EP1364204A1 - Conceptions de marqueurs pour detection a l'aide d'une technique optique non lineaire selective en surface - Google Patents

Conceptions de marqueurs pour detection a l'aide d'une technique optique non lineaire selective en surface

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Publication number
EP1364204A1
EP1364204A1 EP01952798A EP01952798A EP1364204A1 EP 1364204 A1 EP1364204 A1 EP 1364204A1 EP 01952798 A EP01952798 A EP 01952798A EP 01952798 A EP01952798 A EP 01952798A EP 1364204 A1 EP1364204 A1 EP 1364204A1
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label
nonlinear
active
target
particle
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Joshua S. Salafsky
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • 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
    • 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
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/35—Non-linear optics
    • G02F1/353—Frequency conversion, i.e. wherein a light beam is generated with frequency components different from those of the incident light beams
    • G02F1/3534—Three-wave interaction, e.g. sum-difference frequency generation
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/35—Non-linear optics
    • G02F1/37—Non-linear optics for second-harmonic generation

Definitions

  • second harmonic-active moieties when coupled to a protein as a label, can render the protein detectable at an interface by second harmonic generation.
  • the second harmonic-active label was an oxazole dye derivative which could be covalently coupled to surface amines or sulfhydryls on the protein's surface.
  • Single- molecule labels may not provide enough scattering cross-section and the target objects - such as cells, proteins, viruses or nucleic acids - may have only one or several sites available for labeling and these are typically at random orientations to each other, further reducing the net cross-section. Because the second harmonic labels used to date may not be sufficient to monitor many processes of interest, designs of labels with significantly higher hyperpolarizabilities (eg., second harmonic cross-sections) are needed.
  • the present invention offers a number of designs of second-harmonic active labels with high hyperpolarizabilities. Designs are proposed which involve the use of non-centrosymmetric metallic particles, oxazole dyes, linear chains of nonlinear-active dyes and solid scaffolds on which to build nonlinear active moieties, nanocrystals or nanoparticles. These high-cross-section (hype ⁇ olarizability) labels may then be used to label viruses, cells, proteins, nucleic acids or other particles, especially biological particles ("bioparticles").
  • One means of determining whether a particular molecule or particle is a candidate for use as a nonlinear-active label is by studying it using second harmonic generation at an air-water interface. For instance, in the case of particles, if the particles assemble at the air- water interface in a manner which gives a net orientation ofthe particles (on a length scale ofthe coherence length) the layer of particles will generate second harmonic light. Another means of doing this is by measuring a sample of a suspension ofthe particles and detecting the hyper-rayleigh scattering. Yet another means is by EFISH (Electric-field induced second harmonic generation). EFISH can be used to determine if a candidate molecule or particle is nonlinearly active.
  • EFISH Electro-field induced second harmonic generation
  • Electric field induced second harmonic is well known in the field of nonlinear optics.
  • the effect can be used to measure the hype ⁇ olarizabilty of molecules in solution by using a dc field to induce alignment in the medium, and allowing SHG to be observed. This type of measurement does not require that the particle themselves be ordered at an interface, but does require that the particles be nonlinear active.
  • the use of linkers which couple the labels to their targets can be made long enough so that the orientation ofthe targets at the interface does not significantly affect the orientation ofthe label. Because the intensity ofthe nonlinear light generated will depend on the net orientation ofthe labels at the interface - and the orientation ofthe targets at an interface can be difficult to control (i.e., the targets may even be randomly oriented at the interface) - the use of linkers can separate the labels sufficiently from the targets so that the orientation ofthe targets does not necessarily determine the orientation ofthe labels, resulting in a net orientation of labels.
  • Complementary refers to the topological and chemical compatibility of interacting surfaces between two biological components, such as with a ligand molecule and its receptor (also referred to in the prior art as: 'molecular recognition').
  • the receptor and its ligand can be described as complementary, and, furthermore, the contacts' surface characteristics are complementary to each other.
  • Bio may include any naturally occurring or modified particles or molecules found in biology, or those molecules and particles which are employed in a biological study. Examples of these include, but are not limited to, a biological cell, protein, nucleic acids, antibodies, receptors, peptides, small molecules, oligonucleotides, carbohydrates, lipids, liposomes, polynucleotides and others such as drugs, toxins and genetically engineered protein or peptide.
