WO1997012238A1

WO1997012238A1 - Particle enhanced spectroscopic detection

Info

Publication number: WO1997012238A1
Application number: PCT/US1996/015729
Authority: WO
Inventors: Kenneth David Hughes
Original assignee: Georgia Tech Research Corporation
Priority date: 1995-09-28
Filing date: 1996-09-27
Publication date: 1997-04-03
Also published as: EP0876606A1; EP0876606A4; AU7383296A

Abstract

A method for increasing the signal levels (50) of solutions containing emissive molecules. The increase in signal level (50) is derived from an interaction between small particles (20) and the excitation electromagnetic radiation (42). The method may be used in different optical geometries and in both flow and static applications.

Description

PARTICLE ENHANCED SPECTROSCOPIC DETECTION

Cross-Reference to Related Application This application claims priority to provisional application Serial No. 60/004,438, filed on September 28, 1995, which application is hereby incorporated in its entirety by this reference.

Background ofthe Invention 1. Field ofthe Invention 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. When the emissive molecules are fluorophores, they 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. As a result, 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.

2. Description ofthe Background Art

The use of electromagnetic emission from molecules is used routinely to qualitatively and quantitatively examine solutions containing chemical species from biological and synthetic origin. This physical phenomena which requires an excitation source and an appropriate electromagnetic radiation detector has been the basis for many chemical assays and numerous commercial instruments are available for making these measurements. These commercial instruments range in size from small hand held devices to large benchtop models. These instruments are vital tools in many fields of science including biomedical and environmental monitoring and have a direct impact on society.

Typical performance characteristics germane to all instruments include speed, simplicity, selectivity or specificity, detection limit, and cost. In many cases 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.

Summary ofthe Invention

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. When the 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.

Recent work by N.M. Lawandry, et al., reported in "Laser Action in Strongly Scattering Media," Nature, vol. 368, p. 436 (March 31. 1994), describes developments in laser gain media and generating photonic devices that can deliver laser radiation.

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.

Thus, while 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. 145 at 228 (April 1994), reported that possible applications of Lawandry's work included: (1) dermatological creams to help remove skin discolorations upon excitation ofthe cream; (2) reflective paints; and (3) possible creation 6 ofthe particle to alter the difference in refractive index between the particle surface and the solution.

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. Generally, 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.

In one embodiment ofthe invention, 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. In another embodiment of the invention, 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. Although a variety of emissive molecules, particles and solutions may be used with the present invention, 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.

Surface moφhology of particles used in the present invention is not critical to its operability. For instance, the particles may be formed in any geometrical structure (e.g., spheres, rectangles, triangles, etc.). However, it may be possible to tune the increase of intensity ofthe emissions in a specific system by coating or otherwise modifying the surface 8 specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope ofthe invention will become apparent to those skilled in the art from the description herein of the invention. Brief Description ofthe Drawings

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.

Generally, 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.

It is accordingly an object ofthe present invention to provide a method for enhancing measurement signals of electromagnetic radiation emitted by emissive molecules that involves enhancing emissions from a test solution containing these molecules.

It is an additional object ofthe invention to provide a method for enhancing such emissions by introducing particles, for instance inert particles of millimeter or submillimeter size, into a test solution to enhance emissions resulting from excitation of the solution when it is exposed to electromagnetic radiation.

It is a further object ofthe invention to provide a apparatus for introducing such particles and measuring the resulting increased emissions.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the additional disclosure below and in the drawings. However, it should be understood that the drawings, detailed description, and 10 fluorogenic molecule, such as diacetyl fluorescein, is attached to the surface ofthe particle. This attachment may be accomplished using the procedures described in U.S.

Patent Application Serial No. 08/327,286, the entire contents of which are hereby incoφorated by reference. Both ofthe situations discussed above may be implemented using several different embodiments. In one embodiment, 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. In a second embodiment, the particles are incoφorated into the chromatographic mobile phase. In a yet third embodiment, 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. In a fourth embodiment, 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. 6, 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. When chemically identical particles at 0.5 or 0.7 microns are used, as shown Fig. 5, 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.

