm of gold, solution colour changes is still study hotspot.But, because the nano particle state easily produces severe jamming to the target molecule detection signal to the high dependency of solution physicochemical property and complex biological sample matrix, therefore, how realizing the quick separation of nano particle in numerous and diverse biological sample matrix and direct, the accurately detection of target molecule, is an important front edge problem in current nanosecond medical science and bioanalysis field.
At present, separate that nano particle generally adopts at a high speed or the methods such as gradient centrifugation, film dialysis or ultrafiltration, electrophoresis, exclusion chromatography, nano-sized surface modification, solvent evaporates, organic reagent phase transformation, the shortcoming of its existence is troublesome poeration, time and effort consuming; The agents useful for same cost is higher, and equipment therefor complexity and energy consumption are high, and economic benefit is lower.
Recently, research find to adopt the inorganic salts electrolyte can selective precipitation go out collaurum be mixed with difform nano Au particle in liquid (
Chem. Commun., 2011,47 (14): 4180-4182), yet the method can not effectively be got rid of impurity in complex biological sample the detection of object is disturbed, be difficult to also meet that the nm of gold that faces simultaneously in biological sample analysis is separated and many pre-treatment requirements such as determinand purification, thereby can't directly use more effective instrument means such as chromatogram to do mensuration accurately to target components.
Summary of the invention
Technical matters to be solved by this invention is, provides a kind of easy and simple to handle, and cost is low, can realize simultaneously that nm of gold is separated and determinand is purified, thus the purification method of the biological sample based on the short phase transformation separation of inorganic salts nm of gold of Accurate Determining target components.
The technical scheme that the present invention solves its technical matters employing is: the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts comprises the following steps:
(1) by the volume of the biological sample that contains nm of gold: the volume of hydrophilic organic solvent=1:(0.5-10) (preferred 1:0.8-2, more preferably 1:1) ratio adds hydrophilic organic solvent to mix in biological sample, obtains biased sample;
The described biological sample that contains nm of gold, include but not limited to animal and human's body fluid, tissue homogenate, plant extraction liquid, microorganism extract, external biological sample, described external biological sample comprises cell, subcellular fraction system, vitro recombination enzyme and antigen-antibody reaction system;
(2) by the volume of the biological sample that contains nm of gold: (the preferred 1:0.08-2 of the quality of inorganic salts=1:(0.05-5), more preferably 1:0.1) ratio adds inorganic salts in biased sample, ultrasonic dissolution 1-3 minute, then with the vortex oscillator, shake 1-3 minute;
(3) ℃ (the preferred 0-10 ℃ in temperature-20～20, more preferably 4 ℃) lower standing 8-15 minute, or after 2500-12000 rev/min of centrifugal 4-10 minute, get the upper organic phase clear liquid, be directly used in the analysis of liquid chromatography sample introduction, the subnatant that contains nm of gold moves in new centrifuge tube with the ultrasonic redissolution of pure water.
Further, described hydrophilic organic solvent can be the hydrophilic organic solvents such as acetonitrile or acetone, preferred acetonitrile.
Further, described inorganic salts can be one or more the potpourri in sodium chloride (particle of right≤5 nm gold coagulation effect is better), magnesium sulphate (the particle gold coagulation effect to 85-100 nm is better), sodium sulphate, magnesium chloride, the potpourri of preferred sodium chloride and magnesium sulphate, the more preferably potpourri of the sodium chloride of mass ratio 1:1 and magnesium sulphate.
Biological sample solution and the hydrophilic organic solvents such as acetonitrile or acetone that the present invention first will contain nm of gold dissolve each other, make the dilution of biological sample matrix and sex change, be conducive to separating and the dispersion of object to be measured in solvent of nano particle and sample, then introduce the inorganic salts electrolyte, utilize it to induce the collaurum coagulation and the double effect of the phase-splitting extraction of saltouing, through stand at low temperature or centrifugal, aqueous solution (containing nm of gold) and hydrophilic organic solvent (containing target components) are separated into the up and down two-phase from the single-phase system of dissolving each other again.Because low temperature can be accelerated water-acetonitrile system phase-splitting, the short time, standing can reaching fully separated.
