Patent Application
20140371106
The present invention relates to compositions, including fluorescent compositions and lasing compositions. The invention particularly relates to programmable biological substrates, which fluoresce and/or lase, and which have a wide variety of different applications. The invention extends to use of the fluorescent compositions and lasing compositions comprising programmable biological substrates in fabricating lasers, and in various biological imaging applications, such as in assays.
Structural biology has shown that the function of molecular machines, including proteins and RNAzymes, is determined by their structure and that structural details on the angstrom scale can be decisive. The structure and consequently the function of a protein can be modified by mutations to the gene coding for the protein or by post-translational modifications to the protein surface. As such, biology at the molecular scale is readily programmable because new or modified functionality can be conferred to biological systems by making genetic mutations or chemical modifications to proteins.
Scientists engaged in tree field of nanotechnology look to make novel devices on the scale of nanometres, which is the same length scale as molecular biology. These scientists have begun looking towards the programmable nature of biology to take advantage of biological structure and complexity. So far, the interface between molecular biology and nanotechnology has been centred on the development of virus hybrid systems. Examples of virus hybrid devices include lithium batteries, cobalt wires, light harvesting devices, magnetic memory storage devices and ordered arrays of quantum dots in viral films.
M13 has been the substrate of choice for much of this research because it has a number of desirable attributes. M13 will only infect F′ pilus strains of E. coli and typical yields from overnight shakeflask production are of the order 5×1011 phage per ml. The wild type phage forms long, thin rod like particles with dimensions 9300 Å by 65 Å, as shown in
Without prior genetic mutation, the major coat proteins of M13 can be modified by direct functionalization of the amino (N-terminus and lysine), carboxylic acid (glutamic acid and aspartic acid) and phenolic (tyrosine) groups exposed at the surface. The pKa of the amino group at the N-terminus is 7.6-8.0, but the pKa of the lysine amino group is 9.3-9.5. As such, the N-terminus can be selectively modified in reactions buffered at lower pH. M13 has been found to be stable when incubated in solutions of pH 3.0 to 11.0 and when heated to temperatures of 80° C. M13 can be exposed to concentrations of at least 20% methanol, ethanol, 1-propanol, acetonitrile or N, N-dimethylformamide and remain infective. Often M13 can be exposed to much higher concentrations of solvent. For instance, helper phage strain pG8H6 can be exposed to 99% acetonitrile.
The optical properties of rod-like viruses modified with dyes and catalysts have been studied with a view to making light harvesting devices. The practice of making nanoantennae from biological scaffolds represents an effort to use biology in a domain usually dominated by physicists. The transfer of energy between dyes on biological scaffolds via FRET is an established phenomenon. Accordingly, time-resolved fluorescence spectroscopy of these systems reveals a reduction in the lifetime of the excited state of the scaffolded dyes. The overall fluorescence intensity of M13 labelled with N,N,N′,N′-tetramethylrhodamine or Zn(II)-porphyrin derivatives decreases as more dyes are conjugated to the surface because of the close proximity of dyes labelled to the N-terminus and lysine residues of the same coat protein. However, in other systems labelling does not lead to fluorescence quenching. For instance, another virus, cowpea mosaic virus (CPMV), was labelled with fluorescein derivatives to a high density with no observable quenching and used for intravital vascular imaging. Rhodamine and porphyrin molecules are both bulkier than fluorescein molecules so their wavefunctions are more likely to overlap on the surface of biological scaffolds when there is a high density of conjugated dyes. A more common practice is to use dye-labelled viruses in biosensors or biological assays. For these practices, the physics of the dyes labelled to the biological substrate is not the primary concern of the research.
Filamentous bacteriophage fd, a close relative of M13, has been labelled with fluorescein measure the self diffusion of rod-like viruses in the nematic phase using fluorescence microscopy. The labelled viruses were observed to be relatively photostable, though this is attributed to the mixing of the samples in an anti-oxygen solution. In the biosensor field, fluorescently stained LG1 phage and a PP01 phage displaying green fluorescent protein fusions have been used to detect E. coli O157:H7 cells. Labelled fd can be taken up by human B cells and their localization observed with confocal microscopy. Additionally, MS2 bacteriophage has been labelled with dyes and used as a tracer and a T7 phage was hybridized with a europium complex to create a new fluorescent probe for imaging and bioassay's. Fluorescently stained viruses can be used to identify specific bacterial strains in a microbial community.
