GB2433589A

GB2433589A - Quantum dots which enable luminescence signals to be detected simultaneously with Raman signals generated by the moiety coupled to the quantum dots

Info

Publication number: GB2433589A
Application number: GB0608223A
Authority: GB
Inventors: Benjamin Richard Horrocks
Original assignee: University of Newcastle, The; Newcastle University of Upon Tyne
Current assignee: University of Newcastle, The; Newcastle University of Upon Tyne
Priority date: 2006-04-26
Filing date: 2006-04-26
Publication date: 2007-06-27
Anticipated expiration: 2026-04-26
Also published as: GB0608223D0; GB2433589B; WO2007125300A1

Abstract

A composition forming a quantum dot probe comprising a semiconductor nanoparticle or semiconductor nanoparticle attached to a chemical or biological molecule conjugate, such semiconductor being selected to provide a luminescence signal upon being excited by incident radiation from an external energy source, the composition characterized in that the semiconductor nanoparticle is selected to exhibit a significant Stokes shift such that the luminescence signal has an upper threshold wavenumber significantly less than the wavenumber of the incident radiation, and further configured to be in close proximity with a metal nanoparticle to allow the enhancement and simultaneous detection of a second signal, preferably a Raman spectrum, which is characteristic of the attached molecule and or the molecules with which it interacts or in the vicinity of the quantum dot probe, that is detectable substantially simultaneously with said luminescence signal.

Description

IMPROVEMENTS TO QUANTUM DOTS, QUANTUM DOT PROBES AND

METHODS OF USE THEREOF

Field of the Invention

The present invention relates to quantum dots, quantum dot probes and methods of use thereof and particularly, but not exclusively, the invention relates to improved quantum dot probes suitable for, but not limited to, use in investigating and assessing the biochemistry of living systems such as cells. More specifically the invention relates to improved quantum dots which enable luminescence signals to be detected simultaneously with a second signal which enables specific chemical and biochemical events to be detected in the vicinity of the probe.

Background to the Invent ion

In the context of analytical science, labels are molecules or small particles which enable the researcher to track the movement and spatial distribution of an analyte (substance or molecule of interest). We use the term probe to mean a molecule or particle that acts as a label, but which may also provide information about chemical composition and reactions in the vicinity of the probe by virtue of some specific signal. There is a need in biological research for methods to label molecules such as proteins and nucleic acids in order to track these * S S * S * S SI SI * S S. * . S * S 155 5 * I SS * S us S S * S S.. S..

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molecules as they carry out their biological function. By doing this, the researcher hopes to understand how such molecules carry out their biological function, how these processes are organized and where they are located within the cell. This type of study is frequently performed by biologists on cultured or isolated cells and tissues with the aim of developing new understanding and therapeutic strategies for treatment of disease.

The current technology is often based on luminescent probes, e.g., organic dyes (examples include fluorescein, rhodamine and their derivatives) and green fluorescent protein (GFP, a naturally occurring luminescent protein).

(Luminescence is used throughout the text because it is the general term for emission of light, whereas fluorescence is specifically the light emitted upon a spin-allowed electronic transition typical of organic molecules). The luminescence of the probe is generated by irradiation with a focused laser at a wavelength that matches the absorption spectrum of the probe. The luminescence spectrum is determined by the separation of the electronic energy levels of the probe. Desirable characteristics of probes include absence of toxicity towards the cell, bright luminescence, stability under continuous irradiation (that is, the label does not become bleached) and emission of luminescence at wavelengths where the cell does not strongly absorb, e.g., red light.

The spatial distribution of such luminescence probes can be easily determined by a fluorescence microscope, often operated in confocal mode. The limitations of organic dyes and GFP include Photobleaching of the dye, high levels of background luminescence, PH-dependent variations in the luminescence and difficulties in quantitation due to self- quenching effects. In addition, all luminescence probes suffer from limitations arising from the broad and relatively featureless nature of luminescence spectra. The requirement for individual dyes to be excited using different wavelengths of light also makes it difficult to * . S * S *I* S a * * * S S S 5 *, a S S *S S * ** * S S * S S* S S S S. p p *a image a number of different biological molecules simultaneously. Although it is possible to multiplex labels (i.e. provide multiple signals) with different emission wavelengths, this is typically limited to 4-5 colours to avoid overlap of the spectra. Further, multiplexing techniques fundamentally do not address the lack of detailed molecule-specific features in the luminescence spectrum: unlike Raman spectra, luminescent spectra cannot be used to identify an unknown molecule.

Increasingly quantum dots are being used in place of fluorescent dyes or as complementary tools for bioimaging applications in order to overcome many of the problems previously described. Quantum dots are semiconductor nanopartjcles with physical dimensions less than the radius of the bulk exciton. The resulting quantum confinement effect endows the nanopartjc].es with unique optical properties that offer several distinct advantages over the conventional luminescent dyes and stains. Most notably, the bandgap and the luminescence can be tuned all the way across the visible spectrum simply by adjusting the particle size: smaller particles luminesce at shorter wavelengths.. As well as producing quantum yields comparable to the brightest conventional dyes available, quantum dots also exhibit excellent chemical and photo stability. Their resistance to photo bleaching and longevity renders them particularly useful for longer term studies and allows researchers to watch cellular processes unfold. In recent years quantum dots have been tested in a range of biotechnological applications including DNA array technology, ilnmunofluorescence, cell and animal biology.

The coupling of quantum dots to proteins (Brunchez et al., 1998; Chan & Nie, 1998; Mattoussi et al., 2000), oligonucleoje (Mitchell et al., 1999; Pathak et al., 2001) and small molecules has permitted selective targeting to areas of interest. Quantum dot probes have now been used to successfully label a range of biological molecules including membrane proteins (Sukhanova et al., * S I * S S I i I a * * * * I I S S I I. I S * I S * , S * S. S I.SS 2004; Wu et a]..,, 2003; Akerman et al,, 2002), microtubules (Wu et al., 2003), actin (Wu et al., 2003; Brunchez et al., 1998), nuclear antigens (Wu et al., 2003) and cell surface receptors (Wu et al., 2003; Lidke et al., 2004.

US patent no. US 6326144 represents one of a number of patents in the name of the Quantum Dot Corporation and relates to a composition comprising a fluorescent semiconductor nanocrystal core associated to a compound such as a nucleic acid or antibody.

Quantum dots of all Common semiconductor materials have been produced, however, the best characterjsed and most widely used in applications are those based on heavy metal chalcogenjdes, especially cadmium selenide (CdSe). The luminescence of bare CdSe particles is strongly influenced by chemical reactions at the particle surface, e.g., loss of Cd2' by oxidation, and quantum dot labels often comprise a coating of a wider bandgap semiconductor, called the shell, to protect the luminescent core. CdSe core I ZnS shell nanoparticles are frequently employed as luminescent labels. Recently research has focused on fabrication of and use of silicon based nanoparticles or quantum dots. A reason for this is that silicon quantum dots emit a strong luminescence signal. In addition, silicon is relatively inert and therefore less toxic to cells than competing tecbnologies, e.g., cadmium selenide nanopartjcles.

Silicon quantum dots also luminesce orange-red at particle diameters as small as 2 nm, this is desirable because smaller particles are likely to cause less cellular disruption and, at longer wavelengths, the background fluorescence / absorption from components of the cell is minimal. An example of biocompatjble fluorescent silicon nanoparticles and in-vivo imaging methods IS international patent (PCT) publication number W02004].08902 in the name of Visen Medical Inc. (US). Other examples include the paper by Lars H. Lie et al, 2002 and the paper by Lars H. Lie et al, 2004.

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The ease of multiplexing and optical barcoding is one of the major advantages provided by the tunable luminescence wavelength of quantum dots over traditional luminescent probes. Multiplexing is also facilitated by the narrower emission spectra of quantum dots which minimises spectral overlap and permits the simultaneous resolution of multiple coloured probes using only a single wavelength of light for excitation of the luminescence. By simply altering the particle size of quantum dots the emission wavelengths can be tuned from the blue to red regions of the spectrum (Brunchez et al., 1998). The work of Nie and coworkers (Han et al., 2001) describes the development of polystyrene beads linked to capture molecules which are embedded with numerous CdSe quantum dots capable of producing multiple colour and intensity combinations.

Although this technique is particularly useful in applications such as microarray technology it is of limited value in living cells due to potential spectral overlap between coding and target signals, and the need for conventional assay methodologies such as immunoass ys to determine the identity of unknown analytes.

Although luminescence provides a convenient and sensitive technique for mapping spatial distribution, luminescence spectra whether obtained using quantum dots or using traditional fluorescent dyes/stains do not in themselves provide much chemical or biochemical information. By this it is meant that little or no information is provided as regards the identity of the chemical or biochemical species interacting with or in the.vicinity of the probe.

