Classifications

• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
  • B01J13/0004—Preparation of sols
  • B01J13/0008—Sols of inorganic materials in water
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
  • B01J13/0004—Preparation of sols
  • B01J13/0043—Preparation of sols containing elemental metal
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F24—HEATING; RANGES; VENTILATING
  • F24S—SOLAR HEAT COLLECTORS; SOLAR HEAT SYSTEMS
  • F24S70/00—Details of absorbing elements
  • F24S70/10—Details of absorbing elements characterised by the absorbing material
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F28—HEAT EXCHANGE IN GENERAL
  • F28D—HEAT-EXCHANGE APPARATUS, NOT PROVIDED FOR IN ANOTHER SUBCLASS, IN WHICH THE HEAT-EXCHANGE MEDIA DO NOT COME INTO DIRECT CONTACT
  • F28D15/00—Heat-exchange apparatus with the intermediate heat-transfer medium in closed tubes passing into or through the conduit walls ; Heat-exchange apparatus employing intermediate heat-transfer medium or bodies
  • F28D15/02—Heat-exchange apparatus with the intermediate heat-transfer medium in closed tubes passing into or through the conduit walls ; Heat-exchange apparatus employing intermediate heat-transfer medium or bodies in which the medium condenses and evaporates, e.g. heat pipes
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F28—HEAT EXCHANGE IN GENERAL
  • F28D—HEAT-EXCHANGE APPARATUS, NOT PROVIDED FOR IN ANOTHER SUBCLASS, IN WHICH THE HEAT-EXCHANGE MEDIA DO NOT COME INTO DIRECT CONTACT
  • F28D15/00—Heat-exchange apparatus with the intermediate heat-transfer medium in closed tubes passing into or through the conduit walls ; Heat-exchange apparatus employing intermediate heat-transfer medium or bodies
  • F28D15/02—Heat-exchange apparatus with the intermediate heat-transfer medium in closed tubes passing into or through the conduit walls ; Heat-exchange apparatus employing intermediate heat-transfer medium or bodies in which the medium condenses and evaporates, e.g. heat pipes
  • F28D15/04—Heat-exchange apparatus with the intermediate heat-transfer medium in closed tubes passing into or through the conduit walls ; Heat-exchange apparatus employing intermediate heat-transfer medium or bodies in which the medium condenses and evaporates, e.g. heat pipes with tubes having a capillary structure
• • 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
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • 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
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
  • B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E10/00—Energy generation through renewable energy sources
  • Y02E10/40—Solar thermal energy, e.g. solar towers

Definitions

• This invention relates to the manufacture of particle structures for analyte detection. Specifically, the invention relates to the manufacture of particle structures having receptor molecules attached to resonance domains within the particle structures. More specifically, the invention relates to the use of particle structures having receptor molecules for the detection of analytes using Raman spectroscopy. Description of Related Art
• ligand analyte
• chromatography mass spectroscopy
• nucleic acid hybridization nucleic acid hybridization
• receptor a receptor molecule
• the basis for specificity of these methods is conferred by a receptor molecule can bind in a specific fashion to the ligand molecule, thereby creating a bound complex.
• the bound ligand can be assayed.
• DNA deoxyribonucleic acid
• mRNA messenger ribonucleic acid
• DNA can contain important information about the genetic makeup of an organism, and mRNA can be an important indicator of which genes are active in a specific physiological or pathological condition and what proteins may be created as a result of gene activation. Additionally, the direct detection of proteins can be important to the understanding ofthe physiological or pathological condition of an individual.
• DNA is made of a double helix of two strands, each of which is composed of a series or "sequence" of nucleotide bases.
• the bases found in DNA include adenine, thymine, cytosine and guanine.
• One strand of the double helix has a sequence of the nucleotides that can be transcribed into mRNA, herein termed a "reading strand,” and the other strand has a sequence of bases, each of which is complementary to the base in the position corresponding in the reading strand. For every adenine in the reading strand, a thymine is present in the other strand.
• a guanine is present in the other strand.
• a cytosine and a thymine is found in the other strand.
• Ribonucleic acid has a similar structure as DNA, except that thymine is typically replaced by the base uracil. However, uracil is complementary to adenine, and thus, hybridization of RNA can occur with DNA. Because the information content of nucleic acids resides significantly in the sequence of the units that make up the nucleic acid, purely chemical methods that can detect only the presence of nucleotide bases are of limited usefulness. Thus, methods for detecting the presence of specific DNA or RNA relies upon the characterization ofthe sequence of bases of that nucleic acid. A. Hybridization Detection of Nucleic Acids
• nucleic acids and proteins Many different methods are currently in use for the detection of nucleic acids and proteins, but those methods can be time-consuming, expensive, or poorly reproducible.
• the detection of specific nucleic acid sequences in DNA or RNA molecules can be accomplished using hybridization reactions, wherein an analyte DNA or RNA molecule is permitted to attach to a complementary sequence of DNA.
• a complementary DNA molecule can be attached to a supporting matrix, and the bound DNA and matrix is herein termed a "substrate.” Exposing an analyte nucleic acid to a complementary substrate DNA can result in the formation of a relatively stable hybrid. Detection ofthe duplex DNA hybrid is characteristically carried out using methods that can detect labeled DNA analytes.
• the labeling is typically performed using radioactive, spin resonance, chromogenic or other labels, which are attached to the analyte molecules.
• unbound analyte can be removed and the bound, or specific, analyte can be detected and quantified.
• mRNA molecules having a specific sequence For example, to detect a mRNA molecule having a specific sequence using current methods, naturally occurring, or “native" mRNA is typically converted to a complementary DNA (“cDNA") molecule using an enzyme called “reverse transcriptase” under conditions that incorporate a labeled nucleotide into the cDN A.
• cDNA complementary DNA
• reverse transcriptase reverse transcriptase
• the bound ligand can be detected using a radiometric technique such as scintillation counting, fluorescence or spin resonance, depending on the type of label used.
• PCR polymerase chain reaction
• PCR requires DNA polymerase enzymes to amplify the cDNA. Some DNA polymerases can insert incorrect bases into a growing strand of newly synthesized cDNA.
• the recognition of ceratin cDNA by DNA polymerase and primers used for PCR can vary depending on the specific sequences of DNA in the sample to be amplified. This variation can result in non-proportional amplification of different cDNA molecules. Subsequent amplification of an strand having an incorrect sequence can result in the presence of several different cDN A sequences in the same sample. Thus, the accuracy and sensitivity of analysis of cDNA using PCR can be compromised. Additionally, for medical diagnostic or forensic purposes, it can be very important for results of tests to be available rapidly. Commonly used methods for detection of specific nucleic acid sequences can be too slow for therapeutic or forensic uses. Thus, there is a need for rapid, accurate measurement of nucleic acid sequences.
• Raman spectroscopy involves the use of electromagnetic radiation to generate a signal in an analyte molecule.
• Raman spectroscopic methods have only recently been developed to the point where necessary sensitivity is possible. Raman spectroscopic methods and some ways of increasing the sensitivity of Raman spectroscopy are described herein below.
• Raman scattering when incident photons having wavelengths in the near infrared, visible or ultraviolet range illuminate a certain molecule, a photon of that incident light can be scattered by the molecule, thereby altering the vibrational state of the molecule to a higher or a lower level.
• the vibrational state of a molecule is characterized by a certain type of stretching, bending, or flexing ofthe molecular bonds. The molecule can then spontaneously return to its original vibrational state. When the molecule returns to its original vibrational state, it can emit a characteristic photon having the same wavelength as the incident photon. The photon can be emitted in any direction relative to the molecule. This phenomenon is termed "Raleigh Light Scattering.”
