Patent Grant
9063007
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The present invention relates to luminescence systems for irradiating bioanalytical instrumentation including wells containing chemicals for inducing reactions or detecting reactants or products of chemical reactions. The bioanalytical instrumentation can include a light source and fiber optic systems for irradiating analytes within capillaries with selected wavelengths of light and detecting luminescence produced by the analytes within the capillaries.
The micro titer plate reader has been a workhorse for bioanalytical testing for decades. It enables the facile and rapid interrogation of an array of chemical reactions. Typically 8 by 12 well formats of 96 wells are filled with reagents and or products of a colorimetric or fluorescent reaction. Higher order formats include multiples of 96 wells. Such micro-titer plates are exposed to light of a desired wavelength and the interaction of the reacting species with the light is recorded.
Reactions may be immunochemical, enzyme based, polymerizations, intercollations or any of the varied molecular biological and biochemical systems investigated in the biochemists' laboratory. The interactions may include and are not limited to absorbance, transmittance, scatter and fluorescence. Micro titer plate readers are designed to generate one or more wavelengths of interest. Typically the light is generated using a wide spectrum source, arc lamps and halogen bulbs with numerous filters or gratings are common design components.
Biochemical reactions formatted in either homogeneous or heterogeneous based detection platforms are also performed in miniaturized systems, e.g. on micro fluidic chips and micro arrays. In miniaturized systems, reaction volumes are contained within channels and/or with carefully modified surface chemistry to allow for a plethora of chemical and biological analysis in small reaction volumes. The chemistry is typically interrogated using confocal microscopy or imaging to assess the extent of higher density features. Tens to hundreds of thousands of enzyme, immunochemical, nucleic acid and protein reactions can be followed simultaneously. Lamp and lasers power the various detection systems designed to report on the extent of reaction.
One now commonplace procedure performed in the bioanalytical laboratory is the polymerase chain reaction (PCR). The technique has become fundamental to molecular biology. It is one of a family of methods (i.e. reverse transcriptase PCR) for synthesizing a given quantity of pre-selected biopolymer. PCR functions on DNA. In a typical experiment, the DNA of interest is separated into two complementary strands. The ends of each strand bind to a primer at the end where the synthesis begins. The addition of the DNA polymerase initiates the synthesis of a complementary strand on each single strand creating a doubling of the amount of DNA. The process is repeated until a sufficient number of DNA segments have been synthesized. A unique temperature profile is used to advance the reaction through the phases of separation (melting), primer binding (annealing), and replication (extension). While the PCR technique has become a workhorse of the biotechnologist due to the enhanced sensitivity it offers over blotting techniques, PCR is not ideally suited to quantitation. Small differences between sample sizes can become huge differences in final amplified material after multiple doublings.
A typical PCR reaction can be seen in three phases: an early lag phase, an exponential growth phase and a plateau region. The lag phase is a consequence of the sensitivity of the instrument and the background signal of the probe system used to detect the PCR product. The exponential growth phase commences when sufficient product has accumulated to be detected by the specific instrument. During this exponential growth the amplification is described by Tn=Ti(E)n, where Tn is the amount of target sequence at cycle n, Ti is the initial amount of target material, and E is the efficiency of amplification. In the final plateau phase the amplification efficiency drops as product competes more effectively with primers for annealing and the amount of enzyme becomes limiting. The exponential equation no longer holds in the plateau phase. Most of the quantitative information is found in the exponential cycles but the exponential cycles typically comprise only 4 or 5 cycles out of 40. For traditional PCR methods, identifying the exponential cycles requires the reaction be split into multiple reaction tubes that are assayed for PCR product after varying numbers of cycles. This requires either assaying many tubes or having a fairly good idea of the answer before the experiment is begun. Once the position of this phase is determined the experimental phase can be compared to known standards and the copy number can be calculated.
Instrumentation advancements have made real-time monitoring of PCR reactions possible. Thermocycling is carried out using standard techniques known to those skilled in the art, including rapid cycling PCR. Fluorescence monitoring at each cycle for quantitative PCR was developed by Higuchi et al., “Simultaneous Amplification and Detection of Specific DNA Sequences,” Bio. Technology, 10:413-417, 1992, which is herein expressly incorporated by reference in its entirety. Ethidium bromide was used as the fluorescent entity. Fluorescence was acquired once per cycle for a relative measure of product concentration. The cycle where observable fluorescence first appeared above the background fluorescence correlated with the starting copy number, allowing the construction of a standard curve. Alternatively PCR amplification may be conducted with fluorescently labeled hybridization probes. The hybridization probe system comprises two oligonucleotide probes that hybridize to adjacent regions of a DNA sequence wherein each oligonucleotide probe is labeled with a respective member of a fluorescent energy transfer pair. In this embodiment, the presence of the target nucleic acid sequence in a biological sample is detected by measuring fluorescent energy transfer between the two-labeled oligonucleotides. A number of strategies now exist using molecular beacons or intercollating dyes all of which are strategies to increase signal as a function of increasing DNA concentration as the synthesis cycles increase.
