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1.
Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis
The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812±10 μS cm−1), almost double that of active cells (560±20 μS cm−1), while that of dead (autoclaved) M. smegmatis cells was the highest (950±15 μS cm−1) overall. It was also found that at 80 kHz, 900 μS cm−1 dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 μS cm−1 active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted. 相似文献
2.
Olivier Ducloux Elisabeth Galopin Farzam Zoueshtiagh Alain Merlen Vincent Thomy 《Biomicrofluidics》2010,4(1)
A droplet-based micro-total-analysis system involving biosensor performance enhancement by integrated surface-acoustic-wave (SAW) microstreaming is shown. The bioreactor consists of an encapsulated droplet with a biosensor on its periphery, with in situ streaming induced by SAW. This paper highlights the characterization by particle image tracking of the speed distribution inside the droplet. The analyte-biosensor interaction is then evaluated by finite element simulation with different streaming conditions. Calculation of the biosensing enhancement shows an optimum in the biosensor response. These results confirm that the evaluation of the Damköhler and Peclet numbers is of primary importance when designing biosensors enhanced by streaming.It has been pointed out that biosensing performances can be limited by the diffusion of the analytes near the sensing surface.1 In the case of low Peclet number hydrodynamic flows, typical of microfluidic systems, molecule displacements are mainly governed by diffusive effects that affect time scales and sensitivity. To overcome this problem, the enhancement of biosensor performance by electrothermal stirring within microchannels was first reported by Meinhart et al.2 Other authors3, 4 numerically studied the analyte transport as a function of the position of a nanowire-based sensor inside a microchannel, stressing on the fact that the challenge for nanobiosensors is not the sensor itself but the fluidic system that delivers the sample. Addressing this problem, Squires et al.5 developed a simple model applicable to biosensors embedded in microchannels. However, the presented model is limited to the case of a steady flow. The use of surface-acoustic waves (SAWs) for stirring in biomicrofluidic and chemical systems is becoming a popular investigation field,6, 7, 8, 9 especially to overcome problems linked to steady flows by enhancing the liquid∕surface interaction.1, 10, 11 The main challenges that need to be addressed when using SAW-induced stirring are the complexity of the flow and its poor reproducibility. However, some technical solutions were proposed to yield a simplified microstreaming. Yeo et al. presented a centrifugation system based on SAW that produces the rotation of the liquid in a droplet in a reproducible way by playing on the configuration of the transducers and reflectors,12 and presented a comprehensive experimental study of the three-dimensional (3D) flow that causes particle concentration in SAW-stirred droplets,13 revealing the presence of an azimuthal secondary flow in addition to the main vortexlike circular flow present in acoustically stirred droplets. The efficiency of SAW stirring in microdroplets to favorably cope with mass transport issues was finally shown by Galopin et al.,14 but the effect of the stirring on the analyte∕biosensor interaction was not studied. It is expected to overcome mass transport limitations by bringing fresh analytes from the bulk solution to the sensing surface.The studied system, described in Fig. Fig.1,1, consists of a microliter droplet microchamber squeezed between a hydrophobic piezoelectric substrate and a hydrophobic glass cover. Rayleigh SAWs are generated using interdigitated transducers (interdigital spacing of 50 μm) laid on an X-cut LiNbO3 substrate.1, 15, 16 The hydrophobicity of the substrate and the cover are obtained by grafting octadecyltrichlorosilane (OTS) self-assembled monolayers (contact angle of 108° and hysteresis of 9°). To do so, the surface is first hydroxylized using oxygen plasma (150 W, 100 mT, and 30 sccm3 O2) during 1 min and then immersed for 3 h into a 1 mM OTS solution with n-hexane as a solvent.Open in a separate windowFigure 1(a) General view of the considered system. (b) Mean value of the measured speeds within the droplet as a function of the inlet power before amplification.When Rayleigh waves are radiated toward one-half of the microchamber, a vortex is created in the liquid around an axis orthogonal to the substrate due to the momentum transfer between the solid and the liquid. This wave is generated under the Rayleigh angle into the liquid.Speed cartographies of the flow induced in the droplet are realized using the particle image tracking technique for different SAW generation powers. To do so, instantaneous images of the flow are taken with a high-speed video camera at 200 frames∕s and an aperture time of 500 μs on a 0.25 μl droplet containing 1 μm diameter fluorescent particles. Figure Figure11 shows the mean speed measured in the droplet as a function of the inlet power. The great dependence of the induced mean speed with the SAW power enables a large range of flow speeds in the stirred droplet. Moreover, the flow was visualized with a low depth of field objective. It was found to be circular and two dimensional (2D) in a large thickness range of the droplet.The binding of analytes to immobilized ligands on a biosensor is a two step process, including the mass transport of the analyte to the surface, followed by a complexation step,(1)with km as the constant rate for mass transport from and to the sensor, and ka and kd as the constant rates of association and dissociation of the complex.At the biosensor surface, the reaction kinetics consumes analytes but their transport is limited by diffusive effects. In this case, the Damköhler number brings valuable information by comparing these two effects. Calling the characteristic time of reaction and diffusion, respectively, τC and τM, the mixing time in diffusion regime can be approximated by τM≈h2∕D with D as the diffusion coefficient and h a characteristic length of the microchannel. Calling RT the ligand concentration on the surface in mole∕m2, the Damköhler number (Da) can