Patent Application: US-201013516103-A

Abstract:
the present invention provides for mixed metal structures that can be deposited on a substrate or free in solution that exhibit several distinctive properties including a broad wavelength range for enhancing fluorescence signatures . further , metal surface plasmons can couple and such diphase coupled luminescence signatures create extra plasmon absorption bands . the extra bands allow for a broad range of fluorophores to couple therefore making more generic substrates with wider reaching applications .

Description:
before the present invention is disclosed and described , it is to be understood that this invention is not limited to the particular process steps and materials disclosed herein as such process steps and materials may vary somewhat . it is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof . it must be noted that , as used in this specification and the appended claims , the singular forms “ a ,” “ an ,” and “ the ” include plural references unless the content clearly dictates otherwise . the term “ biomolecule ” means any carbon based molecule occurring in nature or a derivative of such a molecule . the biomolecule can be in active or inactive form . “ active form ” means the biomolecule is in a form that can perform a biological function . “ inactive form ” means the biomolecule must be processed either naturally or synthetically before the biomolecule can perform a biological function . exemplary biomolecules include nucleic acids , aromatic carbon ring structures , nadh , fad , amino acids , carbohydrates , steroids , flavins , proteins , dna , rna , oligonucleotides , peptide nucleic acids , fatty acids , sugar groups such as glucose etc ., vitamins , cofactors , purines , pyrimidines , formycin , lipids , phytochrome , phytofluor , peptides , lipids , antibodies and phycobiliproptein . fluorophore ,” and “ fluorescence label ,” used interchangeably herein , means any substance that emits electromagnetic energy such as light at a certain wavelength ( emission wavelength ) when the substance is illuminated by radiation of a different wavelength ( excitation wavelength ) and is intended to encompass a chemical or biochemical molecule or fragments thereof that is capable of interacting or reacting specifically with an analyte of interest in a sample to provide one or more optical signals . additionally fluorophore includes both extrinsic and intrinsic fluorophores . extrinsic fluorophore refer to fluorophores bound to another substance . intrinsic fluorophores refer to substances that are fluorophores themselves . exemplary fluorophores include but are not limited to those listed in the molecular probes catalogue which is incorporated by reference herein . representative fluorophores include but are not limited to alexa fluor ® 350 , dansyl chloride ( dns - cl ), 5 -( iodoacetamida ) fluoroscein ( 5 - iaf ); fluoroscein 5 - isothiocyanate ( fitc ), tetramethylrhodamine 5 -( and 6 -) isothiocyanate ( tritc ), 6 - acryloyl - 2 - dimethylaminonaphthalene ( acrylodan ), 7 - nitrobenzo - 2 - oxa - 1 , 3 ,- diazol - 4 - yl chloride ( nbd - cl ), ethidium bromide , lucifer yellow , 5 - carboxyrhodamine 6g hydrochloride , lissamine rhodamine b sulfonyl chloride , texas red ™ sulfonyl chloride , bodipy ™, naphthalamine sulfonic acids including but not limited to 1 - anilinonaphthalene - 8 - sulfonic acid ( ans ) and 6 -( p - toluidinyl ) naphthalen - e - 2 - sulfonic acid ( tns ), anthroyl fatty acid , dph , parinaric acid , tma - dph , fluorenyl fatty acid , fluorescein - phosphatidylethanolamine , texas red - phosphatidylethanolamine , pyrenyl - phophatidylcholine , fluorenyl - phosphotidylcholine , merocyanine 540 , 1 -( 3 - sulfonatopropyl )- 4 -[-. beta .-[ 2 [( di - n - butylamino )- 6 naphthyl ] vinyl ] pyridinium betaine ( naphtyl styryl ), 3 , 3 ′ dipropylthiadicarbocyanine ( dis - c 3 -( 5 )), 4 -( p - dipentyl aminostyryl )- 1 - methylpyridinium ( di - 5 - asp ), cy - 3 iodo acetamide , cy - 5 - n - hydroxysuccinimide , cy - 7 - isothiocyanate , rhodamine 800 , ir - 125 , thiazole orange , azure b , nile blue , al phthalocyanine , oxaxine 1 , 4 ′, 6 - diamidino - 2 - phenylindole ( dapi ), hoechst 33342 , toto , acridine orange , ethidium homodimer , n ( ethoxycarbonylmethyl )- 6 - methoxyquinolinium ( mqae ), fura - 2 , calcium green , carboxy snarf - 6 , bapta , coumarin , phytofluors , coronene , and metal - ligand complexes . representative intrinsic fluorophores include but are not limited to organic compounds having aromatic ring structures including but not limited to nadh , fad , tyrosine , tryptophan , purines , pyrirmidines , lipids , fatty acids , nucleic acids , nucleotides , nucleosides , amino acids , proteins , peptides , dna , rna , sugars , and vitamins . additional suitable fluorophores include enzyme - cofactors ; lanthanide , green fluorescent protein , yellow fluorescent protein , red fluorescent protein , or mutants and derivates thereof . fluorophores with high radiative rates have high quantum yields and short lifetimes . increasing the quantum yield requires decreasing the non - radiative rates k nr , which is often only accomplished when using a low solution temperature or a fluorophore bound in a more rigid environment . the natural lifetime of a fluorophore , τ n , is the inverse of the radiative decay rate or the lifetime which would be observed if their quantum yields were unity . this value is determined by the oscillator strength ( extinction coefficient ) of the electronic transition . hence , for almost all examples currently employed in fluorescence spectroscopy , the radiative