Such instrumentation and fluorescent monitoring techniques have made kinetic PCR significantly easier than traditional competitive PCR. The ease, accuracy and precision of quantitative PCR have all improved by allowing observation of the PCR product concentration at every cycle. In the Roche® Diagnostics embodiment of the kinetic PCR instrument, PCR reactions are conducted using the Light Cycler®, a real-time PCR instrument that combines a rapid thermal cycler with a fluorimeter. Through the use of such a device, PCR product is detected with fluorescence and no additional sample processing, membrane arrays, gels, capillaries, or other analytical tools are necessary. Other PCR instrumentation as known in the art may be used in the practice of the present invention.
Separation by electrophoresis is based on differences in solute velocity in an electric field. The velocity of a charged analyte is a function of its electrophoretic mobility and the applied voltage. The method of electrophoresis is used in a number of different techniques including capillary gel electrophoresis, capillary zone electrophoresis, micellar electrokinetic chromatography, capillary electro chromatography, isotachophoresis and isoelectric focusing.
In general, the mobility of an analyte in a particular medium is constant and characteristic of that analyte. The analytes mobility is a result of two factors. The analyte is attracted to the electrode of opposite charge, pulling it through the medium. At the same time, however, frictional forces try to prevent the analyte moving toward the charge. The balance of these forces determines the actual overall mobility of the analyte. An analytes size, polarity and number of electric charge(s), relative hydrophobicity and ionic strength determine how rapidly an electric field can move the analyte through a medium. A buffer is used to assist the flow of the analyte relative to the field. The buffer's chemical composition, pH, temperature and concentration alter the mobility of the analyte. Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionizable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations containing a positive (+) charge or as anions containing a negative (−) charge. Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode. A small analyte will have less frictional drag than a large analyte and hence move through the medium faster than a large analyte. Similarly, a multiply charged analyte will experience more attraction to the electrode and also move through the medium faster than a singly charged analyte. It is this difference in solute velocities that is responsible for the separating effect in electrophoresis that results in resolution of the species detected.
Gel electrophoresis is a method that separates molecules such as DNA or proteins on the basis of their physical properties. A gel is a solid colloid. Thus, gel electrophoresis refers to the technique in which molecules are forced to cross a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the electric field and thus the driving force for the migration of the analyte. During electrophoresis, molecules are forced to move through the pores in the gel when the electrical current is applied. Their rate of migration, through the induced electric field, depends on the strength of the field, their charge, their size and the shape of the molecules, the relative hydrophobicity of the molecules, and on the ionic strength and temperature of the buffer in which the molecules are moving.
One use of gel electrophoresis is the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis, after being cut with various restriction enzymes. Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this way. Another use is the isolation and purification of individual DNA fragments containing interesting genes, which can be recovered from the gel with full biological activity.
Capillary Zone Electrophoresis (CZE) replaces the gel in gel electrophoresis with the combination of a buffer and a solid support contained within the capillary. In CZE, the analyte must move through the solid support contained within the capillary under the action of the buffer, which is charged by the applied electric field. The buffer's chemical nature, pH, temperature, concentration and the presence of surfactant additives can be selected to assist in fully resolving (i.e., spatially separating different analytes in the capillary with respect to the time from introduction of the sample) different analytes in space (position in the capillary) with respect to time. Analytes separated by CZE can be detected based on absorption or fluorescence. Detection can be carried out using on-column or fiber optic Z-cells.
In addition to electrophoretic techniques, separation of molecules can be carried out in the absence of an applied field using chromatographic techniques. In liquid chromatography, the molecule dissolved in a buffer can still be charged, but rather than an electric field creating the driving force, molecule migration is dependent on the flow of the buffer. Frictional forces due to the interaction of the molecule with a solid support present in a column, act to prevent the molecule from moving with the buffer. The molecule's size, hydrophobicity, and ionic strength determine how rapidly the buffer can move the molecule through a medium. The buffer's chemical composition, pH, temperature and concentration together with the nature of the solid support dispersed in the column alter the mobility of the molecule. High performance liquid chromatography (HPLC) utilizes pumps to increase the flow of buffer through the columns resulting in high column backpressure, improved resolution, increased flow rates and reduced analysis times. By reducing the diameter of the column and/or increasing the length of the column the resolution can be improved. However, a problem with narrower columns (milli bore or micro bore) involves detection of the eluted species. As the diameter of the capillary in the narrow bore HPLC systems is further reduced, only a small number of molecules are available for detection in a small-defined area.
Microfluidic systems comprised of microfluidic chips, automated reagent delivery apparatus and detection instrumentation are designed to minimize the users' effort in reagent delivery, reagent dilution and/or mixing, initiating chemical reactions and detecting those chemical reactions in small volumes within highly automated environments. Among the numerous applications that exist, fluorescence is a commonly used detection format. It is a sensitive and robust method for detecting enzyme assays, immunoassays, polymerase chain reaction (PCR), quantitative PCR, genomic sequencing among many other important chemical reactions. Both homogeneous and heterogeneous reactions are suited to such devices and analysis is not limited by whether the reaction takes place in free solution or on a solid support or within a narrow pore. Often microfluidic devices are produced by etching, molding or embossing channels and wells into solid substrates (glass, silicon, plastic, etc.). Numerous layers of the device can be fabricated and then the layers assembled to form the final analysis tool. Channels can be etched in single or multiple dimensions enabling more complicated chemical separation and detection. Such devices can be used to introduce reagents directly onto the chip or interfaced with automation equipment for such purposes. Like all fluorogenic detection, these systems require an excitation source.
