WO1997012238A1 - Detection spectroscopique amelioree par des particules - Google Patents

Detection spectroscopique amelioree par des particules Download PDF

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Publication number
WO1997012238A1
WO1997012238A1 PCT/US1996/015729 US9615729W WO9712238A1 WO 1997012238 A1 WO1997012238 A1 WO 1997012238A1 US 9615729 W US9615729 W US 9615729W WO 9712238 A1 WO9712238 A1 WO 9712238A1
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Prior art keywords
particles
ofthe
solution
molecules
particle
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PCT/US1996/015729
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English (en)
Inventor
Kenneth David Hughes
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Georgia Tech Research Corporation
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Priority to AU73832/96A priority Critical patent/AU7383296A/en
Priority to EP96936099A priority patent/EP0876606A4/fr
Publication of WO1997012238A1 publication Critical patent/WO1997012238A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/84—Preparation of the fraction to be distributed
    • G01N2030/8429—Preparation of the fraction to be distributed adding modificating material
    • G01N2030/8441—Preparation of the fraction to be distributed adding modificating material to modify physical properties

Definitions

  • the present invention relates to a method of enhancing the detectable emission from emissive molecules (fluorophores, fluorogenic molecules, phosphors, or molecules capable of scattering electromagnetic radiation) in solution through the introduction of millimeter and sub-millimeter size particles.
  • emissive molecules fluorophores, fluorogenic molecules, phosphors, or molecules capable of scattering electromagnetic radiation
  • the emissive molecules may be added to the solution in a form that itself fluoresces, or as a fluorogenic material that is converted to a fluorescent material by the action of an enzyme or by another chemical reaction.
  • the present invention may be used to enhance the detectable emissions of fluorophores used in chromatographic separations, immunoassays, studies of reaction or enzymatic kinetics, chemical sensing of ion concentrations, or protein or DNA analysis.
  • Typical performance characteristics germane to all instruments include speed, simplicity, selectivity or specificity, detection limit, and cost.
  • chemical separation instruments/methods are included in the detection and quantitation of the targeted chemical species. Further development of this technology must occur if chemical/biological assays and the associated devices are to become more sensitive, rapid, and less expensive. 4 of porous solid lasing mediums that could be applied to television tubes to create bright. vivid displays.
  • the present invention comprises the addition of chemically inert particles in the millimeter and sub-millimeter size range to a solution containing emissive molecules, which may be fluorophores or fluorogenic (generating fluorophores under suitable conditions) molecules, phosphors, or molecules capable of scattering (such as Raman scattering) whose emission of electromagnetic radiation is to be detected by conventional spectroscopic-detection equipment.
  • emissive molecules which may be fluorophores or fluorogenic (generating fluorophores under suitable conditions) molecules, phosphors, or molecules capable of scattering (such as Raman scattering) whose emission of electromagnetic radiation is to be detected by conventional spectroscopic-detection equipment.
  • concentration ofthe particles in the solution reaches a critical concentration the emission ofthe molecules detected by the spectroscopic-detection equipment dramatically increases, desirably by a factor in the range of 1 to 20, preferably 5 to 20, times.
  • the excitation power of the excitation source which applies the electromagnetic radiation to the solution which typically may be a laser, light emitting diode, or broadband source coupled with a filter, appears to have little or no effect on the increased emissions. While not wishing to be bound by any theory, it is believed that the critical concentration at which increased emissions occur is related to the size ofthe added particles. As the particles get smaller, the critical concentration at which the enhancement effect is observed increases.
  • the present invention is a simple, nontoxic. inexpensive way to increase detection signal levels to an extent as large as or greater than 20 fold. It is applicable to detecting the presence of emissive molecules in any known solvents. It can also be combined with any or all ofthe other signal enhancement methods described above.
  • the invention is based upon use of small diameter particles that "trap" exciting electromagnetic radiation in the quasi-ordered array of particles, thus serving to increase the pathlength and/or magnitude of electromagnetic radiation interacting with the sample.