decay rate is essentially constant . the modification and control of the radiative rate have also been referred as radiative decay engineering ( rde ), or “ lightening rod ” fluorescence enhancement effect . for example , enhanced intrinsic dna fluorescence above metallic particles has recently been observed , which is typically not readily observable because of dna &# 39 ; s very low quantum yield of less than 10 − 4 . the second favorable “ lightening rod ” effect also increases the fluorescence intensity by locally enhanced excitation . in this case , emission of fluorophores can be substantially enhanced irrespective of their quantum yields . the reduction in lifetime of a fluorophore near a metal is due to an interaction between the fluorophore and metal particle , which enhances the radiative decay rate ( quantum yield increase ) or depending on distance , d − 3 , causes quenching . it should be noted that lifetimes of fluorophores with high quantum yields ( 0 . 5 ) would decrease substantially more than the lifetimes of those with low quantum yields ( 0 . 1 and 0 . 01 ). a shorter excited - state lifetime also allows less photochemical reactions , which subsequently results in an increased fluorophore photostability . notably , the use of low quantum yield fluorophores would lead to much larger fluorescence enhancements ( i . e . 1 / q 0 ) and could significantly reduce unwanted background emission from fluorophores distal from the silvered assay . fluorophore photostability is a primary concern in many applications of fluorescence . this is particularly true in single molecule spectroscopy . a shorter lifetime also allows for a larger photon flux . the maximum number of photons that are emitted each second by a fluorophore is roughly limited by the lifetime of its excited state . for example , a 10 ns lifetime can yield about 10 8 photons per second per molecule , but in practice , only 10 3 photons can be readily observed . the small number of observed photons is typically due to both photo - destruction and isotropic emission . if a metal surface decreases the lifetime , one can obtain more photons per second per molecule by appropriately increasing the incident intensity . on the other hand , the metal - enhanced fluorescence provides enhanced intensity , while simultaneously shortening the lifetime . that is , it may be possible to decrease the excitation intensity , yet still see a significant increase in the emission intensity and photostability . the emission enhancement may be observed at distances according to the type of fluorophore to be detected and the type , shape of the metal material , noting a difference between a film and a metallic island or colloid . for example , emission enhancement may be observed when a fluorophore distances about 5 nm to about 200 nm to metal surfaces . preferable distances are about 5 nm to about 30 nm , and more preferably , 5 nm to about 20 nm to metal surfaces . at this scale , there are few phenomena that provide opportunities for new levels of sensing , manipulation , and control . in addition , devices at this scale may lead to dramatically enhanced performance , sensitivity , and reliability with dramatically decreased size , weight , and therefore cost . attaching of the fluorophore to a probe may be achieved by any of the techniques familiar to those skilled in the art . for example , the fluorophore may be covalently attached to the bimolecular probe by methods disclosed in u . s . pat . no . 5 , 194 , 300 ( cheung ) and u . s . pat . no . 4 , 774 , 189 ( schwartz ). in another embodiment , the assay system of the present invention provides for detecting and separating at least two target pathogen by choosing fluorophores such that they possess substantially different emission spectra , preferably having emission maxima separated by greater than 10 nm , more preferably having emission maxima separated by greater than 25 nm , even more preferably separated by greater than 50 nm . when differentiation between the two fluorophores is accomplished by visual inspection , the two dyes preferably have emission wavelengths of perceptibly different colors to enhance visual discrimination . when it is desirable to differentiate between the two fluorophores using instrumental methods , a variety of filters and diffraction gratings allow the respective emission maxima to be independently detected . any chemiluminescent species may be used in the present invention that provides for a chemical reaction which produces a detectable reaction ( observed emission ) wherein the excited state responsible for the observed emission including , but not limited to the following excitation mechanisms : r •+ r ′•→ r − r + hv ( single bond formation ( radical - radical reaction )) • r •+• r •′→ r ═ r + hv ( double bond formation ( radical - radical reaction )) ro 2 • r •+ o 2 → r + hv r + + → r + hv ( electron capture ) examples of suitable chemiluminescence detector molecules include but without limitation , peroxidase , bacterial luciferase , firefly luciferase , functionalized iron - porphyrin derivatives , luminal , isoluminol , acridinium esters , sulfonamide and others . a recent chemiluminescent label includes xanthine oxidase with hypoxanthine as substrate . the triggering agent contains perborate , a fe - edta complex and luminol . choice of the particular chemiluminescence labels depends upon several factors which include the cost of preparing labeled members , the method to be used for covalent coupling to the detector molecule , and the size of the detector molecules and / or chemiluminescence label . correspondingly , the choice