Patent Application
20120142552
1. Field of Invention
The present invention relates generally to methods and systems for detecting a pathogen, and more particularly, to methods and systems using metal-enhanced fluorescence due to interactions of fluorophores with metallic particles and/or surfaces.
2. Description of the Related Art
The 2001 terror attacks in the United States clearly demonstrated a need for rapid detection systems for unequivocal identification of bio-warfare/bio-terrorism agents such as Bacillus Anthracis, the causative agent of anthrax. The ability to accurately identify biological threat agents in real time will enable first responders and clinicians to make informed decisions about the most appropriate countermeasures.
Bacillus anthracis, the virulent, endospore-forming bacterium notorious for its recent use as a bioterror weapon, has plagued humans and livestock for many years [34]. The bacterium was intimately associated with the founding of the sciences of bacteriology and immunology, highlighted by Pasteur's famous demonstration of vaccine protection of sheep at Pouilly-le-Fort, France in 1881 [35]. Since then, little attention has been focused on understanding the biology of the organism, save for the fact that it possesses properties that make it ideally suited as a biological weapon. It forms heat resistant spores that are easy to produce using commercially available technology and can infect via the aerosol route. It has been reported that at the time of the Gulf War, Iraq produced large quantities of anthrax spores and had deployed SCUD/Al-Hussein missiles equipped with biological weapons warheads [36].
Bacillus anthracis is the only obligate pathogen within the genus Bacillus, which comprises the Gram-positive aerobic or facultatively anaerobic spore-forming, rod-shaped bacteria. It is frequently convenient to class B. anthracis informally within the B. cereus group, which, on the basis of phenotype, comprises B. cereus, B. anthracis, B. thuringiensis and B. mycoides [37]. It is not possible to discriminate between species in this group based on 16S rRNA sequences. However, amplified fragment length polymorphism (AFLP) and multiple-locus VNTR (variable number tandem repeat) analysis (MLVA analysis) have provided clear evidence that B. anthracis can be distinguished reliably from other members of the bacilli [38,39]. In practical terms, the demonstration of virulence constitutes the principle point of difference between typical strains of B. anthracis and those of other anthrax-like organisms [40].
While Bacillus anthracis can kill a broad range of animals, other members of the Gram-positive Bacillus genus are typically soil-dwellers that cause, at worst, mild opportunistic infections. B. anthracis is a member of the B. cereus group of very closely-related, ubiquitous, soil bacteria [41,42] phylogenetically separate from the other completely sequenced Bacillus genomes, B. subtilis [41] and B. halodurans [43]. The nature of the virulence of individual strains in the B. cereus group is often determined by genes carried on large plasmids. For instance, B. thuringiensis strains are distinguished by their production of plasmid-encoded insecticidal crystal toxins (δ-endotoxins) of different specificity [44]. For B. anthracis, genes for the anthrax toxin complex and poly-D-glutamic acid capsule are found on plasmid pX01 (181.6 kb) [45] and pX02 (96 kb) [46] respectively. While the plasmid genes are necessary for full virulence, the contributions of chromosomal determinants to the complex pathogenesis of anthrax are still largely unknown.
The US anthrax bio-terrorism attack in the autumn of 2001 offered a tragic “proof of principle” of the danger of B. anthracis and has spurred numerous efforts by the biomedical community to improve forensics and medical countermeasures against the bacterium. In the key area of agent detection and warning, major advances have been made. For example, systems capable of detecting aerosolized agents have been developed and deployed based on DNA (BASIS) and antibody based technologies (Portal Shield). The ground breaking work of Department of Energy (DOE) researchers looking at aerosol samples from 11 major US cities found that nonpathogenic, close relatives of B. anthracis could be detected year round and that their abundance varied with the season [47]. However, experience gained from the field has shown that differentiating threat agents, particularly B. anthracis from other nonpathogenic dose relatives is challenging.
When compared to other bacterial genomes, most B. anthracis proteins have their highest level of similarity to other Bacillus genomes (B. subtilis (2065 (36%)) and B. halodurans (1082 (19%)). Most B. anthracis chromosomal proteins have homologs in the draft sequence of the B. cereus 10987 genome, confirming the very close relationship between these organisms. There are 642 genes in B. anthracis without matches in B. subtilis, B. halodurans or B. cereus 10987, but these are mostly small hypothetical proteins. Only 43 have a predicted function and these numbers may be lower when the completed B. cereus 10987 genome is available. These genes may define unique phenotypic characteristics of B. anthracis, which could be potentially of great interest in regard to virulence.
