CN102030316A

CN102030316A - Extracting and purifying method of cadmium selenide quantum dot in yeast cells

Info

Publication number: CN102030316A
Application number: CN 201010562410
Authority: CN
Inventors: 谢志雄; 庞代文; 张鹏; 李勇
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2010-11-26
Filing date: 2010-11-26
Publication date: 2011-04-27
Anticipated expiration: 2030-11-26
Also published as: CN102030316B

Abstract

The invention discloses an extracting and purifying method of cadmium selenide quantum dots in yeast cells. The invention comprises the steps of 1) yeast cell wall lysis: centrifuging yeast cell culture solution of synthesized cadmium selenide quantum dots, resuspending precipitation in buffer solution for cell lysis, adding cell lytic enzyme, and carrying out cell wall lysis by means of oscillating and incubating; 2) yeast cell crushing: centrifugally collecting the processed cells, weight dropping in the buffer solution for cell lysis, adding protease inhibitor, and ultrasonically crushing the cell; 3) rough separation of the cadmium selenide quantum dot: centrifuging the processed sample, and collecting supernate; 4) fine separation of the cadmium selenide quantum dot: adding dodecyl sodium sulfate into the collected supernate, centrifugally collecting the supernate, and filtering, ultra-filtrating and washing the supernate; and 5) cadmium selenide quantum dot purification: centrifuging ficoll by means of density gradient, ultra-filtrating and washing to obtain purified cadmium selenide quantum dots. The invention is simple in method, high in extraction ratio, stable in product and safe, and does not refer to poisonous reagent and the dangerous reagent.

Description

CdSe quantum dots extraction, purification process in the yeast cell

Technical field

The invention belongs to microbiology, biological chemistry, nanoscale science and technology field, the extraction, the purification process that more specifically relate to synthetic CdSe quantum dots in a kind of brewing yeast cell are applicable to the separation preparation of the interior synthetic other types quantum dot of other microorganism cellss or other nano materials.

Background technology

Quantum dot is a kind of novel nano-material that grows up recent years, its synthetic classical chemical process such as metallic compound-element organic synthesis method, the inorganic synthesis method of water that always adopts, and being synthesized is exactly purer quantum dot.1989 NatureOn reported the earliest can be in microorganism synthesizing cadmium sulfide quantum dot, biosynthesizing quantum dot development after this rapidly, dissimilar quantum dots also are synthesized in cell in succession.

The relative traditional chemical synthesis method of the method for biosynthesizing quantum dot is with the obvious advantage, but unique limiting factor is that the synthetic quantum dot is not easy to be extracted effectively in cell.Quantum dot can utilize magnetic force to separate with cellular component unlike the magnetic corpusculum.The synthetic quantum dot is wrapped up and modifies by biomacromolecule in the cell, the stability of quantum dot depends on the stable of these biomacromolecules, in case these parcels or the biomacromolecule of modifying outside quantum dot are destroyed, the synthetic quantum dot has not just existed yet in the cell so.Therefore how guaranteeing under the prerequisite that quantum dot stability, natural structure are not destroyed, extraction of synthetic quantum dot and purifying in the cell are being come out effectively " tenderness ", is a major challenge that the synthetic field of current quanta point biological faces.

Yeast is the eukaryotic microorganisms that a class has cell walls, and this laboratory was published in 2009 Advanced Functional MaterialsPaper (2009,19:2359-2364) in, reported and utilized yeast success synthetizing CdSe quantum dots in viable cell, and carried out preliminary quantum dot and extracted preparation.Because yeast cells wall is comparatively tough and tensile, quantum dot extracts preparation condition must be comparatively gentle, otherwise fluorescence losses is bigger.The traditional method of abolishing cell walls such as methods such as multigelation, high pressure homogenization, microwave method, not good to the yeast cells wall crushing effect, and more violent method as: alkaline lysis can destroy quantum dot in the cell.Therefore must develop a kind of mild condition, cytoclasis degree height, not influence quantum dot fluorescence character again, and can keep the yeast cell cleavage method of quantum dot stability.

Except cell walls cracked technical problem, it is that quantum dot separates with cyto-architectural that the interior synthetic quantum dot of pair cell extracts another limiting factor.The synthetic quantum dot combines closely with organoid in the cell, even if cell destroyed they also be combined in securely on organoid or the cell debris.The synthetic quantum dot extracts in the sophisticated cell of one cover, purification scheme is not only this green of biosynthesizing quantum dot, the eco-friendly method of further developing, and is also significant to the research of the application of quantum dot and biosynthesizing quantum dot mechanism.

Summary of the invention

The objective of the invention is to be to provide the method for synthetic quantum dot in a kind of microorganism cells, mainly is to utilize biochemical method to extract and the interior synthetic CdSe quantum dots of purifying yeast cell.This separation purification process is easy and simple to handle, the extraction yield height, and product is stable, and importantly experiment safety does not relate to any poisonous or adventurous reagent, all can finish at general biology laboratory.

