Debanjan Das

A microfluidic device for continuous manipulation of biological cells using dielectrophoresis.

The present study demonstrates the design, simulation, fabrication and testing of a label-free continuous manipulation and separation micro-device of particles/biological cells suspended on medium based on conventional dielectrophoresis. The current dielectrophoretic device uses three planner electrodes to generate non-uniform electric field and induces both p-DEP and n-DEP force simultaneously depending on the dielectric properties of the particles and thus influencing at least two types of particles at a time. Numerical simulations were performed to predict the distribution of non-uniform electric field, DEP force and particle trajectories. The device is fabricated utilizing the advantage of bonding between PDMS and SU8 polymer. The p-DEP particles move away from the center of the streamline, while the n-DEP particles will follow the central streamline along the channel length. Dielectrophoretic effects were initially tested using polystyrene beads followed by manipulation of HeLa cells. In the experiment, it was observed that polystyrene beads in DI water always response as n-DEP up to 1MHz frequency, whereas HeLa cells in PBS medium response as n-DEP up to 400kHz frequency and then it experiences p-DEP up to 1MHz. Further, the microscopic observations of DEP responses of HeLa cells were verified by performing trapping experiment at static condition.

Buckling assisted and lithographically micropatterned fully flexible sensors for conformal integration applications

Development of flexible sensors/electronics over substrates thicker than 100 μm is of immense importance for its practical feasibility. However, unlike over ultrathin films, large bending stress hinders its flexibility. Here we have employed a novel technique of fabricating sensors over a non-planar ridge topology under pre-stretched condition which not only helps in spontaneous generation of large and uniform parallel buckles upon release, but also acts as stress reduction zones thereby preventing Poisson’s ratio induced lateral cracking. Further, we propose a complete lithography compatible process to realize flexible sensors over pre-stretched substrates thicker than 100 μm that are released through dissolution of a water soluble sacrificial layer of polyvinyl alcohol. These buckling assisted flexible sensors demonstrated superior performance along different flexible modalities. Based on the above concept, we also realized a micro thermal flow sensor, conformally wrapped around angiographic catheters to detect flow abnormalities for potential applications in interventional catheterization process.

Effect of electrode geometry on voltage reduction in EWOD based devices

Electrowetting(EW) has been used sophistically in the fields of biomedical and optical applications. The major problem posing challenge in EW is to reduce its operating voltage. The study consist of understanding the effect of device parameters that affect EW phenomenon to reduce the actuation voltage with optimum velocity. Using EDEW 2.0 simulation tools [8] and Surface Evolver software [9, 11] EW is simulated. By varying the different parameters (electrode length, gap between electrodes, dielectric thickness, shape of electrode edge and dielectric constant) the corresponding energy curves (surface energy versus centroid position) are obtained. The main goal is to reduce the voltage. It is observed that voltage bears inverse relation with the surface energy and thus actuation voltage can be reduced by reducing the surface energy. There is direct influence of Electrodes parameters in reducing the surface energy.

Evaluation of single cell electrical parameters from bioimpedance of a cell suspension

The present study introduces a simple and detailed analysis technique to extract the electrical properties of a single cell from the impedance spectroscopy data from a group of cells in suspension, leading to a more reliable and cost effective diagnosis process for disease detection. The existing method for bioimpedance measurement, by trapping a single cell in a microchannel, is quite a complex process and suffers from localized joule heating. Considering that biological cells show their natural characteristics and functionality in a colony of similar cells rather than in an individual environment, the extraction of single cell electrical parameters from the impedance measurement of a group of suspended cells may provide more reliable and effective information. Experimental and theoretical analyses were performed to extract single cell permittivity, conductivity, membrane capacitance and cytoplasm resistance, utilizing the established Maxwells mixture theory. The bioimpedance of the suspended HeLa cells was characterized with a controlled volume fraction of cells in the suspension, and the measurement was performed by varying the voltage to investigate the change in permittivity and conductivity of the HeLa cells. The proposed technique showed the membrane capacitance and cytoplasm resistance of a single HeLa cell to be in the 1.8 nF cm−2 and 35 kΩ cm2 ranges, respectively. Analysis of the measured impedance data also reveals that the relative permittivity and conductivity of a single HeLa cell is a function of the applied potential and frequency.

Nanomechanical signatures of oral submucous fibrosis in sub-epithelial connective tissue

Oral sub-mucous fibrosis (OSF), a potentially malignant disorder, exhibits extensive remodeling of extra-cellular matrix in the form of sub-epithelial fibrosis which is a possible sequel of assaults from different oral habit related irritants. It has been assumed that micro/nanobio-mechanical imbalance experienced in the oral mucosa due to fibrosis may be deterministic for malignant potential (7-13%) of this pathosis. Present study explores changes in mechanobiological attributes of sub-epithelial connective tissue of OSF and the normal counterpart. The atomic force microscopy was employed to investigate tissue topography at micro/nano levels. It documented the presence of closely packed parallel arrangement of dense collagen fibers with wide variation in bandwidth and loss of D-space in OSF as compared to normal. The AFM based indentation revealed that sub-epithelium of OSF tissue has lost its flexibility with increased Youngs modulus, stiffness, adhesiveness and reduced deformation of the juxta-epithealial connective tissue towards the deeper layer. These significant variations in nano-mechanical properties of the connective tissue indicated plausible impacts on patho-physiological microenvironment. Excessive deposition of collagen I and diminished expression of collagen III, fibronectin along with presence of α-SMA positive myofibroblast in OSF depicted its pathological basis and indicated the influence of altered ECM on this pathosis. The mechanobiological changes in OSF were corroborative with change in collagen composition recorded through immunohistochemistry and RT-PCR. The revelation of comparative nanomechanical profiles of normal oral mucosa and OSF in the backdrop of their structural and cardinal molecular attributes thus became pivotal for developing holistic pathobiological insight about possible connects for malignant transformation of this pre-cancer.

Study of PDMS as Dielectric Layer in Electrowetting Devices

An electrowetting-on-dielectric (EWOD) device by using Polydimethylsiloxane (PDMS) as dielectric layer has been fabricated. Aluminium metal film is used for EWOD electrode fabrication. Teflon AF 1600 thin film is coated to improve hydrophobicity and reduce liquid sticking (found in PDMS surface). Both PDMS and Teflon layer is deposited by spin coating. Direct coating of Teflon, on top of PDMS layer, results in poor quality film, because of inherent hydrophobic nature of PDMS. So, Oxygen plasma treatment of PDMS surface is carried, before Teflon coating. Movement of water droplet (conductivity: 250 μ S/m) is obtained at 150 V DC voltage supply. Present study demonstrates a simple, cost and time effective fabrication procedure for EWOD device.

Fragmental Frequency Analysis Method to Estimate Electrical Cell Parameters From Bioimpedance Study

This paper introduces an alternate approach utilizing the fragmental frequency analysis method for analyzing bioimpedance data to estimate electrical cell parameters. Impedance of cervical cancer cells (HeLa) in phosphate buffer saline media is measured using an electric cell-substrate impedance sensing (ECIS) device and impedance analyzer. The measured impedance data were visualized by modeling an equivalent electrical circuit of the system considering the dominancy of individual parameters of the ECIS system and analyzed in different frequency zones. The present approach eliminates the convergence inaccuracy in fitting the experimental impedance data with fitting software arising due to invalid initial conditions, and a large number of data points. This method provides high-frequency characterization, modeling of ECIS system, knowledge of effect of ECIS model parameters with frequency, and an alternate way to calculate model parameters.

Metabolomics reveals perturbations in endometrium and serum of minimal and mild endometriosis

Endometriosis is a common benign gynecological disease, characterized by growth and proliferation of endometrial glands and stroma outside the uterus. With studies showing metabolic changes in various biofluids of endometriosis women, we have set upon to investigate whether endometrial tissue show differences in their metabolic profiles. 1H NMR analysis was performed on eutopic endometrial tissue of women with endometriosis and controls. Analysis was performed on spectral data and on relative concentrations of metabolites obtained from spectra using multivariate and univariate data analysis. Analysis shows that various energy, ketogenic and glucogenic metabolites have significant altered concentrations in various stages of endometriosis. In addition, altered tissue metabolites in minimal and mild stages of endometriosis were explored in serum of these patients to assess their role in disease diagnosis. For Stage I diagnosis alanine was found to have 90% sensitivity (true positives) and 58% specificity (true negatives). For Stage II diagnosis alanine, leucine, lysine, proline and phenylalanine showed significant altered levels in serum. While sensitivity of these serum metabolites varied between 69.2–100% the specificity values ranged between 58.3–91.7%. Further, a regression model generated with this panel of serum markers showed an improved sensitivity and specificity of 100% and 83%, respectively for Stage II diagnosis.

Wavelet-based multiscale analysis of bioimpedance data measured by electric cell-substrate impedance sensing for classification of cancerous and normal cells.

The paper presents a study to differentiate normal and cancerous cells using label-free bioimpedance signal measured by electric cell-substrate impedance sensing. The real-time-measured bioimpedance data of human breast cancer cells and human epithelial normal cells employs fluctuations of impedance value due to cellular micromotions resulting from dynamic structural rearrangement of membrane protrusions under nonagitated condition. Here, a wavelet-based multiscale quantitative analysis technique has been applied to analyze the fluctuations in bioimpedance. The study demonstrates a method to classify cancerous and normal cells from the signature of their impedance fluctuations. The fluctuations associated with cellular micromotion are quantified in terms of cellular energy, cellular power dissipation, and cellular moments. The cellular energy and power dissipation are found higher for cancerous cells associated with higher micromotions in cancer cells. The initial study suggests that proposed wavelet-based quantitative technique promises to be an effective method to analyze real-time bioimpedance signal for distinguishing cancer and normal cells.

Bioimpedimetric analysis in conjunction with growth dynamics to differentiate aggressiveness of cancer cells

Determination of cancer aggressiveness is mainly assessed in tissues by looking at the grade of cancer. There is a lack of specific method to determine aggressiveness of cancer cells in vitro. In our present work, we have proposed a bio-impedance based non-invasive method to differentiate aggressive property of two breast cancer cell lines. Real-time impedance analysis of MCF-7 (less aggressive) and MDA-MB-231 cells (more aggressive) demonstrated unique growth pattern. Detailed slope-analysis of impedance curves at different growth phases showed that MDA-MB-231 had higher proliferation rate and intrinsic resistance to cell death, when allowed to grow in nutrient and space limiting conditions. This intrinsic nature of death resistance of MDA-MB-231 was due to modulation and elongation of filopodia, which was also observed during scanning electron microscopy. Results were also similar when validated by cell cycle analysis. Additionally, wavelet based analysis was used to demonstrate that MCF-7 had lesser micromotion based cellular activity, when compared with MDA-MB-231. Combined together, we hypothesize that analysis of growth rate, death resistance and cellular energy, through bioimpedance based analysis can be used to determine and compare aggressiveness of multiple cancer cell lines. This further opens avenues for extrapolation of present work to human tumor tissue samples.