Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria

Probst C, Grünberger A, Wiechert W, Kohlheyer D. Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria. Micromachines. 2013;4(4):357-369.Microfluidics has become an essential tool in single-cell analysis assays for gaining more accurate insights into cell behavior. Various microfluidics methods have been introduced facilitating single-cell analysis of a broad range of cell types. However, the study of prokaryotic cells such as Escherichia coli and others still faces the challenge of achieving proper single-cell immobilization simply due to their small size and often fast growth rates. Recently, new approaches were presented to investigate bacteria growing in monolayers and single-cell tracks under environmental control. This allows for high-resolution time-lapse observation of cell proliferation, cell morphology and fluorescence-coupled bioreporters. Inside microcolonies, interactions between nearby cells are likely and may cause interference during perturbation studies. In this paper, we present a microfluidic device containing hundred sub-micron sized trapping barrier structures for single E. coli cells. Descendant cells are rapidly washed away as well as components secreted by growing cells. Experiments show excellent growth rates, indicating high cell viability. Analyses of elongation and growth rates as well as morphology were successfully performed. This device will find application in prokaryotic single-cell studies under constant environment where by-product interference is undesired

OPTIMAL CONTROL OF OBJECTS ON THE MICRO- AND NANO-SCALE BY ELECTROKINETIC AND ELECTROMAGNETIC MANIPULATION: FOR BIO-SAMPLE PREPARATION, QUANTUM INFORMATION DEVICES AND MAGNETIC DRUG DELIVERY

In this thesis I show achievements for precision feedback control of objects inside micro-fluidic systems and for magnetically guided ferrofluids. Essentially, this is about doing flow control, but flow control on the microscale, and further even to nanoscale accuracy, to precisely and robustly manipulate micro and nano-objects (i.e. cells and quantum dots). Target applications include methods to miniaturize the operations of a biological laboratory (lab-on-a-chip), i.e. presenting pathogens to on-chip sensing cells or extracting cells from messy bio-samples such as saliva, urine, or blood; as well as non-biological applications such as deterministically placing quantum dots on photonic crystals to make multi-dot quantum information systems. The particles are steered by creating an electrokinetic fluid flow that carries all the particles from where they are to where they should be at each time step. The control loop comprises sensing, computation, and actuation to steer particles along trajectories. Particle locations are identified in real-time by an optical system and transferred to a control algorithm that then determines the electrode voltages necessary to create a flow field to carry all the particles to their next desired locations. The process repeats at the next time instant. I address following aspects of this technology. First I explain control and vision algorithms for steering single and multiple particles, and show extensions of these algorithms for steering in three dimensional (3D) spaces. Then I show algorithms for calculating power minimum paths for steering multiple particles in actuation constrained environments. With this microfluidic system I steer biological cells and nano particles (quantum dots) to nano meter precision. In the last part of the thesis I develop and experimentally demonstrate two dimensional (2D) manipulation of a single droplet of ferrofluid by feedback control of 4 external electromagnets, with a view towards enabling feedback control of magnetic drug delivery to reach deeper tumors in the long term. To this end, I developed and experimentally demonstrated an optimal control algorithm to effectively manipulate a single ferrofluid droplet by magnetic feedback control. This algorithm was explicitly designed to address the nonlinear and cross-coupled nature of dynamic magnetic actuation and to best exploit available electromagnetic forces for the applications of magnetic drug delivery

Self-Cleaning Interfaces of Polydimethylsiloxane Grafted with pH-Responsive Zwitterionic Copolymers

International audienceSelf-cleaning surfaces allow the reversible attachment and detachment of microorganisms which show great promise in regards to their reusability as smart biomaterials. However, a widely used biomaterial such as polydimethylsiloxane (PDMS) suffers from high biofouling activity and hydrophobic recovery that results in decreased efficiency and stability. A current challenge is to modify and fabricate self-cleaning PDMS surfaces by incorporating antifouling and pH-sensitive properties. To address this, we synthesized a zwitterionic and pH-sensitive random poly(glycidyl methacrylate-co-sulfobetaine methacrylate-co-2-(dimethylamino)ethyl methacrylate) polymer, poly(GMA-co-SBMA-co-DMAEMA). In this work, chemical modification of PDMS was done by grafting onto poly(GMA-co-SBMA-co-DMAEMA) after surface activation via UV and ozone for 90 min to ensure the formation of covalent bonds necessary for stable grafting. The PDMS grafted with G20-S40-D40 exhibit antifouling and pH-sensitive properties by mitigating fibrinogen adsorption, blood cell adhesion, and releasing 98% adhered E. coli bacteria after immersion at basic pH. The grafting of poly(GMA-co-SBMA-co-DMAEMA) presented in this work shows attractive potential for biomedical and industrial applications as a simple, smart, and effective method for the modification of PDMS interface

An Insulating Constriction Dielectrophoresis Based Microfluidic Device for Protein Biomolecules Concentration

