Cancer 2013 May-18

Quantification of ovarian cancer markers with integrated microfluidic concentration gradient and imaging nanohole surface plasmon resonance

 Analyst, 2013; 138(5), 1450-1458.

Carlos Escobedo, Yu-Wei Chou, Mohammad Rahman, Xiaobo Duan, Reuven Gordon, David Sinton, Alexandre G. Brolo and Jacqueline Ferreira*


INTRODUCTION: Nanohole arrays in metal films constitute a new generation of surface plasmon resonance (SPR) biosensors that can be easily integrated into miniaturized devices, such as microfluidic chips. Additionally, nanohole-based biosensors allow the use of simple optical arrangements involving low cost light sources and detectors. The detection scheme may be based on the measurements of changes in the intensity of a monochromatic light transmitted through the nanohole arrays due to molecular adsorption. The main advantage of this detection scheme is the possibility of multiple analyte detection in a single acquired image (multiplexing), in addition to the label-free real-time quantitative detection, which is common to all types of SPR sensors. All the advantages afforded by a nanohole-based biosensor integrated in microfluidic systems suggest that these devices can be explored for the early detection of diseases, such as cancer. The early detection is critical to effective treatment and survival of patients.

METHODS: The quantification of ovarian cancer biomarkers (PAX8 and r-PAX8) concentrations in the sample stream was achieved by SPR imaging via normal transmission of monochromatic light on a CCD detector array. As the amount of bound analyte to the nanohole arrays is proportional to the analyte concentration, the transmitted light through the nanohole arrays in different microchannels also varies. These intensity variations are tracked and the determinations of unknown analyte concentrations are achieved by comparison with the standard curve during a real-time measurement.

RESULTS: A comparison of the normalized intensities of the images obtained after non‑specific immobilization of PAX8 with the ones obtained for the calibration curve reveled a concentration of ca. 24.85 ± 0.05 mg.mL-1. The effective refractive index associated with the biomolecular layer was estimated to be 1.57. The normalized intensities from the arrays for two different r-PAX8 sample concentrations (detected through their bioaffinity with PAX8) were analyzed and related to their concentrations in solution. A minimum intensity was observed for the most concentrated solution of r‑PAX8. The concentrations of the unknown samples were calculated by comparison to the concentration of the calibration curve obtained in real‑time. The experimentally determined concentrations for r-PAX8 were 1.54 ± 0.74 mg.mL-1 and 6.47 ± 0.46 mg.mL-1 for the diluted and concentrated r-PAX8 samples, respectively. The deviation of these concentration values relative to that expected values from the unknown samples was about 23% in both samples.

CONCLUSION: SPR-based biosensor integrated into a micro-fluidic concentration gradient generator was demonstrated for the quantitative assessment of ovarian cancer biomarkers. The ovarian cancer biomarkers were quantified using a microfluidic concentration gradient integrated with the nanohole array sensor platform. This allows the generation of an “in situ” calibration curve simultaneously with the analysis. The system was tested using “unknown” samples prepared in our laboratory by different experimenters. The limit of detection for r-PAX8 protein achieved in this work was about 5 nM within a dynamic range of about 1 order of magnitude. The dynamic range was limited by the linear range of the dilution chip used to generate the calibration curve. The logical next step for this technology is its direct implementation using clinical samples. Towards that goal, the current system would benefit from improved instrumentation (implementing temperature control, low-noise detectors, and highly stable light sources). Importantly, the proof-of-concept operation demonstrated in this work emphasizes the potential of the nanohole technology for parallel, label-free and real-time quantification of biological interactions.


Supplement picture:

Jacqueline Ferreira-1

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