Bioplasmonic paper-based assay for perilipin-2 non-invasively detects renal cancer

Abstract

Renal cell carcinoma (RCC) has poor survival prognosis because it is asymptomatic at an early, more curative stage. Recently, urine perilipin-2 (PLIN-2) was demonstrated to be a sensitive and specific biomarker for the noninvasive, early detection of RCC and an indispensable indicator to distinguish cancer from a benign renal mass. However, current Western blot or ELISA PLIN-2 assays are complicated, expensive, time-consuming or insensitive, making them unsuitable for routine analysis in clinical settings. Here we developed a plasmonic biosensor based on the high refractive index sensitivity of gold nanorattles for the rapid detection of PLIN-2 in patient urine. The paper-based plasmonic assay is highly sensitive and has a dynamic range of 50 pg/ml to 5 μg/ml PLIN-2. The assay is not compromised by variations in urine pH or high concentrations of interfering proteins such as albumin and hemoglobin, making it an excellent candidate for routine clinical applications. The urine PLIN-2 assay readily distinguished patients with pathologically proven clear cell carcinomas of various size, stage and grade (55.9 [39.5, 75.8] ng/ml, median [1st and 3rd quartile]) from age-matched controls (0.3 [0.3, 0.5] ng/ml), patients with bladder cancer (0.5 [0.4, 0.6] ng/ml) and patients with diabetic nephropathy (0.6 [0.4, 0.7] ng/ml). Urine PLIN-2 concentrations were roughly proportional to tumor size (Pearson coefficient 0.59). Thus, this cost-effective and label-free method represents a novel approach to conduct a non-invasive population screen or rapid differential diagnosis of imaged renal masses, significantly facilitating the early detection and diagnosis of RCC.

Trial registration: ClinicalTrials.gov NCT00851994 NCT01538823.

Keywords: bioplasmonic assay; cancer biomarkers; cancer diagnostics; renal cancer.

Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Schematic illustration of plasmonic paper manufacture. Gold nanorattles (AuNRTs) coated with cetyltrimethylammonium chloride (CTAC) were functionalized with monoclonal antibody by means of a bifunctional polyethylene glycol (PEG). The functionalized AuNRTs were then coated on filter paper and non-specific binding sites neutralized by further PEGylation. The paper-bound functionalized AuNRTs then bind specific analyte; in this case, perilipin-2 (PLIN-2) and cause a red shift in the localized surface plasmon resonance (LSPR) for the AuNRTs.

Figure 2. A)

A) Representative localized surface plasmon resonance spectra of anti-PLIN-2 functionalized AuNRTs (black), functionalized AuNRTs incubated with urine from a healthy volunteer (red), and functionalized AuNRTs incubated with urine from a patient with reanl clear cell carcinoma (blue). B) Box and whisker plot of the urine PLIN-2 concentrations of 20 control patients, 20 patients with renal cell cancer, 8 patients with bladder cancer and 10 patients with diabetic nephropathy. The median with whiskers and boxes depicting the interquartile range is shown. The concentration of each patient is depicted as a solid dot or a plus sign. The plus signs are outliers more than 1.5 times but less than 3.0 times the interquartile range. Patients with renal cell cancer have significantly higher urine PLIN-2 concentrations than that of other cohorts (P<0.001 by Kruskal-Wallis). C) Urine PLIN-2 concentration of patients with renal cell cancer as a function of tumor size. The Spearman correlation coefficient is 0.59 (P=0.009). Regression line (solid). 95% prediction interval (dashed lines). D) Urine PLIN-2 concentration determined by plasmonic paper vs relative absorbance by Western blot. Dashed line of identity. Spearman correlation 0.94 (P<0.001).

Figure 2. A)

A) Representative localized surface plasmon resonance spectra of anti-PLIN-2 functionalized AuNRTs (black), functionalized AuNRTs incubated with urine from a healthy volunteer (red), and functionalized AuNRTs incubated with urine from a patient with reanl clear cell carcinoma (blue). B) Box and whisker plot of the urine PLIN-2 concentrations of 20 control patients, 20 patients with renal cell cancer, 8 patients with bladder cancer and 10 patients with diabetic nephropathy. The median with whiskers and boxes depicting the interquartile range is shown. The concentration of each patient is depicted as a solid dot or a plus sign. The plus signs are outliers more than 1.5 times but less than 3.0 times the interquartile range. Patients with renal cell cancer have significantly higher urine PLIN-2 concentrations than that of other cohorts (P<0.001 by Kruskal-Wallis). C) Urine PLIN-2 concentration of patients with renal cell cancer as a function of tumor size. The Spearman correlation coefficient is 0.59 (P=0.009). Regression line (solid). 95% prediction interval (dashed lines). D) Urine PLIN-2 concentration determined by plasmonic paper vs relative absorbance by Western blot. Dashed line of identity. Spearman correlation 0.94 (P<0.001).

Figure 2. A)

A) Representative localized surface plasmon resonance spectra of anti-PLIN-2 functionalized AuNRTs (black), functionalized AuNRTs incubated with urine from a healthy volunteer (red), and functionalized AuNRTs incubated with urine from a patient with reanl clear cell carcinoma (blue). B) Box and whisker plot of the urine PLIN-2 concentrations of 20 control patients, 20 patients with renal cell cancer, 8 patients with bladder cancer and 10 patients with diabetic nephropathy. The median with whiskers and boxes depicting the interquartile range is shown. The concentration of each patient is depicted as a solid dot or a plus sign. The plus signs are outliers more than 1.5 times but less than 3.0 times the interquartile range. Patients with renal cell cancer have significantly higher urine PLIN-2 concentrations than that of other cohorts (P<0.001 by Kruskal-Wallis). C) Urine PLIN-2 concentration of patients with renal cell cancer as a function of tumor size. The Spearman correlation coefficient is 0.59 (P=0.009). Regression line (solid). 95% prediction interval (dashed lines). D) Urine PLIN-2 concentration determined by plasmonic paper vs relative absorbance by Western blot. Dashed line of identity. Spearman correlation 0.94 (P<0.001).

Figure 2. A)

A) Representative localized surface plasmon resonance spectra of anti-PLIN-2 functionalized AuNRTs (black), functionalized AuNRTs incubated with urine from a healthy volunteer (red), and functionalized AuNRTs incubated with urine from a patient with reanl clear cell carcinoma (blue). B) Box and whisker plot of the urine PLIN-2 concentrations of 20 control patients, 20 patients with renal cell cancer, 8 patients with bladder cancer and 10 patients with diabetic nephropathy. The median with whiskers and boxes depicting the interquartile range is shown. The concentration of each patient is depicted as a solid dot or a plus sign. The plus signs are outliers more than 1.5 times but less than 3.0 times the interquartile range. Patients with renal cell cancer have significantly higher urine PLIN-2 concentrations than that of other cohorts (P<0.001 by Kruskal-Wallis). C) Urine PLIN-2 concentration of patients with renal cell cancer as a function of tumor size. The Spearman correlation coefficient is 0.59 (P=0.009). Regression line (solid). 95% prediction interval (dashed lines). D) Urine PLIN-2 concentration determined by plasmonic paper vs relative absorbance by Western blot. Dashed line of identity. Spearman correlation 0.94 (P<0.001).