  • a ligand is a molecule that is recognized by a particular receptor.
  • ligands that can be studied by this invention include, but are not restricted to, antagonists or agonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g. opiates, steroides, etc.), lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.
  • antagonists or agonists for cell membrane receptors include, but are not restricted to, antagonists or agonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g. opiates, steroides, etc.), lectins, sugars, oligonucleotides, nucleic acids
  • Receptor A molecule that has an affinity for a given ligand. Receptors may be naturally occurring or man-made molecules. Also, they can be used in an unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, to a binding partner, either directly or via a specific binding substance. Examples of receptors which can be employed by this invention include, but are not limited to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes and organelles.
  • Receptors are occasionally referred to in the art as anti-ligand. As the term receptors is used herein, no difference in meaning is intended.
  • a "Ligand Receptor Pair" is formed when two macromolecules have combined through molecular recognition to form a complex.
  • receptors which can be investigated by this invention include but are not restricted to:
  • Microorganism receptors Determination of ligands which bind to receptors, such as specific transport proteins or enzymes essential to survival of microorganisms, is useful in developing a new class of antibiotics. Of particular value would be antibiotics against opportunistic fungi, protozoa, and those bacteria resistant to the antibiotics in current use.
  • Enzymes For instance, one type of receptor is the binding site of enzymes such as the enzymes responsible for cleaving neurotransmitters; determination of ligands which bind to certain receptors to modulate the action ofthe enzymes which cleave the different neurotransmitters is useful in the development of drugs which can be used in the treatment of disorders of neurotransmission.
  • enzymes such as the enzymes responsible for cleaving neurotransmitters
  • determination of ligands which bind to certain receptors to modulate the action ofthe enzymes which cleave the different neurotransmitters is useful in the development of drugs which can be used in the treatment of disorders of neurotransmission.
  • the invention may be useful in investigating the ligand-binding site on the antibody molecule which combines with the epitope of an antigen of interest; determining a sequence that mimics an antigenic epitope may lead to the development of vaccines of which the immunogen is based on one or more of such sequences or lead to the development of related diagnostic agents or compounds useful in therapeutic treatments such as for autoimmune diseases (e.g., by blocking the binding ofthe "self antibodies).
  • nucleic Acids Sequences of nucleic acids may be synthesized to establish DNA or RNA binding sequences.
  • Catalytic Polypeptides Polymers, preferably polypeptides, which are capable of promoting a chemical reaction involving the conversion of one or more reactants to one or more products. Such polypeptides generally include a binding site specific for at least one reactant or reaction intermediate and an active functionality proximate to the binding site, which functionality is capable of chemically modifying the bound reactant. Catalytic polypeptides are described in, for example, U.S.
  • Hormone receptors examples include, e.g., the receptors for insulin and growth hormone. Determination ofthe ligands which bind with high affinity to a receptor is useful in the development of, for example, an oral replacement ofthe daily injections which diabetics must take to relieve the symptoms of diabetes, and in the other case, a replacement for the scarce human growth hormone which can only be obtained from cadavers or by recombinant DNA technology. Other examples are the vasoconstrictive hormone receptors; determination of those ligands which bind to a receptor may lead to the development of drugs to control blood pressure.
  • Opiate receptors Determination of ligands which bind to the opiate receptors in the brain is useful in the development of less-addictive replacements for mo ⁇ hine and related drugs.
  • Ion channel proteins or receptors or cells containing ion channel receptors.
  • Surface-selective refers to a non-linear optical technique such as second harmonic generation or sum/difference frequency generation in which, by symmetry, only a non-centrosymmetric surface (comprising array, substrate, solution, biological components, etc.), is capable of generating nonlinear light.
  • Array Refers to a substrate or solid support on which is fabricated one type, or a plurality of types, of biological components in one or a plurality of known locations. This includes, but is not limited to, two-dimensional microarrays and other patterned samples. Other terms in the prior art which are often used interchangeably for 'array' include: gene chip, gene array, biochip, DNA chip, protein chip and microarray.
