WO1994018552A1 - Multiplexed fluorescence detector system for capillary electrophoresis - Google Patents

Abstract

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially monitor separations in multiple capillaries. The multiplexing approach involves laser (40) irradiation of a sample in a plurality of capillaries (20) through optical fibers (15) that are coupled individually with the capillaries (20). The array (20) is imaged orthogonally through a microscope (60) onto a charge-coupled device camera (50) for signal analysis.

Description

ML JT 1PI J_XED FIJJOR SCENCK DFTFCIOR SYST M FOR ΓAPΠ I AKY T Γ^ PP ESTS

Statement of Government Rights

The present invention was made with Government support under Grant No. W7405-ENG-82 awarded by the U.S. Department of Energy. The Government has certain rights in the invention. Field of the Invention

The present invention relates to fluorescence detector systems for capillary electrophoresis. Specifically, the present invention relates to fluorescence detector systems with the ability to substantially simultaneously excite fluorescence and to substantially simultaneously monitor separations in multiple capillaries. Background of the Invention

Electrophoresis is an electrochemical process in which molecules with a net charge migrate in a solution under the influence of an electric current. Traditionally, slab gel electrophoresis has been a widely used tool in the analysis of genetic materials. See, for example, G.L. Trainor, Anal. Chem.. £2, 418-426 (1990). Recently, capillary electrophoresis (CE) has emerged as a powerful separations technique, with applicability toward a wide range of molecules from simple atomic ions to large DNA fragments. In particular, capillary gel electrophoresis (CGE) has become an attractive alternative to slab gel electrophoresis (SGE) for biomolecule analysis, including DNA sequencing. See, for example, Y. Baba et al., Trends in Anal. Chem.. H, 280-287 (1992). This is generally because the small size of the capillary greatly reduces Joule heating associated with the applied electrical potential. Furthermore, CGE produces faster and better resolution than slab gels. Because of the sub-nanoliter size of the samples involved, however, a challenging problem in applying this technology is the detecting and monitoring of the analytes after the separation. Currently, sophisticated experiments in chemistry and biology, particularly molecular biology, involve evaluating large numbers of samples. For example, DNA sequencing of genes are time consuming and labor intensive. In the mapping of the human genome, a researcher must be able to process a large number o samples on a daily basis. If many capillaries of CE can be conducted and monitore simultaneously, i.e., multiplexed, cost and labor for such projects can be significantl reduced. Attempts have been made to sequence DNA in slab gels with multiple lane to achieve multiplexing. However, slab gels are not readily amenable to a high degre of multiplexing and automation. Difficulties exist in preparing uniform gels over a larg area, maintaining reproducibility over different gels, and loading sample wells Furthermore, difficulties arise as a result of the large physical size of the medium, th requirement of uniform cooling, large amounts of media, buffer, and samples, and lon run times for extended reading of nucleotides. Unless gel electrophoresis can be highl multiplexed and run in parallel, the advantages of capillary electrophoresis canno produce substantial gain in shortening the time needed for sequencing the huma genome.

Significantly, the capillary format is in fact well suited for multiplexing The substantial reduction of Joule heating per lane makes the overall cooling an electrical requirement more manageable. The cost of materials per lane is reduce because of the smaller sample sizes. The reduced band dimensions are ideal fo excitation by laser beams and for imaging onto solid-state array detectors. The use o electromigration injection, i.e., applying the sample to the capillary by an electrical field provides reproducible sample introduction with little band spreading and with littl labor.

Among the techniques used for detecting target species in capillar electrophoresis, laser-excited fluorescence detection so far has provided the lowes detection limits. Therefore, fluorescence detection has been used for the analysis o chemicals, especially macromolecules in capillary electrophoresis. For example, Zar et al. (U.S. Patent No. 4,675,300) discussed a fluoroassay method for the detection o macromolecules such as genetic materials and proteins in capillary electrophoresis. Yeung et al. (U.S. Patent No. 5,006,210) presented a system for capillary zon electrophoresis with laser-induced indirect fluorescence detection of macromolecules, including proteins, amino acids, and genetic materials. Systems such as these generally involve only one capillary. There hav been attempts to implement the analysis of more than one capillary simultaneously i the electrophoresis system, but the number of capillaries has been quite small. Fo example, S. Takahashi et al., Proceedings of Capillary Electrophoresis Symposium. December, 1992, referred to a multi-capillary electrophoresis system in which DN fragment samples were analyzed by laser irradiation causing fluorescence. This method, however, relies on a relatively poor focus (large focal spot) to allow coupling to only a few capillaries. Thus, this method could not be applied to a large number o capillaries, such as 1000 capillaries, for example. This method also results in relatively low intensity and thus poor sensitivity because of the large beam. Furthermore, detection in one capillary can be influenced by light absorption in the adjacent capillaries, thus affecting accuracy.

Attempts have been made to perform parallel sequencing runs in a set o up to 24 capillaries by providing laser-excited fluorometric detection and coupling a confocal illumination geometry to a single laser beam and a single photomultiplier tube. See, for example, XC. Huang et al., Anal. Chem.. 61, 967-972 (1992), and Anal. Chem.. £4, 2149-2154 (1992). However, observation is done one capillary at a time and the capillary bundle is translated across the excitation/detection region at 20 mm/sec by a mechanical stage. There are features inherent in the confocal excitation scheme that limit its use for very large numbers (thousands) of capillaries. Because data acquisition is sequential and not truly parallel, the ultimate sequencing speed is generally determined by the observation time needed per DNA band for an adequate signal-to-noise ratio. Having more capillaries in the array or being able to translate the array across the detection region faster will not generally increase the overall sequencing speed. To achieve the same signal-to-noise ratio, if the state-of-the-art sequencing speed of 1000 nucleotides/hour/lane is used, the number of parallel capillaries will have to be reduced proportionately regardless of the scan speed. Moreover, the use of a translational stage can become problematic for a large capillary array. Because of the need fo translational movement, the amount of cycling and therefore bending of the capillaries naturally increases with the number in the array. It has been shown that bending of th capillaries can result in loss in the separation efficiency. This is attributed to distortion in the gel and multipath effects. Sensitive laser-excited fluorescence detection als requires careful alignment both in excitation and in light collection to provide fo efficient coupling with the small inside diameter (i.d.) tubing and discrimination of stra light. The translational movement of the capillaries thus has to maintain stability to th order of the confocal parameter (around 25 μm) or else the cylindrical capillary wall will distort the spatially selected image due to misalignment of the capillaries in relatio to the light source and photodetector. Further, the standard geometry for excitation perpendicular to the axis o the capillary requires the creation of an optically clear region in the capillary. This makes the capillary fragile and complicates the preparation of capillaries for use. Moreover, the excitation path length, and hence the fluorescence signal, is restricted t the small diameter of the capillary. Therefore, there is a need for a fluorescenc detection system that can be utilized to analyze a large number of samples substantiall simultaneously without bulky equipment and unduly complicated procedures such as careful alignment.