Traditional laboratory based methods have exploited these differences but can take several days to produce results. In the context of a biological attack this is an unacceptable delay if resulting casualties are to be minimized. The need for “real-time” (<60 mins) detection has lead to the development of technologies based on DNA (PCR) and protein (antibody) targets [37]. PCR and reverse transcriptase PCR assays have been reported for detecting anthrax [1,2] in air samples [5], anthrax spore detection by flow cytometry [6], microsonication to disrupt bacterial spores [7] and real-time devices for PCR analysis [8]. However, these advances are not considered simple or monetarily reasonable, and therefore limit their potential as field-deployable, emerging technologies for use in ultra-sensitive pathogen detection.
Other methods include use of a detection label such as a linked fluorescent dye molecule, such as fluorescein isothiocyanate, rhodamine, Cascade blue, that absorb electromagnetic energy in a particular absorption wavelength spectrum and subsequently emit visible light at one or more longer (i.e., less energetic) wavelengths. The fluorescent molecules (fluorophores) can be detected by illumination with light of an appropriate excitation frequency and the resultant spectral emissions can be detected by electro-optical sensors or light microscopy. A wide variety of fluorescent dyes are available and offer a selection of excitation and emission spectra. Unfortunately, detection methods that employ fluorescent labels are of limited sensitivity for a variety of reasons. Firstly, the lifetime of the fluorescence emission is usually short, on the order of 1 to 100 ns. Further, the limit of detection of from typical fluorophores is limited by the significant background noise contributed by nonspecific fluorescence and reflected excitation light. Additionally, organic dye fluorophores are susceptible to photolytic decomposition of the dye molecule (i.e., photobleaching). Thus, even in situations where background noise is relatively low, it is often not possible to integrate a weak fluorescent signal over a long detection time, since the dye molecules decompose as a function of incident irradiation in the UV and near-UV bands.
Thus, there is a need for a detection method and system using fluorophores that identifies the pathogenic agent and preferably able to differentiate between multiple agents, that does not suffer from the problems of the prior art and does not require any amplification steps, such as in PCR or ELISA.
In one aspect, the present invention relate to a DNA sequence based micro-assay approach with metallic-fluorophore combinations to provide superior high sensitivity detection, low copy number detection and single copy-number detection.
In another aspect, the present invention relates to a method for detecting the class A agent anthrax using a single-copy number detection method without the need for any amplification steps that can also be applied to the detection of plague, smallpox, tularemia, botulism (botulinum toxin) or any other organism where the DNA sequence is known.
In yet another aspect, the present invention relates to a method for detecting a pathogen in a sample, the method comprising:
In a further aspect, the present invention provides a method for detecting a pathogen in a sample, the method comprising:
In still a further aspect, the above-described method further comprises a metal colloid attached to the free capture DNA sequence probe and positioned for sandwiching the fluorophore between the metal colloid and immobilized metal particles on the surface substrate.
Another aspect of the present invention relates to an assay system comprising:
Yet another aspect relates to an assay system for detecting a target pathogen comprising:
The present invention further comprises a detection device for detecting fluorescence 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, 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.
A further aspect of the present invention, relates to a kit for detecting a target pathogen in a sample, the kit comprising
Other aspects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.
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.
As used hereinafter the terms fluorescent dye, fluorochrome, or fluorophore are used interchangeably and bear equivalent meanings Fluorophore used in this invention are preferably of the general class known as cyanine dyes, with emission wavelengths between 550 nm and 900 nm. These dyes may contain methine groups and their number influences the spectral properties of the dye. The monomethine dyes that are pyridines typically have blue to blue-green fluorescence emission, while quinolines have green to yellow-green fluorescence emission. The trimethine dye analogs are substantially shifted toward red wavelengths, and the pentamethine dyes are shifted even further, often exhibiting infrared fluorescence emission.