  • the particles have a diameter on the order of one millimeter or smaller, preferably in the submicron (e.g., nanometer) and micron range.
  • the particles may be either polydisperse or monodisperse, but typically polydisperse particles are used.
  • the invention can be applied to the analysis of static solutions as well as flowing solutions (as in separation instruments) of any volume.
  • antigens, antibodies, etc. Fluorophores are also used to monitor enzyme or reaction kinetics by studying the reaction of an enzyme or reactant that results in the conversion of a fluorogenic material into a fluorophore. Tagging of amino acids or nucleotides with fluorophores is also used in protein and DNA analysis, respectively. Fluorophores are also used in the chemical sensing of ion concentrations, such as calcium, magnesium and hydrogen ion (pH). When emission levels are low and difficult to detect, variations in the emission are difficult to quantify effectively.
  • ion concentrations such as calcium, magnesium and hydrogen ion (pH).
  • Lawandry, et al. describes generating stimulated fluorescence emission using titanium particles with aluminum oxide coatings. However, these particles were specifically designed to not aggregate in methanol solutions due to the desire of Lawandry to obtain a lasing effect, complicating and limiting the applicability ofthe described technique. Lawandry et al. do not teach or suggest aqueous systems. Further, Lawandry, et al. teaches using high concentrations of fluorescent dye in non-aqueous solution to generate radiation with characteristics similar to laser radiation. That is done by injecting pulsed radiation through the use of "front-face" excitation (i.e., the pulsed radiation is injected at an incident angle directly into the face ofthe cuvette containing the solution) as a preferred optical geometry for their application.
  • front-face excitation i.e., the pulsed radiation is injected at an incident angle directly into the face ofthe cuvette containing the solution
  • Lawandry, et al. describe techniques for generating lasing action through coated titanium particle injection of concentrated dye solutions, there is no description of using this technique to enhance the detection of chemical species and improve sensor sensitivity. Indeed, the Lawandry, et al. application, with its front face geometry, concentrated dye solutions, and use of non-aggregating particles to increase lasing activity, simply is not suited for sensor, chemical separation, or chemical reaction analysis applications. Practical applications of this technique for enhancing lasing efficiency through particle injection are not described. However, a related article by I. Peterson in Science News, vol.
  • the chemical composition ofthe particles also is not critical to the invention's operability, and the particles chosen can be inert, catalytic or reactive with one or more emissive molecules, or with fluorogenic molecules, as mentioned above.
  • a method and system of enhancing detectable emissions from emissive molecules in solution involves first providing a sample and then adding chemical reagents to the sample. Next, particles ranging in size from about 10 nanometers to about 1 millimeter, more particularly about 100 nanometers to about 10 microns are introduced into the sample. As pointed out above, the particles may be monodisperse or polydisperse.
  • Sufficient particles should be introduced to bring the particle density to a range from about 10 particles/milliliter to about 10 20 particles/milliliter.
  • the sample is then excited by exposing it to electromagnetic radiation, such as light emitted by a laser, light emitting diode, or broadband lamp, of a suitable wavelength and intensity, and the emissions from the excited sample are collected and measured.
  • the present invention can be used to detect the emission or scattering of electromagnetic radiation, such as fluorescence or phosphorescence using a fluorophore or phosphor concentration range at or below that used in current clinical or environmental methods using spectroscopic detection. For instance, detecting concentrations of single molecules is possible, however, a concentration of 10- 18 moles per liter is the current practical limit due to the costs involved.
  • the method can be applied to solutions that are static or flowing, that use aqueous or nonaqueous solvents, or combinations thereof, and can use a sample of any volume between or surrounding the particles or structures used to generate the effect.
  • the type and size ofthe container are not critical, and these solutions may be contained in any size vessel (e.g.. a cuvette or capillary) that is transparent to the exciting and emitting radiation. Exciting electromagnetic radiation may be provided for any discrete duration of time or duty cycle, or it may be continuous.
