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WO2005107404A2 - Procedes et systemes d’amelioration nanoparticulaire de signaux - Google Patents

Procedes et systemes d’amelioration nanoparticulaire de signaux Download PDF

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Publication number
WO2005107404A2
WO2005107404A2 PCT/US2005/015344 US2005015344W WO2005107404A2 WO 2005107404 A2 WO2005107404 A2 WO 2005107404A2 US 2005015344 W US2005015344 W US 2005015344W WO 2005107404 A2 WO2005107404 A2 WO 2005107404A2
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Prior art keywords
solution
detecting molecule
molecule
reporting
reporting entity
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PCT/US2005/015344
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English (en)
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WO2005107404A3 (fr
Inventor
Samer Al-Murrani
Stephen J. Fonash
Matthew R. Henry
Ali Kaan Kalkan
Daniel Krissinger
Terry Rager
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The Penn State Research Foundation
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Publication of WO2005107404A2 publication Critical patent/WO2005107404A2/fr
Publication of WO2005107404A3 publication Critical patent/WO2005107404A3/fr

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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
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • nucleic acids DNA or RNA
  • Many diagnostic tests are based upon recognizing specific genetic sequences, and cloning. Probes with reporting entities are typically used in such tests and one of the most commonly applied reporting entities utilizes fluorescence.
  • DNA Microarrays whether they are glass, plastic, membrane or Affymetrix® type arrays all use the same basic principal where a DNA sequence (cDNA or oligonucleotide) that corresponds to a particular gene is immobilized on a solid surface support.
  • a DNA sequence cDNA or oligonucleotide
  • an EST- expressed sequence tag the end result is a surface that contains thousands of sequences, each corresponding to a unique gene, that form an "array”.
  • RNA samples are taken from all patients and the white blood cells are isolated and total RNA is extracted using standard protocols.
  • the RNA is first tested for quality and quantity and then it is used in a reaction to produce cDNA or cRNA (for Affymetrix® chips, for example).
  • the resultant cDNA or cRNA sample is also checked for quality and quantity and used in a labeling reaction.
  • the labeling reactions will differ based on the type of array used and may be dual color fluorescent-based (glass slide arrays), single color biotin and avidin-based (such as, for example, Affymetrix® chips) or radioactivity-based (plastic and membrane arrays).
  • the arrays or chips are hybridized to the array or chip, the arrays or chips are. then washed and scanned, light signals are generated and quantified based on how much complementary labeled DNA or RNA present in the probe sample bound to each spot on the microarray.
  • the image file generated from each scanned array is then used to convert the signal intensities of each individual spot to numbers using specialized software supplied with the scanners and a data file is produced for each array that gives the information pertaining to how much signal was detected from each spot on the array, including levels of local background and other information.
  • each of the samples that are being compared are labeled with a different fluorophor each with a different emission wavelength (e.g., Cy3 and Cy5 or Alexa® 555 and Alexa® 645).
  • a different fluorophor each with a different emission wavelength (e.g., Cy3 and Cy5 or Alexa® 555 and Alexa® 645).
  • the difference between the red channel fluorescence (Alexa® 645 or Cy5) and the green channel fluorescence (Alexa® 555 or Cy3 ) the amount of a given gene transcript present in the mRNA from the non-responder leukemic patient group relative to the amount of that same gene transcript present in the mRNA from the responder leukemic patient group can be inferred.
  • Microarrays allow scientists to monitor the transcription, or expression levels, of thousands of genes in parallel fashion. Although this optical signal technology has revolutionized biological science and the way many experiments are conducted, it is important to note that its sensitivity could be enhanced further. With the current technology only genes that are present at medium or high levels of expression are detectible simply because these are the genes that have enough transcripts present at any given time to reach the signal detection threshold of most commercially available scanners. This means that genes that are expressed at low levels will go undetected; therefore, blinding researchers to what could be major contributors to the phenotype under study.
  • RNA that is available from different sources is insufficient for microarray analysis, such as when the use of Laser Capture Microdissection (LCM) is necessary, or when the source of RNA is a surgical biopsy.
