before explaining the disclosed embodiment of the present invention in detail it is to be understood that the invention is not limited in its application to the details of the particular arrangement shown since the invention is capable of other embodiments . also , the terminology used herein is for the purpose of description and not of limitation . it would be useful to discuss the meanings of some words and abbreviations used herein to explain the invention in greater detail . ng stands for nanogel , which is the gel - like substance formed by the interconnection of nanoparticles . np stands for nanoparticles which have a particle size from approximately 10 nm to approximately 50 nm si is used herein to mean silicon dioxide , which is also commonly known as “ silica .” it is to be understood that the discussions and examples herein are directed to the use of copper compounds as the active ingredient contained in silica nanoparticles that are used to form silica nanogel in a treatment for citrus canker . applicant &# 39 ; s invention is not limited to the use of copper compounds and the treatment of citrus canker , the scope of the invention is to broadly cover the use of silica based nanoformulations that serve as hosts for a broad array of fungicides / bactericides , including , but not limited to , metallic copper ( cu ), copper salts , copper complexes , metallic zinc ( zn ), zinc oxides , zinc salts , metallic silver ( ag ), silver salts , silver complexes , titanium dioxide ( tio 2 ), cerium oxides , magnesium oxides , zirconium oxides , polyethyleneimine ( pei ), carbon , mixed carbon or soot , fullerenes , carbon nanotubes and the like . the present invention involves a unique application of nanoscience and nanotechnology to develop engineered copper cu ( ii ) loaded silica nanogel ( cusing ) material . the nanotechnology allows the manipulation of a silica nanoenvironment around cu , establishing a sustained cu ion release mechanism . the novel cusinp design shown in fig2 , allows association of copper ( cu ) to silica ng in three different forms , enabling sustained cu release . first , electrostatically bound cu ions in the core 10 . second , covalently bound cu in the hydrated particle surface 20 and nanopores 30 . third , surface bound cu as hydroxide / oxide nanoclusters / nanoparticles 40 . the nanogel structure consists of ultra - small nanoparticles ( cusinps ) less than approximately 50 nm in diameter to provide better plant surface coverage and improved adherence properties . all these attractive features will form a solid basis for long - term protection of plants from pathogens using cusing material . due to nanoscale engineering , the cusing of the present invention has the following advantages over the existing cu based compounds : uniform coverage of plant surface because of ultra - small particle size , better adherence property due to gel - like nanostructure , sustained ( long - term ) cu release profile , better control on cu release rate ( adjustable “ soluble ” to “ insoluble ” cu ratio ), more antibacterial / antifungal activity with less amount of cu content , reduced phytotoxic effect because of adjustable “ soluble ” to “ insoluble ” cu ratio and environment - safe due to less cu content , no harmful by - product formation , water - based synthesis , utilization of excess cusing as plant nutrient and minimal possibility of having elevated local cu concentration that could cause environmental toxicity . the synthesis protocol has the following advantages : ( i ) simplicity , ( ii ) water - based , ( ii ) scalable to field applications , ( iii ) single - pot synthesis method , requiring no purification steps and ( v ) concentrated cusing material could be easily diluted for field application . a non - technical person can do this task by adding an appropriate amount of water , thus reducing shipping costs . the method also uses inexpensive raw chemicals and is easily produced in a cost - effective manner . the synthesis of the copper / silica nanogel ( cusing ) was carried out at room temperature via one - step acid - catalyzed sol - gel process using tetraethoxysilane ( teos ), water , ethanol , and cu ( ii ) sulfate . hydrolysis as shown below in equation 4 and condensation reactions in equations 5 and 6 below resulted in the formation of ultra - small (& lt ; 10 nm size ) cu loaded silica nanopaticles ( cusinps ). when maintained in acidic condition for several hours , gelation takes place . fig3 shows formation of cusing where cusinps are interconnected . the overall synthesis process will involve two simple steps : first , the addition of cu ( ii ) salts to the acidic reaction medium in the beginning of nanoparticle synthesis and second , addition of a neutralizing agent , such as , sodium hydroxide ( naoh ) after the synthesis to adjust the ph to 7 . 