of chemiluminescence triggering agent will depend upon the particular chemiluminescence label being used . chemiluminescent reactions have been intensely studied and are well documented in the literature . for example , peroxidase is well suited for attachment to the detector molecule for use as a chemiluminescence . the triggering agent effective for inducing light emission in the first reaction would then comprise hydrogen peroxide and luminol . other triggering agents which could also be used to induce a light response in the presence of peroxidase include isobutyraldehyde and oxygen . procedures for labeling detector molecules , such as antibodies or antigens with peroxidase are known in the art . for example , to prepare peroxidase - labeled antibodies or antigens , peroxidase and antigens or antibodies are each reacted with n - succinimidyl 3 -( 2 - pyridyldithio ) proprionate ( hereinafter spdp ) separately . spdp - labeled peroxidase , or spdp - labeled antigen or antibody is then reacted with dithiothreitol to produce thiol - labeled peroxidase , or thiol - labeled antigen or antibody . the thiol derivative is then allowed to couple with the spdp - labeled antigen or antibody , or spdp - labeled peroxidase . the present invention provides enhanced emissions using metallic structures of elliptical , spherical , triangular , rod - like forms or any geometric form . in exemplary cases , the elliptical islands have aspect ratios of 3 / 2 , and the spherical colloids have diameters of 20 - 60 nm . using known coating techniques , the placement of metallic structures could be controlled precisely , as close as 50 nm apart . further , the metallic structures can be fabricated to form a geometric shape such as triangle , square , oblong , elliptical , rectangle , or any shape that provides at least one apex area of the metallic surface . it is envisioned that the apex area includes not only pointed regions but regions with rounded edges such as found in an oblong or elliptical shape . the apex areas are preferably arranged so that one apex area is opposite from another apex area and aligned to cause the reactive zone to be positioned therebetween . the distances between the apex areas may range from 0 . 01 mm to 5 mm , more preferably from 2 mm to about 3 mm and depending on the size of the required reactive zone . the thickness of the metallic geometric shaped forms ranges from 25 nm to about 1000 nm , and more preferably from about 45 nm to about 250 nm . the present invention further comprises a detection device for detecting emissions including , but not limited to visual inspection , digital ( ccd ) cameras , video cameras , photographic film , or the use of current instrumentation such as laser scanning devices , fluorometers , luminometers , photodiodes , quantum counters , plate readers , epifluorescence microscopes , fluorescence correlation spectroscopy , scanning microscopes , confocal microscopes , capillary electrophoresis detectors , or other light detector capable of detecting the presence , location , intensity , excitation and emission spectra , fluorescence polarization , fluorescence lifetime , and other physical properties of the fluorescent signal . excitation light sources can include arc lamps and lasers , natural sunlight , laser diodes and light emitting diode source , and both single and multiple photon excitation sources . in another embodiment , use of a ti - sapphire laser , laser diode ( ld ) or light emitting diode sources ( leds ) may be used with the rna assay of the present invention . for example , using 2 - photon excitation at 700 - 1000 nm and also using short pulse width (& lt ; 50 pi ), high repetition rate ( 1 - 80 mhz ), laser diode and led ( 1 ns , 1 - 10 mhz ) sources . the enhanced sensitivity of the assay using 2 - photon excitation , as compared to 1 - photon , can be shown by using series dilution with rna , initially with the ti - sapphire system , and later with leds and lds . if a fluorophore absorbs two photons simultaneously , it will absorb enough energy to be raised to an excited state . the fluorophore will then emit a single photon with a wavelength that depends on the fluorophore used and typically in the visible spectra . the use of the ti - sapphire laser with infrared light has an added benefit , that being , longer wavelengths are scattered less , which is beneficial for high - resolution imaging . importantly , there is reduced background signal level gained by using 2 - photon excitation as compared to 1 - photon excitation by utilizing localized excitation near by metallic particles . in one embodiment , the application of low level microwave heating of the sample may be used to speed up any chemical / biochemical kinetics within the system . notably , low level microwaves do not destroy or denature proteins , dna , or rna , but instead heat the sample sufficiently to provide for accelerated kinetics such as binding or hybridization . in addition , the microwaves are not scattered by the metallic structures , which is contrary to most metal objects , such as that recognized by placing a spoon in a microwave oven . microwaves ( about 0 . 3 to about 300 ghz ) lie between the infrared and radiofrequency electromagnetic radiations . it is widely thought that microwaves accelerate chemical and biochemical reactions by the heating effect , where the heating essentially follows the principle of microwave dielectric loss . polar molecules absorb microwave radiation through dipole rotations and hence are heated , where as non - polar molecules do not absorb due to lower dielectric constants are thus not heated . the polar molecules align themselves with the external applied field . in the conventional microwave oven cavity employed in this work , the radiation frequency ( 2450 mhz ) changes sign 2 . 