The most common use of phage in biotechnology is phage display, which is a well-established method of finding peptide sequences with an affinity to a target. Fusion proteins can be expressed on the surface of M13 phage by direct genetic modification of the phage genome or by transforming cells with a phagemid and infecting them with helper phage. Phage display to discover peptides that bind non-biological targets, including GaAs, ZnS, CdS, PbS, Au, Ag, Co and carbon nanotubes, has proven successful and phage display to identify antibody fragments is routine.
Once phage display has identified a phage clone with an affinity to a target, it is possible to use the phage as though it was a primary antibody in immunobiological assays using a conjugated anti-M13 secondary antibody. Alternatively, the phage itself can be fluorescently labelled. For instance, T7 can be genetically engineered to display enhanced yellow fluorescent protein fusions and M13 has been labelled with a fluorescein derivative. The M13 derivatised with fluorescein may have had a structure similar to M13-fluorophage, but no evidence was presented that the construct had enhanced optical properties. The construct did retain its capacity to bind to targets and so could be used as a primary phage conjugate. Horse radish peroxidase (HRP) conjugated protein A is one alternative to secondary antibodies in immunobiological assays because all of the domains of protein A bind to the Fab antibody domain and HRP can catalyse a chemilumeniscent signal.
There are a limited number of examples of biology influencing the design of lasers. Lasers use stimulated emission to emit spatially coherent, monochromatic beams of electromagnetic radiation with narrow divergence. The first laser was the ruby dye laser but since then many types of laser have been developed that are used in a vast number of different applications. For instance, random lasers use multiple scattering to provide optical feedback instead of mirrors. As biological tissues are strong scatterers, it has been possible to demonstrate laser action in dye infused biological tissues. In FRET lasers, the donor is excited and energy is transferred to the acceptor for lasing. A FRET laser has been built using DNA scaffolds to separate the donor and acceptor dyes and lasing was achieved at lower acceptor concentrations than conventional FRET lasers.
To date, biology has not been used to tackle the limitations of dye lasers. Common to all dye lasers is the use of a dye as the gain medium. The dyes are typically organic molecules and examples include rhodamine and fluorescein. One of their main advantages is their availability in a wide range of emission wavelengths. Their broad gain bandwidth is useful for generating short pulses via mode locking and gives them a broad wavelength tunability. Dye lasers might be used if a situation demands a readily tunable laser source or requires either high average powers or high pulse energies.
In most cases, the dye is dissolved in a solvent and a jet sprays the dye through the beam path so that only fresh dye is used. On safety grounds, it would be preferable for the operator not to work with solvents. Although solid state dye lasers have been constructed, the construction of an electrically pumped solid state dye laser system remains an active area of research. Dye lasers have developed a reputation for not being “user friendly” and their application is restricted to a limited number of situations. They are now less common than solid state lasers, which are made from crystals or glasses doped with transition or rare earth metals or semiconductors. Solid state lasers are more stable than dye lasers and can sometimes be electrically pumped.
The most important limitations of laser dyes are related to the excited triplet state of the dye. Under pumping, there is a probability that electrons in the excited singlet state will make the spin forbidden transition to the excited triplet state, as shown in
All of the above notwithstanding, organic dyes remain the molecules of choice for biological imaging, where fluorescent labels are necessary for the confocal microscopy of biomolecules and some biosensors and immunobiological assays. Inorganic quantum dot labels are making inroads, but they must be packaged to mitigate against toxicity, and subsequently functionalized to attach to the sites of interest, yielding moieties much more unwieldy than organic dye molecules. In particular, derivatives of dyes are available which allow the dye to be conjugated to the functional groups on proteins. Nonetheless, the photobleaching of dyes in confocal microscopy can prove problematic in some cases.
Accordingly, there is clearly a need to provide improved light-emitting compositions, which include lasing compositions and fluorescent compositions, which are programmable and which can therefore be used in a range of different biological and physical applications, such as in laser dyes and lasers, and in biological imaging applications, such as assays.
The inventors have developed new types of lasing compositions and fluorescing compounds, which are collectively referred to herein as a “fluorophage”, and which have a wide range of useful applications. The fluorophage compositions described herein comprise an array of dye molecules or light-emitting labels, which are scaffolded onto a programmable biological substrate.
Thus, according to a first aspect, there is provided a lasing composition comprising a biological substrate chemically modified at specific attachment sites with light-emitting labels.
It will be appreciated that a lasing composition is one which is capable of acting as the source of optical gain in a laser. The distance between adjacent light-emitting labels on the biological substrate is preferably such that they are unable to chemically react with each other, but can still allow dipole-dipole interactions to occur between adjacent light-emitting labels.
In some embodiments, compositions of the invention may fluoresce.