A luminescence probe alone therefore does not allow the researcher to identify in detail the biochemical processes that may be occurring at a given position in a cell.

Characterization of the protein-protein interactions within living cells is essential in order to understand the basis of biological processes such as DNA replication, transcription, signalling pathways and cell cycle control.

Protein interactions have been traditionally studied using * * S * * * S S * S S * S S * * S S S S *S* S S * ** * * *s S * * * * * S S S S S SS * *.S S uS techniques such as protein-protein affinity chromatography, immunoprecipitation, sedimentation and gel-filtration (Phizicky & Field, 1995), all of which require cell lysis and are therefore unable to supply any information on where and when the interaction is occurring. Current techniques available for in vivo imaging of molecular events include Fluorescence Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) which allow visualization of the interaction between proteins tagged with two different chromophores (donor and acceptor) when in sufficiently close proximity to permit energy transfer. However, these methods only provide spatial and temporal distance information, cannot be used to target unknown proteins, can only be used if the interacting proteins are separated by small distances, make it difficult to observe proteins present in large complexes and are subject to the usual limitations of fluorescence microscopy previously described. In view of this there is a need to develop biological probes that not only allow determination of distribution in real time but also permit identification of unknown analytes and provide more detailed chemical/biochemical information to be obtained from the medium in which the probes are deployed.

A spectrum that is sufficiently detailed and characteristic to fingerprint' molecules is normally only possible with vibrational or magnetic resonance spectroscopy. Raman scattering is one such vibrational spectroscopic technique which is capable of providing detailed information on the chemical structure of unknown substances. In Raman spectroscopy the incident laser light is scattered with a change in energy (and therefore wavelength) determined by the vibrational modes of the molecule of interest. Since Raman spectra have very narrow linewidths (10 -101 cm) and show many lines that can be assigned to particular groups of atoms, unlike the broad, featureless single band in a typical luminescence * * * * . * S * * S S * S S S * S S S * 555 * S S SS S S 55 * S * S * S * S S S S S. S 555 S *SS spectrum, they can be used to "fingerprint" a molecule or deduce information about the chemical composition of an unknown species. In addition to the high structural/chemical information content of the spectra, Raxnan spectroscopy has a number of advantages over other spectroscopic methods including a high sensitivity to changes in chemical composition and binding, low interference from water and high spatial resolution.

Raman scattering is however a very inefficient process and surface-enhanced Raman scattering (SERS) must be exploited in order to amplify signals to detectable levels. The effect known as SERS is the phenomenon of a strongly increased Raman signal from molecules attached to SERS active substrates such as colloidal silver or gold nanopartjcles, electrodes or evaporated films of these metals. Further enhancement, up to factors of have been obtained by combining the metal nanoparticle with dyes which produce a resonant enhancement by virtue of an electronic transition close to the excitation wavelength: this is the surface enhanced resonance Raman (SERRS) effect. It has been shown to be possible to detect a range of biological molecules using silver or gold nanopartic].es as probes associated with Raman-active dyes (Nie & Emory, 1997; Cao et al., 2002). More recently Raman spectroscopy has been successfully applied to living cells to allow detection of a reporter dye present on colloidal silver and gold nanoparticles in addition to yielding vibrational information from molecules in the vicinity (Kneipp et al., 2005). Such signals can be assigned to the native chemical constituents of a cell including DNA, RNA and proteins, however they are not targeted effectively.

It will be appreciated by those skilled in the art, that much chemical / biochemical information could be produced by deploying techniques which would enable Raman spectra to be collected in combination with the luminescence signal. However, the ability to detect such signals simultaneously, or substantially simultaneously has not * S * S S * S S S S S S S S S S S S S S SIS * S * S S *S S * S S

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SS S 555 S 555 been achieved. Furthermore the prior-art teaches that the simultaneous collection of Raman spectra and luminescence are wholly incompatible: "Since its inception Raman spectroscopy has been plagued by fluorescence, which can overwhelm the Rarnan effect." ("Handbook of Raman Spectroscopy", Chapter 22, pp 865-6, by J. L. Lauer, TIC San Diego, editors I. R. Lewis and H. G. N. Edwards, Marcel-Dekker, NY, 2001.) Although Raman labels are being developed by several workers, (P. Jordan, J. Cooper, G. McNay, F. T. Docherty, W. E. Smith, G. Sinclair and M. J. Padgett "Three-dimensional optical trapping of partially silvered silica microparticles." Opt Lett. 2004, 29, 2488- 2490. S. Nie and S. R. Emory, "Probing Single Molecules and Nanoparticles by Surface-Enhanced Ra.man Scattering.

Science, 1997, 275, 1102-1106.), these methods do not allow the simultaneous acquisition of luminescence data.

Other techniques, such as the Kerr-gate (K. Matousek, M. Towrie, A. Stanley and A. W. Parker, Appl. Spectrosc.

1999, 53, 1485-1489.) have been developed to suppress luminescence when acquiring Raman spectra of normally luminescent samples, but none of these technologies can be used simultaneously with luminescence spectroscopy to track analytes and examine the Raman fingerprint".

The disclosure by Nie S and Emory SR. entitled "Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering" (Science 1997 Feb 21; 275 (5303): 1102-6) describes the use of silver particles to not only enhance the Raman signal of the attached dye as previously described but to also quench the luminescence signal so that the Raman spectra can be collected. Other publications describe the templating of silver onto bulk porous silicon in order to produce large SERS enhancements whilst suppressing the luminescence. In these examples quenching of luminescence is achieved by preparing the silicon under conditions unfavourab].e for luminescence (Lin et al., 2004; Haohao et al., 2004) or fully coating the silicon surface with the metal so that light is unable * S S S * * * * * S S * S S S * . * S * *SS S S S S. S S *S S S S * * S S * S S S S. S *SS S SS* to penetrate and produce a luminescent signal from the underlying semiconductor (Chan et al., 2003).

Throughout this document the terms quantum dot and semiconductor nanopartjcle should be considered interchangeable.

Summary of the Invention

This invention aims to address the aforementioned need in the art and provide a means to produce probes capable of simultaneously generating detectable luminescence and Raman signals. This invention comprises a method for the construction of probes which provide two useful signals simultaneously, i.e., Raman and luminescence spectra. The probes consist of a composition comprising a semiconductor nanopartjcle and a metallic material, optionally further comprising a biological or chemical molecule or composition. The semiconductor has certain characteristic properties described below in order to facilitate the simultaneous acquisition of Raxnan and luminescence spectra. The luminescence produced by the probe originates from the semiconductor and provides a means to track its location. The Raman signal originates from both the probe and its environment; it is enhanced by the electric field of the metal nanoparticle, and provides detailed information on analytes, chemicals or biochemical events present in the vicinity of the probe.

An objective of the present invention is to provide a quantum dot probe such that said probe simultaneously or substantially simultaneously provides both a detectable luminescence signal and a detectable Raman signal when excited by incident radiation.

Another objective of the present invention is to provide a method and means to tag biological or chemical molecules with said quantum dot so that information on their * e S * * * S S * S S S * I S * S S * S **S S p 5 S. S I *S S I I I * I I S S I I PS S **S * S*I behaviour and associated chemical interactions in a medium can be obtained simultaneously or substantially simultaneously from a luminescence signal and a Ranian-based signal.

A further objective of the present invention is to provide a method and means to tag biological or chemical molecules with said quantum dot so that information on their behaviour and associated chemical interactions inside a cell can be obtained simultaneously or substantially simultaneously from a luminescence signal and a Rarnan-based signal.

A further objective of the present invention provides a means to tag said quantum dot with molecules which will target quantum dot to a particular locality within a cell so that information regarding biochemical and chemical events within a specific locality can be obtained simultaneously or substantiality simultaneously from a luminescence signal and a Raman-based signal.

Another object of the present invention is to provide an improved quantum dot probe and an associated methodology that enables biochemical events occurring inside a cell to be determined.

Yet another object of the present invention is to provide a method of spectroscopy that yields a luminescence signal and a Raman- based signal from a quantum dot.

According to a first aspect of the present invention, there is provided a photoluminescent composition for use in proximity to a moiety capable of generating a Raman signal in response to an incident radiation event, said composition comprising; at least one first material configured to emit a detectable luminescence signal when excited by selected incident radiation from an external source; and at least * * * * * * S * S I S S S I S * S S * I *SI I S S I. S S IS S S S *

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one second material adapted to augment or enhance detection of the Raman signal generated by said moiety, said second material being associated with said first material without significant occlusion thereof to permit luminescent emission therefrom, wherein said first material is selected to emit luminescence with an upper threshold wavenumber sufficiently less than the wavenumber of the incident radiation to allow the luminescence and the Raman signal to be detectable substantially simultaneously.

Preferably the first material is a semiconductor nanoparticle.