45 × 10 9 times per second . heating occurs due to the tortional effect as the polar molecules rotate back and forth , continually realigning with the changing field , the molecular rotations being slower than the changing electric field . the dielectric constant , the ability of a molecule to be polarized by an electric field , indicates the capacity of the medium to be microwave heated . thus , solvents such as water , methanol and dimethyl formamide are easily heated , where as microwaves are effectively transparent to hexane , toluene and diethylether . for metals , the attenuation of microwave radiation arises from the creation of currents resulting from charge carriers being displaced by the electric field . these conductance electrons are extremely mobile and unlike water molecules can be completely polarized in 10 - 18 s . in microwave cavity used in the present invention , the time required for the applied electric field to be reversed is far longer than this , in fact many orders of magnitude . if the metal particles are large , or form continuous strips , then large potential differences can result , which can produce dramatic discharges if they are large enough to break down the electric resistance of the medium separating the large metal particles . interestingly , and most appropriate for the new assay platform described herein , small metal particles do not generate sufficiently large potential differences for this “ arcing ” phenomenon to occur . however , as discuss hereinbelow , the charge carriers which are displaced by the electric field are subject to resistance in the medium in which they travel due to collisions with the lattice phonons . this leads to ohmic heating of the metallic structures in addition to the heating of any surface polar molecules . intuitively , this leads to localized heating around the metallic structures in addition to the solvent , rapidly accelerating assay kinetics . in the present invention , microwave radiation may be provided by an electromagnetic source having a frequency in a range between 0 . 3 and 10 ghz and a power level in a range between about 10 mwatts and 400 watts , more preferably from 30 mwatts to about 200 watts . any source , known to one skilled in the art may be used , such as a laser that emits light , wherein light is used in its broad sense , meaning electromagnetic radiation which propagates through space and includes not only visible light , but also infrared , ultraviolet and microwave radiation . thus , a single instrument placed above the surface of the assay can be used to generate the microwave energy and energy to excite fluorescing molecules . the light can be emitted from a fiber continuously or intermittently , as desired , to maintain the metallic particles at a predetermined temperature such that it is capable of increasing the speed of chemical reactions within the assay system . the microwave radiation may be emitted continuously or intermittently ( pulsed ), as desired . in the alternative , microwave energy can be supplied through a hollow wave guide for conveying microwave energy from a suitable magnetron . the microwave energy is preferably adjusted to cause an increase of heat within the metallic material without causing damage to any biological materials in the assay system . although fluorescence , chemiluminescence and / or bioluminescence detection has been successfully implemented , the sensitivity and specificity of these reactions require further improvements to facilitate early diagnosis of the prevalence of disease . in addition , most protein detection methodologies , most notably western blotting , are still not reliable methods for accurate quantification of low protein concentrations without investing in high - sensitivity detection schemes . protein detection methodologies are also limited by antigen - antibody recognition steps that are generally kinetically very slow and require long incubation times ; e . g ., western blots require processing times in excess of 4 h . thus , both the rapidity and sensitivity of small - molecule assays are still critical issues to be addressed to improve assay detection . as such the use of low intensity ultrasound will increase the rapidity of the assay . there are many important assays that can directly benefit from enhanced signal intensities and quicker kinetics . for example , myoglobin concentrations for heart attack patients , patients of toxic shock and pancreatitus . all of these assays are widely used in hospitals emergency rooms with assay times of greater than 30 minutes . thus , the present invention can be used for points - of - care clinical assessment in emergency rooms . thus it would be advantageous to increase speed of any chemical or biochemical reaction by using any device capable of generating and transmitting acoustic energy through any medium to transit ultrasonic atomizing energy . the ultrasonic emitting device can be placed in either the interior of a vessel used in a detection system or positioned adjacent thereto for transmitting energy into the vessel . the device may include components for the traditional electromagnetic stimulation of piezoelectric transducers , ( man - made or naturally occurring ), purely mechanical devices ( such as high frequency air whistles or microphones ), and laser devices . individual components for acoustic energy systems are commercially available from a wide variety of manufacturers , which can be configured