be written as(2)Depending on the type of reaction, the calculation of Da helps determine if a specific biointeraction will benefit from a mass SAW-based microstreaming. If the Damköhler number is low, the reaction is slow compared to mass transport and the reaction will not significantly benefit from microstirring. For example, the hybridization of 19 base single stranded DNA in a microfluidic system with a characteristic length of 500 μm is characterized by a Damköhler number of 0.07 and is therefore not significantly influenced by mass transport. On the contrary, the binding of biotin to immobilized streptavidin is characterized by a Da number of approximately 104. In this case, the stirring solution will significantly improve the reaction rate.COMSOL numerical simulations were carried out to study the efficiency of the SAW stirring in the case of a droplet-based microbioreactor with a diameter of 1 mm. Assuming a 2D flow, the simulated model takes into account the convective and diffusive effects in the analyte-carrying fluid and the binding kinetics on the biosensor surface. This approach was thoroughly developed by Meinhart et al.2On the biosensor surface, the following equations are solved:(3)(4)with c as the local concentration of analytes in the droplet and B as the surface concentration of bound analytes on the biosensor surface. Simulation results show that a depleted zone is formed near the biosensor in the case of an interaction without stirring. This zone is characterized by a low concentration of analytes and results from the trapping of analytes on the biosensor surface, thus creating a concentration gradient on the vicinity of the biosensor. When stirring is applied, the geometry of the depleted zone is modified, as it is pushed in the direction of the flow. The geometry of the depleted zone then depends on many parameters, among which the diffusion coefficient D, the speed distribution of the flow (not only near the biosensor but also in the whole microfluidic system), and the reaction kinetics on the biosensor. In our case, which is assimilated to a simple circular flow, the depleted zone reaches a permanent state consisting of an analyte-poor layer situated in the exterior perimeter of the stirred droplet. The diffusion of analytes is then limited again by diffusion from the inner part of the droplet toward its exterior perimeter (see Fig. Fig.22).Open in a separate windowFigure 2(a) Mean concentration of bound analytes vs time for different mean flow speeds. (b) The obtained concentration profiles with and without circular stirring, t=10 000 s.The initial analyte and receptor concentrations are, respectively, 0.1 nM in the solution and 3.3×10−3 nM m on the biosensor surface, the diffusion coefficient is D=10−11 m2 s−1, and the reaction constants are ka=106 M−1 s−1 and kd=10−3 s−1. Simulations show that the mean concentration of bound analytes highly increases with the flow speed, improving the efficiency of the biosensing device. To evaluate the benefits of in situ microstreaming with SAW, the same simulations were conducted for Da numbers ranging from 104 to 108 M−1∕s, by ranging the diffusion coefficient from 4×10−12 to 4×10−9 m2∕s, and the association coefficient ka from 104 to 108 M−1∕s. The enhancement factor of analyte capture, defined as the ratio of the binding rate with streaming B and the binding rate without streaming B0, is plotted in Fig. Fig.33 for different values of Da. Calculations are done in the case of a mean flow speed of 0.5 mm∕s.Open in a separate windowFigure 3(a) Enhancement factor (defined as the ratio between binding rate with streaming B and binding rate without streaming B0) for different Damkhöler numbers and (b) normalized enhancement factor for different Peclet numbers.One can notice the saturation of the enhancement factor curve for large value of Da to the value of 3.5 for high Da. This can be explained by the fact that for large ka∕Da ratios, the analytes, which normally require penetration in the depleted zone by diffusion, do not have time to interact with the biosensor when they pass in the vicinity of its surface. The efficiency of the streaming is then reduced for large values of Da. In the case of our specific flow configuration, the enhancement factor reaches 3.2 for the interaction of streptavidin on immobilized biotin (Da=103).The reported simulation results can be compared to an experimental value obtained using the droplet-based surface plasmon resonance sensor streamed in situ using SAW reported by Yeo et al.12 By monitoring the streptavidin∕biotin binding interaction on an activated gold slide, they showed that SAW stirring brings an improvement factor of more than 2. This difference can be accounted to the high complexity of the induced 3D flow, which was modeled in a simple manner in our calculations.Other factors must be taken into account when optimizing the improvement factor, such as the flow velocity and the characteristic length of the mixing. To do so, the Peclet number allows the comparison of the convective and diffusive effects.17 For δC a typical variation in concentration on the distance h, the Peclet number is given by(5)A significantly high Peclet number causes a decrease in biosensing efficiency as the analytes do not have enough time to interact with the biosensing surface by diffusion through the analyte-poor layer. On the contrary, the case of a low Peclet number corresponds to the diffusion-limited problem. Therefore, for each Damköhler number, there is a Peclet number optimizing this factor. To illustrate this fact, Fig. Fig.3b3b shows the calculation of the enhancement factor as a function of the Peclet number for a given Da.In this paper, we showed that surface loading of typical analytes on a droplet-based biosensor can be highly increased by SAW microstirring. The system permits the enhancement of the biosensing performances by the continuous renewal of the analyte-carrying fluid near the sensing surface. Thanks to mean flow speeds measured up to 1800 μm∕s, the SAW microstreaming can be beneficial to the biosensing of a large range of analyte∕ligand interactions. In addition to the biosensing performance improvement, such a method can be easily integrated in micro-micro-total-analysis systems, which makes it a convenient tool for liquid handling in future biochips. 相似文献
3.