  • LCD Laser Capture Microdissection
  • users are forced to enzymatically amplify their RNA, which could lead to misrepresentation of the mRNAs present in the sample due to preferential transcription of certain target sequences through mechanisms that are not well understood.
  • NPs nanoparticles
  • SPR Surface Plas on Resonance
  • metal nanoparticles are used to enhance the fluorescence of fluorophores by means of adsorption or attachment of such fluorophores onto metal nanoparticle-coated or nanotextured-metal surfaces.
  • This approach requires manufacturing of pre-coated or pre-textured surfaces. Since optical signal quenching can result if a fluorophore is too close to a nanopartice, a spacer layer is used to prevent quenching of fluorescence by the nanoparticles.
  • the adhesion of the metal to the underlying substrate as well as to the probed bio-molecules must be strong enough to avoid detachment of molecules/nanoparticles as well as to avoid nanoparticle aggregation during chemical processes such as the hybridization or the washing step in DNA microarrays. Furthermore, for larger molecules like DNA or proteins, the enhancement in fluorescence by this approach will be significant only at the point of contact on the substrate surface. These problems limit the use of nanoparticle-coated or metal-textured surfaces. [0013] Therefore, a simple method for increasing the sensitivity of the microarray assays which enhances the detectable optical signal generated from each spot on the array and which does not require the NPs to be attached or adhered to a surface is needed. Such a method would be of great value.
  • the system of this invention includes one or more nanoparticles, one or more reporting entities, and, a molecule probe (also referred to as a detecting molecule) solution into which the reporting entities and the nanoparticles are incorporated; whereby the nanoparticles enhance a sensing function due to the reporting entities.
  • exemplary molecule probe solutions include, but are not limited to, DNA, RNA, or proteins in a solution.
  • Figure 1 (top panel) is a pictorial representation of the fluorescence observed in a conventional application;
  • Figure 1 (middle panel) is a pictorial representation of the fluorescence enhancement observed with the use of 20 nm diameter Au particles added to the solution;
  • Figure 1 is another pictorial representation of the fluorescence enhancement observed with the use of 20 nm diameter Au particles added to the solution;
  • Figure 2 is another pictorial representation of the fluorescence observed with the use of Au and Ag nanoparticles of various diameters added to the solution either separately or in combination;
  • Figure 3 is a pictorial representation of the fluorescence response obtained using fluorescently labeled Arabidopsis cDNA to probe a human oligonucleotide array;
  • Figure 4 is a schematic graphical representation of the reaction mechanism for a reactive functionalized nanoparticle
  • Figure 5 is a pictorial representation of the fluorescence enhancement observed with the use of non- reactive functionalized particles
  • Figure 5 is a pictorial representation of the fluorescence enhancement observed with the use of reactive functionalized particles of the same size as used in Fig. 5 (top panels);
  • Figure 6 is a schematic pictorial representation of the reactive functionalized gold to fluorophore distance;
  • Figure 7 is another pictorial representation of the fluorescence enhancement observed with the use of reactive functionalized particles;
  • Figure 8 is another pictorial representation of the results of Fig. 7;
  • Figure 9 is another pictorial representation of the results of Fig. 7;
  • Figure 10 is a schematic pictorial representation of dUTP with two acceptor sites.
  • RNA DNA
  • antibodies enzymes, factors, cell membrane receptors, proteins or peptides.
  • the molecule under test can be in active or inactive form.
  • An "active form” molecule is in a form that can perform a biological function.
  • An “inactive form” molecule is one that cannot perform a biological function. Usually, it can be processed either naturally or synthetically in order for the molecule to perform a biological function.
  • test molecules include, but are not limited to, nucleic acids, aromatic carbon ring structures, NADH, FAD, amino acids, carbohydrates, steroids, flavins, proteins, DNA, RNA, oligonucleotides, peptide nucleic acids, fatty acids, sugar groups such as glucose etc., vitamins, cofactors, purines, pyrimidines, formycin, lipids, phytochrome, phytofluor, peptides, lipids, antibodies and phycobiliproptein.