to particular applications and frequency ranges . ( see thomas directory of american manufacturers , photonics buyer &# 39 ; s guide , 1996 , microwave and rf , and electronic engineer &# 39 ; s master catalogue ). any oscillator or signal generator that produces a signal with predetermined characteristics such as frequency , mode , pulse duration , shape , and repetition rate may be used to generate acoustic frequencies for applying to the system of the present invention . various oscillators or signal generators can be commercially purchased from a wide variety of manufacturers and in a variety of designs configured to particular applications and frequencies . applicable transducers will include types that produce an acoustic wave within a range of frequencies ( broadband ) or for one specific frequency ( narrowband ) for frequencies ranging from hertz to gigahertz . the acoustic delivery system will be variable depending on the application . for example , acoustic energy waves can be transmitted into liquid or solid source material either by direct contact of the source material with a transducer , or by coupling of transmission of the acoustic wave through another medium , which is itself in direct contact with the source material . if the source material is a liquid , a transducer can be placed in the liquid source material , or the walls of the vaporization vessel can be fabricated of a material that acts as a transducer thereby placing the liquid source material in direct contact with the transducer . additionally , an acoustic energy emitting device may be positioned on the exterior of a system container for transmitting the appropriate energy . if the source material is a solid , a transducer can be placed in direct contact with it or the solid source material can be placed in a gas or liquid that is used as a coupling agent . in the preferred acoustic frequencies any system that generates acoustic energy may be utilized . preferably , the output of the ultrasonic generator is of a sufficient frequency to provide a movement flow within the system vessel to move molecules to the source of binding or reaction site without causing a large increase of heat in the system . for example , using the power output of 0 . 5 to 50 w at a frequency of 10 to 200 khz , and more preferably from about 20 to 60 khz and most preferably at about 40 khz . to obtain the maximum transfer of acoustical energy from one medium to another , the characteristic acoustical impedance of each medium is preferably as nearly equal to the other as possible . the matching medium is sandwiched between the other two and should be the appropriate thickness relative to the wavelength of the sound transmitted , and its acoustical impedance r should be nearly equal to ( r 1 : r 2 ). any impedance matching device that is commercially available can be utilized in the present invention . the system may include ultrasonic vessels wherein at least a section of the vessel includes a transducer such as a piezoelectric transducer to generate acoustic vibrations . such transducers can be located in the bottom of a vessel or in a plate whereon a vessel may be placed . further such transducers can be placed at different levels on the vessel walls to enhance fluid flow within the vessel . materials . toluene , fluorescein , rose bengal , n - acetyl - tryptophan - amid ( nata ) and phenylalanine ( phe ), were purchased from sigma . the concentration of nata and phe were determined by measuring the optical density of the solutions using extinction coefficients of e 280 = 5 , 300 m − 1 cm − 1 and e 257 = 195 m − 1 cm − 1 , respectively ( 26 ; 27 ). bovine serum albumin ( bsa ) was purchased from sigma . to determine the concentration of the protein in solutions , an extinction coefficient of e 280 = 43 , 824 m − 1 cm − 1 was used . aluminum wire , 99 % pure , for thermal vapor deposition onto glass slides was purchased from the kurt j . lesker company , material group ( clairton , pa .). chromophores , including amino acids and protein , bsa , were dissolved in te buffer , ph 7 . 4 . measurements of absorption and fluorescence . absorption spectra of the fluorophores , protein intrinsic chromophores and bsa were measured using a varian spectrophotometer in quartz b7 1 - cm path - length cuvettes . fluorescence spectra of the fluorophores and bsa were recorded using a cary eclipse spectrofluorimeter ( varian , inc ., usa ) at room temperature . the solutions were sandwiched between a quartz slide and a glass slide with deposited metal ( s ) ( silver , aluminum and their mixture ) and for the control sample , containing no metal , between quartz / glass slides . time - resolved fluorescence decay measurements . the fluorescence intensity decay functions of the fluorescein chromophore on metal slides of different composition and thickness , and on glass ( mef control sample ) were measured using a tempro fluorescence lifetime system ( horiba jobin yvon , usa ). the reference cell contained colloidal silica , sm - 30 ludox solution , used as a control ( zero lifetime ). measurements were performed at room temperature . determination of fluorescein excited state lifetimes ( τ i ) and corresponding amplitudes ( α i ) were undertaken using the tempro fluorescence lifetime system software , das 6 . the emission intensity decays were analyzed in terms of the multiexponential model : [ t ] = ∑ i ⁢ α i ⁢ exp ⁡ [ - t / τ i ] ( 1 ) where α i are the amplitudes , the sum of which equals to 1 . 