Summary of the Invention The present invention provides a fluorescence detection system for capillary electrophoresis wherein a laser can be used to substantially simultaneously excite fluorescence in multiple capillaries and a detector can substantially simultaneously monitor analyte separations by detecting the fluorescence in a plurality of separation capillaries. This multiplexing approach involves laser illumination of a bundle of optical fibers that are coupled individually with the capillaries in a capillary array. The coupling can be done by inserting at least one optical fiber into each capillary that contains sample. It can also be accomplished by placing the optical fiber adjacent to and perpendicular in relation to the capillary. The fluorescence of the array o capillaries is focused orthogonally, i.e., perpendicularly in relation to the length of the capillaries, through a lens such as a microscope or camera lens and imaged onto a charge-coupled-device camera for signal analysis. The technique can be used fo monitoring as many capillaries as desired, from at least 2 to more than 1000 capillarie The multiplexed fluorescence detection system contains an array of least two (but possibly thousands) of capillaries, each preferably having an insid diameter of about 20-500 microns, and an array of at least two (but possibly thousands of optical fibers of a suitable outside diameter. Each capillary has an annular wall, a intake end, and an exit end. A more preferable inside diameter of each capillary i about 40-100 microns. The multiplexed fluorescence detection system contains a lase for generating coherent light of a wavelength suitable for exciting fluorescence in fluorescent species and a means for detecting the fluorescent light emitted by th fluorescent species through the wall of each capillary.

An argon laser is a preferred means for generating coherent light in th present invention, although any laser with the proper power and wavelength fo excitation of the fluorescent species can be used. A beam from the laser is focuse through a microscope objective onto the ends of the optical fibers distal to th capillaries. Light is therefore transmitted axially, i.e., parallel to the length of th capillaries, into the cores of the capillaries by means of the optical fibers. Alternatively light is transmitted through the capillary wall of each of the capillaries with an optica fiber placed perpendicular in relation to each capillary. Each capillary has a detectio zone where the fluorescence is transmitted through the capillary wall. The detectio zones of the capillaries are imaged onto a charged-coupled device imaging system, o (CCD) camera, through a microscope. Data is extracted from the memory of the CC camera and analyzed by a computer. Instead of a CCD system, a charge injectio device based (CID) imaging system can also be used. The present invention can be applied to a direct fluorescence system o an indirect fluorescence system. In a direct fluorescence system, the presence of a targe fluorescent species is detected by a change, typically an increase in the fluorescenc recorded by the detector as the target fluorescent species passes the detection zone. I an indirect fluorescence system, the buffer solution contains a background fluorophore As the target analyte species traverses the detection zone, laser induced fluorescenc results in either displacement or ion pairing of an ionic analyte with the fluorophore.

The present invention can be implemented utilizing an array of capillaries containing preferably at least about 100 capillaries, and more preferably at least about 500 capillaries, and most preferably at least about 1000 capillaries. It is necessary to select a material of construction for the capillaries such that at least part of each capillary is translucent to light of a wavelength of about 200-1,500 nm, so that fluorescent light can pass through the capillary wall. Inorganic materials such as quartz, glass, fused silica, and organic materials such as teflon and its related materials, polyfluoroethylene, aramide, nylon, polyvinyl chloride, polyvinyl fluoride, polystyrene, polyethylene and the like can be used.

Any conventional fluorescence labels such as a salicylate, dansyl chloride, ethidium bromide, rhodamine, fluorescein, fluorescein isothiocyanate, and the like can be used. To eliminate or reduce photochemical bleaching (i.e., a reduction in the fluorescent properties of fluorophores) of the fluorescent labels, an organic absorber, such as Orange G, which is a dye, can be used to reduce bleaching of the fluorophores before they reach the detection zone of the capillaries. In the case of a partial internal reflection system, a bend can be made in the capillary after the detection zone to prohibit any further light travel through the liquid core. Spacers are preferably placed between each of the separation capillaries to reduce crosstalk, which is the interference between the separation capillaries due to the emission of fluorescence light in the adjacent capillaries. Fibers, capillaries, thin pieces of paper of dark color and the like can be used as spacers.

This multiplexed detection system can be used for analyzing macromolecules such as proteins, amino acids, polypeptides, carbohydrates, polysaccharides, oligonucleotides, nucleic acids, RNAs, DNAs, bacteria, viruses, chromosomes, genes, organelles, fragments, and combinations thereof. This invention is also equally applicable whether a gel is used in the capillary electrophoresis system or not. A method for detecting macromolecules, such as biological molecule using a multiplexing approach is also provided. According to this method, samples a introduced, optionally with fluorophores, into capillaries of a capillary array in capillary electrophoresis system. Coherent light emitted by a laser is transmitte through optical fibers coupled to the capillaries. The change in fluorescence emitted b the fluorescent species is detected orthogonally by a CCD or CID camera through microscope.

Brief Description of the Drawings Figure 1 is schematic representation of a preferred multiplexed detectio system in capillary electrophoresis utilizing axial irradiation.

Figure 2 is a schematic representation of a portion of an interface betwee capillaries and optical fibers.

Figure 3 is a schematic representation of an alternative multiplexe detection system in capillary electrophoresis wherein orthogonal irradiation is used. Figure 4 shows graphs of fluorescence intensity versus time in thre different capillaries. These graphs show crosstalk between separation channels in th absence of spacers. The eluted fluorophore is 3,3'-diethylthiadica_τxκ.yanine iodid (DTDCI). The elution times are different in the three graphs due to variations amon the capillaries. The scale is deliberately magnified. Figure 5 shows graphs of fluorescence signals from 10 differe capillaries. These graph show the results of simultaneous electrophoretic separations o riboflavin and fluorescein. The concentrations of riboflavin and fluorescein were 5 10^"5 M and 6 x 10^'7 M, respectively. The injection time at 7500 volts was 5 second The time axis is in minutes. Detailed Description of the Invention

The present invention provides a fluorescence detector system, i.e fluorescence detection system, for capillary electrophoresis wherein a laser ca substantially simultaneously excite fluorescence in analyte in multiple capillaries and detector can substantially simultaneously monitor separations of analytes, i.e., targ species, in a plurality of capillaries. Herein, "a plurality" means at least two. In thi system, an excitation laser is coupled to a plurality of optical fibers that are in turn individually coupled with the capillaries. The technique of the present invention can be used for monitoring as many capillaries as desired, even up to thousands of capillaries and more. Furthermore, data collection rates are much faster in this system than in conventional CGE. Significantly, the present invention allows for future increases in sequencing rates as permitted by advances in optics and capillary technology.

In capillary electrophoresis (CE), typically analytes, i.e., target species, are detected by measuring laser-induced fluorescence emitted by the target species. Fluorescence is a phenomenon in which an atom or molecule emits light when passing from a higher to a lower electronic state. A fluorescent species is excited by absorption of electromagnetic radiation of a proper wavelength. It emits light of a longer wavelength when it passes from the high energy state to a low energy state. The time interval between absorption and emission of energy is extremely short, typically within a range of about 10^' θ^"3 seconds. Some analyte molecules such as DNAs can be fluorescent naturally and gives off native fluroescences when irradiated with a light of suitable wave length. Other molecules, such as proteins, may need to be tagged with fluorophores to become fluorescent. The present invention is a system for detection of such laser-excited fluorescence. This invention can also be used for the detection of phosphorescence in which the time interval between absorption and admission of energy is much longer. As used herein, the term "fluorescence" includes fluorescent phenomenon as well as phosphorescent phenomenon.