• a molecule having an altered vibrational state can return to a vibrational state different from the original state after emission of a photon. If a molecule returns to a state different from the original state, the emitted photon can have a wavelength different from that ofthe incident light. This type of emission is known as "Raman Scattering" named after C. V. Raman, the discoverer of this effect. If, a molecule returns to a higher vibrational level than the original vibrational state, the energy ofthe emitted photon will be lower (i.e., have longer wavelength) than the wavelength of the incident photon.
• This type of Raman scattering is termed “Stokes-shifted Raman scattering.” Conversely, if a molecule is in a higher vibrational state, upon return to the original vibrational state, the emitted photon has a lower energy (i.e., have a shorter wavelength). This type of Raman scattering is termed “anti-Stokes-shifted Raman scattering.” Because many more molecules are in the original state than in an elevated vibrational energy state, typically the Stokes- shifted Raman scattering will predominate over the anti-Stokes-shifted Raman scattering. As a result, the typical shifts of wavelength observed in Raman spectroscopy are to longer wavelengths. Both Stokes and anti-Stokes shifts can be quantitized using a Raman spectrometer.
• surface enhanced Raman scattering When Raman active molecules are excited near to certain types of metal surfaces, a significant increase in the intensity of the Raman scattering can be observed. The increased Raman scattering observed at these wavelengths is herein termed “surface enhanced Raman scattering.”
• the metal surfaces that exhibit the largest increase in Raman intensity comprise minute or nanoscale rough surfaces, typically coated with minute metal particles.
• nanoscale particles such as metal colloids can increase intensity of Raman scattering to about 10 6 times or greater, than the intensity of Raman scattering in the absence of metal particles. This effect of increased intensity of Raman scattering is termed "surface enhanced Raman scattering.”
• the mechanism of surface enhanced Raman scattering is not known with certainty, but one factor can affect the enhancement.
• Electrons can typically exhibit a vibrational motion, termed herein "plasmon" vibration. Particles having diameters of about 1/lOth the wavelength of the incident light can contribute to the effect. Incident photons can induce a field across the particles, and thereby can alter the movement of mobile electrons in the metal. As the incident light cycles through its wavelength, the induced motion of electrons can follow the light cycles, thereby creating an oscillation of the electron within the metal surface having the same frequency as the incident light. The electrons' motion can produce a mobile electrical dipole within the metal particle.
• plasma vibrational motion
• incident light can cause groups of surface electrons to oscillate in a coordinated fashion, thereby causing constructive interference of the electrical field so generated, creating an area herein termed a "resonance domain.”
• the enhanced electric field due to such resonance domains therefore can increase the intensity of Raman scattering and thereby can increase the intensity of the signal detected by a Raman spectrometer.
• surface enhanced resonance Raman scattering The combined effects of surface enhancement and resonance on Raman scattering is termed “surface enhanced resonance Raman scattering.”
• the combined effect of surface enhanced resonance Raman scattering can increase the intensity of Raman scattering by about 10 14 or more. It should be noted that the above theories for enhanced Raman scattering may not be the only theories to account for the effect. Other theories may account for the increased intensity of Raman scattering under these conditions.
• an analyte molecule can have a reporter group added to it to increase the ability of an analytical method to detect that molecule.
• Reporter groups can be radioactive, flourescent, spin labeled, and can be inco ⁇ orated into the analyte during synthesis.
• reporter groups can be introduced into cDNA made from mRNA by synthesizing the DNA from precursors containing the reporter groups of interest.
• other types of labels such as rhodamine or ethidium bromide can intercalate between strands of bound nucleic acids in the assay and serve as reporter groups of hybridized nucleic acid oligomers.
• Patent No: 5,567,628, both incorporated herein fully by reference, provide an analyte that has been labeled using a Raman active label and an unlabeled analyte in the test mixture.
• the above- described methods rely upon the introduction of a Raman active label, or "reporter” group, into the analyte molecule.
• the reporter group is selected to provide a Raman signal that is used to detect and quantify the presence ofthe analyte.
• one object of this invention is the development of spectroscopic methods that do not rely on labeling of analyte molecules.
• Another object of this invention is the development of methods for manufacturing and the manufacture of particle structures for optical detection methods including fluorescence, SERS and SERRS
• compositions useful for analyte detection of the present invention can use particle structures that are designed to enhance electromagnetic signals, including Raman signals Particle structures may be fractal, random or ordered
• particle structures can be generated using chemical methods using linkers
• Such linked particle structures can be designed and manufactured to have desired properties, including but not limited to selection of wavelengths of incident electromagnetic radiation that permit the generation of enhanced Raman signals to permit sensitive detection of a variety of analytes
• Raman and other electromagnetic signals can be detected for analytes without the need for incorporation of electromagnetically active labels into analyte molecules
• Methods of these embodiments as used for Raman spectroscopic methods are herein termed "reverse Raman spectroscopy" or "RRS " Upon binding ofthe analyte to the receptor and removal of unbound analyte, the analyte can provide the detectable Raman signal for detection and/or quantification and/or identification
• oligonucleotide receptor molecules can be made that have sequences complementary to the specific sequences to be identified, but lack a typical component ofthe analyte molecule
• adenine can be replaced by 2,6,-di-aminopurine ("2, 6
• deuterium can be used to replace H 2 O incertain synthesis of certain receptors.
• peptide nucleic acids herein termed "PNA" can be used in place of phosphate- and sugar- containing nucleic acids.
• surfaces are created that promote the surface enhancement effect of SERS.
• Raman enhancing surfaces are made that inco ⁇ orate receptors locally at resonance domains, thereby increasing the sensitivity of Raman spectroscopic detection.
• systems for analysis of biologically significant moieties are provided, wherein a particle structure, receptor and analyte are exposed to incident electromagnetic radiation, and the Raman spectrum of the complexes are used to detect and/or quantify the amounts of analyte present.
• receptors can be attached to or placed near resonance domains, thereby concentrating the productive signal and increasing the sensitivity of detection of analytes.
• Certain embodiments provide for the attachment of receptors to resonance domains selectively, thereby decreasing the effects of analyte-receptor complexes at other locations.
• fractal particle structures can be used to enhance a Raman signal generated in the presence of an analyte, thereby providing methods for detection of signals with increased sensitivity.
• Figure 1 is a drawing depicting particle structures of this invention used for spectroscopy.
• Figure 2 depicts particle structures of this invention that has been subjected to photoaggregation.
• Figures 3a - 3c depict a strategy of this invention for chemically linking particles to form particle structures of this invention.
• Figures 4a - 4d depict a strategy of this invention for linking pairs of particle pairs together using linker molecules, and the manufacture of particle structures of this invention.
• Figure 5 depicts another embodiment of this invention in which the linker groups are comprised of aryl di-isonitrile groups.
• Figures 6a - 6e illustrates a photolithographic method for manufacturing particle structures of this invention.
• Figures 7a -7b depicts particle structures of this invention as in Figure 2 additionally having receptors.
• Figure 7a depicts two particle structures of this invention having oligonucleotide receptor molecules comprising adenine residues ("A") attached to resonance domains.
• Figure 7b depicts two particle structures of this invention having oligonucleotide receptor molecules, similar to that depicted in Figure 7a, wherein the oligonucleotide receptor molecules comprises 2, 6- diaminopurine ("AP") residues substituted for adenine residues.
• AP 2, 6- diaminopurine
• Figure 8 depicts a portion of a particle structure of this invention having AP- substituted oligonucleotide receptors shown binding to thymine residues ("T") of analyte molecules.