Preferably the second material is a metallic material.

As the resultant luminescence signal and Rarnan signal will occur at significantly different wavenumbers this will allow both of the signals to be detected substantially simultaneously without the luminescence overwhelming the Raman signal. Also, the second material does not occlude the first material such that both luminescence of said semiconductor nanoparticle and the Raman signal remain

detectable.

Preferably the wavenuirther of the upper threshold of said luminescence signal is at least 500 cm' less than the wavenumber of the incident radiation.

Selecting a semiconductor nanoparticle that exhibits luminescence with an upper threshold of 500 cm' less than the wavenumber of the incident radiation ensures that the luminescence differs sufficiently from the Raman signal to avoid overlapping signals.

Preferably said Raman-based signal occurs at a wavenuinber in the range of 500 cm' to 3500 cm1 from the wavenuinber of the incident radiation.

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* S * S * S S S S S * S I S 5 III I I S S. I * 55 5 S S S * S S S S S S I ISI S 5.1 Preferably said semiconductor nanoparticle comprises an indirect bandgap semiconductor nanoparticle.

Preferably said semiconductor nanoparticle comprises at least one atom of an element selected from Group IV of The Periodic Table of The Elements.

Preferably said Group IV element is selected from the set of elements comprising Silicon (Si), Germanium (Ge) and their alloys.

Preferably said at least one metallic material comprises an element selected from the set comprising Silver (Ag), Gold (Au) and Copper (Cu).

Preferably said photoluminescent composition further comprises an additional compound of interest.

Preferably the additional compound of interest is a molecular compound.

Preferably said molecular compound is comprised of a molecule that comprises at least one carbon atom.

Preferably a molecule of said molecular compound is attached to said semiconductor nanoparticle via a covalent bond.

Preferably said molecular compound is covalently bonded to said semiconductor via a link comprising said at least one carbon atom.

Preferably said link comprising said at least one carbon atom is of the form X-C, where X is an atom of said semiconductor, and C is a carbon atom of a molecule of said compound.

* S S S S * S S S S S S S S S * S S S S 55. S S S 5 * 55 5 * S S * S S S S S S ** * *5* a Alternatively said link comprising said at least one carbon atom is of the form X-O-C, where X is an atom of said semiconductor, a is an oxygen atom and the group -0-c constitutes a part of a molecule of said compound.

A further alternative is that said link comprising said at least one carbon atom is of the form X-O-X--C, where Xis an atom of said semiconductor, o is an oxygen atom and the group -O-x-c constitutes a part of a molecule of said compound.

Optionally said molecular compound has an affinity for a biological target.

Optionally said molecular compound is a biological compound.

Preferably said biological compound is selected from the set comprising: a peptide; a nucleic acid; a carbohydrate; a protein; an enzyme; an antibody; and an oligonucleotide.

Most preferably said biological compound is selected from the set comprising: a ribonucleotide and derivatives thereof; a deoxyribonucleoje and derivatives thereof; and a dideoxyribonucleotide and derivatives thereof.

According to a second aspect of the present invention there is provided a method of performing spectroscopic analysis comprising the steps; * selecting a composition according to the first aspect of the invention; * deploying the composition to an area of interest; * exciting said composition with by incident radiation from an external source;

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* S *I * S S S I * * S S * I 555 I S S II S I *s I S S I * I S I SI S * S 555 S *.* detecting the radiation spectrum emitted Preferably the method further comprises the step; * analysing the radiation spectrum emitted to obtain further information about the area of interest.

Preferably when detecting the radiation spectrum both a luminescence signal and a Raznan signal are detected.

Preferably the incident radiation comprises light from any appropriate source.

Preferably said incident radiation comprises an Ar ion laser.

Brief DescriDtiofl of the Drawings For a better understanding of the invention and to show how the same may be carried into effect, there will now be described by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which: Fig. 1 schematically illustrates, in accordance with a preferred embodiment of the invention, desirable characteristics of a suitable absorption spectrum for enabling detection of both luminescence and Rainan spectra from a quantum dot molecular conjugate; The absorbance spectrum 101 is that for a silicon-based quantum dot; and Fig. 2A schematically illustrates fabrication of a preferred embodiment of a quantum dot probe as configured in accordance with the present invention; In the embodiment illustrated the quantum dot probe comprises a biomolecule attached to a surface enhanced silicon based quantum dot to form the conjugate and combined a metal nanoparticle that enhances the Raman signals (202); and a S S S * I * S S S S S I S * S I a I IIS I S S S I Is a a I I * a a S *II S S SI.

Fig. 2B schematically illustrates the process of obtaining spectroscopic measurements using a quantum dot probe of the type illustrated in Figs. 1 and 2A; and Fig. 3 schematically illustrates the basic steps involved in on-chip solid-phase synthesis of quantum dot conjugates (303) of the type that may be configured in accordance with the present invention; and Fig. 4 schematically illustrates the combination of Ag/Au flanoparticles with silicon quantum dot conjugates (Q-Si-DNA) by simple mixing of the two to produce an optimal enhancement of the Raman signal and retain the luminescence of the Q-Si; and Fig. 5 schematically illustrates another method of associating Ag/Au nanoparticles with a silicon based quantum dot conjugate. The method involves attaching an Au or Ag nanopartjcle to Q-Si-DNA using DNA hybridization; and Fig. 6 schematically illustrates a further method of associating Ag/Au nanoparticles with a silicon based quantum dot, the method involving attachment of an Au or Ag nanoparticle to Q-Si-DNA via binding of the metal nanopartjcle to a thiol-terinjnated Q-Si particle; and Fig. 7. shows actual spectra obtained from the following compositions: 20 nm diameter Ag colloid alone; Q-Si-DNA alone; and Q-Si--DNA mixed with 20 rim diameter Ag colloid.

The 488 run line of an argon ion laser was used to provide the excitation and the spectrum was acquired using a Witec (tllm, Germany) CRM200 confocal Raman microscope.

Fig. 8. shows another spectrum of Q-Si-DNA mixed with 20 run diameter Ag colloid in which various Raman bands have been assigned.

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Detailed Description

There will now be described by way of example a specific modes contemplated by the inventors. In the following description numerousspecific details are set forth in order to provide a thorough understanding. it will be apparent however, to one skilled in the art, that the present invention may be practiced without limitation to these specific details. In other instances, well known methods and structures have not been described in detail so as not to unnecessarily obscure the description. First we describe the materials employed and the rationale for the choice of materials, then we describe practical aspects of obtaining simultaneous Raman and Luminescence spectra with quantum dot probes configured to enable such signals to be detected, next we describe the construction of the probes and finally we show experimental data that demonstrates a working probe.

Notation Quantum dot structures may be conveniently referred to using a shothand notation. The notation "Q-X" refers to a quantum dot structure or probe comprising the chemical element, species or group "X". For example "Q-Si" refers to a quantum dot comprising the element Silicon. Another example is "Q-Si-DNA" which refers to a quantum dot probe wherein the quantum dot comprises silicon and such that the dot is attached to the biological rnacromoiecule deoxyribonucleic acid (DNA).

Materials The preferred semiconductor nanoparticle. Indirect gap materials have a weak absorption at photon energies just greater than the bandgap because the transition is dipole forbidden and requires the simultaneous absorption/emission of a phonon (quantised lattice * . . . * , * * * * o * I P * P I I *?I P I IP * I *P i * P

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I. * III I SIP vibration). In the case of bulk Si, the indirect band gap is about 1.]. eV, but there is also a direct gap at about 3.4 eV. In Si nanoparticles the gaps are somewhat larger, especially the indirect gap which is increased from the bulk value to energies corresponding to the orange/red part of the spectrum. Light is strongly absorbed for photon energies 3.4 eV and emitted strongly at a value close to the band gap at about 1.9 eV (in the particles employed in this description). The difference, 3.4 -1.8 = 1.5 eV corresponds to a Stokes shift of 12,000 cm1. In practice, the Stokes shift may be smaller because the excitation photon energy is usually fixed by the available lasers. In our case we have employed the 488 nm line of a standard Ar+ ion laser; the photon energy of the excitation laser is therefore 2.55 eV and the observed Stokes shift is about 5800 cm1, sufficient to detect the full range of Rainan signals of chemical interest (<3500 cm 1) Direct gap materials such as CdSe and CdS might also be used as the semiconductor because they can give large Stokes shifts, but are distinctly less advantageous because the Stokes shift is smaller than for indirect gap materials and the semiconductor particles may absorb some of the Raman-scattered light.