Fluorescence is measured by a detector at a detection zone of a capillary. During the process of electrophoresis, as a fluorescent species traverses the detection zone, it is excited by an incident laser beam. In a direct fluorescence detection system, either the target species is fluorescent, or it is transformed into a fluorescent species by linking with a fluorophore. The passing of the fluorescent species across the detection zone results in a change, typically an increase in the fluorescence that is detectable.

It is also possible to employ other schemes of fluorescence. For example, instead of producing an increase in fluorescence it is possible to have a change in the spectral characteristics of the fluorescent species. The change in the emitted fluorescence spectrum can be detected as a change in the intensity of light of particul wavelengths. Another applicable system is the use of indirect fluorometry. Such system is described by Yeung et al. in U.S. Patent No. 5,006,210. In this method, fluorescent ion or fluorophore is added to a buffer solution as a principal component the buffer. As these fluorophore components in the buffer pass the detection zone, las induced fluorescence results in chemical reaction of the ionic analyte with t fluorophore. This produces a change in fluorescence, a signal which can be detected a camera through a microscope.

The Detection System Figure 1 is a schematic representation of the application of t multiplexed detection system 100 in capillary electrophoresis. Container 12 contains buffer solution 25 that is in fluid communication with each individual capillary 20 an array 23 of capillaries 20 of the electrophoresis system 100. Each capillary 20 h a high voltage end 13 and a low voltage end 33 (see Figure 2). The high voltage en 13 of each capillary 20 is immersed in buffer 25 and the low voltage end 33 of eac capillary 20 is in contact with buffer 30 in container 32. Buffer 30 is at groun potential in this embodiment. A sample to be analyzed is injected into each capillar 20 at the high voltage end 13. The samples migrate through the array 23 of individu capillaries 20 and flow out of the low voltage end 33 of each capillary 20 into buff 30.

For the sake of simplicity, the voltage source equipment for applying th high potential to the capillaries is not shown. In this embodiment, the high potential en of the cap illary electrophoresis system is at the entrance end of the capillary. Herei the high voltage end of each capillary is also referred to as the intake end, and the lo voltage end as the outflow end. For specifics regarding a description of the componen and operation of capillary electrophoresis systems, see, for example, H. Swerdlow et al Anal. Chem.. £3, 2835-2841 (1991).

The system also includes a bundle 10 of optical fibers 15 wherein eac optical fiber 15 is coupled to an individual capillary 20 by insertion into the outflow en 33 of the capillary 20. Each optical fiber 15 has a first end 42 and a second end 7 Excitation laser 40 is coupled into the individual optical fibers 15 of bundle 10 at fir ends 42 by means of a microscope objective 45. First ends 42 of the fibers are dist to the capillaries 20. The laser 40 and the microscope objective 45 are positioned that the laser beam, represented by arrow 47, is focused onto the ends 42 of the optic fibers 15. The capillaries 20 with optical fibers 15 inserted at the outflow ends 33 the capillaries 20 are arranged in an array 102, which is held in a fixed position on stage 52 under an objective 65 of a microscope 60. The capillaries 20 as well as t optical fibers 15 are held firmly in place in an array 102 by guides (not shown). T fluorescent light emitted from the fluorescent species in the array 102 of capillaries 2 through capillary walls 104 is imaged orthogonally through the microscope 60 by mea of an adapter 55 onto a charge-coupled device camera 50 for signal analysis.

Figure 2 (not to scale) shows the array 102 of capillaries 20 with optic fibers 15 inserted at the outflow end 33 of the capillaries 20 in capillary electrophoresi The outflow end 33 of the capillaries 20 are aligned substantially evenly in the field the microscope 60. Optical fibers 15 are positioned into the capillaries 20 so that t second ends 70 of the optical fibers 15 are also aligned substantially evenly. Capillari 80 with black coating are placed between each of capillaries 20 that are used f separation, to act as spacers for reducing the crosstalk between the separation capillari 20. Detection zone 90 is the area of a capillary 20 before the end 70 of the optic fiber. The detection zones 90 of the capillaries 20 are arranged such that the capillar images are detected by the pixel columns of the camera 50 (not shown) coupled to th microscope 60.

Referring again to Figure 1, the microscope objective 45 is a means f collimaiing the laser beam 47 onto the ends of a plurality of optical fibers 1 Depending on the power of the laser 40, the diameter of the laser beam 47, and th number and size of the optical fibers 15, the collimating means 45 can either b focusing or diverging the laser beam 47 to irradiate the ends 42 of the optical fibers 1 If the laser has a narrow beam of high power and the number of optical fibers is larg the microscope objective 45 can be used to diverge and spread the laser beam 47 evenl over the ends 42 of the optical fibers 15. If the laser 40 has a wide beam 47 of lo power, it may be necessary to use the microscope objective 45, to focus the beam 4 onto the ends 42 of the optical fibers 15.

The light source used for excitation of the fluorophores in the sample of interest is a means for generating a coherent light, or a laser. The wavelength of th laser radiation is selected to match the excitation wavelength of the particula fluorophore. Suitable fluorescent species typically absorb light at a wavelength of abou 250-700 nm, preferably about 350-500 nm, and emit light at a wavelength of abou 400-800 nm.

Preferably a laser that can deliver about 0.05-10 mW per fiber, and mor preferably about 0.1 -0.5 mW per fiber is used, although lasers with power outside thes ranges can be employed. Any appropriate laser of the proper wavelength and energ can be used. A commonly used laser is an Argon laser that produces coherent light o 488 nm. Such lasers are commercially available from, for example, Cyonics of Sa Jose, California. Any appropriate optical fibers can be used for illuminating the sample in the capillaries. Optical fibers are selected based on cost, size, and the attenuation o light intensity related to length. Most typical commercial optical fibers of th appropriate diameter are suitable. The selection of the size of the optical fiber i dependent on the capillary inside diameter (i.d.). The fibers should be small enough t facilitate smooth insertion into the capillaries. The fibers should not be so large however, that they hinder substantially the flow of analytes and buffer in the capillaries Preferably, the reduction of flow rate due to the presence of the optical fiber is less tha about 50%. Generally the size of the optical fiber is not critical as long as the detectio zone is well irradiated. The fibers and the capillaries are held in place by guides Generally, however, the friction between the fiber and the capillary is adequate to affi the fibers in place so that fluid motion or minor vibration does not cause a fiber to mov in relation to the capillary. Although in this embodiment each capillary is coupled t one optical fiber, more than one fiber can be used to irradiate each capillary. There ca be a slight pressure differential because of the restricted cross section of the capillar bore. Therefore, the overlap region should be short compared to the length of th capillary. Typically, the overlap is about 0.1-2 cm. Optical fibers are commercial available from, for example, Edmond Scientific Co., Barrington, NJ. The insertion an optical fiber to the end of the capillary reduces contributions from the surfaces of t optical fiber to the separation. Capillaries are often coated with a polymer, typically polyimide. fluorescence detection, the capillary wall and the polyimide coating fluoresce if they a irradiated by the laser light. In certain cases, this may interfere with the sma fluorescence signal from the analytes. Thus, the preferred embodiment of the prese invention focuses the excitation laser beam directly into the capillary core axially b focusing the laser beam into an optical fiber, which has been inserted into the separatio capillary. Because of the unique light transmitting property of the optical fiber, lig is efficiently transmitted to the liquid core.

