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Microfluidic pillar array sandwich immunofluorescence assay for ocular diagnostics
James V Green1, Dawei Sun, Ali Hafezi-Moghadam
1Department of Chemical Engineering, Northeastern University, Boston, MA, 02115, USA.
Insights
A novel microfluidic device improves diagnosis of eye diseases like uveitis and primary intraocular lymphoma (PIOL). This sensitive assay detects critical biomarkers in ocular fluids, surpassing current limitations for accurate patient stratification.
Area of Science:
- Ophthalmology
- Biomedical Engineering
- Analytical Chemistry
Background:
- Uveitis and primary intraocular lymphoma (PIOL) present diagnostic challenges due to similar symptoms and low sensitivity of current diagnostic methods.
- Existing techniques for analyzing ocular fluid biomarkers have a diagnostic yield below 20% due to inadequate sensitivity.
- Accurate diagnosis is crucial for effective treatment and improved patient outcomes in these distinct ocular conditions.
Purpose of the Study:
- To develop and validate a novel microfluidic device for sensitive detection of biomarkers in ocular samples.
- To overcome the limitations of current diagnostic techniques for uveitis and PIOL.
- To enable accurate quantification of cytokine concentrations in ocular fluids for improved disease diagnosis and management.
Main Methods:
- A simple sandwich immunofluorescence assay (sIFA) microfluidic device was designed and fabricated.
- The device utilizes antibody-coated pillars for capture of target molecules, followed by detection with biotinylated antibodies and fluorescent avidin.
- Cytokine concentrations were measured in aqueous humor samples from rats with induced uveitis and human cataract patients.
Main Results:
- The sIFA device demonstrated high sensitivity, achieving single-digit femtomolar detection limits for cytokines.
- Results from ocular fluid samples correlated well with conventional protein quantification techniques.
- The assay surpassed the detection limits of current state-of-the-art immunoassay techniques for human samples.
Conclusions:
- The developed sIFA microfluidic device offers a sensitive and cost-effective alternative for diagnosing ocular diseases.
- Its high sensitivity and potential for automation facilitate translation to clinical settings for improved patient diagnosis.
- This technology enables precise mapping of cytokine profiles in vitreous biopsies, advancing diagnostic capabilities in ophthalmology.
Abstract:
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 fluorescent 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.
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