Single‐step CE for miniaturized and easy‐to‐use system

We developed a novel single‐step capillary electrophoresis (SSCE) scheme for miniaturized and easy to use system by using a microchannel chip, which was made from the hydrophilic material polymethyl methacrylate (PMMA), equipped with a capillary stop valve. Taking the surface tension property of liquids into consideration, the capillary effect was used to introduce liquids and control capillary stop valves in a partial barrier structure in the wall of the microchannel. Through the combined action of stop valves and air vents, both sample plug formation for electrophoresis and sample injection into a separation channel were successfully performed in a single step. To optimize SSCE, different stop valve structures were evaluated using actual microchannel chips and the finite element method with the level set method. A partial barrier structure at the bottom of the channel functioned efficiently as a stop valve. The stability of stop valve was confirmed by a shock test, which was performed by dropping the microchannel chip to a floor. Sample plug deformation could be reduced by minimizing the size of the side partial barrier. By dissolving hydroxyl ethyl cellulose and using it as the sample solution, the EOF and adsorption of the sample into the PMMA microchannel were successfully reduced. Using this method, a 100‐bp DNA ladder was concentrated; good separation was observed within 1 min. At a separation length of 5 mm, the signal was approximately 20‐fold higher than a signal of original sample solution by field‐amplified sample stacking effect. All operations, including liquid introduction and sample separation, can be completed within 2 min by using the SSCE scheme.     

Micromixing of miscible liquids in segmented gas-liquid flow

We present an integrated microfluidic system that achieves efficient mixing between two miscible liquid streams by introducing a gas phase, forming a segmented gas-liquid (slug) flow, and completely separating the mixed liquid and gas streams in a planar capillary separator. The recirculation motion associated with segmented flow enhances advection in straight microchannels without requiring additional fabrication steps. Instantaneous velocity fields are quantified by microscopic particle image velocimetry (muPIV). Velocities in the direction normal to the channel amount to approximately 30% of the bulk liquid velocity inside a liquid segment. This value depends only weakly on the length of a liquid segment. Spatial concentration fields and the extent of mixing (EOM) are obtained from pulsed-laser fluorescence microscopy and confocal scanning microscopy measurements. The mixing length is reduced 2-3-fold in comparison with previously reported chaotic micromixers that use three-dimensional microchannel networks or patterned walls. Segmented gas-liquid microflows allow mixing times to be varied over several orders of magnitude between milliseconds and second time scales.     

Estimation of aqueous solubility for a diverse set of organic compounds based on molecular topology

An accurate and generally applicable method for estimating aqueous solubilities for a diverse set of 1297 organic compounds based on multilinear regression and artificial neural network modeling was developed. Molecular connectivity, shape, and atom-type electrotopological state (E-state) indices were used as structural parameters. The data set was divided into a training set of 884 compounds and a randomly chosen test set of 413 compounds. The structural parameters in a 30-12-1 artificial neural network included 24 atom-type E-state indices and six other topological indices, and for the test set, a predictive r2 = 0.92 and s = 0.60 were achieved. With the same parameters the statistics in the multilinear regression were r2 = 0.88 and s = 0.71, respectively.     

Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis

A laser-induced fluorescence detection system coupled with a highly sensitive silicon-intensified target (SIT) camera is successfully applied to the imaging of a band for DNA fragment labeling by fluorescence dye in a microchannel, and to the visualizing of the separation process on a microfabricated chip. We demonstrated that an only 6 mm separation channel is sufficient for the separation of triplet repeat DNA fragment and DNA molecular marker within only 12 s. The separation using the microfabricated capillary electrophoresis device is confirmed to be at least 18 times faster than the same separation carried out by conventional capillary electrophoresis with 24.5 cm effective length. The use of a short capillary with 8.5 cm effective length is also efficient for fast separation of DNA; however, the microchip technology is even faster than capillary electrophoresis using a short capillary.     

Improvement of proteolytic efficiency towards low-level proteins by an antifouling surface of alumina gel in a microchannel

A microfluidic reactor has been developed for rapid enhancement of protein digestion by constructing an alumina network within a poly(ethylene terephthalate) (PET) microchannel. Trypsin is stably immobilized in a sol-gel network on the PET channel surface after pretreatment, which produces a protein-resistant interface to reduce memory effects, as characterized by X-ray fluorescence spectrometry and electroosmotic flow. The gel-derived network within a microchannel provides a large surface-to-volume ratio stationary phase for highly efficient proteolysis of proteins existing both at a low level and in complex extracts. The maximum reaction rate of the encapsulated trypsin reactor, measured by kinetic analysis, is much faster than in bulk solution. Due to the microscopic confinement effect, high levels of enzyme entrapment and the biocompatible microenvironment provided by the alumina gel network, the low-level proteins can be efficiently digested using such a microreactor within a very short residence time of a few seconds. The on-chip microreactor is further applied to the identification of a mixture of proteins extracted from normal mouse liver cytoplasm sample via integration with 2D-LC-ESI-MS/MS to show its potential application for large-scale protein identification.     