Light based detection systems utilizing the processes described above have long been workhorses of chromatography systems and reaction vessels including microarray scanners, microtiter plate readers, DNA sequencers, PCR and Q-PCR instruments, fluorescent microscopes, flow cytometery instruments and lab on a chip devices used in drug discovery and other life-sciences research. Light sources are integral components of these bioanalytical tools. However, the lamps and lasers that power these bioanalytical systems have presented engineering and cost constraints that limit sensitivity, reproducibility and robustness.
The present invention consists of one or more light sources in the form of a luminescent light pipe referred to herein as a lamp, in conjunction with relay optics for luminescence collection from an analyte forming a luminescence system for a volume interrogation apparatus wherein the interaction of light with a chemical species located within or supported on a solution volume can be the measure of the presence or quantitation of an analyte. Luminescence is defined as light not generated by high temperature alone, typical of incandescence, including but not limited to fluorescence and phosphorescence. Where high temperatures are defined as above approximately 2000° K. The analyte can be part of a reaction involving species including biopolymers such as, oligonucleotides (DNA, RNA iRNA, siRNA), proteins (including antibodies, enzymes, agonists, antigens, hormones, toxins), oligosaccharides and non polymeric species such as steroids, lipids, phospholipids, small organic signaling molecules (e.g., retinoic acid), pesticides and non peptidic toxins, hormones and antigens.
In alternative embodiments of the present invention, a lamp, in conjunction with relay optics for luminescence collection, form a flexible and efficient luminescence system for a capillary/fluorescence apparatus. In an embodiment of the invention, a plurality of light sources and fiber optic systems separately and simultaneously irradiate a plurality of capillaries with selected wavelengths of light and the fluorescence produced by the molecules flowing within the capillaries can be separately and simultaneously detected.
While lamps and lasers are key components in the biochemical reactor instrument design each is best suited to unique applications with compromises based on inherent performance traits. Typically lamps produce broad-spectrum spontaneous emission but due to their large angular output collection efficiency is poor. Large power densities are difficult to attain; moreover accessing discrete wavelengths using filters and lenses results in dramatic power losses. Lasers can produce large power outputs at discrete wavelengths based on stimulated emission; however, intensity and spatial modulation is difficult and costly. As well, the number of available discrete wavelengths is limited. The design and cost effective production of bench top and point-of-care analyzers is limited by this current pool of light generators.
Traditional lamps and lasers are the most frequently employed light generators in bioanalytical microfluidic instrumentation. While lamps and lasers are key components in the instrument design each is best suited to unique applications with compromises based on inherent performance traits. Typically lamps produce broad-spectrum spontaneous emission but due to their large angular output, collection efficiency is poor. Further, large power densities are difficult to attain. Moreover, accessing discrete wavelengths using filters and lenses results in dramatic power losses. Lasers can produce large power outputs at discrete wavelengths based on stimulated emission, however, intensity and spatial modulation is difficult and costly. In addition, the number of available discrete wavelengths is limited. The design and cost effective production of bench top and point-of-care analyzers is limited by the current light generators.
Lamp
In various embodiments of the present invention, a lamp emits wavelengths of light, which excite fluorescence from photosensitive targets in the sample of interest. In various embodiments of the present invention, a lamp can be in the form of a tube, rod, or fiber of varying or constant diameter and varying or constant curvature. The cross section can be circular, square or rectangular and can be constant or varying. In various embodiments of the present invention, a constituent light pipe can be made of glass, plastic, single or multiple inorganic crystal(s), or a confined liquid. In various embodiments of the present invention, a pipe either contains or can be coated with a layer or layers containing, a narrow band luminescent material such as organic or inorganic compounds involving rare earths, transition metals or donor-acceptor pairs. In various embodiments of the present invention, a lamp emits confined luminescence when excited by IR, UV, or visible light from an LED, laser, fluorescent tube, arc lamp, incandescent lamp or other light source. In an embodiment of the present invention, a lamp operates through the process of spontaneous emission, which results in a much larger selection of available wavelengths than can be available for efficient stimulated emission (laser action). In an alternative embodiment, electrons or other radiation are used to excite the activator and thereby the light pipe.
In an embodiment of the invention, light pipes will incorporate organic luminescent material referred to as plastic scintillators, doped or activated plastic optical fibers or wavelength shifting plastics as described by Albrecht, M. et al., in “Scintillators and Wavelength Shifters for the Detection of Ionizing Radiation”, Nuclear Science Symposium Conference Record, 2003 IEEE, Volume 2, Issue: 19-25 Oct. 2003 1086 and Pla-Dalmau, A. et al., “Low-cost extruded Plastic Scintillator”, Nuclear Instruments and Methods in Physics Research A, 466 (2001) 482 both of which are hereby expressly incorporated by reference in their entireties. These materials are commercially available through Kuraray, Saint-Gobain and Eljen Technology. The host material can be typically made from polymethylmethacrylate or acrylic (PMMA), polyvinyltoluene, or polystyrene. There are numerous fluorescing dopants available enabling these materials to emit across the visible from 400 to 650 nm. The best dopant for the Argon Ion laser replacement is K27. The plastic material can take on any geometric shape including uncladded fiber, cladded fiber, tubes and rods with circular, square or rectangular cross section which can be constant or varying. The tubes and rods can have constant or varying curvature and have a constant or varying diameter.