Characterization and separation of Cryptosporidium and Giardia cells using on-chip dielectrophoresis
Narayanan Unni H Hartono D Yue Lanry Yung L Mah-Lee Ng M Pueh Lee H Cheong Khoo B Lim KM 《Biomicrofluidics》2012,6(1):12805-1280514
Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage). 相似文献
4.
Epshteyn AA Maher S Taylor AJ Holton AB Borenstein JT Cuiffi JD 《Biomicrofluidics》2011,5(4):46501-465016
The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 μm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane 相似文献
5.
Camilla Luni Hope C. Feldman Michela Pozzobon Paolo De Coppi Carl D. Meinhart Nicola Elvassore 《Biomicrofluidics》2010,4(3)
This work presents the development of an array of bioreactors where finely controlled stirring is provided at the microliter scale (100–300 μl). The microliter-bioreactor array is useful for performing protocol optimization in up to 96 parallel experiments of hematopoietic stem cell (HSC) cultures. Exploring a wide range of experimental conditions at the microliter scale minimizes cost and labor. Once the cell culture protocol is optimized, it can be applied to large-scale bioreactors for stem cell production at the clinical level. The controlled stirring inside the wells of a standard 96-well plate is provided by buoyancy-driven thermoconvection. The temperature and velocity fields within the culture volume are determined with numerical simulations. The numerical results are verified with experimental velocity measurements using microparticle image velocimetry (μPIV) and are used to define feasible experimental conditions for stem cell cultures. To test the bioreactor array’s functionality, human umbilical cord blood-derived CD34+ cells were cultured for 7 days at five different stirring conditions (0.24–0.58 μm∕s) in six repeated experiments. Cells were characterized in terms of proliferation, and flow cytometry measurements of viability and CD34 expression. The microliter-bioreactor array demonstrates its ability to support HSC cultures under stirred conditions without adversely affecting the cell behavior. Because of the highly controlled operative conditions, it can be used to explore culture conditions where the mass transport of endogenous and exogenous growth factors is selectively enhanced, and cell suspension provided. While the bioreactor array was developed for culturing HSCs, its application can be extended to other cell types. 相似文献
6.
Buchegger W Haller A van den Driesche S Kraft M Lendl B Vellekoop M 《Biomicrofluidics》2012,6(1):12803-128039
In this study, the pre-steady state development of enzymatic bioreactions using a microfluidic mixer is presented. To follow such reactions fast mixing of reagents (enzyme and substrate) is crucial. By using a highly efficient passive micromixer based on multilaminar flow, mixing times in the low millisecond range are reached. Four lamination layers in a shallow channel reduce the diffusion lengths to a few micrometers only, enabling very fast mixing. This was proven by confocal fluorescence measurements in the channel’s cross sectional area. Adjusting the overall flow rate in the 200 μm wide and 900 μm long mixing and observation channel makes it possible to investigate enzyme reactions over several seconds. Further, the device enables changing the enzyme/substrate ratio from 1:1 up to 3:1, while still providing high mixing efficiency, as shown for the enzymatic hydrolysis using β-galactosidase. This way, the early kinetics of the enzyme reaction at multiple enzyme/substrate concentrations can be collected in a very short time (minutes). The fast and easy handling of the mixing device makes it a very powerful and convenient instrument for millisecond temporal analysis of bioreactions. 相似文献
7.
Understanding the biological feeding strategy and characteristics of a microorganism as an actuator requires the detailed and quantitative measurement of flow velocity and flow rate induced by the microorganism. Although some velocimetry methods have been applied to examine the flow, the measured dimensions were limited to at most two-dimensional two-component measurements. Here we have developed a method to measure three-dimensional two-component flow velocity fields generated by the microorganism Vorticella picta using a piezoscanner and a confocal microscope. We obtained the two-component velocities of the flow field in a two-dimensional plane denoted as the XY plane, with an observation area of 455×341 μm2 and the resolution of 9.09 μm per each velocity vector by a confocal microparticle image velocimetry technique. The measurement of the flow field at each height took 37.5 ms, and it was repeated in 16 planes with a 2.50 μm separation in the Z direction. We reconstructed the three-dimensional two-component flow velocity field. From the reconstructed data, the flow velocity field [u(x,y,z),v(x,y,z)] in an arbitrary plane can be visualized. The flow rates through YZ and ZX planes were also calculated. During feeding, we examined a suction flow to the mouth of the Vorticella picta and measured it to be to 300 pl∕s. 相似文献
8.