In a fourth set of experiments, 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. Detailed Description ofthe Specific Embodiments As discussed above, the present invention is applicable to a wide variety of systems using different emissive molecules, different solvents, and different particles and particle sizes. 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, poφhyrins, 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

First Embodiment:

Emissive molecules that are dissolved or suspended in solution with appropriate concentrations of particles yield increases in emission intensity.

Aqueous solutions containing micromolar and nanomolar concentrations of emissive dyes, such as rhodamine and fluorescein, have been used to demonstrate this invention. These concentrations are quite typical of solution concentrations analyzed in clinical and environmental investigations. It is also quite common to produce calibration curves in fluorescent assays, and this idea was used to demonstrate the sensitivity (signal increase) resulting from the injection of particles. In these demonstrations, experiments are conducted with coherent continuous radiation at 488 nanometers from an argon-ion laser. Standard four milliliter quartz fluorescence cuvettes were used. A ninety degree optical geometry was used for the measurement. This is a standard optical geometry in many commercial instruments. The instrument used in this work incoφorated 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.

Experiments were conducted by initially measuring the fluorescence signal from the cuvette containing the solution of dye and no particles. After this was completed, microliters of a concentrated particle solution were injected into the cuvette. Injection of different volumes generated solutions with different particle concentrations. Again, the fluorescence signal was measured with the detector. An increase in fluorescence intensity was observed. By varying the particle concentration over a typical application range, it was observed that the enhancement ofthe signal maximizes at particle concentrations dependent on the particle diameter.

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.

Example Embodiments A number of exemplary embodiments ofthe present invention are described below. These descriptions represent summaries of a number of experiments.

groups. 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. Those skilled in the art will recognize that other catalytic and solution based homogeneous chemical reactions can be conducted in solution around and between the particles. Specifically, in these experiments 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. First, the enzyme in a suitable buffer, in this case TRIS buffer, is placed in a standard four milliliter cuvette. Next, particles are injected into the cuvette to provide the signal amplification capability. The particles are inert with respect to the enzymes. Finally, the fluorogenic substrate is injected into the cuvette. Immediately upon introduction, fluorescein molecules are produced. Excitation and fluorescence signal measurement as described in the first embodiment is used for measurement ofthe fluorescent signal. Typically micromolar concentrations of fluorescein diacetate are used with nanomolar concentrations ofthe enzyme. In this application 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.

Fourth Embodiment:

In some applications the particles may serve two puφoses: as a carrier of chemical reagents and as a catalytic surface for increasing the reaction rate of chemical reactions. Those skilled in the art will recognize that many metallic particles can serve as catalysts, and that chemical reactions of all types can be conducted at surface of particles.

In a demonstration of this embodiment, 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.

Second Embodiment:

Emissive molecules can be attached to the surface of sub-millimeter and sub-micron particles or incoφorated 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.

In order to demonstrate this application, 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. The same instrumentation and procedure as described in the first embodiment was used in these experiments. 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.

Third 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

Measuring System Figures 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. In any event, 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. Optionally, 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.

Figure 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. Alternatively, 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, 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. In each of these examples, the fluorescein "base" molecule remains attached to the particle surface after reaction with the enzyme.

Experiments utilizing these particles are carried out in a way similar to that which is described in the third embodiment. The difference is that the fluorogenic substrate (fluorescein diacetate) and the particles are injected simultaneously into the cuvette containing enzyme and buffer. Fluorescence signals are measured as described in the previous embodiments. In these systems the rate of enzyme activity (substrate turnover) was increased due to the attachment ofthe fluorogenic substrate to the surface. This is a possible advantage over embodiments where the fluorogenic substrate is not attached to the particle surface.

