FIELD OF THE INVENTION The present invention relates to protein detection and quantification using microfabricated devices and capillaries. In particular, the present invention provides methods for separation and detection, in a microfabricated device or capillary, of proteins in a sample without requiring pre-or post-column protein labeling or modification for eventual laser-induced fluorescence (LIF) detection.

BACKGROUND OF THE INVENTION Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the workhorse of clinical laboratories interested in protein sizing. SDS-PAGE offers simple methodologies; parallel processing capabilities and accurate mass determination of complex protein samples. More recently, capillary electrophoresis (CE) has begun to supplant the more traditional gel electrophoresis in the biomedical and clinical settings.

This can be attributed to order-of-magnitude decreases in separation times, on-line detection, automation, low sample volumes, and the ability to parallel process in 96- capillary array instruments. In fact, the replacement of SDS-PAGE with a capillary counterpart was addressed early in the genesis CE technology by Cohen and Karger (J.

Chromatogr. 1987, 397, 409-417) as an alternative to gels.

Many have further developed CE-SDS analysis by moving away from a"gel-in-a- <BR> <BR> capillary"approach and toward non-gel sieving polymers or"physical gels. "Dextran, non-crosslinked polyacrylamide (PAA), polyethylene oxide (PEO), and hydroxypropyl cellulose (HPC) have all been used in CE-SDS analysis. Ganzler et al. (Anal. Chem.

1992, 64, 2665-2671) improve sensitivity and ease-of-use in developing dextran and PEO as sieving polymers which reduced the background noise for UV absorbance detection in order to demonstrate detection limits as low as 500 ng/mL (10% dextran; 2,000, 000 molecular weight; S/N-2) with a myoglobin standard. However, analysis of myoglobin in dog plasma could only yield a limit-of-detection of 15 . g/mL. This reduction in sensitivity was attributed to the complexity of the sample.

Bean and Lookhart (J. Agric. Food Chem. 1999, 47, 4246-4255) significantly reduced the complexity of CE-SDS analysis of wheat proteins by focusing on self-coating sieving polymers. These polymers, including PAA and PEO, sufficiently suppressed EOF provided the appropriate capillary conditioning is utilized.

Others focus on electrophoresis in general (not particular to proteins). U. S. Pat.

No. 5, 126,021 to Grossman discloses a capillary electrophoresis element which includes a capillary electrophoresis tube containing a low viscosity uncharged polymer solution, for separating nucleic acids.

U. S. Pat. No. 5,264, 101 to Demorest et al. discloses the use of a hydrophilic polymer solution, which is characterized by a molecular weight of 20 to 5,000 kD, and a charge between 0. 01 and 1% as measured by the molar percent of total monomer subunits to total polymer subunits, where the charge is opposite to the charge of the surface of the capillary in which the polymer is used. This opposite charge of the polymer is reported

to result in an interaction between the polymer and the capillary wall to reduce electroosmotic flow within the capillary.

U. S. Pat. Nos. 5,552, 028 and 5,567, 292, both to Madabhushi et al. , disclose the use of a uncharged, water soluble, silica adsorbing polymer in a capillary electrophoresis system to reduce or eliminate electroosmotic flow.

While there has been much success with respect to protein analysis on capillaries, translating these separations to the microchip platform has been limited, particularly for protein detection and quantification. Although micro-chip-based electrophoresis has been developed for DNA (see e. g. , U. S. Patent Nos. 5,948, 227; 6,042, 710; and 6,440, 284; all to Dubrow; and U. S. Patent Nos. 6,337, 740 and 6,590, 653, both to Parce), the problem of protein detection and quantification on microchip platforms can be attributed to the difficulty in developing UV detection on microchips, due to physical limitations such as path length and coupling of the light in and out, or more practical limitations such as the cost of quartz to produce chips with low UV absorbance, and the reliance on non-trivial labeling procedures for laser-induced fluorescence (LIF) detection.

Until recently, protein analysis on chips has relied on either detection using natural fluorescence or time-consuming pre-column tagging with fluorescent labels. Conversely, size-based analysis of DNA on microchips is commonplace due to the ease with which DNA can be labeled with intercalating dyes during separation. Unlike DNA analysis, protein detection on microchips has not had an analogous labeling approach until recently (Jin et al. Anal. Chem. 2001, 73, 4994-4999; and Bousse et al., Anal. Chem. 2001. 73, 1207-1212). The work by Bousse et al. involves inclusion of a proprietary fluorescent dye which bound to protein-SDS complexes and SDS micelles during a microchip-based

separation that is equivalent to SDS-PAGE. Inclusion of the dye at high concentrations in the separation buffer results in low sensitivity attributing to the high background fluorescence resulting from dye-bound SDS micelles. In order to alleviate this problem, a dilution step prior to detection is utilized to reduce the SDS concentration to sub- miceller levels. Conversely, Jin et al. uses a fluorescent dye, NanoOrange@, which also binds to SDS-micelles and protein-SDS complexes, however, a dilution step was not required prior to detection, and separation and detection is possible on both the capillary and microchip platforms. In contrast, the work by Bousse et al. is limited to the microchip due to the microchannel structure necessary for the dilution step.

