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A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.

ABSTRACT: Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays, and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microscale benefits of rapid magnetic separation. This system comprises magnetic nanoparticles and polymer-antibody (Ab) conjugates that transition from hydrophilic nanoscale reagents to microscale aggregates in response to temperature stimuli. The binary reagent system was benchmarked against Ab-labeled Dynabeads in terms of biomarker isolation kinetics, assay speed, and reagent needs. Surface plasmon resonance (SPR) measurements showed that polymer conjugation did not significantly alter the Ab's binding affinity or kinetics. ELISA analysis showed that the unconjugated Ab, polymer-Ab conjugates, and Ab-labeled Dynabeads exhibited similar equilibrium dissociation constants (Kd), ?2 nM. However, the binary reagent system isolated HIV p24 antigen from spiked serum specimens (150 pg/mL) much more quickly than Dynabeads, which resulted in shorter binding times by tens of minutes, or about 30-50% shorter overall assay times. The binary reagent system showed improved performance because the Ab molecules were not conjugated to large, solid microparticle surfaces. This stimuli-responsive binary reagent system illustrates the potential advantages of nanoscale reagents in molecule and cell isolations for both research and clinical applications.

SUBMITTER: Nehilla BJ

PROVIDER: S-EPMC6750004 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.

Nehilla Barrett J BJ Hill John J JJ Srinivasan Selvi S Chen Yen-Chi YC Schulte Thomas H TH Stayton Patrick S PS Lai James J JJ

Analytical chemistry 20161014 21

Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays, and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microsc ...[more]

PMID: 27686335

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Project description:Immunoassays have been translated into microfluidic device formats, but significant challenges relating to upstream sample processing still limit their applications. Here, stimuli-responsive polymer-antibody conjugates are utilized in a microfluidic immunoassay to enable rapid biomarker purification and enrichment as well as sensitive detection. The conjugates were constructed by covalently grafting poly(N-isopropylacrylamide) (PNIPAAm), a thermally responsive polymer, to the lysine residues of anti-prostate specific antigen (PSA) Immunoglobulin G (IgG) using carbodiimide chemistry via the polymer end-carboxylate. The antibody-PNIPAAm (capture) conjugates and antibody-alkaline phosphatase (detection) conjugates formed sandwich immunocomplexes via PSA binding in 50% human plasma. The complexes were loaded into a recirculating poly(dimethylsiloxane) microreactor, equipped with micropumps and transverse flow features, for subsequent separation, enrichment, and quantification. The immunocomplexes were captured by heating the solution to 39 °C, mixed over the transverse features for 2 min, and washed with warm buffer. In one approach, the assay utilized immunocomplex solution that was contained in an 80 nL microreactor, which was loaded with solution at room temperature and subsequently heated to 39 °C. The assay took 25 min and resulted in 37 pM PSA limit of detection (LOD), which is comparable to a plate ELISA employing the same antibody pair. In another approach, the microreactor was preheated to 39 °C, and immunocomplex solution was flowed through the reactor, mixed, and washed. When the specimen volume was increased to 7.5 μL by repeating the capture process three times, the higher specimen volume led to immunocomplex enrichment within the microreactor. The resulting assay LOD was 0.5 pM, which is 2 orders of magnitude lower than the plate ELISA. Both approaches generate antigen specific signal over a clinically significant range. The sample processing capabilities and subsequent utility in a biomarker assay demonstrate the opportunity for stimuli-responsive polymer-protein conjugates in novel diagnostic technologies.

Dataset Information

A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.

Publications

A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.

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