On-chip solid phase extraction coupled with electrophoresis using modified magnetic microspheres as stationary phase

A poly(dimethylsiloxane)(PDMS)/glass hybrid microchip for on-line solid phase extraction (SPE) and electrophoresis separation has been developed and evaluated. The SPE microchannel was crossed to the electrophoresis microchannel. All the microfluidic channels were etched on the glass substrate. The magnetic microspheres were coated with hydroxyl-terminated poly-dimethylsiloxane (PDMS-OH) serving as extraction phase, which could be conveniently immobilized into the sample pretreatment channel by magnetic field. The PDMS-OH microspheres were mobilized into and out of the pretreatment channel by injection flow. The 0.1 μmol/L solution of fluorescence isothiocyanate (FITC)-labeled phenylalanine (Phe) was electrically injected into the SPE channel and extracted onto the PDMS-OH microspheres bed. The enriched FITC-labeled Phe was electrically eluted by 9 mmol/L sodium acetate containing 10% acetonitrile and electrically driven into the electrophoresis channel and then separated. The preconcentration factor could reach 87.5 after sufficient extraction. A linear preconcentration curve was obtained with the initial FITC-labeled Phe concentration ranging from 6 nmol/L to 300 nmol/L (R ²=0.9922) with 200 s loading time. The detection limit (S/N=3) for the FITC-labeled Phe was 3 nmol/L.     

Classification of cell types using a microfluidic device for mechanical and electrical measurement on single cells

This paper presents a microfluidic system for cell type classification using mechanical and electrical measurements on single cells. Cells are aspirated continuously through a constriction channel with cell elongations and impedance profiles measured simultaneously. The cell transit time through the constriction channel and the impedance amplitude ratio are quantified as cell's mechanical and electrical property indicators. The microfluidic device and measurement system were used to characterize osteoblasts (n=206) and osteocytes (n=217), revealing that osteoblasts, compared with osteocytes, have a larger cell elongation length (64.51 ± 14.98 μm vs. 39.78 ± 7.16 μm), a longer transit time (1.84 ± 1.48 s vs. 0.94 ± 1.07 s), and a higher impedance amplitude ratio (1.198 ± 0.071 vs. 1.099 ± 0.038). Pattern recognition using the neural network was applied to cell type classification, resulting in classification success rates of 69.8% (transit time alone), 85.3% (impedance amplitude ratio alone), and 93.7% (both transit time and impedance amplitude ratio as input to neural network) for osteoblasts and osteocytes. The system was also applied to test EMT6 (n=747) and EMT6/AR1.0 cells (n=770, EMT6 treated by doxorubicin) that have a comparable size distribution (cell elongation length: 51.47 ± 11.33 μm vs. 50.09 ± 9.70 μm). The effects of cell size on transit time and impedance amplitude ratio were investigated. Cell classification success rates were 51.3% (cell elongation alone), 57.5% (transit time alone), 59.6% (impedance amplitude ratio alone), and 70.2% (both transit time and impedance amplitude ratio). These preliminary results suggest that biomechanical and bioelectrical parameters, when used in combination, could provide a higher cell classification success rate than using electrical or mechanical parameter alone.     

Estimation of aqueous solubility of chemical compounds using E-state indices.

The molecular weight and electrotopological E-state indices were used to estimate by Artificial Neural Networks aqueous solubility for a diverse set of 1291 organic compounds. The neural network with 33-4-1 neurons provided highly predictive results with r(2) = 0.91 and RMS = 0.62. The used parameters included several combinations of E-state indices with similar properties. The calculated results were similar to those published for these data by Huuskonen (2000). However, in the current study only E-state indices were used without need of additional indices (the molecular connectivity, shape, flexibility and indicator indices) also considered in the previous study. In addition, the present neural network contained three times less hidden neurons. Smaller neural networks and use of one homogeneous set of parameters provides a more robust model for prediction of aqueous solubility of chemical compounds. Limitations of the developed method for prediction of large compounds are discussed. The developed approach is available online at http://www.lnh.unil.ch/~itetko/logp.     

Zeolite nanoparticle modified microchip reactor for efficient protein digestion

An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol-gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon-oxygen-silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s(-1). Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL min(-1), which affords a very short reaction time of less than 5 s.     