0 . the acid reaction medium is formed with any inorganic acid , such as hydrochloric ( hcl ), sulfuric ( h 2 so 4 ), nitric ( h 2 no 3 ) and the like . the neutralizing agent is any compound with an alkaline ph , such as sodium hydroxide , ammonium hydroxide , potassium hydroxide and the like . gelation of sol particles will take place over time . the viscosity of the resulting gel can be easily controlled by adjusting the ph and timing between these two simple synthesis steps . this is a simple one - pot , a few hours long synthesis technique that requires no purification . because of this simplicity , large scale , multi - ton scale production of cusing is feasible . the process is environmentally - safe because it is a water - based “ green synthesis ” technique , resulting in no harmful by - products , both silica and cu are naturally found in the environment , cusing will have “ fixed ” cu at low levels , and cusing will not elevate local cu levels in the soil as it will be consumed as a plant nutrient . the method also uses readily available inexpensive chemicals for the synthesis of cusing . to study the binding of ionic cu to sing , cusing is synthesized in acidic ph reaction media , such as a 1 % hydrochloric acid solution ; however , a neutralizing agent is not added . the cusing material is collected by centrifugation to remove the supernatant containing any free cu ions . the cusing is washed several times with deionized ( di ) water . in this cusing material , cusinp surface will be free of cu hydroxide / oxide precipitate and cu will be found only in its ionic form and is hereafter identified as “ cusinp - clean .” the cusing ( nanogel ) consists of inter - connected ultra - small uniform size cusinps ( particles ), as shown in fig2 , forming a gel - like structure , as shown in fig3 . these cusinps will have core - shell nanostructure . the core 10 will consist of cu ion loaded silica and the shell 20 will support cu hydroxide / cu oxide nanoclusters 40 that will be deposited on the surface of the shell 20 . the cusinp size is tunable and the particle size is variable from 10 nm to 50 nm by controlling nucleation and growth process . the rationale of varying particle size is to control the surface to volume ratio . larger size particle will have less surface area but more core volume . this will provide a unique opportunity to manipulate the ratio of ionic cu in the cusinp core 20 to the surface - bound ionic cu 40 . it should also be noted that amorphous silica is highly porous , consisting of numerous hydrated nano - channels 30 . the channel diameter is typically 1 - 2 nm . water and ethanol molecules populate the plurality of nano - channels ( bound via hydrogen bonding ) and provide a unique nanoenvironment to cu ions . it should be noted that the polarity and the hydrogen bonding capacity can be easily tuned within the nano - channel 30 by varying ethanol to water ratio , providing a good control over cu ion release from the channels . the core 10 of the particle has a reduced polar environment and is deprived of solvent molecules . this environment will facilitate the formation of cu salt nanocrystals where both cu and sulfate ions will be present in a reduced polar environment , as shown in fig2 . in cusing , surface - bound ionic cu will populate the particle surface as well as at the particle - particle interface . therefore , the unique cusinp design provides three different cu nanoenvironments , ( i ) electrostatically held core 10 cu ; i . e . ionic cu at the ng core , ( ii ) covalently bound ionic cu within nano - channels 30 , at the particle surface 20 and at the interface and ( iii ) deposited cu on the particle surface in its hydroxide / oxide form 40 ; the surface form 40 of cu will be highly exposed to the surrounding environment . the cu present in these three nanoenvironments will establish different cu release kinetics that can be controlled in a sustained manner . the cusing is expected to have very uniform coverage on plant surface due to smaller particle size . again , much improved surface adherence property is expected because of gel - like nanostructure of cusing ( as it takes the advantage of combined adherence property of individual cusinps in the network ). the novel cusing design provides more soluble (“ free and active ”) cu for improved