Murat Gel

Force sensing submicrometer thick cantilevers with ultra-thin piezoresistors by rapid thermal diffusion

One of the most important requirements for a cantilever-type sensor to obtain high force sensitivity is small thickness. By using current micromachining technology it is possible to produce cantilevers of submicrometer thickness. Where self-sensing piezoresistive cantilevers with submicrometer thickness are concerned, it is necessary to use a technology which can create ultra-thin (<100 nm) piezoresistors on a cantilever surface. This work demonstrates for the first time the application of a relatively simple, rapid thermal diffusion method by using spin-on glass film to fabricate sub-100 nm piezoresistors on an ultra-thin single-crystal silicon cantilever. Compared to other shallow junction fabrication methods, which involve implantation or deposition of a doped layer, this method is advantageous since no damage is created in the crystal structure and no toxic gas or hazardous material is used during the process. Besides, this technique can be applied by using low-cost rapid annealers, which can be readily found in most laboratories. By using this method, piezoresistive cantilevers with stiffness in the range of 0.001 N m−1 with sub-100 nm thick piezoresistors are fabricated, and a complete characterization of the fabricated cantilevers is performed.

Dielectrophoretic cell trapping and parallel one-to-one fusion based on field constriction created by a micro-orifice array

Micro-orifice based cell fusion assures high-yield fusion without compromising the cell viability. This paper examines feasibility of a dielectrophoresis (DEP) assisted cell trapping method for parallel fusion with a micro-orifice array. The goal is to create viable fusants for studying postfusion cell behavior. We fabricated a microfluidic chip that contained a chamber and partition. The partition divided the chamber into two compartments and it had a number of embedded micro-orifices. The voltage applied to the electrodes located at each compartment generated an electric field distribution concentrating in micro-orifices. Cells introduced into each compartment moved toward the micro-orifice array by manipulation of hydrostatic pressure. DEP assisted trapping was used to keep the cells in micro-orifice and to establish cell to cell contact through orifice. By applying a pulse, cell fusion was initiated to form a neck between cells. The neck passing through the orifice resulted in immobilization of the fused cell pair at micro-orifice. After washing away the unfused cells, the chip was loaded to a microscope with stage top incubator for time lapse imaging of the selected fusants. The viable fusants were successfully generated by fusion of mouse fibroblast cells (L929). Time lapse observation of the fusants showed that fused cell pairs escaping from micro-orifice became one tetraploid cell. The generated tetraploid cells divided into three daughter cells. The fusants generated with a smaller micro-orifice (diameter approximately 2 mum) were kept immobilized at micro-orifice until cell division phase. After observation of two synchronized cell divisions, the fusant divided into four daughter cells. We conclude that the presented method of cell pairing and fusion is suitable for high-yield generation of viable fusants and furthermore, subsequent study of postfusion phenomena.

Microorifice-Based High-Yield Cell Fusion on Microfluidic Chip: Electrofusion of Selected Pairs and Fusant Viability

Microorifice-based fusion makes use of electric field constriction to assure high-yield one-to-one fusion of selected cell pairs. The aim of this paper is to verify feasibility of high-yield cell fusion on a microfluidic chip. This paper also examines viability of the fusant created on the chip. We fabricated a microfluidic chip to fuse selected cell pairs and to study postfusion behavior. We used a self-forming meniscus-based fabrication process to create microorifice with a diameter of 2-10 ¿m on the vertical walls in a microfluidic channel. When 1 MHz was applied to electrodes located on both sides of the microorifice, dielectrophoretic force attracted the cells toward microorifice to form a cell pair. Once the cells get into contact, fusion pulse was applied. Real time imaging of cells during fusion and cytoplasmic dye transfer between cells indicated success of cell fusion. We found that when high frequency voltage for dielectrophoresis was swept from 1 MHz to 10 kHz in 100 ¿s, cell fusion was initiated. The effective electric field strength was 0.1-0.2 kV/cm. We analyzed viability by imaging fusant going into cell division phase after 48 h of incubation. We conclude that fabricated microfluidic chip is suitable for high-yield one-to-one fusion and creation of viable fusants. This technology should be a useful tool to study fusion phenomena and viability of fusants, as it allows imaging of the cells during and after the fusion.