Fifth Embodiment:

The detection of emissive molecules in flow streams is the basis of many detectors utilized in chemical separation instruments. Based upon the disclosure above, it should be clear to anyone skilled in the art that having particles and emissive molecules in a flow stream of solvent will generate the same signal enhancements as discussed above. With respect to chemical separations it is quite common to label or tag a mixture of non-emissive chemical species with a fluorescent species and then to separate the mixture. Analysis of proteins, amino acids, DNA, RNA, and the individual base pairs by labeling is quite common and well known in the art. Passing a flow stream containing these tag species or species that do not require labeling because they are naturally emissive is covered by the present invention.

Sixth Embodiment:

In this embodiment, 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

What is claimed is:

1. A method of enhancing detectable emissions or scattering from emissive molecules in solution, the method 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.

2. The method of claim 1 , wherein said particles are of micron or submicron size.

3. The method of claim 1 , wherein the emissive molecule is a fluorophore.

4. The method of claim 1, wherein the emissive molecule is a fluorogenic molecule that is converted into a fluorophore in the presence of said particles.

5. The method of claim 1, wherein the emissive molecule is a phosphor.

6. The method of claim 1, wherein the emissive molecule is a molecule that is capable of scattering electromagnetic radiation.

7. The method of claim 1 wherein the solution comprises a flowing fluid stream eluting from a chromatographic column, and the particles are introduced into this column downstream of said chromatographic column.

8. The method of claim 1 wherein the solution comprises a chromatographic mobile phase, the particles are introduced into the chromatographic mobile phase prior to or during passage through the column. which are increased because ofthe presence ofthe particles 20 within the fluid flow 62, are collected by the detector 80.

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.

The foregoing is provided for puφoses of explanation and disclosure of preferred embodiments ofthe present invention. For instance, the type, size and concentration of particles can vary greatly depending on the desired emissions increase, yet still fall within the following claims. Further modifications and adaptations to the described embodiments will be apparent to those skilled in the art and may be made without departing from the scope or spirit ofthe invention and the following claims.

Claims

20

19. The method of claim 1, wherein the particles comprise an inorganic material.

20. The method of claim 1 , wherein the particles comprise a mixture of particles made of an organic material and particles of an inorganic material, or particles composed of both organic and inorganic materials.

21. The method of claim 1 wherein the excitation energy is produced by a device selected from the group consisting of: a laser; a light emitting diode; a broadband source and an optical filter that passes only a selected band of wavelengths.

22. The method of claim 21 , wherein the excitation energy is applied via a laser.

23. The method of claim 21 , wherein the excitation energy is applied via a light emitting diode.

24. The method of claim 21 , wherein the excitation energy is applied via a broadband lamp.

25. The method of claim 1 further comprising the step of observing the results of the increased emissions, wherein observation ofthe increased emissions can be made at any angle relative to the incidence ofthe excitation source on the sample.

9. The method of claim 1 wherein the solution comprises a flowing fluid stream and the particles are introduced as a permanent fused fixture through which the fluorophore molecules pass, and which is placed into the flow stream.

10. The method of claim 1 wherein the solution comprises a batch sample within a vessel into which particles are introduced.

11. The method of claim 1 wherein the particles are organic polymeric particles.

12. The method of claim 11 , wherein the organic polymer is selected from the group consisting of polystyrene, polymethylmethacrylate, divinylbenzene, acrylic polymer, butylmethylmethacrylate, and polyvinyl acetate.

13. The method of claim 1 wherein the particles are chemically inert in the solution.

14. The method of claim 13 further comprising the step of separating the particles from the solution.

15. The method of claim 1 wherein the mean diameter ofthe particles ranges from about 1 nanometer to about 1 millimeter.

16. The method of claim 15, wherein the mean diameter of the particles is in the range of about 100 nanometers to about 10 microns.

17. The method of claim 11 wherein the density ofthe particles within the solution ranges between about 10 particles/milliliter and about 10 20 particles/milliliter.

18. The method of claim 17 wherein the concentration of the particles in the solution ranges from about 100 million to 100 billion particles per milliliter.