Biological molecules and nanoparticle conjugates. The combination of any nanoparticle covalently bound to a biological molecule is referred to as a conjugate. The systems described below are designated Q-Si-DNA, Q-Si-protein, Q-Si-PNA and Q-Si-peptide conjugates. The phrase biological molecule is used below to indicate one of deoxyribonucleic acid (DNA), protein (including enzymes and antibodies), peptide nucleic acid (PNA), peptide and ribonucleic acid RNA or a chemical derivative of one of these. The interactions of the conjugate with natural partners in the cell are of direct interest to biologists * * * S I * SI. IS I IS* * S I S S III I S S I... *5 S I I S * .. S I S I I a. 5 5 SIl and may also be used to drive the conjugate to a particular location in the cell.

DNA. Short lengths of single stranded DNA -oligonucleot ides -can be covalently attached to the silicon particles by direct, automated chemical synthesis of DNA on the porous silicon support before it is broken-up into nanoparticles. Sonication is required to break-up the porous silicon layer into Q-Si bearing one or more DNA molecules. This procedure produces DNA molecules anchored at the 3' end. Q-Si-DNA nanoparticles have been characterjsed by gel-electrophoresis by cleaving the DNA from the silicon and the DNA has been shown to remain intact during the formation of the Q-Si-DNA conjugates.

Figures 4-6 show schematics of such Q-Si-DNA particles. It is also possible to chemically immobiljse pre-formed oligonucleotjdes on the particles.

Proteins,Enzymes, Antibodies, PNA and peptides. Molecules bearing one or more primary amine groups, e.g., lysine residues in proteins, N-terminus of peptide nucleic acids (PNA) or the N terminus or lysine residues of peptides, can be anchored on porous silicon via a Schiff base chemistry developed by the inventors. An organic monolayer is formed on the porous silicon which bears aldehyde functional groups at the opposite side of the monolayer from the Si-C bond which covalently anchors the molecule to the porous silicon. These aldehyde groups react with primary amines (e.g., the external lysine residues of proteins) in neutral, aqueous solution (in ca. 1 h) in the presence of 1 mol dzn sodium cyanoborohydride (NaCNBH3) to form a covalent C-N link between the monolayer and the protein/PNA/peptide. The porous silicon can then be broken-up into Si nanoparticles bearing one or more protein/PNA/peptide molecules by sonication. This methods allows enzymes, antibodies and any protein bearing lysine residues to be conjugated to the Si nanoparticle. S * S

* S S S S * * * S S * S S S S 555 5 5 S S. 5 5 *S 5 5 5 5 * S S S S S S S 555 S 555 Simultaneous Rainan and Luminescence Spectroscopy with Quantum dot Probes In accordance with the present invention a method is provided to allow Raman-based and luminescence spectra to be acquired simultaneously from the same semiconductor nanoparticle probe. The prevailing view in the field of Raman spectroscopy is that samples which are luminescent pose a problem for the Raman spectroscopist and that the luminescence must be suppressed or attenuated for satisfactory Raman spectra to be obtained. However, the inventors have shown that this is not true in the particular case of certain semiconductor nanoparticles.

The inventors have exploited this to develop probes with a novel molecular architecture comprising a semiconductor nanoparticle, and metal nanoparticle or semiconductor nanoparticle, metal nano particle and biological molecule configured to enable luminescence and Raman based signal to be collected simultaneously or substantially simultaneously at the same time Those skilled in the art will be aware that the Raman effect is much weaker than luminescence. Typically one photon from 106 incident photons is Raman-scattered compared to quantum efficiencies which may approach 100% for luminescence probes. For this reason, the prior art would teach the Raman spectra as generated in accordance with semiconductor nanoparticles would be swamped by the luminescent signal.

The inventors decided to try to configure a quantum dot probe which would enable both signals to be collected at the same time. Surprisingly they found this was possible with a quantum dot configured in accordance with the present invention based on the realisation that in contrast to the usual expectation for luminescence spectra, the position of the peaks in Raman spectrum depends on the excitation wavelength. Thus it is the * S I S I * S S S S * S S S S * S S S S 555 5 5 5 5 5 S. S S 5 5 * S * S * S S S. S SSS * ..* relative position of the Raman peaks to the incident light which is fixed. By configuring the probe in accordance with the invention and selection of the appropriate excitation wavelength, the Raman signal is suitably amplified and positioned in the spectral gap relative to of the luminescence signal to be detectable at the simultaneously or substantially simultaneously. The method may suitably employ the same equipment as would be used for confocal Raman microscopy: a confocal optical microscope with a Raman filter and a spectrograph with a CCD detector.

Examples of suitable metal nanoparticies include but are not limited to silver, gold and copper. The detailed mechanism of the enhancement remains a topic of debate in the scientific community, but there is general agreement that two factors are important: the influence of the metal on the electronic structure of adsorbed molecules and a longer range electromagnetic enhancement (A. Campion and P. Kambhampati, Chemical Society Reviews, 1998, 27, 241- 250). The electromagnetic part of the enhancement allows the experimenter to amplify Raxnan signals from the environment of the metal nanoparticle, not just those molecules chemically bonded directly to the particle. The range of this effect may extends tens of nanometres from the metal surface; for a single molecule at a distance, d, from a metal particle of radius, r, the enhancement falls off as Er! (r+d) 12 (A. Campion and P. Kainbhampati, Chemical Society Reviews, 1998, 27, 241-250., pp242-243).

Collectively these effects are known to those ski-lied in the art by the acronym "SERS" -Surface Enhanced Raman Spec troscopy.

By the term "Raman-based spectra" it is meant herein spectra that are generated according to the Raman effect whether it be the Raman effect per se or an enhanced Raman effect or some other Raman spectroscopy effect such as, for example, anti-Stokes Raman spectroscopy. Anti-Stokes a * . a * . a * * S a S S S * a S S S *SP S S S S. S S *S S S S S * * S S S S S * S.. S 555 Raman spectroscopy can usefully be employed in a given system since it further separated the Raman signal from the fluorescence signal. In addition to the SERS effect described above, another Ranian enhancement technique that is suitable for implementing the present invention is the Surface-Enhanced Resonance Raman Scattering (SERRS) effect. SERRS is the combination of resonant enhancement of Raman signals (RR) owing to the proximity of the excitation energy to an electronic transition in the system and the SERS effect described above.

In accordance with a preferred embodiment of the present invention the semiconductor from which the quantum dot is made must possess a number of characteristics in order to produce a suitably configured probe. Such semiconductor nanoparticles must emit luminescence at a wavelength much longer than that which excites the luminescence, that is, they must show a large Stokes shift. The reason for this is that the Raman spectrum always occurs at wavelengths slightly longer than the excitation wavelength, therefore if the Stokes shift of the luminescence is small, the two signals will overlap in the spectrum and the Raman signal will be swamped. Since Raman signals appear at a fixed energy with respect to the photon energy of the excitation light, these considerations can be made precise and general by considering the Stokes shift in terms of energy, or equivalently, wavenumber. If the Stokes shift is greater than 500 cm-i, then Raman signals at shifts <500 cm-i may be detectable; this holds irrespective of the excitation wavelength. Useful Raman signals from chemical groups lie in the range 500-3500 cm-i, the upper limit of the range therefore defines the minimum desirable Stokes shift and the lower limit defines the minimum usable Stokes shift.

In order for the Stokes shift of the semiconductor nanoparticle to be large, the desirable absorption * S * * * * S I * S S S I * * I a * III S I S S. S * ** S * S * * S S S S S S. S *SS * S..

spectrum for the nanoparticle should have the form illustrated in Fig. 1. The absorbance of the particle rises slowly above a threshold photon energy near to that of the transition responsible for the luminescence and the absorbance becomes large at energies much greater than this threshold. Suitable materials for providing the above characteristics have been determined to be indirect bandgap semiconductors. Elements selected from Group IV of The Periodic Table of the Elements, such as Silicon (Si), Germanium (Ge) and their alloys are considered to represent the best mode for carrying out the invention.

Some other materials such as CdSe, which are direct gap semiconductors, can show quite large Stokes shifts, but their absorbance near the threshold is significant and the Raman spectrum occurs in a region where the particles absorb strongly; they may also be used in place of Si in our invention, but are not optimal.

In figure 1 the absorbance spectrum 101 is that for a silicon-based -quantum dot attached to a molecule of a compound. The Figure illustrates absorbance or emission intensity on the vertical axis 102 versus wavenumber in cm (or photon energy in eV) along the horizontal axis 103.