Dielectrophoresis‐assisted massively parallel cell pairing and fusion based on field constriction created by a micro‐orifice array sheet

In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two‐dimensional micro‐orifice array. The sheet is sandwiched by a pair of micro‐chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size‐independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two‐dimensional orifice arrangement at the pitch of 50 μm realizes simultaneous fusion of 6×103 cells on a 4 mm diameter chip, and the fusion yield of 78–90% is achieved for various sizes and types of cells.

Real-time, continuous detection of maltose using bioluminescence resonance energy transfer (BRET) on a microfluidic system

We have previously shown that a genetically encoded bioluminescent resonance energy transfer (BRET) biosensor, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a variant of Renilla luciferase (RLuc2) at the C-terminus, has superior sensitivity and limits of detection for maltose, compared with an equivalent fluorescent resonance energy transfer (FRET) biosensor. Here, we demonstrate that the same MBP biosensor can be combined with a microfluidic system for detection of maltose in water or beer. Using the BRET-based biosensor, maltose in water was detected on a microfluidic chip, either following a pre-incubation step or in real-time with similar sensitivity and dynamic range to those obtained using a commercial 96-well plate luminometer. The half-maximal effective concentrations (EC50) were 2.4×10(-7)M and 1.3×10(-7) M for maltose detected in pre-incubated and real-time reactions, respectively. To demonstrate real-time detection of maltose in a complex medium, we used it to estimate maltose concentration in a commercial beer sample in a real-time, continuous flow format. Our system demonstrates a promising approach to in-line monitoring for applications such as food and beverage processing.

Sub-micron thick high sensitive piezoresistive cantilevers by boron etch stop and argon implantation

A new method far improving piezoresistive response of thin (<1 /spl mu/m) cantilevers with Argon implantation is described. Argon implantation is used to damage atomic structure of one side of the cantilever. Test cantilevers have thickness of less than 500 nm while length and width is 60 /spl mu/m and 30 /spl mu/m respectively. For a total implanted dose of 35 /spl times/ 10/sup 15/ ions/cm/sup 2/ maximum displacement sensitivity obtained is 8.24 /spl times/ 10/sup -7/ [1//spl Aring/] which is 82% of the theoretical maximum. Force sensitivity is found to be 3.5 /spl times/ 10/sup -3/ [1//spl mu/N].

Mechanically Controlled Quantum Contact With On-Chip MEMS Actuator

We report fabrication and characterization of a mechanically controlled quantum point contact with on-chip electrostatic actuators. Two metallic tips separated with a nano gap are integrated with a micro mechanism, which can be actuated by applied voltage. The electrostatic actuation of the micro mechanism allows the control of separation gap at nanometer level. The gap variation at nanometer level is calibrated with the devices operated in transmission electron microscope. Furthermore, performance of the device is demonstrated by measuring conductance quantization while separating the tips in contact as well as electrical measurements by using tips coated with self assembled monolayers (1-4 benzene dithiol) and conjugated Ru complex

Progress in bio-manufacture of platelets for transfusion

Abstract Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

Fabrication of micro pore arrays in free standing COC membranes and their application for in vitro barrier tissue models

This paper reports a low-cost fabrication method for mass production of free standing cyclic olefin copolymer (COC) membranes embedded with micro pore arrays. We show that the method creates suspended membranes with thickness of 2μm and micro pores having diameter ranging from 3-5μm. We integrated the membrane in a microfluidic system designed to mimic the endothelial barrier in bone marrow sinusoids. The device was tested with HUVEC and supported their attachment and proliferation on the membrane.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.