Harvey et al. (J. Chromatogr. B 2001, 754, 345-356) have used NanoOrangee as an on-column dynamic labeler of proteins in CE under non-denatured conditions, achieving a limit of detection of 212 ng/mL for human serum albumin.

Liu et al. (Anal. Chem. 2000,72, 4606-4613) utilized the same fluor for post- column labeling of non-denatured proteins on a microchip with a unique cross-t mixing design positioned prior to the detection point with sensitivity as low as 1. 21 jig/mL.

SUMMARY OF THE INVENTION The present invention describes the development of conditions more amenable to dynamic labeling with a fluorescent dye, particularly a merocyanine dye, especially those <BR> disclosed in U. S. Patent No. 5,616, 502 to Haughland et al. , which is incorporated herein by reference, for capillary and microchip electrophoresis-SDS analysis. Buffer components including sieving polymer, ionic strength, and SDS concentration were considered with a mind towards increasing detection sensitivity. These conditions were

translated to the microchip platform for rapid protein separation, detection, and quantification.

The present invention further provides a method for laser-induced fluorescence (LIF) detection of proteins in capillary electrophoresis or microfabricated devices without pre-or post-column modification of the proteins. The method consists of incorporating a fluorescent dye, particularly a merocyanine dye, into the matrix, buffer, or sample. When the dye is used in the matrix or buffer, the proteins, denatured by SDS, binds to the fluorescent dye through hydrophobic interaction.

When SDS is not present to promote dye-protein interaction, "trasient denaturing" can be used to optimize uptake of the fluorescent dye by the protein. Heat shock labeling involves labeling with the fluorescent dye, on-capillary or on-microchip, by heat denaturing the protein in the absence of SDS or other surfactants. Heating the native protein causes the protein to unfold, which allows the fluorescent dye to incorporate into the protein during the refolding process upon cooling which allows for detection of the protein.

Once bound to the fluorescent dye, the protein can be detected when the dye is excited by a radiation source, such as an argon-ion laser. This method simplifies sample treatment yet exploits detection sensitively of LIF detection. Further, the fluorescent dye binds to proteins on a per-mass basis when capillary and microchip conditions are equivalent to SDS-PAGE, which allows for protein quantification.

The methods of the present invention are sufficiently sensitive to detect proteins at concentrations of less than 1 llg/mL with a detection limit of about 500 ng/mL (signal/noise = 3).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the separation of an 8-component protein-sizing ladder at a total concentration of 32 ug/mL. Separation buffer was 250 mM Tris, 250 mM CHES, pH 8.7 with 0. 1 % w/v SDS and 5% w/v 100,000 MW PEO. Capillary was 50 micron diameter, 27 cm long (20 cm to detection window). Sample was injected for 10 seconds at a field strength of 370 V/cm. Separation field strength was 370 V/cm.

Figure 2 shows A) the separation observed when dye is added to the sample, while none is included in the separation buffer. Conditions, save for dye inclusion in the separation buffer, as in Figure 1; and B) the separation observed when dye is included in both the sample and the separation buffer.

Figure 3 shows a comparison in peak intensity between 0.04% w/v SDS containing separation buffer and 0.1% w/v SDS containing separation buffer on a capillary. Injection and separation fields as described in Figure 1. Sample is the 8- component sizing ladder at a total protein concentration of 16 tg/mL. Sample is injected using a cross-t configuration at-lOOV/cm. Sample is separated at-300V/cm.

Figure 4 shows separation of the 8-component ladder on-chip with 0.04% w/v containing separation buffer using the 0.04% SDS containing separation buffer, 0.8% merocyanine dye included. Sample concentration-64 mg/mL total Figure 5 shows: Lower trace-microchip electropherogram of bovine serum albumin (66 kDa) using 0.04% w/v SDS separation buffer with 0.8% v/v merocyanine dye; and Upper trace-separation of BSA with 1.6% merocyanine dye added to sample prior to separation.

Figure 6, shows a sample matrix components comparison between capillary and microchip: A) 25 mM Tris-CHES, ImM DTT on capillary; B) 25 mM Tris-CHES, 1mM DTT on microchip; C) SDS containing sample matrix on capillary; D) SDS containing sample matrix on microchip; E) SDS containing sample matrix with dye included on capillary; F) SDS containing sample matrix with dye included on microchip; G) 25 mM Tris-CHES, 1mM DTT with dye included on capillary; and H) 25 mM Tris-CHES, 1 mM DTT with dye included on microchip.

