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RESEARCH PROFILES Microfluidic IEF without carrier molecules
JONGYOON HAN
tein or peptide in the mass spectrometry and could manipulate the magnitude Isoelectric focusing (IEF), either in analysis,” says Han. “To get rid of them, and polarity of the diffusion potential combination with SDS-PAGE or by itby varying the sheath buffer. Tris-HCl you have to go through extensive proself, is widely used to fractionate combuffer ions generated a larger potential cessing, which can decrease your sigplex protein mixtures. However, the than sodium phosphate buffer ions benal.” Coupling IEF directly to MS can technique requires carrier ampholytes, cause of a larger difference in the diffuresult in excessive contamination of the which are small molecules that can insion coefficients. Even though strong mass spectrometer, he adds. terfere with subsequent analysis. In this electric fields were created by issue of Analytical Chemistry (a) High-concentration Low-concentration the diffusion of buffer ions, (pp 3528– 3536), Jongyoon phosphate buffer phosphate buffer the investigators showed that “Jay” Han and colleagues at (1.0 mM, pH 6.0) (200 mM, pH 8.0) superimposition of an external the Massachusetts Institute of Sample (pl markers 5.1 and 7.2) transverse electric field imTechnology describe a novel proved the sorting efficiency ampholyte-free IEF system for the high-molecular-weight that runs on nothing but (b) protein R-phycoerythrin. standard buffers. Han and colleagues are The new system consists Diffusion potential currently working on stacking of a continuous-flow PDMS two sorting devices in series microfluidic chip in which a to collect specific pI groups, stream of sample solution in – and eventually they want to a low-concentration buffer is Distance x x couple the technique with flanked by a stream of higher100 µm MS. “Our idea is to separate concentration buffer. Because the sample into two groups the diffusion coefficients of first, one group containing the buffer anions and cations molecules with a pI larger differ, a diffusion potential pl marker 5.1 than the buffer pH and the develops at the laminar-flow pl marker 7.2 boundary, generating an elec(a) Schematic of a binary mixture of pI markers separated at the lami- other group containing those with a pI lower than the tric field. The researchers show nar-flow boundary between streams of differently concentrated buffer pH. Then we titrate that over the width of a micro- phosphate buffers with different pH values in a microfluidic channel. the output stream into a fluidic channel (10–50 µm), the (b) Fluorescence image of separated streams of pI markers. slightly lower pH, send it into field strength is large enough to the next device, and do another separaThe investigators first studied the set charged biomolecules in motion. tion,” explains Han. separation of binary mixtures of IEF Exploiting diffusion potentials to The new technique has its limitations. markers with isoelectric points (pIs) bemove molecules around in microfluidic channels is not a new idea (Electrophore- tween 5.1 and 9.0. The samples were in- The resolution of the device is ~0.1 pH units, ~1 order of magnitude below cursis 2002, 23, 2642–2652), but Han and jected into one of two arrangements: a rent top-of-the-line IEF systems. Howbinary flow in which the sample stream colleagues have taken the notion a step ever, a resolution >0.1 pH units is not was adjacent to a sheath buffer stream, further and coupled the system with a really necessary for sample preparation, or a tri-flow in which the sample was in pH gradient. Because the laminar-flow Han says, adding that resolution is not the center flanked by a stream of sheath boundaries prevent mixing of sample the only important aspect in this applicabuffer on either side. Han and coland sheath buffer streams, the researchtion. “High sample throughput, ease of leagues imaged the separation with an ers established a gradient simply by operation, and the absence of amphoinverted epifluorescence microscope using two buffers with different pH lytes are all very important factors when equipped with a CCD camera. In both values. Their technique eliminated the sorting schemes, the mixtures were sepa- it comes to downstream analysis. Our need for carrier ampholytes, which prodevice, if further developed, could do rated into distinct, focused streams acduce pH gradients because of differa continuous-flow separation at a high cording to their pI values, and no exterences in their pK values. flow rate that allows you to process benal field was necessary. “[Current] IEF systems require a tween 1 and 100 µL of sample with reaThe researchers obtained similar revery high concentration of ampholytes, sonably good resolution.” a sults for a binary mixture of recombiand these molecules are always going to —Nicole Branan nant GFP and FITC-labeled ovalbumin critically interfere with the target pro3488
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