B. Saboktakin Rizi K. Braasch E. Salimi M. Butler G. E. Bridges D. J. Thomson 《Biomicrofluidics》2014,8(6)
One of the main uses of adenosine triphosphate (ATP) within mammalian cells is powering the Na+/K+ ATPase pumps used to maintain ion concentrations within the cell. Since ion concentrations determine the cytoplasm conductivity, ATP concentration is expected to play a key role in controlling the cytoplasm conductivity. The two major ATP production pathways within cells are via glycolysis within the cytoplasm and via the electron transport chain within the mitochondria. In this work, a differential detector combined with dielectrophoretic (DEP) translation in a microfluidic channel was employed to observe single cell changes in the cytoplasm conductivity. The DEP response was made sensitive to changes in cytoplasm conductivity by measuring DEP response versus media conductivity and using double shell models to choose appropriate frequencies and media conductivity. Dielectric response of Chinese hamster ovary (CHO) cells was monitored following inhibition of the mitochondria ATP production by treatment with oligomycin. We show that in CHO cells following exposure to oligomycin (8 μg/ml) the cytoplasm conductivity drops, with the majority of the change occurring within 50 min. This work demonstrates that dielectric effects due to changes in ATP production can be observed at the single cell level. 相似文献
9.
The capability of the AC dielectrophoresis (DEP) for on-chip capture and chaining of microalgae suspended in freshwaters was evaluated. The effects of freshwater composition as well as the electric field voltage, frequency, and duration, on the dielectrophoretic response of microalga Chlamydomonas reinhardtii were characterized systematically. Highest efficiency of cell alignment in one-dimensional arrays, determined by the percentage of cells in chain and the chain length, was obtained at AC-field of 20 V mm−1 and 1 kHz applied for 600 s. The DEP response and cell alignment of C. reinhardtii in water sampled from lake, pond, and river, as well as model media were affected by the chemical composition of the media. In the model media, the efficiency of DEP chaining was negatively correlated to the conductivity of the cell suspensions, being higher in suspensions with low conductivity. The cells suspended in freshwaters, however, showed anomalously high chaining at long exposure times. High concentrations of nitrate and dissolved organic matter decrease cell chaining efficiency, while phosphate and citrate concentrations increase it and favor formation of longer chains. Importantly, the application of AC-field had no effect on algal autofluorescence, cell membrane damage, or oxidative stress damages in C. reinhardtii. 相似文献
10.
We have studied the contraction and extension of Vorticella convallaria and its mechanical properties with a microfluidic loading system. Cells of V. convallaria were injected to a microfluidic channel (500 μm in width and 100 μm in height) and loaded by flow up to ∼350 mm s−1. The flow produced a drag force on the order of nanonewton on a typical vorticellid cell body. We gradually increased the loading force on the same V. convallaria specimen and examined its mechanical property and stalk motion of V. convallaria. With greater drag forces, the contraction distance linearly decreased; the contracted length was close to around 90% of the stretched length. We estimated the drag force on Vorticella in the channel by calculating the force on a sphere in a linear shear flow. 相似文献
11.
This Special Topic section of Biomicrofluidics contains original contributions that were presented at the 82nd Colloid and Surface Science Symposium, which took place on 15–18 June 2008 at North Carolina State University. The Symposium covered a wide range of topics that are relevant to the fundamentals of fluidics and their application to biological systems.The recent interest in microfluidics and nanofluidics is constantly increasing due to the numerous applications that these techniques have to offer. They have been used for chemical and biomolecular sensing, separation of charged analytes, and single DNA molecule manipulation. These applications were facilitated by the significant increase in the range of advanced microfabrication and nanofabrication techniques. Improving and extending the range of applicability of micro- and nanofluidic techniques also requires better fundamental understanding of the physics of the transport at small length scales. The transport of fluids and solutes in microchannels and nanochannels usually occurs at very small Reynolds regime. The typical length scale and the surface forces (electrostatic, van der Waals, hydrophobic, hydration, etc.) may be comparable to the size of the channels. All these features often require the development of new experimental techniques and approaches for theoretical analysis.The importance and the substantial recent interest in micro- and nanofluidics prompted the organization of a special session on Electrokinetic Phenomena and Microfluidics as part of the program at the 82nd Colloid and Surface Science Symposium at North Carolina State University in June 2008. The collected papers in this issue of Biomicrofluidics cover some of the very important fundamental and engineering aspects of electrokinetic phenomena in micro- and nanofluidic channels. These include molecular dynamics simulation of biomolecules in confined spaces, analysis of the electric double layer effects on the fluid flow in nanochannels, hydrodynamic resistance to droplet motion in microchannels, electrophoresis in nanocomposite gels, and microfluidics for nanoparticle fabrication. The paper by Srivastava et al.1 explores the possibility of using microfluidics for fabrication of Janus nanofibers. Chang2 presented a theoretical analysis of the electro-osmosis on a salt-free microchannel by simultaneously solving the nonlinear Poisson–Boltzmann equation for the electrostatic potential distribution and the Navier–Stokes equations for the fluid flow. Trahan and Doyle3 reported theoretical analysis of DNA molecule interaction with obstacles in a microchannel. Finally, Labrot et al.4 presented studies on the droplet hydrodynamic resistance in a microfluidic channel.We hope that the reader will find the papers useful and informative. 相似文献
12.