Figure 7 shows a separation of solid-phase extraction purification of proteins from human semen.

Figure 8 shows comparison of CZE analysis of human sera to partially-denatured sera using the 0. 04% merocyanine dye containing run buffer. Sera is diluted 1-500 in 0.5% SDS, with 1% merocyanine dye included. Separation conditions are as shown in Figure 4. Figures 8A and 8B show the normal serum profile, 8C and 8D the profile of a sample with elevated p-region, and 8E and 8F the profile with an elevated y-region.

Figure 9 shows a pictorial representation of capillary and microchip electrokinetic injection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention provides methods for electrophoretically separating, detecting, and quantifying proteins in a capillary or microfabricated device.

Microfabricated or microfluidic devices are used to perform the separation of the present invention."Microfabricated"or"microfluidic,"as used herein, refers to a system or device having fluidic conduits or microchannels that are generally fabricated at the

micron to submicron scale, e. g. , typically having at least one cross-sectional dimension in the range of from about 0.1 u. m to about 500 um. The microfluidic system of the invention is fabricated from materials that are compatible with the conditions present in the particular experiment of interest. Such conditions include, but are not limited to, pH, temperature, ionic concentration, pressure, and application of electrical fields. The materials of the device are also chosen for their inertness to components of the experiment to be carried out in the device. Such materials include, but are not limited to, glass, quartz, silicon, and polymeric substrates, e. g. , plastics, depending on the intended application.

In certain embodiment, the devices can include an optical or visual detection element. In this case, the devices are generally fabricated, at least in part, from transparent materials to allow, or at least, facilitate detection and/or detection.

Alternatively, transparent windows of, e. g. , glass or quartz, may be incorporated into the device for these types detection elements.

The device generally comprises a solid substrate, typically on the order of a few millimeters thick and approximately 0.2 to 12.0 centimeters square, microfabricated to define at least one inlet reservoir, at least one outlet reservoir, and a microchannel flow system, preferably a network of flow channels, extending from the at least one inlet reservoir to the at least one outlet reservoir. Typically, the device contains at least a main electrophoresis channel and at least one intersection channels forming a cross-t intersection with the main electrophoresis channel. More than one cross-t intersections may be appropriate for injecting various samples, buffers, and reactants.

A variety of manufacturing techniques are well known in the art for producing microfabricated channel systems. For example, where such devices utilize substrates commonly found in the semiconductor industry, manufacturing methods regularly employed in those industries are readily applicable, e. g. , photolithography, wet chemical etching, chemical vapor deposition, sputtering, electroforming, etc. Similarly, methods of fabricating such devices in polymeric substrates are also readily available, including injection molding, embossing, laser ablation, LIGA techniques and the like. Other useful fabrication techniques include lamination or layering techniques, used to provide intermediate microscale structures to define elements of a particular microscale device.

To provide appropriate electric fields, for electrophoresis and for fluid movement such as sample injection, the system generally includes a voltage controller that is capable of applying selectable voltage levels, sequentially or, more typically, simultaneously, to each of the reservoirs, including ground. Such a voltage controller is implemented using multiple voltage dividers and multiple relays to obtain the selectable voltage levels. Alternatively, multiple independent voltage sources are used. The voltage controller is electrically connected to each of the reservoirs via an electrode positioned or fabricated within each of the plurality of reservoirs. Use of electrokinetic transport to control material movement in interconnected channel structures was described, e. g. , in WO 96/04547 to Ramsey, which is incorporated by reference.

Modulating voltages are concomitantly applied to the various reservoirs to affect a desired fluid flow characteristic, e. g. , continuous or discontinuous (e. g. , a regularly pulsed field causing the sample to oscillate direction of travel) flow of labeled components toward a waste and/or collection reservoir. Particularly, modulation of the

voltages applied at the Various reservoirs can move and direct fluid flow through the interconnected channel structure of the device.

Another way to control flow rates is through creation of a pressure differential.

For example, in a simple passive aspect, a cell suspension is deposited in a reservoir or well at one end of the channel, and at sufficient volume or depth, that the cell suspension <BR> <BR> creates a hydrostatic pressure differential along the length of the channel, e. g. , by virtue of its having greater depth than a well at an opposite terminus of the channel. Typically, the reservoir volume is quite large in comparison to the volume or flow through rate of <BR> <BR> the channel, i. e. , 1 uL reservoirs or larger as compared to a 100 um channel cross section.

Another pressure based system is one that displaces fluid in the microfluidic channel <BR> <BR> using, e. g. , a probe, piston, pressure diaphragm, or any other source capable of generating a positive or negative pressure.

Alternatively, a pressure differential is applied across the length of the channel.

