Patent Application: US-59326808-A

Abstract:
the present invention provides methods for the manufacturing of metal nanowires using protein fibrils as biotemplates . the methods comprise use of a solvent providing a dual effect by promoting the formation of protein fibrils of suitable size as well as acting as a reducing agent . the invention further provides metal nanowires .

Description:
the method used for nanowire production is outlined in fig1 . according to the method of the invention two critical events , i . e . metal reduction and assembly of the nanowire within the biotemplate mould occur concomitantly . a key factor in this procedure is the use of a solvent exhibiting a dual effect , both promoting the formation of protein fibrils of suitable size , and acting a reducing agent , the solvent being exemplified by 2 , 2 , 2 - trifluoroethanol ( tfe ). firstly , it is demonstrated that the solvent reduces ionic silver into colloidal form following the reaction ( 1 ): the reaction was monitored by changes of the absorbance spectra of the solution ( fig2 a ), which were also corroborated by the colour developing from fully uncoloured to yellow / brown 15 . the samples were analysed by afm , demonstrating the formation of uniform silver nanoparticles . their size depended on the reduction time ; 1 . 2 nm particles were formed after 2 hours and 35 nm after 72 hours , respectively ( fig2 b - 2 e ). secondly , the solvent induces the lateral assembly of individual protein fibrils into larger and thicker scaffolds , which trap silver in inter - fibrillar space and serve a role of the moulds . before treatment with tfe the amyloid fibrils were characterised by 2 - 4 nm height in afm images ( fig3 b ), exhibiting typical amyloid tinctorial features such as thioflavin t dye binding 16 , 17 ( fig3 a ). after incubation with tfe in the presence and absence of silver ions fibrils become thicker with ca 6 - 9 nm height ( fig3 c , 3 e ), which corroborated with a slight decrease in thioflavin t binding due to a decrease in number of exposed thioflavin t binding sites ( fig3 a ). in the fibrillar sample containing silver the yellow - brown colour was developed , indicating silver reduction . in order to verify formation of silver nanowires , the proteinaceous moulding scaffolds were digested by proteinase k . in the sample without silver , the amyloid was fully removed after 24 hours of proteinase treatment . this was confirmed by the drop of the thioflavin t signal to the level of free dye in solution ( fig3 a ) and by afm ( fig3 e ). in the sample where the silver nanowires were cast in the amyloid scaffold , the nanowires were released and observed by afm ( fig3 d ), while the thioflavin t signal also disappeared , indicating the degradation of amyloid structures ( fig3 a ). the nanowires were characterised by having a diameter of 1 - 2 . 5 nm and a variable length from 0 . 5 to ca . 2 μm ( fig3 ). the nanowires were straight or banded and sometimes a few nanowires were twisted around each other ( fig3 d , fig4 ). in control experiments the amyloids in the presence of agno 3 were treated with the reducing agents n , n - dimethylformamide ( dmf ) or citric acid . while both agents initiated the formation of silver nanoparticies 9 , 15 , they did not induce fibril thickening , but rather caused their fragmentation to shorter species of 0 . 5 - 1 μm length and 2 - 4 nm in height ( fig5 ). consequently , after the treatment with proteinase k all amyloid material was removed but the silver nanowires were not detected . this indicates that fibrillar lateral assembly and formation of the structured , hollow fibrillar interface is essential for silver moulding , whiles the individual fibrils with a common diameter of 2 nm and less do not possess such a cavity . thus , naturally occurring proteinaceous amyloid polymers assembled into larger scaffolds can be used for casting metal nanostructures . this has been utilised for this purpose an abundant and cheep material such as hen lysozyme . the limit in biotemplating producing the ultra thin silver nanowire of ca . 1 nm diameter has been reached , which is significantly below the dimensions attainable by standard electronic manufacturing processes and also one order of magnitude thinner than nanowires fabricated using other proteinaceous scaffolds 8 , 9 . the method according to the present invention allows removing the moulding scaffold by proteinase digestion compared to other approaches , in which the protein polymers remain within the formed metal wires 8 . for the first time it is surprisingly demonstrated that a fluorinated alcohol , tfe , can be used as a reducing agent and has potential applications in manufacturing metal nanoparticles . the major advantage of the methods according to the invention is the use of a solvent such as tfe , which compared to other combinations of solvents and reducing agents induces formation and stabilization of protein fibril scaffold simultaneously with metal ion reduction . less suited reducing agents , such as citric acid and dmf causes fragmentation of the protein fibrils without the formation of metal nanowires . the process of biotemplating can be used as a tool to get an insight on the properties of the scaffold itself , according to the present invention on amyloid . by analysing the x - ray diffraction pattern of fibrillar amyloids , perutz and co - authors suggested that the amyloid fibrils are probably built of a few concentric cylindrical β - sheets containing the hollow part filled with water 18 . this hypothesis was supported by production of synthetic dipeptide amyloid - like nanotubes and filling them with silver of ca . 20 nm diameter 9 . however , the nmr - based analysis of amyloid fibrils of aβ peptide and prions indicate the dense packing within fibrillar interior , which rules out the cavity in the protofilament core 19 , 20 . in this light the data directly demonstrate the absence of the hollow part within the individual amyloid fibrils , unlike synthetic peptide nanotubes , prior their assembly into the larger scaffold . the methods according to the invention can be used to form nanowires from a noble metal , such as silver , gold , platinum , and palladium , or other suitable metal such as copper . the methods according to the invention can be used to produce nanoparticles with controlled dimensions . hen egg white lysozyme was purchased from sigma ( usa ). amyloid was produced by incubation of protein in 20 mm glycine buffer at ph 2 . 5 , 57 ° c . proteinase k was purchased from fermentas ( lithuania ). dmf , tfe and citric acid were purchased from fluka ( germany ). thioflavin t binding amyloid assay was carried out as described previously 13 . fluorescence measurements were performed on a jasco spectrofluorometer fp 6500 , using excitation at 440 nm and measuring emission between 460 and 520 nm . afm measurements were performed on a pico plus microscope ( molecular imaging , usa ) in a tapping mode using a 100 μm scanner with acoustically driven cantilevers operating at a resonance frequency in 320 - 370 khz range . scanning resolution was 512 × 512 pixels . the scanning was performed in trace and retrace to avoid the scan artefacts . specimens were diluted in miliq water , incubated on freshly cleaved mica for 10 min , subsequently washed 2 × 200 μl with miliq water and dried over night at room temperature . the parameters of nanostructures were measured by using a scanning probe image processor software ( image metrology , denmark ). the stock of 1m agno 3 in miliq water was diluted to a final concentration of 20 mm with the excess of appropriate reducing agents such as tfe , dmf or citric acid and subsequently incubated at 57 ° c . for 72 hours . reduction of silver was estimated by measuring absorbance spectra at 300 - 700 nm on a lambda 40 spectrophotometer ( perkin elmer , usa ) as described previously 15 . amyloid fibrils were sedimented for 30 min at 12000 g on a minicentrifuge ( eppendorf , germany ). the pellet was resuspended in 100 μl miliq water and sedimented again . agno 3 was added to the fibrillar pellet to 20 mm final concentration and the sample was incubated at room temperature for 24 h . the fibrils were centrifuged and an access of reducing agent was added to the pellet , following incubation for 48 h at 57 ° c . the complexes were centrifuged , resuspended at 100 mm tris - 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