In addition to fluorophores discussed above, related dyes can be further selected from cyclobutenedione derivatives, substituted cephalosporin compounds, fluorinated squaraine compositions, symmetrical and unsymmetrical squaraines, alkylalkoxy squaraines, or squarylium compounds. Some of these dyes can fluoresce at near infrared as well as at infrared wavelengths that would effectively expand the range of emission spectra up to about 1,000 nm. In addition to squaraines, i.e., derived from squaric acid, hydrophobic dyes such as phthalocyanines and naphthalocyanines can also be selected to operate at longer wavelengths. Other classes of fluorophores include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine, 5-Hydroxy Tryptamine (5-HT), Acid Fuhsin, Acridine Orange, Acridine Red, Acridine Yellow, Acriflavin, AFA (Acriflavin Feulgen SITSA), Alizarin Complexon, Alizarin Red, Allophycocyanin, ACMA, Aminoactinomycin D, Aminocoumarin, Anthroyl Stearate, Aryl- or Heteroaryl-substituted Polyolefin, Astrazon Brilliant Red 4 G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, BOBO 1, Blancophor FFG Solution, Blancophor SV, Bodipy F1, BOPRO 1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbocyanine, Carbostyryl, Cascade Blue, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH-CH3, DAPI, Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Eosin, Erythrosin ITC, Ethidium Bromide, Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue, Haematoporphyrin, Hoechst 33258, Indo-1, Intrawhite Cf Liquid, Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine, Nile Red, Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin EBG, Oregon Green, Oxazine, Oxazole, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, Propidium Iodide, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT, Rose Bengal, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine G Extra, Tetracycline, Texas Red, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, TOTO 1, TOTO 3, True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, XRITC, YO PRO 1, or combinations thereof.
One skilled in the art would know which one to select among such fluorescence dyes as long as the desired emission and absorption properties as well as their hydrophobic properties are appropriate. The spectral properties of the fluorescent dyes should be sufficiently similar in excitation wavelengths and intensity to fluorescein or rhodamine derivatives as to permit the use of the same.
Attaching of the fluorophore to the immobilized and/or free biomolecular 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 biomolecular 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.
The technology used in the disclosed assay is “Metal-Enhanced Fluorescence” (MEF), or in the alternative Surface-Enhanced Fluorescence (SEF), whereby metallic particles can interact with fluorophores (by modifying their free-space conditions), producing ultra-bright fluorophores, which are both more photostable and can emit approximately 106 more photons per fluorophore before photodestruction.
MEF is enhanced by further exploiting another complimentary property of the metal-fluorophore combinations, namely the “enhanced rate of excitation” (Em), where a red-pulsed laser diode and light emitting diode can serve as sources for two-photon excitation of labeled fluorophores. Such excitation sources and assay also provide an inexpensive field-deployable anthrax detection system. Up to about 1010 fold enhancement in the fluorescence signal may be obtained when using two-photon excitation, allowing the detection of single copies of anthrax DNA.
Two assays are disclosed in
In Assay 2, the fluorophore labeled capture DNA sequence also has an attached silver colloid. The fluorescence signal in this assay is further enhanced by the presence of silver colloids above and below the fluorophore upon hybridization to the target DNA sequence.
It is important to include a stable, non-transferable chromosomal target [49] for the target pathogen DNA sequence and importantly, the chromosomal gene targets must be usable to discriminate B. anthracis and other members of the B. cereus group [37,50].
Knowledge about fluorescence is based on measurements of the spectroscopic properties of fluorophores that upon excitation, radiate into a homogeneous and non-conducting medium, typically referred to as free space. These spectral properties are well described by Maxwell's equations for a radiating oscillating dipole. However, the interactions of an emitting dipole with physical objects can be considerably more complex, as known from antenna and receiver design. The size and shape of an antenna are designed with the goal of directing the radiation and accounting for its interactions with the earth's surface. A fluorophore is also like an antenna, but one which oscillates at high frequency and radiates short wavelengths. Local effects are not usually seen because of the small size of fluorophores relative to the experimental apparatus.
In most spectroscopic measurements, the solutions or medium is transparent to the emitted and sampling radiation. However there are several important exceptions to the free space condition. One well-known example is Surface Enhanced Raman Scattering (SERS) [9-14]. It is known that the presence of a metallic surface can enhance the Raman signals by factors of 103 to 108, and reports of even larger enhancements have appeared. The presence of a nearby metal film, island or particle can also alter the emission properties of fluorophores. The most well known effect is the quenching of fluorescence by a near-by metal. The emission of fluorophores within 50 Å of a metal surface is almost completely quenched. This effect is used in fluorescence microscopy with evanescent wave excitation. For a fluorophore located on a cell membrane and near the quartz-water interface the fluorescence emission is quenched, allowing selective observation of the emission from a fluorophore in the cytoplasmic region of the cell which is more distant from the solid-liquid interface [15]. In addition to quenching, metal surfaces or particles can cause significant increases in fluorescence. Remarkably, depending on the distance and geometry, metal surfaces or particles can result in enhancement factors of up to 1000 for the fluorescence emission [16-18]. Fluorophores near a metal film are not expected to emit isotropically, but rather the emission is directed into selected directions which are dependent on the sample configuration and the nature of the metallic surface [19-24]. In addition to directionality, the decay times of fluorophores are altered by the metal. In fact the lifetimes of fluorophores placed at fixed distances from a continuous metallic-surface oscillate with distance [21].