Although the preferred embodiment is to irradiate the sample axially, viable application of the present invention is to irradiate orthogonally, i.e., perpendicul to the capillary 20 length. The illiirninating ends of the optical fibers 15 are located o the opposite side of the array 102 of capillaries 20 in relation to the microscope 60 an the camera 50. Optical fibers 15 are again coupled individually to the capillaries 2 Figure 3 (not to scale) illustrates this scheme. A camera 50 (not shown) is coupled t a microscope objective 65 in substantially the same manner as in Figure 1 for detectin fluorescence. Optical fibers 15 are oriented at an angle that is perpendicular t capillaries 20 but is 45° in relation to the light path between the capillaries 20 and th microscope objective 65. (For the purpose of simplicity, the electrophoretic equipmen camera, laser and microscope body are not shown. They can be the same as in Figu 1.) Again, as in the previously described embodiment, the capillaries 20 as well as th optical fibers 15 are held by guides (not shown in Figure 3) and affixed in place t prevent movement. Although 45° is preferred, the angle of incident light on the surfac of the capillary 20 can be varied as long as care is taken to reduce stray laser light fro interfering with the fluorescent light as detected by the camera. Although not show in Figure 3, spacers similar to those in Figure 2 can be used for reducing stray ligh The size of the optical fiber 15 can be selected so that the fibers 15 and spacers 80 ma be conveniently held in place.

The laser/capillary interface of individually coupling optical fibers t capillaries 20 represented by Figure 3 is also advantageous because alignment of th optical fiber 15 with the laser beam 47 can be made permanent. Changing th separation capillary is then simplified. With fibers or capillaries of larger than 50 μ alignment does not even require the use of a microscope. Because there is no movin part, once the alignment is made, no further alignment is needed. Single-mode optic fibers with diameters down to 5 μm are presently available. Therefore capillaries wit inside diameters of slightly larger diameters may be utilized in electrophoresis.

The emitted fluorescent light that passes through the capillary wall 10 is detected by a charge transfer device imaging system 50 through coupling with microscope 60. Any appropriate microscope or camera lens system may be used so lon as it adequately transmits the image of the separation zone of the capillary array to th imaging camera. A Bausch and Lomb Stereo Zoom 7 binocular microscope wit camera extension is suitable.

The present invention is capable of multiplexing more than 1000, e.g. 1024, capillaries in, for example, a DNA sequencing run. This number is based on th number of column elements already available in modern solid-state imaging devices The principle is the same as an embodiment of an optical system device based on 1 capillaries. Fiber bundles with up to 1000 fibers are readily available at a low cost.

For an embodiment of 1024 capillaries, an argon ion laser of 1-5 Wa power or any appropriate laser of suitable power and wavelength can be used t illuminate one end 42 of the fiber bundle, conveniently distributing about 0.4-2 mW o light to each fiber. The other end 70 of the fiber bundle 10 can be fanned out into a fla sheet on a guide (not shown), which is a set of parallel grooves to fix the location o each fiber 15 to maintain a constant spacing. Similarly, 1024 separation capillaries ca also form a flat sheet on a suitable guide. Insertion of the fibers 15 (smaller than 5 μm) into the capillaries 20 (about 75 μm i.d.) as in the preferred embodiment, o positioning the optical fibers 15 at a 45° angle to the capillaries 20 as in anothe embodiment, can be readily accomplished. The imaging optics 50 can be a standar distortion-free camera lens, matching each of the optical windows on the capillaries 2 to each pixel column on the detector. Several rows of pixels can be binned, i.e., th data are added together before transferring to storage, to provide increased dynami range without degrading resolution. The data rate of CCD cameras, even in the high sensitivity, slow-scan mode, can be around 10 Hz. Since all channels are monitored a all times, true multiplexing is achieved.

Both charge injection devices (CID), and charged coupled devices (CCD), are charge transfer devices (CTD) containing semiconductor material. In CTD, an individual detector contains several conductive electrodes and a region fo photogenerated charge storage. A photon striking the semiconductor material in th device creates a mobile hole which is collected as a positive charge under an electrode The number of photons striking the device is counted by transferring the charges. I the CCD, the charge from each detector element is moved to a charge sensing amplifie by sequentially passing the charge from one detector element to the next adjacen detector element. The CCD is designed in such a way that it is necessary to shi through all the detector elements in the entire detector before proceeding to the nex exposure. As a consequence, the reading of data in a CCD detector cannot be done i a random access manner. However, the amplifier gives a very high signal to nois output.

In a CID, there are two different kinds of electrodes, a collectio electrode and a sense electrode. The photogenerated charge is kept in the detecto element by potential barriers that prevent the charge from migrating along the electrode The charge that is kept at the collection electrode can be transferred to the sens electrode. This transfer induces a voltage change on the sense electrode which can b measured. Thus, during the readout process, the charge is not altered in the detectio element. This nondestructive readout enables a random access to the charge stored i the detector elements. In CID, the nondestructive reading of charge can be analyzed b a computer system to determine the proper process exposure time for each emission lin of the spectrum. Therefore, data rate can be increased. Data storage is als substantially reduced because the information can be evaluated before binning or readin multiple frames, which is a way of reducing background noise by summation of dat from multiple measurements. Also, once a base of a DNA molecule is identified, th other 3 lanes in the set of 4 need not be read or subjected to data processing to analy for the other three bases. Imaging design can also include color filters to accommodat the advanced techniques such as the 4-color sequencing process utilizing different part of the CID detector. For references for CID and CCD, see J.V. Sweedler et al., Ana Chem.. .0, 282A-291 A (1988), and P.M Appereson et al. Anal. Chem..6ϋ, 327A-325 (1988). A wide range of fluorescence labels can be used. A researcher would b able to select the proper label for the particular target species and the fluorescenc system used. Common fluorescence labels include materials such as salicylate, 3,3' diethylthiadicarbocyanine iodide (DTDCI), dansyl chloride, fluorescein, fluorescei isothiocyanate, ethidium bromide, rhodamine, and the like. Commonly used fluoresce dyes that can be used for tagging target species include FAM, JOE, TAMRA, which ar rhodamine derivatives. Such dyes are commercially available from, for exampl Applied Biosystem, Foster City, CA.