Hence, according to a second aspect, there is provided a fluorescent composition comprising a biological substrate and an array of spaced apart dye molecules attached thereto, wherein the distance between adjacent dye molecules is such that they are unable to chemically react with each other, but can allow dipole-dipole interactions to occur between adjacent dye molecules.
It will be appreciated that a fluorescent composition is one which is capable of spontaneous emission of a photon resulting from a transition from an excited singlet state to the ground singlet state.
It is preferred that, following attachment to the substrate, the light-emitting labels in the lasing composition of the first aspect, and the dye molecules in the fluorescent composition of the second aspect, are unable to chemically react with each other, preferably when excited. If the light-emitting labels or dye molecules are too close together they quickly photobleach because they can chemically react with each other. Advantageously, as described in the Examples, the inventors have demonstrated that the compositions of the invention have a significantly reduced photobleaching rate compared to free light-emitting labels or dye molecules in solution, because the light-emitting labels or dye molecules are too far apart from each other for them to exchange electrons.
It is also preferred that, following attachment, the distance between adjacent light-emitting labels and dye molecules is such that dipole-dipole interactions can occur between adjacent light-emitting labels of dye molecules. Such dipole-dipole interactions are important as they provide alternative deactivation pathways for the molecules (e.g. light-emitters or dyes) to return to the ground state from the excited triplet state. It will be appreciated that the triplet state quenches lasing and can also result in photobleaching. Advantageously, as described in the Examples, the triplet state lifetime of the compositions of the invention is dramatically reduced because of the high number, of neighbouring light-emitters or dye molecules sufficiently within range of each other for sufficient dipolar interactions to occur, which enhances triplet state quenching. Thus, the compositions of the invention are much more stable, and significantly minimise photobleaching.
The inventors have shown that the light-emitting labels or dye molecules can be scaffolded with nanoscale precision onto the surface of the biological substrate, as they will only covalently bond to particular chemical groups (i.e. the attachment sites). Since biological substrates are programmable, the chemical nature of the substrate can be readily modified by making either genetic mutations or post-translational modifications thereto. Consequently, the position of the t-emitting labels or dye molecules on the substrate can be “programmed” to form arrays with different optical properties. As the substrate is programmable, it is also possible to introduce genetic modifications that cause protein fusions to be displayed on the surface of either the lasing composition of the first aspect, or the fluorescent composition of the second aspect. These protein fusions may be used to bind the biological substrate to surfaces, or other biomolecules, or non-biological nanoparticles. In one embodiment, the substrate may comprise (or be modified to comprise) one or more cysteine residues each with an exposed thiol group. The thiol group may be positioned so that it can act as a reducing agent towards the scaffolded dye molecules. For example, 2-Mercaptoethylamine (MEA) is effective at reducing the photobleaching rate of the dye, fluorescein. The thiol group on MEA is responsible for its properties as a reducing agent. Therefore, adding a plurality of thiol groups to the surface of the substrate helps to reduce photobleaching.
Preferably, the light-emitting labels or dye molecules are attached to specific, spaced-apart attachment sites, which are disposed along the structure of the biological substrate. The attachment sites may be amino acids, or a side chain thereof. These potential attachment sites are preferably regularly spaced apart (i.e. ordered) along the substrate. In other words, the distance between adjacent potential attachment sites may be substantially the same along the substrate. The positions of the potential attachment sites may create a repeating pattern along the substrate. Preferably, the potential attachment sites are not randomly arranged along the substrate. The light-emitting labels or dye molecules may not self-assemble to form a monolayer on the substrate.
It will be appreciated that whether or not interactions occur between the light-emitting labels or dye molecules on the surface of the biological substrate depends on several factors, including the bulkiness of the light-emitter or dye molecule itself, the separation of the attachment sites along the structure of the biological substrate and the rotational freedom of the light-emitters or dye molecules, as well as the intrinsic electronic properties of the light-emitters and the substrate, or the dyes and the substrate. Thus, what constitutes as being too close and too far spacing along the substrate depends on the size of the light-emitting label or dye molecule. The average molecular diameter of a suitable light-emitter or dye molecule may be between about 0.1 nm and 5 nm, or between 0.2 nm and 5 nm, or between about 0.3 nm and 3 nm, or between about 0.5 nm and 2 nm.
The inventors believe that dipole-dipole interactions may be weak between light-emitting labels or dye molecules with an intermolecular separation greater than about 10 nm or 15 nm. However, if the separation is less than about 1 nm, then the light-emitting labels or dye molecules may chemically react unless otherwise hindered. Therefore, in some embodiments, the average distance between adjacent light-emitting labels or dye molecules may be between about and 15 nm, or between about 1 nm and 10 nm, or between 1 nm and 7 nm, or between 1 nm and 5 nm, or between 1 nm and 3 nm. In other embodiments, the average distance between adjacent light-emitting labels or dye molecules may be between about 2 nm and 15 nm, or between about 2 nm and 10 nm, or between about 2 nm and 7 nm, or between 2 nm and 5 nm, or between 2 nm and 3 nm.
