WO2007125300A1 - Points quantiques permettant de detecter des signaux de luminescence en meme temps que des signaux raman - Google Patents

Points quantiques permettant de detecter des signaux de luminescence en meme temps que des signaux raman Download PDF

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WO2007125300A1
WO2007125300A1 PCT/GB2007/001489 GB2007001489W WO2007125300A1 WO 2007125300 A1 WO2007125300 A1 WO 2007125300A1 GB 2007001489 W GB2007001489 W GB 2007001489W WO 2007125300 A1 WO2007125300 A1 WO 2007125300A1
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raman
luminescence
photoluminescent composition
composition
semiconductor
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Ben Horrocks
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University Of Newcastle Upon Tyne
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • 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.
  • 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) .
  • probe 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.
  • probe There is a need in biological research for methods to label molecules such as proteins and nucleic acids in order to track these 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.
  • luminescent probes e.g., organic dyes (examples include fluorescein, rhodamine and their derivatives) and green fluorescent protein (GFP, a naturally occurring luminescent protein) .
  • organic dyes examples include fluorescein, rhodamine and their derivatives
  • GFP green fluorescent 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.
  • all luminescence probes suffer from limitations arising from the broad and relatively featureless nature of luminescence spectra.
  • Quantum dots are semiconductor nanoparticles with physical dimensions less than the radius of the bulk exciton.
  • the resulting quantum confinement effect endows the nanoparticles with unique optical properties that offer several distinct advantages over the conventional luminescent dyes and stains .
  • 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..
  • 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.
  • quantum dots have been tested in a range of biotechnological applications including DNA array technology, immunofluorescence, cell and animal biology.
  • the coupling of quantum dots to proteins 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 . , 2004; Wu et al . , 2003; Akerman et al .
  • 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 characterised and most widely used in applications are those based on heavy metal chalcogenides , especially cadmium selenide (CdSe).
  • CdSe cadmium selenide
  • the luminescence of bare CdSe particles is strongly influenced by chemical reactions at the particle surface, e.g., loss of Cd 2+ 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 / 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.
  • silicon is relatively inert and therefore less toxic to cells than competing technologies, e.g., cadmium selenide nanoparticles.
  • 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.
  • PCT international patent
  • WO2004108902 in the name of Visen Medical Inc.
  • Other examples include the paper by Lars H. Lie et al , 2002 and the paper by Lars H. Lie et al, 2004.
  • 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 immunoassays to determine the identity of unknown analytes .
  • 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.
  • Raman scattering is one such vibrational spectroscopic technique which is capable of providing detailed information on the chemical structure of unknown substances.
  • 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° - 10 1 cm “1 ) and show many lines that can be assigned to particular groups of atoms, unlike the broad, featureless single band in a typical luminescence spectrum, they can be used to "fingerprint" a molecule or deduce information about the chemical composition of an unknown species .
  • Raman 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.
  • SERS surface-enhanced Raman scattering
  • 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 nanoparticles, electrodes or evaporated films of these metals.
  • quantum dot and semiconductor nanoparticle should be considered interchangeable .
  • 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 nanoparticle 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 Raman 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 behaviour and associated chemical interactions in a medium can be obtained simultaneously or substantially simultaneously from a luminescence signal and a Raman- 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 Raman- 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.
  • 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.
  • the first material is a semiconductor nanoparticle .
  • the second material is a metallic material.
  • the second material does not occlude the first material such that both luminescence of said semiconductor nanoparticle and the Raman signal remain detectable.
  • the wavenumber of the upper threshold of said luminescence signal is at least 500 cm "1 less than the wavenumber of the incident radiation.
  • said Raman-based signal occurs at a wavenumber in the range of 500 cm “1 to 3500 cm “1 from the wavenumber of the incident radiation.
  • said semiconductor nanoparticle comprises an indirect bandgap semiconductor nanoparticle.
  • said semiconductor nanoparticle comprises at least one atom of an element selected from Group IV of The Periodic Table of The Elements.
  • Group IV element is selected from the set of elements comprising Silicon (Si) , Germanium (Ge) and their alloys .
  • said at least one. metallic material comprises an element selected from the set comprising Silver (Ag) , Gold (Au) and Copper (Cu) .
  • said photoluminescent composition further comprises an additional compound of interest.
  • the additional compound of interest is a molecular compound.
  • said molecular compound is comprised of a molecule that comprises at least one carbon atom.
