title: An embedded microretroreflector-based microfluidic immunoassay platform.
abstract: We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 μm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, USA. willson@uh.edu.
authors.forename: Balakrishnan
authors.initials: B
authors.lastname: Raja
authors.affiliation: Department of Electrical and Computer Engineering, University of Houston, Houston, Texas, USA.
authors.forename: Carmen
authors.initials: C
authors.lastname: Pascente
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, USA. willson@uh.edu.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Knoop
authors.affiliation: Department of Electrical and Computer Engineering, University of Houston, Houston, Texas, USA.
authors.forename: David
authors.initials: D
authors.lastname: Shakarisaz
authors.affiliation: Department of Electrical and Computer Engineering, University of Houston, Houston, Texas, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Sherlock
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, USA. willson@uh.edu.
authors.forename: Steven
authors.initials: S
authors.lastname: Kemper
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, USA. willson@uh.edu.
authors.forename: Katerina
authors.initials: K
authors.lastname: Kourentzi
authors.affiliation: Advanced Systems Engineering and Deployment, Sandia National Laboratories, Livermore, California, USA.
authors.forename: Ronald F
authors.initials: RF
authors.lastname: Renzi
authors.affiliation: Department of Biotechnology and Bioengineering, Sandia National Laboratories, Livermore, California, USA.
authors.forename: Anson V
authors.initials: AV
authors.lastname: Hatch
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Olano
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Bi-Hung
authors.initials: BH
authors.lastname: Peng
authors.affiliation: Department of Electrical and Computer Engineering, University of Houston, Houston, Texas, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Ruchhoeft
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, USA. willson@uh.edu and Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Campus Monterrey, Monterrey, Nuevo León, Mexico.
authors.forename: Richard
authors.initials: R
authors.lastname: Willson
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000378:metabolism; D057205:Automation, Laboratory; D018914:Cells, Immobilized; D017076:Computer-Aided Design; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D018189:Immunomagnetic Separation; D056656:Lab-On-A-Chip Devices; D057230:Limit of Detection; D060328:Magnetic Phenomena; D008853:Microscopy; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D055616:Microtechnology / Q000379:methods; D019904:Polymethyl Methacrylate / Q000737:chemistry; D000075082:Proof of Concept Study; D015203:Reproducibility of Results; D020114:Rickettsia conorii / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D013499:Surface Properties
chemical_list: D055910:Antibodies, Immobilized; D019904:Polymethyl Methacrylate