  • the metal nanoparticles used in the present invention can be spheroid, ellipsoid, or of any other geometry.
  • Exemplary metals include, but are not limited to, rhenium, ruthenium, rhodium, palladium, silver, copper, osmium, iridium, platinum, and gold and combinations thereof.
  • the nanoparticles used in the invention are referred to as metal nanoparticles, nanoparticles of any composition having a conductivity that will support the generation of surface plasmons (and their fields) such that signals from a reporting entity are enhanced can be used.
  • nanoparticles refers to colloidal nanoparticles, non-colloidal nanoparticles, capped/terminated (e.g., citrate coated) nanoparticles, nanoparticles with organic groups attached to their outer surface for a bonding or spacing role (also referred herein as functionalized reactive nanoparticles) and other nanoparticles.
  • Fluorophores are examples of reporting entities.
  • Reporting entity refers to compounds or molecules (not genes) used to generate a labeling signal.
  • fluorophore means any substance that emits electromagnetic energy (light) at a specific wavelength (emission wavelength) when the substance is illuminated by radiation of a different wavelength (excitation wavelength).
  • Extrinsic fluorophores refers to structures where fluorophores are bound to another substance.
  • Intrinsic fluorophores refer to substances that are fluorophores themselves.
  • fluorophores include but are not limited to Alexa Fluor® 350, dansyl Chloride (DNS-C1), 5- (iodoacetamida) fluoroscein (5-IAF); fluoroscein 5- isothiocyanate (FITC), tetramethylrhodamine 5-(and 6- )isothiocyanate (TRITC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 7-nitrobenzo-2-oxa-l, 3,-diazol-4-yl chloride (NBD-C1), ethidium bromide, Lucifer Yellow, 5-carboxyrhodamine 6G hydrochloride, Lissamine rhodamine B sulfonyl chloride, Texas Red TM sulfonyl chloride, BODIPYTM, naphthalamine sulfonic acids including but not limited to l-anilinonaphthalene-8- sulfonic acid (
  • Representative intrinsic fluorophores include but are not limited to organic compounds having aromatic ring structures including but not limited to NADH, FAD, tyrosine, tryptophan, purines, pyrirmidines, lipids, fatty acids, nucleic acids, nucleotides, nucleosides, amino acids, proteins, peptides, DNA, RNA, sugars, and vitamins. Additional suitable fluorophores include enzyme-cofactors; lanthanide, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, blue fluorescent protein or mutants and derivates thereof. It must also be noted that in recent years there has been a growing interest in using quantum dots, typically semiconductor nanoparticles/nanocrystals, as fluorophores due to their superior emission and structural/chemical stability. This type of fluorophore is within the scope of this invention.
  • the labeling signal radiated by the reporting entity can also be produced by Raman scattering.
  • Raman scattering is inelastic scattering of light from matter.
  • the particles of light which are photons, interact with the vibrational modes of a molecule.
  • photons either absorb (energy gain) or emit (energy loss) these vibrational modes .
  • the scattered photon energy shows gains or losses at certain frequencies in the form of sharp peaks. In other words the scattered light's frequency is shifted from that of incident light. This change is termed the Raman shift.
  • the frequency (or energy) of these sharp peaks corresponds to vibrational modes in the molecules causing the scattering. Since different molecules or materials have different vibrational modes at different energies, the Raman peaks are a characteristic of the molecule or material that scatters the light. Raman spectroscopy may also be used to identify (trace) the "labeling molecules" or "reporter molecules”. Tracing of the labeling molecules or reporter molecules from their Raman signal has advantages over tracing them from their fluorescence signal. Unlike fluorescence bands, Raman peaks are very sharp, so they are much easier to resolve when several different reporter molecules are used.
  • Raman scattering reporter entities of this invention can be chosen from organic or inorganic molecules, or semiconductor, polymer nanoparticles/nanocrystals.