  • a molecule of said molecular compound is attached to said semiconductor nanoparticle via a covalent bond.
  • said molecular compound is covalently bonded to said semiconductor via a link comprising said at least one carbon atom.
  • 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.
  • said link comprising said at least one carbon atom is of the form X-O-C, where X is an atom of said semiconductor, O is an oxygen atom and the group -0-C constitutes a part of a molecule of said compound.
  • said link comprising said at least one carbon atom is of the form X-O-X-C, where X is 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.
  • said molecular compound has an affinity for a biological target .
  • said molecular compound is a biological compound.
  • 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.
  • 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.
  • the method further comprises the step;
  • both a luminescence signal and a Raman signal are detected.
  • the incident radiation comprises light from any appropriate source.
  • said incident radiation comprises an Ar ion laser.
  • 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 Raman spectra from a quantum dot molecular conjugate;
  • the absorbance spectrum 101 is that for a silicon-based quantum dot;
  • Fig. 2A schematically illustrates fabrication of a preferred embodiment of a quantum dot probe as configured in accordance with the present invention
  • 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);
  • Fig. 2B schematically illustrates the process of obtaining spectroscopic measurements using a quantum dot probe of the type illustrated in Figs. 1 and 2A;
  • 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.
  • Fig. 4 schematically illustrates the combination of Ag/Au nanoparticles 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;
  • 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 nanoparticle to Q-Si-DNA using DNA hybridization;
  • 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 nanoparticle to a thiol-terminated Q-Si particle;
  • 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 nm diameter Ag colloid.
  • the 488 nm line of an argon ion laser was used to provide the excitation and the spectrum was acquired using a Witec (Ulm, Germany) CRM200 confocal Raman microscope; and
  • Fig. 8. shows another spectrum of Q-Si-DNA mixed with 20 nm diameter Ag colloid in which various Raman bands have been assigned; and Fig. 9. shows a confocal luminescence image of HeLa cells which have been exposed to silicon quantum dots.
  • 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”.
  • Q-Si refers to a quantum dot comprising the element Silicon.
  • Q-Si-DNA refers to a quantum dot probe wherein the quantum dot comprises silicon and such that the dot is attached to the biological macromolecule deoxyribonucleic acid (DNA) .
  • 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 vibration ' ) .
  • the indirect band gap is about 1.1 eV, but there is also a direct gap at about
  • 1.5 eV corresponds to a Stokes shift of 12,000 cm “1 .
  • the Stokes shift may be smaller because the excitation photon energy is usually fixed by the available lasers.
  • the photon energy of the excitation laser is therefore 2.55 eV and the observed Stokes shift is about 5800 cm “1 , sufficient to detect the full range of Raman signals of chemical interest ( ⁇ 3500 cm " x ).
  • 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.
  • Bio 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 and may also be used to drive the conjugate to a particular location in the cell.
  • Short lengths of single stranded DNA oligonucleotides - 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 characterised 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 immobilise pre-formed oligonucleotides on the particles.
  • 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.
  • 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 dm "3 sodium cyanoborohydride (NaCNBH 3 ) 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.
  • 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.
  • 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
  • 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 relative position of the Raman peaks . to the incident light which ' is fixed.
  • 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.
  • suitable metal nanoparticles 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 Raman 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 [r/ (r+d) ] 12 .
  • SERS Surface Enhanced Raman Spectroscopy
  • 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 sortie other Raman spectroscopy effect such as, for example, anti-Stokes Raman spectroscopy.
  • Anti-Stokes Raman spectroscopy can usefully be employed in a given system since it further separated the Raman signal from the fluorescence signal.
  • another Raman 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.
  • 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.
  • the Stokes shift is greater than 500 cm-1, then Raman signals at shifts ⁇ 500 cm-1 may be detectable; this holds irrespective of the excitation wavelength.
  • Useful Raman signals from chemical groups lie in the range 500-3500 cm-1, the upper limit of the range therefore defines the minimum desirable Stokes shift and the lower limit defines the minimum usable Stokes shift.
  • the desirable absorption 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.
  • 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.
  • a typical wavenumber of the emission maximum (104) is 15,380 cm “1 (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.
  • luminescence signal 104 centres on 15,380 cm "1 (650 nm) in the example.
  • the 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 wavenumbers) is termed the Stokes 1 shift.