  • Label refers to a nonlinear-active moiety, particle or molecule which can be attached (covalently or non-covalently) to a molecule, particle or phase (e.g., lipid bilayer) in order to render the latter more nonlinear optical active.
  • the labels are pre-attached to the molecules or particles and unbound or unreacted labels separated from the labeled entities before a measurement is made.
  • Linker A molecule which serves to chemically link (usually via covalent bonds) two different objects together.
  • a linker can be used to couple targets to non-linear active particles or moieties, targets to nonlinear-active derivatized particles, surface layers to targets, surface layers to nonlinear-active particle or moieties, etc.
  • a linker may be a homobifunctional or heterobifunctional cross-linker molecule, a biotin-streptavidin couple wherein the biotin is attached to one ofthe two objects and the streptavidin to the other, etc.
  • Many linkers are available commercially, for example from Pierce Chemical Inc., Sigma- Aldrich, Fluka, etc.
  • the term 'tether', 'spacer' or 'cross-linker' is also used with the same meaning.
  • Nonlinear refers herein to those optical techniques capable of transforming the frequency of an incident light beam (called the fundamental).
  • the nonlinear beams are the higher order frequency beams which result from such a transformation, e.g. second harmonic, etc.
  • second harmonic sum frequency or difference frequency generation
  • the nonlinear beams are generated coherently.
  • SHG second harmonic generation
  • two photons ofthe fundamental beam are virtually scattered by the interface to produce one photon ofthe second harmonic.
  • Target refers herein to a particle or molecule to be labeled with a nonlinear-active moiety, in order to render said particle or molecule for study by a nonlinear-active technique at an interface of interest.
  • Biological targets may include the following: a protein, oligosaccharide, peptide, nucleic acid, liposome, small molecule, oligonucleotide, liposome, or biological cell, liposome, receptor, antibody, antigen, peptide, receptor, drug, enzyme, ligand, carbohydrate.
  • Attached refers herein to biological components which are either prepared or engineered in- vitro to be attached to some surface, via covalent or non-covalent means, including for example the use of linker molecules; or are naturally part ofthe surface such as in the example of membrane receptors embedded in cell membranes, liposomes, tissues, organs (in-vitro or in- vivo) or supported lipid bilayer membranes.
  • Centrosymmetric A molecule or material phase is centrosymmetric if there exists a point in space (the 'center') through which an inversion (x,y,z) -- (-x,-y,-z) of all atoms is performed that leaves the molecule or material unchanged. A non-centrosymmetric molecule or material lacks this center of inversion. For example, if the molecule is of uniform composition and spherical or cubic in shape, it is centrosymmetric.
  • Nucleic Acid Analog A non-natural nucleic acid which can function as a natural nucleic acid in some way.
  • a Peptide Nucleic Acid PNA
  • PNA Peptide Nucleic Acid
  • the PNAs can hybridize to natural nucleic acids via base-pair interactions.
  • Another example of a Nucleic acid analog can be one in which the base pairs are non-natural in some way.
  • Binding Affinity or Affinity The specific physico-chemical interactions between binding partners, such as a probe and target, which lead to a binding complex (affinity) between them.
  • the binding reaction is characterized by an equilibrium constant which is a measure ofthe energetic strength of binding between the partners.
  • Specificity in a binding reaction implies that probe-target binding only occurs appreciably with specific binding partners - not any at random.
  • the protein Immunoglobulin G (IgG) has a specific binding affinity for protein G and not for other proteins.
  • the term 'molecular recognition' is used to describe the binding affinity between components.
  • Electrically Charged or Electric Charge Defined herein as net electric charge on a particle or molecule, which confers a mobility (velocity) of said particle or molecule in an electric field.
  • the net charge could be part of a molecular moiety such as phosphate group on nucleic acid backbones, side-chains of amino acid residues in proteins, lipid head groups in membrane lipids or cellular membranes, etc.
  • the charge can be positive or negative and would determine the direction of mobility ofthe particle or molecule if said particle or molecule is placed in an electric field of a given orientation (direction of positive to negative electric potential).
