Fluid Flow Dependency in Immunoselective Cell Capture via Liquid Biopsy.

Liquid biopsy targets rare cells that overexpress disease-specific membrane markers and capture these cells via immunoaffinity. The diagnosis efficiency of liquid biopsy can be impaired by the presence of healthy adherent cells also expressing the same biomarkers. Here, we investigated the effect of settling times and rinsing flow rates on the efficiency of EpCAM-based immunocapture using both simulation and experiments with three different cell types. Cell-surface adhesion forces and shear rates were calculated to define the range of rinsing flow rates to test experimentally. Healthy adherent cells did not adhere to blocked immunofunctionalized surfaces within the timeframe of the experiment; however, healthy EpCAM positive cells did bind to the surface to some extent. The greatest difference in capture efficiency was obtained using a high rinsing flow rate of 25 mL/min following 40 min static incubation, indicating that optimizing rinsing flow rates could be a viable option to capture, more specifically, cancer cells overexpressing EpCAM.

Cancer cell detection device for the diagnosis of bladder cancer from urine.

Bladder cancer is common and has one of the highest recurrence rates. Cystoscopy, the current gold standard diagnosis approach, has recently benefited from the introduction of blue light assisted photodynamic diagnostic (PDD). While blue light cystoscopy improves diagnostic sensitivity, it remains a costly and invasive approach. Here, we present a microfluidic-based platform for non-invasive diagnosis which combines the principle of PDD with whole cell immunocapture technology to detect bladder cancer cells shed in patient urine ex vivo. Initially, we demonstrate with model cell lines that our non-invasive approach achieves highly specific capture rates of bladder cancer cells based on their Epithelial Cell Adhesion Molecule expression (>90%) and detection by the intensity levels of Hexaminolevulinic Acid-induced Protoporphyrin IX fluorescence. Then, we show in a pilot study that the biosensor platform successfully discriminates histopathologically diagnosed cancer patients (n = 10) from non-cancer controls (n = 25). Our platform can support the development of a novel non-invasive diagnostic device for post treatment surveillance in patients with bladder cancer and cancer detection in patients with suspected bladder cancer.

Plasma enabled devices for the selective capture and photodynamic identification of prostate cancer cells.

Prostate cancer is the second most common cancer in men and the second leading cause of male cancer deaths. The current blood test for detecting prostate cancers measures prostate-specific antigen. It has many limitations including a very high rate of false positives. Herein, prostate-specific membrane antigen (PSMA) based immunocapture and hexaminolevulinate (HAL) based photodetection are integrated into a new diagnostic device designed to selectively identify whole prostate cancer cells from voided urine with the aim of providing an accurate noninvasive alternative to current diagnosis methods. Prestained, prostate cancer cells spiked in urine samples at concentrations ranging from 1500 to 2000 cells/ml were captured with 89% sensitivity and 95% specificity. HAL, a cancer specific photosensitizer, was then used to circumvent the need for prestaining. Optimum HAL incubation conditions were identified (50 µM at 37 °C for 2 h) where the mean HAL-induced fluorescence intensity of LNCaP cells was three times that of healthy PNT2 cells, thus providing an independent way to discriminate captured cancer cells from background metabolites. Combining anti-PSMA immunocapture with HAL-induced fluorescent detection, 86% sensitivity and 88% selectivity were achieved, thereby proving the validity of the dual-method for the selective photospecific detection of prostate cancer cells.

Bladder Cancer Cell Capture: Elucidating the Effect of Sample Storage Conditions on Capturing Bladder Cancer Cells via Surface Immobilized EpCAM Antibody.

The gold standard to detect bladder cancer, cystoscopy, is an invasive procedure requiring ambulant hospitalization, thus presenting an obstacle for routine diagnosis. We aim to develop a noninvasive detection method as an alternative that selectively captures shed cancer cells in the patient's urine via surface-immobilized anti-EpCAM antibody. However, the urine sample storage conditions prior to analysis affect the subsequent cancer cell capture rates by the device. In this study, we investigate the capture rates of HT1197 and HT1376 bladder cancer cells in different media (fresh and aged urine as well as PBS) and storage temperatures prior to analysis (37 and 4 °C) as well as in the presence of adjuvants in the medias (free antibodies and cell debris). Capture efficiencies decreased in as little as 1 h of the sample being incubated at 37 °C in all media studied here. Furthermore, cell debris played a strong part in reducing the capture efficiency. From the data, we conclude that storing the sample at 4 °C resulted in the best capture efficiency if storage of more than 1 h was required, which gave valuable insights for this sensor's translation from laboratory to real-world applications.

A platform for selective immuno-capture of cancer cells from urine.

Urothelial cancers are amongst the 10 most common types of cancer and represent a major health problem worldwide. Current urinary diagnostic tests for urothelial cancer are expensive and have limited sensitivity and specificity. In this work, proofs of concept for a selective cancer cell capture platform are presented with the aim to achieve the first generation of specific urinary tests for the detection of cancer cells in urine specimen. The unique reactivity of plasma deposited polyoxazoline was used to covalently bind cancer specific antibodies in microchannels. Cancer cells dispersed in patient urine were successfully captured with up to 99% selectivity and 100% sensitivity over a wide range of cell concentrations. The streamlined two steps preparation process of the capture platform represents an important advance in medical diagnostics, with broader potential applications.

Synergic bactericidal effects of reduced graphene oxide and silver nanoparticles against Gram-positive and Gram-negative bacteria.

Reduced graphene oxide (rGO) is a promising antibacterial material, the efficacy of which can be further enhanced by the addition of silver nanoparticles (nAg). In this study, the mechanisms of antibacterial activity of rGO-nAg nanocomposite against several important human pathogenic multi-drug resistant bacteria, namely Gram-positive coccal Staphylococcus aureus and Gram-negative rod-shaped Escherichia coli and Proteus mirabilis are investigated. At the same concentration (100 µg/ml), rGO-nAg nanocomposite was significantly more effective against all three pathogens than either rGO or nAg. The nanocomposite was equally active against P. mirabilis and S. aureus as systemic antibiotic nitrofurantoin, and significantly more effective against E. coli. Importantly, the inhibition was much faster in the case of rGO-nAg nanocomposite compared to nitrofurantoin, attributed to the synergistic effects of rGO-nAg mediated contact killing and oxidative stress. This study may provide new insights for the better understanding of antibacterial actions of rGO-nAg nanocomposite and for the better designing of graphene-based antibiotics or other biomedical applications.