For silicon nanoparticles employed by the inventors, a typical wavenumber of the emission maximum (104) is 15,380 cm' (equivalent to a wavelength of 650 nm or a photon energy of 1.9 eV), the exact value being determined by the semiconductor particle size and being independent of the excitation energy or wavelength. Thus luminescence signal 104 centres on 15,380 cm1 (650 run) in the example. In the example the excitation wavelength 105 has been selected as 488 run (wavenurnber= 20, 490 cm1), but the useful range may be considered to comprise wavenuinbers that are at least 500 cin1 greater than that of the emission so as to enable the Raman signal to be reliably be detected. 488 run is suitable since it is >5000 cm1 away from the wavelength of the peak of the luminescence signal for silicon and is in fact a strong line obtained using an Argon ion laser. The S S * S * * S S S I S S * * * S S I I 555 5 S S IS S * IS S S S S * S S S S S S S. 5 *SS 5 5** threshold illustrated at 106 is the photon energy at which the semiconductor material (silicon) starts to absorb radiation. Region 107 generally designates that part of the spectrum that lies between the excitation wavelength and the threshold, the size of this region (in wavenuinbers) is termed the Stokes' shift. In accordance with the present invention, region 107 is required to exhibit only weak absorption so that light of within this region of the spectrum is neither absorbed by the quantum dots nor emitted as luminescence. This facilitates detection of the Raman scattered light in the spectral gap 107. The peaks illustrated generally at 108 are the Raritan scattered light. The Raman spectral peaks shown are mainly due to the molecule to which one or more quantum dots are attached to. However a quantum dot will itself provide a Raritan signal as part of the overall Raman signal produced by a given quantum dot-molecular conjugate. Each Raman signal peak appears red-shifted with respect to the excitation radiation by a constant characteristic of the vibration mode of molecule that is responsible for the Raman scattering.

The dashed line 109 represents a typical absorption spectrum for a direct bandgap semiconductor material such as, for example, CdSe or Ga1s. From the threshold point 106 the spectrum 109 shows a rapidly increasing absorbance with respect to increasing wavenumber and therefore the illustrated Raritan spectrum 108 is not easily detectable for a quantum dot system comprising such a semiconductor.

This is in sharp contrast to spectrum 101 for an indirect bandgap material where the absorbance is shown as increasing only slowly with decreasing wavelength from the threshold until it rises rapidly at higher wavenuinbers near the position of the excitation light in the spectrum.

In accordance with the present invention a primary requirement is that the Stokes' shift 107 is large enough to enable a Raman spectrum to fit between the excitation energy (or wavelength) 105 and the luminescence signal a a a * a * a a I I I S S I I * I S S S 115 a I aS S I IS S I S * S S S S S Se 5 **b 1 threshold 106. In other words the second spectrum, preferably a Raman spectrum, and the luminescence spectrum must occur at sufficiently widely separated wavelengths for the two effects to be detectable simultaneously.

In accordance with the present invention a Rarnan signal will arise from the indirect bandgap semiconductor itself, but in biological applications this is not of particular interest. The main signal of interest is that derived from the molecule to which the semiconductor nanoparticle is attached in a particular semiconductor nanoparticle-molecular conjugate that is being utilised. In the best mode contemplated the excitation wavelength is that derived from an Argon laser (488 nm) and the spectral gap should be configured for a particular quantum dot/probe such that the highest wavelength components of the Rainan spectrum preferably occur at least 500 cm' from the threshold wavelength 106.

Rainan scattered light/radiation from molecular species is shifted in wavenumbers from the excitation (incident) light by a fixed amount (termed Raman shift) dependent only on the inherent vibrational properties of the molecule. For example C-H stretching vibrations give rise to Raman signals at a shift of 3000 cm' and C-C or C-N at shifts of approximately 1000-1200 cm. Although the Raman shift for a particular molecule, when expressed in wavelengths, will depend on the wavelength of the incident light; the Raman shift, when expressed in wavenumbers, is independent of the incident light or laser used. Most useful Raman signals occur in the range 500 cm' to 3500 cm from the incident light. However in order to cover all possibilities the wave number for the luminescence signal should be at least 500 cm less than the wave number of the incident light. In other words the luminescence is, in accordance with the best mode contemplated, required to occur at wave numbers that are lower than the incident * S S V S * * S V S * S S * * S S S S 555 S I S I. S S St S S 5 5 * S I S S S S S. S 555 S 555 light by an amount greater than or equal to at least 500 wavenurnbers.

In accordance with the principles and methods of the present invention suitably configured quantum dots (or their agglomerates) are provided to act as both luminescence probes (for spatial localization/imaging) and Raman probes (to provide biochemical information). A schematic illustrating an example of such a quantum dot sensor and the fabrication thereof is illustrated in Fig. 2. A silicon based quantum dot is thus fabricated by starting with porous silicon as indicated at 201 and then, as indicated, modifying the surface followed by breaking up the porous silicon layer to create the nanoparticle conjugates 202. In the best mode contemplated conjugate 203 comprises an alkyl-rnodified silicon core (marked Si) that is an efficient fluorophore. The core is also chemically stable in aqueous/biological media, non-toxic and emits photons (in the wavelength region of 600-700 mn) that do not interact strongly with most biological molecules whilst allowing convenient location of the particles by confocal fluorescence microscopy. One or a plurality of the required capture molecule 203 may be attached to the core as illustrated in figures 4 -6.

Examples of typical molecules that may be captured (204) and detected via their Raman signal include: Oligodeoxynucleotides (ssDNA) to capture mRNA, PNA for d5DNA and small ligands or antibodies for protein capture.

In the embodiment illustrated in Fig. 2A the SERS effect is provided by one or a plurality of silver nanoparticles (marked Ag) attached to or otherwise associated with the structural body comprising the Silicon core and the one or more capture molecules. This partial coating or association of silver particles is intended to provide an enhancement of the Raman signal from bound species via the SERS effect or, in combination with suitable chromophores, via the SERRS effect.

* p p p S * p * * * a S S * S * S S S I **I S S S *. S S S* S S S S * S * S S S S * * p e * As illustrated schematically in Fig. 2B the probe may be excited with visible light of short wavelength (blue) 205 such that the probe emits luminescence at much longer wavelengths (orange-red, 207). In this way the intervening (green) spectral region (206) is then substantially free of luminescent background and optically transparent (that is no large absorbance is present) to thereby enable a Rainan signal to be detected.

To implement the methods of the present invention, for example as silicon based quantum dot probes, three basic steps are involved as follows: * Preparation of silicon quantum dot -biomolecule conjugates * Introducing quantum dot probes into cells; * Spectroscopic detection of nucleic acid hybridization and protein binding and various experiments on aspects of cell biology.

* Preparation of silicon quantum dot-biomolecule conjugates: The chemistry required for functionalisation of macroscopic silicon surfaces in order to produce DNA and PNA molecules covalently bound to Si quantum dots are known in the prior art. Such Si-based particles can be produced by electrochemical etching of silicon wafer to form porous silicon (a material consisting of interconnected silicon nanoparticles) under suitable conditions, followed by functionalisation of the porous silicon layer using hydrosilation chemistry and finally the breaking-up of this layer into the desired individual nanoparticle-inolecule conjugates ref [1] and [2) (Fig. 3.). Porous silicon has been synthesized with a range of chemical functionalities on the surface including dimethoxytrityl-protected 11-undecen-1-ol, which may be * * * . * * S S * * S S S I I * S S S S *S* S * S *5 * S US S S S S * I S S S S S S. S 555 * 27 used to start oligodeoxynucleotide synthesis at porous silicon (301). In general, functionalisation of suspended nanoparticles is not straightforward. However the immobilization chemistry may suitably be carried out on-chip, i.e., on the porous silicon layer, prior to cleaving the particles from the porous silicon. Sometimes an additional electrochemical etch at higher current density (302) may be necessary to enable the conjugates (303) to be released from the porous silicon (or Ge) layer by sonication or ref lux in toluene or mesitylene. This technique facilitates complex chemical functionalisation of the nanoparticles since advantageously it allows solid-phase synthetic methods to be used and therefore extensive chromatographic or electrophoretic purifications of the nanoparticles are substantially avoided.

(1) On-chip synthesis of functionalised Q-Si (DNA, PNA) The on-chip synthesis outlined in Fig. 4 is an advantageous over known methods because of the simplicity of solid-phase synthesis and because of the increasing experience of those skilled in the art as regards protein immobilization chemistries on silicon surfaces.

Experimental details for the preparation of Q-Si-DNA conjugates are given below, it will be apparent to those skilled in the art that this method can be adapted to form Q-Si-PNA conjugates (PNA=peptide nucleic acid) and Q-Si-peptide conjugates.

(ii) Preferred approaches to attach quantum dots to molecules In relation to the present invention preferred embodiments as regards the attachment of quantum dots to molecules of a compound, in particular a biomolecular compound, concern chemical linkages in the form of covalent chemical bonds.

In respect of molecules that comprise at least one carbon atom, such as organic chemical and biological molecules, the link is via the at least one carbon example. The best mode contemplated for many applications consists of a link * S S S S * S S S S S * S * S * S S * S 555 5 * S ** S I 55 S S S S * S S S S S S 5S * *** * S..

of the form X-C, where X is an atom of said semiconductor, C is a carbon atom of a molecule of said compound and the hyphen represents a covalent bond between said atoms. Such a link is called an "alkyl" link such that if X is silicon then the structure may be termed "alkylated silicon". In accordance with the present invention X is preferably selected from the Group IV elements of the Periodic Table of the Elements. Thus X may suitably be Silicon or Germanium for example.