Relay Optics
In an embodiment of the present invention, relay optics consist of light pipes, optical fibers, lenses and filters, which optically transport the light from a lamp to one or more capillaries and light pipes, optical fibers, lenses and filters which collect and transport any generated fluorescence to an appropriate detector or array of detectors, in conjunction with adaptors for coupling the excitation light into the capillaries, coupling the emission light out of the capillaries and for enhancing physical discrimination of the excitation and emission. In an embodiment of the present invention, relay optics, including fibers, can be constructed in a loop or as a cavity so that light from a lamp can pass through one or more capillaries multiple times to enhance excitation efficiency.
In an embodiment of the present invention, a number of lamps each emitting one or more color of light can have their constituent light pipes coupled in parallel or in series acting to produce multiple colors simultaneously or in sequence. In an embodiment of the present invention, one or more lamps can illuminate single channels, multiple parallel channels, multiple channels in multiple dimensions, numerous spots along the analysis channel and/or reservoirs connected to the flow streams.
In an embodiment of the present invention, lamps can be irradiated continuously during the measurement process or can be pulsed on and off rapidly to enable time-based detection methods. In an embodiment of the present invention, a lamp can be switched off between measurements, to eliminate the heat output. This can be contrasted with alternatives such as arc lamps or lasers that are unstable unless they are operated continuously.
Luminescence and Collection System
In an embodiment of the present invention, a flexible luminescence and collection system for capillary/fluorescence apparatus allows for a varying number of samples to be analyzed simultaneously. ‘Simultaneously’ is herein defined as occurring close in time. Two light pipes can irradiate two capillaries at the same time and the fluorescence from the molecules in one of the capillaries can be delayed due to physical or chemical effects relating to absorption, phosphorescence and/or fluorescence resulting in a delay in the fluorescence from the molecules in one of the capillaries. This excitation can be still considered to result in ‘simultaneous detection’. In an embodiment of the present invention, a luminescence and collection system can be adjusted for uniform luminescence of multiple capillaries or wells or a large area including numerous wells, spots or channels as ‘detection volumes’. In an embodiment of the present invention, luminescence systems can irradiate an array of channels in an array of capillaries. In an embodiment of the present invention, an array of channels can be etched, molded, embossed into the capillaries. In an embodiment of the present invention, a set of wells intimately connected to fluidic conduits can be stepped along the length of the fluidic conduit such that they can be interrogated at numerous sites for the purposes of creating a map or image of the reacting species.
In an embodiment of the present invention, a luminescence and collection system can irradiate an array of wells, spots and or an array of channels (be they etched, molded or embossed) or a set of wells intimately connected to fluidic conduits such that they can be interrogated at numerous sites for the purposes of creating a map or image of the reacting species.
In an embodiment of the present invention, a luminescence and collection system can irradiate homogeneous reactions within fluidic conduits or reservoirs; to irradiate heterogeneous reactions on the surface of fluidic conduits or reservoirs; to irradiate homogeneous or heterogeneous reactions on the surface of or within the pores of a porous reaction support.
In an embodiment of the present invention, a luminescence and collection system can emit multiple colors as desired. In an embodiment of the present invention, a luminescence and collection system can be pulsed on and off as desired to reduce heat generation. In an embodiment of the present invention, a luminescence and collection system can be pulsed on and off to allow time-based fluorescence detection.
In an embodiment of the present invention, a luminescence and collection system can detect one or a number of reactions within the detected volume or volumes. The narrow band source of the light pipe driven analyzer provides better specificity, higher sensitivity, and lower backgrounds signals. The light pipe driven analyzer easily accommodates multiple wavelengths by additions of serially connected components.
In an embodiment of the present invention, a luminescence and collection system can be pulsed on an off as desired to reduce or control heat generation and to allow time-based fluorescence detection.
In an embodiment of the present invention, luminescence systems can irradiate homogeneous reactions within fluidic conduits or reservoirs. In an embodiment of the present invention, luminescence systems can irradiate heterogeneous reactions on the surface of fluidic conduits or reservoirs. In an embodiment of the present invention, luminescence systems can irradiate homogeneous or heterogeneous reactions on the surface of or within the pores of a porous reaction support.
Other objects and advantages of the present invention will become apparent to those skilled in the art from the following description of the various embodiments, when read in light of the accompanying drawings.
Various embodiments of the present invention can be described in detail based on the following figures, wherein:
The detection volume in the form of a well of fluid, a spot of fluid, a channel containing fluid or a reservoir attached to a channel containing fluid will all be referred to herein as the “detection volume”. The term “detection volume” can also refer to any of the afore mentioned constructs in which the reaction for detection occurs in freely diffusing solution, in a gel or polymer, attached to a surface, contained within a pore, or in some subsection of the entire well volume. As seen in
In an embodiment of the present invention, the source can be coupled to the detection volumes using a special adapter. The adapter simultaneously provides for coupling of fluorescent emissions from the samples to the detection system.
In an embodiment of the present invention, the excitation light and emission light can be separately coupled from the bottom, side, or top of any or all detection volumes as preferred.
