On-chip measurements of cell compressibility via acoustic radiation

Measurements of mechanical properties of biological cells are of great importance because changes in these properties can be strongly associated with the progression of cell differentiation and cell diseases. Although state of the art methods, such as atomic force microscopy, optical tweezers and micropipette aspiration, have been widely used to measure the mechanical properties of biological cells, all these methods involve direct contact with the cell and the measurements could be affected by the contact or any local deformation. In addition, all these methods typically deduced the Young's modulus of the cells based on their measurements. Herein, we report a new method for fast and direct measurement of the compressibility or bulk modulus of various cell lines on a microchip. In this method, the whole cell is exposed to acoustic radiation force without any direct contact. The method exploits the formation of an acoustic standing wave within a straight microchannel. When the polystyrene beads and cells are introduced into the channel, the acoustic radiation force moves them to the acoustic pressure node and the movement speed is dependent on the compressibility. By fitting the experimental and theoretical trajectories of the beads and the cells, the compressibility of the cells can be obtained. We find that the compressibility of various cancer cells (MCF-7: 4.22 ± 0.19 × 10(-10) Pa(-1), HEPG2: 4.28 ± 0.12 × 10(-10) Pa(-1), HT-29: 4.04 ± 0.16 × 10(-10) Pa(-1)) is higher than that of normal breast cells (3.77 ± 0.09 × 10(-10) Pa(-1)) and fibroblast cells (3.78 ± 0.17 × 10(-10) Pa(-1)). This work demonstrates a novel acoustic-based method for on-chip measurements of cell compressibility, complementing existing methods for measuring the mechanical properties of biological cells.     

On-chip CO2 control for microfluidic cell culture

Carbon dioxide partial pressure (P(CO(2))) was controlled on-chip by flowing pre-equilibrated aqueous solutions through control channels across the device. Elevated P(CO(2)) (e.g. 0.05 atm) was modulated in neighboring stagnant channels via equilibration through the highly gas permeable substrate, poly(dimethylsiloxane) (PDMS). Stable gradients in P(CO(2)) were demonstrated with a pair of control lines in a source-sink configuration. P(CO(2)) equilibration was found to be sufficiently rapid (minutes) and stable (days) to enable long-term microfluidic culture of mammalian cells. The aqueous solutions flowing through the device also mitigated pervaporative losses at sustained elevated temperatures (e.g. 37 C), as compared to flowing humidified gas through the control lines to control P(CO(2)). Since pervaporation (and the associated increase in osmolality) was minimized, stopped-flow cell culture became possible, wherein cell secretions can accumulate within the confined environment of the microfluidic culture system. This strategy was utilized to demonstrate long-term (> 7 days) microfluidic culture of mouse fibroblasts under stopped-flow conditions without requiring the microfluidic system to be placed inside a cell culture incubator.     

On-chip generation and reaction of unstable intermediates-monolithic nanoreactors for diazonium chemistry: azo dyes

Monolithic nanoreactors for the safe and expedient continuous synthesis of products requiring unstable intermediates were fabricated and tested by the synthesis of azo dyes under hydrodynamic pumping regimes.     

On-chip magnetically actuated robot with ultrasonic vibration for single cell manipulations

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.     

A microfluidic electroporation device for cell lysis

We demonstrate a micro-electroporation device for cell lysis prior to subcellular analysis. Simple circuit models show that electrical lysis method is advantageous because it is selective towards plasma membrane while leaving organelle membrane undamaged. In addition, miniaturization of this concept leads to negligible heat generation and bubble formation. The designed microdevices were fabricated using a combination of photolithography, metal-film deposition, and electroplating. We demonstrate the electro-lysis of human carcinoma cells in these devices to release the subcellular materials.     

Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation

A highly effective, reagentless, mechanical cell lysis device integrated in microfluidic channels is reported. Sample preparation, specifically cell lysis, is a critical element in 'lab-on-chip' applications. However, traditional methods of cell lysis require purification steps or complicated fabrication steps that a simple mechanical method of lysis may avoid. A simple and effective mechanical cell lysis system is designed, microfabricated, and characterized to quantify the efficiency of cell lysis and biomolecule accessibility. The device functionality is based on a microfluidic filter region with nanostructured barbs created using a modified deep reactive ion etching process. Mechanical lysis is characterized by using a membrane impermeable dye. Three main mechanisms of micro-mechanical lysis are described. Quantitative measurements of accessible protein as compared to a chemically lysed sample are acquired with optical absorption measurements at 280 and 414 nm. At a flow rate of 300 microL min(-1) within the filter region total protein and hemoglobin accessibilities of 4.8% and 7.5% are observed respectively as compared to 1.9% and 3.2% for a filter without nanostructured barbs.     