  • the charge can be non-integer multiples ofthe fundamental unit of charge (q » 1.6 x 10 "19 C) or a fraction ofthe fundamental unit of charge - so-called 'partial charges', well known to those skilled in the art.
  • Electrically Neutral Defined herein as zero net (sum of positive and negative) electric charge on a particle or molecule, which would result in no appreciable mobility (velocity) of said particle or molecule in an electric field.
  • Hype ⁇ olarizability or Nonlinear Susceptibility The properties of a molecule, particle, interface or phase which allows for generation ofthe nonlinear light.
  • the macroscopic nonlinear susceptibility ⁇ (2) is related by an orientational average ofthe microscopic ⁇ hype ⁇ olarizability.
  • the driving electric fields (fundamentals) oscillate at different frequencies (i.e., coi and ⁇ 2 ) and the nonlinear radiation oscillates at the sum or difference frequency ( ⁇ > ⁇ ⁇ ⁇ 2 ).
  • the terms hype ⁇ olarizability, second-order nonlinear polarizability and nonlinear susceptibility are sometimes used interchangeably, although the latter term generally refers to the macroscopic nonlinear-activity of a material or chemical phase or interface.
  • 'nonlinear active' or 'nonlinearly active' used herein also refer to the general property of the ability of molecules, particles, an interface or a phase, to generate nonlinear optical radiation when driven by incident radiation beam or beams.
  • Polarization The net dipole per unit volume (or area) in a region of space. The polarization can be time-dependent or stationary. Polarization is defined as: J " ⁇ (R) dR where an integration ofthe net dipole is made over all volume elements in space dR near an interface.
  • Radiation refers herein to electromagnetic radiation or light, including the fundamental beams used to generate the nonlinear optical effect, or the nonlinear optical beams which are generated by the fundamental.
  • Near-field techniques Those techniques known in the prior art to be capable of measuring or imaging optical radiation on a surface or substrate with a lateral resolution at or smaller than the diffraction-limited distance. Examples of near-field techniques (or near-field imaging) include NSOM (near-field scanning optical microscopy) whereby optical radiation (from fluorescence, second harmonic generation, etc.) is collected at a point very near the surface.
  • NSOM near-field scanning optical microscopy
  • Detecting Refers herein to methods by which the properties of surface-selective nonlinear optical radiation can be used to detect, measure or correlate properties of probe-target binding reactions or effects ofthe binding reactions.
  • the interface can be defined as that region which generates a nonlinear optical signal.
  • Surface layer refers herein to a chemical layer which functionally derivatizes the surface of a solid support.
  • the surface chemical groups can be changed by the derivatization layer according to the particular chemical functionality ofthe derivatizing agent.
  • the solid surface can be derivatized to produce a different chemical functionality which can be presented to nonlinear active moieties or particles, or to targets.
  • a silica bead with negatively charged silanol groups on its surface can be converted to an amine-reactive, amine-containing, etc. surface via organosilane reagents.
  • Conjugated refers herein to the state in which one particle, moiety or molecule is chemically bonded, covalently or non-covalently linked or otherwise attached to a second particle moiety or molecule.
  • the second particle, moiety or molecule is often a target, i.e. a species of interest which must be labeled for detection by a nonlinear optical technique.
  • an oxazole dye 4-[5-methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate also known as 4PyMPO-MeMs
  • 4PyMPO-MeMs 4-[5-methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate
  • 4PyMPO-MeMs 4-[5-methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate
  • R is a hydrogen atom, methyl group, ethyl group or other akyl group.
  • dyes can readily be made into labels, that is, reactive to various functional groups on targets, by using synthetic methods known to one skilled in the art. Furthermore, the following two commercially available dyes (Molecular Probes, Inc.) can be readily conjugated - without further modification - to protein amines [1] or cysteines [2].
  • a solid microparticle or a nanoparticle of size nanometers to microns in scale including, but not limited to, a sphere (latex, polystyrene, silica, etc.) or a strip, offers a surface area which can be derivatized with a nonlinear-active moiety via chemical or electrostatic means so that the entire object has a much higher hype ⁇ olarizability than may be obtained otherwise.