  • 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 Raman 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 Raman 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 GaAs. From the threshold point 106 the spectrum 109 shows a rapidly increasing absorbance with respect to increasing wavenumber and therefore the illustrated Raman 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 wavenumbers near the position of the excitation light in the spectrum.
  • 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 threshold 106.
  • the second spectrum preferably a Raman spectrum
  • the luminescence spectrum must occur at sufficiently widely separated wavelengths for the two effects to be detectable simultaneously.
  • a Raman 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.
  • 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 Raman spectrum preferably occur at least 500 cm "1 from the threshold wavelength 106.
  • Raman 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.
  • C-H stretching vibrations give rise to Raman signals at a shift of 3000 cm “1 and C-C or C-N at shifts of approximately 1000-1200 cm “1 .
  • 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 “1 to 3500 cm “ 1 from the incident light.
  • the wave number for the luminescence signal should be at least 500 cm "1 less than the wave number of the incident light.
  • the luminescence is, in accordance with the best mode contemplated, required to occur at wave numbers that are lower than the incident light by ah amount greater than or equal to at least 500 wavenumbers .
  • 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.
  • conjugate 203 comprises an alkyl-modified 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 run) 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 dsDNA and small ligands or antibodies for protein capture.
  • 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.
  • 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) .
  • 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 Raman signal to be detected.
  • 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-molecule 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-l-ol, which may be used to start oligodeoxynucleotide synthesis at porous silicon (301).
  • functionalisation of suspended nanoparticles is not straightforward.
  • 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.
  • 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 reflux 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.
  • 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.
  • 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 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” .
  • X is preferably selected from the Group IV elements of the Periodic Table of the Elements.
  • X may suitably be Silicon or Germanium for example.
  • the resultant ' link is called an "alkoxy" link and in the best mode contemplated X is silicon.
  • 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).
  • 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 1 different regions of the electromagnetic spectrum and can, in principle, support SERS at those frequencies.
  • 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 be located at a close enough distance to the molecule from which a Raman signal is required.
  • the luminescence of the Q-Si probes prepared as below is typically at about 670 nm and independent of the excitation wavelength/wavenumber .
  • Use of the 488 nm 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 nm are also usable, though the 514 nm line is closer to the emission and less of the Raman spectrum can be observed in the region of the spectrum free of luminescence.
  • a 1 x 1 cm* 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 cm "2 for 5 min 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 Ih on a grease-free glass vacuum line employing Young's taps.
  • 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 refluxing the chip for 8h in a 20 mM solution of dimethoxytrityl-undec-1-enol in toluene under nitrogen. After the reflux, 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 ⁇ mole 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(IIl), nanocrystalline porous silicon and silicon nanopartides” (The Royal Society of Chemistry 2003, Faraday Discuss., 2004, 125, 235-249) .
  • the silicon nanoparticle-DNA conjugates 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.
  • 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.
  • the surface of the porous silicon is chemically modified with a monolayer formed from a thiol containing alkene, 11-undecene-l-triphenylmethanethiol .
  • This is achieved by refluxing the chip under dry nitrogen with -3ml of 11-undecene-l-triphenylmethanethiol diluted in dry mesitylene (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.
  • Porous silicon modified with monolayers containing trityl- protected thiol groups was treated with a solution of Et 3 SiH (1%) , tetrafluoroacetic acid (50%) and dichloromethane (49%) for 1 hour before rinsing with dichloromethane 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.
  • a 50 ⁇ l sample of aqueous Q-Si-DNA was mixed with a 150 ⁇ l portion of 20 nr ⁇ diameter commercial Ag colloid, in a Gilson® pipette tip for ca. 30 seconds (Ag colloid, a suspension of nanoparticles, 20 nm mean diameter from BBI International, product code: EM.SC20).
  • the pre-mixed sample was then deposited on a microscope cover-slip and allowed to air-dry.
  • SERS/Raman spectroscopy was carried out directly on this sample using the 488 nm line of an Argon ion laser in a confocal Raman microscope (Witec, CRM200 , Ulna, Germany) .
  • the grating employed was 150 lines / mm and an integration time of Is .
  • Fig . 7 shows actual spectra obtained from the following compositions :
  • the spectra show the broad luminescence (704) of the silicon nanoparticles at a relative wavenumber of 5500 cm “1 compared to the excitation wavelength of 488 nm from an argon-ion laser. (luminescence peak wavelength ca. 670 nm) .