The light-emitting labels or dye molecules may comprise organic or inorganic light-emitters or dyes. However, organic light-emitting labels or dyes are preferred. Thus, the light-emitting labels or dye molecules may comprise hydrocarbons or hydrocarbon derivatives that possess at least one conjugated double bond. The light-emitting labels or dye molecules preferably absorb wavelengths between 220 urn and 1000 For example, the light-emitting labels or dye molecules may absorb in the visible (i.e. 390-750 nm), the ultraviolet (i.e. 10-400 urn) or the infrared (1 m-750 nm) parts of the spectrum. Thus, the light-emitting labels or the dye molecules may be capable of absorbing between 240 nm and 900 nm, or between 460 nm and 500 nm, or between 900 nm and 3000 nm, or between 3-8 μm (mid-infrared), or between 8-15 μm (long-infrared), or between 15-1000 μm (far-infrared).
Preferably, the light-emitting labels or the dye molecules are adapted to form covalent bonds with the functional groups of an amino acid, which are present in the biological substrate. Suitable light-emitting labels used in the compositions of the invention may be characterised by their capability to emit light via a transition from a higher quantum state to a lower quantum state and a functional group that allows the labels to be covalently attached to a biomolecule without compromising its light emitting capability. Suitable dye molecules used in the compositions of the invention may comprise hydrocarbons or derivatives thereof, which possess at least one conjugated double bond.
The light-emitting labels or the dye molecules may comprise a fluorophore. For example, the light-emitting labels or dye molecules may be members of the xanthene family of dyes, or other optical moieties or light-emitters, such as GFP or quantum dots. The dye molecules comprise Rhodamine or a derivative thereof, such as Rhodamine B, Rhodamine 6G or Rhodamine 123.
As described in the Examples, the light-emitting labels or dye molecules may comprise fluorescein or a derivative thereof. It will be appreciated that fluorescein has many derivatives, including fluorescein isothiocyanate (FITC), NHS-fluorescein or 6-FAM phosphoramidite.
In one embodiment, the lasing composition or fluorescent composition may comprise one type or species of light-emitting label or dye molecule. However, in another embodiment, more than one type or species of light-emitting labels or dye molecules may be scaffolded to the substrate. For example, the composition may comprise one or more type of light-emitting labels or dye molecules from the xanthene family (e.g. fluorescein and Rhodamine, etc.).
The biological substrate may be any surface comprising a biomolecule (e.g. a peptide, protein, nucleic acid, or any combination thereof), which is subjectable to chemical modification for the attachment of the dye molecules to potential attachment sites. The biological substrate may not comprise carbohydrate or lipid. The biological substrate is preferably self-assembled into a well-defined, regular structure. For optimum use in the lasing and fluorescent compositions of the invention, the biological substrate preferably comprises chemical attachment sites that form a regular array therealong. Thus, the biological substrate may be proteinaceous. The biological substrate may comprise a protein-nucleic acid complex or conjugate, for example an RNA- or DNA-protein complex. The biological substrate may comprise a virus or virus-like particle.
Preferably, the biological substrate comprises amino acid residues to which the light-emitting labels or dye molecules may be attached. These amino acids form the potential attachment sites for the light-emitters or dye molecules. The amino acids may be either natural (i.e. the 20 amino acids which form naturally occurring peptides), or non-natural. The substrate may be substantially elongate, or rod-like. For example, the substrate may be at least 500 Å, 1000 Å, 2000 Å, 3000 Å, 4000 Å, 5000 Å, 6000 Å, 7000 Å, or 8000 Å in length.
The biological substrate may comprise a wild-type or mutant biological substrate, including a bacteriophage, actin fiber, bio me compound, or other proteinaceous substrate. The substrate may comprise an RNA-protein complex, which may be expressed in E. coli.
Bacteriophages and viruses, which may be used as the substrate may be filamentous or non-filamentous. The virus may be a plant virus, for example cowpea mosaic virus (CPM) and tobacco mosaic virus (TW), as described in the Examples. The TMV may be expressed in E. coli.