Wherein the nano particle coagulation in biased sample enters lower floor's water, by ultraviolet-visible spectrum absorbance numerical evaluation coagulation clearance, can reach 98%, and lower floor's gathering nm of gold can heavily be dissolved through pure water, and after finding to redissolve by electron microscopic observation, its size form is without marked change; Component to be measured is enriched in upper organic phase, and the organic solvent after separating-purifying (being supernatant liquor) can be directly used in stratographic analysis (extraction recovery reaches more than 95%).
Compared with prior art, the present invention has the following advantages: hydrophilic organic solvent used, inorganic salts cheaply are easy to get, and cost is low; Need not to use complicated separation equipment, low consumption environment protection; Easy fast, strong adaptability, good reproducibility, collaurum, enrichment extraction target compound can be realized simultaneously separating fast from the homogeneous sample solution, the separation of nm of gold in the various biological samples such as blood, tissue homogenate, enzyme, recombinant protein, cell extract and the purification of matrix can be widely used in.
Description of drawings
Fig. 1 is the Electronic Speculum figure of the embodiment of the present invention 2 gained lower floor waters;
Fig. 2 is the high-efficient liquid phase chromatogram of the embodiment of the present invention 2 gained upper strata purified clear liquid direct injected.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
The present embodiment comprises the following steps:
(1) get external drug metabolic enzyme reaction system sample 200 microlitres, add 200 microlitre ice acetonitriles in sample, vortex concussion 2 minutes, obtain biased sample;
The preparation of described external drug metabolic enzyme reaction system sample (representative take the CYP2D6 in Cytochrome P450 as enzyme reaction):
At 135 microlitre phosphate buffer (100 mmol/L, pH7.4) add successively the people's hepatomicrosome (protein concentration is 20 mg/mL) after 5 microlitres thaw in, 10 microlitre specific substrate medicine dextromethorphan (0.4 mmol/L, by the phosphate buffer preparation), 10 microlitre nm of gold aqueous solution (10 nm, 1 mg/mL), mix, 37 ℃ of preincubates 5 minutes; And then add 40 microlitre NADPH solution (50 mmol/L are prepared by phosphate buffer temporarily), 37 ℃ of reactions 10 minutes;
(2) to the potpourri that adds 0.02 g sodium chloride and magnesium sulphate in the biased sample (quality of sodium chloride: the quality of magnesium sulphate=1:1), ultrasonic dissolution 2 minutes, then with vortex oscillator concussion 1 minute;
(3) be placed in standing 10 minutes of the refrigerator of 4 ℃, obtain upper strata organic solution 160 microlitres after phase-splitting, get 10 microlitre supernatant direct injected, to dextromethorphan and metabolic product thereof, go the first dextromethorphan to carry out high performance liquid chromatography-fluoroscopic examination analysis.
Result shows that nano particle separates with sample substrate fully, and repeating sample introduction (30 times) does not affect baseline background, retention time, peak shape and the signal intensity that chromatographic column is separated, and through this recovery of processing latter two object, is 97%.
Embodiment 2
The present embodiment comprises the following steps:
(1) get 200 microlitres and contain the human serum sample of collaurum (100 nm, 0.05 mg/mL), add 200 microlitre ice acetonitriles in sample, vortex concussion 2 minutes, obtain biased sample;
Be added with dextromethorphan medicine and metabolin thereof in described human serum and remove the first dextromethorphan, the concentration of described dextromethorphan medicine is 0.05mmol/L, and described to go the concentration of first dextromethorphan be 8 mmol/L;
(2) add 0.02 g magnesium sulphate in biased sample, ultrasonic dissolution 2 minutes, vortex concussion 1 minute;
(3) under 3000 rev/mins of rotating speeds centrifugal 5 minutes, obtain upper strata organic solution 160 microlitres after phase-splitting, get 10 microlitre supernatant direct injected, go the first dextromethorphan to carry out high performance liquid chromatography-fluorometric assay analysis to dextromethorphan and metabolic product thereof.