  • the absorption and emission energy of these Raman scattering reporter entities should match with the energy of the plasmon resonance in metal particles as well as the laser excitation (resonant Raman scattering)".
  • fluorophores are usually good Raman scattering entities.
  • quantum dots also have stable and very distinctive characteristic Raman signals.
  • nanoparticles and reporting entities are incorporated into a molecule probe solution.
  • the nanoparticles (NPs) enhance the signal arising from the reporting entity (e.g., fluorescence, Raman scattering).
  • the reporting entity e.g., fluorescence, Raman scattering
  • the present invention includes one or more nanoparticles (NPs), one or more reporting entities (in the embodiments shown below, dye molecules), and a detecting molecule (also referred to as a probe molecule) and a solution into which reporting entities and nanoparticles are incorporated.
  • NPs nanoparticles
  • the nanoparticles enhance a labeling signal (fluorescence, in the embodiments shown below, or Raman scattering) due to the reporting entities.
  • the nanoparticles are capable of being bound/incorporated to molecules in the detecting molecule solution.
  • Each one of the reporting entities is bound/incorporated to a molecule in the detecting molecule solution.
  • one or more nanoparticles are capable of being bound/incorporated to the probe molecule in a location different from a location at which the reporting entity is bound/incorporated to the probe molecule in the detecting molecule solution.
  • the nanoparticles are bonded to the test (detecting) molecule by mechanisms including, but not limited to, Van der Waals, ionic, hydrogen, or covalent bonding.
  • One method aspect of the present invention includes incorporating nanoparticles and reporting entities into a detecting molecule solution.
  • Several detailed embodiments are presented herein below in which the nanoparticles and the reporting entities are incorporated into the detecting molecule solution either separately or together and either at the same step or in different steps of the method.
  • DNA is the molecule under test and fluorophores (also referred to as fluorescent tag molecules) are the reporting entities.
  • fluorophores also referred to as fluorescent tag molecules
  • the metal nanoparticles cannot be directly attached to fluorescent tag molecules, since this will quench the fluorescence.
  • the nanoparticles and tags can be separately attached to the DNA. In this manner, the density of particles and fluorophores attached to DNA can be controlled for optimizing the separation between them. This can yield maximum fluorescence enhancement. If the bonding energy between the particle and DNA (or any other molecule to be probed) is larger than the average thermal energy (kT), aggregation or loss of nanoparticles during the hybridization or washing processes will be prevented.
  • kT average thermal energy
  • the tag molecule and the nanoparticle may, in one embodiment, be bridged with a ligand molecule and be attached to the DNA (or any other molecule to be probed, including but not limited to RNA, antibodies, enzymes, factors, cell membrane receptors, proteins or peptides) together. This may be done as long as the bridging molecule blocks charge transfer and prevents quenching of the fluorescence. This will ensure a minimum and well-defined separation between the fluorescent tag and nanoparticle.
  • the characteristic dimension (diameter in the case of substantially spherical nanoparticles) of the nanoparticles is between about 0.3 nm and about 40 nm. However, it should be noted that this characteristic dimension range is not a limitation of this invention.
  • the means of detection can be modified to Raman signals.
  • the electrons in the nanoparticles move in phase. In so doing, they generate an oscillating dipole (or multipole depending on the shape of the particle) that has a resonance condition at a certain frequency (plasmon frequency) at which the amplitude of the oscillating dipole can be excited to a maximum.
  • the plasmon frequency depends on and is tunable by the type of particle material, particle size, shape, and separation, and dielectric constant of the local medium.
  • the plasmon band shifts to lower frequencies (higher wavelengths) and broadens as the particle size is increased, or particle spacing is decreased, or particle aggregation occurs.
  • the embodiments described herein cover a number of metals and materials and include, but are not limited to, plasmon bands occurring between 1.45 to 4 eV corresponding to 900-350 nm, or near- infrared (NIR) to ultraviolet (UV) .
  • NIR near- infrared
  • UV ultraviolet
  • the plasmon band will tail into more of the NIR, while for Al nanoparticles, the plasmon band is found in the UV.