As described in the Examples, the biological substrate may comprise M13 filamentous bacteriophage (M13). Thus, in a preferred embodiment, the fluorescent composition may comprise fluorescein dye molecules scaffolded in ordered arrays on M13 bacteriophage substrate. This class of composition or “fluorophage” has been termed herein as “M13-fluorophage”. The fluorescein derivative dye molecules react specifically to the primary amine groups on the N-terminus and lysine residues on the major coat protein of M13. The fluorescein molecules may be spaced apart by about 1 nm to 3 nm, and by at least 2 nm, which the inventors have shown is a distance which prevents electron transfer between adjacent light-emitting labels or dye molecules, thereby preventing photobleaching. However, dipolar interactions between the dye molecules are still possible, thereby providing alternative deactivation pathways for the dyes to return to the ground state from the excited triplet state.
The effective concentration of the fluorescein derivatives on the surface of M13 is greater than that for the same number of dyes in solution, and so the rate of triplet-triplet annihilation is dramatically increased. Consequently, the excited triplet state lifetime is reduced. Additionally, if the sample is flushed with argon or nitrogen to remove molecular oxygen, then the formation of reduced and semi-oxidized form of the dye would be prevented. Advantageously, by reducing the population of electrons in the excited triplet state, the biological scaffold can be used to make dye lasers with longer pulse lengths and faster repetition rates and lower rates of photobleaching. Therefore, the inventors believe that the application of programmable biological substrates in the composition of the first aspect represents a new paradigm in laser technology.
Thus, in a third aspect, there is provided use of the composition of either the first or second aspect as a laser dye.
In a fourth aspect, there is provided a laser dye comprising the composition of either the first or second aspect.
The lasing or fluorescent composition of the invention may act as a gain medium in the laser dye, or as a saturable absorber. Thus, it will be appreciated that the laser dye acts as a lasing medium. Advantageously, the use of ordered, regularly-spaced, genetically programmable attachment sites on the biological substrate (e.g. a rod-like virus or phage) the lasing or fluorescent composition of the invention allows the laser dye of the fourth aspect to exhibit improved lasing properties and decreased dye degradation, when compared to the use of an amorphous substrate in which the attachment sites are randomly located thereon. As discussed above, this is because the regular spacing of the attachment sites and thus light-emitting molecules or dye molecules along the substrate ensures that the distance between adjacent molecules is such that they are unable to chemically react with each other, but allow dipole-dipole interactions to occur therebetween, thereby avoiding photobleaching.
In fifth aspect, there is provided use of the composition of either the first or second aspect as a saturable absorber.
It will be appreciated that the composition therefore acts as a medium for electromagnetically-induced transparency.
The laser dye may be used in a laser, for example a dye laser.
Thus, in a sixth aspect, there is provided a laser comprising the laser dye of the fourth aspect.
The laser may be selected from the group of lasers consisting of: a plasmonic laser; a single-particle plasmonic laser; a solid state laser; an electrically pumped solid state laser; a dye laser; a liquid dye laser; a tunable laser; a pulse laser; and an ultrashort pulse laser. The laser may be used in laser treatment targeted to a specific cell type.
The laser may be a dye laser. A dye laser may comprise the dye mixed with a solvent, which may be circulated through a dye cell, or streamed through open air using a dye jet. The laser may comprise a high energy source of light to “pump” the liquid dye beyond its lasing threshold. The high energy source may comprise a fast discharge flashlamp or an external laser. The laser may comprise one or more mirrors to oscillate the light produced by the dye's fluorescence, which is amplified with each pass through the liquid dye. An output mirror is normally around 80% reflective, while all other mirrors are usually more than 99% reflective. Alternatively, metallic nanostructures adjacent to or attached to the biological scaffold could provide feedback through surface plasmon resonance. The dye solution may be circulated at high speeds, to help avoid triplet absorption and to decrease degradation of the dye. A prism or diffraction grating may be mounted in the beam path, to allow tuning of the beam.
The inventors have shown that the dye lasers of the invention have several significant advantages over conventional dye lasers. Firstly, the laser dye comprising the fluorescent composition of the first aspect does not photobleach as quickly as conventional dye molecules, and so they do not need to be pumped through the optical cavity. Secondly, the dye could be cast into viral films or blocks to make a solid state gain medium. Thirdly, tunable dye lasers are able to sustain longer pulse lengths than conventional dye lasers. Fourthly, the lasers of the invention overcome the limitations of the previous generation of dye lasers because they do not require dye to be pumped through the optical cavity. Fifthly, mirrorless lasing is achievable with the fluorescent composition of the first aspect at lower pump powers than is possible with free dye molecules in solution.