Nanochannels offer a way to align and analyze long biopolymer molecules such as DNA with high precision at potentially single basepair resolution, especially if a means to detect biomolecules in nanochannels electronically can be developed. Integration of nanochannels with electronics will require the development of nanochannel fabrication procedures that will not damage sensitive electronics previously constructed on the device. We present here a near-room-temperature fabrication technology involving parylene-C conformal deposition that is compatible with complementary metal oxide semiconductor electronic devices and present an analysis of the initial impedance measurements of conformally parylene-C coated nanochannels with integrated gold nanoelectrodes.No two cells are exactly alike, either in terms of their genome, the genomic epigenetic modification of the genome, or the expressed proteome.1 The genomic heterogeneity of cells is particularly important from an evolutionary perspective since it represents the stages of evolution of a population of cells under stress.2 Because of the important variances in the genome that occur from cell to cell, it is critical to develop genomic analysis technologies which can do single-cell and single molecule genomic analysis as an electronic “direct read” without intervening amplification steps.3, 4, 5, 6, 7, 8 In this paper, we present a technique which uses conformal coverage of nanochannels containing nanoelectrodes using a room-temperature deposition of parylene-C, a pin-hole-free, excellent electrical insulator with low autofluorescence.9 This procedure should open the door to integration of many kinds of surface electronics with nanochannels. One of the most difficult aspects in introducing electronics into nanochannel technology is the sealing of nanochannel so that the electrodes are not compromised by harsh chemicals or high temperatures. There are various methods to form nanochannels containing nanoelectrodes, including wafer bonding techniques,10 removal of sacrificial materials,11 and nonuniform sputtering deposition.12 Methods employing a sacrificial layer removal show the greater compatibility to electronic integration, but current methods to remove sacrificial materials require either high temperatures11 or harsh chemicals.13, 14The basic device consisted of 12 mm long, 100 nm wide, 100 nm high nanochannels interrogated by 22 pairs of 30 nm wide gold nanoelectrodes. The outline of the fabrication process is shown in Fig. Fig.1.1. The fabrication process was carried out on a standard 4 in. single-side polished p-type ⟨100⟩ silicon wafer with 100 nm of dry thermal oxide on the top as an insulating layer, which also helped the wetting of the nanochannels. The first step involved nanofabrication of the 25 nm thick nanoelectrodes on the SiO2 top of the wafer using electron beam lithography (EBL). External gold connection pads were constructed using standard metal lift-off techniques and photolithography to connect to the nanoelectrodes. A Raith E-Line e-beam writing system (Raith USA, Ronkonkoma, NY) was used to expose polymethyl methacrylate (PMMA) for metal lift-off. Figure Figure1a1a shows a scanning electron microscopy (SEM) image of the nanoelectrodes. The 100 nm sealed nanochannels were constructed using sacrificial removal techniques. We used EBL to expose a 100 nm thick film of PMMA over the gold nanolines in the region around the nanolines, leaving behind lines of unexposed sacrificial layer of PMMA. We next evaporated 25 nm of SiO2 over the nanolines to improve the surface wetting properties of nanochannel and then conformally coated with 4 μm thick of parylene-C [poly(chloro-p-xylylene)] using a Specialty Coating Systems model PDS 2010 parylene coating system (SCS Systems, Indianapolis, IN). Access holes for the gold electrodes and the feeding channels were etched through by oxygen plasma and 1:10 buffered oxide etchant. To avoid autofluorescence induced in parylene by an active plasma15 and ambient UV radiation,16 it is important not to expose the remaining parylene with plasma and to keep the samples in the dark. The sacrificial removal of PMMA in the nanochannels was done in four steps: (1) soaking the chip in 55 °C 1165 MicroChem resist remover (MicroChem, Newton, MA) for 36 h, (2) room-temperature soaking in 1,2-dichloroethane for 12 h, (3) soaking in room-temperature acetone for 12 h, and (4) drying the nanochannels by critical point drying (CPD-030, BAL-TEC AG, Principality of Liechtenstein), which served to prevent the collapse of the nanochannel resulting from surface tension of the acetone.Open in a separate windowFigure 1(a) SEM image of gold nanoelectrodes; scale bar is 200 nm. (b) 100×100 nm2 PMMA nanoline is written over the gold nanoelectrodes by exposure of the surrounding PMMA. (c) Parylene-C conformal coating over the PMMA nanoline. PMMA is dissolved and parylene-C etched by reactive ion etching.Conductance measurements were done using ac techniques. The ac impedance Ztot of an insulating ionic fluid such as water between electrodes is a complex subject.17 The most general model for the complex impedance of an electrode in ionic solution is typically modeled as the Randle circuit, which is shown in Fig. Fig.22.17 There are two major contributions to the imaginary part of the impedance: the capacitance of the double layer (Cdl), which is purely imaginary and has no dc conductance, and the impedance due to charge transfer resulting in electrochemical reactions at the electrode∕electrolyte interface, which can be modeled as a contact resistor (RCT), which is given by the Butler–Volmer equation, which describes the I-V characteristic curve when electrochemical reactions occur at the electrode,18 in series with a complex Warburg impedance (ZW) which represents injected charge transport near the electrode;19 more details can be found in Ref. 20. Since we applied a 10 mV rms ac voltage with no dc offset