For example, a pressure source is optionally applied to one end of the channel, and the applied pressure forces the material through the channel. For example, pressure applied at the inlet reservoir would force the cell mixture contained therein through the <BR> <BR> microchannel, and into the outlet reservoir. The pressure is optionally pneumatic, e. g. , a<BR> pressurized gas or liquid, or alternatively a positive displacement mechanism, i. e. , a plunger fitted into a material reservoir, for forcing the material along through the channel.

Pressure can, of course, also be due to electrokinetic force, thermal expansion, or a variety of other methods and devices.

Alternatively, a vacuum source (i. e. , a negative pressure source) is applied to a reservoir at the opposite end of the channel to draw the suspension through the channel.

A vacuum source can be placed in the outlet reservoir to draw a cell suspension from the inlet reservoir. Pressure or vacuum sources are optionally supplied external to the device or system, e. g. , external vacuum or pressure pumps sealably fitted to the inlet or outlet of<BR> the channel, or they are internal to the device, e. g. , microfabricated pumps integrated into the device and operably linked to the channel, such as those disclosed in WO 97/02357 to Anderson et al. , which is incorporated herein by reference.

Flow control is important for injecting samples and appropriate buffers into the main electrophoresis channel from crossing channel (s). Certainly, electric field is also required for electrophoresis in the main channel.

The main electrophoresis channel may or may not be filled with a sieving matrix.

However, for some embodiments, it is preferred that a sieving matrix be used. The matrix can be, but is not limited to, dextran, polyacrylamide (PAA), polyethylene oxide (PEO), hydroxypropyl cellulose, and/or combinations thereof.

The proteins can be subjected to electrophoresis in their native, partially denatured, or in completely denatured form. The proteins can be denatured by a surfactant, such as routinely done with SDS; reducing agent, such as 2-mercaptoethanol and/or dithiothreitol (DTT); pH; heat; and/or other methods known in the art. In a preferred embodiment the protein is bound to SDS at a ratio of 0-1.4 g SDS/g protein.

The protein-SDS complex is then heated for about 10 minutes at about 95-100°C. If the proteins are to be analyzed in their reduced form, a reducing agent can be added prior to heating. When using SDS, the SDS concentration is preferably below the micelle concentration (Cmc). Depending on the ionic character of the system, the SDS concentration is preferably about 0.5 to about 8mM

The proteins are detected using a fluorescent dye, particularly a merocyanine dye.

In a preferred embodiment, the dyes disclosed by U. S. Patent No. 5,616, 502 to Haughland et al. , which is incorporated herein by reference, is most appropriate for the present invention. Several fluorescent dye properties are highly desirable for use with the present invention. First, the dye binds preferentially to SDS-protein complex, when compare to its binding with free SDS in solution. The binding of the fluorescent dye to free SDS is preferably at least 10% lower than its binding to SDS-protein complex, resulting in low background fluorescence. Second, the fluorescent dye binds to the protein through hydrophobic and/or electrostatic interaction. Although the interaction is hydrophobic, the protein needs not be hydrophobic. Only the region of the protein that binds to the fluorescent dye has to be hydrophobic, while the overall protein is not required to be hydrophobic.

The fluorescent dye can be in the sample, the matrix, or the buffer. Importantly, however, the fluorescent dye must come into contact with the proteins on-column, i. e. , no pre-or post-column modification. If the fluorescent dye is used in the buffer and/or the matrix, it should be in concentrations of about 0.01-25%, more preferably about 0.1%- 1.0%, and most preferably about 0.2-0. 8 % (v/v). The fluorescent dye preferably has an excitation wavelength of about 450-500 nm, most preferably about 470-480 nm, and an emission wavelength of about 520-660 nm, most preferably about 570-600 nm.

The matrix is selected so that it has little of no binding with the fluorescent dye.

Preferably, the binding of the fluorescent dye to the matrix should be at least 10% lower than its binding to the protein or the SDS-protein complex, resulting in low background fluorescence.

Preferably, the protein is SDS-denatured when electrophoresis is performed with the dye in the buffer or the matrix. The addition of the fluorescent dye to the separation buffer and sample matrix allows for a simple method for non-covalent labeling of protein-SDS complexes for sizing. The fluorescent dye binds to the SDS-protein complex on a per-mass basis regardless of the type of protein under denaturing conditions. This is very important because it allows for protein quantification. SDS binds to proteins in a per-mass fashion: for every gram of protein, about 1.4 grams of SDS is bound to the peptide backbone. This amounts to a single SDS molecule binding to the backbone of two amino acids. For the present invention, it is preferable that SDS is used at the sub-micellular concentrations (<Cmc).

In an embodiment of the invention, the protein can also be injected into the electrophoresis column/microfabricated device in its native form. In this approach, the proteins are labeled on-column and then ran under completely non-denaturing conditions.

