Silicon photonic microring resonators for quantitative cytokine detection and T-cell secretion analysis

Abstract

The ability to perform multiple simultaneous protein biomarker measurements in complex media with picomolar sensitivity presents a large challenge to disease diagnostics and fundamental biological studies. Silicon photonic microring resonators represent a promising platform for real-time detection of biomolecules on account of their spectral sensitivity toward surface binding events between a target and antibody-modified microrings. For all refractive index-based sensing schemes, the mass of bound analytes, in combination with other factors such as antibody affinity and surface density, contributes to the observed signal and measurement sensitivity. Therefore, proteins that are simultaneously low in abundance and have a lower molecular weight are often challenging to detect. By employing a more massive secondary antibody to amplify the signal arising from the initial binding event, it is possible to improve both the sensitivity and the specificity of protein assays, allowing for quantitative sensing in complex sample matrices. Herein, a sandwich assay is used to detect the 15.5 kDa human cytokine interleukin-2 (IL-2) at concentrations down to 100 pg/mL (6.5 pM) and to quantitate unknown solution concentrations over a dynamic range spanning 2.5 orders of magnitude. This same sandwich assay is then used to monitor the temporal secretion profile of IL-2 from Jurkat T lymphocytes in serum-containing cell culture media in the presence of the entire Jurkat secretome. The same temporal secretion analysis is performed in parallel using a commercial ELISA, revealing similar IL-2 concentration profiles but superior precision for the microring resonator sensing platform. Furthermore, we demonstrate the generality of the sandwich assay methodology on the microring resonator platform for the analysis of any biomolecular target for which two high-affinity antibodies exist by detecting the approximately 8 kDa cytokine interleukin-8 (IL-8) with a limit of detection and dynamic range similar to that of IL-2. This work demonstrates the first application of silicon photonic microring resonators for detecting cellular secretion of cytokines and represents an important advance for the detection of protein biomarkers on an emerging analytical platform.

Figure 1

Representative sandwich assay response and schematic for a single microring optical resonator functionalized with capture anti-IL-2 antibody. An anti-IL-2 antibody-modified microring resonator is initially incubated in buffer (time before A), then a 50 ng/mL solution of IL-2 is introduced to the ring (A) resulting in a ~15 pm net shift in resonance wavelength after 30 min of binding. Quantitative signal enhancement is then achieved by introducing an anti-IL-2 detection antibody (B), which gives a ~40 pm net shift in resonance wavelength after a 15 min incubation. The sensor is regenerated with a low-pH buffer rinse (C) prior to returning to buffer (D) for subsequent IL-2 analyses.

Figure 2

Real-time monitoring of resonance wavelength shifts of an anti-IL-2 antibody-functionalized microring during exposure to a variety of known (0, 0.10, 0.26, 0.64. 1.60, 4, 10, 25, and 50 ng/mL) and two unknown solutions (Unknowns A and B) in BSA-PBS. Following a 30-min exposure to each IL-2 concentration, anti-IL-2 detection antibody is flowed over the ring (dashed lines), and the secondary net shift after 15 minutes is used for quantitation. A BSA-PBS sample without IL-2 produced no secondary anti-IL-2 binding signal, as shown between time points 238 and 253 minutes. After each secondary detection, the microring surface is regenerated by a low-pH glycine rinse, and the sample chamber returned to BSA-PBS to achieve a stable baseline before subsequent sample injections. The signal is corrected for drift by subtracting the shift from an adjacent control ring that is not functionalized with anti-IL-2 but is introduced to identical conditions throughout.

Figure 3

Concentration-response plot of the average control-ring-corrected net shift as a function of IL-2 concentration, as determined from 15 microring resonators. Following 30-min incubation in IL-2 standard solutions prepared in BSA-PBS, the net shift arising from detection antibody binding is measured after 15 min for each ring. The plot is fit with a quadratic regression, and the displayed equation is used to successfully quantitate solutions with unknown IL-2 concentrations (relative positions of unknowns depicted on curve with red X). The inset in the lower right corner is an expanded view of the low concentration range below 4 ng/mL. Error bars represent the 95% confidence interval, n = 15 rings.

Figure 4

Temporal Jurkat IL-2 secretion profile. Aliquots were taken at 8-hr intervals over a 24-hr period to compare IL-2 secretion from non-stimulated (squares) and PMA/PHA-stimulated (triangles) Jurkat T-cells. Results were obtained in parallel by using the microring resonator sandwich assay (A) and an IL-2 ELISA (B). Error bars represent the 95% confidence interval for n = 15 (A) and n = 3 (B).