Result shows that nano particle separates with sample substrate fully, repeat sample introduction (30 times) and do not affect baseline background, retention time, peak shape and the signal intensity that chromatographic column is separated, the recovery of object dextromethorphan is 96%, and it is 98% that object goes the recovery of first dextromethorphan.
Embodiment 3
The present embodiment comprises the following steps:
(1) get saccharomyces cerevisiae extract sample 200 microlitres, add 200 microlitre acetone in sample, vortex concussion 2 minutes, obtain biased sample;
The preparation of described saccharomyces cerevisiae extract sample: at 140 microlitre saccharomyces cerevisiae microsome phosphate buffer (protein concentration 20 mg/mL, 100 mmol/L, pH7.4) add successively 10 microlitre specific substrate medicine dextromethorphan (0.4 mmol/L in, by the phosphate buffer preparation), 10 microlitre nm of gold aqueous solution (10 nm, 1 mg/mL), mix, 37 ℃ of preincubates 5 minutes; And then add 40 microlitre NADPH solution (50 mmol/L are prepared by phosphate buffer temporarily), 37 ℃ of reactions 10 minutes;
(2) add 0.06 g sodium chloride in biased sample, ultrasonic dissolution 2 minutes, then with the vortex oscillator, shook 1 minute;
(3) be placed in the refrigerator of 4 ℃, standing 10 minutes, obtain upper strata organic solution 140 microlitres after phase-splitting, get 10 microlitre supernatant direct injected, go the first dextromethorphan to carry out high performance liquid chromatography-fluoroscopic examination analysis to dextromethorphan and metabolic product thereof.
Result shows that nano particle separates with sample substrate fully, and repeating sample introduction (30 times) does not affect baseline background, retention time and peak shape and the signal intensity that chromatographic column is separated, and object dextromethorphan and metabolic product thereof go the recovery of first dextromethorphan to be 96%.
Claims (7)
1. the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts, is characterized in that, comprises the following steps:
(1) in the volume of the biological sample that contains nm of gold: the volume of hydrophilic organic solvent=1:(0.5-10) ratio adds hydrophilic organic solvent to mix in biological sample, obtains biased sample;
The described biological sample that contains nm of gold, comprise animal and human's body fluid, tissue homogenate, plant extraction liquid, microorganism extract and external biological sample, wherein the external biological sample comprises cell, subcellular fraction system, vitro recombination enzyme and antigen-antibody reaction system;
(2) in the volume of the biological sample that contains nm of gold: the quality of inorganic salts=1ml:(0.05-5) ratio of g adds inorganic salts in biased sample, ultrasonic dissolution 1-3 minute, then with vortex oscillator concussion 1-3 minute;
(3) standing 8-15 minute under temperature-20～20 ℃, or after 2500-12000 rev/min of centrifugal 4-10 minute, get the upper organic phase clear liquid and be directly used in the analysis of liquid chromatography sample introduction, the subnatant that contains nm of gold moves in new centrifuge tube with the ultrasonic redissolution of pure water.
2. the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts according to claim 1, it is characterized in that: described hydrophilic organic solvent is acetonitrile or acetone.
3. the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts according to claim 1 and 2 is characterized in that: described inorganic salts are one or more the potpourri in sodium chloride, magnesium sulphate, sodium sulphate, magnesium chloride.
4. the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts according to claim 3, it is characterized in that: described inorganic salts are the sodium chloride of mass ratio 1:1 and the potpourri of magnesium sulphate.
5. the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts according to claim 1 and 2 is characterized in that: step (1), and the described volume that contains the biological sample of nm of gold: the volume of hydrophilic organic solvent=1:(0.8-2).
6. the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts according to claim 1 and 2 is characterized in that: step (2), the described volume that contains the biological sample of nm of gold: the g of the quality of inorganic salts=1ml:(0.08-2).