The technical solution adopted for the present invention to solve the technical problems is:

Extraction, the purification process of synthetic CdSe quantum dots the steps include: in a kind of yeast cell

1) cracking of yeast cells wall: getting, the interior quantum dot synthetic method of yeast cell nutrient solution 40-60 mL[yeast cell of synthetizing CdSe quantum dots was published in referring to this laboratory in 2009 Advanced Functional MaterialsPaper (2009,19:2359-2364)], the centrifugal 1-3 min of 4000 g, collecting cell precipitation is resuspended in the cell lysis buffer solution (Lysis buffer), by 4000 U/10 ¹⁰The amount of cell adds cytase (Lyticase), 30 ℃ of shaking tables, 100 r/min vibration hatch 4-6 h to solution becomes transparent till;

2) cell that the fragmentation of yeast cell: the centrifugal 9-11 min of 10000 g, collect 1) step process is crossed is resuspended in an amount of (24-36 mL) cell lysis buffer solution (Lysis buffer), adds proteinase inhibitor (Cocktail); 400 W(, 2 s that work, 2 s intermittently, 200 times; Stop 10 s per 20 times) the ultrasonication lysing cell;

3) roughing out of CdSe quantum dots: with 2) sample crossed of step process is drawn the yellow supernatant that contains quantum dot in the centrifugal 9-11 min of 4000 g, will contain the crude extract and the cell debris initial gross separation of quantum dot;

4) segmentation of CdSe quantum dots from: to 3) to add sodium lauryl sulphate (SDS) to final concentration in the yellow supernatant of centrifugal collection in the step be 2%, shake up gently, room temperature (identical below 20-25 ℃) leaves standstill 9-11 min, the centrifugal 28-32 min of 10000 g, get supernatant with 0.22 mm membrane filtration, carry out ultrafiltration, washing with 50 K MWCO ultrafiltration pipes then;

5) purifying of cadmium selenide amount: with 4) sample that obtains in the step carries out ficoll 400(Ficoll 400) density gradient centrifugation, the centrifugal 3-5 h of 113000 g carefully takes out the yellow layering after the density gradient centrifugation; 50 K MWCO ultrafiltration pipes carry out ultrafiltration, washing, promptly obtain the CdSe quantum dots of purifying.

The present invention compared with prior art has the following advantages and effect:

Employed reagent is all nontoxic non-stimulated among the present invention, do not relate to used oxidation tri-n-octyl phosphine inflammable, explosive, toxic reagents such as (TOPO) in the chemosynthesis quantum dot, so present method can not cause harm to the people, and it is not high to requirement for experiment condition, all can operate safely at general biology laboratory, related biological chemistry extracting and purifying method is safer, the grain pattern that the CdSe quantum dots that extracts is kept perfectly under Electronic Speculum, and uniform particle diameter, energy spectrum analysis are selenium and cadmium element.What is more important, but the quantum dot that the present invention extracted keeps stronger photoluminescent property 4 ℃ of prolonged preservation.

Description of drawings

Fig. 1 is the yeast cell fluorescence microscope synoptic diagram after a kind of cytase is handled 5 h.

Yeast cell is after cytase is handled 5 h, and most cell wallss are destroyed, the cell that has cracking.

Fig. 2 is the fluorescence microscope synoptic diagram after a kind of supersound process.

The postdigestive cell of cytase is through 400 W supersound process 200 times, and cell is fractured into fragment, and quantum dot is not destroyed, and fluorescence still exists.

Fig. 3 is the separation and purification synoptic diagram of the synthetic CdSe quantum dot of a kind of brewing yeast cell.

A roughing out effect.Quantum dot is opened with a large amount of cell debriss and a small amount of not broken cellular segregation.

The B segmentation is from effect.After adding sodium lauryl sulphate, the sample after centrifugal, filtering with microporous membrane and ultrafiltration.

Result behind the C density gradient centrifugation purifying.CdSe quantum dots after the density gradient centrifugation distributes comparatively concentrated.

Fig. 4 is the transmission electron microscope synoptic diagram of CdSe quantum dots sample behind a kind of purifying.

Sample behind the purifying is dripped on the copper mesh that is covered with ultrathin carbon films, and transmission electron microscope (Tecnai G2 20) 200 KV observe, and can observe the comparatively nano particle material of homogeneous of a large amount of particle diameters.

Embodiment

Extraction purifying with synthetic CdSe quantum dots in the yeast saccharomyces cerevisiae is an example below, further sets forth the present invention with reference to accompanying drawing.Be published in according to this laboratory Advanced Functional MaterialsPaper (2009,19:2359-2364) in the yeast cell of the synthetic good CdSe quantum dots of method preparation.

