Localization and quantification of radical production in cavitating flows with luminol chemiluminescent reactions

Hydrodynamic cavitation experiments in microfluidic systems have been performed with an aqueous solution of luminol as the working fluid. In order to identify where and how much reactive radical species are formed by the violent bubble collapse, the resulting chemiluminescent oxidation reaction of luminol was scrutinized downstream of a constriction in the microchannel. An original method was developed in order to map the intensity of chemiluminescence emitted from the micro-flow, allowing us to localize the region where radicals are produced. Time averaged void fraction measurements performed by laser induced fluorescence experiments were also used to determine the cavitation cloud position. The combination void fraction and chemiluminescence two-dimensional mapping demonstrated that the maximum chemiluminescent intensity area was found just downstream of the cavitation clouds. Furthermore, the radical yield can be obtained with our single photon counting technique. The maximum radical production rates of 1.2*10⁷ OH/s and radical production per processed liquid volume of 2.15*10¹⁰ HO/l were observed. The proposed technique allows for two-dimensional characterisation of radical production in the microfluidic flow and could be a quick, non-intrusive way to optimise hydrodynamic cavitation reactor design and operating parameters, leading to enhancements in wastewater treatments and other process intensifications.     

On-line cysteine modification for protein analysis: new probes for electrochemical tagging nanospray mass spectrometry

A series of electrogenerated selective electrophiles based on substituted benzoquinones has been characterized as tags for l-cysteine and cysteine residues in proteins. The electrophiles are generated electrochemically from the corresponding hydroquinones. It is shown from mass spectrometry analysis that the electrogenerated benzoquinone can tag the biomolecules. The rate constants pertaining to the addition of l-cysteine onto the electrogenerated benzoquinones have been determined using electrochemical techniques. The substitution patterns have been unraveled leading to the assessment of site-specific rate constants. It is shown that the rate constants are primarily dependent on the electronic nature of the substituents as expressed by the Hammett substitution constant. The apparent tagging yields observed for l-cysteine in nanospray mass spectrometry experiments do not correspond to the yields expected from the electrochemical study, as the ionisation efficiencies are highly dependent on the tag. Finally, the on-line tagging has been tested using β-lactoglobulin A and myoglobin. Based on these results, it is concluded that the tagging reaction is selective towards cysteine when it takes place in the nanospray interface. The results show that the methodology presented can be used for a rapid characterization and identification of reactive sites in biomolecules.     

Effect of Pressure on Fluid Damping in MEMS Torsional Resonators with Flow Ranging from Continuum to Molecular Regime

High quality factor of dynamic structures at micro and nano scale is exploited in various applications of micro electro-mechanical systems (MEMS) and nano electro-mechanical system. The quality factor of such devices can be very high in vacuum. However, when vacuum is not desirable or not possible, the tiny structures must vibrate in air or some other gas at pressure levels that may vary from atmospheric to low vacuum. The interaction of the surrounding fluid with the vibrating structure leads to dissipation, thus bringing down the quality factor. Depending on the ambient fluid pressure or the gap between the vibrating and the fixed structure, the fluid motion can range from continuum flow to molecular flow giving a wide range of dissipation. The relevant fluid flow characteristics are determined by the Knudsen number which is the ratio of the mean free path of the gas molecule to the characteristic flow length of the device. This number is very small for continuum flow and reasonably big for molecular flow. In this paper, we study the effect of fluid pressure on the quality factor by carrying out experiments on a MEMS device that consists of a double gimbaled torsional resonator. Such devices are commonly used in optical cross-connects and switches. We only vary fluid pressure to make the Knudsen number go through the entire range of continuum flow, slip flow, transition flow, and molecular flow. We experimentally determine the quality factor of the torsional resonator at different air pressures ranging from 760 Torr to 0.001 Torr. The variation of this pressure over six orders of magnitude ensures required rarefaction to range over all flow conditions. Finally, we get the variation of quality factor with pressure. The result indicates that the quality factor, Q, follows a power law, Q ∝P ^–r , with different values of the exponent r in different flow regimes. In the second part of the paper, we propose the use of effective viscosity for considering velocity slip conditions in solving Navier–Stokes equation numerically. This concept is validated with analytical results for a simple case and then compared with the experimental results presented in this paper. The study shows that the effective viscosity concept can be used effectively even for the molecular regime if the air-gap to length ratio is sufficiently small (h ₀/L<0.01). As this ratio increases, the range of validity decreases.     

Single-molecule fluorescence detection in microfluidic channels—the Holy Grail in μTAS?

