Patent Application: US-200913058655-A

Abstract:
a method of producing sulfur - free nanoparticles involves growing yeast in a growth medium containing a source of an element in a bio - reducible oxidation state , and , precipitating nanoparticles containing the element in a lower oxidation state ) than the oxidation state of the element in the source . the method advantageously can provide substantially spherical nanoparticles at high production efficiencies .

Description:
a wild - type strain of baker &# 39 ; s yeast , saccharomyces cerevisiae , was isolated ( selected after being grown in a petri dish ) and used in all experiments . all chemicals were of analytical grade and compatible with cell cultures . an elan ™ drc ii icpms ( pe - sciex , thornhill , on , canada ) equipped with a ryton ™ spray chamber and cross - flow nebulizer was used for the detection of selenium and sulfur . hydrogen was used as a collision gas and icpms parameters , nebulizer gas flow , rf power , lens voltages , and hydrogen gas flow , were optimized daily to get the best s / n ratio for s and se . a hewlett - packard hp 6890 gc ( agilent technologies canada inc ., mississauga , on , canada ) fitted with a db - 5ms column ( iso - mass scientific inc ., calgary ab , canada ) was used for the separation of methionine ( met ) and selenomethionine ( semet ) in the derivatized yeast extracts . detection was achieved with an hp model 5973 mass - selective detector ( ms ). a cem ( matthews , n . c .) mds - 2100 microwave digester equipped with teflon ™ vessels was used for closed vessel high pressure decomposition of yeast for total se and s determination . as commercially prepared yeast growth media , such as yepd , are usually based on fungus peptone or yeast extracts , they contain many sources of sulfur and possibly selenium . therefore , a synthetically defined medium optimized for s . cerevisiae was prepared to control sulfur during yeast growth . all prepared media had the same chemical constituents except that sources of sulfur and selenium were varied . salts ( mgcl 2 , nacl , cacl 2 ), nitrogen source ( nh 4 cl ), phosphorus source ( kh 2 po 4 ) and carbohydrate source ( dextrose ) were mixed together in an appropriate volume of water to obtain the final concentrations shown in table 1 . this solution was then autoclaved at 121 ° c . for 35 min and stored aseptically . after the mixture cooled , previously prepared vitamins , trace elements and amino acid solutions were added with syringes through 0 . 2 μm sterile filters to reach the final concentrations detailed in table 1 . the latter were added to the medium through syringes and filters after the media was autoclaved to prevent their denaturation . supplementary additions of selenium compounds to the medium for nanoparticle formation were also made through syringes and 0 . 2 μm sterile filters . all manipulations of glassware containing growth media and / or yeast were performed inside a laminar flow hood and the manipulating tools were sterilized with a flame or in the autoclave . yeast cells were stabilized using glutaraldehyde buffer ( 0 . 1m phosphate buffer at ph 6 . 7 containing 4 % glutaraldehyde ) for chemical fixation . after incubation ( 5 min ) and centrifugation ( 1500 g , 4 min ), the supernatant is discarded and the cells are re - suspended in 1 ml of glutaraldehyde buffer diluted twice . the cells are incubated overnight at 4 ° c . after centrifugation , the supernatant is replaced by 1 . 5 ml of deionised water , incubated in water ( 10 min ) and centrifuged . this re - suspension / centrifugation is repeated 3 times . finally , the cells are re - suspended in 1 ml of deionised water . yeast was grown in batch conditions ( an erlenmeyer flask ) in a fully defined synthetic growth medium comprising the medium described above including 0 . 08 mm cysteine and 0 . 4 mm sodium selenate or semet . the growth medium was free of sulfur sources except for the cysteine . the temperature was held at 28 ° c . and the flask shaken at 150 rpm for up to five days . under these conditions the yeast metabolized inorganic selenium ( sodium selenate in which se is in the + 6 oxidation state ) forming selenomethionine ( a non - canonical amino acid ) which is incorporated into the yeast proteome in the place of methionine . under these growth conditions , total se concentration in the yeast was 2 . 