The effects of metallic particles and surfaces on fluorophores are due to at least three known mechanisms as described in
The distance dependence of fluorescence enhancements and those of quenching may be determined by standard methods disclosed herein.
In fluorescence, the spectral observables are governed by the magnitude of Γ, the radiative rate, relative to the sum of the non-radiative decay rates, knr such as internal conversion and quenching. In the absence of metallic particles or surfaces, the quantum yield, Q0 and fluorescence lifetime τ0 are given by:
Fluorophores with high radiative rates have high quantum yields and short lifetimes Increasing the quantum yield requires decreasing the non-radiative rates knr, which is often only accomplished when using low solution temperatures or a fluorophore binding in a more rigid environment. The natural lifetime of a fluorophore, τN, is the inverse of the radiative decay rate or the lifetime, which would be observed if the quantum yield were unity. This value is determined by the oscillator strength (extinction coefficient) of the electronic transition [31]. The extinction coefficients of chromophores are only very slightly dependent on their environment. Hence for almost all examples currently employed in fluorescence spectroscopy, the radiative decay rate is essentially constant.
The concept of modifying the radiative decay rate of fluorophores is unfamiliar to most spectroscopists. It is therefore intuitive to consider the novel effects of fluorescence enhancement due to metal particles, m, by assuming an additional radiative rate, Γm, as shown in
These equations result in important predictions for a fluorophore near a metal surface. As Γm increases, the fluorescence quantum yield increases while the lifetime decreases, as shown in
The plots in
The reduction in lifetime of a fluorophore near a metal is due to an interaction between fluorophore and metal particle, which enhances the radiative decay rate (quantum yield increase) or depending on distance, d−3, causes quenching.
Fluorophore photostability is a primary concern in many applications of fluorescence. This is particularly true in recent trends in single molecule spectroscopy. A shorter lifetime also allows for a larger photon flux. The maximum number of photons that are emitted by a fluorophore each second is roughly limited by the lifetime of its excited state. For example, a 10 ns lifetime can yield about 108 photons per second per molecule, but in practice only 103 photons can readily be observed [15]. The small number of observed photons is typically due to both photodestruction and isotropic emission. If the metal surface decreases the lifetime then one can obtain more photons per second per molecule by appropriately increasing the incident intensity. On the other hand, the metal enhanced fluorescence effects of the present invention enhances intensity while simultaneously shortening the lifetime. Decreases in the excitation intensity will still result in increases in the emission intensity and therefore photostability.
The ability to increase the radiative decay rate suggests that any chromophore, even non-fluorescent species such as bilirubin, fullerenes, metal-ligand complexes or porphyrins could display usefully high quantum yields when appropriately placed near a metal surface.
The effects of metal surface-fluorophore interactions are highly dependent upon distance and the nature of the metal surface. The emission enhancement is observed when fluorophore distances near 5-50 nm to the metal surfaces, c.f.
In one preferred embodiment of the present invention, a fluorophore is positioned between multiple silver colloids. In the assay of the present invention, (
As part of the anthrax assay of the present invention, sequences with specificity for the anthrax toxin gene PA (protective antigen) were used, and further it is noted that a bioinformatics approach may be used to identify additional, unique, chromosomally located, Anthrax specific target sequences.