This method is referred to as"transient denaturing. "This involves labeling with the fluorescent dye on-capillary or on-microchip by denaturing the protein in the absence of SDS or other surfactants. This is preferably carried out on-column in a zone that can be heated, either by Joule heating provided the appropriate buffer conditions and electric fields are utilized or by inputting energy (laser, IR, microwave, and the like). Heating the native protein causes it to unfold. Adding the fluorescent dye in the heated zone it will incorporate the dye into the protein during the refolding process upon cooling (the heated zone disperses over time as equilibrium is achieved). Alternatively, the dye and protein can exist in separate zones, with the dye outside of the heated zone and, during electrophoresis, the unfolded protein is allowed to migrate out of the heated zone, into

the dye zone which may or may not be at a lower temperature-refolding of the protein, thus leads to incorporation of the dye. The binding of the fluorescent dye to the protein would be a combination of interaction with hydrophobic residues, the peptide backbone, and just being in the right place at the right time and being caged within the refolded protein. While the refolded protein may not necessarily take on the original tertiary structure, it will have bound sufficient dye for detection and be amenable to separation.

During electrophoresis, the proteins can be detected and/or quantify through the optical or visual detection element on the microfabricated device. In an embodiment, a light source, such as a laser, which emits light in the wavelengths known to induce fluorescence of the fluorescent dye, is focused onto the optical or visual detection element. For example, the excitation/emission wavelengths for merocyanine dye are 450-500/520-660 nm, respectively. Thus, the 488 nm line of an Argon-ion laser can be used for excitation of the fluorophore and emission can be filtered through a 590DF35 band-pass filter for dection of fluorescence. Various lenses and optics can be used to focus and gather excited and emitted lights. The emitted light can be detected and quantify by a photoreceptor, such as a photomultiplier tube, a photodiode, a CCD array, and the like, and can be converted into electrical signals to be sent to a microprocessor for data recording and analysis.

Example 1-Experimental Method Apparatus Beckman P/ACE 5510 (Beckman Instruments, Inc. , California) with a LIF detector was used to obtain capillary electrophoresis data. The microchip electrophoresis

system was assembled in-house. A laser beam of 488 nm was emitted from an Argon Ion laser source (Laser Physics, Utah) and reflected to the beam splitter (505DRLP02 ; Omega Optical, Vermont) set 45° to the incident beams and collected onto the channel of electrophoretic microchip by a microscope objective (16x/numerical aperture, 0.32 ; Melles Griot, New York). Two mirrors (Thor Labs, Inc. , New Jersey) positioned parallel to each other were set after the laser source to level up the laser beam to the beam splitter.

Fluorescence emitted by the sample was collected by the same microscope objective and focused by a 150-mm lens onto a Tu-Can PMT (Hamamatsu, New Jersey) filtered by a 590nm bandpass filter (590DF30, Omega Optical) set before PMT. The data collection was processed via a program written in Labview.

Reagents Tris [hydroxymethyl] aminomethane (TRIS), 2- [N-cyclohexylamino] ethane sulfonic acid (CHES), Dextran (2,000, 000 MW), and Hydroxyethylcellulose (250,000 MW) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium dodecylsulfate and an 8-component protein ladder obtained from Bio-Rad (Hercules, CA). Polyethylene oxide (100,000, 200,000, and 600,000 MW) was purchased from Acros (Pittsburgh, PA).

Fluorescent dye (merocyanine dye) was purchased from Molecular Probe (Oregon) as NanoOrange @ and was used according to the product instruction manual. The merocyanine dye stock solution (500X) was added to the separation buffer at 1X concentration unless otherwise specified.

Microchip Fabrication

The microchip Was fabricated by using standard photolithography and wet chemical etching technique. A film mask containing the microchip design was prepared on a negative film using an image setter. The microchip design, a traditional cross-T type, was transferred onto the glass wafer (Nanofilm, California) with positive photoresist by UV exposure. The channels were etched with HF solution. Etched plate was thermally bonded to a drilled cover plate in a programmable furnace (Ney Dental Inc. , California). The configuration of a single channel microchip is 7.5 cm from injection cross to the outlet, 0.5 cm from injection cross to inlet and sample and sample waste.

Capillary and Microchip Separations Unless otherwise specified, capillaries and microchips were flushed for 10 minutes with 1M HN03, followed by a 12 minute rinse with 1X merocyanine dye containing run buffer. Proteins (dissolved in 25 mM Tris-CHES, 0.1% SDS, 1mM DTT and heated at 94°C for 10 minutes then cooled to room temperature) were electrokinetically injected into the capillary or microchip. Separations were performed under reverse polarity (inlet was cathode) with field strength of 370 V/cm.