Other known chemistries that are suitable for conjugating molecules to semiconductor particles of the type of concern in connection with the present invention include the following: * A link of the form X-O-C, where X is an atom of the semiconductor, 0 is an oxygen atom and the group -0-C constitutes a part of a molecule of the particular compound. The resultant link is called an "alkoxy" link and in the best mode contemplated X is silicon.

* A link of the form X-O-X-C, where X is an atom of the semiconductor, 0 is an oxygen atom and the group -O-x-c constitutes a part of a molecule of the particular compound.

Those skilled in the art will understand that it can also be expected that sometimes the particular molecules under consideration will adsorb onto the surface of the semiconductor nanoparticle. The terms used are physisorption when the adsorption does not involve a covalent bond (and is therefore weak) and chemisorption when a covalent bond is involved.

(iii) Coupling of Q-Si and Ag/Au In order to enhance the Raman signals there are various ways of incorporating Ag or Au on the Q-Si particles. The following are meant as examples and are not meant to limit * * S * S * S S S S S S S * S * S S S S 155 S S S S. S I 55 5 5 5 5 * * . S I S S S *.5 S **S the invention. This can be done simply by mixing the Q-Si particles with metal nanoparticles. The metal nanoparticles which are in close proximity to the semiconductor nanoparticle / conjugate then enhance the Raman signal of molecules in the vicinity of the semiconductor nanoparticle I conjugate. A second method that may be employed is to attach a semiconductor nanoparticle-DNA conjugate to a metal nanoparticle-DNA conjugate bearing a complementary DNA sequence using DNA hybridisation (Fig. 5). In this instance it is expected that any individual semiconductor nanoparticle would be conjugated to more than one DNA molecule, permitting free unhybridised DNA molecules to interact with the targeted biological system under study. Another method of attaching the metal nanoparticle to the semiconductor nanoparticle / conjugate is via covalent chemistry (Fig. 6), e.g. by a metal-sulphur bond between a thiol group naturally present in the biomolecule (cysteine residues in proteins) or deliberately introduced (5'-thiolated DNA).

To determine suitability of metals that may be used to enhance Raman spectroscopic signals by the SERS effect the following considerations should be taken into account.

When the complex frequency-dependent dielectric function of a metal particle satisfies a resonance condition: Re(c1) = where C1 is the complex dielectric function of the metal particle and c2 is the dielectric function of the surrounding medium, excitation of the surface plasmon by the incident light greatly enhances the local field experienced by molecules close to the particle surface. As will be appreciated by those skilled in the art the latter equation is for a spherical particle and the numerical factor of 2 would be different for other geometries, though the basic physics remains the same (Alan Campion and Patanjali Kambhampati, Surface-enhanced Raman * a * * S * S S S S S S S S S * S S S S *SS S S S S. S 5 IS 5 5 S S * .5 5 55* S. S 555 S SSS Scattering, Chemical Society Reviews, 1998, volume 27, p242) The coinage metals (mainly Ag, Au and Cu) and the alkali metals are suitable for SERS because the resonance condition is satisfied at the visible frequencies commonly used for Raman spectroscopy. Other metals have their surface plasmon resonances in different regions of the electromagnetic spectrum and can, in principle, support SERS at those frequencies. In addition, the imaginary part of the dielectric function (which measures losses in the solid) for the coinage and alkali metals is very small at the resonance frequency. Low loss materials sustain sharper and more intense resonances than those where scattering and other dissipative mechanisms are important.

To provide the desired SERS effect the metal particles must simply belocated at a close enough distance to the molecule from which a Raman signal is required.

* Introducing quantum dot probes into cells There are various methods known to those skilled in the art that can be used to introduce quantum dot sensors into cells. The following are given as examples and are not meant to be limiting: including microinjection, electroporation, fusion of quantum dots in vesicles, and chemical/biochemical techniques for disruption of the cell membrane. The fusion method involves utilization of cationic lipid mixtures and Q-Si/ micelles, which on attachment to negatively charged cell surfaces, undergo endocytosis or fuse with the endosome. A more venturesome strategy may make use of Q-Si-protein conjugates using polypeptides from organisms which have evolved to invade living cells without immediate damage to the cell. The process is mediated by defined invasin proteins that can alone confer the ability to internalise. The structures of these invasins are known which makes them highly amenable to further engineering for attachment on the surface of Q-Si. The Q-Si is thus internalized by a process which makes * a * . a * S * S * S I S I * * a a a S 155 5 S S *. a a a. S S S S a a a S S a I as a.as a S.' cells phagocytotic even if they do not normally show this behaviour. The Q-Si will then be transported into internal membrane compartments. Additional proteins on their surface may then direct them to their final destination and methods can be developed to enable the Q-Si to enter the cytoplasm. Here targeting signals adopted by bacterial toxins may be used, which have been shown to direct proteins to specific cellular compartments.

* Spectroscopic detection of hybridization and protein binding The luminescence of the Q-Si probes prepared as below is typically at about 670 nm and independent of the excitation wavelength/wavenuinber. Use of the 488 run line from an argon ion laser in a standard confocal Raman microscope allows collection of both the luminescence and Raman signals by a standard spectrograph as configured on such a microscope. Other lines from the argon ion laser, e.g., 514 nm and 457 run are also usable, though the 514 run line is closer to the emission and less of the Raman spectrum can be observed in the region of the spectrum free of luminescence.

Experimental: The following example is put forth so as to provide those skilled in the art with a detailed description of how to work the invention and make the quantum dot probes. In accordance with the method and principles of the present invention the method of enabling simultaneous detection of Raman and luminescence spectra from the same sample of a material composition to be investigated has been demonstrated using the following: (a) silicon nanoparticles as the luminescence label; with (b) a short strand of DNA attached as the biomolecule and co-deposited on a glass slide with a commercially available (Ag colloid 20 run diameter * * S * * * * S S * S S S S * * S S S S *SS S S S. S S 55 5 5 I S

S SI S S S I

S I.e 5 5.5 from BBI International, product code: EM.SC2O) silver (Ag) colloid to provide the SERS effect.

Example

Preparation of silicon nanoparticle-DNA conjugates: Q-Si-DNA as in Fig. 4.

A 1 x 1 cm2 piece of Si (p-type, boron-doped, 5-15)cm resistivity, oriented <100>) is cut from a wafer: this chip is then galvanostatically etched at 75 mA cm2 for 5 mm in an electrolyte consisting of 48% HF(aq) and ethanol in 1:1 ratio. The anodic etch forms a layer of luminescent porous silicon on the chip surface. The porous layer is dried under vacuum for lh on a grease-free glass vacuum line employing Young's taps.

Next, the surface of the porous silicon is chemically modified with a monolayer formed from a bifunctional molecule, dimethoxytrityl-undec-1-enol. This is achieved by ref luxing the chip for 8h in a 20 inN solution of dimethoxytrityl-undec-1-enol in toluene under nitrogen.

After the ref lux, the chip is washed with toluene and dried under vacuum for 1 h. This chip is then ready for automated solid-phase DNA synthesis.

Synthesizer Protocol: parameters set appropriate for 1 pmole column quantities, final DMT off. The synthesizer was an Applied Biosystems Expedite model with a column assembly modified in-house as described in Lie L. H. et al entitled "Immobilisation and synthesis of DNA on Si(111), nanocrystalline porous silicon and silicon nanoparticles" (The Royal Society of Chemistry 2003, Faraday Discuss., 2004, 125, 235-249).

Sequence synthesized:Q-Si-3' -G-C-G-T-A-C-T-A-T-C-A-G-T-C-A-G-A-T -5' * S S * S * S * S S S I S S *

S I S S I III S S S

S. S I IS 5 S S S * I I * S S S 5 555 5 555 After oligosynthesis, protecting groups were removed using gaseous methylamine for -20 mm. The chip is then washed with ethyl acetate (x3) to remove any residual material from the synthesis and protecting groups / methylamine.

The silicon nanoparticle-DNA conjugates (Q-Si-DNA) were then removed from chip surface by scraping the surface with Microlance 3 needle. The Q-Si-DNA can then be suspended in the solvent of choice, e.g., water, with sonication if required.

Preparation of silicon nanoparticle-DNA conjugates: Q-Si-DNA as in Fig. 5.

The Q-Si-DNA conjugates are prepared as above, but instead of mixing with bare Ag nanoparticles, they are mixed with Ag or Au nanoparticles bearing the complementary DNA strand that hybridises with that on the Q-Si-DNA.

Preparation of silicon nanoparticle-DNA conjugates: Q-Si-DNA as in Fig. 6.

