Proteomics Center–

Microfluidic eTag Separation for Proteomic Applications

Recent Publications

Abstract
In collaboration with P.J Utz of the Stanford Medical Center and ACLARA Biosciences, we have developed a microfluidic capillary electrophoresis method for multiplex detection and quantification of both DNA-DNA and protein-protein interactions using ACLARA's eTagTM Assay system. The major motivation for this work is multiplex detection for proteomics applications, which require elucidation of the pathogenic mechanisms contributing to conditions such as blood and autoimmune diseases. Microfluidic capillary electrophoresis is an elegant diagnostic tool for this process as it requires sample volumes of order 1uL or less, and requires analysis times of 100sec or less. On chip devices also have the potential of parallel architecture of one or more assays. To date, we have demonstrated simultaneous on-chip separation of 13 reaction markers called eTagsTM which are used for RNA detection. We use a borosilicate microfluidic chip containing a 30um cross channel geometry using 100nm concentration eTagTM samples. Our ultimate goal is to integrate the entire eTagTM Assay process on a microchip.


Background
ELECTROKINETIC FLOW BASICS
Capillary Electrophoresis is a technique for separating charged samples by their electromigration rate, or drift velocity as characterized by their electrophoretic mobility ( ), in an electric field. Electrophoretic mobility is defined as the molecule's drift velocity per unit electric field. These drift velocities are used to separate and detect multiple species. Advective dispersion and diffusion causes sample bands to broaden and ultimately limit the resolution of the technique. To date, our microchip-based separation schemes have a signal-to-noise ratio of over 50 and can achieve separation in under a minute. Figure 1 shows a simplified schematic of an electrophoretic separation in a cross-geometry microchip.

eTag Technology
EtagsTM are fluorescent labels engineered with distinctive electrophoretic mobilities and used to achieve multiplex detection of molecular binding events. These molecules are developed by ACLARA Biosciences Inc. eTagsTM can be covalently attached to multiple proteins simultaneously. One advantage of this assay is that affinity agents such as antibodies and aptamers are not immobilized on surfaces, as required with other multiplexing technologies, and this allows assays to be preformed in solution. An outline of AN eTag assay is depicted in Figure 2:


Figure 2: Individual proteins are incubated in individual wells with two corresponding antibodies, one conjugated to an eTag, one to molecular "scissors", and the binding reaction continues until equilibrium is reached (a). The molecular scissors molecules are then activated by exposing the wells to infrared (IR) raditation, releasing the fluorescent eTagTM (b-c). The eTags are then separated by microCE and quantitated using standard analysis software.


Experimental Setup


 

Figure 3. (a,c) A commercially available borofloat glass Microfluidic channel (Micralyne, Canada) was used in these experiments. The channels have a depth of 30 m and a nominal width of 50 m. The channels of the cross are 85 mm and 10 mm long. Buffer solution seeded with 200 nM eTags was injected into the eTag well, while the rest of the channel was filled with the unseeded background buffer. A 9 mM Borate buffer was used for the working fluid to minimize the effects of Joule heating. A high voltage supply (Micralyne, Canada) connected to platinum electrodes controlled the migration of the eTag-seeded fluid towards the eTag waste well. In the second step the eTags are injected into the longer channel and separated, as schematically depicted in Figure 1. Image (c) depicts actual image of microfluidic chip attached to platinum electrodes. (b,d) The experimental setup consisted of an inverted, epifluorescent microscope (Olympus IX70) fitted with 20X and 10X magnification objectives with a numerical aperture (NA) of 0.5 and 0.4, respectively. Images were captured through 532 excitation 488 emission filter cube (Chroma, XPSPEC452), and recorded using a cooled 12-bit CCD camera (Roper Scientific, MicroMAX). The camera controller adjusted the shutter to the illumination source to minimize photobleaching. The frame rate was maximized using 30x30 pixels on-chip binning, to create binned pixel dimensions of 16.8 x 16.8 um in the image plane. A function generator controlled the frame rate of the camera. (d) shows digital photo of experimental setup, depicting the Intensified CCD camera, high voltage power supply, computer controller, inverted microscope, electrode connections and microfluidic chip.


Results

Figure 4. An injection and separation sequence of 200 nM solutions of eTagTM (ACLARA Biosciences). Images (a) through (d) are 120 ms exposures separated by 200 ms. In Figure (a), the sample is injected applying 0.5 kV to the eTag well and grounding the eTag waste well (refer to figure 3a). The sample volume at the intersection is "pinched" by applying 0.5 kV at the buffer well and 1.9 kV at the waste well. Once a steady flow condition is achieved, the voltages are switched to inject a small sample plug into the separation channel (b). During this separation phase, the voltages applied at the buffer well and waste well are 2.4 kV and ground, respectively. The sample remaining in the injection channel is "retracted" from the intersection by applying 1.4 kV to both eTag wells. The electrokinetic injection introduces an approximately 400 pl volume of the homogenous sample mixture into the separation channel. In (c), this difference in electrophoretic mobilities results in a separation of the two eTags into distinct analyte bands, as seen in figures (c) and (d).

Figure 5. Electropherogram showing signal peaks associated with 13 released eTags. These separations were preformed on a microfluidic cross channel geometry chip. Experimental conditions and chemistry are as follows: 10mM borate buffer as the background electrolyte, 200 nM eTagTM reporters in buffer, and the voltage scheme described in figure 4. Images were captured on a CCD camera with 15x15 on-chip binning with a frame rate of 20Hz and exposure time of 150ms. Fluorescence images through a 488nm excitation 532nm emission filter cube (Chroma, Inc.) and a 10X (NA 0.5, WD 10mm) Olympus objective were taken 10mm downstream of the injection. Image were background subtracted to optimize SNR. Analysis was preformed using Win32 software (Roper Scientific) and MATLAB. Figure shows an unexpected peak with an intensity greater than 4000 units possibly due to multiple eTagsTM crystallized into a solid particle.

 

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