• the analyte molecules have adenine residues ("A") that provide a Raman or other electromagnetic signal for detection.
• Figure 8b depicts a matrix of this invention, having defined areas thereon with particle structures within each area.
• Figures 9a -9b are graphs illustrating the principle of this invention involving the use of an oligonucleotide receptor not having adenine in Raman spectroscopic detection of oligonucleic acids that contain adenine.
• Figure 9c is a graph showing the Raman spectrum of guanine.
• Figures 10a - 10c depict a methods for manufacturing nested particle structures of this invention.
• Figure 10a depicts two particles having complementary oligonucleic acid sequences aligned to hold the particles in relationship with each other.
• Figure 10b depicts a first-order nested particle structure of this invention.
• Figure 10c depicts a second-order nested particle structure of this invention.
• Figures 1 1a - l lg depict methods for manufacturing biochips of this invention.
• Figure 1 la depicts a substrate used for subsequent attachment of particle structures.
• Figure 1 lb depicts a substrate as in Figure 1 la having thiol groups.
• Figure 1 lc depicts particles of different sizes used to manufacture particle structures of this invention.
• Figure 1 Id depicts a group of nested particle structures of this invention.
• Figure 1 le depicts a group of chemically linked particle structures of this invention.
• Figure 1 If depicts a portion of a biochip of this invention having nested particle structures as in Figure 1 Id attached to a substrate.
• Figure 1 1 g depicts a portion of a biochip of this invention having chemically linked particle structures as in Figure l ie attached to a substrate.
• Figures 12a - 12d depict embodiments of this invention having chemically linked particle structures and/or rods.
• Figure 12a depicts two rods useful for enhancement of electromagnetic signals.
• Figure 12b depicts a rod as shown in Figure 12a having analyte receptors.
• Figure 12c depicts a portion of a biochip of this invention having rods with analyte receptors applied to a substrate.
• Figure 12d depicts a portion of a biochip of this invention having rods with receptors and chemically linked particles structures of this invention applied to a substrate.
• Figure 13a - 13b depict alternative embodiments of this invention.
• FIG. 13a depicts a top view of a portion of a biochip of this invention having rods/receptors aligned end-to end and within channels inscribed in a substrate, with and without particles.
• Figure 13b depicts a cross-sectional view through a portion of a biochip of this invention as described in Figure 13 a.
• analyte as used herein means molecules, particles or other material whose presence and/or amount is to be determined. Examples of analytes include but are not limited to deoxyribonucleic acid ("DNA”), ribonucleic acid (“RNA”), amino acids, proteins, peptides, sugars, lipids, glycoproteins, cells, sub-cellular organelles, aggregations of cells, and other materials of biological interest.
• RNA ribonucleic acid
• fractal as used herein means a structure comprised of elements, and having a relationship between the scale of observation and the number of elements, i.e., scale-invariant. By way of illustration only, a continuous line is a 1 -dimensional object.
• a plane is a two-dimensional object and a volume is a three-dimensional object.
• the dimension is less than one.
• the fractal dimension is V_.
• the fractal dimension ofthe plane is between one and 2.
• the fractal dimension is 1.5.
• Vz of the points of a solid are missing, the fractal dimension is 2.5.
• the structure of objects appears to be similar, regardless ofthe size of the area observed.
• fractal structures are a type of ordered structures, as distinguished from random structures, which are not ordered.
• fractal associate means a structure of limited size, comprising at least about 100 individual particles associated together, and which demonstrates scale invariance within an area of observation limited on the lower bound by the size ofthe individual particles comprising the fractal associate and on the upper bound by the size ofthe fractal associate.
• fractal dimension means the exponent D of the following equation: N « R D , where R is the area of observation, N is the number of particles, and D is the fractal dimension.
• fractal particle associates means a large number of particles arranged so that the number of particles per unit volume (the dependent variable) or per surface unit changes non-linearly with the scale of observation (the independent variable)
• label means a moiety having a physicochemical characteristic distinct from that of other moieties that permit determination ofthe presence and/or amount of an analyte of which the label is a part
• labels include but are not limited to fluorescence, spin-resonance, radioactive moieties Also known as reporter group.
• linker means an atom, molecule, moiety or molecular complex having two or more chemical groups capable of binding to a surface and permitting the attachment of particles together to form groups of particles
• the simplest linker connects two particles A branched linker may link together larger numbers of particles
• order structures means structures that are non-random
• particle structures as used herein means a group of individual particles that are associated with each other in such a fashion as to permit enhancement of electric fields in response to incident electromagnetic radiation
• particles include metals, metal-coated polymers and fullerenes
• films or composites comprising particles on a dielectric surface or imbedded in a dielectric material
• percolation point means a point in time on a conductive surface or medium when the surface exhibits an increase in conductance, as measured either via surface or bulk conductance in the medium.
• One way to measure surface or "sheet” conductance is via electric probes applied to the surface.
• Radar array reader means a device having a light source and a light detector.
• Raman signal means a Raman spectrum or portion of Raman spectrum.
• Raman spectral feature means a value obtained as a result of analysis of a Raman spectrum produced for an analyte under conditions of detection.
• Raman spectral features include, but are not limited to,
• Radar spectroscopy means a method for determining the relationship between intensity of scattered electromagnetic radiation as a function ofthe frequency of that electromagnetic radiation.
• Radar spectrum means the relationship between the intensity of scattered electromagnetic radiation as a function ofthe frequency of that radiation.
• Random structures as used herein means structures that are neither ordered nor fractal. Random structures appear uniform regardless ofthe point and scale of observation, wherein the scale of observation encompasses at least a few particles.
• receptor means a moiety that can bind to or can retain an analyte under conditions of detection.
• resonance means an interaction with either incident, scattered and/or emitted electromagnetic radiation and a surface having electrons that can be excited by the electromagnetic radiation and increase the strength ofthe electric field ofthe electromagnetic radiation.
• resonance domain means an area within or in proximity to a particle structure in which an increase in the electric field of incident electromagnetic radiation occurs.
• reporter group means label.
• RRS reverse Raman spectroscopy
• scaling diameter means a relationship between particles in a nested structure, wherein there is a ratio (scaling ratio) of particle diameters that is the same, regardless ofthe size ofthe particles.
• SERS surface enhanced Raman spectroscopy
• SERRS surface enhanced resonance Raman spectroscopy
• compositions and methods of this invention represent improvements over the existing methods for spectroscopic methods for detection and quantification of analyte molecules.
• the compositions and methods can be desirable for use in conjunction with infrared spectroscopy, fluorescence spectroscopy, surface plasmon resonance, Raman spectroscopy, mass spectroscopy or any other method utilizing excitation of an analyte by electromagnetic radiation.
• Certain embodiments of this invention are based upon Surface Enhanced Raman Spectroscopy ("SERS”), Surface Enhanced Resonance Raman
• This invention includes methods for manufacturing Raman active structures having specific analyte receptor molecules attached to those structures.
• the invention also includes methods for detecting analytes using Raman spectroscopy, reverse Raman spectroscopy, compositions useful for reverse Raman spectroscopy, and arrays and test kits embodying Raman spectroscopic methods.
• the structures that are desirable for use according to the methods of this invention include structures of small particles in structures, herein termed particle structures, which includes as a subset, fractal associates.
• Particle structures can be characterized by having physical and chemical structures that enable oscillations of electrons to be in resonance with incident and outgoing electromagnetic radiation.
• the Raman active structures desirable for use according to this invention can include any structure in which Raman signals can be amplified.
• the following discussion regarding metal fractal structures is not intended to be limiting to the scope ofthe invention, but is for pu ⁇ oses of illustration only.