  • the location ofthe emissive molecule relative to the particles is not critical, and the method is also applicable where the emissive molecules interact in any manner with the
  • An optical geometry is utilized that can include, but is not limited to, ninety degree, front face, and epi-fluorescence (reflecting light from a source to the solution by a dichroic mirror). Either pulsed radiation or continuous exciting radiation may applied using such optical geometry. Use of continuous radiation does provide the signal enhancement effect.
  • this invention aims to provide a method for increasing the signal level of solutions of emissive molecules. Preferred embodiments are disclosed that involve the use of fluorescent molecules as the emissive molecules.
  • compounds that are themselves fluorophores are used. This embodiment may be used, for example, in chromatographic or immunoassay applications, where the fluorophores are desirably dissolved in a solution separate from the small particles.
  • a fluorogenic molecule is used that generates a fluorophore under a certain set of conditions. This embodiment may be used where it is desired to determine whether that set of conditions exists, e.g., in the study ofthe kinetics of an enzyme or reaction that converts the fluorogenic molecule to the fluorophore, or in the chemical sensing of ion concentrations.
  • This embodiment may use either dissolved or suspended fluorogenic molecules in solution, or may involve the binding of fluorogenic molecules to the surfaces ofthe small particles. In either case, the fluorophore is generated in the presence ofthe particles.
  • a particular embodiment includes using organic based polymeric particles, such as those made from polystyrene, for enhancing the emissive signal originating from aqueous solutions (which may contain portions of organic solvents, surfactants, modifiers, or other suitable components).
  • Dilute concentrations of a combination of different emissive molecules may be used, where the emissive molecules dissolve to form a homogenous phase in the solution or another emissive (or fluorogenic) molecule is attached to the particle surfaces to form a heterogeneous phase that enhances the spectroscopic signal observed from the molecules in solution.
  • the particles may be formed in any geometrical structure (e.g., spheres, rectangles, triangles, etc.).
  • Figure IA is a schematic diagram of a ninety-degree optical geometry using a solution according to the prior art.
  • Figure IB is a schematic diagram of a ninety-degree optical geometry using a solution according to the present invention.
  • Figure 2A is a schematic diagram of a front face optical geometry using a solution according to the prior art.
  • Figure 2B is a schematic diagram of a front face optical geometry using a solution according to the present invention.
  • Figure 3 A is a schematic diagram of analysis of a flowing fluid using a solution according to the prior art.
  • Figure 3B is a schematic diagram of analysis of a flowing fluid using a solution according to the present invention having particles dispersed therein.
  • Figure 3C is a schematic diagram of analysis of a flowing fluid according to an embodiment ofthe invention wherein the particles are fused to form a rigid structure.
  • Figure 4 A is a schematic diagram of an apparatus and method according to the present invention using a ninety-degree optical geometry.
  • Figure 4B is a schematic diagram of an apparatus and method according to the present invention using a front face optical geometry.
  • Figure 5 is a schematic diagram of a detection apparatus for use with a flow stream according to the present invention.
  • Figure 6 is a graph demonstrating a representative level of signal enhancement as a function of particle diameter and particle concentration.
  • Figure 7 is a graph, based on the information of Figure 6, demonstrating a linear relationship between particle diameter and concentration for given signal enhancement, at maximum emission. surface or interior ofthe particles or structures, e.g., by diffusing into the interior ofthe particles.
  • the present invention is applicable to use with enhancing the signal received from emissive molecules in applications such as immunoassays, monitoring of enzymatic or chemical activity or reactions, protein or DNA analysis, chemical sensing of ion concentrations, and any other analytical procedure where irradiation of a sample and measurement or determination of emission of electromagnetic radiation is used.
  • the specifics of these procedures are generally known in the art, and are not critical to the operation ofthe present invention, since the process ofthe present invention is applicable to a wide variety of different solutions and emissive molecules.