7. the purification method that short phase transformation separates the biological sample of nm of gold based on inorganic salts according to claim 1 and 2, it is characterized in that: step (3), described standing temperature is 0-10 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101058284A CN102662015B (en) | 2012-04-12 | 2012-04-12 | Biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101058284A CN102662015B (en) | 2012-04-12 | 2012-04-12 | Biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102662015A CN102662015A (en) | 2012-09-12 |
| CN102662015B true CN102662015B (en) | 2013-11-20 |
Family
ID=46771547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101058284A Expired - Fee Related CN102662015B (en) | 2012-04-12 | 2012-04-12 | Biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102662015B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111001184A (en) * | 2019-12-25 | 2020-04-14 | 中国科学院长春应用化学研究所 | Method for efficiently inducing water-organic solvent mixed solution phase separation by inorganic salt |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5772888A (en) * | 1995-07-27 | 1998-06-30 | Massachusetts Institute Of Technology | Separation and/or concentration of an analyte from a mixture using a two-phase aqueous micellar system |
| US6437101B1 (en) * | 1999-05-07 | 2002-08-20 | Akzo Nobel N.V. | Methods for protein purification using aqueous two-phase extraction |
| CN1589246A (en) * | 2001-09-20 | 2005-03-02 | 江崎格力高株式会社 | Method for extracting and method for purifying effective substance |
| CN101915818A (en) * | 2010-08-13 | 2010-12-15 | 东北农业大学 | LC-MS/MS method for determining milbemycin oxime content of animal plasma |
| CN102353571A (en) * | 2011-09-20 | 2012-02-15 | 苏州东奇生物科技有限公司 | Viscous biological sample purifier and pre-processing method of viscous biological sample |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011159537A2 (en) * | 2010-06-15 | 2011-12-22 | The Regents Of The University Of California | Method and device for analyte detection |
-
2012
- 2012-04-12 CN CN2012101058284A patent/CN102662015B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5772888A (en) * | 1995-07-27 | 1998-06-30 | Massachusetts Institute Of Technology | Separation and/or concentration of an analyte from a mixture using a two-phase aqueous micellar system |
| US6437101B1 (en) * | 1999-05-07 | 2002-08-20 | Akzo Nobel N.V. | Methods for protein purification using aqueous two-phase extraction |
| CN1589246A (en) * | 2001-09-20 | 2005-03-02 | 江崎格力高株式会社 | Method for extracting and method for purifying effective substance |
| CN101915818A (en) * | 2010-08-13 | 2010-12-15 | 东北农业大学 | LC-MS/MS method for determining milbemycin oxime content of animal plasma |
| CN102353571A (en) * | 2011-09-20 | 2012-02-15 | 苏州东奇生物科技有限公司 | Viscous biological sample purifier and pre-processing method of viscous biological sample |
Non-Patent Citations (12)
| Title |
|---|
| Coupling of acetonitrile deproteinization and salting-out extraction with acetonitrile stacking for biological sample clean-up and the enrichment of hydrophobic compounds (porphyrins) in capillary electrophoresis;Qi Li et al;《Electrophoresis》;20061130;第27卷(第21期);第4219-4229页 * |
| Jun Zhang et al.Salting-out assisted liquid/liquid extraction with acetonitrile: a new high throughput sample preparation technique for good laboratory practice bioanalysis using liquid chromatography–mass spectrometry.《Biomedical Chromatography》.2009,第23卷(第4期), |
| Nucleotide-Mediated Size Fractionation of Gold Nanoparticles in Aqueous Solutions;Wenting Zhao et al;《Langmuir》;20100518;第26卷(第10期);第7405-7409页 * |
| Qi Li et al.Coupling of acetonitrile deproteinization and salting-out extraction with acetonitrile stacking for biological sample clean-up and the enrichment of hydrophobic compounds (porphyrins) in capillary electrophoresis.《Electrophoresis》.2006,第27卷(第21期),第4219–4229页. |