in our measurements, electrochemical reactions are negligible, which means no electrochemical charge transfer occurred and as a result RCT goes to infinity. We have drawn a gray box around the elements connected to the Warburg impedance branch of the circuit to show that they are negligible in our analysis.Open in a separate windowFigure 2The equivalent circuit of the nanoelectrodes in contact with water lying atop an insulating SiO2 film which covers a silicon substrate. The elements in the gray boxes can be ignored in our measurements since there is no hydrolysis at low voltage, while the elements within the dotted box are coupling reactances to the underlying p-doped silicon wafer.In the case of no direct charge injection, the electrodes are coupled by the purely capacitive dielectric layer impedance Cdl to the solvent and are also coupled capacitively by the dielectric SiO2 film capacitance Cox to the underlying p-doped silicon semiconductor. We model the semiconductor as a purely resistive material with bulk resistivity ρSi. The value of Cdl∕area is on the order of ϵϵoκ, where ϵ is the dielectric constant of water (about 80) and κ is the Debye screening parameter of the counterions in solution: ,20 where is the bulk ion concentration of charge zi. At our salt molarity of 50 mM (1∕2 Tris∕Borate∕EDTA (TBE) buffer), Cdl is approximately 30 μF∕cm2 using 1∕κ∼1 nm.In Fig. Fig.3,3, we show the ac impedance measurements between pairs nanoelectrodes for both dry and TBE buffer wet nanochannels. The electrodes are capacitively coupled to the underlying silicon substrate through an oxide capacitor Cox. We model the doped silicon wafer as pure resistors, so there is an R1 that connects both Cox, and each Cox is connected to the ground with an R2. Curve fitting was done by using the 3SPICE circuit emulation code (VAMP Inc., Los Angeles, CA). We therefore obtained the following parameters for the dry curve: Cox=1.32 nF, R1=17.5 μΩ, and R2=32.8 kΩ. R1 is not sensitive in the fit as long as it is smaller than the impedance of Cox. Given ρSi of the wafer of 1–10 Ω cm, R2 should be on the order of 103 Ω, which is slightly smaller than our fitting results. The same parameters for the wafer coupling parameters were then used for fitting the impedance measurements for wet channels. For TBE buffer solution in the nanochannel, curve fitting yields Cdl=50 pF and Rsol=105 Ω. However, given the dimension of our nanochannels, we should get a transverse resistance R∼109 Ω. One possible explanation for this difference is that the evaporated SiO2 film which was put over the PMMA is porous and allows buffer to penetrate the oxide film,21 but given that the film is only 25 nm thick this would at most increase the cross section by one order of magnitude. However, it is known that there is a high fractional presence of mobile counterions associated with the charged channel walls.22 To calculate exact conductance contribution from the surface charges is a tricky business, but since the surface-to-volume ratios in our nanochannels are much greater than the slits, a larger conductance enhancement can be expected, and more work needs to be done.Open in a separate windowFigure 3ac impedance spectra of TBE buffer solution in a transchannel measurement between adjacent pairs of nanoelectrodes separated by 135 μm. The red circles are data for a dry channel and the solid red line is the fit to the model shown in the upper right hand corner. The green squares and dashed green line are for a nanochannel wet with TBE buffer.We have presented a way to fabricate a nanochannel integrated with electrodes. This technology opens up opportunities for electronic detection of charged polymers. With our techniques to fabricate nanoelectrodes with nanochannels, it should be possible to include integrated electronics with nanofludics, allowing the electronic observation of a single DNA molecule at high spatial resolution. However, the present design has problems. Most of the ac went through the silicon wafer instead of the solution. To enhance the sensitivity, we need either to increase the ratio of current going through the liquid to the current going through the wafer or to have a circuit design that picks up the changes in Cdl and Rsol. 相似文献
13.
A seven channel polystyrene (PS) microchip has been constructed using a micromilling machine and a high-temperature assembling. Protein A (PA) has been immobilized by a passive sorption on the microchannel walls. Two bioaffinity assays with human immunoglobulin G (hIgG) as a ligand have been carried out. (i) PA as the receptor and fluorescently labeled hIgG (FITC-hIgG) as the ligand, (ii) PA as the receptor with hIgG as the quantified ligand and fluorescently labeled goat anti-human IgG (FITC-gIgG) as the secondary ligand. One incubation step of the assays took only 5 min instead of hours typical for enzyme-linked immunosorbent assay applications. Calibration curves of the dependence of a fluorescence signal on the hIgG concentration in a sample have been obtained in one step due to a parallel arrangement of microchannels. A mathematical model of the PA-FITC-hIgG complex formation in the chip has been developed. The values of the kinetic constant of the PA-FITC-hIgG binding (kon=5.5 m3 mol−1 s−1) and the equilibrium dissociation constant of the formed complex (Kd≤3×10−6 mol m−3) have been obtained by fitting to experimental data. The proposed microchip enables fast evaluation of kinetic and equilibrium constants of ligand-receptor bioaffinity pairs and the ligand quantification. As the use of microfluidic chips for immunoassays is often limited by price, we used procedures and chemicals that allow for an inexpensive construction and operation of the microdevice, e.g., temperature assembling as a fabrication technique, detection via an ordinary digital camera, nonspecific polystyrene as a substrate, passive sorption of biomolecules as an immobilization technique, etc. 相似文献
14.
