Aptamer-conjugated graphene oxide membranes for highly efficient capture and accurate identification of multiple types of circulating tumor cells

Abstract

Tumor metastasis is responsible for 1 in 4 deaths in the United States. Though it has been well-documented over past two decades that circulating tumor cells (CTCs) in blood can be used as a biomarker for metastatic cancer, there are enormous challenges in capturing and identifying CTCs with sufficient sensitivity and specificity. Because of the heterogeneous expression of CTC markers, it is now well understood that a single CTC marker is insufficient to capture all CTCs from the blood. Driven by the clear need, this study reports for the first time highly efficient capture and accurate identification of multiple types of CTCs from infected blood using aptamer-modified porous graphene oxide membranes. The results demonstrate that dye-modified S6, A9, and YJ-1 aptamers attached to 20-40 μm porous garphene oxide membranes are capable of capturing multiple types of tumor cells (SKBR3 breast cancer cells, LNCaP prostate cancer cells, and SW-948 colon cancer cells) selectively and simultaneously from infected blood. Our result shows that the capture efficiency of graphene oxide membranes is ~95% for multiple types of tumor cells; for each tumor concentration, 10 cells are present per milliliter of blood sample. The selectivity of our assay for capturing targeted tumor cells has been demonstrated using membranes without an antibody. Blood infected with different cells also has been used to demonstrate the targeted tumor cell capturing ability of aptamer-conjugated membranes. Our data also demonstrate that accurate analysis of multiple types of captured CTCs can be performed using multicolor fluorescence imaging. Aptamer-conjugated membranes reported here have good potential for the early diagnosis of diseases that are currently being detected by means of cell capture technologies.

Scheme 1. (A) Schematic Representation Showing Aptamer-Conjugated Porous Graphene Oxide Membrane-Based Separation and Capture of Multiple Types of CTCs from Infected Blood and (B) Schematic Representation Showing Fluorescence Imaging of Multiple Types of CTCs Captured by Graphene Oxide Membranes Using a Dye-Conjugated Aptamer

Figure 1

(A) Schematic representation showing the stepwise chemical formation of 3D graphene oxide. (B) Photograph showing the formation of a porous graphene oxide membrane from aptamer-bound graphene oxide foam. (C) Scanning electron microscopy image of a graphene oxide membrane, which indicates a pore size of 20–40 μm. (D) Energy-dispersive X-ray spectroscopy mapping showing the presence of C and O in 3D network membranes.

Figure 2

(A) TEM image showing that SW-948 colon tumor cells are captured by the membranes. (B) Fluorescence image showing that LNCaP tumor cells are not captured by the Alexa Fluor 488 dye-modified YJ-1 aptamer-attached membrane. (C) Fluorescence image showing that a huge amount of SW-948 colon tumor cells is captured by the membranes. The observed blue fluorescence is due to the presence of the Alexa Fluor 488 dye-attached aptamer on the SW-948 cancer cell surface. (D) Number of CEA positive cells in the supernatant and membranes in the absence of the YJ-1 aptamer. (E) Number of CEA positive cells in the supernatant and membranes in the presence of the YJ-1 aptamer. (F) Number of PSMA positive cells in the supernatant and membranes when PSMA positive LNCaP colon cancer cell-infected blood was passed through the YJ-1 aptamer-attached membranes. (G) Plot that demonstrates the biocompatibility of our membranes.

Figure 3

(A) Fluorescence image showing that a huge amount of SK-BR-3 breast cancer cells is captured by the Cy2-modified S6 aptamer-attached membranes. (B) Fluorescence image showing that the capture efficiency is approximately zero when SW-948 colon cancer cell-infected blood is used for capture by the membranes. (C) Number of HER2 positive cells in the supernatant and membranes when HER2 positive SK-BR-3 breast cancer cell-infected blood was passed through the membranes. (D) Number of CEA positive cells in the supernatant and membranes. (E) Percent of HER2 positive cells captured by S6 aptamer-attached membranes, when citrated whole rabbit blood infected with (10 cells/mL) cancerous and (10⁵ cells/mL) normal cells was used.

Figure 4

(A) Fluorescence image showing that the bioconjugated porous graphene oxide membrane is capable of capturing different types of tumor cells from infected blood. (B) Fluorescence image demonstrating that no cells are captured when a normal skin HaCaT cell is used. (C) Number of HER2 positive, PSMA positive, and CEA positive cells in membranes. (D) Number of HER2 positive, PSMA positive, and CEA positive cells in the supernatant.