The possibility for altering the radiative decay rate was demonstrated by measurements of the decay times of europium (Eu3+) positioned at various distances from a planar silver mirror using Langmuir-Blodgett films [51-54]. In a mirror the metal layer is continuous and thicker than for a semi-transparent film. The lifetimes of Eu3+ oscillate with distance from the metal yet still remain a single exponential at each distance, as shown in
Also the decay is no longer a single exponential on the silver island films [55]. The silver islands had the remarkable effect of increasing the intensity 5-fold while decreasing the lifetime 100-fold. Such an effect can only be explained by an increase in the radiative decay rate, c.f. equations 3 and 4. In this sample geometry an inert coating between the islands prevented Eu3+ chelates between the islands from being emissive. The 5-fold increase in the quantum yield of Eu(ETA)3 resulted in an apparent quantum yield of 2.0, which by definition is impossible. Hence this additional enhancement must be due to an increase in the local excitation field (Em on
It was found that dramatic increases occur for metallic colloids rather than planar-mirrored surfaces. For example, Raman signals are dramatically enhanced by metal colloids or islands [56,57]. Surface enhanced fluorescence was investigated by using low (Rose Bengal Q0=0.02) and high quantum yield fluorophores (Rhodamine B, RhB, Q0=0.48) and silver island metal films, as shown in
Several other fluorophores of different well-characterized free space quantum yields were examined, including Erythrosin B, [Ru(bpy)2]2 +, Basic Fucisin, [Ru(phn)2dppz]2+ as shown in
Complimentary to above discussed 1-photon results and interpretations, it was shown localized two-photon excitation of Rhodamine B (RhB) fluorescence occurring near metallic silver islands increases fluorescence emission intensity. This increase, as shown in
In addition to metallic particles and/or colloids modifying a fluorophores radiative decay rate, they are also known to increase excitation rates by concentrating the incident light [59,60]. The maximum enhancement in the incident electric field has been calculated to be a factor of 140 in the vicinity of appropriately sized metallic ellipsoids [61]. Since the incident intensity is the square of the incident field strength then for a 1-photon process, enhancements in excitation rates by a factor of up-to 2×104 are possible. It is this phenomenon that one can typically attribute to increases in observed apparent quantum yields near metallic particles to greater than unity. However, a much more dramatic enhancement is possible for multiphoton excitation. For a two-photon absorption process the rate of excitation is proportional to the square of the incident intensity. This suggests that two-photon excitation could be enhanced by a factor of 3.8×108. Such an enhancement in the excitation rate is thought to provide selective excitation of fluorophores near metal islands or colloids, even if the solution contains a considerable concentration of other fluorophores that could undergo two-photon excitation at the same wavelength, but are more distant from the metals surface,
For the surface anthrax assay described herein and shown in
Fluorophores can undergo a finite number of excitation and emission event cycles prior to photodecomposition. This is a particularly important consideration in single molecule detection and fluorescence microscopy, where the signal level is limited by photodecomposition. For photostable molecules like tetramethyl-rhodamine, photodecomposition occurs after about 105 event cycles, where less stable fluorophores will degrade after significantly fewer event cycles.
The d−3 quenching effect which occurs for fluorophores positioned less than 50 Å close to the surface will be first considered. Assuming there is no change in the radiative rate, Γm=0, the emission intensity will decrease due to quenching near the surface. However, it is known that quenching by RET results in increased photostability, because the fluorophores spend less time in the excited state, avoiding potential opportunities for photochemical reactions. Given the increase in photostability, the fluorophore can undergo more excitation/emission event cycles prior to photodecomposition, but the reduced quenched lifetime can cancel out this effect, resulting in a similar number of observed photons or fluorophore detectability.
Suitably fabricated metallic particles, with regard to shape and size, can result in a maximum 140-fold amplification of the local intensity [59], which is likely to result in a larger observed fluorescent -intensity at a given illumination. Given that the radiative rate is not altered, then the lifetime will remain the same. Also, since there is no quenching (lifetime remains unchanged), essentially the same overall number of photons will be emitted until the fluorophore degrades. One possible effect is that the fluorophore may display ground state depletion at lower incident intensity of the excitation light. Since the intensity is proportional to the squared field strength, appropriately designed metallic particles may result in an additional 10,000-fold increase in fluorescence intensity. Enhanced excitation rates are likely to increase the extent of fluorophore photodecomposition. Moreover, given the enhancements, it may be expected that the same emission intensity will be observed from fluorophores, but by using a 10,000 times weaker power excitation source, which may be desirable in some biological assay studies.
Further, it has been predicted that the location of a fluorophore at a suitable distance from a metallic surface can result in over a 103 increase in radiative decay rate which at the same illumination intensity and combined with an enhanced excitation rate, would produce a remarkable cumulative 107 increase in fluorescence intensity. In terms of photostability and detectability, an increase in the radiative decay rate is expected to result in dramatic increases in the number of photons observed from each fluorophore. The decreased lifetime is expected to increase the photostability of fluorophores and therefore allow a greater excitation/emission cyclic rate. Hence the combined effect of an increased quantum yield and reduced fluorescence lifetime would result in substantially more detected photons per fluorophore.
