Multiplexed, rapid point of care platform to quantify allergen-specific IgE

Abstract

Variation of probe immobilization on microarrays hinders the ability to make high quality, assertive and statistically relevant conclusions needed in the healthcare setting. To address this problem, we have developed a calibrated, compact, inexpensive, multiplexed, dual modality point-of-care detection platform that calibrates and correlates surface probe density measured label-free to captured labeled secondary antibody, is independent of chip-to-chip variability, and improves upon existing diagnostic technology. We have identified four major technological advantages of our proposed platform: the capability to perform single spot analysis based on the fluorophore used for detection, a 10-fold gain in fluorescence signal due to optimized substrate, a calibrated, quantitative method that uses the combined fluorescent and label-free modalities to accurately measure the density of probe and bound target for a variety of systems, and a compact measurement platform offering reliable and rapid results at the doctor's office. Already, we have formulated over a 90% linear correlation between the amount of probe bound to surface and the resulting fluorescence of captured target for IgG, β-lactoglobulin, Ara h 1 peanut allergen, and Phl 5a Timothy grass allergen.

Fig. 1

Fluorescence intensity values for SiO₂ thicknesses of 100nm and 320nm compared to a microscope slide. Over all wavelengths, 100nm oxide has more than 2-fold emission enhancement compared to glass for a broad range of fluorophores. To compare, 320nm oxide enhances Cy3 and Cy5 emitters about 1.9 times compared to glass.

Fig. 2

Fluorescence versus bound target to spot label-free measurement for rabbit IgG (left) amd B-lactoglobulin(right). For IgG calibration of Chip 3, Q1 and Q2 analyze spot to spot variability. Results show that CaFE platform yields calibrated, linear responses from chip-to-chip for a variety of proteins.

Fig. 3

Fluorescence versus spotting concentration compared to fluorescence (a) versus serum bound to spot label-free measurement (b). Results show that CaFE platform yields calibrated, linear responses for allergy testing analysis compared to traditional “semi-quantifiable” analysis. For the CaFE measurement of Ph1 Timothy grass allergen, the last point was removed from the calibration due to saturation of bound serum.

Fig. 4

Fluorescence signal of labeled IgE versus bound target to spot label-free measurement to investigate calibrating chip-to-chip variability for peanut (left) and timothy grass (right) allergens. Results show that CaFE platform yields calibrated, linear responses despite chip-to-chip for a variety.