Microfluidic pillar array sandwich immunofluorescence assay for ocular diagnostics

• Published in: Biomedical microdevices Vol. 13; no. 3; pp. 573 - 583
• Main Authors: Green, James V., Sun, Dawei, Hafezi-Moghadam, Ali, Lashkari, Kameran, Murthy, Shashi K.
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.06.2011; Springer Nature B.V
• Subjects: Aged; Animals; Aqueous Humor - metabolism; Biological and Medical Physics; Biomedical Engineering and Bioengineering; Biophysics; Biopsy; Cataract - diagnosis; Cataract - metabolism; Cataract - pathology; Cytokines - metabolism; Dimethylpolysiloxanes - chemistry; Endotoxins - toxicity; Engineering; Engineering Fluid Dynamics; Eye diseases; Eye Diseases - chemically induced; Eye Diseases - diagnosis; Eye Diseases - metabolism; Eye Diseases - pathology; Fluid dynamics; Fluorescent Antibody Technique - instrumentation; Humans; Hydrodynamics; Immunoassay; Male; Medical diagnosis; Microelectromechanical systems; Microfluidic Analytical Techniques - instrumentation; Middle Aged; Nanotechnology; Rats; Uveitis - chemically induced; Uveitis - diagnosis; Uveitis - metabolism; Uveitis - pathology; Immunoassay; Primary intraocular lymphoma; Microfluidic; Uveitis
• ISSN: 1387-2176; 1572-8781
• DOI: 10.1007/s10544-011-9528-4

Uveitis and primary intraocular lymphoma (PIOL) are diseases associated with the invasion of lymphocytes into various regions of the eye, accompanied by expression of inflammatory cytokines. While these diseases are very different in terms of survivability and treatment options they have similar symptoms that make accurate diagnosis challenging. Furthermore, the diagnostic yield with state-of-the-art techniques for cell and cytokine analysis of vitreous and aqueous humor samples is under 20% due to inadequate sensitivity. This paper describes a simple sandwich immunofluorescence assay (sIFA) microfluidic device that is capable of identifying important analytes in ocular biopsies as a potential alternative to current diagnostic approaches. Detection is accomplished by capture of the target molecules on antibody-coated, vertical, oval shaped pillars in a microfluidic device followed by a biotinylated detection antibody and finally florescent avidin for target molecule quantification. Cytokine concentration measurements were carried out on aqueous humor samples from rats with endotoxin-induced uveitis as well as human cataract patients. Results correlated well with conventional protein quantification techniques and additionally, measurements from the human samples surpassed detection limits of current state-of-the-art immunoassay techniques. The single-digit femtomolar range of detection of this sIFA system provides lower limits of detection when compared to traditional techniques and allows for the mapping of the cytokine content of vitreous biopsies with detection limits that have yet to be realized using cost effective microfluidics. Furthermore, the relative simplicity of the device design, fabrication and ability to automate makes it easily translatable from the laboratory to a clinical setting.