Example 2-Results and Discussion Evaluation of Sieving Polymers Our initial goal in developing a separation buffer more amenable to labeling with merocyanine dye was the difficulty in handling the commercially available system, specifically with respect to viscosity. Loading of separation buffer in both capillaries and

microchips was'difficult. In addition, this buffer provided a high background fluorescence and sensitivity was not enhanced by going from UV to LIF detection. A number of polymers were evaluated in this work, including dextran, PEO, HPC, and HEC. The ideal choice proved to be PEO for a number of reasons; primarily is low contribution to background fluorescence in the presence of merocyanine dye, self-coating capabilities, and low viscosity relative to the other evaluated sieving matrices.

Briefly, as indicated by Bean and Lookhart (J. Agric. Food Chem. 1999, 47, 4246-4255), dextran is an adequate sieving polymer for protein SDS analysis; however, it is not a self-coating polymer and requires other means of EOF suppression in CE-SDS analysis. HPC proved to be incompatible with on-column labeling, though Hu et al. (J.

Chromatogr. A. 2000. 894, 291-296) have demonstrated it as a suitable sieving polymer for protein sizing with fluor-labeled analytes. PEO and HEC allowed for near identical separating power and contribution to background fluorescence, but PEO was less viscous and thus chosen over HEC.

Figure 1 illustrates the capillary separation of an eight-component protein sizing ladder (32 pg/mL total protein concentration) using 5% PEO, 100,000 molecular weight (MW), 250 mM Tris-CHES, 0.1% SDS separation buffer; conditions similar to those described here have been utilized by several groups for CE-SDS analysis, i. e. , 10,14.

Interestingly, addition of dye to the sample matrix is detrimental to resolution and sensitivity. Figure 2a shows the electropherogram for a 100 llg/mL ladder with 0.2% merocyanine dye added to the sample matrix after heat denaturing of the sample and no dye included in the run buffer. A single peak is observed that we attribute to merocyanine dye saturated SDS micelles. Unlike work by Harvey et al. (Electrophoresis,

1998, 19, 2169-2174), there SDS was included in the sample matrix below critical micelle concentration (Cmc) for labeling with Sypro Red, we found it necessary that the SDS concentration need to be >Cmc to ensure good resolution of the protein ladder. No peaks corresponding to the protein ladder are observed; we attribute this to SDS-micelles binding all available merocyanine dye in the sample leaving none for binding to SDS- protein complexes. Including dye in both the sample and the separation buffer results in the same system peak and poorly resolved ladder components (Figure 2b).

Effect of SDS One consequence of maintaining SDS above Cmc in the run buffer besides good resolution is that it increases the background fluorescence and reduces the availability of fluor for dynamic binding to protein-SDS complexes. Data presented in Table 1 indicates that merocyanine dye binds strongly to micellized SDS and as hypothesized in the previous section may make fluor unavailable for binding to protein-SDS complexes.

TABLE 1. The contribution to background fluorescence made by various combinations of buffer components all containing 0.2% merocyanine dye. Measurements were made on a Beckman 5510 with an LIF detector by flowing the solution by the detector window. Solution RFU 250 mM Tris CHES 1 0. 1% SDS 1.0% SDS 195. 4 250 mM Tris CHES, 0. 1% SDS 196 250 mM Tris CHES, 5.0% PEO 13. 6 250 mM Tris CHES, 0.1% SDS, 5.0% PEO 201. 1 250 mM Tris CHE'S, 0.08% SDS, 5.0% PEO 194. 7 250 mM Tris CHES, 0.06% SDS, 5. 0% PEO 201. 3 250 mM Tris CHES, 0.04% SDS, 5.0% PEO 176. 0 250 mM Tris CHES, 0.02% SDS, 5.0% PEO 105. 1

Merocyanine dye was added to various combinations of the separation buffer components to determine their contribution to the background fluorescence. As expected, SDS in solution does increase the background fluorescence, which we attribute to the increased hydrophobic character of the buffer system. Interestingly, the background fluorescence of 1% SDS is approximately 20 times higher than that of 0.1% SDS (3.4 mM) -not the 10-fold increase that would be expected if SDS concentration was the sole contributor. We expect that the 20-fold increase is due to micellization of SDS creating a stable hydrophobic pocket for binding the dye. Inclusion of SDS at 0.1 % to 250 mM Tris-CHES results in the same 20-fold increase in background fluorescence as seen with 1 % SDS. It is known that SDS micellization can be reduced to between 1 and 3 mM in high ionic strength buffer; we believe that is the case in our system. Addition of PEO to the 250 mM Tris-CHES 0. 1% SDS separation buffer results in a small increase in background fluorescence, compared to the contribution of SDS micelles makes, this is a negligible increase.