  • the present invention is particularly applicable to chromatography applications.
  • separations instruments such as chromatographs use some form of fluid flow to move chemical species through columns or flow lines, which may be packed or coated with a selected chemical or solid that interacts with the solution.
  • the fluid flow leaves the column and enters a detection region or vessel, such as a detection cell, where the presence of emissive molecules is to be detected.
  • Electromagnetic radiation such as light, is passed through the cell or the cell is otherwise excited in order to allow measurement of resulting emissions using a standard detector.
  • the particles may be introduced directly into the eluant of a chromatographic flow stream. This post-column addition avoids the loss or separation ofthe particles in the column itself.
  • the particles are inco ⁇ orated into the chromatographic mobile phase.
  • a permanent fixture of fused microparticles is placed into the flow stream, with the fluorophore molecules passing therethrough.
  • This fixture may be prepared from glass particles by chemically bonding the silanol groups thereof or by thermal fusing, or may be prepared by crosslinking polymer particles in a way that channels for fluid flow exist.
  • the fixture may also be prepared using particles having pores or channels therein.
  • an aliquot of particles is injected into the solution in a cuvette system.
  • Figures 6 and 7 shows the observed emission of fluorescence when inert polystyrene particles of known particle size are introduced into a fluorescein solution and that solution is irradiated with microwatt levels of 488 nanometer electromagnetic radiation. Starting with 0.2 micron particles, as shown in Fig.
  • the percent increase in emission remains flat until about a concentration of about 1 billion particles per milliliter is obtained, whereupon the percent increase in emission dramatically increases until a concentration of about 100 billion particles per milliliter is obtained, where the emission is almost 9 times the emission at baseline.
  • the breakaway from the baseline occurs at a concentration near 1 billion particles per milliliter and rises very quickly to a maximum in the range of 8 to 10 times the baseline emission.
  • the particles selected are 1.0 micron particles, chemically identical to the smaller particles described above, and the maximum emission increase is observed at about 100 million particles per milliliter, at which it is about 7 to 8 times the baseline intensity.
  • This fourth example is also shown in Fig. 6.
  • the emissive molecules used herein may include not only molecules capable of emitting electromagnetic radiation, such as fluorophores or phosphors, but also molecules capable of scattering electromagnetic radiation, such as organic or organometallic molecules, and compounds that react to form fluorophores and phosphors.
  • Suitable fluorophores for use in the present invention include fluorescein, rhodamine, and coumarine, and derivatives thereof.
  • Suitable derivatives of fluorescein include esters or carbohydrates thereof.
  • Suitable derivatives of coumarines include amino acid derivatives thereof.
  • Suitable derivatives of rhodamines include rhodamine derivatives suitable as oxidation probes.
  • Suitable phosphors include rare earth metals, po ⁇ hyrins, phthalocyanines, and other compounds known to exhibit phosphoresence.
  • Suitable particles for use in the present invention may include those made from organic compounds, in particular organic polymeric compounds, as well as those made from inorganic compounds and mixtures thereof.
  • Exemplary organic polymeric compounds suitable for use in the present invention include polystyrenes, acrylates, such as polymethylmethacrylates (PMMA) and butylmethylmethacrylate (BMA), acrylic polymers, divinylbenzene, and polyvinylacetate (PVA).
  • the fluorescein molecule is one of several fluorophore molecules that have important chemical and biological applications and the techniques for detecting such fluorophores in the analytical lab by the emission caused by an excitation source, particularly a broadbased source or laser, are well-known and documented.
  • Figures 1-3 show schematically generally two different situations in which the observed effect ofthe present invention is operable. In the first situation, fluorescent molecules are detected as they move past a point in a flow system, where excitation energy is directed at the flow stream, and emission ofthe molecules is observed. When particles of a size and concentration according to the present invention are introduced into the flow stream, the detected emission is greatly enhanced. In the second situation, the fluorophore or a 12
  • Emissive molecules that are dissolved or suspended in solution with appropriate concentrations of particles yield increases in emission intensity.