In an embodiment of the present invention, the optical fibers used for transferring the excitation or emission can be made of glass, plastic, a confined liquid or any other type of light pipe. The coupling adapter can be made of glass, plastic, a confined liquid or any other suitable material.
In another embodiment of the present invention, two excitation transfer fibers and the lamp can be connected in a loop so that light can pass repeatedly around the loop until absorbed by the detection volume. In another embodiment the coupling adapter can contain reflective regions which reflect unused excitation light back into the relay fiber or which form a cavity so that unused light passes repeatedly through the detection volume.
In other embodiments of the present invention, the excitation or emission light can be coupled to fibers using small lenses with or without a larger relay or projection lens.
In other embodiments of the present invention, the specific geometry of the source, fibers, wells and detection elements can be changed to any practicable arrangement. Lenses used can consists of multiple elements, of both positive and negative power, and can contain glass and/or plastic elements. In alternative embodiments Fresnel lenses or a diffractive optics can be used.
In various embodiments of the present invention, the light from separate lamps can impinge on separate detection volumes or the light from one lamp can excite multiple detection volumes at once.
In an embodiment of the present invention, the number of samples illuminated can be varied by varying the number of lamps, which are active. The lamp can be activated during measurements and turned off at other times to minimize the heat generated.
In an embodiment of the present invention, each lamp contains a luminescent praseodymium doped YAG single crystal fiber or other doped single crystals or rod of the same diameter as the delivery fiber. In another embodiment each lamp contains a luminescent praseodymium doped glass fiber or rod of the same diameter as the delivery fiber. In another embodiment, each lamp contains a luminescent doped plastic optical fiber or rod of the same diameter as the delivery fiber. The fibers and rods can have a circular, square or rectangular cross section. Also, the fiber or pipe diameter can be smaller or larger than the delivery fiber and then optically coupled to the delivery fiber using for example a tapered cone.
In another embodiment of the present invention, some lamps can contain alternate materials to allow for the generation of other colors, including infrared and ultraviolet. In one embodiment, these lamps of alternate colors are connected in parallel so that different colors are imaged to different detection volumes. In another embodiment the alternate color lamps are connected in series so that the light of each color passes through the constituent light pipes of lamps of different colors so that each detection volume can be illuminated by light of one or more alternate colors at any given time. In another embodiment the lamp can contain one or more materials capable of producing luminescence at more that one wavelength. As an example, multiple rare earth metal atoms can be doped into a glass host and multiple organic emitters can be doped into a plastic host. In this embodiment, different pump sources such as different color LEDs can be turned on or off to cause the production of the different colors.
In other embodiments of the present invention, relay fibers can be used to direct different wavelengths of light on a detection volume at different positions thereby allowing simultaneous detection of different species present in the detection volume. These different excitation fibers can be positioned to allow detection of species at earlier or later times during the procession of the reaction. In this or other embodiments emission can be collected from more than one region of the detection volume.
In other embodiments of the present invention, the lamp can contain a luminescent fiber of larger or smaller diameter than the delivery fiber with provision for efficient coupling of the two.
In another embodiment of the present invention, the lamp can contain a larger diameter hollow fluorescent tube, which can be “necked down” in diameter to match the delivery fiber.
In another embodiment of the present invention, the lamp can contain a large fluorescent rod, which can be coupled to more than one delivery fiber.
In various embodiments of the present invention, the fibers, rods or tubes form light pipes that can be coated with one or more layers of luminescent material in thick or thin film form. Praseodymium or other rare earth doped lanthanum oxysulfides can be utilized as the film.
In another embodiment of the present invention, the lamp can contain a tube, which contains within it a luminescent material in powder, liquid or other form.
In various embodiments of the present invention, the luminescent light pipe can be of an appropriate cross sectional shape and can be free standing or constructed on a substrate.
Potential luminescent materials suitable for use in this invention include, but are not limited to, CRT or lamp phosphors including all of the lanthanides doped into lanthanum, yttrium, or gadolinium oxides or oxysulfides, or other phosphors with suitable emissions. One can easily generate a wide range of colors based on readily available and known phosphor chemistries. This wide range of colors matches the numerous widely accepted and commonly used fluorophors for bioanalytical applications. Other suitable materials include all of the lanthanides doped into a glass, an organic material containing one of the lanthanides, or a confined solution containing lanthanides.
In alternative embodiments of the present invention, the lamp can be switched on and off rapidly so that a time varying excitation can be produced. The color of the excitation can also be rapidly varied. These rapid variations in excitation can be used in conjunction with time-based detection to increase system sensitivity or to allow for the discrimination of differing numbers, types, or states of fluorescence targets.
In an embodiment of the present invention, the luminescent fiber or fibers of praseodymium doped glass or doped plastic optical fiber are excited (pumped) by an array of LEDs with strong emission near 450 nm in wavelength as shown in
Another lamp embodiment uses an outer waveguide to deliver pump energy to the luminescent pipe. The pipe can be located at the center of the outer or pump waveguide and LEDs are located at either end. The pump waveguide may be filled with a solid, liquid, or gas whose refractive index can be lower than the index of the luminescent pipe. The outer surface of the pump waveguide may be metalized to minimize losses. The luminescent pipe can be positioned in any orientation of the pipe. This orientation can be chosen to maximize the absorption of the excitation light inside the pipe. The luminescent material can be formed into any shape including fibers. More than one pipe emitting more than one pump wavelength can use the same pump waveguide. Different luminescent pipes can be excited by activating different excitation LEDs.
