Anton Simeonov Ph.D. National Institute of Health

Researchers introduce a microfluidic, single-channel, multistage immunoblotting strategy toward scalable protein isoform analysis.

ASSAY & Drug Development Technologies offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents one article that was analyzed in the “Literature Search and Review” column, a paper published in Proceedings of the National Academy of Sciences USA titled “Microfluidic integration for automated targeted proteomic assays.” Authors of the paper are Hughes AJ, Lin RK, Peehl DM, and Herr AE.

Abstract from PNAS USA

A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of protein isoforms would accelerate development of effective protein-based diagnostics.

Toward scalable protein isoform analysis, we introduce a microfluidic “single-channel, multistage” immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting.

In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (~1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients.

The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories.


Despite advances in enzyme-linked immunosorbent assay technologies, new, drastically simplified protocols and instruments are needed for both basic research and clinical diagnostic applications to at least partially match the speed and efficiency of protein testing to those presently available for DNA. The new microfluidic platform developed by Amy Herr’s group promises to deliver an improvement in detection sensitivity and simplification of protocols in combination with streamlined instrument design.

The platform is centered on the use of three-dimensional (3D) photoreactive hydrogels, in combination with electrophoretic transport; thus, diffusion distances are minimized and binding site densities are maximized, ultimately also leading to purported hundredfold gains in analyte capture efficiency. To enable capture and separation, the authors developed a 3D photoclickable hydrogel matrix, termed a light-activated volume-accessible gel (LAVAgel), a copolymer based on (N-[3-[(4-benzoylphenyl)formamido]propyl] methacrylamide (see Figure 1).

Figure 1. Design and operation of the microfluidic LAVAgel assay for high specificity protein isoform analysis. (A) Glass microfluidic device with microchannels linking two fluid reservoirs (dye added for clarity). Scale bar: 2 mm. (B) The 80-min five-stage immunoprobing assay is completed in a single microchannel. (C) Schematic of microchannel cross-section depicting principle of the LAVAgel: Analytes are electrophoresed through the reactive nanoporous hydrogel, exposed to UV, and covalently immobilized. Scale bar: 5 µm. (D) Schematic of reaction between polypeptide backbone and pendant LAVAgel benzophenone groups. Ph denotes phenyl group. For clarity, the electrophilic triplet state of benzophenone, hydrogen abstraction, and radical intermediates are omitted.

Initially, radical polymerization produces the sieving gel contained in the microchannel. Upon brief illumination with ultraviolet (UV) light, the gel further crosslinks both in an interstrand fashion and towards C-H bonds within the proteins’ side chains, essentially switching its state from a molecular sieve to an immobilization scaffold. Using electrophoretic type transport in combination with the light-switchable LAVAgel, the chip enables automatic execution of isoelectric focusing (IEF) for separation of protein isoforms, immobilization of separated proteins, probing of immobilized proteins with affinity reagents, along with the requisite washing steps (see Figure 1). Protein analytes are separated through IEF on a pH gradient formed by a mixture of polyprotic amino carboxylic acids (see Figure 2).

Subsequent to the IEF, UV light is applied to the chip in order to induce photoimmobilization of the separated proteins to the light-activatable copolymer. After immobilization, labeled antibody probes are brought in contact with the immobilized analytes and the sandwich complexes formed are then washed by the use of electrophoretic transport (see second figure). Optimization of the chip’s running conditions necessitated the establishment of zero-flow IEF conditions (commonly referred to as “parking of peaks”) because longer UV irradiation was found to result in the desired greater fraction of the analyte being captured but was also accompanied by unwanted diffusion-induced peak broadening.

Finally, the authors used the new platform to perform analyses of prostate-specific antigen isoforms originating from cultured cells, as well as patient sera, and obtained results comparable to those derived through the use of the significantly more laborious traditional IEF protocols. It is anticipated that this encouraging first report will be followed by an account of a more advanced complete instrument to allow adaptation of this technology by a large number of potential users.

Figure 2. Characterization of protein isoforms using the single-microchannel 80-min LAVAgel immunoblot. Fluorescence micrographs show (A) loading and IEF of a CE540-labeled protein ladder with 617 nM green WT GFP, and (B) IEF readout via UVexcitation. (C) After UV gel photoactivation, the pH gradient is washed out with retention of a portion of each WT GFP isoform. (D, E) Antibody probing of WT GFP with 100 nM Texas red-labeled polyclonal antibody (pAb*) demonstrates specificity and low-background. RFU, relative fluorescence units; CytC, cytochrome C; LCL, lentil lectin; Mb, myoglobin; CA, carbonic anhydrase; BLG, ß-lactoglobulin; GOx, glucose oxidase.

Anton Simeonov works at the NIH.

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