Fast immunoassay for microfluidic western blotting by direct deposition of reagents onto capture membrane

Arvin, Natalie E.; Dawod, Mohamed; Lamb, Don T.; Anderson, Jocelyn; Furtaw, Michael D.; Kennedy, Robert T.

doi:10.1039/d0ay00207k

Cited by 12 publications

(12 citation statements)

References 49 publications

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“…In the paper-based open microfluidic system, the entire separation-based protein analysis workflow (sample preparation, sample injection, protein electrophoresis, protein transfer, and immunoprobing) can be completed within only 1 h, significantly faster than slab-gel systems. Recent ap-proaches for automated antibody application in membranebased immunoblotting workflows were shown to dramatically decrease antibody consumption and time demand [20,58]. These techniques might be implemented into the open microfluidic platform to further accelerate the workflow and improve its performance.…”

Section: Discussionmentioning

confidence: 99%

“…Several strategies have been proposed to allow for immunoprobing upon separation in enclosed microchannels, including protein immobilization to the capillary wall (and flushing the capillaries with antibody solutions [9]), bead-based immunoassays in CE [10], electrophoretic antibody loading [11][12][13][14][15], and reversible microchip bonding [16]. Alternatively, the capillary/microchip outlet can be interfaced with a conventional Western Blot membrane (laterally moving at a constant speed) to deposit the separated proteins onto the membrane for immunoprobing [17][18][19][20]. Apart from the difficulties associated with immunoprobing, microchip fabrication also requires dedicated equipment and cleanroom facilities not available to all researchers, thereby increasing the costs and limiting the access to flexible, customized designs.…”

Section: Introductionmentioning

confidence: 99%

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Paper‐based open microfluidic platform for protein electrophoresis and immunoprobing

et al. 2021

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Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10-to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Paper‐based open microfluidic platform for protein electrophoresis and immunoprobing

et al. 2021

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“…It is desirable to expand using CE /MCE in these laboratories to increase the speed and throughput of protein/peptide analysis, especially for large proteomic/peptidomic studies. The commonly used Western blotting technology can also be performed in CE and MCE format [ 74 , 75 ].…”

Section: Fundamentals Of Microfluidicsmentioning

confidence: 99%

Microfluidics for Peptidomics, Proteomics, and Cell Analysis

et al. 2021

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Microfluidics is the advanced microtechnology of fluid manipulation in channels with at least one dimension in the range of 1–100 microns. Microfluidic technology offers a growing number of tools for manipulating small volumes of fluid to control chemical, biological, and physical processes relevant to separation, analysis, and detection. Currently, microfluidic devices play an important role in many biological, chemical, physical, biotechnological and engineering applications. There are numerous ways to fabricate the necessary microchannels and integrate them into microfluidic platforms. In peptidomics and proteomics, microfluidics is often used in combination with mass spectrometric (MS) analysis. This review provides an overview of using microfluidic systems for peptidomics, proteomics and cell analysis. The application of microfluidics in combination with MS detection and other novel techniques to answer clinical questions is also discussed in the context of disease diagnosis and therapy. Recent developments and applications of capillary and microchip (electro)separation methods in proteomic and peptidomic analysis are summarized. The state of the art of microchip platforms for cell sorting and single-cell analysis is also discussed. Advances in detection methods are reported, and new applications in proteomics and peptidomics, quality control of peptide and protein pharmaceuticals, analysis of proteins and peptides in biomatrices and determination of their physicochemical parameters are highlighted.

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“…Similarly, in 2020, Arvin et al carried out a fast immunoassay for microfluidic Western blotting by direct deposition of reagents onto the capture membrane. Proteins separated by microchip electrophoresis can be trapped on the membrane by dragging the membrane which greatly reduces the separation and transfer time of Western blotting to a few minutes (within 1 hour) [101]. To make up for the shortcoming of the most methodologies and procedures used for the detection of glycomarkers, that is, too many experienced techniques and trained steps, in 2016, Lesur et al conducted an experiment in which the addition of lectin in the electrophoresis buffer was used in CGE to enable lectin affinity electrophoresis, and the liquid-phase binding assay facilitated the formation of the microfluidic-based automated immunoanalyzer [103].…”

Section: Electrophoresis Microfluidic System In Immunoassaysmentioning

confidence: 99%

Application of Microfluidics in Immunoassay: Recent Advancements

Shi

Peng

Yang

et al. 2021

Journal of Healthcare Engineering

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In recent years, point-of-care testing has played an important role in immunoassay, biochemical analysis, and molecular diagnosis, especially in low-resource settings. Among various point-of-care-testing platforms, microfluidic chips have many outstanding advantages. Microfluidic chip applies the technology of miniaturizing conventional laboratory which enables the whole biochemical process including reagent loading, reaction, separation, and detection on the microchip. As a result, microfluidic platform has become a hotspot of research in the fields of food safety, health care, and environmental monitoring in the past few decades. Here, the state-of-the-art application of microfluidics in immunoassay in the past decade will be reviewed. According to different driving forces of fluid, microfluidic platform is divided into two parts: passive manipulation and active manipulation. In passive manipulation, we focus on the capillary-driven microfluidics, while in active manipulation, we introduce pressure microfluidics, centrifugal microfluidics, electric microfluidics, optofluidics, magnetic microfluidics, and digital microfluidics. Additionally, within the introduction of each platform, innovation of the methods used and their corresponding performance improvement will be discussed. Ultimately, the shortcomings of different platforms and approaches for improvement will be proposed.

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Fast immunoassay for microfluidic western blotting by direct deposition of reagents onto capture membrane

Abstract: To further improve the speed and miniaturization of a complete Western blot, a microscale immunoassay with direct deposition of immunoassay reagents has been developed with the flow deposition of antibodies.

Cited by 12 publications

References 49 publications

Paper‐based open microfluidic platform for protein electrophoresis and immunoprobing

Paper‐based open microfluidic platform for protein electrophoresis and immunoprobing

Microfluidics for Peptidomics, Proteomics, and Cell Analysis

Application of Microfluidics in Immunoassay: Recent Advancements

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