Titration of the SDS concentration from 0. 1 % to 0% SDS results in no appreciable change in background fluorescence until a shift from 0.06% to 0.04%, where the signal drops from 200 relative fluorescence units (RFU) to 175 RFU. At this concentration SDS is not completely micellized, thus making more dye available for

protein-SDS complex binding. Using the newly defined separation buffer, we analyzed the protein ladder on a capillary with a total protein concentration of 16 pg/mL and compare the results to the separation obtained using the 0.1% SDS containing separation buffer (Figure 3). Resolving power is only marginally effected, there is no obvious effect on separation time, and most importantly the signal is increased 2. 5-fold, with no appreciable change in the noise level. A limit-of-detection of 500 nanograms/mL (S/N = 3) was determined for bovine serum albumin on a capillary using these separation conditions.

Microchip Electrophoresis-Effect of Dye in Sample Buffer In contrast to the detrimental effect observed in capillaries, increasing the dye concentration increased the sensitivity of the microchip system. The dye concentration was, therefore, titrated from 0 to 2.0% in the sample matrix to explore the effect of this variable. It was found that addition of 1.6% dye to the sample matrix in combination with 0.8% dye in the separation buffer, allowed for a CLOD of 500 ng/mL BSA with a signal/noise of 8. No improvements in signal-to-noise were observed with dye concentration in excess of 1.6% v/v. Accepting a minimal signal/noise limit of 3, one can calculate a CLOD of less than 200 ng/ml under these conditions. This represents approximately a 5-fold improvement in microchip limits of detection as reported by Bousse et al.

Electropherograms from the microchip and capillary systems were further compared in an attempt to understand the differences between the separations. Injection of 25 mM Tris-CHES, 1 mM DTT in the absence of SDS produces the same profile in

capillaries and the microchips. A baseline drop, always observed upon injection and separation of a sample matrix, a followed by a peak which could be attributed to dye- bound SDS micelles (Figures 6A-B). Injection of the normal sample matrix, which has a higher SDS concentration (0. 1% SDS) than the separation buffer (0. 04%) in two system peaks in both the capillary and microchip separations. One of these peaks 0 again attributed to the dye-bound SDS-micelles (MC-dye), the other attributed to monomeric SDS bound dye (SDS-dye). Figures 6C-D illustrate, however, that the appearance of the system peaks M drastically between the two systems. On capillaries the SDS-dye peak dominates and there, two baseline perturbations, the first between the SDS-dye and MC-dye peaks (due to the sample matrix), while the second after the MC-dye peak and is attributed to a region that dye trailing the MC-dye peak. On the microchip, the MC-dye and the SDS-dye peak tiW similar in magnitude, the sample matrix-induced drop in baseline is not resolved (Figure 6D), but the dip associated with the absence of dye following the MC-dye peak is observed. The less obvious nature of this drop can be attributed to the higher concentration of dye used in the microchip experiments.

Conducting these same experiments with dye in the sample matrix markedly different profiles for the capillary and microchip systems. Repeating the experiments with SDS containing sample matrix, also containing 1.6% dye, the capillary separation showed only the MC-dye peak and the slight dip expected for the sample matrix [Figure 6E]. As expected, the dip attributed to absence of dye following the MC-dye peak is not present due to the excess of dye in the sample matrix, but there is also no SDS-dye peak.

This is ascribed to the induced micellization of SDS in the sample matrix in the presence

of the dye. The'corresponding microchip experiment shows an increase in the MC-dye peak but maintains the SDS-dye peak (Figure 6F). This is not unexpected due to the differences between the electrokinetic injections seen in capillaries and microchips.

Injection of 25 mM Tris-CHES, 1 mM DTT containing 1.6% dye in the absence of SDS produces two peaks with the microchip and only one with the capillary. The peaks observed on the microchip are attributed to SDS-and MC-dye, respectively (Figure 6H).

Inclusion of dye in 25 mM Tris-CHES, 1 mM DTT with no SDS produces an anomalous peak tentatively identified as SDS-dye on the capillary [Figure 6G]. The dye is net neutral at the pH of the sample and separation buffers (pH = 8.7), thus, if included in sample matrix, it should not be injected in the absence of a carrier anion, such as SDS (demonstrated in 6E). This is the result of micellized dye, causing charge condensation to a net negative species, which is capable of being injected. Upon injection, the dye- micelle would bind free SDS in the separation buffer to produce the observed SDS-dye peak.

This separation system exhibits the fundamental difference in capillary versus microchip electrokinetic injections-with capillaries, sample is injected into the separation channel with partial separation of sample components during the injection, while on microchips, sample is injected across the separation channel with a truly representative sample present in the cross-t (after a particular time) (Figure 9). The microchip injection to result in a concentrating of SDS-dye and MC-dye (as seen in Figures 6E, F, and H) on both sides of the sample matrix (the side towards the inlet and <BR> <BR> the side towards the outlet) -in capillaries, the side of the sample matrix facing the outlet is disrupted during injection and the concentrating effect is seen only on the side towards

the inlet. Ideally, one would compare a capillary pressure injection with the microchip electrokinetic injection; unfortunately due to the viscosity of the separation buffer, pressure injection results are not reproducible. What this means, is that for LIF detection of proteins using dye, the conditions for achieving maximum sensitivity are system dependent.

