the materials used in the preferred embodiments and applications of the present invention include : 1 . nanosilicate platelets ( nsp ): cation exchange capacity ( cec )= 1 . 20 mequiv / g ; having a single - layered or dual - layered structure in water ; isoelectric point ( ie )= ph 6 . 4 ; almost 100 % inorganic ; available by exfoliating montmorillonite ( na + - mmt ); as described in : u . s . pat . no . 7 , 125 , 916 , u . s . pat . nos . 7 , 094 , 815 , 7 , 022 , 299 , and 7 , 442 , 728 or u . s . publication no . 2006 - 0287413 - a1 . 2 . hectorite : product of co - op chemical co . ( japan ), swn ®, was used , synthetic layered silicate clay , a kind of bentonite , cationic exchange capacity ( cec )= 0 . 67 mequiv / g . 3 . agno 3 : used for exchanging or replacing na + between layers of the clay and for providing silver ions to be reduced to ag nanoparticles ( agnps ). 4 . methanol : ch 3 oh , 95 %, a weak reducing agent , used to reduce the silver ions to agnps at 30 ˜ 150 ° c .. 5 . ethylene glycol ( eg ): c 2 h 4 ( oh ) 2 , a weak reducing agent , used to reduce the silver ions to agnps at 30 ˜ 150 ° c .. 6 . microorganisms : ( 1 ) acinetobacter baumannii : including ordinary , multidrug - resistant and silver - resistant strains , provided by dr . huang chieh - chen of national chung hsing university , department of life sciences , taiwan . ( 2 ) escherichia coli : isolated from wild colonies and used as type culture of gram - negative bacteria ; provided by dr . lin chun - hung of animal technology institute taiwan . ( 3 ) escherichia coli j53 : used as control groups to the silver - resistant strain j53pmg101 , having no silver - resistant plasmid pmg101 , provided by prof . c . m . che , department of chemistry , the university of hong kong . ( 4 ) silver - resistant escherichia coli j53pmg101 : having silver - resistant plasmid pmg101 , provided by dr . anne 0 . summers , department of microbiology , the university of georgia , athens , us . the suspensions of bacteria cultured overnight were added into a fresh luria - bertani ( lb ) liquid media at a volume ratio of 1 / 100 for culturing for about three hours . absorbance ( od 600 ) of the suspensions of bacteria after culturing were determined with a spectrophotometer , and the suspensions having od 600 values ranging between 0 . 4 to 0 . 6 were selected as the standard suspensions of bacteria . 1 . synthetic fluorine mica : mica , product of co - op chemical co . ( japan ), code number somasif me - 100 , with cationic exchange capacity ( cec )= 1 . 20 mequiv / g . 2 . laponite : synthetic layered silicate clay with cationic exchange capacity ( cec )= 0 . 69 mequiv / g . 3 . [ m ii 1 − x m iii x ( oh ) 2 ] intra [ a n − . nh 2 o ] inter : synthetic layered double hydroxide with ionic exchange capacity in the range of 2 . 0 to 4 . 0 mequiv ./ g , m ii is a two - valence metal ion , for example , mg , ni , cu and zn ; m ii is a three - valence metal ion , for example , al , cr , fe , v and ga ; a n − is an anion , for example , co 3 2 − , no 3 − . first , the swn solution ( 1 wt %) and the agno 3 solution ( 1 wt %) were prepared . the agno 3 ( aq ) ( 3 . 4143 g ) was then slowly added into the swn solution ( 30 g ) so that the ag + / cec equivalent ratio was 1 . 0 / 1 . 0 and the ag + / swn weight ratio was about 7 / 93 . the solution immediately bacame light yellow . into this solution , methanol ( meoh , about 6 ˜ 8 ml ) was added and the solution remained in light yellow . by means of ultrasonic mixing and water bath at 70 ˜ 80 ° c ., the reaction began and the color changed . after vibration , the product agnp / swn was achieved . the agnp / swn solution was diluted to 60 μm ( 0 . 01 wt %), 600 μm ( 0 . 1 wt %) and 1 . 2 mm ( 0 . 2 wt %), respectively , for tests of inhibiting bacterial growth . first , the nsp solution ( 1 wt %) and the agno 3 solution ( 1 wt %) were prepared . the agno 3 ( aq ) ( 3 . 5160 g ) was then slowly added into the nsp solution ( 30 g ) so that the ag + / cec equivalent ratio was 1 . 0 / 1 . 