0 , and τ i are the decay times . the fractional contribution of each component to the steady - state intensity can be given as : the mean lifetime of the chromophore excited state was calculated using the following equation : 〈 τ 〉 = ∑ i = 1 n ⁢ α i × τ i ( 4 ) where n is a number of fluorescence decay components in the total decay function . the values of the amplitudes and decay times were determined using nonlinear least - squares impulse reconvolution with a goodness - of - fit χ 2 criterion . numerical fdtd simulations . the 2d computational simulations of the electric field intensities and near - field distributions around metal nanoparticles were undertaken for three systems : a ) two 250 nm silver nanoparticles separated by 40 nm free space ; b ) 30 nm aluminum nanoparticle ( np ) and c ) their mixture — 30 nm aluminum np is centered between two 250 nm silver nps , separated by 40 nm , using the finite difference time domain ( fdtd ) method . tfsf ( total field scattered field ) sources are used to divide the computation area or volume into total field ( incident plus scattered field ) and scattered field only regions . the incident p - polarized electric field was defined as a plane wave with a wave - vector that is normal to the injection surface . using fdtd solution software ( lumerical , inc . http :// www . lumerical . com ), the simulation region was set to 700 × 450 nm 2 with a mesh accuracy of 5 . to minimize simulation times and maximize resolution of field enhancement regions around the particle arrangement , a mesh override region is set to 1 nm around the nanoparticles . the overall simulation time was set to 50 fs and calculated over a broad wavelength range , using known permittivity values and refractive indices of silver and aluminum . the wavelength dependence of the nps extinction , and cross - sections of its components , absorption and scattering , were calculated using lumerical software script . to simulate effects of water polarity on plasmon resonance spectra , the background index was set to the corresponding refractive index of water , 1 . 333 . preparation of silver - island films ( sifs ). silver - island films were prepared according to the procedure found in reference ( 3 ). deviations in sif thickness were reduced by using a fresh selection of silane - prep ™ slides . the slides were stored in a vacuum between sifs preparations to reduce possible oxidation . thermal vapor deposition of aluminum onto glass slides and on silver coated slides ( sifs ) was performed using an auto 306 vacuum coater instrument , equipped with sqm - 160 rate / thickness monitor ( boc edwards , usa ). thickness of the deposited aluminum on glass slides and on slides coated with silver nanoparticles ( sifs ) ranged from 2 to 16 nm , as measured using the quartz - crystal microbalance . real - color photographs of the aluminum and mixed ( aluminum + silver ) slides , used in this study , are shown in fig1 . to study the effects of mixed - metal substrates ( mms ) on enhanced fluorescence intensities , aluminum was thermally evaporated onto glass substrates containing preformed silver - island films ( sifs ), where individual both sifs and aluminum slides also served as control samples . as a function of aluminum deposition , the slides are seen to become increasingly optically dense as evidenced by the photographs within fig1 , left to right . afm images for 2 nm aluminum deposits on glass , fig2 a , show surfaces comprised of small “ rice like ” nanoparticles , more evident in the phase contrast image of fig2 a right . as the thickness of the aluminum is increased , fig2 b , the surface appears much more continuous . in contrast , afm images of sifs deposits on glass show much bigger island deposits , consistent with recent reports , fig3 a ( 28 ). however , when 2 nm aluminum is deposited on the sifs , we see the “ rice like ” structures effectively coating the sifs , fig3 b , the phase contrast images , right , showing the aluminum nanostructured texture over the sifs . the horizontal line scan of fig3 b also shows an increase in surface roughness between the sifs and coated sifs samples , c . f ., fig3 a right and 3 b right images . similarly , fig1 to 17 , show aluminum deposits on sifs for 8 , 12 and 16 nm of aluminum respectively . absorption spectra of the samples , fig4 , shows that both 2 and 8 nm aluminum coatings appear optically mirror like , with a somewhat flat absorbance until the deep uv , where the band at 250 nm is indicative of the plasmon absorbance of aluminum . the sifs absorption spectra shows the typical strong plasmon resonance bands from 380 to 500 nm ( 28 ), which both broadens and increases in optical density for the thicker coatings of aluminum on the sifs . this is consistent with the color photographs in fig1 , which also become less transparent for thicker aluminum coatings . to understand these colorimetric and optical density changes , finite difference time domain ( fdtd ) simulations was undertaken , in essence numerical simulations to both explain and account for the experimental observations . fig5 shows a region - of - interest ( roi ) which has been selected from the afm images of fig3 . interestingly , the enlarged roi clearly shows the aluminum “ rice like ” deposits ( region y ) on the sifs ( region x ). fig6 shows the respective theoretical electrical field simulations for the model . for the case of just two silver nanoparticles , fig6 c , a modest e - field intensity is seen between the nanoparticles , considerably greater in magnitude than for the single aluminum particle shown in fig6 b . fig6 a shows the significantly enhanced electric field for the case of two silver nanoparticles and one centered aluminum nanoparticle . it is important to note the y - axis