4 mg / g ( as dry weight ). about 65 - 70 % of the total se was in the form of selenomethionine , as determined using electrospray and inductively coupled plasma mass spectrometry , replacing about one out of four methionines . however , using electrospray and inductively coupled plasma mass spectrometry the remaining 30 % of se could not be accounted for . with yeast grown on semet - containing media full replacement of met with semet was observed and virtually 100 % of the total se in these samples was in the form of semet . transmission electron microscopy ( tem ) studies of microtomed thin sections of yeast grown in sulfur - free sodium selenate - containing media indicated the presence of electron dense spots in the cells , indicating the presence of nanoparticles . energy - dispersive x - ray spectrometry ( eds ) analysis was used to determine elemental composition of these deposits . eds analysis revealed that the composition of these nanoparticles is elemental se , i . e . se in the 0 oxidation state . the nanoparticles are spherical having an average size in the 50 nm range . transmission electron microscopy ( tem ) images depicted in fig1 a ( scale at bottom left corner is 1 μm ) and fig1 ( scale at bottom left corner is 20 nm ) show the se nanoparticles identified in the cells at various magnification . the presence of fringes indicated that the selenium particles may be composed of nanocrystals . however the chemically and mechanically intensive sample preparation required for tem could raise questions about contamination or potentially the formation of such deposits during the chemical treatment or sectioning process . additionally , the eds analysis of the cells allowed only detection of highly concentrated se spots , i . e . se nanoparticles ( fig2 ). in contrast the tem analysis of yeast grown on semet showed no nanoparticle formation . this is expected because in these samples all the selenium is in the form of semet and semet is replacing met in the yeast proteome . interestingly the only location in the cell where any se could be detected with eds is the nuclear membrane . a second synthetically defined growth medium optimized for s . cerevisiae was prepared to control sulfur during yeast growth . all prepared media had the same chemical constituents except that sources of metal for nanoparticle production were varied . salts ( mgcl 2 , nacl , cacl 2 ), nitrogen source ( nh 4 cl ), phosphorus source ( kh 2 po 4 ) and carbohydrate source ( dextrose ) were mixed together in 850 ml of water to obtain the final concentrations shown in table 2 . this solution was then autoclaved at 121 ° c . for 45 min and stored aseptically . after the mixture cooled , previously prepared vitamins , trace elements and amino acid solutions were added with syringes through 0 . 2 μm sterile filters to reach the final concentrations detailed in table 2 . the latter were added to the medium through syringes and filters after the medium was autoclaved to prevent their denaturation . supplementary additions of metal compounds to the medium for nanoparticle formation were also made through syringes and 0 . 2 μm sterile filters . all manipulations of glassware containing growth media and / or yeast were performed inside a laminar flow hood and the manipulating tools were sterilized with a flame or in the autoclave . yeast cells were stabilized using glutaraldehyde buffer for chemical fixation . 50 ml of the medium containing the growing yeast is collected and centrifuged ( 3000 × g ) for 3 min . the supernatant is discarded , the cells resuspended in the same volume of doubly distilled water ( ddw ) and centrifuged again ( 3000 × g ) for 3 min . this resuspension / centrifugation in ddw is repeated 3 times . it is then resuspended in 10 ml ddw . a volume of 100 μl of the sample is mixed with 900 μl of a 0 . 1 m phosphate buffer ph 6 . 7 containing 4 % glutaraldehyde and allowed to incubate for 5 minutes . then , it is centrifuged at 2000 × g for 4 min , the supernatant discarded and the cells resuspended in 1 ml of a 0 . 05 m phosphate buffer ( ph 6 . 7 ) containing 2 % glutaraldehyde . the sample is incubated overnight at 4 ° c ., then centrifuged ( 3000 × g ) for 4 min . the supernatant is discarded , the cells resuspended with 1 . 5 ml of ddw , incubated in water for 10 min and centrifuged again ( 3000 × g ) for 4 min . this resuspension / centrifugation in ddw is repeated 3 times , and the cells resuspended in 1 ml ddw . trial 1 : yeast was grown on 50 ml of defined medium 2 for 26 hours at 150 rpm at a temperature of 28 ° c . or 25 ° c . various metal compounds were then added with syringes through a 0 . 2 μm sterile filter to different batches of the medium to achieve final concentrations as follows : ag ( i ) 10 ppm ; au ( iii ) 10 ppm ; pt ( ii ) 10 ppm ; te ( vi ) 4 ppm ; se ( iv ) 4 ppm . after about 60 hours of yeast growth , formation of nanoparticles was confirmed for ag by visual inspection of a color change in the growth medium . no color changes in the growth media for au , pt , te and se were observed , probably due to the low concentrations of metals used , but nanoparticle formation was confirmed through microscopic observations . trial 2 : yeast was grown on defined medium 2 for 10 hours at 150 rpm at a temperature of 28 ° c . or 25 ° c . various metal compounds were then added to different 50 ml batches of the medium to achieve final concentrations as follows : ag ( i ) 10 ppm ; au ( iii ) 10 ppm ; pt ( ii ) 10 ppm ; pd ( ii ) 10 ppm ; se ( iv ) 5 ppm . the ph of the metal solutions was adjusted to 4 with nh 4 oh , except for au which was adjusted to 2 - 3 . after about 60 hours of yeast growth , no color changes in the growth media were observed , again probably due to the low concentrations of metals used , but nanoparticle formation was confirmed through microscopic observations . sabouraud dextrose broth is a commercially available growth medium for yeast which comprises 20 . 