• Metal colloids can be composed of noble metals, specifically, elemental gold or silver, copper, platinum, palladium and other metals known to provide surface enhancement.
• a dilute solution containing the metal salt is chemically reacted with a reducing agent.
• Reducing agents can include ascorbate, citrate, borohydride, hydrogen gas, and the like. Chemical reduction ofthe metal salt can produce elemental metal in solution, which combine to form a colloidal solution containing metal particles that are relatively spherical in shape.
• a solution of gold nuclei is made by preparing a 0.01% solution of NaAuCl 4 in water under vigorous stirring. One milliliter ("ml") of a solution of 1% sodium citrate is added. After 1 minute of mixing, 1 ml of a solution containing 0.075 % NaBH 4 and 1% sodium citrate is added under vigorous stirring. The reaction is permitted to proceed for 5 minutes to prepare the gold nuclei having an average diameter of about 2 nm). The solution containing the gold nuclei can be refrigerated at 4° C until needed.
• This solution can be used as is, or can be used to produce particles of larger size (e.g., up to about 50 nm diameter), by rapidly adding 30 ⁇ l ofthe solution containing gold nuclei and 0.4 ml of a 1% sodium citrate solution to the solution of 1% HAuCl 4 3H 2 O diluted in 100 ml H 2 O, under vigorous stirring. The mixture is boiled for 15 minutes and is then cooled to room temperature. During cooling, the particles in the solution can form fractal structures. The resulting colloid and/or fractal particle structures can be stored in a dark bottle.
• Deposition of enhancing particles on dielectric surfaces including glass can generate films that can enhance electromagnetic signals.
• Such films can be as thin as about 10 nm.
• the distribution of electric field enhancement on the surface of such a film can be uneven.
• Such enhancing areas are resonance domains. Such areas can be particular useful for positioning receptors for analyte binding and detection.
• one way to manufacture enhancing structures is to treat the surface until "percolation points" appear. Methods for measuring sheet resistance and bulk resistance are well known in the art.
• Example 2 Manufacture of Metal Particles and Fractal Structures Using Laser Ablation
• laser ablation is used to make metal particles.
• a piece of metal foil is placed in a chamber containing a low concentration of a noble gas such as helium, neon, argon, xenon, or krypton. Exposure to the foil to laser light or other heat source causes evaporation ofthe metal atoms, which, in suspension in the chamber, can spontaneously aggregate to form fractal or other particle structures as a result of random diffusion.
• a noble gas such as helium, neon, argon, xenon, or krypton.
• the colloidal metal particles can be deposited onto quartz slides as described in Examples 1 or 2. Other films can be made that inco ⁇ orate random structures or non-fractal ordered structures in similar fashions.
• Example 3 Manufacture of Quartz Slides Containing Gold Fractal Structures
• Quartz slides (2 5 cm x 0 8 cm x 0 1 cm) are cleaned in a mixture of
• Figure 1 depicts a particle structure suitable for use with the methods of this invention.
• the particles are arranged in a scale-invariant fashion, which promotes the formation of resonance domains upon illumination by laser light.
• ordered non-fractal structures and random structures can be generated. These different types of structures can have desirable properties for enhancing signals associated with detection of analytes using electromagnetic radiation.
• particles can be attached together to form structures having resonance properties.
• the particles can be desirable to have the particles being spheres, ellipsoids, or rods.
• ellipsoidal particles it can be desirable for the particles to have a long axis (x), another axis
• x be from about 0.05 to about 1 times the wavelength ( ⁇ ) ofthe incident electromagnetic radiation to be used.
• x can be less than about 4 ⁇ , alternatively, less than about 3 ⁇ , alternatively less than about 2 ⁇ , in other embodiments, less than about l ⁇ , and in yet other embodiments, less than about
• the ends ofthe rods can be either flat, tapered, oblong, or have other shape that can promote resonance.
• the particle pair can be desirable for the particle pair to have an x dimension to be less than about 4 ⁇ , alternatively, less than about 3 ⁇ , alternatively less than about 2 ⁇ , in other embodiments, less than about l ⁇ , and in yet other embodiments, less than about Vi ⁇ .
• pairs of particles, rods, rods plus particles together can be used. The arrangement of these elements can be randomly distributed, or can have a distribution density that is dependent upon the scale of observation in a non-linear fashion. In other embodiments, rods can be linked together end-to end to form long structures that can provide enhanced resonance properties.
• a suspension of particles can be desirable.
• the suspended particles can have dimensions in the range of about Vi ⁇ to about 1 millimeter (mm).
• a researcher or developer can satisfy many needs, including, but not limited to selecting the absorbance of electromagnetic radiation by particle elements, the nature ofthe surface selected, the number of resonance domains, the resonance properties, the wavelengths of electromagnetic radiation showing resonance enhancement, the porosity ofthe particle structures, and the overall structure ofthe particle structures, including, but not limited to the fractal dimensions ofthe structure(s).
• Photoaggregation can be used to generate particle structures that have properties which can be desirable for use in Raman spectroscopy. Irradiation of fractal metal nanocomposites by a laser pulse with an energy above a certain threshold leads to selective photomodification, a process that can result in the formation of "dichroic holes" in the abso ⁇ tion spectrum near the laser wavelength (Safonov et al., Physical Review Letters 80(5): 1102- 1 105 (1998), inco ⁇ orated herein fully by reference). Selective photomodification ofthe geometrical structure can be observed for both silver and gold colloids, polymers doped with metal aggregates, and films produced by laser evaporation of metal targets.
• optical modes formed by the interactions between monomers in fractal are localized in domains that can be smaller than the optical wavelength of the incident light and smaller than the size ofthe clusters of particles in the colloid.
• the frequencies ofthe optical modes can span a spectral range broader than the abso ⁇ tion bandwidth ofthe monomers associated with plasmon resonance at the surface.
• other theories may account for the effects of photomodification of fractal structures, and this invention is not limited to any particular theory for operability.
• Photomodification of silver fractal aggregates can occur within domains as small as about 24 x 24 x 48 nm 3 (Safonov et al., Physical Review Letters 80(5): 1102-1 105 (1998), inco ⁇ orated herein fully by reference).
• the energy absorbed by the fractal medium can be localized in a progressively smaller number of monomers as the laser wavelength is increased. As the energy absorbed into the resonant domains increases, the temperature at those locations can increase. At a power of 11 mJ/cm 2 . light having a wavelength of 550 nm can produce a temperature of about 600 K (Safonov et al., Physical Review Letters 80(5): 1 102-1 105 (1998), incorporated herein fully by reference).
• photoaggregation can be accomplished by exposing a metal colloid on a surface to pulses of incident light having a wavelengths in the range of about 400 nm to about 2000 nm.
• the wavelength can be in the range of about 450 nm to about 1079 nm.
• the intensity ofthe incident light can be in the range of about 5 mJ/cm 2 to about 20 mJ/cm 2 .
• the incident light can have a wavelength of 1079 nm at an intensity of 1 1 mJ/cm 2 .
• Fractal aggregates that are especially useful for the present invention can be made from metal particles having dimensions in the range of about 10 nm to about 100 nm in diameter, and in alternative embodiments, about 50 nm in diameter.
• a typical fractal structure of this invention is composed of up to about 1000 particles, and an area ofthe aggregate typically used for large-scale arrays can have a size of about 100 ⁇ m x 100 ⁇ m.
• Figure 2 depicts a particle structure that have been photoaggregated and that are suitable for use with the methods of this invention. Local areas of fusion ofthe metal particles can be observed (circles).