Capillaiy Electrophoresis In capillary electrophoresis, a buffer filled capillary is suspended betwee two reservoirs filled with buffer. An electric field is applied across the two ends of th capillary. The electrical potential that generates the electric field is in the range o kilovolts. Samples are typically introduced at the high potential end and under th influence of the electrical field. The same sample can be introduced into man capillaries, or a different sample can be introduced into each capillary. Typically a array of capillaries are held in a guide and the intake ends of the capillaries are dippe into vials that contain samples. Obviously each vial can contain the same or differe samples as the other vials. After the samples are taken in by the capillaries, the end of the capillaries are removed from the sample vials and submerged in a buffer whic can be in a common container or in separate vials. The samples migrate toward the lo potential end. When the samples leave the capillary zones after migrating through th capillary, they are detected by a detector. Techniques for capillary electrophoresis ar well known in art. The above-mentioned references, such as Huang et al, Yeung et al Zare et al, and Baba et al, are illustrative of the exemplary equipment and procedure for such techniques. The channel length for capillary electrophoresis is selected such that i is effective for achieving proper separation of species. Generally, the longer th channel, the greater the time a sample will take in migrating through the capillary an thus the species may be separated from one another with greater distances. However longer channels would also contribute to the band broadening and lead to excessiv separation time. Generally, for capillary electrophoresis, which includes CZE and CGE the capillaries are about 10 cm to about 5 meters long, and preferably about 20 cm t about 200 cm long. In capillary gel electrophoresis, the more preferred channel lengt is about 10 cm to about 100 cm long.

The capillaries should be constructed of material that is sturdy an durable so that it can maintain its physical integrity under repeated use under norma conditions for capillary electrophoresis. It should be constructed of nonconductiv material such that high voltages can be applied across the capillary without generatin excessive heat. It is also necessary that part of the capillary be translucent so that th fluorescent light that is emitted by the fluorescent species can be transmitted across th capillary wall to the detection system. Inorganic materials such as quartz, glass, fuse silica, and organic materials such as teflon (polytetrafluoroethylene and fluorinate ethylene/propylene polymers), polyfluoroethylene, aramide, nylon (polyamide), polyviny chloride, polyvinyl fluoride, polystyrene, polyethylene and the like may be used. Th capillary is translucent to light of a wavelength of about 200-1,500 nm, preferably abou 350-800 nm. In a case where the capillary is not translucent to such light, the detectio zone of such a capillary is preferably made of material that is translucent to such light.

To achieve total internal reflection, the choice of the electrophoreti buffer may have to be limited. A buffer with a lower refractive index is employed t provide total reflectance. Materials other than fused silica may be used for th separation capillary so that such buffers with lower refractive indices may be applicable One distinct advantage of the axial-beam technique (as represented b Figures 1 and 2) is that it allows the supportive coating on the capillary to remain intact Most fused silica capillaries have a polyimide coating that is known to emit a broa fluorescence when exposed to wavelengths of light under 600 nm. If a through-the-wal excitation scheme (as represented by Figure 3) is used without first removing thi coating the fluorescence background may mask the weak analyte signal at lo concentrations. It is therefore preferable that the detection zone of the capillary has n coating. The polymer coating may be removed by any method known in the art, fo example, by boiling in sulfuric acid for a period of time. In electrophoresis, the buffer is typically selected so that it would aid i the solubilization or suspension of the species that are present in the sample. Typicall such a liquid, an electrolyte, which contains both anionic and cationic species, contain about 0.005-10 moles per liter of ionic species and preferably about 0.001-0.5 mole pe liter of ionic species. Examples of an electrolyte for a typical electrophoresis syste are mixtures of water with organic solvents and salts. Representative material that ca be mixed with water to produce appropriate electrolytes includes inorganic salts suc as phosphates, bicarbonates and borates, and organic acids such as acetic acids propionic acids, citric acids, chloroacetic acids and their corresponding salts and the like lower alkyl amines such as methyl amines, lower alcohols such as ethanol, methanol and propanol; lower polyols such as the lower alkane diols; nitrogen containing solvent including acetonitrile, pyridine, and the like; lower ketones such as acetone and methy ethyl ketone; and lower alkyl amides such as dimethyl formamide, N-methyl and N-ethy formamide, and the like. The above ionic and electrolyte species are given fo illustrative purposes only. A researcher would be able to formulate electrolytes fro the above-mentioned species and in combination with other species such an amino acids salts, alkalies, etc. to produce suitable support electrolytes for using capillar electrophoresis systems.

Reducing Interference In the present invention, light may be propagated through a capillary b partial or total internal reflection. The invention is equally applicable to either case In total internal reflection, no stray ligjit from the quartz wall or the polyamide coati reaches the camera. Total internal reflection is advantageous in certain fluorescen excitation applications because all the light entering the capillary within a certain critic angle will propagate with little loss. This results in less noise due to capillary misalig ment and a detector response that should be independent of the location of the analy band. The capillary can thus be bent without attenuating the excitation beam. An adde advantage in axial-beam fluorescence excitation with total internal reflection is that the is very little (in principle, none) scattered light originating from the capillary wall. Thi should allow the use of capillaries with intact polyimide coatings without problems interference due to absorption or greatly increased fluorescence background. Anoth benefit from the axial-beam geometry is a longer absorption path length compared t irradiation across the capillary. This increases the interaction length from 75 μm to mm to provide increased fluorescence intensity.

Increased laser power is advantageous in providing a larger analyte signa However, fluorophores are easily bleached, i.e., their fluorescing characteristic destroye by the laser beam, even at the milliwatt level, negating any increase in excitatio intensity. Furthermore, in the axial-beam arrangement, fluorophores may be completel bleached before they reach the detection zone because of the residence time in th excitation beam. A novel way to increase the incident laser power without bleachin the compounds of interest is to add an inert absorber that does not react chemically wit the sample or the buffer contained in the capillary. Suitable absorbers include Orang G, Cresol Red, or any other nonfluorescing dye molecules. In a capillary containing a absorber producing an absorbance of 0.6 per cm of path length, for example, th incident laser intensity is reduced to 50% in 5 mm, typically the distance between th end of the optical fiber and the detection zone. Over a distance of about 4 cm, lig intensity is reduced to less than 0.5% of its original value. Thus if a detection devic is placed close to the point where excitation light is introduced, an analyte ban traveling from the opposite end of the capillary will interact with very little light unt it is near the detection zone. Therefore photochemical bleaching is greatly reduced. may be necessary to isolate the detection zone from the end of the optical fibe however, so that specular reflection and fluorescence from the fiber can be discriminate against.

In the case of partial internal reflection, only a small fraction of th incident laser intensity generally remains in the capillary after 1 cm of travel. A smal bend can be made in the capillary after the detection region to prohibit any further ligh travel. This renders the use of an organic absorber unnecessary, because in partia internal reflection, the incident laser light exits the capillary at the bend.

Because interested bands of analytes are not passing by the detection zon continuously, an expert system can be used to predict when the bands are approachin the detection zone. Only during such time where the bands are near to the detection zone would the laser irradiation be needed.

An additional electronic shutter in the beam path may be used to open synchronously with the CCD shutter, allowing light to be transmitted to the capillary only during the period of data collection, and blocking the incident beam during the time when no signal is collected. With the implementation of an expert system for data collection only when interested data is being produced, signal collection time and thus photochemical damage can be greatly reduced.