  • a size and separation range of the nanoparticles of a certain metal or material may be selected in order to produce enhanced local fields for effecting optical response (fluorescence, Raman) enhancement.
  • the fluorescent signal intensities of several dyes are enhanced using gold and silver NPs.
  • the gold and silver NPs have been shown to have SPR absorptions of approximately 510 nm and 420nm, respectively.
  • the dyes tested include:
  • Alexa® 555 molecular probes
  • the nanoparticles were added to the solution containing the cDNA probe prior to the hybridization step.
  • the nanoparticles were introduced to target DNA by mixing the Au solution into Cy3/Cy5 dye-coupled cDNA dissolved in a formamide hybridization buffer. The hybridization was carried out for 16 hours.
  • the density of particles and tag molecules (reporting entities) attached to DNA is controlled to optimize the separation between them. Details of the protocol utilized to obtain the result shown in figures 2b-21 are given in Appendix I.
  • the protocol given in Appendix I includes the conventional protocol for incorporating the reporting entity into the cDNA.
  • Figures 3a-3d depict the fluorescence response obtained using fluorescently labeled Arabidopsis cDNA to probe a human oligonucleotide array in the absence of nanoparticles and in the presence of nanoparticles.
  • Figures 3a-3d illustrate the fact that the addition of NPs does not alter the specificity of the probe-target interaction.
  • Fluorescently labeled Arabidopsis cDNA was used to probe a human oligonucleotide array with several Arabidopsis spots embedded into each human sub-array as controls .
  • the presence of the nanoparticle combination enhances the fluorescent signal detected.
  • Figure 4 gives a schematic representation of the mechanism of attachment of reactive functionalized NPs (such as those described in US Patent No. 5,728,590 and in US patent number 5,521,289, both of which are incorporated by reference herein) to an aminoallyl-modified nucleotide.
  • the functionalized reactive nanoparticle utilized in this embodiment of this invention has been functionalized so that it will attach by substantially the same reaction undergone by the fluorophores during dye coupling to the cDNA.
  • This reaction mechanism whereby a linking molecule, sulfo-N- hydroxysuccinimi.de ester (sulfo-NHS) in the embodiment shown in figure 4, reacts with a primary a ine, is illustrated in simplified form in this figure ( Figure 4).
  • the reactive functionalized nanoparticle shown in Figure 4 may, in one embodiment, be directly incorporated as a modified base analog doing the reverse transcription of the mRNA template into cDNA.
  • Figures 5a-5d depict the fluorescence enhancement observed with the use of gold nanoparticles, which attach to the test molecule by some mechanism, such as vanderwalls bonding.
  • Nanogold® with no reactive group was used as the nanoparticle.
  • gold nanoparticles of 1.4 nm diameter were used to obtain the results of Figs. 5a-5d. It should be noted that this diameter is not a limitation of this invention; nanoparticles of different diameter and compositions can be utilized.
  • the method of this invention utilized to obtain the embodiment and data shown in Figures 5g-5h, includes the step of adding the functionalized nanogold particles to the reaction at the same time as the fluorescent dye is added. Therefore, both the nanoparticle and the fluorophore "compete" with one another for available binding sites on the cDNA. Protocols used in this embodiment are given in Appendix II. This method of competitively incorporating functionalized reactive NPs and fluorophores in a controlled manner throughout the length of the cDNA molecules shows evidence of reproducible signal enhancement ( Figures 5g-5h). The data in Figure 5 also indicate that utilizing functionalized reactive NPs results in an average enhancement of total intensity of 0.91 O.M.
  • Figure 7 Shown in Figure 7 are results obtained in simulations where low level gene expression was simulated by using suboptimal amounts of cDNA in the labeling reactions.
  • the signal generated from each of 100 genes having the lowest detectible fluorescent signal intensity was compared in the control condition ( Figure 7; point # 1 on the x-axis), which included no NPs, to the signal generated from the same genes when increasing amounts of functionalized NPs (IX; 4X and 16X) were added in a competitive manner ( Figure 7; points # 2, 3 and 4 on the x-axis respectively) .
