title: Point-of-care vertical flow allergen microarray assay: proof of concept.
abstract: BACKGROUND
  Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of <10 min before imaging and data analysis.
  
  
  METHODS
  Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of ≤10 min. Microarray images were captured by a consumer-grade flatbed scanner.
  
  
  RESULTS
  A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was <14%. The observed concordance with a clinical assay, ImmunoCAP, was R(2) = 0.89 (n = 31).
  
  
  CONCLUSIONS
  In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing.
authors.affiliation: Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden;
authors.forename: Thiruppathiraja
authors.initials: T
authors.lastname: Chinnasamy
authors.affiliation: BIOS Lab on a Chip group, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands;
authors.forename: Loes I
authors.initials: LI
authors.lastname: Segerink
authors.affiliation: Thermo Fisher Scientific, Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Nystrand
authors.affiliation: Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden;
authors.forename: Jesper
authors.initials: J
authors.lastname: Gantelius
authors.affiliation: Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden; helene.andersson.svahn@scilifelab.se.
authors.forename: Helene
authors.initials: H
authors.lastname: Andersson Svahn
mesh_terms: D000485:Allergens / Q000032:analysis*; D003124:Colorimetry; D006046:Gold / Q000737:chemistry; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood*; D053768:Metal Nanoparticles / Q000737:chemistry; D019095:Point-of-Care Systems; D040081:Protein Array Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000485:Allergens; D007073:Immunoglobulin E; D006046:Gold