intensity scales for each respective image . the significant increases in electric field strength are further visualized when considering the respective normalized plot of fig6 and fig1 . in these figures , one only sees an electric field in the top portion of the images , i . e . for the two silver and one aluminum nanoparticle constructs . it has been previously postulated that the mechanism underpinning mef to be comprised of both an enhanced absorption ( i . e . enhanced electric field effect ) as well as an enhanced plasmon coupling component , the extent of mef luminescence enhancement underpinned by the spectral overlap of a fluorophores &# 39 ; emission spectra with the plasmon - scattering component of a nanoparticles &# 39 ; extinction spectra ( 16 ). this second mode of fluorescence enhancement has recently been experimentally verified , ( 16 ), and manifests itself by a shorter system luminescence / fluorescence lifetime , the surface plasmons in essence radiating the coupled quanta , in a system which is coupled in both the ground and excited state ( 16 ). subsequently to further understand the plasmon - coupling component in mef mixed metal substrates , further simulations have been undertaken fig7 . fig7 shows the extinction spectra ( a ), absorption ( b ) and scattering components ( c ) for the 3 - particle model . interestingly , all three spectra are typically broader for the 2 ag and 1 al nanoparticle system i . e . that considered in our roi , fig5 . surprisingly , deconvolution of the respective spectra shows the presence of a new plasmon resonance band at ≈ 540 nm , not present in the plasmon absorption spectra of the two individual metals themselves , fig8 . at this time it is believed that the new resonance band is due to the coupling and high - frequency dephased resonance of the surface plasmons from both metal types , not unlike the dephasing of similar resonances for identical metals , which have been shown to couple up to 2 . 5 times their diameter ( 6 ). interestingly , the roi afm image of fig5 , clearly shows the same particles are within this geometrical coupling consideration . it is worth noting that this model considers particles which are spatially separated and no model has been considered for the case of the al directly coated onto the sifs , which is far more complex , both sample embodiments present in fig5 . to test the utility of the mixed metal substrates for mef , both traditional and intrinsic fluorescent chromophores were considered , fig9 . for a solution of fluorescein sandwiched between the mixed metal substrates and a blank slide , further enhanced luminescence signatures can be seen as compared to the mef from the individual metals . this finding can also be observed visually in the color photographs of fig1 , and suggests that mixed - metal substrates are a much better choice for applications in mef , as compared to the widely used silver substrates . a similar result as for fluorescein can be seen for rose bengal , see fig1 . as briefly mentioned earlier , mef affords for both enhanced luminescence intensities and reduced fluorophore lifetimes . these observations are empirically underpinned by modifications to the classical far - field ( greater than 1 wavelength of light away ) rate equations . for a fluorophore in the far - field condition the free - space quantum yield , q 0 , is given by : q 0 = γ γ + k nr ( 5 ) τ 0 = 1 γ + k nr ( 6 ) where γ is the radiative rate , τ 0 is the free space lifetime and k nr are the non - radiative rates . in this free - space condition , any changes in a fluorophores &# 39 ; radiative rate , invariably results in the quantum yield and lifetime , q 0 and τ 0 respectively , changing in unison . however for mef , geddes has shown that these classical far - field considerations can be rewritten for the near - field condition ( 3 ), such that : q m = γ + γ m γ + γ m + k nr ( 7 ) τ m = 1 γ + γ m + k nr ( 8 ) where q m and t m modified quantum yields and lifetimes respectively . to test whether mixed - metal substrates follow these near - field approximations , which have been shown to hold for numerous reports of single metals ( 3 ; 19 ), the time - resolved fluorescence decay times were measure , fig2 and table 1 , as shown below . deconvolution analysis ( 5 ) of the decays in fig2 , shows the greatest reduction in both amplitude weighted and mean lifetime , table 1 , consistent with the maximum fluorescein fluorescence enhancement shown in fig9 and 10 . this finding is completely consistent with current mef thinking ( 4 ) and equations 7 and 8 . from equations 7 and 8 , it can be readily seen that an increase in the system radiative rate , γ m , provides for both an enhanced quantum yield , i . e . observed fluorescence intensity along with a reduced decay time ( lifetime ), consistent with our experimental observations . it is well - known that in classical far - field fluorescence spectroscopy , shorter fluorescence lifetimes are indicative of fluorophores with more enhanced photostabilities , due to these molecules spending less time in highly reactive excited states . subsequently , the mixed metal substrates using fluorescein were tested . by measuring the steady - state intensity vs time ( i . e . photostability ), one typically observes a greater photon flux from the mixed metal substrates , as can be seen in fig1 top , where the photon flux of the sample is proportional to the intergraded area under the respective curves . when the samples are excitation adjusted to reflect the same initial steady - state emission intensity , a further significantly improved photostability can be seen