0 and the ag + / nsp weight ratio was about 7 / 93 . the na + ions between the clay layers were replaced with the ag + ions and the solution turned into a cream color . into this solution , ethylen glycol ( eg , about 0 . 1 ˜ 5 ml ) was added and the solution was still in cream color . by means of ultrasonic mixing and water bath at 40 ˜ 80 ° c ., the reaction began and the color changed . after vibration , the product agnp / nsp was obtained . the agnp / nsp solution was diluted to 60 μm ( 0 . 01 wt %), 600 μm ( 0 . 1 wt %) and 1 . 2 mm ( 0 . 2 wt %), respectively , for tests of inhibiting bacterial growth . in the above agnp / clay composites , clay served as carriers for adsorbing the agnps to kill ordinary bacteria and multidrug - resistant bacteria . the agnps had a particle size of about 20 to 30 nm . measured with inductively coupled plasma - mass spectrometry ( icp - ms ), the silver ions in the agnp / clay composite solution ( 0 . 1 wt %) had a concentration of about 120 to 190 ppb . in the present invention , the tests of inhibiting bacterial growth were performed by adding the water solutions of silver nitrate , agnp / swn or agnp / nsp of different ratios into the uncoagulated lb solid culture media to prepare 100 mm lb solid cultere media of different concentrations . the standard suspensions of bacteria ( each 100 μl ) were spread on the lb solid media including silver nitrate of different concentrations with sterilized glass beads to culture at 37 ° c . for 16 hours . the numbers of colonies were determined by dividing the plate into 8 or 16 areas wherein one area was selected to count the colonies thereon . the total number of colonies was obtained by multiplying the number of colonies on the selected area with the number of the areas . results were as follows , wherein the mock group without treatment was relatively set as 100 % and the colony ratios (%) could be used to estimae the inhibition effects (= 100 %− the colony ratio ). as shown in fig1 , for acinetobacter baumannii ( ab ) without drug - resistance , growth could not be effectively inhibited in silver nitrate ( 8 μm ), 90 % could be inhibited in silver nitrate ( 40 μm ) and all could be inhibited in silver nitrate ( 200 μm ). for the silver - resistant acinetobacter baumannii strains ( 1 - 52 , 2 - 10 , 51 - 76 , 53 - 49 ), only 50 ˜ 80 % were inhibited in silver nitrate ( 2000 . the concentration of silver ions had to be as high as 1 mm for all of the bacteria to be inhibited . as shown in fig2 , for escherichia coli ( j53 strain ) without drug - resistance , growth could not be effectively inhibited in silver nitrate ( 8 μm ), 90 % could be inhibited in silver nitrate ( 40 μm ) and all could be inhibited in silver nitrate ( 200 μm ). for the silver - resistant escherichia coli ( j53pmg101 ), only about 80 % were inhibited in silver nitrate ( 200 μm ). the concentration of silver ions had to be as high as 1 mm for almost all of the bacteria to be inhibited . as shown in fig3 , for acinetobacter baumannii ( ab ) without drug - resistance , growth could not be effectively inhibited in agnp / swn ( 60 μm ) and all could be inhibited in agnp / swn ( 600 μm ). for the silver - resistant acinetobacter baumannii strains ( 1 - 52 , 2 - 10 , 51 - 76 , 53 - 49 ), only 50 ˜ 80 % were inhibited in agnp / swn ( 600 μm ). even in agnp / swn ( 1 . 2 mm ), about 5 % of the bacteria could still be live . as shown in fig4 , for escherichia coli ( j53 strain ) without drug - resistance , growth could not be effectively inhibited in agnp / swn ( 60 μm ) and all could be inhibited in agnp / swn ( 600 μm ). for the silver - resistant escherichia coli ( j53pmg 101 strain ), only 50 ˜ 80 % were inhibited in agnp / swn ( 600 μm ). even in agnp / swn ( 1 . 2 mm ), 10 % of the bacteria could still be live . as shown in fig5 , for acinetobacter baumannii ( ab ) without drug - resistance , growth could not be effectively inhibited in agnp / nsp ( 60 μm ) and all could be inhibited in agnp / nsp ( 600 μm ). for the silver - resistant acinetobacter baumannii strains ( 1 - 52 , 2 - 10 , 51 - 76 , 53 - 49 ), only 50 ˜ 80 % were inhibited in agnp / nsp ( 600 μm ). the bacteria could be completely inhibited in agnp / nsp ( 1 . 