CN102662015B - Biological sample purification method based on inorganic salt promoted phase transition and separation of nano-gold - Google Patents

Abstract

The method for purifying biological samples based on inorganic salt-promoted phase change separation of nano-gold comprises the following steps: (1) according to the volume of biological samples containing nano-gold: the volume of hydrophilic organic solvent = 1: (0.5-10) to the Add a hydrophilic organic solvent to the biological sample and mix to obtain a mixed sample; (2) add the inorganic salt to the mixed sample according to the volume of the biological sample containing nano-gold: the mass of the inorganic salt = 1: (0.05-5), Sonicate for 1-3 minutes, then vortex for 1-3 minutes; (3) Stand still at a temperature of -20 to 20°C, or centrifuge to separate layers, and take the upper organic phase clear liquid. The invention does not need complex reagents and separation equipment, is low-consumption and environmentally friendly, is simple and fast, has strong adaptability and good repeatability, and can simultaneously realize the separation of nano gold in biological samples and the sufficient purification of substrates.

Description

Purification method of biological samples based on inorganic salt-promoted phase change separation of nano-gold

technical field

The invention relates to a method for purifying biological samples, in particular to a method for purifying biological samples based on inorganic salts promoting phase change to separate nano gold.

Background technique

In recent years, gold nanoparticles have been widely used in the field of biomedicine due to their unique physical and chemical properties. In the field of bio-nano analysis technology, the homogeneous biosensing technology based on the color change of the solution caused by the dispersion/aggregation of gold nanoparticles is still a research hotspot. However, due to the high dependence of the state of nanoparticles on the physical and chemical properties of the solution and the complex biological sample matrix is likely to seriously interfere with the detection signal of target molecules, how to achieve rapid separation of nanoparticles in complex biological sample matrices and direct and accurate detection of target molecules Detection is an important frontier topic in the field of nanomedicine and bioanalysis.

At present, high-speed or gradient centrifugation, membrane dialysis or ultrafiltration, electrophoresis, size-exclusion chromatography, nano-surface modification, solvent volatilization, and organic reagent phase conversion are generally used to separate nanoparticles. The disadvantages are that the operation is troublesome, time-consuming and laborious; The cost of the reagent used is high, the device used is complex and consumes high energy, and the economic benefit is low.

Recently, studies have found that the use of inorganic salt electrolytes can selectively precipitate gold nanoparticles of different shapes in the colloidal gold mixed preparation solution ( Chem. Commun ., 2011, 47 (14): 4180-4182 ), however, this method cannot effectively exclude complex Impurities in biological samples interfere with the detection of target substances, and it is also difficult to meet the pretreatment requirements of nano-gold separation and analyte purification in the analysis of biological samples, so it is impossible to directly use more effective instruments such as chromatography to detect target components. Make accurate measurements.

Contents of the invention

The technical problem to be solved by the present invention is to provide a biological sample based on inorganic salt-promoted phase change separation of nano-gold that is easy to operate, low in cost, and can simultaneously realize the separation of nano-gold and the purification of the analyte, thereby accurately measuring the target component. Purification method.

The technical solution adopted by the present invention to solve the technical problem is: a method for purifying biological samples based on inorganic salt-promoted phase transition separation of nano-gold, comprising the following steps:

(1) According to the volume of the biological sample containing nano-gold: the volume of the hydrophilic organic solvent = 1: (0.5-10) (preferably 1:0.8-2, more preferably 1:1) ratio, add hydrophilic to the biological sample Mix with a neutral organic solvent to obtain a mixed sample;

The biological samples containing gold nanoparticles include but not limited to animal and human body fluids, tissue homogenates, plant extracts, microbial extracts, in vitro biological samples, and the in vitro biological samples include cells, subcellular systems, and in vitro recombinant enzymes. And antigen-antibody reaction system;

(2) According to the volume of the biological sample containing nano-gold: the mass of the inorganic salt = 1: (0.05-5) (preferably 1:0.08-2, more preferably 1:0.1), add the inorganic salt to the mixed sample, and ultrasonically Dissolve for 1-3 minutes, then shake with a vortex shaker for 1-3 minutes;

(3) Stand at a temperature of -20-20°C (preferably 0-10°C, more preferably 4°C) for 8-15 minutes, or centrifuge at 2500-12000 rpm for 4-10 minutes, then take the upper organic phase to clear The solution was directly used for liquid chromatography injection analysis, and the lower layer containing gold nanoparticles was transferred to a new centrifuge tube and reconstituted with pure water ultrasonically.

Further, the hydrophilic organic solvent may be acetonitrile or acetone and other hydrophilic organic solvents, preferably acetonitrile.

Further, the inorganic salt can be sodium chloride (better for gold coagulation effect of ≤ 5 nm particles), magnesium sulfate (better for gold coagulation effect of 85-100 nm particles), sodium sulfate, magnesium chloride One or more mixtures, preferably a mixture of sodium chloride and magnesium sulfate, more preferably a mixture of sodium chloride and magnesium sulfate with a mass ratio of 1:1.

In the present invention, the biological sample solution containing nano-gold is miscible with hydrophilic organic solvents such as acetonitrile or acetone, so that the biological sample matrix is diluted and denatured, which is beneficial to the separation of nanoparticles and samples and the dispersion of the target object to be measured in the solvent. Then introduce an inorganic salt electrolyte, use its dual effects of inducing colloidal gold coagulation and salting out phase separation extraction, and then stand at low temperature or centrifuge to separate the aqueous solution (containing nano-gold) and hydrophilic organic solvent (containing the target component) It is separated into upper and lower phases from a single miscible system. Since the low temperature will accelerate the phase separation of the water-acetonitrile system, complete separation can be achieved by standing still for a short time.

Among them, the nanoparticles in the mixed sample coagulate and sink into the lower water phase, and the coagulation removal rate can reach 98% according to the absorbance value of the ultraviolet-visible spectrum. There is no significant change in size and shape; the components to be tested are enriched in the upper organic phase, and the separated and purified organic solvent (that is, the supernatant) can be directly used for chromatographic analysis (the extraction recovery rate is over 95%).

Compared with the prior art, the present invention has the following advantages: the hydrophilic organic solvents and inorganic salts used are cheap and easy to obtain, and the cost is low; there is no need to use complicated separation equipment, low consumption and environmental protection; simple and fast, strong adaptability and good repeatability , can simultaneously realize rapid separation of colloidal gold, enrichment and extraction of target compounds from homogeneous sample solutions, and can be widely used in the separation and matrix of nano-gold in various biological samples such as blood, tissue homogenate, enzymes, recombinant proteins, and cell extracts purification.

Description of drawings

Fig. 1 is the electron micrograph of embodiment of the present invention 2 gained lower floor aqueous phases;

Fig. 2 is the high-efficiency liquid phase chromatogram of direct sample injection of the supernatant purified supernatant obtained in Example 2 of the present invention.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1

This embodiment includes the following steps:

(1) Take 200 microliters of the in vitro drug metabolism enzyme reaction system sample, add 200 microliters of glacial acetonitrile to the sample, and vortex for 2 minutes to obtain a mixed sample;

Preparation of samples of the in vitro drug metabolism enzyme reaction system (represented by CYP2D6 in cytochrome P450):

In 135 microliters of phosphate buffer (100 mmol/L, pH 7.4), 5 microliters of thawed human liver microsomes (protein concentration: 20 mg/mL) and 10 microliters of specific substrate drugs were added sequentially. Methorphan (0.4 mmol/L, prepared by phosphate buffer), 10 microliters of nano-gold aqueous solution (10 nm, 1 mg/mL), mixed well, pre-incubated at 37°C for 5 minutes; then added 40 microliters of NADPH Solution (50 mmol/L, temporarily prepared from phosphate buffer), react at 37°C for 10 minutes;

(2) Add 0.02 g of a mixture of sodium chloride and magnesium sulfate to the mixed sample (mass of sodium chloride: mass of magnesium sulfate = 1:1), ultrasonically dissolve for 2 minutes, and then shake for 1 minute with a vortex shaker;

(3) Put it in a refrigerator at 4°C for 10 minutes, obtain 160 microliters of the upper layer organic solution after phase separation, take 10 microliters of the supernatant and inject directly into the sample, and detect dextromethorphan and its metabolite nordextromethorphan Perform high-performance liquid chromatography-fluorescence detection analysis.

The results show that the nanoparticles and the sample matrix are completely separated, and repeated injections (30 times) do not affect the baseline background, retention time, peak shape and signal intensity of the chromatographic column separation. After this treatment, the recoveries of the two targets are both 97%. .

Example 2

This embodiment includes the following steps:

(1) Take 200 microliters of human serum samples containing colloidal gold (100 nm, 0.05 mg/mL), add 200 microliters of glacial acetonitrile to the samples, and vortex for 2 minutes to obtain mixed samples;

Add dextromethorphan medicine and its metabolite nordextromethorphan in described human serum, the concentration of described dextromethorphan medicine is 0.05mmol/L, the concentration of described nordextromethorphan is 8 mmol/L;

(2) Add 0.02 g of magnesium sulfate to the mixed sample, ultrasonically dissolve for 2 minutes, and vortex for 1 minute;

(3) Centrifuge at a speed of 3000 rpm for 5 minutes, obtain 160 microliters of the upper layer organic solution after phase separation, take 10 microliters of the supernatant and directly inject the sample, and carry out dextromethorphan and its metabolite nordextromethorphan HPLC-fluorometric analysis.