AC Faradaic reactions have been reported as a mechanism inducing non-ideal phenomena such as flow reversal and cell deformation in electrokinetic microfluidic systems. Prior published work described experiments in parallel electrode arrays below the electrode charging frequency (fc), the frequency for electrical double layer charging at the electrode. However, 2D spatially non-uniform AC electric fields are required for applications such as in plane AC electroosmosis, AC electrothermal pumps, and dielectrophoresis. Many microscale experimental applications utilize AC frequencies around or above fc. In this work, a pH sensitive fluorescein sodium salt dye was used to detect [H+] as an indicator of Faradaic reactions in aqueous solutions within non-uniform AC electric fields. Comparison experiments with (a) parallel (2D uniform fields) electrodes and (b) organic media were employed to deduce the electrode charging mechanism at 5 kHz (1.5fc). Time dependency analysis illustrated that Faradaic reactions exist above the theoretically predicted electrode charging frequency. Spatial analysis showed [H+] varied spatially due to electric field non-uniformities and local pH changed at length scales greater than 50 μm away from the electrode surface. Thus, non-uniform AC fields yielded spatially varied pH gradients as a direct consequence of ion path length differences while uniform fields did not yield pH gradients; the latter is consistent with prior published data. Frequency dependence was examined from 5 kHz to 12 kHz at 5.5 Vpp potential, and voltage dependency was explored from 3.5 to 7.5 Vpp at 5 kHz. Results suggest that Faradaic reactions can still proceed within electrochemical systems in the absence of well-established electrical double layers. This work also illustrates that in microfluidic systems, spatial medium variations must be considered as a function of experiment time, initial medium conditions, electric signal potential, frequency, and spatial position. 相似文献
15.
Srinivas Velugotla Steve Pells Heidi K. Mjoseng Cairnan R. E. Duffy Stewart Smith Paul De Sousa Ronald Pethig 《Biomicrofluidics》2012,6(4)
Assessment of the dielectrophoresis (DEP) cross-over frequency (fxo), cell diameter, and derivative membrane capacitance (Cm) values for a group of undifferentiated human embryonic stem cell (hESC) lines (H1, H9, RCM1, RH1), and for a transgenic subclone of H1 (T8) revealed that hESC lines could not be discriminated on their mean fxo and Cm values, the latter of which ranged from 14 to 20 mF/m2. Differentiation of H1 and H9 to a mesenchymal stem cell-like phenotype resulted in similar significant increases in mean Cm values to 41–49 mF/m2 in both lines (p < 0.0001). BMP4-induced differentiation of RCM1 to a trophoblast cell-like phenotype also resulted in a distinct and significant increase in mean Cm value to 28 mF/m2 (p < 0.0001). The progressive transition to a higher membrane capacitance was also evident after each passage of cell culture as H9 cells transitioned to a mesenchymal stem cell-like state induced by growth on a substrate of hyaluronan. These findings confirm the existence of distinctive parameters between undifferentiated and differentiating cells on which future application of dielectrophoresis in the context of hESC manufacturing can be based. 相似文献
16.
Magnetotactic bacteria (MTB) are capable of swimming along magnetic field lines. This unique feature renders them suitable in the development of magnetic-guided, auto-propelled microrobots to serve in target molecule separation and detection, drug delivery, or target cell screening in a microfluidic chip. The biotechnology to couple these bacteria with functional loads to form microrobots is the critical point in its application. Although an immunoreaction approach to attach functional loads to intact MTB was suggested, details on its realization were hardly mentioned. In the current paper, MTB-microrobots were constructed by attaching 2 μm diameter microbeads to marine magnetotactic ovoid MO-1 cells through immunoreactions. These microrobots were controlled using a special control and tracking system. Experimental results prove that the attachment efficiency can be improved to ∼30% via an immunoreaction. The motility of the bacteria attached with different number of loads was also assessed. The results show that MTB can transport one load at a velocity of ∼21 μm/s and still move and survive for over 30 min. The control and tracking system is fully capable of directing and monitoring the movement of the MTB-microrobots. The rotating magnetic fields can stop the microrobots by trapping them as they swim within a circular field with a controllable size. The system has potential use in chemical analyses and medical diagnoses using biochips as well as in nano/microscale transport. 相似文献
17.