Sample enrichment or molecules concentration has been considered as an essential step for sample processing and biomarker detection recently in many applications involving miniaturized devices aiming at biosensing and bioanalysis, including the development of specialized tests for the detection of specific proteins and antibodies in human blood with the help of microfluidic and lab-on-a-chip devices. Among all the means involved to achieve this aim, dielectrophoresis (DEP) is increasingly employed in molecule manipulation and concentration because it is non-destructive and ensures high efficiency. However, there are still constraints on implementing the required functions using the dielectrophoresis technique in the devised micro-scale structures with high throughput, as well as the technical challenge in integration of sensors and concentration units for low-abundance molecular detection.;In the present work, we demonstrated a methodology to achieve protein concentration utilizing the combination effects of electrokinetics and low frequency insulator-based dielectrophoresis (iDEP) generated within a microfluidic device, in which a submicron constricted channel was fabricated using DNA molecular combing and replica molding. This fabrication technique, avoids using e-beam lithography or other complicated nanochannel fabrication methods, provides an easy and low cost approach with the flexibility in controlling channel dimensions to create highly constricted channels embedded in a microfluidic device. With theoretical analysis and experiments, we demonstrated that albumin--fluorescein isothiocyanate conjugate (FITC-BSA) protein molecules can be significantly concentrated to form an arc-shaped band near the constricted channel under the effects of negative dielectrophoretic force and DC electrokinetic forces within 2-3 minutes. It was also observed that the amplitudes of the applied DC and AC electric fields, AC frequencies, as well as suspending medium conductivities had strong effects on the concentration responses of the FITC-BSA molecules, including the concentrated area and position, intensities of the focused molecules, and concentration speed.;Our method demonstrated in the thesis provides a simple and flexible approach for quickly concentrating protein molecules by controlling the applied electric field parameters. The iDEP device reported in this thesis can be used as a stand-alone sensor or worked as a pre-concentration module integrated with biosensors for protein biomarker detections. Furthermore, low frequency dielectrophoresis provides practical uses for integrating the concentration module with a portable biosensing system

Micro- and nanotechnology for cell biophysics

Procedures and methodologies used in cell biophysics have been improved tremendously with the revolutionary advances witnessed in the micro- and nanotechnology in the last two decades. With the advent of microfluidics it became possible to reduce laboratory-sized equipment to the scale of a microscope slide allowing massive parallelization of measurements with extremely low sample volume at the cellular level. Optical micromanipulation has been used to measure forces or distances or to alter the behavior of biological systems from the level of DNA to organelles or entire organisms. Among the main advantages is its non-invasiveness, giving researchers an invisible micro-hand to “touch” or “feel” the system under study, its freely and very often quickly adjustable experimental parameters such as wavelength, optical power or intensity distribution. Atomic force microscopy (AFM) opened avenues for in vitro biological applications concerning with single molecule imaging, cellular mechanics or morphology. As it can operate in liquid environment and at human body temperature, it became the most reliable and accurate nanoforce-tool in the research of cell biophysics. In this paper we review how the above three techniques help increase our knowledge in biophysics at the cellular level

Acoustic Microfluidic Separation Techniques and Bioapplications: A Review

Microfluidic separation technology has garnered significant attention over the past decade where particles are being separated at a micro/nanoscale in a rapid, low-cost, and simple manner. Amongst a myriad of separation technologies that have emerged thus far, acoustic microfluidic separation techniques are extremely apt to applications involving biological samples attributed to various advantages, including high controllability, biocompatibility, and non-invasive, label-free features. With that being said, downsides such as low throughput and dependence on external equipment still impede successful commercialization from laboratory-based prototypes. Here, we present a comprehensive review of recent advances in acoustic microfluidic separation techniques, along with exemplary applications. Specifically, an inclusive overview of fundamental theory and background is presented, then two sets of mechanisms underlying acoustic separation, bulk acoustic wave and surface acoustic wave, are introduced and discussed. Upon these summaries, we present a variety of applications based on acoustic separation. The primary focus is given to those associated with biological samples such as blood cells, cancer cells, proteins, bacteria, viruses, and DNA/RNA. Finally, we highlight the benefits and challenges behind burgeoning developments in the field and discuss the future perspectives and an outlook towards robust, integrated, and commercialized devices based on acoustic microfluidic separation

Microsc Microanal

Abstract A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to documen

Particle Capture Devices and Methods of Use Thereof

The present invention provides a device and methods of use thereof in microscale particle capturing and particle pairing. This invention provides particle patterning device, which mechanically traps individual particles within first chambers of capture units, transfer the particles to second chambers of opposing capture units, and traps a second type of particle in the same second chamber. The device and methods allow for high yield assaying of trapped cells, high yield fusion of trapped, paired cells, for controlled binding of particles to cells and for specific chemical reactions between particle interfaces and particle contents. The device and method provide means of identification of the particle population and a facile route to particle collection