• particle structures can be made using chemical methods.
• metal particles can be either made according to methods described above, or alternatively can be purchased from commercial suppliers (NanoGram Inc., Fremont, California).
• the particles can be joined together to form first-order structures, for example, pairs of particles.
• the first-order structures can be joined together to form second-order structures, for example, pairs of particle pairs.
• third-order fractal structures can be made by joining second-order structures together.
• each particle can be attached to a linker molecule via a thiol or other type of suitable chemical bond.
• the linker molecules then can be attached to one another to link adjacent colloid particles together.
• the distance between the particles is a function ofthe total lengths of the linker molecules. It can be desired to select a stoichiometric ratio of particles to linker molecules. If too few linker molecules are used, then the array of particles will be too loose or may not form at all.
• the first step comprises adding linker molecules to individual particles under conditions that do not permit cross-linking of particles together.
• a linker can comprise an oligonucleotide having a reactive group at one end only.
• the reactive end ofthe oligonucleotide can bind with a metal particle, thereby forming a first particle-linker species, and having a free end ofthe linker.
• the ratio of linker molecules to particles can be selected, depending on the number of linker molecules are to be attached to the particle.
• a second linker can be attached to another group of particles in a different reaction chamber, thereby resulting in a second linker-particle species, again with the linker having a free end
• the different linker-particle species can be mixed together and the linkers can attach together to form "particle pairs" joined by the linker molecules
• Figure 3a to 3c illustrates methods for manufacturing fractal structures of this invention
• metal particles 10 are formed using methods previously described
• Short linkers 20 have chemically active ends that are capable of binding to metal particles 10
• linker 20 has sulfhydryl ("SH") groups at each end ofthe linker 20
• metal particles 10 When combined, metal particles 10 bind with the SH ends of linkers 20 to form particle pairs 30
• Figure 3b illustrates the steps that can be used to form clusters of particle pairs Particle pairs 30 are reacted with medium-length linkers 40 to form clusters 50
• Figure 3c illustrates the steps that can be used to form nanoscale fractal structures of this invention Clusters 50 are reacted with long linkers 60 to form nanoscale fractal structure 70
• nucleic acids can be used as linkers, based upon the ability of DNA to form hybrids with nucleic acids comprising complementary sequences DNA ligases or other mechanisms can be used to join the linkers together to form a complete linker between metal particles
• Figure 4 depicts, in general, the linkage of metal particles to form particle pairs using linkers having binding domains
• Figure 4a depicts two metal particles (M), each having a linker molecule (LI or L2) having a desired length, comprising inter-linker binding domains (BD1 and BD2)
• the inter-linker binding domains are unbound
• Figure 4b depicts the particles shown in Figure 4a after binding ofthe inter-linker binding domains to form a particle pair.
• the linkers are nucleic acids
• the binding domains can have complementary sequences, such that the nucleotide residues can form stable hybrid complexes with each other, thereby linking the metal particles together as a pair.
• the sequence of BD1 can be polyjadenine] for example, A 10 .
• the sequence of BD2 can be poly[thymidine], for example, T 10 .
• a 10 can hybridize to T 10 , thereby forming a stable hybrid.
• the lengths ofthe binding domains can be any convenient length that permits the formation of a stable hybrid.
• BD 1 and BD2 can be selected to be complementary to a third nucleic acid, herein termed a "bridge nucleic acid" or (“BNA”), comprising two sequences, one complementary to BD1 and an other complementary to BD2.
• BNA bridge nucleic acid
• the portion of BNA complementary to BD1 can form a stable hybrid ofthe first metal particle Ml with LI and BNA attached thereto.
• the portion ofthe BNA that is complementary to BD2 of L2 is free to hybridize to BD2.
• the BD2 can bind to that portion ofthe BNA complementary to BD2, forming a stable particle pair.
• Figure 4d depicts an alternative particle pair in which the inter-linking molecules are attached by way of their ends.
• the size ofthe nanoscale structures should have average dimensions in the range of about 20 nm to about 500 nm In alternative embodiments, the dimensions can be in the range of about 50 nm to about 300 nm, and in other embodiments, in the range of about 100 to about 200 nm, and in yet other embodiments, about 150 nm
• the linking can be carried out using an aryl di-thiol or di-isonitrile molecules
• Figure 5 depicts the structure of a class of linkers having thiol (SH) groups at each end
• linker can have a central area having ethylbenzene moieties, where n is a number between 1 and about 10,000
• the ratio of length for each subsequent pairs of linkers can be in the range of about 2 to about 20 Alternatively, the ratios of lengths of subsequent pairs of linkers can be in the range of about 3 to about 10, and in other embodiments, about 5 In certain other embodiments, the ratio of linker lengths in successive orders can be non-constant, thus resulting in the manufacture of an ordered, non-fractal structure
• the ratio of LI L2 L3 can be in the range of about 1 2 4 Alternatively, the ratio can be about 1 5 25, and in yet other embodiments, the ratio can be about 1 20 400
• the ratio between LI and L2 and from L2 to L3 need not be the same
• the ration of LI L2 L3 can be 1 3 20, or alternatively, 1 20 40 3.
• suspensions of fractal particle associates can be used, for example, to provide a structure in solution that can bind or retain analytes for detection using methods of this invention.
• the size of fractal particle associates can be in the range of from hundreds of nanometers to mm dimensions.
• the fractal associates can comprise a number of particles arranged by means of chemical linkers. The number of particles per fractal associate can be as few as about 100 particles, or alternatively, thousands can be used to form a fractal associate. By increasing the number of particles in a fractal associate, the increase in the void size increases by a greater proportion.
• Nested Fractal structure for example, comprises a core of a large particle, surrounded by a "halo" of smaller particles, and each ofthe smaller particles is surrounded by a "halo" of even smaller particles. (See Example 6). Nested fractal structures can be especially useful for generation of essentially uniform fractal surfaces for enhanced analyte detection. It can be desirable to include large excesses of smaller particles compared to larger particles for each successive step. For example, it can be desirable to have excess of smaller particles in the range of about 10 to about 1000 times the number of larger particles.
• particle structures can be manufactured using lithographic methods known in the semiconductor manufacturing arts
• an image ofthe particle structure to be made can be made and stored in a computer memory
• Each point defining the particle structure can be represented by a single location within the memory
• the memory device can then direct the projection of a beam of electromagnetic radiation, electrons, or other particles locally onto a suitable surface
• the beam can create site on the surface for the subsequent formation of a metal particle at desired locations
• FIG. 6 depicts several steps in the lithographic manufacture of a particle structure of this invention
• Figure 6a shows an image 600 of a desired distribution of nanoparticles
• the image is stored in a computer memory, in which each particle is represented by a pair of reference coordinates, one x and one y for each point
• Figure 6b depicts a substrate for nanoparticle structure 610 comprising a gold substrate 615 having a film of hexadecanethiol 620 on which the nanoparticle structure is to be manufactured
• Figure 6c illustrates the placement ofthe tip 635 of a scanning tunneling microscope (STM) over the gold substrate 615 at a point stored in the computer memory Electrons emitted from the tip 635 ofthe STM can interact with the hex
• STM scanning tunneling microscope
• nanoparticle structure 650 ( Figure 6e).
• traditional semiconductor masks can be used to direct the location of nanoparticle structures on substrates. Regardless ofthe method used, the result obtained will provide for resonance properties ofthe structures.
• the receptor chosen to be attached to particle structures of this invention will depend on binding properties ofthe desired analyte. For example, to detect and quantify nucleic acid sequences, it can be desirable to use oligonucleotide receptors. Oligonucleotide receptors can hybridize to analyte nucleotide sequences, thereby producing a bound ligand. Alternatively, if desired, one can use an antibody directed against a nucleotide sequence to bind the nucleic acid.