antifungal / antibacterial activity even though it will have less metallic cu content per gram of material . during spray application in the field , some amount of cusing will be deposited on the soil that will serve as cu - based nutrient for the plant . reduced cu content in cusing will thus be used for dual purposes ; first as an essential micronutrient and secondly , as a fungicide / bactericide while minimizing cu related toxicity in the environment . the binding of cu to sing was clearly evident from the blue color appearance of the product shown in fig4 a . the product is water - dispersible . upon centrifugation , a blue residue was obtained and the supernatant was clear as shown in fig4 b . in contrast , silica ng when prepared in the absence of cu ions showed characteristic white color of silica . to evaluate how well cu was bound to silica ng , cusing was treated with ethylenediamine tetraacetic acid ( edta ), a strong chelator for cu ions . within several minutes , a water - soluble cu - edta complex formed as shown in fig4 c . upon centrifugation , we obtained white color residue of silica ng and blue color supernatant of cu - edta complex as shown in fig4 d . these results clearly demonstrate that the binding of cu to silica is not as strong as in cu - edta complex . therefore , the loaded cu could be slowly released in its ionic form from the cusing . samples were characterized by transmission electron microscopy ( tem ), x - ray diffraction ( xrd ), x - ray photoelectron spectroscopy ( xps ), and energy dispersive spectroscopy ( eds ). the size of cusing was determined using transmission electron microscopy ( tem ). the cusing consisted of ultra - small (˜ 10 nm ) size inter - connected nanoparticles ( nps ), forming a gel - like nanostructure as shown in fig3 . x - ray diffraction ( xrd ) was used to determine crystallinity ( if any ) for cusing and compared the data with kocide ® 3000 . xrd pattern clearly distinguishes cusing from kocide ® 3000 . as shown in the fig5 , kocide ® 3000 was primarily crystalline cu hydroxide / oxide material whereas cusing was purely amorphous in nature . the scanning electron microscopy - energy dispersive spectroscopy ( sem - eds , an elemental analysis technique ) showed that the amount of cu ( in atomic percentage , qualitative estimation ) present in kocide ® 3000 and cusing is 54 at . % and 13 at . %, respectively . the oxidation state of cu in cusing was determined by the x - ray photoelectron spectroscopy ( xps ). xps is able to provide information on the oxidation state of each component in the sample as well as the composition of the sample surface . high resolution regional spectra of the key elemental composition provide detailed individual component information such as chemical shift and intensity changes , indicating changes of bonding among individual elements . high - resolution xps spectra of copper ( cu ) 2p , oxygen ( o ) 1s and siica ( si ) 2p3 are shown in fig6 . the cu 2p peaks at binding energy 933 . 3 ev was attributed to the cu 2 + environment . broad satellite peaks at higher binding energies also suggest the presence of cu 2 + ionic states . the higher binding energy component located at 935 . 2 ev was attributed to cu ( oh ) 2 species . the o 1s and si 2p3 peaks were located at binding energy of 532 . 1 ev and 103 . 1 ev respectively . the binding energy of si 2p matches well with the data published by national institute of standard and technology of usa that is the si in the cusing sample is in the form of sio 2 . semi - quantitative compositional analysis is possible by comparing xps and eds data , since xps is a surface sensitive method and eds is a bulk method of compositional analysis . the atomic ratios of si / cu from xps and eds were determined by integrating the peak areas and dividing by the sensitivity factors . the si / cu ratios from eds and xps data were identified as 2 . 3 and 7 . 