Microchip Electrophoresis-Detection Sensitivity and Range The buffer system described in Figure 3 was utilized in a traditional cross-T injection configuration microchip for on-chip protein analysis. On-chip sensitivity was improved by going to a 0.8% merocyanine dye concentration. Interesting, use of this dye concentration in capillary separations resulted in increases in the noise levels with no appreciable increase in signal. Figure 4 illustrates the protein ladder (64 ug/mL) as separated on a microchip. A log (MW) versus 1/MT plot was associated with an W value of 0.9957, indicating that the ability to size proteins on the microchip is retained. The sensitivity attained on-chip was comparable to that observed on a capillary with a detection limit of 1 « ag/mL for BSA (S/N = 3) (Figure 5, lower trace). While capillary experience suggested that addition of dye to the sample is detrimental to sensitivity, we felt that given the enhanced sensitivity afforded by including more dye in the run buffer on-chip it would be interesting to see what the addition of dye to the sample matrix produced. In fact, addition of 1.6% merocyanine dye to the sample matrix while retaining 0.8% merocyanine dye in the separation buffer results in detection of 500 ng/mL BSA with a signal/noise of 8 (Figure 5, upper trace).

In additi'on to a highly very sensitive detection limits, detector response is linear with respect to concentration. BSA at various concentrations was included in sample matrix and electrophoresed. The peak area of BSA was determined and correlated to concentration. We found that detector response was not linear with respect to concentration and there was variation in peak area for the same BSA concentration greater than 5%. We attributed these variations to run-to-run differences in chip alignment. We proceeded to add a second protein to the sample at a fixed concentration (myosin, 20 g/mL). The peak area of BSA was normalized to the peak area for myosin and correlated to BSA concentration. We found that the normalized peak area is linear with concentration between 1-50 g/mL for BSA, with an R2 value equal to 0.9600.

These results demonstrate nearly order-of-magnitude response linearity with respect to concentration. We also found that merocyanine dye does binding to proteins in a per- mass fashion. We will have a figure, which demonstrates that, merocyanine dye binds in a per-mass fashion to proteins. This implies a limit of detection that is consistent for all proteins regardless of mass.

Microchip Electrophoresis-Clinical Applications In order to test the effectiveness of our labeling technique, we investigated separation of proteins extracted from various biological sources. Figure 7 shows the protein sizing profile observed from a sample of proteins extracted from human semen.

In our laboratory research in DNA and protein purification from biological samples is underway, with particular interest in solid-phase extraction. In this process, the sample is dissolved in an 8M Guanadine HCl buffer and is flowed through a silica bed, in the form

of a sol-gel. Biological components (i. e. proteins, DNA, lipids) stick to the silica surface.

The proteins and other cellular components (i. e. lipids and sugars) are washed off of the silica surface in a 40% solution of isopropanol. DNA is eluted from the sol-gel using aqueous buffer such as Tris-EDTA. The sample from Figure 7 was diluted 1: 10 in sample matrix and heat denatured. Since the sample is a crude extract, it has in the range of 10,000 individual proteins and peptides at various concentrations. In order to reduce complexity of the microchip electropherogram, dye was not included in the sample (addition of dye to the sample matrix would result in a more sensitive separation). The electropherogram shows a series of primary peaks on top of what appears to be a drifting baseline; in fact they are low abundance proteins not well resolved in this separation. It is important to note that the analyzed sample is over a 1-to-10000-fold dilution of the original semen sample.

The utility of merocyanine dye labeling under CE-SDS separation conditions is not limited to samples for purely sizing purposes. A sample can be dissolved in SDS containing sample matrix, however without heat denaturing proteins tend not to unfold.

Thus, SDS simply binds to the protein surface, imparting some charge, but not enough for an accurate sized-based separation. By dissolving the sample in SDS-containing solution you also facilitate the binding of dye to the sample, by imparting some hydrophobic character to the protein surface. We utilized partially-denatured conditions (dissolved in SDS, but no heat denaturing) in the analysis of human serum in hopes of detecting abnormal serum profiles. Figure 8 shows the traditional CZE analysis and microchip CE-SDS analysis for 3 human serum samples. Figure 8A and 8B show the normal serum profile, 8C and 8D the profile of a sample with elevated p-region, and 8E

and 8F the profile with an elevated y-region. Diagnostic capabilities for obvious serum disorder are maintained using the partially denaturing microchip separation. Further studies need to be performed in order to determine whether or not the microchip separation is sensitive enough to pick up subtler difference between normal and abnormal sera.

Although certain presently preferred embodiments of the invention have been specifically described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the various embodiments shown and described herein may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of law.