| Salting-out assisted liquid/liquid extraction with acetonitrile: a new high throughput sample preparation technique for good laboratory practice bioanalysis using liquid chromatography–mass spectrometry;Jun Zhang et al;《Biomedical Chromatography》;20090430;第23卷(第4期);第419-425页 * |
| Shape separation of colloidal gold nanoparticles through salt-triggered selective precipitation;Zhirui Guo et al;《Chemical Communications》;20111231;第47卷(第14期);第4180-4182页 * |
| Wenting Zhao et al.Nucleotide-Mediated Size Fractionation of Gold Nanoparticles in Aqueous Solutions.《Langmuir》.2010,第26卷(第10期), |
| Zhirui Guo et al.Shape separation of colloidal gold nanoparticles through salt-triggered selective precipitation.《Chemical Communications》.2011,第47卷(第14期), |
| 李章万等.色谱分析中生物样品的前处理方法.《药物分析杂志》.1996,第16卷(第1期), |
| 生物样品中6种抗凝血鼠药的测定;蔡增轩等;《中国卫生检验杂志》;20081031;第18卷(第10期);第2006-2008,2145页 * |
| 色谱分析中生物样品的前处理方法;李章万等;《药物分析杂志》;19960131;第16卷(第1期);第55-59页 * |
| 蔡增轩等.生物样品中6种抗凝血鼠药的测定.《中国卫生检验杂志》.2008,第18卷(第10期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102662015A (en) | 2012-09-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103454360B (en) | Ultrafiltration and UPLC-MS/MS (ultra-high performance liquid chromatography tandem mass spectrometry) method for measuring concentration of free docetaxel in human plasma | |
| CN103149308B (en) | Pretreatment method for detecting BPA (bisphenol A) in environment samples | |
| CN104017800A (en) | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof | |
| CN102839169B (en) | Kits for extracting enterovirus RNA and corresponding method for extracting and purifying enterovirus RNA | |
| CN104962553A (en) | Using method for extracting virus DNA by using micro-nucleic acid releasing agent | |
| CN105136957A (en) | Detection method for simultaneously measuring OXC in human plasma and metabolite MHD and MHD-G | |
| CN103710338A (en) | Kit for extracting DNA (deoxyribonucleic acid) from white cells in human whole blood | |
| CN112834631A (en) | Single-cell single-tube sample preparation and single-cell proteomics analysis method | |
| CN103191581B (en) | Method for separating/gathering trace ciprofloxacin in food | |
| CN102662015B (en) | Biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold | |
| CN105385680B (en) | For the reagent that DNA and RNA is extracted simultaneously, extracting method and purposes | |
| CN104330516A (en) | Method for simultaneously determining content of allantoin and adenosine in Chinese yams | |
| CN103184214A (en) | Hepatitis B virus nucleic acid rapid extraction reagent | |
| Ramos-Garcia et al. | Isolation and lipidomic screening of human milk extracellular vesicles | |
| CN102539546A (en) | Methods for detecting content of free carnitine or content of total carnitine in body fluid | |
| CN113533548B (en) | Method for detecting 1-vinyl imidazole in chemical product | |
| CN103163225A (en) | High-sensitivity quantitative detection kit for cyclosporine A in whole blood and preparation method thereof | |
| CN107505422B (en) | Method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time | |
| CN101880706A (en) | Direct fungus detection tachypleus amebocyte lysate box and method | |
| CN103940918A (en) | A method of simultaneously detecting the content of artesunate and the content of dihydroartemisinin in animal blood plasma | |
| Rahimi et al. | Development of a dispersive liquid-liquid microextraction method based on solidification of a floating organic drop for the determination of beta-carotene in human serum | |
| CN103954717A (en) | Method for testing hemoglobin concentration by utilizing liquid chromatogram tandem mass spectrum | |
| CN109254104A (en) | The detection method of residues of organophosphate pesticides in a kind of grape wine | |
| CN112014198B (en) | Kit and method for enriching endogenous low-molecular-weight proteins and polypeptides | |
| Hsu et al. | Isolating cells from blood using buoyancy activated cell sorting (BACS) with glass microbubbles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131120 Termination date: 20170412 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |