A practical review on the measurement tools for cellular adhesion force

Cell cell and cell matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion

Development of planar patch clamp with potentiometric calcium ion-selective electrode

Ion channels are proteins in cell lipid bilayer membranes and act as pores which can adopt closed and open states, thus gating the flow of ions in and out of the cell. Patch clamp technology has been the proven standard for fundamental studies of ion channel activities. However, the technique has some basic limitations: low throughput, time consuming nature of its process, need of highly skilled personnel and inability to identify ionic composition of electrophysiological events. Many different materials and fabrication methods have been introduced to replace traditional patch clamp setup to overcome limitations.^ In this dissertation, a planar patch clamp device with calcium ion-selective electrode is developed in miniaturized form for high throughput cell electrophysiology, and screening of ion channel modulators as potential drug targets in an in vitro format. Femtosecond laser-drilling technique is newly introduced to fabricate the patch-pore and new design of planar ion-selective electrode is suggested for calcium ion measurement. By integrating a standard patch clamp electrophysiological interface with calcium ion-selective electrode on a single platform, it is possible to directly identify the ionic component of a whole-cell potential recording. This system is innovative because the focus is not entirely on increasing experimental throughput, but instead offers information on user specified target ion activities through ion channels

Developing an Optomechanical Approach for Characterizing Mechanical Properties of Single Adherent Cells

Mechanical properties of a cell reflect its biological and pathological conditions including cellular disorders and fundamental cellular processes such as cell division and differentiation. There have been active research efforts to develop high-throughput platforms to mechanically characterize single cells. Yet, many of these research efforts are focused on suspended cells and use a flow-through configuration. Therefore, adherent cells are detached prior to the characterization, which seriously perturbs the cellular conditions. Also, methods for adherent cells are limited in their throughput. My study is aimed to fill the technical gap in the field of single cell analysis, which is a high-throughput and non-invasive mechanical characterization of single adherent cells. I developed a multi-modal platform to mechanically characterize single adherent cells. The platform is based on optomechanical principle, which induces least perturbation on the cells and does not require cell detachment. Besides, multiple measurements can be performed on a single cell to track its mechanical behavior over time. Proposed platform can expand our understanding on the relationship between mechanical properties and cellular status of adherent cells. Single adherent cells are characterized optomechanically using the vibration-induced phase shift (VIPS). VIPS is a phase shift of apparent velocity of a vertically vibrating substrate measured with laser Doppler vibrometer (LDV), when the measurement laser passes through an adherent cell or any transparent objects on the substrate. The VIPS and height oscillation of a single cell on a vibrating substrate have negative correlation with the cell stiffness. An analytical model is established which demonstrates relationship between cell’s mechanical properties and its VIPS. With the VIPS measurements, at multiple frequencies on large population of cells, the statistical significant difference in the cell stiffness is confirmed after exposure to various drugs affecting cytoskeleton network. Also, a 3-dimensional finite element model is developed to extract the cell stiffness from VIPS. VIPS technique is used to reconstruct the detailed oscillation pattern of transparent objects such as water microdroplets and intracellular lipid droplets on a vibrating substrate, which can give us better understanding of mechanical behavior of biological transparent objects. In addition, using VIPS measurement mechanical interaction between extracellular matrixes (ECMs) and adherent cells is studied. Statistical significant difference in bonding straight of single cells and different ECMs is demonstrated

Techniques to stimulate and interrogate cell–cell adhesion mechanics

Cell–cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell–extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell–cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell–cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell–cell adhesion from cell pairs to monolayers

ENHANCED NANOPORE DETECTION VIA DIFFUSION GRADIENTS AND OPTICAL TWEEZERS

Nanopore-based resistive pulse sensing represents an important class of single-molecule measurements. It provides information about many molecules of interest (i.e. DNA, proteins, peptides, clusters, polymers, etc.) without the need for labeling. Two experiments that are especially well suited for studying with nanopore sensors are DNA sequencing and DNA-protein force measurements. This thesis will describe progress that has been made in both areas. DNA sequencing has become an active area of research for stochastic single-molecule sensing, with many researchers striving for the ultimate goal of single-molecule de novo DNA sequencing. One intriguing method towards that goal involves the use of a DNA exonuclease or polymerase enzyme, which when attached close to the mouth of a pore, leads to cleavage of individual DNA nucleotide bases for loading into the pore for sensing. Though this method seems promising, the end goal has been elusive because the nucleotide motion is dominated by diffusion over the relevant length scales. This limits the likelihood of the cleaved nucleotide entering the pore to be characterized. The first part of this thesis will describe a method for addressing this problem, where it is shown that increasing the nucleotide capture probability can be achieved by lowering the bulk diffusion coefficient relative to the pore diffusion coefficient. The second part of this thesis will describe the design and implementation of a new type of sensor that combines a biological nanopore experimental apparatus with optical tweezers. The goal of this apparatus is to develop a means to independently measure the force on a charged molecule inside of the pore. The setup will be thoroughly described, and preliminary results showing that it is possible to optically trap a micron sized bead within a few microns of an isolated biological nanopore while simultaneously making current measurements through that pore will be presented. This will enable force measurements on DNA molecules tethered to the bead, which opens the door for the study of molecular force interactions between DNA and biological nanopores, DNA-bound protein interactions that cause diseased states, and controlled translocation of DNA through biological nanopores

Bacterial Chemotaxis in an Optical Trap

An optical trapping technique is implemented to investigate the chemotactic behavior of a marine bacterial strain Vibrio alginolyticus. The technique takes the advantage that the bacterium has only a single polar flagellum, which can rotate either in the counter-clock-wise or clock-wise direction. The two rotation states of the motor can be readily and instantaneously resolved in the optical trap, allowing the flagellar motor switching rate to be measured under different chemical stimulations. In this paper the focus will be on the bacterial response to an impulsive change of chemoattractant serine. Despite different propulsion apparati and motility patterns, cells of V. alginolyticus apparently use a similar response as Escherichia coli to regulate their chemotactic behavior. Specifically, we found that the switching rate of the bacterial motor exhibits a biphasic behavior, showing a fast initial response followed by a slow relaxation to the steady-state switching rate . The measured can be mimicked by a model that has been recently proposed for chemotaxis in E. coli. The similarity in the response to the brief chemical stimulation in these two different bacteria is striking, suggesting that the biphasic response may be evolutionarily conserved. This study also demonstrated that optical tweezers can be a useful tool for chemotaxis studies and should be applicable to other polarly flagellated bacteria

Monitoring single heart cell biology using lab-on-a- chip technologies

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states