2 mm ), which indicated that agnp / nsp performed better than agnp / swn . the reason could be that the single - layered nsp provides larger contact area than swn constructed with 8 to 10 layers . as shown in fig6 , for escherichia coli ( j53 strain ) without drug - resistance , growth could not be effectively inhibited in the agarose gel including agnp / nsp ( 60 μm ) and all could be inhibited in agnp / swn ( 600 μm ). for the silver - resistant escherichia coli ( j53pmg101 ), only about 80 % were inhibited in agnp / nsp ( 600 μm ). the bacteria could be completely inhibited in agnp / nsp ( 1 . 2 mm ), which indicated that agnp / nsp performed better than agnp / swn . the reason could be that the single - layered nsp provides larger contact area than swn constructed with 8 to 10 layers . according to the analysis of the composite ( 600 μm , 0 . 1 wt %), the silver ions were present in a concentration of only 150 ppb ( about 1 ˜ 1 . 5 μm ) in the upper clear liquid . since the silver ions of such concentrations could not kill bacteria , the composite of the present invention could not inhibit growth of bacteria through the dissociated silver ions . therefore , the bacteria must have been killed through lots of radicals which could destroy cell membranes thereof live / dead baclight kit ( invitrogen ) was used to determine whether a cell is live or dead . all cells could be stained with cyto9 , but only the damaged cells of bacteria could be stained with propidium iodide ( pi ). by combing these two stain reagents , the live cells could be distinguished from the dead . the bacteria were stained at room temperature with slow vibration , at about 50 rpm . at certain intervals , the cells were monitored with a microscope ( oil immersion ). fig7 shows the percentages of the dead cells among all the bacteria cells : the bacteria treated with agnp / swn after 72 hours were about 38 ± 6 . 8 % dead , and the bacteria treated with swn were about 10 % dead . when the cells generated radicals , for example , reactive oxygen species ( ros ), dcfh - da ( 2 ′, 7 ′- dichlorofluorescin - diacetate ) would be oxidized to dcf ( dichlorofluorescin ) and emit fluorescent light . brightness of the fluorescent light was proportional to the amount of the radicals . in the present invention , dcfh - da ( 10m ) was applied to the bacteria which were observed under microscope at the 0 . 5th , 1st and 2nd hours . percentages ( pi + / cyto9 + cells %) of the bacteria generating fluorescent light to the total bacteria could be estimated . escherichia coli strains treated with agnp / swn and swn were monitored . the microscope images indicated that the strains emit more green fuorecent light after being treated with swn or agnp / swn ( 300 μm , 0 . 05 wt %) for 2 hours ; and the strains emit more red fuorecent light after being treated with swn or agnp / swn ( 600 μm , 0 . 1 wt %) for 24 and 48 hours . fig8 showed the percentages of the cells generating radicals ros : about 40 . 3 ± 10 . 2 % for the bacteria treated with agnp / swn after 2 hours , and less than 10 % for the bacteria treated with swn . according to the above assays , effects of the composite of the present invention in inhibiting bacteria were factually achieved by the radicals ros generated by bacteria . the present invention provides an composite of agnps which can effectively kill bacteria in lower silver ion concentrations , particularly the silver - resistant strains . the present invention also verifies that the composite kills the bacteria by radicals but not dissociated silver ions , so that side effects of the silver ions can be significantly decreased .
the present invention provides a method of using a composite of spherical silver nanoparticles and layered inorganic clay , in particular nanosilicate platelets , for inhibiting the growth of silver - resistant bacteria . the layered inorganic clay serves as carriers of the silver nanoparticles and disperses them . the composite has a particle size of about 5 nm to 100 nm . the silver nanoparticles can be dispersed in an organic solvent or water .