Bela Goyal Sudip Kumar Datta Altaf A. Mir Saidaiah Ikkurthi Rajendra Prasad Arnab Pal 《Indian journal of clinical biochemistry : IJCB》2016,31(2):224-230
The estimation of electrolytes like sodium (Na+), potassium (K+) and chloride (Cl−) using direct and indirect ion-selective electrodes (ISE) is a routine laboratory practice. Interferents like proteins, triglycerides, drugs etc. are known to affect the results. The present study was designed to look into the effect of increasing glucose concentrations on estimation of Na+, K+ and Cl− by direct and indirect ISE. Pooled sera was mixed with glucose stock solution (20 g/dL) prepared in normal saline to obtain glucose concentrations ranging from ~100 to ~5000 mg/dL. Na+, K+ and Cl− levels were estimated by direct and indirect ISE analyzers and results were statistically analysed using ANOVA and Pearson’s correlation. Similar experiment was also performed in 24 h urine sample from healthy subjects. Significant difference was observed between Na+ and Cl− measurements by direct and indirect ISE, with indirect ISE values being consistently higher than direct ISE. Besides this, significant difference was observed amongst Na+ and Cl− values from baseline values obtained by indirect ISE at glucose concentrations ≥2486 mg/dL. However, no such difference was observed with direct ISE. Na+ and Cl− estimation by indirect ISE showed significant negative correlation with glucose concentration, more so, above ~2000 mg/dL. K+, however, showed no significant difference with varying glucose. Similar results were observed in 24 h urine samples with a significant difference observed amongst Na+ and Cl− values at ≥2104 mg/dL glucose. Thus we conclude that high glucose concentrations interfere significantly in estimation of Na+ and Cl− by indirect ISE in serum as well as urine.
Electronic supplementary material
The online version of this article (doi:10.1007/s12291-015-0522-0) contains supplementary material, which is available to authorized users. 相似文献18.
Shau-Chun Wang Hsiao-Ping Chen Yi-Wen Lai Lai-Kwan Chau Yu-Chun Chuang Yi-Jie Chen 《Biomicrofluidics》2007,1(3)
A novel microstirring strategy is applied to accelerate the digestion rate of the substrate Nα-benzoyl-L-arginine-4-nitroanilide (L-BAPA) catalyzed by sol-gel encapsulated trypsin. We use an ac nonlinear electrokinetic vortex flow to stir the solution in a microfluidic reaction chamber to reduce the diffusion length between the immobilized enzyme and substrate in the solution. High-intensity nonlinear electroosmotic microvortices, with angular speeds in excess of 1 cm∕s, are generated around a small (∼1.2 mm) conductive ion exchange granule when ac electric fields (133 V∕cm) are applied across a miniature chamber smaller than 10 μl. Coupling between these microvortices and the on-and-off electrophoretic motion of the granule in low frequency (0.1 Hz) ac fields produces chaotic stream lines to stir substrate molecules sufficiently. We demonstrate that, within a 5-min digestion period, the catalytic reaction rate of immobilized trypsin increases almost 30-fold with adequate reproducibility (15%) due to sufficient stirring action through the introduction of the nonlinear electrokinetic vortices. In contrast, low-frequency ac electroosmotic flow without the granule, provides limited stirring action and increases the reaction rate approximately ninefold with barely acceptable reproducibility (30%). Dye molecules are used to characterize the increases in solute diffusivity in the reaction reservoir in which sol-gel particles are placed, with and without the presence of granule, and compared with the static case. The solute diffusivity enhancement data show respective increases of ∼30 and ∼8 times, with and without the presence of granule. These numbers are consistent with the ratios of the enhanced reaction rate. 相似文献
19.
The study investigates the use of CO2 laser to induce glass strip peeling off to form microchannels on soda lime gass substrate. The strip peeling exhibits a strong dependence on the energy deposition rate on the glass surface. In spite of the vast difference in the combination of laser power and scanning speed, when the ratio of the two makes the energy deposition rate in the range 3.0-6.0 J/(cm2 s), the temperature rising inside glass will be above the strain point and reach the softening region of the glass. As a result, glass strip peeling is able to occur and form microchannels with dimensions of 20-40 μm in depth and 200-280 μm in width on the glass surface. Beyond this range, higher energy depsotion rate would lead to surface melting associated with solidification cracks and lower energy deposition rate causes the generation of fragment cracks. 相似文献
20.
S. Pandey Ravi Ranjan R. M. Mishra Sw. Pandey R. Saxena 《Indian journal of clinical biochemistry : IJCB》2012,27(3):314-317
Hemoglobin D exist in four form; HbD trait, HbD-thalassemia, HbD sickle cell and HbD homozygous. HbD trait and HbD homozygous generally asymptomatic condition but when HbD co-inherit with thalassemia and sickle cell anemia, produces clinically significant conditions like chronic hemolytic anemia. Here we present a case of HbD Punjab with α 3.7 kb deletion and IVS-1-5 β-thalassemia across a family. Diagnosis of HbD patient was performed by high performance liquid chromatography and complete blood count was measured by automated cell analyzer. Molecular study for common alpha deletions done by Gap-PCR while beta thalassemia mutation identified by ARMS-PCR. Case was clinically significant due to the inheritance of HbD/β+thalassemia genotype. Thus observed case behaved like thalassemia intermedia due to co-existence of α 3.7 deletions with IVS 1-5 β-thalassemia mutation in HbD Punjab patient. 相似文献