Extraction, the purification process of synthetic quantum dot in a kind of microorganism cells, its step is as follows:

1, the cracking of yeast cells wall: get yeast cell nutrient solution 50 mL of synthetizing CdSe quantum dots, centrifugal 2 min of 4000 g, the collecting cell precipitation is resuspended in the 30 mL cell lysis buffer solution (Lysis buffer), by 4000 U/10 ¹⁰The amount of cell add cytase (Lyticase, Sigma, L4025), 30 ℃, 100 r/min vibration hatch 5 h to solution becomes transparent till;

The related solution component is as follows:

Cell lysis buffer solution (Lysis buffer)

1.5 mL 1 mol/L pH 7.5 phosphoric acid buffers

4.5?mL 4?mol/L?Sorbitol

24?mL H ₂O

1 mol/L phosphoric acid buffer pH 7.5

80?mL 1?mol/L?K ₂HPO ₄

19.8?mL 1?mol/L?KH ₂PO ₄

Be mixed to pH 7.5

2) fragmentation of yeast cell: centrifugal 10 min of 10000 g, collect the postdigestive cell precipitation of cytase in the step 1), cell precipitation is resuspended in the 4.5 mL cell lysis buffer solution, add proteinase inhibitor (Cocktail, Roche, 04693132001, usage quantity is with reference to specification sheets); 4 ℃, 400 W(, 2 s that work, 2 s intermittently, 200 times; Stop 10 s per 20 times) the ultrasonication lysing cell;

3) sample after handling the roughing out of CdSe quantum dots: with step 2) is in centrifugal 10 min of 4000 g, and careful absorption contains the yellow supernatant of quantum dot, and supernatant is the CdSe quantum dots crude extract;

4) segmentation of CdSe quantum dots from: in the yellow supernatant that step 3) is collected, add sodium lauryl sulphate (SDS, Sigma) to final concentration be 2%, shake up gently, room temperature leaves standstill 10 min, and centrifugal 30 min of 10000 g carefully collect supernatant, with 0.22 mm filter membrane the supernatant of collecting is filtered to 50 K MWCO ultrafiltration pipe (Millipore then, Amicon Ultra-15) upper strata is diluted to 10 mL with redistilled water, centrifugal 10 min of 5000 g; With redistilled water upper solution is diluted to 10 mL, centrifugal 10 min of 5000 g repeat once;

5) purifying of CdSe quantum dots: the sample of ultrafiltration gained in the step 4) is carried out ficoll 400(Ficoll 400, Pharmacia, 17-0300-01) density gradient centrifugation, ficoll density gradient are 8%, 15%, 30%, 45%(is mass volume ratio), centrifugal 4 h of 113000 g.Behind centrifugal the finishing, draw the yellow solution that contains CdSe quantum dots, be diluted to 10 mL, add to 50 K MWCO ultrafiltration pipe (Millipore, Amicon Ultra-15) upper stratas, centrifugal 10 min of 5000 g with redistilled water with syringe needle; With redistilled water upper solution is diluted to 10 mL, centrifugal 10 min of 5000 g repeat once, promptly obtain the CdSe quantum dots of purifying.

More than describing is explanation of the invention, is not limitation of the invention, and institute of the present invention restricted portion is referring to claim, and under the situation of spirit of the present invention, the present invention can make any type of modification.

Claims

1. extraction, the purification process of the interior synthetic quantum dot of microorganism cells the steps include:

1) cracking of yeast cells wall: get the yeast cell nutrient solution of synthetizing CdSe quantum dots, the centrifugal 1-3 min of 4000 g, the collecting cell precipitation is resuspended in the cell lysis buffer solution, by 4000 U/10 ¹⁰The amount of cell adds cytase, 30 ℃ of shaking tables, 100 r/min vibration hatch 4-6 h to solution becomes transparent till;

2) cell that the fragmentation of yeast cell: the centrifugal 9-11 min of 10000 g, collection 1) step process is crossed is resuspended in the cell lysis buffer solution, adds proteinase inhibitor; 400 W ultrasonication lysing cell;

4) segmentation of CdSe quantum dots from: to 3) to add sodium lauryl sulphate to final concentration in the yellow supernatant of centrifugal collection in the step be 2%, shake up gently, room temperature leaves standstill 9-11 min, the centrifugal 28-32min of 10000 g, get supernatant with 0.22 mm membrane filtration, carry out ultrafiltration, washing with 50 K MWCO ultrafiltration pipes then;

5) purifying of CdSe quantum dots: with 4) sample that obtains in the step carries out ficoll 400 density gradient centrifugations, the centrifugal 3-5 h of 113000 g, the yellow layering after the taking-up density gradient centrifugation; 50 K MWCO ultrafiltration pipes carry out ultrafiltration, washing, promptly obtain the CdSe quantum dots of purifying.