  • the highest signal intensity observed corresponds to the addition of 16X functionalized NPs ( Figure 7; point # 4 on the x-axis).
  • Figure 8 Using the direct non-competitive approach (Figure 8; IX nanoparticle labeled dTTP) resulted in the detection of a total of 6831 genes or a 57.8% increase over control ( Figure 8).
  • Figure 9 The same data presented in Figure 8 is shown using a cluster diagram in Figure 9. In this figure ( Figure 9) it is easier to visualize the increase in the number genes detected and the intensity of the signal generated from each gene (represented by a horizontal line in each column) under the different conditions .
  • fluorophores and nanoparticles can be covalently attached at regular, predictable positions throughout the cDNA probe sequence.
  • One embodiment of the method for this attachment includes constructing a heteroconjugate molecule that consists of a dUTP that has been modified so as to contain an a ine modifier in addition to a thiol modifier as two acceptor sites ( Figure 10).
  • the amine modifier will serve as a specific attachment point for the fluorophore molecule
  • the thiol modifier will serve as a specific attachment point for a nanoparticle.
  • the enhanced fluorescence observed from molecules in the close vicinity of metal nanoparticles is attributable to the enhanced absorption of the light excitation as well as the enhanced emission rate as a result of enhanced near fields.
  • Another advantage of the enhanced emission rate is a decrease in photobleaching resulting from a shortening of the characteristic time a fluorescent molecule spends at the excited state.
  • RNA synthesis was accomplished through reverse transcription. This process was performed by adding 5 ⁇ g of total RNA dissolved in ddH20 and Oligo-d(T)20 Primers to a sterile microcentrifuge tube.
  • nuclease-free water was added to bring the final volume within the tube to 15.5 ⁇ L.
  • the tube was then incubated at 752C for 8 minutes and at the conclusion of this time it was immediately placed on ice for 1 minute to allow for the primers to bind to the appropriate sites on the mRNA strand.
  • the following reagents were then added to the microcentrifuge tube: 3 ⁇ L of StrataScript Buffer, lOx (Stratagene, CA) ; 2 ⁇ L of aa-dNTP mix, 50x; 2 ⁇ L of StrataScript RT (50 U/ ⁇ L) (Statagene, CA) and 8.9 ⁇ L of nuclease-free H20.
  • the aa-dNTP mixture is an aqueous solution containing 10 ⁇ L of the following nucleotides which the cDNA is constructed of: dATP, dCTP, and dGTP, each at a concentration of lOOmM.
  • dATP dATP
  • dCTP dCTP
  • dGTP dGTP
  • 6 ⁇ L of dTTP and 3 ⁇ L of amine-allyl conjugated dUTP (aa-dUTP) both at lOOmM concentration, were added to the mix.
  • the amine-allyle group will be utilized later to link a fluorochrome to the cDNA as a means for detecting the presence of cDNA following hybridization.
  • the microcentrifuge tube containing the aforementioned reagents was incubated at 42sc for 1 hour.
  • the synthesized Complementary DNA was then isolated and concentrated through the use of Zymo Clean & Concentrator-5TM columns (Zymo Research, CA) .
  • Zymo Clean & Concentrator-5TM columns Zymo Research, CA
  • 1 mL of Zymo DNA binding buffer Zymo Research, CA
  • the solution containing the cDNA to be purified is added to the tube and gently mixed by inverting the tube several times .
  • This solution was then added to a Zymo Clean and Concentrator-5TM column with a collection tube attached to the bottom and was spun in a centrifuge at 5,000 rpm.
  • the eluted solution was disposed of and 600 ⁇ L of Zymo Wash Buffer (Zymo Research, CA) was added to the column.
  • the column was spun at 5,000 rpm for 1 minute and the solution collected in the tube was discarded. This step was repeated to remove any residual wash solution from the column.
