Label-free quantitation of a cancer biomarker in complex media using silicon photonic microring resonators

Abstract

Recent advances in label-free biosensing techniques have shown the potential to simplify clinical analyses. With this motivation in mind, this paper demonstrates for the first time the use of silicon-on-insulator microring optical resonator arrays for the robust and label-free detection of a clinically important protein biomarker in undiluted serum, using carcinoembryonic antigen (CEA) as the test case. We utilize an initial-slope-based quantitation method to sensitively detect CEA at clinically relevant levels and to determine the CEA concentrations of unknown samples in both buffer and undiluted fetal bovine serum. Comparison with a commercial enzyme-linked immunosorbent assay (ELISA) kit reveals that the label-free microring sensor platform has a comparable limit of detection (2 ng/mL) and superior accuracy in the measurement of CEA concentration across a 3 order of magnitude dynamic range. Notably, we report the lowest limit of detection to date for a microring resonator sensor applied to a clinically relevant cancer biomarker. Although this report describes the robust biosensing capabilities of silicon photonic microring resonator arrays for a single parameter assay, future work will focus on utilizing the platform for highly multiplexed, label-free bioanalysis.

Figure 1

(A) Schematic diagram illustrating the principle of microring optical resonator biosensing, including a representative transmission spectrum. (B) Top-view scanning electron micrograph image of a microring resonator and linear waveguide, visible through an annular opening in the fluoropolymer cladding layer.

Figure 2

Schematic showing surface functionalization. (A) Silicon surface of microring sensors prior to modification. (B) APTES reacts with the surface siloxane groups to generate an amino-terminated surface. (C) S-HyNic reacts with primary amines to create a HyNic-displaying surface. (D) Addition of 4FB-modified antibodies results in hydrazone bond formation between the 4FB moieties on the antibodies and the HyNic moieties on the surface.

Figure 3

(A) Real-time monitoring of the shift in resonance frequency for twelve microrings within the same sample flow chamber during organic modification via reaction with APTES. The microrings were initially submerged in 95% ethanol solution and a 2% solution of APTES injected at t = 6.5 min. The silane was flushed from the chamber and microrings returned to 95% ethanol after 10 min. (B) Real-time shift in resonance frequency from five individual microrings during covalent immobilization of antibody onto the sensor surfaces. The 4FB-tagged anti-CEA antibody was added at t = 10 min and removed (sample chamber returned to acetate buffer) at t = 210 min.

Figure 4

Time-resolved detection of CEA using five anti-CEA-functionalized microrings alongside five control microrings that were not functionalized with antibody. Following exposure to CEA, the antibody surface was regenerated by exposure to glycine buffer for two minutes before returning to BSA-PBS.

Figure 5

Real-time monitoring of resonance frequency shifts of an anti-CEA antibody-functionalized microring upon exposure to increasing concentrations of CEA in BSA-PBS. After exposure to antigen, the antigen-antibody interaction was disrupted with glycine buffer, regenerating the original sensor surface, and the sample chamber was returned to BSA-PBS to reestablish the sensor baseline.

Figure 6

Real-time, label-free detection of CEA using microring resonators. (A) Overlay of three time-resolved association curves for the same ring at each concentration of CEA. The colored traces are tangent lines to the association curve at t = 0 and are used to determine the initial slope of sensor response. (B) Concentration-response calibration plot of the initial slope of sensor response versus CEA concentration upon introduction of antigen standard solutions. The dashed box in the corner of the graph represents the range shown in panel (C) for measurement of CEA concentrations of unknown samples. (C) Overlay of the unknown solutions on a concentration-response calibration plot for CEA as determined by the initial slope method covering a dynamic range comparable to a commercial ELISA.

Figure 7

(A) Example sensor response following addition of CEA in 100% FBS. Initial slope is determined by using a linear fit of the baseline to subtract the drifting baseline from the change in signal caused by addition of CEA in FBS. (B) Overlay of the unknown solution on the concentration-response calibration plot for CEA in 100% FBS. The concentration was determined to be 61 ± 23 ng/mL, which is in good agreement with a commercial ELISA assay.