Microchannel Capillary Electrophoresis

In this section we describe initial efforts to miniaturize conventional capillary electrophoresis experiments. Microfluidic devices were fabricated that replicated the essential qualities of capillary systems. These early microflu-idic chips incorporated electrokinetic sample handling protocols to facilitate easy sample manipulation within the microfluidic environment. Additional improvements over conventional capillary systems were realized through decreased detection limits and decreased analysis time.

One of the first steps towards miniaturizing conventional biological assays and developing a pTAS was the work of Harrison et al. [29]. Over a decade ago, these researchers micromachined a glass substrate with 30 pm wide channels for use in separating two charged, fluorescent molecules. The experiment was essentially the microfluidic analog of capillary electro-phoresis. Their results indicated that glass substrates and microfabrication techniques could effectively be used to miniaturize capillary electropho-resis equipment. Furthermore, their results showed that microfluidics offered excellent fluid and sample handling characteristics. Rather than using pressure in conjunction with separate sample vials, they were able to introduce samples from on-chip reservoirs with electroosmotic pumping. Electrokinetic sample handling is significantly simpler than pressure-based methods and is very compatible with microfluidic systems.

They report their separation efficiency in terms of the theoretical number of plates (a common chromatographic measure of separation efficiency), and obtain a maximum number of theoretical plates for calcein of 35,000. This is comparable to conventional capillary electrophoresis methods. Finally, they estimate that their microfluidic separation system performs nearly ideally in the sense that the only measurable dispersion is due to the finite size of the detector and the size of the injected sample plug. Joule heating and analyte-wall interactions play no role in band broadening.

Another effort to miniaturize conventional capillary electrophoresis experiments was conducted by [30]. One of their first reports describes a 2.5 cm2 glass chip upon which a serpentine channel was etched. This unique geometry enables much longer microfluidic channels to be made on a much smaller footprint. In addition to using a new geometry to minimize the overall chip-area, the group describes techniques for minimizing band dispersion caused by inefficient injection methods. In order to minimize the width of the injected analyte plug, it is necessary to apply appropriate voltages to all reservoirs, not just the sample loading and sample loading waste reservoirs. The separation reservoirs must be biased with voltage (as opposed to floated relative to ground) to prevent leakage during the loading phase. The group showed that these serpentine devices could be used to separate two fluorescent dyes as depicted in Fig. 3.7.

Shortly after their work in 1992, Harrison et al. reported results showing the separation of six amino acids (the monomer units of proteins) in similar microfluidic channels [31]. Results of the amino acid separation experiment are shown in Fig. 3.8. The reported separation efficiency was 40,000 to 75,000 theoretical plates, as compared to 400,000 theoretical plates reported in conventional capillary electrophoresis experiments [32]. While the microfluidic system's separation efficiency is an order of magnitude lower than the capillary system, the microfluidic-based experiment takes about 15 sec while the conventional experiment takes about 15 min.

In 1995, Jacobsen et al. used fused quartz microchips to electrophoreti-cally separate metal ions bound to 8-hydroxyquinoline-5-sulfonic acid [33]. The researchers used zinc, cadmium, and aluminum, all of which possessed a net negative charge in solution. Because the negative charge on the metals would cause the metal electrophoretic mobility to be in the opposite direc tion as the bulk fluid electroosmotic mobility, the researchers coated the channels with linear acrylamide. This coating, which is applied by flowing acrylamide through the channels and covalently linking it to the negatively charged fused quartz surface prior to electrophoresis experiments, has the effect of suppressing the zeta potential which in turn essentially eliminates electroosmotic flow of the bulk fluid. When only negatively charged analytes are used in electrophoresis experiments, it is generally recommended to eliminate the bulk electroosmotic flow to increase analyte mobility and decrease analysis time. The researchers were able to separate the three metal complexes in about 15 sec. Owing to the very low background fluorescence of the fused quartz microchip, quantities of metals as low as 46, 57, and 30 ppb for Zn, Cd, and Al respectively. Similar experiments done with con-

Figure 3.7 A five panel collage of optical micrographs of the serpentine device described in [30], reprinted with permission from [30], Copyright 1994 American Chemical Society. (a) bright field image of the intersection of the loading and separation channels. The beginning of the serpentine separation channel extends to the bottom and bottom-left of the image. (b)-(e) 1 sec interval fluorescent images of plug injection and separation. (b) Loading fluorescent dye through the intersection. (c) The plug just after injection into the serpentine separation channel. (d) The plug has separated into its two constituents, rhodamine (faster) and sulforhodamine (slower). (e) The samples are fully resolved. Note that the slight back-bias along the loading channel has drawn the sample in the loading channels away from the intersection, preventing sample leakage into the separation channel.