Crosstalk between the separation channels is another important design consideration in multiplexed detectors. This is the interference of the fluorescence between neighboring capillaries. Such interferences must be eliminated or reduced to an acceptable level before applications such as DNA sequencing can be practiced. Crosstalk has two causes: signal light reflections from the walls of adjacent capillaries and scattered light from optics inside the microscope. When capillaries are placed side by side, false peaks are observed in nearby capillaries. Figure 4 shows such false peaks. In Figure 4, the tracings are derived from electropherograms of capillaries No. 6, 9, and 11 of an array of thirteen separation capillaries. Peaks A and B are reflections of the fluorescence signal in capillary 9. C and D arise from the signal in capillary 11, and E represents interference from capillary 6. Crosstalk can be reduced by placing spacers, which are the same 150 μm outside diameter (o.d.) capillaries coated with black ink, between each of the separation capillaries. Such black capillaries are illustrativ examples. Other suitable spacers may certainly be devised using the above principle The fluorescence, once imaged onto the CCD camera, is extracted fro the memory of the CCD camera and analyzed using a computer. A knowledgeabl person will be able to select the proper software, computer program and algorithm f the analysis of the data. Such data may also be used for the control of th electrophoresis system or the detection system. For example, valves or pumps may b operated by control signals generated by the computer based on the analysis of th capillary electrophoresis results. Proper calibration methods can be used to compensate for slight variation in migration times from one capillary to the next. An example of such a method is th utilization of a migration index as described in T.T. Lee et al. Anal. Chem. £2, 2842 2848 (1991). This method involves separate current measurements in each capillary If series resistors are connected to each of the capillaries, the resulting voltage can b monitored sequentially by an AD converter and a desktop computer. Measurement every 0.1 second are sufficient for calibration compensations. Even for 1000 capillaries using commercially available serial interface boards, such calibration would still be relatively simple task. A knowledgeable person would be able to configure computer and electronic components, and utilize software systems to compensate for variations b proper calibrations.

The present invention may be used for the separation and measuremen of the species present in samples of biological, ecological, or chemical interest. O particular interest are macromolecules such as proteins, polypeptides, saccharides an polysaccharides, genetic materials such as nucleic acids, polynucleotides, carbohydrates cellular materials such as bacteria, viruses, organelles, cell fragments, metabolites, drugs and the like and combinations thereof. Protein that are of interest include proteins tha are present in the plasma, which includes burnin, globulin, fibrinogen, blood clottin factors, hormones, and the like. Other interesting proteins which may be separated usin the capillary electrophoresis systems are interferons, enzymes, growth factors, and th like. Of particular interest are the group of macromolecules that are associated with th genetic materials of living organisms. These include nucleic acids and oligonucleotide such as RNA, DNA, their fragments and combinations, chromosomes, genes, as well a fragments and combinations thereof. Other chemicals that can be detected using th present invention include, but is not limited to: pharmaceuticals such as antibiotics agricultural chemicals such as insecticides and herbicides.

The proper selection of the capillary electrophoresis system is importan for the separation of the species in the samples. An important factor to be considere is the plugging of the system. For example, samples containing cell fragments or cell such as bacteria, large chromosomes, DNAs, RNAs may result in the plugging o capillaries containing gel in capillary gel electrophoresis. A scientist would be able t select the proper capillary electrophoresis system for the particular species of interest The present invention is equally applicable to capillary gel electrophoresis (CGE) wherein a gel or a similar sieving medium is present in the capillaries, and capillar zone electrophoresis (CZE), wherein no gel is used. Depending on the particular targe species, different electrophoresis systems may be used. Details about the selection o proper channel size, channel length, material construction for the capillaries, solvents, electrolytes, gels, etc. may be varied by the person conducting the analysis.

Several important advantages are realized by the present invention. First, truly simultaneous multiplexing of capillary electrophoresis can be achieved because th CCD camera monitors all capillaries simultaneously, resulting in data rates fast enoug for sequencing at greater than about 1 nucleotide, i.e., base, per second per lane. This translates to 1000 bases per second for a system with 1000 capillaries, which already meets the goal of sequencing rate necessary for the Human Genome initiative. Second, there are no moving parts and the injection (high voltage) end of the capillary bundl can be freely manipulated without affecting alignment. Third, the 5 mW excitation lase simply irradiates the entrance of the optical fiber bundle without critical alignment o the optics to achieve distribution of energy into each capillary. Fourth, although ther are variations in the excitation energies reaching each capillary, these variations can b compensated for by calibration in the same way the individual CCD pixels ar normalized. Fifth, the variations in absolute and relative migration times for the targe compounds can be adjusted for by using a migration index and an adjusted migratio index. Sixth, variations in the relative peak heights and areas among the capillaries fo the same injected sample concentration can be corrected by the T.T. Lee et al. Anal. Chem. 61, 2842-2848 (1991) calibration method to reduce the bias to less than 5%, which is adequate for DNA sequencing.

The following examples are offered to further illustrate the various specific and preferred embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the scope of the present invention. Examples

Example 1 Excitation. A schematic diagram of the multiplexing apparatus is shown in Figures 1 and 2. Approximately 500 optical fibers (0.0051 cm o.d, P31735, Edmond Scientific, Barrington, NJ) were grouped together and inserted into a 0.32 cm i.d. heat shrinking tube. After heating, a razor blade was used to cleave the heat shrink casing and the enclosed fibers. This fiber bundle was inserted into the laser beam path by a microscope objective (Bausch and Lomb, Rochester, NY 16 mm, 10X, BM2888). The 488 nm laser beam (2011-30SL, Cyonics, San Jose, CA) was partially focused by the objective to provide a beam spot size that overlaps the desired number of fibers in the illumination zone. A total laser power of 5 mW was thus divided among 10-12 optical fibers. The free end of each illuminated fiber was inserted 1.5 cm into a different fused silica capillary as discussed previously.

Detection. The capillary array in the detection region was imaged onto a charge-coupled device (CCD) camera (Photometries, Tucson, AZ, Series 200) through the camera extension of a binocular microscope (Bausch and Lomb, Stereo Zoom 7). Each of the optical windows on the capillaries was matched to a pixel column of the camera. The CCD camera was operated in a 25 to 1 column binning mode. This allowed compression of 200 columns and 375 rows of image data into an 8 x 375 array. Data extraction from the CC200 memory and analysis was carried out on a PC- compatible 80386-based computer equipped with an IEEE interface. Each frame of data, corresponding to a 0.1 second CCD exposure taken every 0.9 second, was store in an individual file. The total storage space for the 400 data files collected during run was under 4 megabytes. Using a simple BASIC algorithm, the intensities o selected 3 x 5 element regions representing each separation capillary were summed. These time dependent sums were then plotted as electropherograms.

