A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.
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.
Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.
Barrett J
BJ
Nehilla
Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.
John J
JJ
Hill
Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.
Selvi
S
Srinivasan
Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.
Yen-Chi
YC
Chen
Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.
Thomas H
TH
Schulte
Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.
Patrick S
PS
Stayton
Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.
James J
JJ
Lai
D015415:Biomarkers / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006678:HIV / Q000302:isolation & purification; D016655:HIV Core Protein p24 / Q000032:analysis / Q000097:blood*; D015658:HIV Infections / Q000097:blood; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018796:Immunoconjugates / Q000737:chemistry*; D007202:Indicators and Reagents; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D055503:Protein Multimerization; D020349:Surface Plasmon Resonance / Q000379:methods*; D013696:Temperature
D015415:Biomarkers; D016655:HIV Core Protein p24; D018796:Immunoconjugates; D007202:Indicators and Reagents; D058185:Magnetite Nanoparticles; D011108:Polymers
J Infect Dis. 2012 Feb 15;205(4):521-4
22207652
Langmuir. 2008 Oct 21;24(20):11577-86
18816013
Bioconjug Chem. 2010 Oct 20;21(10):1820-6
20845976
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Dec 15;877(32):4079-89
19926541
Biotechnol Prog. 2007 Nov-Dec;23(6):1513-6
17949107
Annu Rev Biophys Biomol Struct. 1997;26:541-66
9241429
Ann Biomed Eng. 2014 Nov;42(11):2322-32
24874602
Mol Immunol. 1987 Oct;24(10):1055-60
3683403
Anal Biochem. 2008 Jun 15;377(2):209-17
18405656
J Lab Autom. 2013 Jun;18(3):206-11
23015519
J Biochem Biophys Methods. 2003 Sep 30;57(3):213-36
14512156
J Acquir Immune Defic Syndr. 2010 Dec;55(4):413-9
20811289
Lab Chip. 2009 Jul 21;9(14):1997-2002
19568666
Langmuir. 2007 Jun 19;23(13):7385-91
17503854
J Virol. 1992 Nov;66(11):6547-54
1404602
Anal Biochem. 2008 Oct 15;381(2):270-2
18644343
Anal Chem. 2011 Dec 15;83(24):9197-200
22070407
Nucleic Acids Res. 1997 Aug 1;25(15):2979-84
9224596
J Biomed Mater Res A. 2010 Apr;93(1):175-88
19536838
J Clin Virol. 2011 Dec;52 Suppl 1:S51-5
21983253
Sci Transl Med. 2011 Aug 10;3(95):95ra73
21832238
Biomed Microdevices. 2013 Jun;15(3):539-51
23420191
J Am Chem Soc. 2004 Jun 16;126(23):7330-5
15186170
Acta Biomater. 2012 Oct;8(10):3596-605
22732383
ACS Macro Lett. 2013 Feb 19;2(2):132-136
35581774
J Immunol Methods. 2010 Jan 31;352(1-2):13-22
19854197
J Pharm Biomed Anal. 2010 Jan 5;51(1):252-4
19679421
Methods. 2013 Dec 1;64(2):137-43
23806645
Biomacromolecules. 2012 Sep 10;13(9):2945-51
22861824
ACS Nano. 2012 Aug 28;6(8):6776-85
22804625
J Immunol Methods. 1997 Jan 15;200(1-2):155-9
9005954
Lab Chip. 2010 Nov 21;10(22):3130-8
20882219
ACS Appl Mater Interfaces. 2009 Jul;1(7):1492-503
20355953
ACS Appl Mater Interfaces. 2015 Mar 25;7(11):6062-9
25710198
J Biochem Biophys Methods. 2003 Jan 31;55(1):87-94
12559591
J Immunol Methods. 2007 Dec 1;328(1-2):53-8
17884083
J Mol Recognit. 2000 Nov-Dec;13(6):388-407
11114072
Int J Pharm. 2005 Aug 26;300(1-2):102-12
16009513
Biosens Bioelectron. 2009 Mar 15;24(7):2143-8
19157844
Lab Chip. 2013 Mar 21;13(6):1172-81
23370785
Biotechnol Adv. 2013 Dec;31(8):1374-85
23747736
Eur Polym J. 2015 Aug 1;69:532-539
26365998