  • nonlinear-active dyes can be assembled on silica bead surfaces via electrostatic interactions (dye is positively charged, silica surface is negatively charged) and the entire bead, if derivatized with target-reactive linkers, can then function as a nonlinear active label.
  • the nonlinear active moieties are assembled on the solid surface so that phase interference between moieties is small, the overall hype ⁇ olarizability will scale nonlinearly (eg., quadratically) in their number.
  • the solid particle can vary in shape and its size can range from nanometers to microns in scale.
  • Linkers which allow attachment to the target object e.g., cells, viruses, proteins, nucleic acid
  • the nonlinear active moieties will ideally all have the same orientation, or the same orientation with respect to the solid particle, for an optimal scattering cross-section.
  • the particles to be used include, but are not limited to, polystyrene beads and silica beads, both readily commercially available.
  • the solid particles can be surface derivatized using a variety of chemistries available in the prior art.
  • Nonlinear-active moieties are covalently coupled either to the solid particles or to a derivatized layer.
  • the nonlinear-active moieties themselves can contain linkers for making the covalent attachment, if necessary.
  • polystyrene beads can be derivatized with dextran, lactose or amines (the latter case for example, via chloromethyl groups with ethylenediamine).
  • Silica can be derivatized using organofunctional silanes, for example using trichlorosilanes or other functional silanes (such as methoxy, a ine, or other functional groups), to produce surfaces with a variety of chemical functionalities. The surfaces ofthe derivatized beads can then be reacted with a nonlinear active moiety via appropriate chemistry.
  • Nonlinear active moieties can also be electrostatically bound to a micron- or nanometer-sized particle surface. This has been demonstrated in the prior art with charged nonlinear active moieties using silica or polystyrene beads and malachite green or oxazole dyes.
  • a charged nonlinear active moiety an organic dye for example, can be oriented at a counter-charged microparticle surface, thus allowing for a net hype ⁇ olarizability ofthe object when using an appropriate geometry.
  • An example of an appropriate geometry is a microparticle sphere where the diameter is approximately the wavelength of the fundamental light, i.e.
  • a commercially available oxazole dye with functionality for binding to amines (l-(3- (succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-yl)pyridinium bromide (PyMPO-SE) can be coupled to beads with amine or surface groups.
  • Spherical silica beads ( ⁇ 200 nm diameter) are derivatized with surface amines using 3-aminopropyltrimethoxysilane according to means well known in the prior art.
  • the PyMPO dye is then reacted with the beads, covalently linking the dye to the surface ofthe beads.
  • amines can be left unreacted with the dye by varying the reaction conditions. These unreacted amines can then be covalently coupled to a heterobifunctional crosslinking agent N(4-Azidosalicylamido)Butyl 3'(2'Pyridyldithio)Propionamide (available from Pierce Chemical, Inc.).
  • the crosslinker allows covalent attachment to amines and sulfhydryls. The sulfhydryl-functional end is available for reaction with sulfhydryl groups on the target's surface.
  • Silica beads (-200 nm, roughly spherical) are reacted with a low concentration of 3- aminopropyltrimethoxysilane or 3-aminooctyltrimethoxysilane so that only -5-10% ofthe surface silanols become covalently coupled to the silane agent. These amine groups are then reacted with the amine-reactive homobifunctional crosslinker Disuccinimidyl glutarate (DSG, Pierce Chemical) to create amine-reactive linkers on -5-10% ofthe bead surface.
  • DSG amine-reactive homobifunctional crosslinker
  • the beads are then incubated with 4-[5- methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate (also known as 4PyMPO-MeMs), a positively charged dye which binds electrostatically to the charged silanols on the surface and orients to some degree.
  • 4PyMPO-MeMs 4-[5- methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate
  • the excess dye is removed from the beads by centrifugation.
  • the electrostatic adso ⁇ tion can be sufficiently high in some cases to immobilize the charged dye, even in the absence of a bulk concentration of it.
  • the beads can then be attached covalently to target objects containing amines using the amine-reactive tethers.
  • Fig. 1 illustrates in (A) two possibilities for an organic nonlinear-active molecule or moiety. (B) illustrates some ofthe various possibilities involving linkers, solid objects ('scaffolds') and non-linear active components, the components being molecules, particles, proteins or moieties which are nonlinear active.