The fluorescent composition of the invention may be used to construct solid state or liquid dye lasers with protein fusions that enable electrical, optical and chemical reactions to pump energy into the laser. Also, aside from their scaffolding properties, the M13 phage forms viral cast films, and this property can be readily exploited to build a solid state dye laser. Additionally, the fluorescent composition of the invention may be cast onto an organic polymer film that conducts electricity so that there is near field energy transfer between the organic polymer and the fluorophage film, and such a system would be capable of electrically pumped laser action. A pump laser would act as a source by pumping the fluorescent composition (e.g. a single fluorophage) to just under its threshold power. A probe laser would act as a gate by causing the single pumped fluorophage to emit stimulated emission. The emission could be collected at the drain. The laser may therefore be a polymer laser. The fluorescent composition may be a dye doped in a polymer matrix of a polymer laser.
The composition may be used in a wide range of biomedical applications, for example a protein microarray. For example, the inventors have also shown that the lasing composition of the first aspect and the fluorescent composition of the second aspect may be effectively used in biosensors and in biological assays, for detecting small molecules.
Therefore, in a seventh aspect, there is provided use of the composition of either the first or second aspect as a biosensor, or in biological imaging applications.
Therefore, in an eighth aspect, there is provided a biosensor or biolabel comprising the composition of either the first or second aspect.
The biosensor may be a protein microarray biosensor. The composition may be used in an assay. The composition may be fused to a protein or other biological entity or small molecule, or substrate. Thus, the fluorescent composition may comprise a fusion protein attached thereto. As described in the Examples, in one embodiment, the g3p coat protein of the M13-fluorophage may be genetically engineered to display the BR-domain from protein A. The BB-M13 fluorophage may bind IgG antibodies and be used in the same way as secondary antibodies to detect proteins in biological assays, including Western blots and ELISA. The main advantage of this would be greater sensitivity to lower protein concentrations and a more quantifiable, repeatable emission compared to using the chemiluminescence from an enzyme-based secondary antibody system.
It will be appreciated that in a conventional assay, the fluorophore emits light as spontaneous emission in all directions. However, in an assay involving the use of the fluorescent composition of the invention, the composition can emit light as stimulated emission preferred direction. This is possible because the composition is more resistant to immediate photobleaching. Advantages of using stimulated emission in this manner are a superior signal to noise ratio, and a narrower bandwidth emission.
The inventors have found that the fluorescent composition may be assembled into larger structures, including liquid crystals and films.
Thus, in a ninth aspect, there is provided a liquid crystal structure or film comprising the composition of either the first or second aspect.
Furthermore, in a tenth aspect, there is provided use of the liquid crystal structure or film according to the ninth aspect, as a display.
In an eleventh aspect, there is provided a display comprising the composition of either the first or second aspect.
For example, the display may be a display for any computation, control or communications devices (e.g. a mobile phone). Advantageously, the display is more energy efficient because stimulated emission can be used instead of spontaneous emission.
Furthermore, the composition may be used as a component optical computer.
Thus, in a twelfth aspect, there is provided an optical computer comprising the composition of either the first or second aspect.
All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:—
a illustrates the M13 major coat protein assembly, and
a shows fluorescence spectra, and
As described in the Examples below, the inventors have developed a composition (referred to herein as a “fluorophage”) in which a light-emitting label or dye molecule (e.g. fluorescein) is conjugated to a programmable biological substrate (e.g. M13 bacteriophage, or Tobacco Mosaic Virus). The light-emitting labels or dye molecules are regularly spaced-apart to form an array on the biological substrate, which thereby acts as a scaffold structure holding the dye molecules in position. The inventors have analysed the light-emitting and/or fluorescence characteristics of the fluorophage, and have used it as a dye in a dye laser, and instead of molecular dyes for biosensing and biological imaging applications. The lasing properties of the fluorophage make an excellent candidate as a light-emitting composition in a wide range of applications.
The dye used was 5-carboxyfluorescein, succinimidyl ester (5-FAN′, SE) powder (Invitrogen). Herein, the term “dye” and “light-emitting label” are used interchangeably. It was dissolved in spectroscopic grade dimethyl sulfoxide (DMSO) (VWR) to a final concentration of 10 mg/ml. Referring to
19 μl of the 5-FAM, SE stock dye was added to 100 μl of the M13 phage stock and left for one hour at 37° C. with shaking in the dark to form the M13-fluorophage. The reaction was conducted at pH 7.5 to encourage single labelling of the coat proteins. The reaction was quenched with 1 M TRIS pH 8.5. The reaction was subjected to centrifugation to remove any insoluble reaction products or polymerised dye. The sample was then precipitated using 5% PEG-8000/200 mM NaCl and resuspended. The sample was subjected to further rounds of precipitation and resuspension until the supernatant was colourless by sight. The sample was then buffer-exchanged using a Zeba desalt spin column (Thermo Scientific) and subjected to a final precipitation with 5% PEG-8000/200 mM NaCl to concentrate the sample.