  • the instrument used in this work inco ⁇ orated a single stage spectrograph CCD detector and approximately 500 microwatts of excitation laser power. Plano-convex optics were used to introduce the excitation radiation into the cuvette and to collect the fluorescence emission from the cuvette and focus it on the detector.
  • Figure 6 provides the relationship between signal enhancement, particle size, and density for experiments that used a fixed micromolar concentration of fluorescein dye in water. This figure summarizes many experiments which involved monitoring the complete emission spectrum ofthe micromolar solution of fluorescein with various particle densities and diameters. Clearly the magnitude ofthe enhancement approaches the same value, but at different particle densities. As expected, as particle size increases the number of particles required for enhancement.
  • Figure 7 indicates that a linear relationship between particle If the particles selected are chemically inert in the solution, as is often desirable to maintain the integrity of the system being analyzed, the particles can be physically separated from the solution after the emission test, thereby regenerating the original solution. These particles can be removed using known filtration techniques. The particles that are preferred for this technique are readily available commercially, and are sold by particle size and extent of dispersion (monodisperse vs. polydisperse).
  • Figure 6 summarizes many experiments that involved monitoring the complete emission spectrum of a micromolar solution of fluorescein with various particle densities and diameters. Fluorescence signals were generated with continuous radiation from an argon-ion laser (488.0 nm). Fluorescence was detected in a ninety-degree geometry with a single stage spectrograph/CCD detector and approximately 500 microwatts of laser power. Clearly the magnitude ofthe enhancement approaches the same value for different particle diameters, but at different particle densities. As particle size increases, the number of particles required for enhancement decreases.
  • Figure 7 shows a linear relationship exists between particle diameter and number density for generating the intensity enhancement. Data for this plot was obtained by extrapolating the final limiting particle concentration of each curve in Figure 6 to zero percent intensity. While not wishing to be bound by any theory, it is believe that this particle is due to electromagnetic scattering ofthe radiation.
  • the fluorescent species that is generated by the enzyme in this case fluorescein, is dissolved in solution and free to fluoresce as described in the first embodiment.
  • fluorescein is dissolved in solution and free to fluoresce as described in the first embodiment.
  • a fluorescein diacetate derivative is used as a fluorogenic substrate for investigating the chemical activity of pig liver esterase.
  • This enzyme was obtained from Sigma Chemical Company and used in diluted form.
  • the enzyme in a suitable buffer in this case TRIS buffer, is placed in a standard four milliliter cuvette.
  • particles are injected into the cuvette to provide the signal amplification capability.
  • the particles are inert with respect to the enzymes.
  • the fluorogenic substrate is injected into the cuvette.
  • fluorescein molecules are produced.
  • Excitation and fluorescence signal measurement as described in the first embodiment is used for measurement ofthe fluorescent signal.
  • micromolar concentrations of fluorescein diacetate are used with nanomolar concentrations ofthe enzyme.
  • the kinetic curve that is typically produced by investigating the chemical activity ofthe enzyme is obtained.
  • the presence ofthe particles greatly increase the signal observed at initial time periods and increases the overall sensitivity ofthe measurement. After the experiment the particles can be collected for reuse by filtering with common papers and membranes and the original enzyme and fluorogenic substrate solution recovered.
  • the particles may serve two pu ⁇ oses: as a carrier of chemical reagents and as a catalytic surface for increasing the reaction rate of chemical reactions.
  • a carrier of chemical reagents and as a catalytic surface for increasing the reaction rate of chemical reactions.
  • metallic particles can serve as catalysts, and that chemical reactions of all types can be conducted at surface of particles.
  • particles with a diameter of 0.933 microns were synthesized with a surface coating of fluorescein diacetate.