  • amines eg., proteins, viruses, cells, oligonucleotides, nucleic acids, etc.
  • Linear chains of nonlinear active moieties which are aligned in a manner to maximize the overall scattering cross-section and have functionality for attaching to target objects would be useful as nonlinear-active labels.
  • rigid nonlinear active dyes can be coupled in a head-to-tail polymeric fashion, with each monomer of dye oriented in the same direction.
  • dyes such as the oxazole can be created so that they can covalently connect to each other in a head-to-tail fashion, and with a coupling moiety at one end only for attachment to the target object. For example, as depicted in Fig.
  • Prior art shows that metallic nanoparticles and clusters, ranging from about 1 nm to 25 or more microns in size, can be derivatized and conjugated to biomolecules for use in staining for electron microscopy, x-ray scattering and other applications.
  • Prior art also shows that non-centrosymmetric metal nanoparticles can exhibit extremely high hype ⁇ olarizabilities (3,5,9).
  • Another aspect ofthe present invention therefore is to use non-centrosymmetric metal nanocrystals or nanoparticles as labels for nonlinear optical studies.
  • a variety of shapes and sizes of metal nanoparticles are available in the prior art.
  • To use these particles as labels one must derivatize them for conjugation to a target biological component or particle. Either the labels must be derivatized with linkers; or the targets must be derivatized with linkers allowing for their coupling to the labels (the labels can be derivatized if necessary).
  • Fig. 2 depicts some ofthe various combinations of linkers and metallic or semiconductor particles.
  • the particles can be both centrosymmetric or non-centrosymmetric. If centrosymmetric, they must be joined together in clusters to create a composite particle which is overall non-cenfrosyrnmetric; or they must be greater than or equal to 10% ofthe wavelength ofthe fundamental light used in the nonlinear optical technique.
  • Non-centrosymmetric gold particles can be prepared by lithographic means (according to means found in references 2 and 5) or by synthetic methods (according to means found in reference 7). These can then be derivatized with X — R — SH where -SH is a sulfhydryl moiety, R is an alkyl chain and X is a terminal group suitable for conjugation to amines or sulfhydryls.
  • the gold particles can be derivatized, for example, with HS-(CH 2 ) ⁇ 5 -COOH (obtained commercially) according to means well known in the prior art.
  • the carboxyl groups on the derivatized particle can then be made amine- reactive by reaction with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, Molecular Probes, Inc.) according to protocols supplied by Molecular Probes.
  • EDAC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • the particles are now amine-reactive and can be conjugated directly to protein amines, or to protein sulfhydryls via a variety of heterobifunctional crosslinkers which are commercially available (Pierce Inc., Rockford, IL.)
  • carboxyl group ofthe functional alkylthiol listed above can be coupled to oligonucleotides or nucleic acids via similar synthetic means.
  • a tris(aryl) phosphine ligand bearing a single primary amine is mixed with fris ⁇ -N-methylcarboxamidophenyl) in a ratio 1:5 to derivatize the gold particle as described in U.S. Pat. No. 5,521,289, which reference is inco ⁇ orated by reference herein.
  • the gold particles are then reacted with N methoxycarbonylmaleimide (NMCM) in DMSO, mixed and incubated at 0 degrees C for 30 minutes.
  • NMCM N methoxycarbonylmaleimide
  • the maleimido-gold particles can be separated from unreacted NMCM on a gel filtration column.
  • the maleimido-gold particles can then be reacted with a variety of biological moieties including amines, sulfhydryls and carboxylic acids by using cross-linking agents if necessary.
  • a tris(aryl)phosphine ligand bearing a single nonlinear active moiety eg., oxazole dye
  • a tris(aryl)phosphine ligand bearing a single nonlinear active moiety eg., oxazole dye
  • the tris(aryl)phosphine ligand and tris(p-N-methylcarboxamidophenyl) as described in U.S. Pat. No. 5,521,289 to produce particles which contain nonlinear active molecules as well as the means to make them reactive with biological molecules.
  • silica beads can be used (readily available from a number of commercial sources).