Separation. Silica capillaries (Polymicro Technologies, Phoenix, AZ, 75 μm i.d, 150 μm o.d, #TSP075150, length = 27 cm) were prepared by removing 1 cm of polyamide coating with boiling sulfuric acid 25 cm from the injection end. Equilibration consisted of flushing with 0.1 M NaOH followed by filling with ninning buffer ( 10 mM bicarbonate adjusted to about pH = 9) and applying a 5 kV potential for at least 10 hours prior to fiber insertion. Solutions of riboflavin (BioRad) and fluorescein (Eastman Kodak) were prepared in ninning buffer. The electrophoretic separation was driven at +7.5 kV using a high voltage power supply (Glassman, Whitehouse Station, NJ, model PS/MJ30P0400-11) with a platinum electrode at the high voltage end and a chrommel electrode at ground. Injection of samples was by the standard method by placing the intake ends of the capillaries in a sample container. When injection of sample is complete, the capillaries were removed from the sample container and placed in the buffer reservoir.

Other Techniques Employed. In the present case, with partial internal reflection, only a small fraction of the incident laser intensity remained in the capillary after 1 cm of travel. A small bend was made in the capillary after the detection region to prohibit any further light travel. This obviated the need for an organic absorber. An additional electronic shutter in the beam path, which opened synchronously with the CCD shutter, blocked the incident beam during the 80% of time when no signal was being collected. This greatly reduced photochemical damage in the detection region. The lack of photochemical bleaching was confirmed by comparing peak heights in single capillaries with different amounts of cumulative exposure to laser excitation.

To reduce crosstalk, spacers, which were the same 150 μm o.d. capillaries coated with black ink, were placed between each of the separation capillaries. Thus crosstalk was reduced to well below 1% of the observed signal. Results. Electropherograms for ten parallel separations of riboflavin an fluorescein are shown in Figure 5. The retention times for the two components we similar to those in control experiments (not shown). This indicated that th electroosmotic flow normally present in an open capillary is not significantly disturbe by fiber insertion. The individual retention times in the 10 capillaries varied noticeabl The relative standard deviations (RSD) for the migration times of riboflavin (t,) and f the migration times of fluorescein (t->) were about 3% and 5.5% respectively. Since th 10 capillaries were unrelated, even the relative migration times of fluorescein (tyt- showed a 4% RSD. The calculated mobilities (μ) of fluorescein (related to l/t₂-l/t- gave an RSD of 5%. Repeat experiments showed similar results. This range of RS was the result of the uncontrolled nature of the surfaces inside each capillary, variation in capillary i.d, variations in fiber-optic o.d, and differences in the length of insertio of the fiber. However, for a given capillary, run-to-run reproducibility was good. Th ranges of RSD for t,, t ^/t,, and μ are 0.4% to 1.1%, 0.4% to 3.4%, 0.4% to 2.4%, an 0.4% to 2.4% respectively. These were similar to typical run-to-run variations i capillary electrophoresis in the absence of temperature control and for constant voltag operation.

Figure 4 also shows large variations in the peak areas among the 1 parallel capillaries. The RSD ranged from 70% for riboflavin (A-), 95% for fluorescei (A₂), and 29% for the relative areas (A-j/A,). Large differences are expected due t nonuniform coupling of excitation energy into each capillary and variations in pixe sensitivities across the CCD. These dominated over the variations of individual capillar surfaces and geometries. For a given capillary, run-to-run (RSD ranges) for A,, A₂, an A₂/A_] was 6% to 28%, 6% to 29%, and 3.6% to 17% respectively. Injection bias wa responsible for these variations. These were more pronounced than variations i migration times because factors such as unequal time constants for electromigratio injection, ubiquitous injection, and the particular characteristics of the capillary entranc affected the injected amount but not the migration times. It is interesting to note th the peak areas were not correlated with the migration times. This indicated th photochemical bleaching was not important in these experiments. These variation should not be considered as shortcomings of the present invention because they woul be present in conventional cap illary electrophoresis systems. Moreover, these variation can be corrected by proper calibration procedures. Also, internal standards for each o the unknowns in the sample can be used for proper calibrations. For a discussion o the significance of the RSDs and calibration, see T.T. Lee et al. Anal. Chem.. 64, 1226 1231 (1992).

Example 2 In this example, a DNA sequencing experiment similar to Huang's i done. See, XC. Huang et al. Anal. Chem.. M, 967-972 (1992). One hundre capillaries are to be used. Zero-cross-linked polyacrylamide gel-filled capillaries ar prepared using a procedure described by Cohen et al, Proc. Natl. Acad. Sci.. U.S.A.. £ >, 9660-9663 (1988). A detection window is made on the capillary near to the exit en by removing the polyimide coating with boiling in sulfuric acid as in Example 1. Th inner wall of the capillaries is then treated with a bifunctional reagent and then vacuu siphon filled with a gel solution as described by Huang. Chain-terminated Ml 3mpl DNA fragments are made with fluorescein-tagged primer (FAM, Applied Biosystems, Foster City, CA) as described by MA Quesada et al, BioTechniques. 19(10). 616-625 (1991).

The same type of capillaries and optical fibers as Example 1 are used. Optical fibers are inserted into the capillaries in the same manner as in Example 1. The fibers are inserted to such a distance (about 0.5 cm) inside the capillaries so that th fluorescent light may be readily detected by the detection system. A 50 mW lase generating 488 nm light is used. The size of the capillary and the fiber, the equipmen and operation of the electrophoresis system, and the detection system are as describe in Example 1.

Example 3 In this example, DNA sequencing is done in the same manner as i Example 2. The irradiation of the samples, however, is orthogonal rather than axial As is shown in Figure 3, the illuminating ends of the optical fibers are on the side o the array of capillaries that is away from the microscope and the camera. Again, th outside diameters the capillaries are 150 microns. Optical fibers of 50 microns are use The optical fiber is oriented at an angle that is perpendicular to the length of t capillary but is 45° in relation to the light path between the capillary and the camer The same equipment as in Example 2 is used for the electrophoresis, coherent lig generation, and imaging.

All patents, patent documents and publications cited hereinabove a incorporated by reference herein. All percentages given are weight percents exce indicated otherwise.

Claims

WHAT S CLAIMED IS:

1. A multiplexed fluorescence detection system for use with capilla electrophoresis to detect target species in a sample comprising:

(a) an array of at least two capillaries each having an intake end, outflow end, and an annular wall wherein at least a portion of the wall translucent to light having a wavelength of about 200-1,500 nm, the intake e being in fluid communication with the sample so that the sample is drawn in the capillary;

(b) an array of at least two optical fibers, each optical fiber having first and second end, the second end of each optical fiber being coupled to capillary in the array of capillaries; and

(c) means for generating coherent light positioned to direct t coherent light onto the first end of the optical fibers, wherein the coherent lig is of a wavelength suitable to cause fluorescence in target species present in t capillaries.

2. The detection system of claim 1 further comprising means f substantially simultaneously detecting changes in fluorescent emission by the targ species through the annular wall of each capillary.

3. The detection system of claim 2 wherein the optical fiber is coupled t the capillary by insertion of the second end of the optical fiber into the outflow end the capillary, the optical fiber being of a suitable diameter for affixing the fiber insi the capillary without substantially hindering the sample flow.

4. The detection system of claim 1 wherein the means for generati coherent light includes collimating means for distributing the coherent light into the fir end of the optical fibers in a substantially uniform manner.

5. The detection system of claim 1 wherein at least one of the capillari contains a gel that is suitable for capillary gel electrophoresis systems.