Development of a laser-induced cell lysis system

A novel cell lysis system was developed that is based on laser-induced disruption of bacterial and yeast cells. It will find application as a rapid, efficient and clean sample preparation step in bioanalytical detection systems. Using E. coli as our model analyte, we optimized cell lysis with respect to optimal laser wavelength, lowest energy input requirements, RNA release from the cells, and potential protein damage. The optimized system was finally applied to the lysis of four additional microorganisms. All experiments were carried out with about 2000 cells per sample or less. Initially, lysis was determined by the detection of cell survival after laser treatment using standard microbiological techniques, (i.e., cells were grown on nutrient agar plates). Then, actual release of mRNA from the cells was proven. Wavelengths investigated ranged from 500 nm to 1550 nm. An average power of 100 mW for the lasers was shown to be sufficient to obtain cell lysis at wavelengths above 1000 nm, with optimal wavelengths between 1250 nm and 1550 nm. Since water absorbs energy at those wavelengths, it is assumed that laser exposure results in an instantaneous increase of the cell temperature, which causes rupture of the cell membrane. Second, damage to protein solutions treated under optimized laser-lysis conditions was also studied. Using a pure solution of horseradish peroxidase as a model protein, no loss in enzyme activity was observed. Thus, it was concluded that damage to intracellular proteins is unlikely. Third, RNA release was tested using an E. coli specific RNA biosensor. Release of RNA was not detected from untreated cells, but laser-treated E. coli cells displayed significant RNA release due to laser-induced cell lysis. Finally, lysis of M. luteus, B. subtilis, B. cereus, and S. cerevisiae were investigated under optimized conditions. In all cases, laser-induced lysis of the cells was confirmed by determination of cell survival. Hence, laser-induced cell lysis is an efficient procedure that can be used for sample preparation, without damage to macromolecules, in bioanalytical detection systems for microorganisms. Miniaturized lasers and miniaturized cell-lysis chambers will create a simple, field-usable cell lysis system and allow the application of laser-induced cell lysis in micro Total Analysis Systems.     

On-chip generation of microbubbles as a practical technology for manufacturing contrast agents for ultrasonic imaging

This paper presents a new manufacturing method to generate monodisperse microbubble contrast agents with polydispersity index (sigma) values of <2% through microfluidic flow-focusing. Micron-sized lipid shell-based perfluorocarbon (PFC) gas microbubbles for use as ultrasound contrast agents were produced using this method. The poly(dimethylsiloxane) (PDMS)-based devices feature expanding nozzle geometry with a 7 microm orifice width, and are robust enough for consistent production of microbubbles with runtimes lasting several hours. With high-speed imaging, we characterized relationships between channel geometry, liquid flow rate Q, and gas pressure P in controlling bubble sizes. By a simple optimization of the channel geometry and Q and P, bubbles with a mean diameter of <5 microm can be obtained, ideal for various ultrasonic imaging applications. This method demonstrates the potential of microfluidics as an efficient means for custom-designing ultrasound contrast agents with precise size distributions, different gas compositions and new shell materials for stabilization, and for future targeted imaging and therapeutic applications.     

Determination of ultra-trace amounts of arsenic(III) by flow-injection hydride generation atomic absorption spectrometry with on-line preconcentration by coprecipitation with lanthanum hydroxide or hafnium hydroxide

A time-based flow-injection (FI) procedure for the determination of ultra-trace amounts of inorganic arsenic(III) is described, which combines hydride generation atomic absorption spectrometry (HG-AAS) with on-line preconcentration of the analyte by inorganic coprecipitation-dissolution in a filterless knotted Microline reactor. The sample and coprecipitating agent are mixed on-line and merged with an ammonium buffer solution, which promotes a controllable and quantitative collection of the generated hydroxide on the inner walls of the knotted reactor incorporated into the FI-HG-AAS system. Subsequently the precipitate is eluted with 1 mol 1(-1) hydrochloric acid, allowing ensuing determination of the analyte via hydride generation. The preconcentration of As(III) was tested by coprecipitation with two different inorganic coprecipitating agents namely La(III) and Hf(IV). It was shown that As(III) is more effectively collected by lanthanum hydroxide than by hafnium hydroxide, the sensitivity achieved by the former being approximately 25% better. With optimal experimental conditions and with a sample consumption of 6.7 ml per assay, an enrichment factor of 32 was obtained at a sample frequency of 33 samples h(-1). The limit of detection (3sigma) was 0.003 microg 1(-1) and the precision (relative standard deviation) was 1.0% (n = 11) at the 0.1 microg 1(-1) level.     