3 , respectively , confirming that cu is distributed throughout the cusing . assessment of antibacterial activity of cusing was performed using e - coli as a model system . disc diffusion assay , considered as one of the most common microbiological methods for the evaluation of potential antibiotic agents , was used to evaluate antimicrobial activity of cusing material . specifically , e . coli ( strain 8739 from atcc ) grown on nutrient agar plates were incubated with filter paper discs containing equal volume and cu concentration of aqueous solutions of either kocide ® 3000 50 , cu sulfate ( ph 7 ) 60 , cusing ( ph 7 ) 70 or pure silica nanoparticles ( nps ) ( ph 7 ) ( not shown ). after an overnight incubation at 37 ° c ., we found that the cusing exhibited very significant antimicrobial activity , as shown in fig7 a , and indicated by a marked zone of clearance extending away from the corresponding disc 70 . notably , the activity of the cusing was more pronounced than that of the cu sulfate solution on disc 60 , as shown in fig7 b and kocide ® 3000 on disc 50 , shown in fig7 c even though the cu content in cusing is about 4 times less than the cu content in kocide ® 3000 ( as determined by the sem - eds measurements ). the area of no e . coli growth surrounding the filter paper disc 50 with kocide ® 3000 is approximately half the area of no e . coli growth for the disc 70 with cusing , which confirms the efficiency of the cusing formulation with one - fourth of the copper content , the cusing formulation has twice the antimicrobial activity as kocide ® 3000 . it should be noted that pure silica ng , prepared without the cu ions , did not show any antibacterial activity . assessment of the adherence property of cusing was qualitatively evaluated on a lemon plant . the plant was obtained from a home depot ® hardware store in orlando , fla . in a typical experimental procedure , a first spraying of an aqueous suspension of cusing and kocide ® 3000 was uniformly sprayed at a distance of 6 inches over half a dozen pre - selected experimental leaves , assigned for each sample , using a hand - held spray bottle . the excess liquid dripped off the leaf surface . at this stage , digital images were taken of the leaf surface sprayed with kocide ® 3000 , cusinp and cusing , the adherence results are recorded in table ii below . the sprayed liquid was allowed to completely dry and again images were taken of the dried leaf surface sprayed with kocide ® 3000 and cusing . about 2 hours later , natural rain water was sprayed continuously for approximately 5 minutes onto the same experimental leaves and the leaves with a continuous spraying of rain water were allowed to dry completely . at this stage , digital images of the dry leaves having a first spraying of kocide ® 3000 , cusinp and cusing and a second spraying of rain water are evaluated visually for adherence of antibacterial / antifungal agent to the surface of the leaf . a scale of 1 to 5 is used to evaluate adherence . the results clearly indicate the superior adherence property of the cusing suspension over kocide ® 3000 . as expected , cusing showed more uniform coverage on the leaf surface . interestingly , the cusing application to a lemon tree leaf withstood very well the heavy rain fall and thunderstorm that was experienced continuously for five days and is shown in fig8 ; an enlarged image of a lemon tree leaf with a marbled and visually discernable protective covering of cusing . most of the kocide ® 3000 formulation washed away after a couple of minutes of rain water spray . to further evaluate the adherence property of cusing , a fluorescence technique was used . lemon tree leaves were tagged with fluorescein isothiocyanate ( fitc ), a green emitting fluorescent dye . the dye covalently attached to the silica . the purpose of fluorescence tagging was to monitor the sing adherence property more sensitively using fluorescence measurements . an array of spots was created on the experimental leaf shown in fig9 a with fluorescent sing material and allowed to dry completely under a dark environment to avoid photobleaching of fitc dyes . using a handheld uv light source ( 366 nm multiband excitation ), green fluorescence spots from the leaf surface were clearly noticed before the rain water spraying . fig9 b is a drawing of the fluorescent image showing the fluorescent cusing spots in the same pattern as the leaf in fig9 a . after approximately 3 hours , natural rain water was sprayed for about 5 minutes onto the leaf in fig9 b , the spray was allowed to dry completely again in the dark and the dry leaf is shown in fig9 c , with several cusing spots on the surface . using a handheld uv light source ( 366 nm multiband excitation ), green fluorescence spots from the leaf surface were clearly noticed after the rain water spraying . fig9 d is a drawing of the fluorescent image showing the fluorescent cusing material still adhering to the leaf in fig9 c in a similar pattern as shown on the leaf in fig9 c . the fluorescent images in fig9 b and 9 d confirm that the cusing material is not washed off the leaf when subjected to rain water spraying . the huge surface area of the nanogel ( ng ) materials is indeed a contributing factor towards releasing adequate amount of “ soluble ” cu . the silica - nanogel ( sing ) matrix is porous and the surface is highly hydrated , as disclosed by s . santa et al . in “ fluorescence lifetime measurements to determine the core - shell nanostructure of fitc - doped silica nanoparticles : an optical approach to evaluate nanoparticle photostability ,” journal of luminescence , 2006 . 