The results showed that the nanoparticles and the sample matrix were completely separated, repeated injections (30 times) did not affect the baseline background, retention time, peak shape and signal intensity of the chromatographic column separation, the recovery rate of the target dextromethorphan was 96%, and the target was removed. The recovery rate of dextromethorphan was 98%.

Example 3

This embodiment includes the following steps:

(1) Take 200 microliters of Saccharomyces cerevisiae extract sample, add 200 microliters of acetone to the sample, and vortex for 2 minutes to obtain a mixed sample;

Preparation of the Saccharomyces cerevisiae extract sample: 10 microliters of specific substrate drugs were sequentially added to 140 microliters of Saccharomyces cerevisiae microsomal phosphate buffer (protein concentration 20 mg/mL, 100 mmol/L, pH 7.4) Dextromethorphan (0.4 mmol/L, prepared by phosphate buffer), 10 microliters of nano-gold aqueous solution (10 nm, 1 mg/mL), mix well, and pre-incubate at 37°C for 5 minutes; then add 40 microliters NADPH solution (50 mmol/L, temporarily prepared from phosphate buffer), react at 37°C for 10 minutes;

(2) Add 0.06 g of sodium chloride to the mixed sample, ultrasonically dissolve it for 2 minutes, and shake it with a vortex shaker for 1 minute;

(3) Put it in a refrigerator at 4°C and let it stand for 10 minutes. After phase separation, 140 microliters of the upper organic solution was obtained. Take 10 microliters of the supernatant and inject it directly. Fen was analyzed by high performance liquid chromatography-fluorescence detection.

The results showed that the nanoparticles and the sample matrix were completely separated, repeated injections (30 times) did not affect the baseline background, retention time, peak shape and signal intensity of the column separation, the target dextromethorphan and its metabolite nordextromethorphan The recoveries were all 96%.

Claims (7)

1. A method for purifying biological samples based on inorganic salts promoting phase change separation of nano-gold, characterized in that, comprising the following steps: (1) According to the volume of the biological sample containing nano-gold: the volume of the hydrophilic organic solvent = 1: (0.5-10) ratio, add the hydrophilic organic solvent to the biological sample and mix to obtain a mixed sample; The biological samples containing gold nanoparticles include animal and human body fluids, tissue homogenates, plant extracts, microbial extracts, and in vitro biological samples, wherein in vitro biological samples include cells, subcellular systems, in vitro recombinant enzymes, and antigen-antibody reaction system; (2) According to the volume of the biological sample containing nano-gold: the mass of the inorganic salt = 1ml: (0.05-5) g, add the inorganic salt to the mixed sample, ultrasonically dissolve it for 1-3 minutes, and then shake it with a vortex shaker 1-3 minutes; (3) Stand still at a temperature of -20-20°C for 8-15 minutes, or centrifuge at 2500-12000 rpm for 4-10 minutes, take the upper organic phase clear liquid and directly use it for liquid chromatography sample analysis, containing The lower layer of gold nanoparticles was transferred to a new centrifuge tube and reconstituted with pure water sonication. 2. The purification method of biological samples based on inorganic salt-promoted phase transition separation of nano-gold according to claim 1, characterized in that: the hydrophilic organic solvent is acetonitrile or acetone. 3. according to claim 1 and 2, based on the purification method of the biological sample of inorganic salt promoting phase change separation nano gold, it is characterized in that: described inorganic salt is one of sodium chloride, magnesium sulfate, sodium sulfate, magnesium chloride species or a mixture of several species. 4. the method for purifying biological samples based on inorganic salt-promoted phase change separation of nano-gold according to claim 3, characterized in that: the inorganic salt is a mixture of sodium chloride and magnesium sulfate with a mass ratio of 1:1. 5. according to claim 1 or 2, based on the purification method of the biological sample of inorganic salt promoting phase change separation nano-gold, it is characterized in that: step (1), the volume of described biological sample containing nano-gold: hydrophilicity The volume of organic solvent=1:(0.8-2). 6. according to claim 1 or 2, based on the purification method of the biological sample of inorganic salt promoting phase change separation of nano-gold, it is characterized in that: step (2), the volume of the described biological sample containing nano-gold: the volume of inorganic salt Mass=1ml:(0.08-2)g. 7. The method for purifying biological samples based on inorganic salt-promoted phase transition separation of gold nanoparticles according to claim 1 or 2, characterized in that in step (3), the standing temperature is 0-10°C.

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