• DNA binding proteins can be used.
• specific promoter-binding proteins can be used as receptors.
• peptide nucleic acids can be used to bind native nucleic acids.
• antibodies and other, specific protein binding molecules can be used. Once the type of analyte is chosen, the specific receptor molecule and the conditions for its attachment to the fractal array can be determined Additionally, antibodies directed against low molecular weight analytes can be attached to a substrate
• the nucleic acid receptors can advantageously used in a large scale matrix array to measure a large number of analyte sequences simultaneously
• Thiol-derivatized DNA oligomers are synthesized by standard phosphoramidite chemistry according to the methods of Caruthers Gene Synthesis Machines: DNA Chemistry and Its Uses, Science 230 281 -285 ( 1995), inco ⁇ orated herein fully by reference
• Such oligomers are obtained from Dr Keith McKenney of The Institute for Genomic Research (TIGR), Rockville, Maryland, and are prepared according to the methods of Peterlinz et al Observation of Hybridization and Dehybridtzation of Thwl-Tethered DNA Using Two-Color Surface Plasmon Resonance Spectroscopy, Journal American
• the DNA oligomers are selected to be in the range of about 10 - 50 bases in length, although much longer sequences can also be used In other embodiments, the DNA oligomers are in the range of about 15 - 30 bases in length, and in alternative embodiments, the DNA oligomers are about 25 bases in length If the oligomer is too long, the analyte molecule can be too far from the metal surface, and the surface enhancement of Raman resonance can be undesirably low If the oligomer is too short, the specificity of hybridization can be too low Therefore, the length ofthe oligomer is selected to optimize the sequence specificity and resonance enhancement ofthe analyte In situations in which sequence specificity is less important than resonance enhancement, shorter oligomers can be desirable. Conversely, in situations in which a high degree of sequence specificity is desired, longer oligomers can be desirably used.
• Two sets of complementary nucleotide oligomers are synthesized, one set being manufactured using moieties that lack a Raman active component.
• the DNA oligomer is synthesized using 2,6 di-aminopurine instead of adenine.
• PNA peptide nucleic acid
• PNA peptide nucleic acid receptors
• Peptide nucleic acids have an affinity to RNA and DNA comparable to that of DNA, (Griffin (1998); Kyger et al (1998), Igloi (1998); Ratilainen et al. (1998), each reference herein inco ⁇ orated fully by reference), and thus, can form hybridization pairs with mRNA.
• the difference between the chemical structures of PNA and DNA can result in a pronounced difference in their Raman spectra.
• PNA fragments can be obtained from Atom Sciences (Oak Ridge, Tennessee).
• oligomers can be attached to metal surfaces via an alkanethiol covalently linked at the 5' position of single-stranded DNA oligomers according to the methods of Herne, Characterization of DNA Probes Immobilized on Gold Surfaces, Journal American Chemical Society 1 19:8916-8920 (1997), inco ⁇ orated herein fully by reference.
• the attachment can be irreversible, thereby permitting hybridization and dehybridization on the surface (Peterlinz et al., Observation of Hybridization and Dehybridization of Thiol-Dertvatized DNA Using Two Color Surface Plasmon Resonance Spectroscopy. Journal American Chemical Society 119:3401-3402 (1997), inco ⁇ orated herein fully by reference).
• any method can be used that results in the attachment of receptor molecules to metal surfaces and can permit the receptor to maintain the physical characteristics necessary for specific binding to ligands.
• Example 5 Linking of DNA to Colloidal Gold
• the colloidal gold-coated quartz slides of Example 3 can then used as a matrix or substrate for the binding of DNA used for hybridization detection of analyte nucleic acids.
• the gold colloid derivatized slides are placed in 1.0 M KH 2 PO 4 buffer solution, pH 3.8, containing 1.0 ⁇ M thiol-derivatized DNA for a specific amount of time to achieve thiol-tethering of DNA.
• the surface is then passivated by exposing the DNA tethered slides to 1.0 mM mercaptohexanol (HS(CH 2 ) 6 OH) for 1 hour. This treatment eliminates nonspecific binding of polynucleotides. Thorough rinsing with deionized water is required before analysis of hybridization.
• receptors can be localized to resonance domains within particle structures.
• resonant domains can be heated, and that heating can cause partial melting ofthe metal particles.
• the dimensions of resonance domains are smaller than the wavelength ofthe incident light.
• the size ofthe resonance domains generated at certain wavelengths of incident light can be on the order of 1/25 ofthe wavelength ofthe light used in their generation. However, as the wavelength of light becomes longer, the size ofthe resonance domains can become smaller.
• Resonant domains are areas that can exhibit intense resonance, and can produce greater amplification of Raman signals than that possible in unaggregated metal or metal colloid substrates.
• resonance domains that are especially useful for this invention can be made using incident light, which can result in resonance domains comprising between about 4 to about 10 monomer particles.
• the property of particle structures to become locally heated can be used advantageously to localize receptor molecules to those locations.
• a surface containing particle structures is prepared as above.
• a solution containing receptor molecules is then placed on the surface and in contact with the particle structures.
• Pulses of laser light are used to illuminate the surface, and at those locations where resonance domains are created, the local temperature ofthe reaction mixture can reach the threshold for the formation of intermolecular bonds between the particle structures and the receptor, thus attaching the receptor to the particle structures.
• any thermosensitive chemistry for linking the receptors to the substrate can be used.
• the power required to initiate receptor molecule derivatization is less than that needed for photoaggregation. It can be desirable to provide temperatures at the resonance domains in the range of about 0° C to about 500°
• the temperature can be in the range of about 70° C to about 100° C.
• the temperature needed will vary with the threshold temperature required to initiate the linkage ofthe receptor to the metal surface. In certain embodiments, it is desirable that the temperature locally at the resonance domains remain below the temperature at which bond breakage and reversal of the bond between the receptor and the metal surface occurs.
• photosensitive reagents can be used to link the receptor to the particle structures at specific locations.
• a number of such reagents can be obtained from Pierce Products Inc., Rockford,
• photochemical linking agents By the use of different photochemical linking agents, one can link different types of receptors to the same substrate. For example, one can attach DNA and proteins to the same substrate.
• attachment of receptors at resonance domains can be performed using a scanning atomic force microscope (see Hansen et al. "A Technique for Positioning Nanoparticles Using an Atomic
• the capillary contains derivatized receptors which can be deposited onto a surface.
• the surface can be illuminated by incident electromagnetic radiation produced by a laser.
• the resonance increases the intensity ofthe emitted radiation and thereby provides a signal to the optical feedback device to initiate deposition of receptors at those locations, depending upon the intensity of electromagnetic radiation emitted from the surface in response to external illumination provided by the laser.
• Figures 7a and 7b depict embodiments of this invention in which receptor molecules are attached to resonance domains of particle structures.
• Figure 7a depicts an area of a particle structures in which the receptor molecules are native, adenine ("A")-containing oligonucleotides.
• Figure 7b depicts a particle structures similar to that shown in Figure 7a but having the adenine moieties replaced by 2, 6-diaminopurine ("AP").
• Figure 8a depicts the binding of native, complementary oligonucleotide analytes to a particle structures containing receptors as shown in Figure 7b, having adenine replaced by 2, 6-diaminopurine (AP).
• Analyte molecules containing adenine (A) are depicted as hybridizing to the oligonucleotide receptor such that the adenine residues bind to the 2, 6-diaminopurine residues ofthe receptor molecule.
