This study reports a microfluidic-based optical sensing device for label-free detection of circulating tumor cells (CTCs), a rare cell species in blood circulation. Based on the metabolic features of cancer cells, live CTCs can be quantified indirectly through their lactic acid production. Compared with the conventional schemes for CTC detection, this label-free approach could prevent the biological bias due to the heterogeneity of the surface antigens on cancer cells. In this study, a microfluidic device was proposed to generate uniform water-in-oil cell-encapsulating micro-droplets, followed by the fluorescence-based optical detection of lactic acid produced within the micro-droplets. To test its feasibility to quantify cancer cells, experiments were carried out. Results showed that the detection signals were proportional to the number of cancer cells within the micro-droplets, whereas such signals were insensitive to the existence and number of leukocytes within. To further demonstrate its feasibility for cancer cell detection, the cancer cells with known cell number in a cell suspension was detected based on the method. Results revealed that there was no significant difference between the detected number and the real number of cancer cells. As a whole, the proposed method opens up a new route to detect live CTCs in a label-free manner.

Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally defined as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical significance of these cells (i.e., CD45^neg/EpCAM^neg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of fluorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a microfluidic system was proposed. In this study, an ODEP microfluidic system was developed. The optimal ODEP operating conditions and the performance of live CD45^neg/EpCAM^neg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45^neg/EpCAM^neg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45^neg/EpCAM^neg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45^neg/EpCAM^neg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP microfluidic system capable of simply and effectively isolating a specific, rare cell species from a cell mixture.

Single-cell electrical properties (e.g., specific membrane capacitance (C_{specific membrane}) and cytoplasm conductivity (σ_cytoplasm)) have been regarded as potential label-free biophysical markers for the evaluation of cellular status. However, whether there exist correlations between these biophysical markers and cellular status (e.g., membrane-associate protein expression) is still unknown. To further validate the utility of single-cell electrical properties in cell type classification, C_{specific membrane} and σ_cytoplasm of single PC-3 cells with membrane staining and/or fixation were analyzed and compared in this study. Four subtypes of PC-3 cells were prepared: untreated PC-3 cells, PC-3 cells with anti-EpCAM staining, PC-3 cells with fixation, and fixed PC-3 cells with anti-EpCAM staining. In experiments, suspended single cells were aspirated through microfluidic constriction channels with raw impedance data quantified and translated to C_{specific membrane} and σ_cytoplasm. As to experimental results, significant differences in C_{specific membrane} were observed for both live and fixed PC-3 cells with and without membrane staining, indicating that membrane staining proteins can contribute to electrical properties of cellular membranes. In addition, a significant decrease in σ_cytoplasm was located for PC-3 cells with and without fixation, suggesting that cytoplasm protein crosslinking during the fixation process can alter the cytoplasm conductivity. Overall, we have demonstrated how to classify single cells based on cellular electrical properties.