Finally, directional emission, which can be characteristic of surface enhanced fluorescence would allow for an additional factor of 10 or more, (typical spectrofluorometers only detect about 1% of all isotropic fluorescence emission [30]), which combined with a further potential 100 fold enhancement when using two-photon excitation, enhancements of ≈1010 in fluorescence emission intensity may be observed. Using an appropriately optimized silver surface-fluorophore geometry and appropriate assay washing, single capture events (DNA binding) are detectable.
Fabrication of silver islands and silver colloids deposited on quartz slides (low background), with fluorophores positioned at known distances above the metal was accomplished using Langmuir-Blodgett films. Measurements of the metal-fluorophore distances are obtained by using fatty acid spacers such as arachidic acid (C19H39COOH), and by using spin coated polymer films such as polyvinyl alcohol (PVA). Polymer film thickness is determined using simple absorbance measurements and ellipsometry. Coordinating specific enhancement distances for standard fluorophores with specific oligonucleotides (distance), optimal fluorescence enhancement is achieved.
The Langmuir-Blodgett technique provides the most accurate way of controlling film thickness and surface uniformity and was used to obtain the data for Eu3+ in
Another method to vary the long-range distance-dependence of surface enhanced fluorescence is the use of spin coated poly-vinyl alcohol films. This routine technology readily allows for films to be coated onto a variety of surfaces, with varying thickness from >0.1 μm [68,69].
The silver surfaces required for MEF are obtained using silver metal island films, sandwiched films as shown in
Colloids may be prepared as suspensions by citrate reduction of silver or gold. Silver particles are preferably used with shorter wavelengths [76]. Gold colloids can be used with longer wavelength red and NIR fluorophores. Since there is extensive literature on the optical properties of metal particles [77,78] and the effects of interface chemistry on the optical property of colloids [79], the size of the colloids and their homogeneity are readily determined. It is also possible to prepare bimetallic metal nanoparticles [80] or hollow sphere colloids [81]. For a hollow sphere, a fluorophore is positioned in the center of the silvered sphere, which is transparent to the fluorescence emission.
Additionally, pre-formed metal particles or colloids can be bound to glass surfaces by placing functional groups such as cyanide (CN), amine (NH2), or thiol (SH) on a glass or polymer substrate. Silver and gold colloids spontaneously bind to such surfaces with high affinity [82,83]. Colloids bound to the glass surface can then be coated with either Langmuir-Blodgett spacer films or spin-coated PVA to investigate the fluorescence enhancement as a function of distance from the surface. It was found that significant enhancements occur when using glass-bound silver colloids as compared to silver island films. Both silver island and silver colloid films can also be stabilized to reduce long-term oxidation, which reduces the surface plasmon resonances. APS may be used to grow an inert SiO2 layer, from TMOS (Tetramethylorthossilicate) to reduce surface oxidation. The SiO2 coating not only protects the surface, but also maintains the silver surface plasmon resonance's of silver, where only a slight shift in surface plasmon absorption is observed. If desired the silica can then be derivatized by methods used for attaching organic molecules to glass [84]. Further procedures for coating particles with silica have also been developed [85,86].
Surface plasmon absorption is due to the oscillations of free charges at a metal boundary which propagate along the metal surface. These resonances are often excited using evanescent waves. The surface plasmon absorption can give an indication of colloid size,
For detection of a specific single anthrax DNA sequence, one of the DNA oligonucleotide capture sequences is immobilized to the metal or silica (protective layer) surface, as shown in
The design of the silver micro-arrays used to detect Anthrax (as shown in
As discussed above, DNA oligonucleotides may be bound to silver surfaces or particles by using amino or sulfhydryl groups using methods known in the art. The length of the complimentary capture DNA oligonucleotides are determined by the metal enhanced fluorescence experiments with the Langmuir-Blodgett films and spin coated PVA. Suitable DNA sequences for use in the assay are those DNA sequences that bind the 2nd complimentary strand out of the quenching region (>50 Å,
While any fluorophore can be used for the assay system, low quantum yield fluorophores are more desirable because: 1) they produce a greater fluorescence enhancement and therefore increase detectability [26-30] and; 2) the background of the assay is darker, producing a better signal-to-noise ratio and thus provides better assay sensitivity. Also, in using 2-photon excitation for the assay [97-99], fluorescent probes with good 2-photon cross-sections [100] are desirable. Rhodamine 6G and Fluorescein are readily used as 2-photon probes [101] and nucleotides for oligonucleotide synthesis are available labeled with these two fluorophores.