from the mixed - metal substrates . given that absolute luminescence intensity and photostability is paramount in both microscopy ( 29 ) and fluorescence based assays ( 6 ), then mms offer a potentially new solution to this well - recognized old problem . over the last few years there has been interest in the mef literature in developing surfaces for mef in the uv spectral region , particularly for the potential labeless detection of biomolecules , i . e . metal - enhanced fluorescence of intrinsic protein residues , where metals such as aluminum and indium have been reported to date ( 30 , 31 ). to test whether mms would also enhance uv luminescence labels , solutions of phenylalanine and tryptophan were considered , fig1 and 13 respectively . similar to the visible wavelength fluorophores fluorescein and rose bengal , mms also provide for enhanced luminescence of these intrinsic protein residues . interestingly , solutions of bsa ( bovine serum albumin ) also show enhanced intrinsic protein luminescence , fig1 , with bsa known to contain 21 tyrosine and 3 tryptophan residues ( 32 ). further , it was found that the uv enhancing properties of mms are more pronounced as compared to the individual metals , as evidenced by the trends in fig1 - 14 , which could be an effect of the appearance of a new mms plasmon absorption band , as shown in fig8 . fig2 shows that toluene emission is also enhanced on the mms , suggesting the potential use of mms in applications such as scintillation counting ( 33 ), where solvent emission detectability is a primary concern . in scintillation counting for the detection of radiation such as shown in fig2 , the mixed metal surfaces can be used to enhance the emission of toluene which is widely used as a scintillation fluid ( often with other solutes ). hence mixed metals , both the substrates as well as nanoparticle mixes , may be new media for the enhanced detection of radiation signatures . potential applications of the mixed metal structures can be used for the following : a ) to increase the photostability , brightness and dwell time in microscopy and imaging technologies ; b ) on fabrics , textiles and garments for improved visualization , such as safety wear for road side workers or on jogging wear for visualization by traffic ; c ) on electronic imaging cameras , such as ccd cameras , photomultiplier tubes etc . d ) in cosmetics to change the brightness of skin and hair products , for example , ti improves the photostability of these products and to allow the reduced concentration of dyes / pigments in the formulations ; e ) in paints , coatings and inks to enhance brightness as well as protect the material substrates against sun damage ; f ) in lcd and plasma screen televisions , to both increase brightness and yet also alter the spatial distribution of luminescence ( fluorescence , phosphorescence , chemiluminescence etc ); g ) coated in or on light emitting diodes to increase brightness and the spatial distribution of the emitted light ; h ) in bank notes , stock certificates etc as an anti - counterfeiting technology . the angular nature and enhanced luminescence signatures can not be simply duplicated by simple printing or photocopying ; i ) in the generation of solar cells using the plasmonic electricity concept disclosed herein allows for tunable electric currents to be realized , not achievable using a single metal substrate . j ) in a contact lens embodiment to change the spatial distribution of light passing through the lens as well as to change the cosmetic color of the lens ; k ) on and with fiber optics and optical cables to enhance luminescence signatures , increase the extent of light coupling into the fiber as well as increase and tune the extent the magnitude of the evanescent wave above and at the end of the fibers ; l ) to modify plasmon modes in metallic substrates , particles , thin and thick films ; m ) for the generation of singlet oxygen , 1 o 2 , near - to the substrates or near - to the 3d embodiment of the technology , i . e . mixed metal nanoballs . applications include photodynamic therapy and sterilization , disinfection ; n ) for the enhanced generation of superoxide anion radicals , near - to the substrates or near - to the 3d embodiment of the technology , i . e . mixed metal nanoballs , for applications such as in sterilization or disinfection of harmful bacteria such as mrsa , salmonella , stds , anthrax spores etc ; o ) for use enhancing the luminescence of ds - dna sensing probes such as picogreen , ethidium bromide and cyber green to name a few ; p ) as a substrate for the enhanced detection of analytes , using fluorophores sensitive to such analytes such as iodide , chlorine , bromine , calcium , zinc , lithium , copper , and any other element on the period table , ions and compounds thereof ; q ) for use as a surface in voltage - gated immunoassays ; r ) as a substrate for the enhanced detection of e - type or delayed fluorescence ; s ) as a substrate for the enhanced detection of s 2 or other higher excited singlet or triplet states ; t ) as a substrate for the enhanced detection of p - type fluorescence , such as assays using the pyrene chromophore ; and u ) as a coating to enhance the throughput of emission in surface plasmon coupled fluorescence ( spcf ) and surface plasmon coupled emission ( spce ) applications . the contents of all references are incorporated by reference herein for all purposes . 1 . d &# 39 ; agostino , s ., pompa , p . p ., chiuri , r ., phaneuf , r . j ., britti , d . g ., rinaldi , r ., cingolani , r ., and della sala , f . 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