DNA microelectrophoresis using double focus fluorescence correlation spectroscopy

Double focus fluorescence correlation spectroscopy (dfFCS) was used to determine electrophoretic mobilities of short double-stranded DNA (dsDNA)-fragments (75 base pairs (bp) -1019 bp) in microfluidic channels. The electrokinetic flow profile across a microchannel was measured with 1 microm spatial resolution and separated in electroosmotic and electrophoretic contributions. Experiments show that the free solution mobility is independent of DNA length. The diffusion constant is additionally determined by FCS and follows a length dependent rod-diffusion model. We interpret the electrophoretic mobilities using a modified Nernst Einstein relation, which additionally takes Manning condensation and counterion induced hydrodynamic retardation forces into account. In 3% w/v polyethylene oxide (PEO)-network (M(r) 3 .10(5) Dalton) the electrophoretic velocities become size-dependent with a power-law exponent be-tween 0.28 and 0.31. Mixtures of dsDNA-fragments exhibit distinguishable peaks in the dfFCS cross-correlation function. The potential of dfFCS for realtime micro-analysis in terms of speed and spatial resolution is discussed.     

Microdroplet size prediction in microfluidic systems via artificial neural network modeling for water-in-oil emulsion formulation

In this paper, an experimental study and modeling by artificial neural networks were carried out to predict the generated microdroplet dimensionless size in a microfluidic system in order to formulate a water-in-oil emulsion. The various parameters that affect the size of microdroplets (flow rates, viscosities, surface tensions of both the two phases and the diameter of the microchannel) are studied and further grouped into dimensionless numbers; we used these numbers as input to the neural network and the dimensionless length as output. The better neural network architecture has 10 neurons in the hidden layer with a mean square error of 1.4 10^?6 and a determination’s coefficient near 1 value. The relative importance of inputs on the size of the microdroplets has been determined using the Garson algorithm and the results are in good agreement with other works.     

Diffusion dependent cell behavior in microenvironments

Understanding the interaction between soluble factors and cells in the cellular microenvironment is critical to understanding a wide range of diseases. Microchannel culture systems provide a tool for separating diffusion and convection based transport making possible controlled studies of the effects of soluble factors in the cellular microenvironment. In this paper we compare the proliferation kinetics of cells in traditional culture flasks to those in microfluidic channels, and explore the relationship between microchannel geometry and cell proliferation. PDMS (polydimethylsiloxane) microfluidic channels were fabricated using micromolding methods. Fall armyworm ovarian cells (Sf9) were homogeneously seeded in a series of different sized microchannels and cultured under a no flow condition. The proliferation rates of Sf9 cells in all of the microchannels were slower than in the flask culture over the first 24 h of culture. The proliferation rates in the microchannels then continuously decreased reaching 5% of that in the flasks over the next 48 h and maintained this level for 5 days. This growth inhibition was reversible and influenced only by the cell seeding density and the channel height but not the channel length or width. One possible explanation for the observed dimension-dependent cell proliferation is the accumulation of different functional molecules in the diffusion dominant microchannel environment. This study provides insights into the potential effects of the diffusion of soluble factors and related effects on cell behavior in microenvironments relevant to the emerging use of microchannel culture systems.     

Monitoring of chemical reactions within microreactors using an inverted Raman microscopic spectrometer

An inverted Raman microscope spectrometer has been used to profile the spatial evolution of reactant and product concentrations for a chemical reaction within a microreactor operating under hydrodynamic flow control. The Raman spectrometer was equipped with a laser source at wavelength of 780 nm, confocal optics, a holographic transmission grating, and a charge-coupled device (CCD) detector. The microreactor consisted of a T-shaped channel network etched within a 0.5 mm thick glass bottom plate that was thermally bonded to a 0.5 mm thick glass top plate. The ends of the channel network were connected to reagent reservoirs that were linked to a syringe pump for driving the solutions by hydrodynamic pumping within the channels. The microchannels were 221 micro m wide and 73 micro m deep. The synthesis of ethyl acetate from ethanol and acetic acid was investigated as a model system within the microreactor as Raman scattering bands for each reactant and product species were clearly resolved. Raman spectral intensities of each band were proportional to concentration for each species and hence all concentrations could be quantitatively measured after calibration. By scanning specific Raman bands within a selected area in the microchannel network at given steps in the X-Y plane, spatially resolved concentration profiles were obtained under steady-state flow conditions. Under the flow conditions used, different positions within the concentration profile correspond to different times after contact and mixing of the reagents, thereby enabling one to observe the time dependence of the product formation. Raman microscopy provides a useful complementary technique to UV/VIS absorbance and fluorescence methods for the in situ monitoring and analysis of chemical reaction species having their lowest S(0)-S(1) absorption bands too far in the UV to be of use, due to their probable overlap with the bands from other reactant, product and solvent molecules.     