As above, but the surface of the porous silicon is chemically modified with a monolayer formed from a thiol containing alkene, 11-undecene-1-triphenylmethanethiol.

This is achieved by ref luxing the chip under dry nitrogen with -3ml of 11-undecene--1-triphenylmethanethiol diluted in dry inesitylene (5% V/V) for 2 hours. After alkylation, The chips were then rinsed with dichloromethane, acetone and water and dried on filter paper. No DNA is synthesized on the surface and the Q-Si bearing thiol groups are released from the porous silicon by sonication after deprotection of the thiol.

Preparation of 11 -undecene-1 -triphenylmethanethiol (Cli -STr). Triphenylmethanethiol (2.06g, 7.4mmol) was dissolved in anhydrous THF (5Omls) and to this was added a suspension of NaH (0.18g, 7.4znmol) in anhydrous THF (lOmis). The mixture was allowed to stand for 10 minutes * . . p p pP * . * * S S S * S S P 5 SSS P 5 * S. I 55 5. S - * SP S S S I I. P *SS S S** and then 11-bromo-1-undecene (1.63m1, 7.4xnmol) dissolved in THF (lOmls) was added. The reaction was left for 2 hours to react, resulting in a cloudy, off-white solution.

Buchrier filtration yielded an off-white solution, the solvent was extracted using rotary evaporation to leave a yellow oil. Overnight vacuum drying gave off-white crystals which were further purified by column chromatography on silica eluting with chloroform. 54% yield. 1H NMR (300 MHz, CDC13): 6 7.65 (m, 6H, phenyls) 7.44 (m, 9H, phenyls) 6.04 (m, 1H, CH2CH), 5.17 (m, 2H, CH2CH), 2.4 (m, 2H, CH2SC), 2.26 (m, 2H, CH2CHCH2) 1.45 (m, 14H, CH2 alkyl chain) Deprotection of trityl-thiol monolayers on porous silicon.

Porous silicon modified with monolayers containing trityl-protected thiol groups was treated with a solution of Et3S1H (1%), tetrafluoroacetic acid (50%) and dichloromethane (49%) for 1 hour before rinsing with dichioromethane and water.

The Q-Si-thiol nanoparticles are prepared as above, but instead of mixing with bare Ag nanoparticles, they are mixed with Ag or Au nanoparticles bearing the required biological molecule, e.g., thiolated DNA.

Results Rainan spectroscopy of Q-Si-DNA + Ag prepared as in Fig. 4.

A 50 p1 sample of aqueous Q-Si-DNA was mixed with a 150 p1 portion of 20 mu diameter commercial Ag colloid, -in a Gilson pipette tip for Ca. 30 seconds (Ag colloid, a suspension of nanoparticles, 20 mm mean diameter from BBI International, product code: EM.SC2O). The pre-mixed sample was then deposited on a microscope cover-slip and allowed to air-dry. SERS/Ranian spectroscopy was carried out directly on this sample using the 488 mu line of an Argon ion laser in a confocal Raman microscope (Witec, * a e * * * S S S S I S* S * S I * P SI. * S S. I 5 55 5 a I a * a * * S * I *5 *SS I III CRN200, Ulm, Germany). The grating employed was 150 lines / mm and an integration time of is.

Fig. 7 shows actual spectra obtained from the following compositions: * Ag colloid; * Q-Si-DNA (Silicon based quantum dot conjugated with DNA); and * Q-Si-DNA-Ag colloid The spectra show the broad luminescence (704) of the silicon nanoparticles at a relative wavenurnber of 5500 cm' compared to the excitation wavelength of 488 nm from an argon-ion laser. (luminescence peak wavelength ca. 670 run). The sharp peaks (702 & 703) below 3000 cm' are Rainari features due to a combination of the vibration modes of the citrate stabiliser on the Ag colloid (features common to the green and black spectra), the silicon nanoparticle (sharp feature common to the green and red spectra at Ca.

515 cm1) and due to the DNA molecule bound to the silicon (majority of the Raman features). The wavenumber scale below is in relative cm1 with respect to the laser line used to excite the sample (488 run = 20,490 cm1).

Fig. 8. shows another spectrum of a sample, also prepared by the method illustrated in Fig. 4. and for which a large number of Raman features are visible from.fl the components: the first and second order Si bands at ca. 500 and 950 cm', modes due the DNA between 1000 and 1500 cm1, modes due to Si-H bonds on the silicon core surface (2100 cm'), modes due to the alkyl chain of the organic molecule (derived from undecenol) that connects the DNA to the Si core at 1470 cm1 and 3000 cm1, and, finally the intense orange luminescence of the Si core at 670 run.

Although the preferred embodiments of quantum dots and probes as configured in accordance with the present invention and the methods of use thereof have been * S S S a S S SI S S S *I.

a * S a P 555 I *S a a is S S * a * a a S VS* described in relation to applications in cell biology, molecular biology and medicine they are also considered to find application in a number of different or related technological fields. Thus for example the quantum dots may be used as stains or labels in forensic, security and/or a number of other applications. Furthermore the technology is suitable for various applications in the fields of general sensor technology and diagnostics technology.

References Akerman M. E. et al. Nanocrystal targeting in vivo.

PNAS 99 (2002) 12617-12621.

Brunchez Jr. M. et al. Semiconductor nanocrystals as fluorescent biological labels, Science 281 (1998) 2013-2015.

Cao Y. C. et al. Nanoparticles with raman spectroscopic fingerprints for DNA and RNA detection. Science 297 (2002) 1536-1540 Chan W. C. W. & Nie S. M. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science (1998) 281: 2016-2018.

Kneipp J. et al. Optical probes for biological applications based on surface-enhanced raman scattering from indocyanine green on gold nanoparticles. Anal. Chem. 77 (2005) 2381-2385.

Lars H. et al. Preparation and characterisation of luminescent alkylated-silicon quantum dots. Journal of Electroanalytical Chemistry, 538-539 (2002) pp183-190.

I I S I I

* III SI I IS.

S S S I I 555 1 *a 5 I 55 S I I I

IIS I

II * III I III Lidke D. S. et al. Quantum dot ligands provide new insights into erbE/HER receptor-mediated signal transduction. Nature Biotechnology 22 (2004) 198-203.

Lie L. H. et al. Irnmobilisation and synthesis of DNA on Si(111), nanocrystalline porous silicon and silicon nanoparticles. The Royal Society of Chemistry 2003, Faraday Discuss., (2004), 125, 235-249.

Mattoussi H. et al. Self-assembly of CdSe-ZnS quantum dots bioconjugates using an engineered recombinant protein. J. Am. Chem. Soc. 122 (2000) 12142-12150.

Mitchell G. P. et al. Programmed assembly of DNA functionalized quantum dots. J. Am. Chem. Soc. 121 (1999) 8122-8123.

Nie S. & Emory S. R. Probing single molecules and single nanoparticles by surface-enhanced raman scattering.

Science 275 (1997) 1102-1106.

Pathak S. et al. Hydroxylated quantum dots as luminescent probes for in situ hybridization. J. Am. Chem. Soc. 123 (2001) 4103-4104.

Phizicky E. M. & Fields S. Protein-protein interactions: methods for detection and analysis. Microbiology Reviews 59 (1995) 94-123.

Sukhanova A. et al. Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells.

Analytical Biochemistry 324 (2003) 60-67.

Wong X. et al. Immunofluorescent labelling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nature Biotechnology 21 (2003) 41-46.

* * * * a * 0 S S * * * S S S * S S I S *SS I I S

SS S S IS S S S S

* S S S S S S S* I 551 I *SS

Claims

Claims 1. A photoluminescent composition for use in proximity to a

moiety capable of generating a Raman signal in response to an incident radiation event, said composition comprising; at least one first material configured to emit a detectable luminescence signal when excited by selected incident radiation from an external source; and at least one second material adapted to augment or enhance detection of the Raman signal generated by said moiety, said second material being associated with said first material without significant occlusion thereof to permit luminescent emission therefrom, wherein said first material is selected to emit luminescence with an upper threshold wavenumber sufficiently less than the wavenurnber of the incident radiation to allow the luminescence and the Raman signal to be detectable substantially simultaneously.
2. A photoluminescent composition as in Claim 1 wherein the first material is a semiconductor nanoparticle.
3. A photoluminescent composition as in Claims 1 or 2 wherein the second material is a metallic material.
4. A photoluminescent composition as in any of the previous Claims wherein the wavenumber of the upper threshold of said luminescence signal is at least 500 cm' less than the wavenumber of the incident radiation.