• Figure 8b depicts an array comprising numerous cells or defined areas, each of which has particle structures containing a plurality of receptors bound to each defined area, and being specific for a desired analyte.
• the large-scale array shown is a 10 x 10 matrix, with individual cells positionally located within the large-scale array.
• Other array configurations can be desirable, and includes arrays having identifier moieties different from the receptor molecules.
• Identifier moieties can be used to define the position and/or the type of receptor molecule characteristic ofthe particular defined areas.
• Such identifier moieties can include nucleic acids of defined sequence, or can include identifiers produced by combinatorial chemical methods known in the art.
• defined areas can be identified using colored markers.
• a large-scale array containing fractal aggregates can be exposed to a first receptor type and a beam of highly focused incident light can selectively illuminate one or a few specific cells, thereby linking the first receptor to the substrate in only those cells in which fractal aggregates with the first receptor type is desired.
• Beams of highly focused laser light having the necessary dimensions can be routinely produced using of photolithography methods used in semiconductor manufacture.
• the substrate can be washed to remove unbound first receptor type, and a second receptor type can be applied to the substrate.
• Laser light can illuminate different cells to link the second receptor type to fractal aggregates to form fractal aggregates with the second receptor type.
• the process of sequential application of any desired number of different receptor types to different cells in the matrix array can be carried out using the same chemistry of linkage if desired, or different types of chemical linkage can be used.
• the methods above can be fully automated, so that the reproducibility of manufacture of fractal aggregates can be quite high.
• a result of this process is that a matrix array containing a large number of positionally identifiable cells can be manufactured.
• Such arrays can be used to detect and determine sequences of DNA or mRNA, using strategies as described in, for example, U.S. Patent No: 5,925,525, inco ⁇ orated herein fully by reference.
• Detection of analytes includes the use of a Raman reader and a matrix array. Detection can be performed using a pre-manufactured substrate having particle structures atop the substrate.
• the substrate can have cells or defined areas thereon, having a single type of receptor.
• analytes can bind to or be retained by receptors having sufficient affinity.
• the matrix can then be washed to remove unbound analytes, leaving only those analytes that have a sufficient affinity for the receptors to which they are bound.
• the matrix array can then be subjected to analysis using a reader or be performed using a light source focused upon the array, one cell at a time.
• Light is projected at the cell, and reflected, scattered, or re-emitted light can be collected and transmitted to the light detector. Collected light can be analyzed for Raman spectral features, and such features can be compared with Raman features derived from known moieties. Such known spectra can be imported from external databases, which can include information on biological significance of specific analytes.
• Analysis of information can be performed using a computer, which can be associated with a memory device for storing a program to carry out spectral analyses Also, an output device, such as a screen display or a printer can provide information to the user Such comparison can be the basis for determining the amount of analyte in the cell on the matrix array Additionally, changes in the analyte due to the conditions of measurement can be determined, and any artifacts, such as non-specific binding so introduced can be discovered
• detection can be performed under conditions in which resonance of electron transition in analyte molecules does not occur. According to one theory, this situation can be created when the frequency of incident light does not overlap the absorbance band ofthe analyte In these situations, it can be desirable to add a suspension of particles atop the substrate and receptor analyte complexes Enhancement of Raman signals can be sufficient to provide a highly sensitive detection
• a combination of resonance conditions and enhancement provided by particle structures can be desirable to provide high sensitivity
• a Raman array reader can be used to detect and quantify the amount of analyte bound to a cell of a matrix array
• a Raman reader can be sued for parallel, rapid and sensitive detection of analytes by acquiring Raman spectral features of each cell of an array and comparing the spectral features with known spectral features Thus, the existence, identity and amount of an analyte can be determined
• Detection of analytes is advantageously carried out using native analytes
• receptor molecules that are lacking a structural feature ofthe analyte that is responsible for a Raman signal.
• RRS Reverse Raman Spectroscopy
• nucleic acids can be detected advantageously using RRS Several examples of this strategy follow herein below
• nucleic acid analytes using RRS typically involve the use of receptor molecules that are lacking native nucleotide bases
• the nucleotide bases cytosine, uracil, thymine, guanine and adenine each exhibit Raman bands at wavenumbers in the range of about 610 cm "1 to about 800 cm "1
• nucleotide analogs that have no Raman bands in this range can be suitable for use with the methods and compositions of this invention.
• the nucleotide adenine is composed of a purine ring structure that has a characteristic Raman scattering band at 733 cm '1 (Kurokawa et al. Surface- Enhanced Raman Spectroscopic Detection ofC0 2 , S0 3 , and Nucleic Acid Bases Using Polyvinyl Alcohol Film Doped with Ag Fine Particles Analytic Biochemistry 209:247-250 (1993), inco ⁇ orated herein fully by reference.
• a receptor molecule inco ⁇ orates adenine as a base to pair with thymine residues according to Watson-Crick base pairing, there will be a large Raman band observed at 733 cm "1 even though no analyte is adsorbed to the receptor, thus, making the resolution of analyte nucleic acids containing adenine difficult
• an adenine analog can be inco ⁇ orated into the receptor nucleic acid sequence in the place of adenine Any adenine analog that (1) lacks a characteristic Raman band, and (2) can bind to a complementary base according to Watson-Crick base pairing can be used.
• 2, 6-di-aminopurine (2,6 AP) instead of adenine in a nucleic acid sequence, and then inco ⁇ orating that sequence into a fractal array receptor aggregate, the background Raman spectrum does not have the characteristic band at 733 cm "1
• 2, 6 AP does not interfere substantially with its pairing with thymine (Hacia et al , Enhanced High Density Oligonucleotide Array-Based Sequence Analysis Using
• Figures 9a -9b are graphs illustrating the principle of use of an oligonucleotide receptor not having adenine in Raman spectroscopic detection of oligonucleic acids that contain adenine
• Figure 9a depicts a portion of a Raman spectrum of a nucleic acid not having adenine residues or other moieties having a Raman band at 733 cm
• Figure 9b depicts the Raman spectrum obtained upon binding of an oligonucleotide containing adenine to a receptor molecule not having adenine as in Figure 9a, showing the presence of a Raman band at 733 cm
• RRS can be carried out using receptors in which thymine is replaced with any analog which lacks a characteristic Raman band and can form complementary base pairing with a nucleic acid according to Watson-Crick base pairing
• thymidine can be replaced by 5-methyluridine in DNA oligomers attached to the matrix, without losing the capacity to hybridize with complementary bases (Hacia et al , Nucleic Acids Research 26 4975-4982 (1998), inco ⁇ orated herein fully by reference)
• Thymine can also be replaced with 5-(l-propynyl)uridine
• Guanine has a hydrogen atom at position 8 that can be essentially completely replaced (by about 97%) with deuterium
• This deuterium substitution can be carried out by incubation ofthe nucleic acid in D 2 O at 90° C according to the methods of Manor et al An Isotope Edited Classical Raman Difference Spectoscoptc Study ofthe Interactions of Guanine Nucleotides with Elongation Factor Tu andH-Rasp21, Biochemistry 30 10914-10920 (1991), inco ⁇ orated herein fully by reference
• Deuteration of guanine shifts the Raman band from about 1486 cm “1 to about 1463 cm "1 (Manor et al , Biochemistry 30 10914-10920 (1991), inco ⁇ orated herein fully by reference
• Figure 9c depicts a graph of wavenumber in cm “1 and the intensity of Raman scattering observed (in arbitrary units) of a sample containing 500 mg/ml guanine in 10 M KOH Excitation is at 514 nm with a laser power of 1 Watt and the spectrum was acquired within 3 seconds using a charge coupled device (CCD) Peaks in intensity can be observed at about 980 cm “1 , about 1220 cm “1 , about 1240 cm “1 , about 1280 cm “1 ', 1345 cm “1 , about 1390 cm “1 , about 1470 cm “1 and about 1555 cm “1 This pattern of peaks is similar to the pattern previously published Without enhancement, this system can detect guanine in a concentration of about 20 mg/ml
• the level of specificity of an assay of this invention can depend on the pu ⁇ oses ofthe assay. For example, if the aim ofthe assay is the detection of any of a series of related nucleotide sequences, herein termed "homologues," the fidelity ofthe hybridization reaction need not be as high as an assay in which the detection and identification of single nucleotide polymo ⁇ hisms ("SNPs").
