Polysaccharide Multilayer Films in Sensors for Detecting Prostate Tumor Cells Based on Hyaluronan-CD44 Interactions

Abstract

The increasing need for point-of-care diagnosis has sparked the development of label-free sensing platforms, some of which are based on impedance measurements with biological cells. Here, interdigitated electrodes were functionalized with layer-by-layer (LbL) films of hyaluronan (HA) and chitosan (CHI) to detect prostatic tumor cells (PC3 line). The deposition of LbL films was confirmed with atomic force microscopy and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), which featured the vibrational modes of the HA top layer capable of interacting specifically with glycoprotein CD44 receptors overexpressed in tumor cells. Though the CHI/HA LbL films cannot be considered as a traditional biosensor due to their limited selectivity, it was possible to distinguish prostate tumor cells in the range from 50 to 600 cells/µL in in vitro experiments with impedance spectroscopy. This was achieved by treating the impedance data with information visualization methods, which confirmed the distinguishing ability of the films by observing the absence of false positives in a series of control experiments. The CD44-HA interactions may, therefore, be exploited in clinical analyses and point-of-care diagnostics for cancer, particularly if computational methods are used to process the data.

Keywords: CD44 receptor; cancer; hyaluronan; information visualization; layer-by-layer films; sensing.

Figure 1

(A) Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) spectrum of gold substrates functionalized with hyaluronic acid and chitosan. The spectrum for the uncoated substrate was used as the baseline. (B) Assignment of the main bands in the PM-IRRAS spectrum.

Figure 2

(A) Schematic representation of the electrode functionalization. (B) Average measurements of root mean square roughness (R_RMS) and surface potential (SP) for electrodes before and after layer-by-layer (LbL) functionalization. Atomic force microscopy (AFM) images for interdigitated electrodes (C) before and (D) after chitosan (CHI)/hyaluronan (HA) film deposition.

Figure 3

Representative micrographs of (A) TRITC-phalloidin stained PC3 cells attached to a CHI/HA film deposited on a glass substrate and (B) its respective bright field image. Scale bars are 100 µm for both. (C) Representative dark field micrograph of PC3 cells attached to an uncoated glass substrate. Scale bar is 1000 µm.

Figure 4

(A) Capacitance spectra for CHI/HA functionalized electrodes after exposure to different concentrations of tumor cells (50–1500 cells/µL). The spectrum of an uncoated electrode is also shown. (B) Schematic representation of tumor cell adhesion mediated by CD44-HA interaction in CHI/HA functionalized electrodes.

Figure 5

Two-dimensional interactive document mapping (IDMAP) plot for the capacitance spectra for uncoated electrodes and 3.5 CHI/HA-multilayer functionalized electrodes exposed to different tumor cell concentrations (50–1500 cells/µL) and non-specific analytes (glucose (100 mg/dL), ascorbic acid (4.8 mg/mL), and fetal bovine serum (FBS)).