* S. . * S * S S S a a S S * * S S I a. a * . SI S S IS I * I
5 * I I S I I S a* I 115 5 III 5. A photoluminescent composition as in any of the previous Claims wherein said Rarnan-based signal occurs at a wavenuxnber in the range of 500 cm' to 3500 cm1 from the wavenumber of the incident radiation.
6. A photoluminescent composition as in any of Claims 2-wherein said semiconductor nanoparticle comprises an indirect bandgap semiconductor nanoparticle.
7. A photoluminescent composition as in any of Claims 2- 6 wherein said semiconductor nanoparticle comprises at least one atom of an element selected from Group IV of The Periodic Table of The Elements.
8. A photoluminescent composition as in Claim 7 wherein said Group IV element is selected from the set of elements comprising Silicon (Si), Germanium (Ge) and their alloys.

9. A photoluminescent composition as in any of previous 3-8 wherein said at least one metallic material comprises an element selected from the set comprising Silver (Ag), Gold (Au) and Copper (Cu).

10. A photoluzninescent composition as in any of the previous Claims wherein said photoluminescent composition further comprises a molecular compound.

11. A photoluminescent composition as in Claim 10 wherein said molecular compound is comprised of a molecule that comprises at least one carbon atom.

12. A photoluminescent composition as in Claims 10 to 11 wherein a molecule of said molecular compound is attached to said semiconductor nanoparticle via a covalent bond.

* S S S S * S S I S S S * S * S I S * * III S S S *S S S * * S I S

S S S S S S S

SI S SI. S III 13. A photoluminescent composition as in Claims 10 to 12 wherein said molecular compound is covalently bonded to said semiconductor via a link comprising said at least one carbon atom.

14. A photoluminescent composition as in Claim 12 wherein said link comprises said at least one carbon atom is of the form X-C, where X is an atom of said semiconductor, and C is a carbon atom of a molecule of said compound.

15. A photoluminescent composition as in Claims 10 to 12 wherein said link comprising said at least one carbon atom is of the form X-0-C, where X is an atom of said semiconductor, 0 is an oxygen atom and the group -0-C constitutes a part of a molecule of said compound.

16. A photoluminescent composition as in Claims 10 to 12 wherein said link comprising said at least one carbon atom is of the form X-0-X-C, where Xis an atom of said semiconductor, 0 is an oxygen atom and the group -0-X-C constitutes a part of a molecule of said compound.

17. A photoluminescent composition as in Claims 10 to 16 wherein said molecular compound has an affinity for a biological target.

18. A photoluminescent composition as in Claims 10 to 17 wherein said molecular compound is a biological compound.

19. A photoluminescent composition as in Claim 18 wherein said biological compound is selected from the set comprising: a peptide; a nucleic acid; a carbohydrate; a protein; an enzyme; an antibody; and an oligonucleotide.

* S S S S * S S S S S S S S a * S I I S.55 S S SI S S 55 5 S I S

S S S S S U S

S. * 555 * 555 20. A photoluminescent composition as in Claims 18 or 19 wherein said biological compound is selected from the set comprising: a ribonucleotide and derivatives thereof; a deoxyribonucleotide and derivatives thereof; and a dideoxyribonucleotide and derivatives thereof.

21. A method of performing spectroscopic analysis comprising the steps; selecting a composition according to Claims 1 to 20; deploying the composition to an area of interest; exciting said composition with by incident radiation from an external source; detecting the radiation spectrum emitted 22. A method of performing spectroscopic analysis according to Claim 21 further comprising the step; analysing the radiation spectrum emitted to obtain further information about the area of interest.

23. A method of performing spectroscopic analysis according to Claim 21 or 22 wherein when detecting the radiation spectrum both a luminescence signal and a Raman signal are detected.

24. A method of performing spectroscopic analysis according to Claims 21 to 23 wherein said incident radiation comprises light from any appropriate source.

* S S S S * S S S * S S * S * * S S S * Se. S S S S. S S *S S S S S

S S S S S S S

*5 S SS* S *SS 25. A method of performing spectroscopic analysis according to Claims 21 to 24 wherein said incident radiation comprises an Ar (Argon) ion laser.

* S S S S * S S S S * S S S * S S S S S 555 S S S * S S *S S S S 5

S S S S S S S

5* 5 555 S 5.5 Amendments to the claims have been filed as follows Claims 1. A photoluminescent composition for use in proximity to a moiety capable of generating a Raman signal in response to an incident radiation event, said composition comprising; at least one first material configured to emit a detectable luminescence signal when excited by selected incident radiation from an external source; and at least one second material adapted to augment or enhance detection of the Raman signal generated by said moiety, said second material being associated with said first material without significant occlusion thereof :. to permit luminescent emission therefrom, wherein * *** said first material is selected to emit luminescence with an upper threshold wavenumber sufficiently less than the wavenumber of the incident radiation to * allow the luminescence and the Raman signal to be **.

detectable substantially simultaneously. * * ****

2. A photoluminescent composition as in Claim 1 wherein * 1 the first material is a semiconductor nanoparticle.

3. A photoluminescent composition as in Claims 1 or 2 wherein the second material is a metallic material.

4. A photoluminescent composition as in any of the previous Claims wherein the wavenumber of the upper threshold of said luminescence signal is at least 500 cm1 less than the wavenumber of the incident radiation.

L

5. A photoluminescent composition as in any of the previous Claims wherein said Raman-based signal occurs at a wavenumber in the range of 500 cm' to 3500 cm1 from the wavenuinber of the incident radiation.

6. A photoluminescent composition as in any of Claim 2 wherein said semiconductor nanoparticle comprises an indirect bandgap semiconductor nanoparticle.

7. A photoluminescent composition as in any of Claim 2 wherein said semiconductor nanoparticle comprises at least one atom of an element selected from Group IV of The Periodic Table of The Elements.

8. A photoluminescent composition as in Claim 7 wherein said Group IV element is selected from the set of elements comprising Silicon (Si), Germanium (Ge) and :. their alloys. * 20 ****

Si....
9. A photoluminescent composition as in Claim 3 wherein said at least one metallic material comprises an element selected from the set comprising Silver (Ag), Gold (Au) and Copper (Cu). * *

10. A photoluminescerit composition as in any of the previous Claims wherein said photoluminescent composition further comprises a molecular compound.

11. A photoluminescent composition as in Claim 10 wherein said molecular compound is comprised of a molecule that comprises at least one carbon atom.

12. A photoluminescent composition as in Claims 10 to 11 wherein a molecule of said molecular compound is attached to a semiconductor nanoparticle via a covalent bond.

13. A photoluminescent composition as in Claims 10 to 12 wherein said molecular compound is covalently bonded to a semiconductor via a link comprising at least one carbon atom.

14. A photoluminescent composition as in Claim 13 wherein said link comprising at least one carbon atom is of the form X-C, where X is an atom of a semiconductor, and C is a carbon atom of a molecule of said compound.

15. A photoluminescent composition as in Claim 13 wherein said link comprising at least one carbon atom is of the form X-O-C, where X is an atom of a semiconductor, 0 is an oxygen atom and the group -0-C constitutes a part of a molecule of said compound.

16. A photoluminescent composition as in Claim 13 wherein said link comprising at least one carbon atom is of the form X-O-X-C, where Xis an atom of a semiconductor, 0 is an oxygen atom and the group -0-X-C constitutes a part of a molecule of said S. * compound. *.* *

 25 17. A photoluminescent composition as in Claims 10 **** to 16 wherein said molecular compound has an affinity * * S * 1 for a biological target.

18. A photoluminescent composition as in Claims 10 to 17 wherein said molecular compound is a biological compound.

19. A photoluminescent composition as in Claim 18 wherein said biological compound is selected from the set comprising: a peptide; a nucleic acid; a carbohydrate; a protein; an enzyme; an antibody; and an oligonucleotide. (f-L

20. A photoluminescent composition as in Claims 18 or 19 wherein said biological compound is selected from the set comprising: a ribonucleotide and derivatives thereof; a deoxyribonucleotide and derivatives thereof; and a dideoxyribonucleotide and derivatives thereof.

21. A method of performing spectroscopic analysis comprising the steps; selecting a composition according to Claims 1 to 20; deploying the composition to an area of interest; exciting said composition with by incident radiation from an external source; detecting the radiation spectrum emitted * 20 1**'s 22. A method of performing spectroscopic analysis S... according to Claim 21 further comprising the step; *5 analysing the radiation spectrum emitted to obtain further information about the area of interest. S...

23. A method of performing spectroscopic analysis according to Claim 21 or 22 wherein when detecting the radiation spectrum both a luminescence signal and a Rarnan signal are detected.

24. A method of performing spectroscopic analysis according to Claims 21 to 23 wherein said incident radiation comprises light from any appropriate source. 9;,

25. A method of performing spectroscopic analysis according to Claims 21 to 24 wherein said incident radiation comprises an Ar (Argon) ion laser. I. * S a S.. *5*S * * Seas

S S...

I *5* S... * S I.., S. II S * *

I I