• the methods and compositions of this invention are well suited to detecting the presence or absence of a Raman band within a particular cell of a matrix array. Moreover, because the intensity of a characteristic Raman band is increased as the number of bound analytes increases, the methods of this invention can be used to quantify the amounts of analytes in an assay.
• nucleotide-nucleotide hybridization reactions can depend on the conditions of hybridizations, herein termed "stringency.”
• stringency Hybridization conditions are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Harbor Laboratory Press (1989), inco ⁇ orated herein fully by reference
• high stringency refers to conditions in which the temperature of hybridization is about 5° C to about 10° C below the melting temperature ofthe duplex
• the melting temperature TM of an oligonucleotide duplex can be estimated as follows
• T m 81 5 - 16 6(log 10 [Na + ]) + 0 41 (fraction C + G) - (600/N),
• This stringency is herein termed the "stringency threshold.” By comparing the number of mismatches with the stringency threshold, one can determine the relative degrees of homology of nucleic acid sequences without determining the actual sequences.
• Raman Spectroscopy of Analytes can include any device that can produce laser light ofthe wavelengths needed for analysis.
• the T64000 Raman Spectrometer (The Ultimate Raman Spectrometer Instruments S A. Ltd. (UK) can be used.
• Desirable features of a suitable instrument include the ability to position the sample compartment to adjust the sensitivity ofthe spectrum, provides for low frequency measurements, provides adequate spectral resolution, and a liquid nitrogen cooled charged coupled device (“CCD”) detector.
• the spectrometer is suitably equipped with a laser light source comprising a continuous wave, frequency doubled argon laser.
• a suitable laser is the Inova 300 FReD, available from Coherent Inc , Santa Clara, California Laser power for certain embodiments of this invention can be maintained at about 5 milliWatts at 257 nm, or 1 milliWatt at 244 nm, 229 nm and 238 nm
• Such a light source is a continuous- wave titanium sapphire laser
• light in the visible range can be suitable
• the threshold it can be desirable to set the threshold to a lower value, for example, 2 - 5% ofthe maximal Raman signal.
• analyte is determined, subsequent operations can be carried out to provide additional information. For example, if the analysis is to determine the presence of an oligonucleotide having a desired sequence, the intensity of Raman signal from related cells can be compared. If a series of cells contains receptors having overlapping oligonucleotide sequences, as described, for example, in U.S. Patent No: 5,925,525, inco ⁇ orated herein fully by reference, then the presence of analyte in the related cells can provide information concerning the sequence and overall size ofthe particular analyte in question
• a nested particle associates can be made by selecting colloidal solutions of metal gold particles of uniform size, being 10 nm, 40 nm and 240 nm in diameter, respectively.
• Figures 10a - 10c depict the manufacture of a nested particle structure made from such particles.
• Figure 10a depicts a 10 nm gold particle 1004 having a DNA linker 1012 attached thereto.
• 40 nm particle 1008 having DNA linker 1016 being complementary to DNA linker 1012 is attached to particle 1008.
• Mixtures of particles 1004 and 1008 are placed in solution and interact with each other DNA linkers to form a first-order nested structure 1020 as shown in Figure 10b
• Figure 10c depicts a second-order nested particle structure having particles 1004 and 1008 as shown in Figures 10a and 10b, but with the addition of a larger particle 1024 having a diameter of 240 nm, surrounded by first order nested particles 1020 to form second order nested particle 1028 Heating the mixture of first-order or second-order to a temperature less than about 100° C and then cooling the mixture can result in better ordering ofthe nested particles
• Figures 1 la - 1 lg depict alternative embodiments of surfaces having fractal particle structures thereon
• Figure 1 la depicts a substrate 1104 having a top surface 1108
• Figure 1 lb depicts the surface 1008 as shown in Figure 1 la after being activated , resulting in thiol groups 1112 attached to surface 1108
• Figure 1 lc depicts a plurality of particles 1004 being smaller than intermediate particles 1008
• Figure 1 Id depicts second-order nested particle structures 1028 made from first-order nested particles structures 1020 made from the small particles 1004, the intermediate particles 1008 and larger particles 1024
• Figure 1 le depicts chemically linked particle structures 1132 made from small particles 1004 and intermediate particles 1008
• Figure 1 If depicts an electromagnetic signal enhancer 1132 having substrate 1104 with nested particle structures 1028 thereon
• Figure 1 lg depicts an alternative electromagnetic signal enhancer 1040 comprising substrate 1104 with linked particle structures 1132 as shown in
• Figures 12a - 12d depict the manufacture of a biochip having analyte receptors and enhancers.
• Figure 12a depicts two rod-shaped particles 1204.
• Figure 12b depicts the rod-shaped particles shown in Figure 12a and analyte receptors 1208 with connectors 1212. Some ofthe analyte receptors 1208 are shown attached to rod 1024 by connectors 1212 forming receptor-rod complex
• Figure 12c depicts a biochip 1226 comprised of substrate 1220 with linkers 1224 and having receptor-rod complexes 1216 attached thereto.
• Figure 12 d depicts an alternative biochip 1228, similar to biochip 1216 depicted in Figure 12c, but further comprising linked particle structures 1132 as depicted in Figure 1 le.
• Figures 13a and 13b depict two views of additional embodiments 1324 of this invention having receptor-rod complexes and non-nested particles.
• Figure 13a depicts a top view of a biochip having two types of structures.
• structure 1324 On the right side of Figure 13 a, structure 1324 has linearly arranged rods 1204 having receptors 1212 attached thereto as depicted in Figure 12b.
• trenches 1308 are depicted as being present within trenches 1308. Some rods 1204 are shown parallel to each other, and others are shown end-to-end, although other configurations are within the scope of this invention.
• the right side of Figure 13b depicts a cross-sectional view along line A-A' through the embodiment 1324 depicted on the right side of Figure 13 a.
• Trenches 1308 have receptor-rod complexes 1216 therein.
• the trenches 1308 can be either parallel as shown, or can be non-parallel.
• FIG. 13a depicts an alternative biochip, comprising the biochip as depicted in embodiment 1324 but additionally having particles 1320 distributed over the substrate 1304 and the receptor-rod complexes. Particles
• 1320 can be made, for example, by laser ablation.
• FIG. 13b depicts a cross-sectional view along line A-A' ofthe embodiment 1328 as shown in Figure 13a.
• Substrate 1304 has trenches 1308 with receptor-rod complexes 1216 therein, and having particles 1320 over the top ofthe substrate 1304 and receptor-rod complexes 1216.
• the particle structures of this invention can be used in the fields of chemistry and biotechnology for the detection of analytes in complex solutions containing many different species of molecules. Additionally, the methods of this invention can be used for the detection and quantification of analytes using spectroscopic methods, including Raman spectroscopy, fluorescence spectroscopy, immunobiology and mass spectroscopy.

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