Effect of gamma irradiation on cell lysis and polyhydroxyalkanoate produced by Bacillus flexus

Bacillus flexus cultivated on sucrose and sucrose with plant oil such as castor oil produced polyhydroxybutyrate (PHB), a homopolymer of polyhydroxyalkanoate (PHA) and PHA copolymer (containing hydroxybutyrate and hexanoate), respectively. Gamma irradiation of these cells (5–40 kGy) resulted in cell damage and aided in the isolation of 45% and 54% PHA on biomass weight, correspondingly. Molecular weight of PHB increased from 1.5×10⁵ to 1.9×10⁵ after irradiation (10 kGy), with marginal increase of tensile strength from 18 to 20 MPa. At the same irradiation dosage, PHA copolymer showed higher molecular weight increase from 1.7×10⁵ to 2.3×10 ⁵ and tensile strength from 20 to 35 MPa. GC, GC–MS, FTIR and ¹H NMR were used for the characterization of PHA. Gamma irradiation seems to be a novel technique, to induce cross-linking and molecular weight increase of PHA copolymer and aid in easy extractability of intracellular PHA, simultaneously.     

On-chip screening method for cell migration genes based on a transfection microarray

Cell migration plays a major role in a variety of biological processes and a detailed understanding of associated mechanisms should lead to advances in the medical sciences, for example, in drug discovery for cancer therapy. However, the traditional methods used for analysis of cell migration cannot easily be scaled up for high-throughput screening. In this study, we have attempted to develop a novel simple method for high-throughput phenotypic screening for the identification of genes that are required for cell migration. As the appropriate cell line for the method, we found NBT-L2b cells that would be suitable for screening of migration-related genes in our method without influence by other cellular processes. Moreover, the idea for printing both the labeled fibronectin, for identification of the starting region of a cell, and the green fluorescent protein (GFP) expression vector, for identification of cells that had been transfected with siRNA and of the end point of migration, brings a rapid and efficient high-throughput screening procedure. Our new method will lead to an enhanced understanding of cell migration.     

Automatic generation of ultra-pure hydroxide eluent for carbohydrate analysis of environmental samples

High-performance anion-exchange chromatography coupled with pulsed amperometric detection under alkaline conditions has significantly improved our understanding of the function of carbohydrates in cycling of terrestrial carbon. The use of NaOH as an eluent has many advantages for anion analysis, but NaOH solutions are also an excellent trap for atmospheric CO2 resulting in eluent carbonate ion contamination. To minimize the carbonate contamination, eluent generation technology has been marketed that produces ultra-pure hydroxide (OH) eluents on demand. Application of this technology significantly reduced the amounts of OH present in the waste stream and provided faster chromatographic runs with greater sensitivity and precision for determining the monosaccharide composition from environmental samples. Minimizing the salt concentration to < or = 50 mm from acid hydrolysis extractions injected (25 microl injection volume) on column improved reproducibility of analyte retention times and separations. Eluent generation technology eliminated inconsistent OH eluent preparation that is often due to carbonate contamination. The contamination increases retention time variability for monosaccharides released by hydrolysis of biological samples.     

Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. β-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-β-d-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the β-galactosidase—released from the lysed cells—by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

Integrated microfluidic cell culture and lysis on a chip

We present an integrated microfluidic cell culture and lysis platform for automated cell analysis that improves on systems which require multiple reagents and manual procedures. Through the combination of previous technologies developed in our lab (namely, on-chip cell culture and electrochemical cell lysis) we have designed, fabricated, and characterized an integrated microfluidic platform capable of culturing HeLa, MCF-7, Jurkat, and CHO-K1 cells for up to five days and subsequently lysing the cells without the need to add lysing reagents. On-demand lysis was accomplished by local hydroxide ion generation within microfluidic chambers, releasing both proteinacious (GFP) and genetic (Hoescht-stained DNA) material. Sample proteins exposed to the electrochemical lysis conditions were immunodetectable (p53) and their enzymatic activity (HRP) was investigated.     