Separation of charged colloids by a combination of pulsating lateral electric fields and poiseuille flow in a 2D channel

Separation of colloidal particles of different sizes is becoming increasingly important due to rapid developments in the area of proteomics, genetic engineering, drug discovery, etc. In particular, there is a need to accomplish these separations on a microscale in 'lab-on-a-chip' devices. In this paper, we propose a new method for accomplishing separation of charged colloids of different sizes in a microchannel. This method involves a combination of pulses of lateral electric fields and Poiseuille flow in the axial direction. We develop a model for this separation technique and obtain closed form solutions for the mean velocity and the dispersion coefficient for a pulse of molecules introduced into the channel. These expressions are then utilized to determine the channel length and the separation time. For reasonable value of design constants, the proposed technique can separate molecules of different sizes that have diffusivities of 10(-10) and 0.5 x 10(-10) m2/s in 15.7 s in a 3.7 mm long channel. The length and the time increase to 5.45 cm and 231 s if the ratio of the diffusivities is reduced from 2 to 1.2, i.e., the latter diffusivity is increased to 0.835 x 10(-10) m2/s, while keeping all the other parameters the same. If the diffusivities are about 10(-9) m(2)/s, the length and the time for separation are 1 cm and 17.5 s for D1/D2=2, and 16 cm and 269 s for D1/D2=1.2.     

微流控芯片中细胞动态胞吞二氧化硅纳米颗粒的研究

研究了用微流控芯片在体外模拟人体血液流动状态下细胞胞吞二氧化硅纳米粒子的方法和特性. 通过调节储液池的液面差, 使细胞从微通道入口流入并在通道内沉积贴壁生长. 将含有贴壁细胞的微流控芯片放入37 ℃/体积分数5%CO₂的培养箱中, 使细胞培养液连续流过贴壁细胞. 培养24 h后, 在流动的培养液中加入作为荧光标记物的500 nm 粒径的掺杂有异硫氰酸荧光素(FITC)的二氧化硅微球(MSN), 继续培养6 h后, 用荧光显微镜测定细胞胞吞二氧化硅纳米粒子后的荧光强度, 考察了不同流速下细胞对二氧化硅微球摄入量的影响. 结果表明, 在动态条件下, 细胞对二氧化硅微球的吞噬量明显下降, 当流速从0.022 mm/s 增加至0.74 mm/s时, 吞噬量从静态测得值的74.7%下降至7.1%.     

Channel shape effects on the solution-flow characteristics and the liquid/liquid extraction efficiency in polymer microchannel chips.

A polymer microchannel chip with a symmetrical or unsymmetrical zigzag-side-walled structure was fabricated by an imprinting method, and applied to study shape effects on solution flow characteristics as well as on the liquid/liquid extraction efficiency of an aluminium(III) chelate complex (Al-DHAB: DHAB = o,o'-dihydroxyazobenzene) in a microchannel chip. In an unsymmetrical zigzag-side-walled channel chip (us-channel), an oil/water interface was sinusoidal, while that in a symmetrical zigzag-side-walled channel chip (s-channel) was flat as long as the observation was made under an optical microscope. It was demonstrated that the efficiency of the water-to-oil (1-butanol) extraction of Al-DHAB in the microchannel was governed by the contact time between the two phases. As the most important results, furthermore, the extraction efficiency was higher in the us-channel, as compared with that in the s-channel, owing to the sinusoidal liquid/liquid interfacial structure and, therefore, to the high interfacial area between the two phases.     

Optical classification of algae species with a glass lab-on-a-chip

The identification of submillimetre phytoplankton is important for monitoring environmental and climate changes, as well as evaluating water for health reasons. Current standard methods for phytoplankton species identification require sample collection and ex situ analysis, an expensive procedure which prevents the rapid identification of phytoplankton outbreaks. To address this, we use a glass-based microchip with a microchannel and waveguide included on a monolithic substrate, and demonstrate its use for identifying phytoplankton species. The microchannel and the specimens inside it are illuminated by laser light from the curved waveguide as algae-laden water is passed through the channel. The intensity distribution of the light collected from the biochip is monitored with an external photodetector. Here, we demonstrate that the characteristics of the photodiode signal from this simple and robust system can provide significant and useful information as to the contents of the channel. Specifically, we show first that the signals are correlated to the size of algae cells. Using a pattern-matching neural network, we demonstrate the successful classification of five algae species with an average 78% positive identification rate. Furthermore, as a proof-of-concept for field-operation, we show that the chip can be used to distinguish between detritus in field-collected water and the toxin-producing cyanobacterium Cyanothece.