Another embodiment, which emits multiple colors when excited by a linear array of LEDs, is shown in
In another embodiment of the present invention, the excitation can be delivered to each detection volume via a micro-lens array as shown in
In another embodiment of the present invention, the excitation light can enter a single coupling plate which can be designed to distribute light to the individual detection volumes for analysis while simultaneously collecting emitted light for detection as shown in
In other embodiments of the present invention, more than one coupling plate can be used with each plate coupled to a subset of the complete array of detection volumes. These multiple coupling plates can be connected to one excitation source or can be connected to distinct sources. The coupling plate(s) can be made of glass, plastic, a confined liquid or any other suitable material. In an embodiment of the present invention, the plate(s) can provide uniform luminescence to each detection volume, uniformly collect the emission and minimize the emission crosstalk. Excitation uniformity can be increased by applying a surface treatment to the upper surface of the coupling plate. Improved imaging of the emission can be obtained by providing a lens element on the top side of the central section.
In another embodiment of the present invention shown in
In another embodiment of the present invention shown in
In another embodiment of the invention shown in
In other embodiments of the present invention, some of the light sources can emit infrared light and be used to heat detection volumes as part of the analysis process.
As shown in
In an embodiment of the present invention, a luminescent light pipe can consist of a pipe coupled to a transparent fiber. In an embodiment of the present invention, a luminescent pipe can be a continuous fiber, which can directly deliver the luminescence to one or more capillaries or be coupled to a transparent fiber. In an embodiment of the present invention, a luminescent pipe can consist of a luminescent rod.
In an embodiment of the present invention, a coupling optic can contain a filter to narrow excitation spectrum.
In an embodiment of the present invention, the coupling adapter can contain reflective surfaces, which reflect light passing through the capillary back into the capillary. These reflecting surfaces may form a ring cavity or other form of cavity with the result that excitation light passes repeatedly through the flow region of the capillary. In an embodiment of the present invention, the reflective surfaces are designed to enhance both the excitation and emission intensity. In an embodiment of the present invention, the width of a reflective ring can be 1.5 times the diameter of the capillary. In an embodiment of the present invention, the width of a reflective ring can be 1.5 times the spot size. In an embodiment of the present invention, the reflective ring can be 60 microns-100 microns in width. In an embodiment of the invention the spot size can be 40-60 microns.
In an embodiment of the present invention, one or more LED's (207) in parallel are used as a lamp source (see
In an embodiment of the present invention, a source can be coupled to the capillaries using a special adapter assembly for coupling this light into the capillary system. In an embodiment of the present invention, an adapter assembly can also simultaneously provide for coupling of fluorescent emissions from the samples to the detection system.
In various embodiments of the present invention, optical fibers used for transferring the excitation or emission can be made of glass, plastic, a confined liquid or any other type of light pipe. In various embodiments of the present invention, a coupling adapter can be made of glass, plastic or any other suitable material.
In various embodiments of the present invention, a capillary can be used as a light pipe for transferring either the excitation or emission light to or from the active region.
In an embodiment of the present invention, two excitation transfer fibers and the lamp can be connected in a loop so that light can pass repeatedly around the loop until absorbed by the capillary. In an embodiment of the present invention, a coupling adapter can be designed to collimate the excitation light so that it can pass from the fiber on one side of the capillary to be easily collected by the fiber on the opposite side of the capillary. In an embodiment of the present invention, the coupling adapter can contain reflective regions, which reflect unused excitation light back into the relay fiber. In an embodiment of the present invention, the coupling adapter can contain reflective regions, which form a cavity so that unused light passes repeatedly through the flow region of the capillary.
In another embodiment of the present invention, as seen in
In another such embodiment of the present invention, light enters from both ends (540 and 542) of the adapter. In this embodiment of the present invention one or more optical fibers deliver excitation from one or more luminescent pipes to the capillary light pipe adapter from two directions. Light propagates through the adapter and out the opposite fiber. Light can travel back around through one or more luminescent pipes and re-enter the capillaries. The light pipe adapter can be designed to efficiently pipe the light from one end of the pipe to the other.
In various embodiments of the present invention, the light pipe adapter functions to relay the light to any number of capillaries and through multiple reflections to make the luminescence uniform. Therefore, the light (540) piped by the adapter (546) can be transferred to multiple capillaries with great uniformity.
In an embodiment of the present invention, an adapter (546) can be sufficiently wide so that the capillaries fill region that is smaller than the adapter. In another embodiment of the present invention, a light pipe adapter can be narrower than the capillaries. In this case, the light pipe adapter acts as a bridge to carry light from one capillary to the next capillary.
In various embodiments of the present invention, a light pipe adapter can have its surface treated (550) to internally reflect light directly into the multiple flow regions. This treatment can consist of mechanical grooves, holographic patterning and thin film multi-layer dielectrics. This treatment can be made to be wavelength selective allowing the fluorescence emission from the capillaries to preferentially leave the light pipe adaptor at a specific angle. Such treatments are particularly useful when achieving uniformity over a relatively few number of capillaries.