On-chip pH measurement using functionalized gel-microbeads positioned by optical tweezers

This paper demonstrates local pH measurement in a microchip using a pH-sensing gel-microbead. To achieve this, the gel-microbead made of a hydrophilic photo-crosslinkable resin was functionalized with the pH indicator bromothymol blue (BTB). The primary constituent of this photo-crosslinkable resin is poly(ethylene glycol). Gel-microbeads impregnated with BTB were obtained by stirring the mixture solution, which was composed of the resin, BTB, and an electrolyte solution. The gel-microbead is polymerized by UV illumination. The polymerized gel-microbead can be manipulated by optical tweezers and made to adhere to a glass surface. The local pH was measured from the color of the gel-microbead impregnated with BTB by calibrated color information in the YCrCb color space. We succeeded in measuring the local pH value using the pH-sensing gel-microbead by manipulating and positioning it at the desired point in the microchip.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology

Integrating cell lysis and DNA purification process into a micrototal analytical system (microTAS) is one critical step for the analysis of nucleic acids. On-chip cell lysis based on a chemical method is realized by sufficient blend of blood sample and the lyzing reagent. In this paper two mixing models, T-type mixing model and sandwich-type mixing model, are proposed and simulation of those models is conducted. Result of simulation shows that the sandwich-type mixing model with coiled channel performs best and this model is further used to construct the microfluidic biochip for on-line cell lysis and DNA extraction. The result of simulation is further verified by experiments. It asserts that more than 80% mixing of blood sample and lyzing reagent which guarantees that completed cell lysis can be achieved near the inlet location when the cell/buffer velocity ratio is less than 1:5. After cell lysis, DNA extraction by means of a solid-phase method is implemented by using porous silicon matrix which is integrated in the biochip. During continuous flow process in the microchip, rapid cell lysis and PCR-amplifiable genomic DNA purification can be achieved within 20 min. The potential of this microfluidic biochip is illustrated by pretreating a whole blood sample, which shows the possibility of integration of sample preparation, PCR, and separation on a single device to work as portable point-of-care medical diagnostic system.     

The advantages of cell lysis before blood sample preparation by extraction for HPLC propofol analysis

Propofol (2,6-diisopropylphenol) is a short-acting drug with a large volume of distribution and high body clearance. It is suitable both for the induction of anaesthesia by bolus injection and the maintenance of anaesthesia by repeated injections or a continuous infusion. Examining the drug concentration its analysis in whole blood is recommended. This results from the fact that propofol molecules strongly bind with plasma proteins and cellular blood constituents and blood composition variations are observed between individuals or in different disease states or resulting from transfusion etc. In most cases the HPLC analysis follows the extraction of samples. The degree of propofol binding with blood cells can be different, depending on the blood type, and it can change in time, which may affect the results of the analysis. The paper discusses and shows the necessity of blood cell lysis before the extraction procedure. The cell lysis makes possible to determine the total amount of propofol in blood independently of the degree of propofol binding with cellular blood constituents and its changes.     

On-chip fuel cell: micro direct methanol fuel cell of an air-breathing, membraneless, and monolithic design

This paper proposes a novel design for a microfuel cell as an on-chip power source and demonstrates its fabrication and operation to prove the concept. Its simple design is important from the viewpoints of fabrication (e.g., replication), integration, and compatibility with other microdevices. In testing, the prototype cell was able to generate electric power (maximum: ca. 1.4 microW) on methanol without pumps under both neutral and acidic conditions. As for the size, the electrode part of the cell (two cathodes and one anode) is 400 microns in width and 6 mm in length. The evaluation demonstrated that the proposed design is a promising on-chip power source for miniature devices.     

Chlorovinylidenecarbene: generation from 3,3-dichloropropyne by interaction with potassium hydroxide under phase-transfer catalysis conditions and reactions with alkenes

Chlorination of propargyl aldehyde with PCl₅ in CH₂Cl₂ at –20 °C in the presence of catalytic amounts of pyridine afforded dichloromethylacetylene. Reaction of the latter with powdered KOH under phase-transfer catalysis conditions gave chlorovinylidenecarbene due to -elimination of HCl. In the presence of alkenes the carbene undergoes addition at the double bond giving chlorovinylidenecyclopropanes in 20–45 % yields.For the previous communication, see Ref. 1.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1242–1244, July, 1993.