  • a bead with - 200 nm diameter is reacted with 3-aminopropyltrimethoxysilane so that the charged silanols are functionalized to produce an amine-surface.
  • These beads can then be reacted with a mixture of l-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2- yl)pyridinium bromide (PYMPO-SE; Molecular Probes Inc.) and (N-[ -
  • the noncentrosymmetric metal particles can themselves be complexed to a larger object which, in turn, contains linkers for coupling the object to a molecule or particle.
  • the Au particles can be chemically derivatized with SH-X-SH where X is an alkylthiol according to methods well known in the prior art. If a silica sphere is used, its surface can be readily derivatized with amines by using a well known reaction with an aminoalkyltrichlorosilane. The silica surface can then be covalently coupled to the Au particles via a number of commercially available heterobifunctional crosslinkers (Pierce Chemical, Inc.).
  • metallic or semiconductor particles can be coupled to an SHG-active particle (such as oxazole, a stryrl dye, or some other molecule or particle).
  • SHG-active particle such as oxazole, a stryrl dye, or some other molecule or particle.
  • the resonantly enhancing particles are available commercially with a variety of surfa ⁇ e chemistries amenable to coupling to an SH-active molecule such as oxazole (succinimidyl ester, maleimide, etc. offered by Molecules Probes, Eugene, OR). Or the particle-nonlinear-active moiety complex can be constructed according to a number of schemes available in the prior art.
  • groups or chains ofthe metallic particles bound together via linking molecules can be used as labels with additional functional linkers to the targets.
  • Au particles can first be sparsely derivatized with linkers for a variety of targets according to the method ofthe preferred embodiment. The remaining underivatized surface area on the particles can then be used to chemically couple the particles together via prior art chemistry involving dimercapto-alkyl chains
  • the particles can be centrosymmetric (eg., a sphere or a cube shape) if their size (i.e., diameter or edge length, respectively) is larger than about 10% ofthe wavelength of the fundamental light. For instance, a spherical particle of 80 nm diameter is expected to produce a nonlinear response when illuminated with 800 nm wavelength fundamental light.
  • Some proteins are strongly SH-active and can be used as fusion-protein labels for in-situ or in- vivo studies.
  • the gene encoding a protein of interest X can be fused at the N-terminal or C- terminal to a gene encoding one ofthe SH-active proteins.
  • these include, but are not limited to, green fluorescence protein (GFP) and bacteriorhodopsin.
  • GFP green fluorescence protein
  • bacteriorhodopsin Detailed procedures in the prior art exist for creating the fusion of such proteins to another protein of interest (references 12 and 13).
  • GFP an intrinsically fluorescent protein
  • GFP has been used in this way in prior art to monitor gene expression and cellular location ofthe GFP-X construct via fluorescence detection.
  • (5) represents an organic, nonlinear-active moiety or molecule and (X) denotes the functional group which is reactive towards a target, a linker, a surface layer or a solid object.
  • (10) represents an organic, nonlinear-active moiety or molecule and (Y-Z) denotes a linker molecule where (Y) is the group which bonds the linker to the nonlinear active moiety or molecule and Z is the functional group which is reactive towards a target, a linker, a surface layer or a solid object.
  • (15) represents a solid object used as a 'scaffold' whose surface area is used to attach linkers (Y) and nonlinear-active moieties, molecules or particles (X).
  • linker Y is directly attached to the surface groups ofthe solid object and the nonlinear active components (20) are non-covalently adsorbed to the surface ofthe solid object.
  • Functional group (Y) on the linker (25) is reactive towards a target, another linker, or a solid object.
  • (30) represents a solid object used as a 'scaffold' whose surface area is used to attach linkers and nonlinear-active moieties, molecules or particles (40), (35) is a surface derivatized layer and (45) is a linker group.
  • Functional group (Y) on the linker is reactive towards a target, another linker, or another solid object.
  • (50) represents a solid object used as a 'scaffold' whose surface area is used to attach linkers and nonlinear-active moieties, molecules or particles, (55) are the covalently attached nonlinear-active moieties, molecules or particles and (60) represents the linker.
  • (X) is a functional group on the nonlinear-active component which covalently reacts with a surface group on the solid object and (Y) is the functional group on the linker which is reactive towards a target, another linker or another solid object.