The M13-fluorophage samples and free 5-FAM, SE molecular dye samples were loaded into square capillaries with widths of 0.7 mm by capillary action. The fluorophage sample was shaken to move the sample into the middle of the capillary. The loaded capillaries were then left in the dark at 4° C.
Referring to
Fluorescence lifetime imaging microscopy (ELM) was conducted on a Zeiss Axioskop META 510 NLO microscope with a Becker and Hickl FLIM system to test whether the M13-fluorophage prepared in Example 1 has decreased triplet state lifetimes and decreased steady state triplet state populations compared to free dye molecules in solution.
Referring to
In summary, this experiment measures the time taken for electrons in the excited states to return to the ground state. The shorter the time, the more deactivation pathways are available for electrons to return to the ground state. On the fluorophage of the invention there are more deactivation pathways because of the dipole-dipole interactions between neighbouring dyes. These dipole-dipole interactions are important for quenching the triplet state, and these experiments prove that they are there.
Fluorescence spectra and fluorescence time series were acquired using an Olympus Confocal Inverted Laser Scanning Microscope with a 10× objective lens to test whether the M13-fluorophages prepared in Example 1 have decreased photobleaching rates compared to free dye molecules in solution.
Referring to
As shown in
The M13-fluorophage sample without 5% PEG-8000/200 mM NaCl had significantly lower rates of photobleaching than the concentrated molecular dye sample despite having dye molecules scaffolded to a comparable mean separation. This indicates that the dye molecules on the surface of the M13-fluorophage were not able to exchange electrons with neighbouring dye molecules.
The rate of photobleaching is reduced for M13-fluorophage samples containing 5% PEG-8000/200 mM NaCl. PEG-8000 is a molecular crowding agent that causes the local concentration of M13-fluorophage to increase. M13 forms liquid crystals at high concentrations so it is reasonable to anticipate that M13-fluorophage is also capable of forming crystal structures. The hydrogen bonding network between M13-fluorophage in a liquid crystal is likely to prevent fluorophage-fluorophage collisions, which explains why M13-fluorophage in solutions with 5% PEG-8000/200 mM NaCl undergo an even greater reduction in their rate of photobleaching.
Over time, the intensity of the fluorescence would tend towards zero if the dyes were unable to diffuse away from the laser path. However, as the dyes can diffuse away from the laser path, the intensity reaches an asymptote where the rate of fresh dye diffusing into the path of the laser matches the rate of photobleached dye out of the path. This effect is more pronounced for the molecular dyes because they are much smaller than M13-fluorophage and so can more readily diffuse out of the path of the laser beam.
Based on the data produced from Examples 2 and 3, the inventors have shown that the fluorophages of the invention, as prepared by Example 1, can be readily used instead of molecular dyes in dye laser systems. Two types of dye lasers with commercial value are tunable lasers and ultrashort pulse lasers, which are illustrated in
Tunable dye lasers can emit at a broad range of wavelengths, and an example of such a laser 2 is shown in
Tunable lasers 2 are especially useful in applications where scattering is observed as a function of wavelength. Dye lasers can be used to make tunable ultraviolet and infrared lasers sources by frequency mixing in non-linear materials. Applications of tunable dye lasers, where the fluorophage dye cell shown in
With reference to
Ultrashort pulse lasers 20 can be used to measure the relaxation times of atoms and molecules and to monitor chemical reactions. Applications of ultrashort pulse lasers where the fluorophage dye cell can be employed include:—
The inventors have shown that fluorophage dye lasers 2, 20 have a number of major advantages over conventional dye lasers, including:—
(i) The fluorophage dye does not photobleach as quiddy as conventional dye molecules, so they do not need to be pumped through the optical cavity;
(ii) The fluorophage dye could be cast into viral films to make a solid state gain medium;
(iii) Tunable fluorophage dye lasers are able to sustain longer pulse lengths than conventional dye, lasers.
(iv) The previous generation of dye lasers required the operator to pump toxic solvents around the lasers system, and so they developed a reputation for not being “user friendly”. The fluorophage lasers 2, 20 shown in
The market for lasers is very broad because lasers have so many applications. Laser manufacturers, such as Coherent and Spectra-Physics (Newport Corp), are especially keen to acquire and develop technologies that give them an advantage over the competition.
Ti:Sapphire lasers are the industry standard for generating ultrashort pulses. However, even after frequency doubling, these lasers have wavelengths greater than 350 nm, which is too long to excite intrinsic fluorescence in proteins.
