Ultrasensitive melanoma biomarker detection using a microchip SERS immunoassay with anisotropic Au-Ag alloy nanoboxes

Abstract

The detection of circulating biomarkers in liquid biopsies has the potential to provide a non-invasive route for earlier cancer diagnosis and treatment management. Melanoma chondroitin sulfate proteoglycan (MCSP) is a membrane protein characteristic for melanoma cell migration and tissue invasion with its soluble form (sMCSP) serving as a potential promising diagnostic surrogate. However, at the initial disease stage, the detection of sMCSP is challenging because of its low abundance and the required high specificity to analyze sMCSP in complex bodily fluids. Herein, we report a highly sensitive and high-throughput microchip that enables Surface Enhanced Raman Spectroscopy (SERS) immunoassay for parallel detection of up to 28 samples. Key to assay speed and sensitivity is the stimulation of an alternating current-induced nanofluidic mixing that improves target-sensor collision and displacement of non-specific molecules. Anisotropic Au-Ag alloy nanoboxes (NB's) with strong plasmonic hot spots provide single SERS particle sensitivity that enables ultrasensitive sMCSP detection of as low as 0.79 pM (200 pg ml^-1). As a proof of concept study, we investigate the assay performance in simulated melanoma patient samples.

Scheme 1. The methodological approach for melanoma biomarker detection using microchip-SERS platform. (a) Extraction of protein lysate and immunoprecipitation of pure MCSP protein (b) ac-EHD induced nanofluidic mixing for specific MCSP protein capture (c) SERS labelling of target MCSP with the anti-MCSP conjugated SERS nanotags with ac-EHD nanomixing (d) the molecules are excited with laser for Raman scattering and a characteristic peak is obtained at 1075 cm⁻¹, peak height corresponds to the concentration of the target antigen.

Fig. 1. Study of ac-EHD nanofluidic mixing. (a) Effect of ac-EHD induced nanofluidic mixing on antigen incubation time. (b) Comparison of detection sensitivity between static incubation and ac-EHD induced nanofluidic mixing. Each bar or dot represents the average from three trials (n = 3) and error bars represent the standard deviation within each experiment.

Fig. 2. Specificity assay on the microchip platform. (a) Overlaid Raman spectra obtained from the specificity study. The microchip gold electrodes were functionalised with 100 pg ml⁻¹ target anti-MCSP (grey), without primary antibody-no anti-MCSP (green), 100 pg ml⁻¹ non-target PD-L1 antigen (pink), and non-target anti PD-L1 SERS nanotags (orange). (b) Corresponding bar graphs are showing average Raman intensity for the positive and negative controls. Each bar represents the average from three trials (n = 3) and error bars represent the standard deviation within each experiment.

Fig. 3. Sensitivity assay on the microchip platform. (a) Raman spectra for various concentration of MCSP protein spiked in PBS, 50 pg ml⁻¹ (grey), 100 pg ml⁻¹ (violet), 200 pg ml⁻¹ (orange), 500 pg ml⁻¹ (green), and 1000 pg ml⁻¹ (pink). (b) Corresponding bar graphs are showing the average Raman intensity for the various concentration of MCSP protein spiked in PBS. Each bar represents the average from three trials (n = 3) and error bars represent the standard deviation within each experiment.

Fig. 4. Evaluation of microchip-SERS immunoassay on human serum. (a) SERS spectra obtained after spiking target MCSP protein into diluted serum at various concentration, 0 pg ml⁻¹ (positive control, red), 200 pg ml⁻¹ (violet), 400 pg ml⁻¹ (orange), 600 pg ml⁻¹ (green), and 800 pg ml⁻¹ (grey). (b) Scatter plot with regression line for the detected sMCSP microchip-SERS immunoassay in human serum.