  • (65) represents a solid object
  • (70) a surface layer which derivatizes the surface ofthe solid object and presents a functional group to the nonlinear-active components
  • (80) represents a linker which is directly attached to the surface ofthe solid object while (X) denotes the functional group on the nonlinear-active component which is reactive towards the functional group ofthe surface derivatization layer.
  • (e) represents a solid object
  • (90) represents a nonlinear-active component
  • (X) denotes the functional group ofthe nonlinear-active component which allows the latter to be covalently linked to the surface ofthe solid object
  • (Y) denotes the functional group of a linker (95) which is also part ofthe non-linear active component.
  • (Y) is reactive towards a target, another linker or another solid object.
  • Fig. 2 Metallic and Semiconductor Particles
  • a non-centrosymmetric metallic or semiconductor particle (100) is derivatized with linkers containing functional end-groups (Y).
  • the functional groups (Y) is reactive towards a target, another linker or another solid object.
  • the shape of the metallic particle (1) is drawn to emphasize its non-cenfrosymmetric nature.
  • a non-centrosymmetric metallic or semiconductor particle (105) is capable of directly attaching to a target without the need for a linker.
  • non-centrosymmetric particles (110) are attached together covalently via linkers (115) to create a composite particle which is overall non-centrosymmetric.
  • Another linker (120) is used to covalently link the composite to a target or solid object via an end-functional group (Y).
  • non-centrosymmetric particles (122) are adsorbed or aggregated together to create a composite particle which is overall non-centrosymmetric.
  • Linkers (124) containing an end-functional group (Y) are used to link the composite to a target or solid object.
  • centrosymmetric particles (125) are connected to each other via linkers (130) to create a composite particle which is overall non-centrosymmetric.
  • the centrosymmetric particles are covalently linked to each other via linkers, but they can also be adsorbed or aggregated to each other to create the composite.
  • the spherical shape ofthe particles is intended to emphasize their centrosymmetry.
  • Fig. 3 Linear chains of nonlinear active moieties where X indicates a chemically reactive group on a first nonlinear active moiety capable of bonding to Y on a second nonlinear active moiety.
  • the moieties can be assembled into a chain of desired length.

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Abstract

Selon cette invention, la génération d'une fréquence deuxième harmonique, d'une fréquence somme et d'une fréquence différence peut être utilisée pour détecter différents processus, sinon indétectables, à l'aide de marqueurs actifs non linéaires. Ces marqueurs doivent présenter une hyperpolarisabilité aussi élevée que possible. Plusieurs conceptions de grands marqueurs actifs de deuxième harmonique à hyperpolarisabilité sont décrites dans cette invention. Ces marqueurs peuvent être fixés à n'importe quelle molécule cible ou particule cible, ce qui a pour résultat l'obtention de cibles plus actives au niveau optique non linéaire.
EP01952798A 2001-02-01 2001-07-17 Conceptions de marqueurs pour detection a l'aide d'une technique optique non lineaire selective en surface Withdrawn EP1364204A1 (fr)

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EP1601652B1 (fr) * 2003-03-03 2008-07-30 Mycosol, Inc. Sels de pyridinium, composes et procedes d'utilisation
US8114843B2 (en) 2005-11-18 2012-02-14 The Regents Of The University Of California Photoreactive regulator of protein function and methods of use thereof
US9182406B2 (en) 2008-08-04 2015-11-10 Biodesy, Inc. Nonlinear optical detection of molecules comprising an unnatural amino acid possessing a hyperpolarizability
WO2012129347A1 (fr) 2011-03-21 2012-09-27 Biodesy, Llc Classification d'inhibiteurs de kinase à l'aide de techniques optiques non linéaires
WO2016106286A1 (fr) 2014-12-23 2016-06-30 Biodesy, Inc. Fixation de protéines à des interfaces destinées à être utilisées en détection optique non linéaire
EP3455628A4 (fr) 2016-05-09 2019-12-04 Biodesy, Inc. Méthodes et dispositifs de détection d'interactions de protéines membranaires périphériques à l'aide de techniques optiques non linéaires

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