  • the fluorescein diacetate is a fluorogenic substrate and can be attached to the surface of a particle containing amine functional groups through the use of a succinimydal ester functionality resident on the fluorogenic substrate.
  • An amide bond, as described in the second embodiment is formed.
  • volume and density exists for obtaining this signal enhancement. Data for this plot is obtained by extrapolating the final limiting particle concentration of each curve in Figure 5 to zero percent intensity.
  • Emissive molecules can be attached to the surface of sub-millimeter and sub-micron particles or inco ⁇ orated into the interior of these particles. It is quite common to do this for generating fluorescent tags and tracers used in experiments that examine flow characteristics in small tubes or vessels. Excitation of these modified particles can be by pulsed or continuous excitation.
  • fluorescein and rhodamine fluorescent organic molecules were attached to the surface of 0.933 micron diameter particles through the generation of an amide bond.
  • This bond can be produced by reacting a succinimydal ester group initially present on the fluorescent dye with an amine group on the surface of the particle. This reaction is commonly used in chemistry.
  • Particles containing surface coatings ofthe fluorescent dyes were injected into a cuvette containing water. The fluorescent signal was then measured. After calculating the number of fluorescent molecules attached to the particle surface a solution containing the same number of molecules, but now dissolved in the water was prepared. The fluorescence intensity from this solution was compared to the one containing particles with surface bound fluorophore.
  • the signal enhancement observed is similar to that described in the first embodiment.
  • Emissive molecules are often used as a foundation in which to build chemical substrates for monitoring the activity of enzymes or to report the presence of metal ions and other organic molecules such as sugars and vitamins. These derivatized fluorescent molecules are commonly referred to as substrates or indicators. In the case of substrates prepared for the investigation of enzyme activity, fluorescein, rhodamine, and coumarine are often used. Derivatization to the substrate form renders the molecule non-fluorescent until acted upon by the protein or enzyme. We demonstrated an embodiment of this invention by investigating the activity of enzymes that chemically react with ester organic functional 16
  • FIGs 4 and 5 illustrate apparatus for exciting a static solution 30 impregnated with particles 20 and measuring the resulting emissions 50.
  • Figure 4A illustrates a source 40, such as a laser, a broadband light source, or a light emitting diode, with which exciting radiation 42 can be directed onto the particle-impregnated solution 30.
  • Source 40 may be arranged so that the exciting radiation 42 passes through a dispersion element 70 before reaching the particle-impregnated solution 30 that is contained within a vessel transparent to the exciting radiation 42.
  • Dispersion element 70 may be an optical filter, grating or the like used to focus light of a desired wavelength upon the solution 30.
  • exciting radiation 42 stimulates the solution 30 and particles 20 therein in order to cause emissions 50.
  • Emissions 50 which may be amplified by a factor of about 5 to 10, enter a detector 80, which can more easily and accurately detect and analyze emissions 50.
  • another dispersion element 70 may be located between detector 80 and the solution 30 containing particles 20.
  • Figure 4B shows the same components ofthe exciting and measuring system. However, Figure 4A illustrates an exciting and measuring system arranged in a ninety degree optical geometry, whereas Figure 4B illustrates the system arranged in a front face optical geometry.
  • FIG. 5 illustrates a separation system for use with a flowing fluid 62.
  • a reservoir 90 of flowing fluid 62 couples to a pump 92 and an injection element 94 that injects the sample to be separated into the flowing fluid 62.
  • Flowing fluid 62 passes through a separation column 96, which may be packed or coated with a selected chemical or solid that interacts with the fluid 62.
  • the fluid flow 62 leaves the separation column 96 and enters a detection region 98, which may be a vessel or a detection column. Either before entering the separation column 96 or after exiting it, the particles 20 may be introduced into the flowing fluid 62.
  • a solid, fused structure of particles 20 can be placed in the path ofthe flowing fluid 62 in order to contact the particles 20 into the flowing fluid 62.