  • the column was then placed in an appropriately labeled sterile microcentrifuge tube and 8 ⁇ L of dH20 was added to the column. The column was allowed to sit for approximately 30 seconds before being placed in the centrifuge and spinning the sample at 14,000 rpm for 30 seconds. An additional 6 ⁇ L of dH20 was added to the column. After waiting 30 seconds the column was centrifuged at 14,000 rpm for one minute. The eluted solution contains the purified cDNA in 14 ⁇ L of dH20.
  • the 14 ⁇ L solution of aa-cDNA prepared earlier was further concentrated to a volume of 5 ⁇ L using a centrifugal evaporator to which 3 ⁇ L of 300mM NaHC03 was added, which will act as a coupling buffer.
  • the solution containing the probe cDNA was then added to the Alexa dye, mixed thoroughly, and allowed to incubate in the dark at room temperature for one hour. After the incubation period, the cDNA was separated from the uncoupled dye through use of Zymo Clean & Concentrator-5TM column and conventional protocols.
  • the cDNA probe was ready for exposure to a microarray for examination of gene expression within that particular sample of cells.
  • the cDNA probe from the experimental sample and that from the control were thoroughly mixed and the volume of this solution was then concentrated to 15.5 ⁇ L using a centrifugal evaporator.
  • This mixture was heated for 2 minutes at 100sc to denature all the strands of DNA and then cooled by incubating on ice for a period of time not exceeding 3 minutes .
  • the array was prepared by first using a stream of compressed air to blow any particulates off the surface to ensure that the surface of the array was both clean and dry.
  • a Gene Frame® (MWG, Germany) was then carefully fitted over the spotted area on the array.
  • the citrate terminated Au Colloids were added to the solution containing the probe.
  • the mixture containing the probe was then dispensed at one end of the frame using a pipette.
  • a polyester cover slip, provided with the Gene Frame® was positioned over the frame and was carefully brought into contact with the adhesive frame starting over the area where the probe solution was initially added and gradually working to the opposite end of the frame.
  • the arrays were then sealed in hybridization chambers (Corning, NY) and then incubated at 43SC for approximately 19 hours. During this time the tagged cDNA probes will preferentially hybridize to the complementary sequence of DNA, referred to as the target, bound to the surface of the array.
  • the arrays were immediately placed in a bath of 2x (SSC) with 0.1% (SDS) that was pre-warmed to 302C.
  • the bath was placed on an orbital shaker to provide moderate agitation while in the wash solution.
  • the array was washed for 5 minutes in the dark and then transferred to the next wash solution. This wash process was then repeated two more times, first using a solution of lx SSC and then 0.5x SSC.
  • the arrays were then placed in a 50mL conical centrifuge tube and dried by briefly spinning within a centrifuge.
  • the detection and quantification of fluorescently labeled probes bound to the surface of the array was done using a ScanArray Express, microarray scanner (Packard BioScience, MA) equipped with both 543nm and 633nm argon lasers.
  • This system utilized a fixed laser source and confocal optics, with beam splitter and emission filters, to scan arrays that were mounted on a motorized stage controlled by the system.
  • the 543 nm wavelength laser was used to excite probes coupled with Alexa Fluor® 546 (Molecular Probes, OR). Emitted light was passed through a 570nm wavelength high pass emission filter before reaching the detector to eliminate any reflected laser light from interfering with signal detection.
  • Alexa Fluor® 660 (Molecular Probes, OR) coupled probes were excited using the 633 nm wavelength laser while the emitted light was passed through a 670nm wavelength high pass emission filter. All arrays were scanned at a 10 micrometer resolution and the data collected was analyzed using GeneSpring (Silicon Genetics, CA) , a type of microarray analysis software.
  • cDNA is now ready for dye coupling. This product can be stored at -20 C indefinitely. To be sure the cDNA synthesis went well, test 1 uL cDNA reaction by running on the Agilent bioanalyzer.
  • Labeling Buffer When properly stored, Labeling Buffer should be stable for at least 6 months .