The fluorophage of the invention can also be used instead of molecular dyes in a range of biosensing and biological imaging applications. For example, protein fusions can be displayed at the surface of the fluorophage.
Referring to
In the case of a lasing fluorophage, this can offer an entirely new means of obtaining contrast in an image. Organic dyes remain the molecules of choice for biological imaging, where fluorescent labels are necessary for the confocal microscopy of biomolecules and some biosensors and immunobiological assays. Inorganic quantum dot labels are making inroads, but they must be packaged to mitigate against toxicity, and subsequently functionalized to attach to the sites of interest, yielding moieties much more unwieldy than organic dye molecules. In particular, derivatives of dyes are available which allow the dye to be conjugated to the functional groups on proteins. Nonetheless, the photobleaching of dyes in confocal microscopy can prove problematic in some cases.
The inventors have shown that the fluorophage of the invention can make a big impact in the biosensing and biological imaging fields. Advantages of fluorophage dyes over conventional dyes include:—
(i) The fluorophage is less susceptible to photobleaching than conventional dyes;
(ii) The fluorophage is brighter than single dye molecules because there are over a hundred (or thousand) molecular dyes attached to the biological substrate per fluorophage;
(iii) The fluorophage can be programmed to bind to biological and non-biological targets;
(iv) Unlike assays that use enzyme-conjugated secondary antibodies, the amount of fluorescent emission from a fluorophage does not depend on temperature, exposure time or other experimental variables.
Dyes are used ubiquitously as fluorescent labels for assays and confocal microscopy in bio labs worldwide. The fluorophage of the invention represents a better alternative to molecular dyes for many of these applications. Optical based assays are sold by many biotech companies, including Thermo Scientific and Merck. In addition, since a single fluorophage can act as a laser, because fluorophages are less susceptible to photobleaching, this would be revolutionary for biosensing and microscopy, since the signal to noise ratio would be greatly enhanced in comparison to the fluorescence of a single dye molecule.
Preliminary evidence that the compositions of the invention could sustain laser action was acquired. Fluorescein isothiocyanate was titrated into a M13 phage solution prepared as in the previous examples. Reactions were quenched with 1M pH 7 TRIS. Insoluble products were removed through two centrifugation steps. After the second centrifugation step, only a small pellet precipitated to the side of the tube. Fluorophage was separated from smaller molecular weight products by 5% PEG-8000/200 mM NaCl precipitation.
As shown in
A dye laser 100 was built to test whether the composition was capable of sustaining laser action, and this is shown in
Threshold behaviour was observed, which is a characteristic property of a laser, as shown in
The fluorophage concept was demonstrated using M13 because it is a readily available, well-understood biological substrate, and it clearly showed that the M13 could act as a good biological substrate for producing a light-emitting (i.e. lasing) composition. However, the inventors then set out to show that other biological substrates could also be used. Using principles taken from synthetic biology, multiple biological components can be augmented to fabricate an improved biological substrate.
Dyes or light-emitting molecules can be attached to specific sites on the coat protein of a plant virus called Tobacco Mosaic Virus (TMV) such that there is no contact quenching between neighbouring dyes. This has inspired the design and fabrication of a recombinant Tobacco Mosaic Virus-like particle (rTMV) that can be expressed in E. coli.
Two genetic constructs were designed and constructed using synthetic genes and standard molecular cloning techniques. The first construct (shown in
The second construct shown in FIG. 1,4 is a gene containing no ribosome binding sites (rbs) with the TMV coat protein specific origin of assembly sequence flanked by 5′ and 3′ end protection sequences under the control of an L-arabinose induceable pBAD promoter. A 5′ hairpin loop—pHPI7 with the rbs removed—was incorporated for 5′ end protection because it has been demonstrated to improve the half-life of RNA transcripts to nearly 20 min. The 3′ end tRNA-like structure from the native ‘TMAT genome was included in this synthetic construct to offer 3’ end protection. A strong terminator from the Register of Standard Biological Parts, BBa_B0015, ensures homogeneity in the length of RNA transcripts. Restriction sites are available for the cloning of non-coding spacer DNA which would increase the length of the rTMN. However, no spacer DNA was included in the example provided here.
When co-expressed, the RNA transcript and the coat protein form rMTV. The crystal structure of the native virus (shown in
The inventors then used the following steps for the production of an rTMV fluorophage laser. Light emitting molecules, including dyes, having a thiol reactive moiety were attached to the cysteine residue. The reaction schemes are outlined in
| Number | Date | Country | Kind |
|---|---|---|---|
| 1122158.7 | Dec 2011 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2012/053236 | 12/21/2012 | WO | 00 | 6/20/2014 |