The fluorophores are placed in specific locations (distances which provide optimal metal enhanced fluorescence), by incorporation of labeled nucleotides during solid state oligonucleotide synthesis. Alternatively, labeled nucleotides are incorporated using PCR or various methods well known in the art.
Using an inert SiO2 surface coating reduces oxidation effects while still maintaining the surface plasmon absorption of the metal. To reduce oxidation effects, fresh colloids may be coated with inert silica from sol-gel solutions using known procedures [102]. Silane reagents will subsequently place reactive groups on the surface where capture DNA oligonucleotides are attached, as shown in
A panel of B. anthracis isolates are assembled and stored as spore preps at −70° C. in 15% glycerol broth until required. A panel of other members of the B. cereus group are also assembled and stored at −70° C. in 15% glycerol broth until required. This panel contains representatives of strains that in the past have cross-reacted with anthrax specific sequences. When vegetative organisms are required the strains are cultured in brain-heart infusion agar and checked for purity by subculture onto blood agar plates.
Bacterial DNA may be isolated using various DNA isolation methods known in the art. A kit suitable for isolating DNA is the Roche High Pure PCR Template Preparation Kit. DNA concentration is estimated by measuring the absorbance of a solution at 260 nm. An A260 value of 1 is equivalent to a DNA concentration of 50 μg/ml for double stranded DNA and 20 μg/ml for single-stranded DNA. The purity of a sample is assessed by calculating the 260/280 nm absorbance ratio. This is approximately a ratio greater than 1.5 for protein-free DNA samples.
Oligonucleotide sequences should be examined to ensure that the nucleotide sequence does not contain self-complementary stretches that could potentially form stem loops. Complementarity between oligonucleotide pairs is also avoided as this can lead to formation of primer-dimer artifacts. Binding of the oligomer to other regions of the template DNA is avoided by prior comparison of the DNA nucleotide sequence of the template DNA to be amplified for local high percentage match to the primer, using the PRIMER EXPRESS software package from Perkin Elmer ABI. The step of washing the assay surface after target capture will remove any non-hybridized complimentary labeled capture stands if the background fluorescence signal levels from the bulk solution are high.
Previously designed PCR primers used to determine the nucleotide sequence of PA [103] serve as a starting point for the design of oligomers [104].
When compared to other completed bacterial genomes, most B. anthracis proteins have their highest level of similarity to other Bacillus genomes (B. subtilis (2065 (36%)) and B. halodurans (1082 (19%)). Most B. anthracis chromosomal proteins have homologs in the draft sequence of the B. census 10987 genome (this is currently being undertaken by TIGR), confirming the very dose relationship between these organisms. There are 642 genes in B. anthracis without matches in B. subtilis, B. halodurans or B. census 10987 but these are mostly small hypothetical proteins. Only 43 have a predicted function, and these numbers may be lower when the completed B. cereus 10987 genome is available. These genes may define unique phenotypic characteristics of B. anthracis, which could be potentially of great interest in regard to virulence. Using this information immobilized and free DNA oligonucleotides used for probes are designed and screened for sensitivity and specificity for their target sequence.
The surface anthrax assay can use 2-photon excitation at approximately 750-900 nm using short pulse width (<50 ps), high repetition rate (>1 MHz), laser diode sources. Due to the significant enhancements in the excitation rates of fluorophores near metallic surfaces, Em, (
For a bio-terrorism anthrax assay to be useful, it is necessary to detect B. anthracis DNA in environmental (real world) samples in which large amounts of DNA from other related and unrelated micro-organisms is likely to be present. Soil samples may be collected and the total DNA from the soil samples known to contain anthrax. DNA will be extracted using the Ultra Clean Soil DNA Kit (Mo Bio, Solano Beach, Calif.).