  • NANOGOLD AND ALEXA PREPARATION Dissolve each Alexa dye in 2ul DMSO. Dissolve the mono-sulfo-NHS-NANOGOLD reagent in 200 ul deionized water. Use 5 nmol NG reagent to label 1 nmol of amine sites.
  • the succinimide ester is hydrolyzed in aqueous solution. To ensure better solution, dissolve in a small amount (up to 20% of final solution) of dimethyl sulfoxide (DMSO), then make up to 100% with water. If the reagent is still slow to dissolve, the solution may be vortexed
  • Sulf0-succinimido- NANOGOLD® has extinction coefficients at 280 nm of 2.3 X 10 5 M ⁇ cm "1 and at
  • This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions. For cleanup of other enzymatic reactions, follow the protocol as described for PCR samples or use the new MinElute Reaction Cleanup Kit. Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases, and salts using QiAquick spin columns in a microcentrifuge.
  • IMPORTANT Residual ethanol from Buffer PB will not be completely removed unless the flow-through is discarded before this additional centrifugation.
  • Buffer EB (10 mM Tris Cl, pH 8.5) or H 2 0 to the center of the QiAquick membrane and centrifuge the column for 1 min. Repeat.
  • Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at -20*C as DNA may degrade in the absence of a buffering agent.
  • the purified DNA can also be eluted in TE (10 mM Tris Cl, 1 mM EDTA, pH 8.0), but the EDTA may Inhibit subsequent enzymatic reactions .
  • Hybridization B Lifter-Slips® Pre-Calculated Values for Lifter-Slip Hybridization Volumes Component 22mm X 25mm 60mm X 25mm Other
  • NG-dTTP Note. dTTP was omitted from the dNTP mix and replaced with the reacted Nanogold-aminoallyl dUTP conjugate, (dTTP and dUTP are functionally identical)
  • the succinimide ester is hydrolyzed in aqueous solution. To ensur better solution, dissolve in a small amount (up to 20 % of final solution) of dimethyl sufoxid (DMSO), then make up to 100 % with water. If the reagent is still slow to dissolve, the soluti may be vortexed.
  • DMSO dimethyl sufoxid
  • 3P200 random hexamer 2 ug/ul 3P230 (oligo-dT) 2 ug/ul Priming reaction / total RNA vol/rxn
  • RNA( ⁇ 5ug) x ⁇ l oligo dT primer (2ug/uL) 1.0 ⁇ l random N6 primer (2ug/uL) 1.0 ⁇ l dNTP + aa-dUTP/NG-dTTP 2:1 0.6 ⁇ l

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Abstract

Il est prévu des procédés et des systèmes pour utilisation de nanoparticules de métal pour renforcer des signaux optiques (UV, visibles et IR, selon le cas) provenant d’une entité de signalisation. Les procédés et systèmes de la présente invention n’exigent pas que les nanoparticules soient fixées ou collées à une surface, assemblées en matrice ou enduites d’un revêtement d’espacement.
PCT/US2005/015344 2004-05-03 2005-05-03 Procedes et systemes d’amelioration nanoparticulaire de signaux WO2005107404A2 (fr)

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US4849513A (en) * 1983-12-20 1989-07-18 California Institute Of Technology Deoxyribonucleoside phosphoramidites in which an aliphatic amino group is attached to the sugar ring and their use for the preparation of oligonucleotides containing aliphatic amino groups
US5525711A (en) * 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US6361944B1 (en) * 1996-07-29 2002-03-26 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US6582921B2 (en) * 1996-07-29 2003-06-24 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses thereof
US20040099813A1 (en) * 2000-12-21 2004-05-27 Christian Eggeling Method for characterizing samples of secondary light emitting particles
WO2004111602A2 (fr) * 2003-05-16 2004-12-23 University Of Rochester Procedes de colorimetrie et de fluorescence permettant de detecter des oligonucleotides
US20050074779A1 (en) * 2003-10-02 2005-04-07 Tuan Vo-Dinh SERS molecular probe for diagnostics and therapy

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