title: Automated multiplexed ECL Immunoarrays for cancer biomarker proteins.
abstract: Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica-antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10-100 fg mL(-1) were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.
authors.affiliation: †Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
authors.forename: Karteek
authors.initials: K
authors.lastname: Kadimisetty
authors.affiliation: †Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
authors.forename: Spundana
authors.initials: S
authors.lastname: Malla
authors.affiliation: †Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
authors.forename: Naimish P
authors.initials: NP
authors.lastname: Sardesai
authors.affiliation: †Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
authors.forename: Amit A
authors.initials: AA
authors.lastname: Joshi
authors.affiliation: ∥Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP 13565-905, Brazil.
authors.forename: Ronaldo C
authors.initials: RC
authors.lastname: Faria
authors.forename: Norman H
authors.initials: NH
authors.lastname: Lee
authors.affiliation: †Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.|‡Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, United States.|§School of Chemistry, National University of Ireland at Galway, Galway, Ireland.
authors.forename: James F
authors.initials: JF
authors.lastname: Rusling
mesh_terms: D000954:Antigens, Surface / Q000032:analysis; D001331:Automation; D014408:Biomarkers, Tumor / Q000032:analysis*; D055664:Electrochemical Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D043425:Glutamate Carboxypeptidase II / Q000032:analysis; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015850:Interleukin-6 / Q000032:analysis; D007610:Kallikreins / Q000032:analysis; D008163:Luminescent Measurements; D010978:Platelet Factor 4 / Q000032:analysis; D017430:Prostate-Specific Antigen / Q000032:analysis
chemical_list: D000954:Antigens, Surface; D014408:Biomarkers, Tumor; D015850:Interleukin-6; D010978:Platelet Factor 4; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen
references.citation: Adv Mater. 2011 Jun 24;23(24):H151-76
references.pmid: 21567479
references.citation: Nat Med. 2011 Jul 31;17(8):1015-9
references.pmid: 21804541
references.citation: Langmuir. 2010 Sep 21;26(18):15050-6
references.pmid: 20731335
references.citation: J Am Chem Soc. 2006 Aug 30;128(34):11199-205
references.pmid: 16925438
references.citation: Lab Chip. 2012 Jan 21;12(2):281-6
references.pmid: 22116194
references.citation: Biosens Bioelectron. 2011 Jul 15;26(11):4477-83
references.pmid: 21632234
references.citation: Annu Rev Anal Chem (Palo Alto Calif). 2009;2:265-77
references.pmid: 20636062
references.citation: Annu Rev Anal Chem (Palo Alto Calif). 2009;2:359-85
references.pmid: 20636067
references.citation: Lab Chip. 2012 Jun 21;12(12):2118-34
references.pmid: 22344520
references.citation: Lab Chip. 2009 Jan 7;9(1):79-86
references.pmid: 19209338
references.citation: Biosens Bioelectron. 2006 Apr 15;21(10):1887-92
references.pmid: 16330202
references.citation: Anal Chem. 2013 Jun 4;85(11):5304-10
references.pmid: 23635325
references.citation: Anal Chem. 2005 Jan 1;77(1):64-71
references.pmid: 15623279
references.citation: Nature. 2009 Nov 26;462(7272):461-4
references.pmid: 19940916
references.citation: Analyst. 2010 Oct;135(10):2496-511
references.pmid: 20614087
references.citation: Chem Commun (Camb). 2009 Sep 7;(33):4968-70
references.pmid: 19668818
references.citation: Nat Rev Cancer. 2003 Apr;3(4):267-75
references.pmid: 12671665
references.citation: Chem Rev. 2010 Aug 11;110(8):4910-47
references.pmid: 20394378
references.citation: Anal Chem. 2012 Jul 17;84(14):6249-55
references.pmid: 22697359
references.citation: Anal Chem. 2005 Aug 1;77(15):4726-33
references.pmid: 16053282
references.citation: Biometrics. 2008 Mar;64(1):10-9
references.pmid: 17501937
references.citation: Nature. 2006 Jul 27;442(7101):374-80
references.pmid: 16871204
references.citation: Biosens Bioelectron. 2014 Mar 15;53:268-74
references.pmid: 24144557
references.citation: Nature. 2006 Jul 27;442(7101):368-73
references.pmid: 16871203
references.citation: Annu Rev Anal Chem (Palo Alto Calif). 2011;4:251-73
references.pmid: 21417723
references.citation: Anal Chem. 2004 Sep 15;76(18):5379-86
references.pmid: 15362895
references.citation: Anal Bioanal Chem. 2013 Apr;405(11):3831-8
references.pmid: 23307128
references.citation: Nat Rev Drug Discov. 2006 Apr;5(4):310-20
references.pmid: 16582876
references.citation: Nat Rev Cancer. 2008 Apr;8(4):268-78
references.pmid: 18337732
references.citation: J Mater Chem B. 2014 Jan 7;2(1):
references.pmid: 24392222
references.citation: Proteomics. 2003 Jul;3(7):1345-64
references.pmid: 12872236
references.citation: Anal Chem. 2009 Nov 1;81(21):9129-34
references.pmid: 19775154