00 g / l dextrose and 10 . 0 g / l of a mixture of peptic of animal tissue and pancreatic digest of casein ( 1 : 1 ). the final broth is prepared by suspending 30 grams of the medium in one liter of distilled water and mixing well until a uniform suspension is obtained . the mixture is heated with frequent agitation , boiled for one minute , distributed and sterilized at 118 - 121 ° c . for 15 - 45 minutes . the final ph is 5 . 6 ± 0 . 2 at 25 ° c . yeast cells were stabilized using glutaraldehyde buffer for chemical fixation . 50 ml of the medium containing the growing yeast is collected and centrifuged ( 3000 × g ) for 3 min . the supernatant is discarded , the cells resuspended in the same volume of doubly distilled water ( ddw ) and centrifuged again ( 3000 × g ) for 3 min . this resuspension / centrifugation in ddw is repeated 3 times . it is then resuspended in 10 ml ddw . a volume of 100 μl of the sample is mixed with 900 μl of a 0 . 1 m phosphate buffer ph 6 . 7 containing 4 % glutaraldehyde and allowed to incubate for 5 minutes . then , it is centrifuged at 2000 × g for 4 min , the supernatant discarded and the cells resuspended in 1 ml of a 0 . 05 m phosphate buffer ( ph 6 . 7 ) containing 2 % glutaraldehyde . the sample is incubated overnight at 4 ° c ., then centrifuged ( 3000 × g ) for 4 min . the supernatant is discarded , the cells resuspended with 1 . 5 ml of ddw , incubated in water for 10 min and centrifuged again ( 3000 × g ) for 4 min . this resuspension / centrifugation in ddw is repeated 3 times , and the cells resuspended in 1 ml ddw . trial 1 : yeast was grown in sabouraud dextrose broth for 26 hours at 150 rpm at a temperature of 28 ° c . or 25 ° c . various metal compounds were then added with syringes through a 0 . 2 μm sterile filter to different 100 ml batches of the medium to achieve final concentrations as follows : ag ( i ) 40 ppm ; au ( iii ) 20 ppm ; pt ( ii ) 40 ppm ; te ( vi ) 20 ppm ; se ( iv ) 20 ppm . after about 60 hours of yeast growth , formation of nanoparticles was confirmed for all of the metals by visual inspection of a color change in the growth medium and through microscopic observations . trial 2 : yeast was grown in sabouraud dextrose broth for 10 hours ( for pd , ag , au , pt , u , se ) or for 24 . 5 hours ( for cd , zn , pb ) at 150 rpm at a temperature of 28 ° c . or 25 ° c . various metal compounds were then added to different 100 ml batches of the medium to achieve final concentrations as follows : pd ( ii ) 50 ppm ; ag ( i ) 50 ppm ; au ( iii ) 50 ppm ; pt ( ii ) 50 ppm ; u ( vi ) 50 ppm ; se ( iv ) 20 ppm ; cd ( ii ) 50 ppm ; zn ( ii ) 50 ppm ; pb ( iv ) 50 ppm . the ph of the metal solutions was adjusted to 4 with nh 4 oh , except for au which was adjusted to 2 - 3 . after about 60 hours of yeast growth , formation of nanoparticles was confirmed for ag and au by visual inspection of a color change in the growth medium . no color changes in the growth media for other metals were observed , but nanoparticle formation was confirmed through microscopic observations . trial 3 : two replicates of 0 . 1 ml of yeast and two replicates of 0 . 2 ml of yeast were added to four bottles of sabouraud broth . after growing at 25 ° c . ( 150 rpm ) for 16 . 5 h , se ( iv ) was added to the yeast samples ( with syringes through 0 . 2 μm sterile filter ) to reach final concentrations of 20 , 50 , 150 ppm , respectively . 52 . 5 h after addition of se ( iv ), samples were collected and freezing dried . visual inspection of color change in the medium demonstrated that greater quantities of nanoparticles were formed at the higher concentrations compared to the lower concentrations . references : the contents of the entirety of each of which are incorporated by this reference . abelovska l , bujdos m , kubova j , petrerselyova s , nosek j , tomaska l . ( 2007 ) comparison of element levels in minimal and complex yeast media . can . j . microbiol . 53 , 533 - 535 . dameron c t , reese r n , mehra r k , kortan a r , carroll p j , steigerwald m l , brus l e , einge d r . ( 1989 ) biosynthesis of cadmium sulphide quantum semiconductor crystallites . nature . 338 , 596 - 597 . gericke m , pinches a . 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( 2002 ) getting started with yeast . methods enzymol . 350 , 3 - 41 . other advantages that are inherent to the structure are obvious to one skilled in the art . the embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed . variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims .