Nithya Murugesan

Bio: Nithya Murugesan is an academic researcher from Indian Institute of Technology Madras. The author has contributed to research in topics: Biosensor & Thermotaxis. The author has an hindex of 3, co-authored 4 publications receiving 28 citations.

A diffusion based long-range and steady chemical gradient generator on a microfluidic device for studying bacterial chemotaxis

Abstract: Studies on chemotaxis in microfluidics device have become a major area of research to generate physiologically similar environment in vitro. In this work, a novel micro-fluidic device has been developed to study chemo-taxis of cells in near physiological condition which can create controllable, steady and long-range chemical gradients using various chemo-effectors in a micro-channel. Hydrogels like agarose, collagen, etc, can be used in the device to maintain exclusive diffusive flux of various chemical species into the micro-channel under study. Variations of concentrations and flow rates of Texas Red dextran in the device revealed that an increase in the concentration of the dye in the feed from 6 to 18 μg ml−1, causes a steeper chemical gradient in the device, whereas the flow rate of the dye has practically no effect on the chemical gradient in the device. This observation confirms that a diffusion controlled chemical gradient is generated in the micro-channel. Chemo-taxis of E. coli cells were studied under the steady gradient of a chemo-attractant and a chemo-repellent separately in the same chemical gradient generator. For sorbitol and NiSO46H2O, the bacterial cells exhibit a steady distribution in the micro channel after 1 h and 30 min, respectively. From the distribution of bacterial population chemo-tactic strength of the chemo-effectors was estimated for E. coli. In a long microfluidic channel, migration behavior of bacterial cells under diffusion controlled chemical gradient showed chemotaxis, random movement, aggregation, and concentration dependent reverse chemotaxis.

Interplay of chemical and thermal gradient on bacterial migration in a diffusive microfluidic device

Abstract: Living systems are constantly under different combinations of competing gradients of chemical, thermal, pH, and mechanical stresses allied. The present work is about competing chemical and thermal gradients imposed on E. coli in a diffusive stagnant microfluidic environment. The bacterial cells were exposed to opposing and aligned gradients of an attractant (1 mM sorbitol) or a repellant (1 mM NiSO4) and temperature. The effects of the repellant/attractant and temperature on migration behavior, migration rate, and initiation time for migration have been reported. It has been observed that under competing gradients of an attractant and temperature, the nutrient gradient (gradient generated by cells itself) initiates directed migration, which, in turn, is influenced by temperature through the metabolic rate. Exposure to competing gradients of an inhibitor and temperature leads to the imposed chemical gradient governing the directed cell migration. The cells under opposing gradients of the repellant and temperature have experienced the longest decision time (∼60 min). The conclusion is that in a competing chemical and thermal gradient environment in the range of experimental conditions used in the present work, the migration of E. coli is always initiated and governed by chemical gradients (either generated by the cells in situ or imposed upon externally), but the migration rate and percentage of migration of cells are influenced by temperature, shedding insights into the importance of such gradients in deciding collective dynamics of such cells in physiological conditions.

Effect of gold nanoparticles on thermal gradient generation and thermotaxis of E. coli cells in microfluidic device.

Abstract: Bacteria responds to changing chemical and thermal environment by moving towards or away from a particular location. In this report, we looked into thermal gradient generation and response of E. coli DH5α cells to thermal gradient in the presence and in the absence of spherical gold nanoparticles (size: 15 to 22 nm) in a static microfluidic environment using a polydimethylsiloxane (PDMS) made microfluidic device. A PDMS-agarose based microfluidic device for generating thermal gradient has been developed and the thermal gradient generation in the device has been validated with the numerical simulation. Our studies revealed that the presence of gold nanoparticles, AuNPs (0.649 μg/mL) has no effect on the thermal gradient generation. The E. coli DH5α cells have been treated with AuNPs of two different concentrations (0.649 μg/mL and 0.008 μg/mL). The thermotaxis behavior of cells in the presence of AuNPs has been studied and compared to the thermotaxis of E.coli DH5α cells in the absence of AuNPs. In case of thermotaxis, in the absence of the AuNPs, the E. coli DH5α cells showed better thermotaxis towards lower temperature range, whereas in the presence of AuNPs (0.649 μg/mL and 0.008 μg/mL) thermotaxis of the E. coli DH5α cells has been inhibited. The results show that the spherical AuNPs intervenes in the themotaxis of E. coli DH5α cells and inhibits the cell migration. The reason for the failure in thermotaxis response mechanism may be due to decreased F-type ATP synthase activity and collapse of membrane potential by AuNPs, which, in turn, leads to decreased ATP levels. This has been hypothesized since both thermotaxis and chemotaxis follows the same response mechanism for migration in which ATP plays critical role.

E.coli DH5α cell response to a sudden change in microfluidic chemical environment

Abstract: Motile bacteria respond to changing chemical environment by moving towards or away from a particular location. Bacterial migration under chemical gradient is one of the most studied areas in biomedical field. In this work we looked into how bacterial cells respond to sudden change in the microfluidic chemical environment. E.coli DH5α cells were subjected to an attractant gradient (0.1 mM sorbitol - attractant to E.coli cells) and after 120 min the same cells were exposed to an inhibitor (0.1 mM NiSO 4 ) gradient in the same microfluidic device. Our studies revealed that when the E.coli DH5α cells were exposed to 0.1 mM sorbitol, they showed faster chemotaxis towards the attractant (0.1 mM sorbitol) and achieved steady state by 60 min. When we replaced 0.1 mM sorbitol with 0.1 mM NiSO 4 in the device we found that that the E.coli DH5α cells started responding to change in chemical environment within 10 min and achieved steady state at the end of 60 min. This shows that the bacterial cells respond to change in local chemical environment is within few minutes.

A biosensor for monitoring of salt stress in plants

Abstract: A biosensor based on organic electrochemical transistor has been demonstrated for in-vivo and in-vitro monitoring of salt stress in plants. Plant sap flowing through xylem and phloem in a living plant was utilised as electrolyte for the transistor based device in the case of in-vivo monitoring while plant sap extracted in a vial was utilised as electrolyte in the case of in-vitro monitoring. Electrochemical Impedance Spectroscopy (EIS) studies of the biosensor were done in order to understand the variation in channel current as control voltage was varied. Current modulation was observed at the output of the biosensor with changes in ionic concentration in plant sap when a plant was subjected to salt stress, demonstrating the ability of the device to detect changes in ionic concentration in plant sap, establishing this technology as a productive crop phenotyping tool.

High-Throughput Methods in the Discovery and Study of Biomaterials and Materiobiology

Abstract: The complex interaction of cells with biomaterials (i.e., materiobiology) plays an increasingly pivotal role in the development of novel implants, biomedical devices, and tissue engineering scaffolds to treat diseases, aid in the restoration of bodily functions, construct healthy tissues, or regenerate diseased ones. However, the conventional approaches are incapable of screening the huge amount of potential material parameter combinations to identify the optimal cell responses and involve a combination of serendipity and many series of trial-and-error experiments. For advanced tissue engineering and regenerative medicine, highly efficient and complex bioanalysis platforms are expected to explore the complex interaction of cells with biomaterials using combinatorial approaches that offer desired complex microenvironments during healing, development, and homeostasis. In this review, we first introduce materiobiology and its high-throughput screening (HTS). Then we present an in-depth of the recent progress of 2D/3D HTS platforms (i.e., gradient and microarray) in the principle, preparation, screening for materiobiology, and combination with other advanced technologies. The Compendium for Biomaterial Transcriptomics and high content imaging, computational simulations, and their translation toward commercial and clinical uses are highlighted. In the final section, current challenges and future perspectives are discussed. High-throughput experimentation within the field of materiobiology enables the elucidation of the relationships between biomaterial properties and biological behavior and thereby serves as a potential tool for accelerating the development of high-performance biomaterials.

Recent advances in microfluidic devices for bacteria and fungus research

Abstract: Microorganisms are not only common pathogens in clinical practice, but also an important participant in the maintenance of ecological balance, which plays a vital role in food production, microbial industry, and biomedical research etc. The traditional methods for bacterial and fungi research are time-consuming, high-cost, accurate operation required and unable to realize single-cell analysis. Microfluidic technologies have been applied to microorganism studies recently. Microfluidic devices with micro-sized scale and large-scale integration offer many special benefits including low cost, high throughput, and high efficiency in microorganism analysis. In this paper, we review the development and applications of microfluidic devices with respect to bacteria and fungus, and emphasize the advantages over traditional methods. Most crucially, we discuss how microfluidic chip technology contributes to the study of bacteria, fungus and their interactions. Finally, we provide insights into the challenges of bacterial and fungi studies based on microfluidic chip and present future perspectives.

Horizontal 'gene drives' harness indigenous bacteria for bioremediation.

Abstract: Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in Escherichia coli led to degradation of 60–99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E. coli through several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic exchange through nanotubes. Inoculating petroleum-polluted sediments with E. coli carrying the vector pSF-OXB15-p450camfusion showed that the E. coli cells died after five days but a variety of bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that vectors only persist in indigenous populations when under selection pressure, disappearing when this carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.

Steering air bubbles with an add-on vacuum layer for biopolymer membrane biofabrication in PDMS microfluidics.

Abstract: Membrane functionality is crucial in microfluidics for realizing operations such as filtration, separation, concentration, signaling among cells and gradient generation. Currently, common methods often sandwich commercially available membranes in multi-layer devices, or use photopolymerization or temperature-induced gelation to fabricate membrane structures in one-layer devices. Biofabrication offers an alternative to forming membrane structures with biomimetic materials and mechanisms in mild conditions. We have recently developed a biofabrication strategy to form parallel biopolymer membranes in gas-permeable polydimethylsiloxane (PDMS) microfluidic devices, which used positive pressure to dissipate air bubbles through PDMS to initiate membrane formation but required careful pressure balancing between two flows. Here, we report a technical innovation by simply placing as needed an add-on PDMS vacuum layer on PDMS microfluidic devices to dissipate air bubbles and guide the biofabrication of biopolymer membranes. Vacuuming through PDMS was simply achieved by either withdrawing a syringe or releasing a squeezed nasal aspirator. Upon vacuuming, air bubbles dissipated within minutes, membranes were effortlessly formed, and the add-on vacuum layer can be removed. Subsequent membrane growth could be robustly controlled with the flows and pH of solutions. This new process is user-friendly and has achieved a 100% success rate in more than 200 trials in membrane biofabrication.

Microfluidic devices for studying bacterial taxis, drug testing and biofilm formation.

Abstract: Some bacteria have coevolved to establish symbiotic or pathogenic relationships with plants, animals or humans. With human association, the bacteria can cause a variety of diseases. Thus, understanding bacterial phenotypes at the single-cell level is essential to develop beneficial applications. Traditional microbiological techniques have provided great knowledge about these organisms; however, they have also shown limitations, such as difficulties in culturing some bacteria, the heterogeneity of bacterial populations or difficulties in recreating some physical or biological conditions. Microfluidics is an emerging technique that complements current biological assays. Since microfluidics works with micrometric volumes, it allows fine-tuning control of the test conditions. Moreover, it allows the recruitment of three-dimensional (3D) conditions, in which several processes can be integrated and gradients can be generated, thus imitating physiological 3D environments. Here, we review some key microfluidic-based studies describing the effects of different microenvironmental conditions on bacterial response, biofilm formation and antimicrobial resistance. For this aim, we present different studies classified into six groups according to the design of the microfluidic device: (i) linear channels, (ii) mixing channels, (iii) multiple floors, (iv) porous devices, (v) topographic devices and (vi) droplet microfluidics. Hence, we highlight the potential and possibilities of using microfluidic-based technology to study bacterial phenotypes in comparison with traditional methodologies.