  • Electromagnetic radiation such as light
  • Electromagnetic radiation is passed through the detection region 98 or the detection region 98 is otherwise excited in order to cause emissions.
  • Those emissions These particles with surface coatings of fluorogenic substrate have been demonstrated by to be capable of probing metabolism in microorganisms and aquatic organisms and as probes for immune cell function, specifically endo- and phagocytosis, and enzymatic degradation.
  • the fluorescein "base” molecule remains attached to the particle surface after reaction with the enzyme.
  • fluorogenic molecules were included in the solution, rather than attached to the surface ofthe particles. Results similar to those discussed above were obtained. Those skilled in the art will recognize that other fluorogenic materials that become fluorescent in the presence of other catalytic and chemical reactions can be used by bonding them at the particle surfaces or by introducing them into the solution around/between the particles. 18
  • a method of enhancing detectable emissions or scattering from emissive molecules in solution comprising the steps of: introducing millimeter or submillimeter size particles into the solution and irradiating the solution with electromagnetic excitation energy, whereby the interaction between the particles and the excitation energy increases intensity of emissions or scattering.
  • the emissive molecule is a fluorogenic molecule that is converted into a fluorophore in the presence of said particles.
  • the emissive molecule is a molecule that is capable of scattering electromagnetic radiation.
  • Fluid 62 continues flowing through the separation system and ultimately is collected in waste reservoir 91. If the particles are chemically inert in the solution collected in reservoir 91 , they may be separated from the solution through mechanical filtering.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un procédé pour augmenter les niveaux des signaux (50) de solutions contenant des molécules émissives. L'augmentation du niveau du signal (50) découle de l'interaction entre de petites particules (20) et la radiation électromagnétique excitatrice (42). Le procédé peut être utilisé avec différentes géométries optiques, aussi bien dans des applications statiques que dynamiques.
PCT/US1996/015729 1995-09-28 1996-09-27 Detection spectroscopique amelioree par des particules WO1997012238A1 (fr)

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AU73832/96A AU7383296A (en) 1995-09-28 1996-09-27 Particle enhanced spectroscopic detection
EP96936099A EP0876606A4 (fr) 1995-09-28 1996-09-27 Detection spectroscopique amelioree par des particules

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US443895P 1995-09-28 1995-09-28
US60/004,438 1995-09-28

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GB2480640A (en) * 2010-05-26 2011-11-30 Univ Antwerpen Luminescence enhancing beads for bio-imaging

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US4549807A (en) * 1983-10-07 1985-10-29 At&T Bell Laboratories Process for measuring fluorescence
US5242837A (en) * 1990-12-24 1993-09-07 Slovacek Rudolf E Method for the rapid detection of analytes involving specific binding reactions and the use of light attenuating magnetic particles
US5376556A (en) * 1989-10-27 1994-12-27 Abbott Laboratories Surface-enhanced Raman spectroscopy immunoassay

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CA2076709A1 (fr) * 1992-08-24 1994-02-25 Ulrich J. Krull Emission amplifiee par fluorescence pour la transduction chimique
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US4097338A (en) * 1975-01-28 1978-06-27 Akzona Incorporated Fluorimetric demonstration and determination of a reduced coenzyme or derivative in an aqueous system
US4549807A (en) * 1983-10-07 1985-10-29 At&T Bell Laboratories Process for measuring fluorescence
US5376556A (en) * 1989-10-27 1994-12-27 Abbott Laboratories Surface-enhanced Raman spectroscopy immunoassay
US5242837A (en) * 1990-12-24 1993-09-07 Slovacek Rudolf E Method for the rapid detection of analytes involving specific binding reactions and the use of light attenuating magnetic particles

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Title
NATURE, Volume 368, issued 31 March 1994, N.M. LAWANDY et al., "Laser Action in Strongly Scattering Media", pages 436-438. *
See also references of EP0876606A4 *

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AU7383296A (en) 1997-04-17
EP0876606A1 (fr) 1998-11-11

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