117 ( 1 ) pages 75 - 82 . this silica matrix provides a unique environment , allowing the embedding of various metallic ions , as disclosed in u . s . pat . no . 6 , 548 , 264 to tan , et al , as well as molecules without a significant change in physical and chemical properties . characterization studies clearly show that cu ions are successfully embedded within silica matrix and the embedded cu ions are released in a sustained manner . the adherence and slow , sustained release of cu ions provides several other benefits for the present invention . first , when applied to plants , trees and the like , the leaves and / or branches that fall to the ground provide copper , a highly desired nutrient for the soil and in very low levels that are not environmentally harmful . also , when the plants and trees are harvested or removed from agricultural production , the treated vegetation can be used for mulch or composting and provided additional cu nutrients as a fertilizer . another benefit involves controlling mold and mildew growth . the copper ( cu ) loaded silica nanoparticle / nanogel formulations of the present invention have superior antibacterial / antifungal activity for treating areas that favor and foster the growth of mold and mildew ; in addition , a single treatment remains effective for from at least two months to approximately six months because the release of ionic cu occurs in a slow , sustained manner and in quantities that inhibit germination of fungal spores , the primary “ seeds ” responsible for dissemination and reproduction of the fungus or bacterium . because the spore or cell removed from the current infection cycle does not mature nor reproduce in the presence of copper , the fungus or bacterium is effectively killed . bathrooms , kitchens , interior and exterior areas of structures subject to warm , moist conditions remain free of unsightly mold and mildew when treated with the silica - based nanoformulation of the present invention . in conclusion , for the first time , a sustained cu release mechanism is provided using a uniquely designed copper ( cu ) containing silica nanoparticle / nanogel ( sinp / ng ) formulation . the silica - based nanoformulation finds immediate application for citrus canker treatment . additional uses of antibacterial / antifungal compositions containing sinp / ng are for general purpose antibacterial / antifungal agents in agriculture and horticulture , including , but not limited to use on vegetables , flowers , grass , other plants , household applications , fabrics , leathers , plastics , paints and the like . the nanotechnology - enabled cusing design is a “ revolutionary re - invention ” of cu for safe application in modern agriculture in the 21 st century . while the invention has been described , disclosed , illustrated and shown in various terms of certain embodiments or modifications which it has presumed in practice , the scope of the invention is not intended to be , nor should it be deemed to be , limited thereby and such other modifications or embodiments as may be suggested by the teachings herein are particularly reserved especially as they fall within the breadth and scope of the claims here appended .
a silica - based nanoformulation and method is used to treat citrus canker , inhibit the growth of mold and mildew , and add nutrients to soil used for agricultural purposes . the nanotechnology - enabled copper - loaded , silica nanoformulation design is a “ revolutionary re - invention ” of cu for safe application because it provides a formulation with maximum abundance of ionic cu , provides sustained and optimal cu ion release for long - term disease protection , better adherence to plant surfaces and structural surfaces due to gel - based nanostructure of cusing , thus avoiding multiple spray applications and reducing the amount of cu used in comparison to existing cu compounds without compromising antibacterial activity . thus , the silica - based nanoformulation releases copper in non - toxic quantities to the environment and the silica matrix provides an environmentally safe host material with a flexible design that is optimized to provide specific antifungal and antibacterial remediation using infrequent applications .