This study integrated the microfluidic system and ODEP technology for the isolation of CTC clusters from the background leukocytes. The working principle is based on the size difference between the CTC clusters and leukocytes, and thus different magnitude of ODEP force acting on them. ODEP mainly use a controllable light pattern, acting as a virtual electrode, to generate a non-uniform electric field that is in turn utilized to manipulate the electrically-polarized cells. The utilization of ODEP-based mechanism for CTCs isolation has been successfully demonstrated in our previous study [1]. Since 1970, a series of clinical studies have shown that single CTCs may not be the main cause of cancer metastasis, but two or more aggregated CTCs [2, 3]. In order to isolate CTC clusters for back-end analysis. For the biological-based methods (e.g. HBCTC-Chip [4], or CTC-iChip [5]), although CTC clusters can be specifically separated by antibody-based schemes, but the surface-area-to-volume ratio of CTC clusters is relatively low which might affect the binding efficiency of CTC clusters and antibody. Alternatively, some studies proposed physical-based methods to separate CTC clusters (e.g. ISET [6], FMSA [7], Cluster-chip [8]). Although these methods have been demonstrated to effectively isolate the CTC clusters from the background cells mainly based on their size difference, the influence of shear stress on physical size of CTC cluster, or the viability of the cells isolated is still a problem. To address this issue, The key advantages of ODEP mechanism for cell isolation including: (1) no need of complex microfabrication process for constructing microfilter structures, and (2) the reduction of shear stress acting on the cells manipulated. However, the feasibility of using ODEP-based mechanism for the isolation of CTC cluster (i.e. CTC cell aggregates) has not yet been explored. To test its feasibility, a T-shaped microfluidic chip was designed (Fig.1). A virtual microfilter consisting of multiple light patterns was designed at the CTC clusters isolation zone (Fig.1). By continuously moving and rotating the light patterns in the microfilter, the larger CTC clusters can be separated from the background leukocytes, and also transported to the side microchannel (Fig.2). In this work, the optimum ODEP operating conditions (e.g. moving velocity of light pattern) was explored. Results revealed that moving velocity of light pattern that can manipulate the CTC clusters (containing 2-13 cells) was significantly higher than the background of leukocytes (Fig.3). Based on this, the moving velocity of light pattern was set at 100 μm/sec (Fig.3). At a given sample flow rate of 0.5 μl/min, moreover, we found that the rotation speed of light patterns at 14 RPM could significantly increase the purity of CTC clusters isolation (Fig.4). Based on the set operating conditions, the recovery and purity of the isolated CTC clusters were experimentally evaluated to be 70.1 ± 7.1% and 60.8 ± 2.7%, respectively (Fig.4). As a whole, we have established a high purity CTC clusters isolation method that is easy to operate, and is possible to avoid the problem caused by the shear stress acting on the cells or particles.

This study integrated the microfluidic system and ODEP technology for the isolation of CTC clusters from the background leukocytes. The working principle is based on the size difference between the CTC clusters and leukocytes, and thus different magnitude of ODEP force acting on them. ODEP mainly use a controllable light pattern, acting as a virtual electrode, to generate a non-uniform electric field that is in turn utilized to manipulate the electrically-polarized cells. The utilization of ODEP-based mechanism for CTCs isolation has been successfully demonstrated in our previous study [1]. Since 1970, a series of clinical studies have shown that single CTCs may not be the main cause of cancer metastasis, but two or more aggregated CTCs [2, 3]. In order to isolate CTC clusters for back-end analysis. For the biological-based methods (e.g. HBCTC-Chip [4], or CTC-iChip [5]), although CTC clusters can be specifically separated by antibody-based schemes, but the surface-area-to-volume ratio of CTC clusters is relatively low which might affect the binding efficiency of CTC clusters and antibody. Alternatively, some studies proposed physical-based methods to separate CTC clusters (e.g. ISET [6], FMSA [7], Cluster-chip [8]). Although these methods have been demonstrated to effectively isolate the CTC clusters from the background cells mainly based on their size difference, the influence of shear stress on physical size of CTC cluster, or the viability of the cells isolated is still a problem. To address this issue, The key advantages of ODEP mechanism for cell isolation including: (1) no need of complex microfabrication process for constructing microfilter structures, and (2) the reduction of shear stress acting on the cells manipulated. However, the feasibility of using ODEP-based mechanism for the isolation of CTC cluster (i.e. CTC cell aggregates) has not yet been explored. To test its feasibility, a T-shaped microfluidic chip was designed (Fig.1). A virtual microfilter consisting of multiple light patterns was designed at the CTC clusters isolation zone (Fig.1). By continuously moving and rotating the light patterns in the microfilter, the larger CTC clusters can be separated from the background leukocytes, and also transported to the side microchannel (Fig.2). In this work, the optimum ODEP operating conditions (e.g. moving velocity of light pattern) was explored. Results revealed that moving velocity of light pattern that can manipulate the CTC clusters (containing 2-13 cells) was significantly higher than the background of leukocytes (Fig.3). Based on this, the moving velocity of light pattern was set at 100 μm/sec (Fig.3). At a given sample flow rate of 0.5 μl/min, moreover, we found that the rotation speed of light patterns at 14 RPM could significantly increase the purity of CTC clusters isolation (Fig.4). Based on the set operating conditions, the recovery and purity of the isolated CTC clusters were experimentally evaluated to be 70.1 ± 7.1% and 60.8 ± 2.7%, respectively (Fig.4). As a whole, we have established a high purity CTC clusters isolation method that is easy to operate, and is possible to avoid the problem caused by the shear stress acting on the cells or particles.