In an embodiment of the present invention, emission can be collected by fibers mated to each capillary. The collection fiber (544) leads to the detector (not shown) in
In an embodiment of the present invention, the coupling adapter can be made of material which can be index matched to the capillary body, causing the capillary body to function as part of the adapter. In another embodiment of the invention, material of the adapter can be index matched to the flowing liquid inside the capillary. In another embodiment of the present invention, an adapter can replace the capillaries with the flow proceeding through the adapter. In another embodiment, the adapter can consist of a hollow structure filled with an index matching fluid matched to the capillary body or flowing fluid. The adapter can be made using standard etching technologies.
In another embodiment, the light pipe adapter can be used with other light sources including edge emitting LEDs and lasers.
In various embodiments of the present invention, the excitation (340) or emission (336) light can be coupled to fibers with or without a larger relay or projection lens. One example is shown in
In various embodiments of the present invention, the excitation (440) or emission (436) light can be coupled to fibers using small lenses with or without a larger relay or projection lens. One example is shown in
In various embodiments of the present invention, the specific geometry of the source, fibers, capillaries and detection elements can be changed to any practicable arrangement. In various embodiments of the present invention, lenses used can consist of multiple elements, of both positive and negative power, and can contain glass and/or plastic elements. In an embodiment of the present invention, Fresnel lenses can be used. In an embodiment of the present invention, diffractive optics can be used.
In an embodiment of the present invention, the light from separate lamps can impinge on separate capillaries. In an embodiment of the present invention, the light from one lamp can excite multiple capillaries. In an embodiment of the present invention, the light from one lamp can simultaneously excite multiple capillaries.
In an embodiment of the present invention, the number of samples illuminated can be varied by varying the number of lamps, which are active. In an embodiment of the present invention, a lamp will be activated during measurements and turned off at other times to minimize the heat generated.
In an embodiment of the present invention, rare earth activated glass can be used as light pipes. In one embodiment of the invention Terbium (Tb) is used to dope the glass rods used as a light pipe. In another embodiment of the invention, Praseodymium (Pr) is used to dope the glass rods used as a light pipe. Table 1 gives a list of some common rare-earth-dopants in the UV spectra and the examples of emission wavelength ranges.
In an embodiment of the present invention, a lamp can contain alternate materials to allow for the generation of other colors, including infrared and ultraviolet.
A was constructed in which a Tb doped glass rod was the luminescent pipe. The Tb glass rod was surrounded with five GE® Germicidal lamps (model G8T5, which each emit 2.1 W of UV light) positioned equidistant around the luminescent pipe. These UV lamps emitted radiation at a wavelength of 254 nm. At this wavelength, these lamps were germicidal (an agent that is destructive to pathogenic micro-organisms). The output of the light source subsystem containing the Tb doped glass light pipe is shown in
In an alternative embodiment of the invention, a Tb doped glass can be chosen as the luminescent pipe with five LED's positioned equidistant around the luminescent pipe. In an alternative embodiment of the invention, a Tb doped glass can be chosen as the luminescent pipe positioned at one focus of an elliptical cavity with a bar of LED's positioned at the other focus. In an alternative embodiment of the invention, a Pr doped glass can be chosen as the luminescent pipe with five LED's positioned equidistant around the luminescent pipe. In an alternative embodiment of the invention, a Pr doped glass can be chosen as the luminescent pipe positioned at one focus of an elliptical cavity with a bar of LED's positioned at the other focus.
In an embodiment of the present invention, one or more optical fibers connected to one or more capillaries through one or more coupling optics connect with the capillaries at one or more locations in space. In an embodiment of the present invention, a species flowing through a capillary can be first excited at one location and the absorption or second excitation resulting from the first excitation can be measured at a second location. In an embodiment of the present invention, differences in the absorbed or emitted light with respect to space can be detected. In an embodiment of the present invention, differences in the absorbed or emitted light with respect to time can be detected. In an embodiment of the present invention, differences in the absorbed or emitted light with respect to frequency can be detected.
In an embodiment of the present invention, relay fibers can be used to direct different wavelengths of light on a capillary at different positions thereby allowing simultaneous detection of different species present in the flow stream of a capillary. These different excitation fibers can be positioned to allow detection of species at earlier or later times of elution from a capillary. In an embodiment of the present invention, emission can be collected from more than one region of a capillary.
In an embodiment of the present invention, a lamp can contain a luminescent fiber of larger or smaller diameter than a delivery fiber with provision for efficient coupling of the two fibers.
In an embodiment of the present invention, a lamp can contain a larger diameter hollow fluorescent tube, which can be “necked down” in diameter to match a delivery fiber.
In an embodiment of the present invention, a lamp can contain a large fluorescent rod, which can be coupled to more than one delivery fiber.
In an embodiment of the present invention, the fibers, rods or tubes form light pipes are coated with one or more thick layers of luminescent material. In an embodiment of the present invention, the fibers, rods or tubes form light pipes are coated with one or more thin layers of luminescent material. In an embodiment of the present invention, the fibers, rods or tubes form light pipes are coated with one or more thick or alternatively thin layers of luminescent material. Tb, Pr or other rare earth doped lanthanum oxysulfide which can be utilized as a film are examples.
In an embodiment of the present invention, a lamp can contain a tube, which contains within it a luminescent material in powder, liquid or other form.