Figure 3.7 A five panel collage of optical micrographs of the serpentine device described in [30], reprinted with permission from [30], Copyright 1994 American Chemical Society. (a) bright field image of the intersection of the loading and separation channels. The beginning of the serpentine separation channel extends to the bottom and bottom-left of the image. (b)-(e) 1 sec interval fluorescent images of plug injection and separation. (b) Loading fluorescent dye through the intersection. (c) The plug just after injection into the serpentine separation channel. (d) The plug has separated into its two constituents, rhodamine (faster) and sulforhodamine (slower). (e) The samples are fully resolved. Note that the slight back-bias along the loading channel has drawn the sample in the loading channels away from the intersection, preventing sample leakage into the separation channel.

Buffer

Sample

10 nM Amino acids pH 9.0 11.25 kV (1.06 kV/cm) d« = 2.2 cm

Waste Detector

Waste

Figure 3.8 Results showing the separation of six amino acids in a microfluidic device, reprinted with permission from [31], Copyright 1993 AAAS. Peaks 1 and 3-7 are the amino acids. Peak 2 is a reactive by-product involving the fluorescent dye used in the experiments. The unlabeled peak at ~6 sec is not mentioned in the paper. The schematic in the upper right portion of the image shows the channel configuration. Translating the inset names into the terminology of Fig. 3.3(a), 'sample' is '1', 'buffer' is '3', (lower) 'waste' is '2', and (right) 'waste' is '4'. Electrokinetic pumping is used to move analytes throughout the microfluidic network. The separation efficiency of the microchip capillary electrophoresis device is about 50,000 theoretical plates which is comparable to conventional techniques.

Figure 3.8 Results showing the separation of six amino acids in a microfluidic device, reprinted with permission from [31], Copyright 1993 AAAS. Peaks 1 and 3-7 are the amino acids. Peak 2 is a reactive by-product involving the fluorescent dye used in the experiments. The unlabeled peak at ~6 sec is not mentioned in the paper. The schematic in the upper right portion of the image shows the channel configuration. Translating the inset names into the terminology of Fig. 3.3(a), 'sample' is '1', 'buffer' is '3', (lower) 'waste' is '2', and (right) 'waste' is '4'. Electrokinetic pumping is used to move analytes throughout the microfluidic network. The separation efficiency of the microchip capillary electrophoresis device is about 50,000 theoretical plates which is comparable to conventional techniques.

ventional capillary electrophoresis systems result in detection limits from 46 to 613 ppb in times of at least two or three minutes [34].

These initial microchip-based capillary electrophoresis experiments all involved separations of molecules with different charge-to-mass ratios (or quantification of charged molecules in the case of [33]). No sieving matrix was present in the microchannels. When electrophoresis if performed in capillaries or channels with no matrix, the process is typically referred to as free solution electrophoresis. When the charge-to-mass ratio of molecules is different, then free solution electrophoresis is capable of separating the molecules given enough resolution or channel length. For proteins in their native conformation (or amino acids) or for biomolecules that can be selectively bound to "carrier" molecules of different charge-to-mass ratios, free solution electrophoresis is typically sufficient for separation. For DNA or denatured proteins (which have a constant weight and charge molecule bound along the entirety of the polymer), free solution electrophoresis is not capable of effecting length-based separation. A sieving matrix must be introduced to break the so-called charge-to-mass symmetry. Slab gels of agarose or polyacrylamide are typically used to separate DNA and denatured proteins. Agarose, polyacrylamide, and similar substances can be introduced into microfluidic channels creating microfluidic analogues of gel electrophoresis techniques. These gel-filled channels are discussed in the next section.

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