  • the sharp peaks (702 & 703) below 3000 cm “1 are Raman 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 cm "1 ) and due to the DNA molecule bound to the silicon (majority of the Raman features) .
  • 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 all the components: the first and second order Si bands at ca. 500 and 950 cm “1 , modes due the DNA between 1000 and 1500 cm “1 , modes due to Si-H bonds on the silicon core surface (2100 cm “1 ) , modes due to the alkyl chain of the organic molecule (derived from undecenol) that connects the DNA to the Si core at 1470 cm “1 and 3000 cm “1 , and, finally the intense orange luminescence of the Si core at 670 nm.
  • Fig. 9 Confocal luminescence image of HeLa cells which have been exposed to silicon quantum dots in culture medium for 1.5 h (10 microlitres of QSi/ether 2 mL of culture medium) .
  • the right-hand image is the normal optical image with a scale bar corresponding to 21.5 microns and 5 HeLa cells visible in the field of view.
  • the left-hand image is the luminescence image collected in confocal mode and using the 488 nm line of an argon ion laser as excitation source.
  • the wavelength of maximum emission is 650 nm and the image shows that the particles are capable of penetrating the cell membrane and entering the cytosol where they have a slight tendency to collect around the internal membranes and the cell ' nucleus (central bright red spots) .
  • no toxic effects of the silicon quantum dots are observed.
  • quantum dots and probes as configured in accordance with the present invention and the methods of use thereof have been 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.
  • the quantum dots may be used as stains or labels in forensic, security and/or a number of other applications.
  • the technology is suitable for various applications in the fields of general sensor technology and diagnostics technology.

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Abstract

Composition formant une sonde à point quantique comprenant une nanoparticule en semi-conducteur ou une nanoparticule en semi-conducteur attachée à un conjugué moléculaire chimique ou biologique, ledit semi-conducteur étant choisi pour émettre un signal de photoluminescence quand il est excité par un rayonnement incident provenant d'une source d'énergie externe ; composition caractérisée en ce que la nanoparticule en semi-conducteur est choisie parce qu'elle présente un décalage de Stokes important tel que le signal de luminescence a un nombre d'onde de seuil supérieur qui est notablement inférieur au nombre d'onde du rayonnement incident et en ce que la configuration est telle que cette nanoparticule est située à proximité d'une nanoparticule métallique pour permettre un renforcement et une détection simultanée d'un second signal, de préférence un spectre Raman qui est caractéristique des molécules attachées et/ou des molécules avec lesquelles elle interagit, ou à proximité de la sonde à point quantique, qui est détectable essentiellement en même temps que ledit signal de luminescence.
PCT/GB2007/001489 2006-04-26 2007-04-24 Points quantiques permettant de detecter des signaux de luminescence en meme temps que des signaux raman WO2007125300A1 (fr)

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EP2604168A2 (fr) * 2010-08-11 2013-06-19 SNU R&DB Foundation Procédé de détection simultanée de signaux de fluorescence et raman pour plusieurs cibles de signal de fluorescence et raman, et appareil d'imagerie médicale pour la détection simultanée de plusieurs cibles en utilisant le procédé
EP2604168A4 (fr) * 2010-08-11 2014-11-26 Snu R&Db Foundation Procédé de détection simultanée de signaux de fluorescence et raman pour plusieurs cibles de signal de fluorescence et raman, et appareil d'imagerie médicale pour la détection simultanée de plusieurs cibles en utilisant le procédé
US9833145B2 (en) 2010-08-11 2017-12-05 Snu R&Db Foundation Method for simultaneously detecting fluorescence and raman signals for multiple fluorescence and raman signal targets, and medical imaging device for simultaneously detecting multiple targets using the method
US9547014B2 (en) 2011-06-10 2017-01-17 Cornell University Immobilized protein system for rapid and enhanced multiplexed diagnostics
US11549953B2 (en) 2011-06-10 2023-01-10 Cornell University Immobilized protein system for rapid and enhanced multiplexed diagnostics
CN105548130A (zh) * 2016-02-29 2016-05-04 福州大学 一种用于钴离子检测的荧光传感器及其应用方法
CN109694340A (zh) * 2017-10-20 2019-04-30 Tcl集团股份有限公司 表面配体、量子点及其制备方法
WO2021155375A1 (fr) * 2020-01-31 2021-08-05 Cytoveris Inc. Appareil et méthode de détection et de traitement de tissu cancéreux par spectroscopie raman et hyperthermie

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