The present invention may be further applicable to DNA sequencing. While the human genome and other organisms have been sequenced [105,106], there is still a need for faster, cheaper and more sensitive DNA sequencing such as single strand sequencing [107]. This may be accomplished using an exonuclease to sequentially cleave single nucleotides from the strand, labeling them and detecting every one [107]. Such a process requires that every nucleotide must be both labeled and detected, which is even more difficult than single molecule detection, where one tries to find one fluorophore amongst many. Using intrinsic nucleotide fluorescence (enhanced by metal) would allow a new way to sequence DNA. Individual nucleotides are cleaved from a single strand of a polynucleotide and each of the free nucleotides is identified, based upon its characteristic fluorescence emission spectrum and lifetime. Alternatively, using the above detection method but no exonuclease cleavage, individual nucleotides of a single strand of a polynucleotide are identified by scanning a linear strand of the polynucleotide and obtaining a sequence directly from the intact DNA strand.
The assay of the present invention comprises a surface comprising silver colloids or islands. Attached to the surface and/or silver colloids/islands are polynucleotides which are complimentary to a target polynucleotide sequence. These capture polynucleotide sequences are attached at either the 5′ or 3′ end. The assay is performed by adding the target polynucleotide sequence to the assay surface and allowed to hybridize with the capture polynucleotides. Fluorophore-labeled capture polynucleotides are added and hybridize to the target polynucleotide. Unbound target polynucleotide and labeled capture sequence may be removed by washing, but washing is not required. Bound target polynucleotide is detected by metal enhanced fluorescence. The target polynucleotide sequence and capture sequence are preferably single stranded but may be double stranded and may be either deoxyribonucleic acid or ribonucleic acid. Many conditions suitable for hybridizing polynucleotides are known in the art. High stringency conditions or high stringency hybridization conditions are where polynucleotides are hybridized under the following conditions: 6×SSPE, 5× Denhardt's reagent, 50% formamide, 42° C., 0.5% SDS, 100 μg/ml sonicated denatured calf thymus or salmon sperm DNA. Medium stringency conditions or medium stringency hybridization conditions are where polynucleotides are hybridized under the following conditions: 6×SSPE, 5× Denhardt's reagent, 42° C., 0.5% SDS, 100 μg/ml sonicated denatured calf thymus or salmon sperm DNA. Low stringency conditions or low stringency hybridization conditions are where polynucleotides are hybridized under the following conditions: 6×SSPE, 5× Denhardt's reagent, 30° C., 0.5% SDS, 100 μg/ml sonicated denatured calf thymus or salmon sperm DNA. The formulae for the buffers used for hybridizations are: 20×SSPE: 3.6 M NaCl, 0.2 M phosphate, pH 7.0, 20 mM EDTA. 50× Denhardt's reagent: 5 g FICOLL Type 400, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin and water to 500 ml.
It is recognized in the art of nucleotide hybridization that high, medium and low stringency hybridizations can be performed under a variety of different conditions. The provided conditions for performing nucleotide hybridizations are illustrative of the specific hybridizations for high, medium and low stringency conditions. These hybridization conditions are not intended to limit the disclosed method as one of ordinary skill in the art would recognize that the method of the instant invention is not dependent upon the disclosed hybridization conditions but can be achieved using many other different hybridization conditions.
Additionally, several different attached capture polynucleotide sequences may be used, as well as different fluorophore-labeled capture polynucleotide sequences to allow detection of more than one target polynucleotide sequence. Fluorophore-labeled capture polynucleotides of different sequences may be labeled with different fluorophores to allow identification of the different target sequences that may be present in a sample. The fluorophore-labeled capture polynucleotides may also comprise a silver colloid to further enhance fluorescence emission.
The capture immobilized DNA probe may be any length of nucleotides any preferably of a sufficient length of nucleotides to allow interaction of a bound fluorophore with the metallized surface of the substrate. The sequence may be from about 5 to about 300 nucleotides in
While the invention has been described herein with reference to specific features, aspects and embodiments, it will be recognized that the invention may be widely varied, and that numerous other variations, modifications and other embodiments will readily suggest themselves to those of ordinary skill in the art. Accordingly, the ensuing claims are to be broadly construed, as encompassing all such other variations, modifications and other embodiments, within their spirit and scope.
All references discussed herein are hereby incorporated herein by reference for all purposes.
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This application is a divisional application of copending U.S. patent application Ser. No. 10/536,502 filed on Dec. 14, 2005, now U.S. Pat. No. 8,114,598, which in turn claims priority of International Patent Application No. PCT/US2003/038163 on Nov. 26, 2003, which in turn claims priority of U.S. Provisional Patent Application No. 60/429,263 filed on Nov. 26, 2002.
| Number | Date | Country | |
|---|---|---|---|
| 60429263 | Nov 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10536502 | Dec 2005 | US |
| Child | 13372624 | US |