In an embodiment of the present invention, a luminescent light pipe can be of any appropriate cross sectional shape and can be free standing or constructed on a substrate.
Luminescent material is defined as a material which can be activated to luminesce, including glass impregnated with rare earth dopants, glass impregnated with transmetal dopants, organic polymers impregnated with rare earth dopants, organic polymers impregnated with transmetal dopants, inorganic polymers impregnated with rare earth dopants, inorganic polymers impregnated with transmetal dopants, organic emitters, inorganic emitters, CRT phosphors, lamp phosphors and scintillating material. In various embodiments of the present invention, luminescence materials can include one or more combinations of the luminescent material. In various embodiments of the present invention, luminescence materials can include all of the lanthanides doped into lanthanum, yttrium, or gadolinium oxides or oxysulfides, or other phosphors and scintillators with suitable emissions. In various embodiments of the invention, these rare earth dopants are used to generate a wide range of colors based on available and known phosphor and scintillator chemistries. This wide range of colors matches the numerous widely accepted and commonly used fluorophors for bioanalytical applications. In various embodiments of the invention, the light pipe can emit intense UV through to IR emission. Table 3 identifies characteristics of a light source subsystem which enhance the performance for irradiating molecules present in a plurality of capillaries.
In various embodiments of the present invention, a lamp can be switched on and off rapidly so that a time varying excitation can be produced. In an embodiment of the present invention, the color of the excitation can also be rapidly varied. In an embodiment of the present invention, these rapid variations in excitation can be used in conjunction with time-based detection to increase system sensitivity. In an embodiment of the present invention, these rapid variations in excitation can be used to allow for the discrimination of differing numbers, types, or states of fluorescence targets.
In an embodiment of the present invention, the luminescent fibers of Pr doped YAG are excited (pumped) by an array of LEDs with strong emission at 450 nm. In an embodiment of the present invention, the pump source can be replaced with one or more similar devices such as other color LEDs, fluorescent lamps, semiconductor or solid-state lasers, arc lamps, or incandescent lamps.
In an embodiment of the present invention, a lamp operates through the process of spontaneous emission, which results in a much larger selection of available wavelengths than can be available for efficient stimulated emission (laser action).
In addition to chromatography systems and reaction vessels, the light source envisaged in this invention can be adapted for use in a variety of life science research tools including microarray scanners, microtiter plate readers, DNA sequencers, PCR and Q-PCR instruments, fluorescent microscopes, flow cytometery instruments and total analysis systems in the form of lab on a chip devices, optical sensors, medical devices based on luminescence, and miniaturized readers for therapeutic and diagnostic applications.
The foregoing description of the various embodiments of the present invention has been provided for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent to the practitioner skilled in the art. Embodiments were chosen and described in order to best describe the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention, the various embodiments and with various modifications that are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the following claims and their equivalents.
Other features, aspects and objects of the invention can be obtained from a review of the figures and the claims.
Separate and Simultaneous Irradiation: the light source subsystem provides sufficient power to irradiate linear arrays and 2-D arrays of analysis wells or spots in parallel for high density applications.
Modulation: Simple electronics can be used to modulate the light source subsystem at MHz rates. This capability allows for their use in analyses conducted in ambient light conditions. Analyses can be performed that discriminate against background signals and produce enhanced signal to noise ratios.
Moisture and Temperature Insensitivity: This is particularly important for remote sensing applications. The light source subsystem is stable for a very broad range of environmental testing conditions.
Low Heat Production: Analytical complications associated with heat generated by non light source subsystem are eliminated. This is particularly important for biological analyses.
Stability and Robustness: The color purity and intensity of the light source subsystem light output doesn't change as a function of how intensely they are driven nor does it change over time.
Modulation rates up to and even exceeding MHz can be achieved by modulating the excitation source used to activate our glass. In an embodiment of the invention, LEDs are used to excite the luminescent material. Therefore, modulating the LED will result in modulation of the light source output. The circuitry for modulating an LED is well known and typically consists of a square wave, sinusoidal wave or a pulse generator. The output of the generator is then fed to a transistor amplifier circuit which drives the LED.
It is to be understood that other embodiments of the invention can be developed and fall within the spirit and scope of the invention and claims
This application is a continuation of U.S. patent application titled “BIOANALYTICAL INTRUMENTATION USING A LIGHT SOURCE SUBSYSTEM”, application Ser. No. 11/805,185 now U.S. Pat. No. 7,846,391, filed May 21, 2007; which claims the benefit of priority to U.S. Provisional Patent Application titled “CAPILLARY ELECTROPHORESIS LIGHT PIPE”, Application No. 60/802,883, filed May 22, 2006; U.S. Provisional Patent Application titled “WELL LIGHT PIPE”, Application No. 60/831,011, filed Jul. 14, 2006; and U.S. Provisional Patent Application titled “CAPILLARY ELECTROPHORESIS LIGHT SOURCE SUBSYSTEM”, Application No. 60/888,902, filed Feb. 8, 2007; each of which applications are herein incorporated by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20110044858 A1 | Feb 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60802883 | May 2006 | US | |
| 60831011 | Jul 2006 | US | |
| 60888902 | Feb 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11805185 | May 2007 | US |
| Child | 12938224 | US |
