Use of Polyacrylamide Gel Moving Boundary Electrophoresis to Enable Low-Power Protein Analysis in a Compact Microdevice

Duncombe, Todd A.; Herr, Amy E.

doi:10.1021/ac301875e

Cited by 15 publications

(12 citation statements)

References 48 publications

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“…Run buffer (1× Tris-glycine) conductivity in aqueous solution was measured by a B-173 Compact Conductivity Meter (Horiba Scientific, Kyoto, Japan). Run buffer conductivity in the fs PAG structures was estimated to be similar to that of PAG in glass microfluidic chips, as described by Duncombe et al 28 The mobility and diffusivity of the model protein, TI, was estimated as per Herr and Singh 29 and Hughes et al 30 The voltage boundary conditions were set at each terminus of the fs PAG structure. For post-simulation data analysis, we averaged the protein concentrations over the transverse fs PAG lane dimension at the 120 s time point to generate analyte concentration profiles, which were then analyzed in OriginPro 8.0 to calculate the band widths.…”

Section: Materials and Methodsmentioning

confidence: 99%

High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

et al. 2014

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We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch–ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets.

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Section: Materials and Methodsmentioning

confidence: 99%

High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

et al. 2014

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“…Typically, separations with durations of <1 min are completed in 1 mm separation lengths, with plate heights as high as 4.41 × 10 5 plates/m. 15–17 …”

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confidence: 99%

Fabrication of an Open Microfluidic Device for Immunoblotting

et al. 2017

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Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an “enclosed” microchannel for PAGE and blotting to an “open” PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.

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“…This represents the lowest electric field strength reported for electrophoretic separations in the published literature . This is lower than the field strength of 43 V cm −1 achieved using a 9 V battery over 2 mm for the polyacrylamide gel electrophoresis separations of proteins …”

Section: Figurementioning

confidence: 72%

“…Figure S8 shows that a voltage above 18 V would be required to extract berberine from the matrix spot within 24 h. Experimental data using a potential difference of 24 V supplied using two 12 V batteries (A23) confirmed 6 V cm −1 was indeed sufficient to separate berberine away from the matrix, as illustrated in Figure B. This represents the lowest electric field strength reported for electrophoretic separations in the published literature . This is lower than the field strength of 43 V cm −1 achieved using a 9 V battery over 2 mm for the polyacrylamide gel electrophoresis separations of proteins …”

Section: Figurementioning

confidence: 87%

“…[36] This is lower than the field strength of 43 Vcm À1 achieved using a9Vb attery over 2mmf or the polyacrylamide gel electrophoresis separations of proteins. [36] Theb attery-powered set-up shown in Figure 1w as used for extraction in the mail. A0 .1 mLd rop of freshly drawn whole blood spiked with 60 ppm berberine chloride was deposited onto the PIM and allowed to dry for 5min.…”

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confidence: 85%

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In‐Transit Electroextraction of Small‐Molecule Pharmaceuticals from Blood

et al. 2019

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An electrokinetic platform was developed for extracting small-molecule pharmaceuticals from ad ried blood spot. Through the exclusion of liquid reagents and use of low field strength (6 Vcm À1 ), the electroextraction of adrug from ad ried blood spot, deposited on ap olymer inclusion membrane (PIM), could be realised while in transit in the mail. In transit sample preparation provides ap otential solution to in situ sample degradation and maya ccelerate the workflow upon arrival of ap atient sample at the analytical facility.T he electroextraction method was enabled through our discovery of the use of 15-20 mmt hin PIMs as electrophoretic separation medium in absence of liquid reagents.Here,aPIM consisting of cellulose triacetate as polymer base,2 -nitrophenyl octyl ether as plasticizer and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide as carrier was used. The PIM, was packaged with two 12 Vb atteries to supply the separation voltage.Ablood spot containing berberine chloride was deposited and dried before the applying the separation potential, allowing for the electroextraction while the packaged device was shipped in internal mail. Upon arrival in the analytical laboratory,t he PIM was analysed using af luorescence microscope with photon multiplier tube,quantifying the berberine extracted away from the sample matrix. This platform represents an ew opportunity for processing clinical samples during transport to the laboratory,s aving time and manual handling to accelerate the time to result.Personalised medicine,which aims to treat every patient as an individual has been shown to be highly effective with diabetes,b ut expanding the analytical target range is one of the most important challenges of the forthcoming century. [1][2][3][4][5][6][7][8] Aholistic approach to personalised medicine requires ability to detect and quantify as ignificant and diverse range of pharmaceuticals and their metabolites.T he golden standard for point-of-collection testing is the use of immunoassays, however, these frequently suffer from high cross-reactivity given the structural similarity of many pharmaceuticals and their metabolites,h ence few have been accepted for drug monitoring. [9][10][11] Despite recent achievements in Lab on aC hip devices for Point of Care diagnostics, [12,13] significant advances in chemical resolution and sensitivity are required before their widespread use for routine monitoring of pharmaceuticals and their metabolites.Assuch, the capability to detect and quantify as ignificant and diverse range of pharmaceuticals and their metabolites can be anticipated to remain with centralised laboratories commonly using high resolution analytical instrumentation, [14] and this requires the blood and/or urine sample to be sent off for analysis.U pon arrival at the laboratory,t he sample is processed by wetchemistry techniques-manual or with automated instrumentation-and analysed by any number of instruments,with the sample preparation typically the most time-and labourintensive process.The data is th...

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Use of Polyacrylamide Gel Moving Boundary Electrophoresis to Enable Low-Power Protein Analysis in a Compact Microdevice

Cited by 15 publications

References 48 publications

High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

Fabrication of an Open Microfluidic Device for Immunoblotting

In‐Transit Electroextraction of Small‐Molecule Pharmaceuticals from Blood

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