6. A multiplexed fluorescence detection system for use with capillar electrophoresis to detect target species in a sample, comprising:

(a) an array of at least two capillaries each having an intake end, a outflow end, and an annular wall wherein at least a portion of the wall i translucent to light having a wavelength of about 200-1,500 nm, the intake en being in fluid communication with the sample so that the sample is drawn int the capillary; and

(b) means for substantially simultaneously detecting changes i fluorescent emission by the target species through the annular wall of eac capillary.

7. The detection system of claim 6 wherein the means for detecting th fluorescent emission comprises a charged-coupled device based imaging system.

8. The detection system of claim 6 wherein the means for detecting th fluorescent emission comprises a charge injection device based imaging system.

9. A multiplexed method for detecting the presence of fluorescent targ species in a sample in a plurality of capillaries using capillary electrophoresi comprising:

(a) introducing samples containing a fluorescent species into intak ends of a plurality of capillaries, each capillary having an annular wall, and a outflow end, wherein the samples migrate through the capillaries towards th outflow ends; and

(b) irradiating the samples in the capillaries with coherent lig transmitted through optical fibers, the optical fibers having first ends coupled t a coherent light source and second ends individually coupled to the capillarie

10. The method of claim 9 further comprising a step of substantiall simultaneously detecting change in fluorescent emission by the fluorescent speci through the wall of each of the capillaries.

11. The method of claim 9 wherein the step of irradiating compris transmitting coherent light axially into the capillaries by inserting the second ends of th optical fibers into the outflow ends of the capillaries, the optical fiber having a diamet suitable for affixing the fiber in position in the capillary without substantially hinderin sample flow.

12. The method of claim 9 wherein the step of irradiating comprise irradiating coherent light orthogonally into the capillaries through an optical fiber th is coupled adjacent to, and perpendicular in relation to the capillary.

13. The method of claim 9 wherein the step of introducing samples int capillaries comprises introducing a sample into at least one capillary that contains a g that is suitable for capillary gel electrophoresis.

14. A multiplexed method for detecting the presence of fluorescent targ species in a sample in a plurality of capillaries using capillary electrophoresi comprising:

(a) introducing samples containing a fluorescent species into intak ends of a plurality of capillaries, each capillary having an annular wall, and a outflow end, wherein the samples migrate through the capillaries towards th outflow ends; and

(b) substantially simultaneously detecting change in fluoresce emission by the fluorescent species through the wall of each of the capillaries

15. The detection system of claim 14 wherein the means for detecting th fluorescent emission comprises a charged-coupled device based imaging system.

16. The detection system of claim 14 wherein the means for detecting th fluorescent emission comprises a charge injection device based imaging system.

17. A multiplexed method of detecting the presence of a macromolecule i a sample present in a plurality of capillaries in capillary electrophoresis comprising:

(a) linking a fluorophore to the macromolecule in a sample t transform the macromolecule into a fluorescent species with fluorescence th can be excited by light of suitable wavelength;

(b) introducing samples containing fluorescent species into each of plurality of capillaries, each capillary having an annular wall, an intake end an an outflow end; and (c) irradiating each sample in each capillary with coherent light t cause fluorescence of the macromolecule, the coherent light being transmitte through optical fibers individually coupled to the capillaries.

18. The method of claim 17 further comprising a step of substantiall simultaneously detecting changes in fluorescent emission through the wall of each of th capillaries by the fluorescent species using a charge transfer based imaging system.

19. The method of claim 17 wherein the step of linking a fluorophore to th macromolecule comprises linking a fluorophore to the macromolecule in a materi selected from the group consisting of proteins, amino acids, polypeptides, carbohydrate polysaccharides, oligonucleotides, nucleic acids, RNA, DNA, bacteria, viruses chromosomes, genes, organelles, as well as fragments and combinations thereof.

20. The method of claim 17 wherein the step of linking a fluorophore to th macromolecule comprises linking the macromolecule to a fluorophore selected from th group consisting salicylate, 3,3'-diethylthiadicarbocyanine iodide (DTDCI), dansy chloride, fluorescein, fluorescein isothiocyanate, ethidium bromide, rhodamine, an combinations thereof.

21. A multiplexed method of detecting the presence of a macromolecule i a sample present in a plurality of capillaries in capillary electrophoresis comprising: (a) linking a fluorophore to the macromolecule in a sample t transform the macromolecule into a fluorescent species with fluorescence tha can be excited by light of suitable wavelength; (b) introducing samples containing fluorescent species into each of plurality of capillaries, each capillary having an annular wall, an intake end an an outflow end; and

(c) substantially simultaneously detecting changes in fluorescen emission through the wall of each of the capillaries by the fluorescent species.

22. A multiplexed method of detecting the presence of macromolecules i samples present in a plurality of capillaries in capillary electrophoresis comprising:

(a) providing a buffer solution containing a fluorophore;

(b) introducing samples into a plurality of capillaries in a capillar electrophoresis system, each capillary containing the buffer; and

(c) irradiating the samples in the capillaries with coherent light t cause fluorescence, the coherent light being transmitted though optical fiber individually coupled to the capillaries.

23. The method of claim 22 further comprising a step of substantiall simultaneously detecting changes in fluorescent emission through the wall of each th capillaries by the fluorescent species.

24. A multiplexed method of detecting the presence of macromolecules samples present in a plurality of capillaries in capillary electrophoresis, comprising t steps of:

(a) providing a buffer solution containing a fluorophore; (b) introducing samples into a plurality of capillaries in a capilla electrophoresis system, each capillary containing the buffer; and

(c) substantially simultaneously detecting changes in fluoresce emission through the wall of each of the capillaries by the fluorescent speci by using a charge transfer device based imaging system.

25. A multiplexed method of detecting the presence of DNA fragments i samples present in a plurality of capillaries in capillary electrophoresis comprising:

(a) linking a fluorophore to a DNA fragment in a sample to transfor it into a fluorescent species; (b) introducing samples containing fluorescent species into a pluralit of capillaries in a capillary electrophoresis system, the system comprising a array of at least about 100 capillaries each having an inside diameter of abo

20-500 microns, and an annular wall; and

(c) irradiating the samples in the capillaries with coherent light havin a wavelength of about 250-700 nm to cause native fluorescence of the DN fragment, the coherent light being transmitted though optical fibers individuall coupled to the capillaries.

26. The method of claim 25 further comprising a step of substantiall simultaneously detecting change in fluorescence emitted by the fluorescent speci through the wall of each of the capillaries by focusing the fluorescence onto a char transfer device based imaging system.

27. A multiplexed method of detecting the presence of DNA fragments i samples present in a plurality of capillaries in capillary electrophoresis, comprising: (a) linking a fluorophore to a DNA fragment in a sample to transfor it into a fluorescent species;

(b) introducing samples containing fluorescent species into a pluralit of capillaries in a capillary electrophoresis system, the system comprises an arra of at least about 100 capillaries each having an inside diameter of about 20-50 microns, and an annular wall; and

(c) substantially simultaneously detecting change in fluorescenc emitted by the fluorescent species through the wall of each of the capillaries b focusing the fluorescence onto a charge transfer device based imaging system