Both single-molecule detection (SMD) methods and miniaturization technologies have developed very rapidly over the last ten years. By merging these two techniques, it may be possible to achieve the optimal requirements for the analysis and manipulation of samples on a single molecule scale. While miniaturized structures and channels provide the interface required to handle small particles and molecules, SMD permits the discovery, localization, counting and identification of compounds. Widespread applications, across various bioscience/analytical science fields, such as DNA-analysis, cytometry and drug screening, are envisaged. In this review, the unique benefits of single fluorescent molecule detection in microfluidic channels are presented. Recent and possible future applications are discussed.Dedicated to the memory of Wilhelm Fresenius     

Voltammetry Under Microfluidic Control,a Flow Cell Aproach

A generic approach to the design, construction and experimental characterization of novel microelectrochemical reactors (MECR) is presented. Structurally well‐defined rectangular microchannels incorporating electrochemical sensors were fabricated using a propriety photosensitive glass and photolithographic techniques. Microelectrode sensors were produced via evaporation to yield, gold, silver or platinum bands of approximate lengths 10–50 μm. The approach outlined permits cells of dimensions in the range: height 50–100 μm, width 100–500 μm and length 1–3 cm to be accurately constructed, in single or array configurations and were characterized via a voltammetric study utilizing electrolyte solutions containing N,N,N′N′‐Tetramethyl‐1,4‐phenylenediamine. In all cases, the test cells were constructed so that the three dimensional hydrodynamic boundary layer within the cells would significantly influence the reagent transport and therefore the observed current density at the microelectrodes. The current/flow rate relationship observed was analogous to the response of the observed within the macroscopic channel flow cells, where typically the cell design is restricted to configurations where a two dimensional transport analysis can be performed.     

Rapid on-chip postcolumn labeling and high-resolution separations of DNA

When performing genetic analysis on microfluidic systems, labeling the sample DNA for detection is a critical preparation step. Labeling procedures often involve fluorescently tagged primers and PCRs, which lengthen experimental run times and introduce higher levels of complexity, increasing the overall cost per analysis. Alternatively, on-chip labeling techniques based on intercalating dyes permit rapid labeling of DNA fragments. However, as noted in the literature, the stochastic nature of dye-DNA complex formation hinders the native electrophoretic migration of DNA fragments, degrading the separation resolution. In this study, we present a novel method of controllably labeling DNA fragments at the end of the electrophoretic separation channel in a glass microfluidic chip. Permitting the DNA to separate and labeling just before detection, achieves the rapid labeling associated with intercalators while maintaining the high resolution of native DNA separations. Our analyses are completed in minutes, rather than the hours typical of sample prelabeling. We demonstrate an electrophoretic microchip-based intercalator labeling technique that achieves higher resolution performance than reported in the literature to date.     

Micrometer scale gel patterns

A novel method for fabricating micrometer sized gel patterns is described. The presented method involves spin-coating a pre-gel solution on a surface that was chemically treated to modulate its surface energy, creating highly hydrophobic areas on a hydrophilic substrate. Following spin-coating, the gel solution self organizes on the hydrophilic sites. This method offers the advantages of high resolution, self-alignment to pre-patterned electrodes, and a simple straightforward fabrication process. Minimum feature size achieved was approximately 20 μm. The characteristic shrinking and swelling times of gel patterns were measured and found to be around 0.6 s for swelling and 2 s for shrinking (for a 60 μm diameter gel) in agreement with the reduced response time expected for scaled down gel patterns. These results suggest the suitability of these gel patterns as valves or actuators in microfluidic devices. Micron-size gel patterns were also incorporated into microfluidic channels thus demonstrating a new approach to create simple, affordable, microfluidic devices, which incorporate “smart” hydrogels as building elements in a simple fashion.     

Design of a capillary-microreactor for efficient Suzuki coupling reactions

A Pyrex glass capillary (0.4 mm internal diameter) microreactor was developed and used for Suzuki coupling reactions. Capillary-microreactors are more attractive than photolithographic microfluidic devices in terms of simplicity, low cost and ease of handling. Compared with the conventional synthesis procedure, our approach of using a capillary-microreactor offers a convenient and highly efficient means to optimize reaction conditions and the performance of catalysts. The procedure exhibits good precision, reproducibility and high reaction yield for a range of reactants investigated.     

Enhancement of signal-to-noise level by synchronized dual wavelength modulation for light emitting diode fluorimetry in a liquid-core-waveguide microfluidic capillary electrophoresis system

The signal-to-noise level of light emitting diode (LED) fluorimetry using a liquid-core-waveguide (LCW)-based microfluidic capillary electrophoresis system was significantly enhanced using a synchronized dual wavelength modulation (SDWM) approach. A blue LED was used as excitation source and a red LED as reference source for background-noise compensation in a microfluidic capillary electrophoresis (CE) system. A Teflon AF-coated silica capillary served as both the separation channel and LCW for light transfer, and blue and red LEDs were used as excitation and reference sources, respectively, both radially illuminating the detection point of the separation channel. The two LEDs were synchronously modulated at the same frequency, but with 180°-phase shift, alternatingly driven by a same constant current source. The LCW transferred the fluorescence emission, as well as the excitation and reference lights that strayed through the optical system to a photomultiplier tube; a lock-in amplifier demodulated the combined signal, significantly reducing its noise level. To test the system, fluorescein isothiocyanate (FITC)-labeled amino acids were separated by capillary electrophoresis and detected by SDWM and single wavelength modulation, respectively. Five-fold improvement in S/N ratio was achieved by dual wavelength modulation, compared with single wavelength modulation; and over 100-fold improvement in S/N ratio was achieved compared with a similar LCW-CE system reported previously using non-modulated LED excitation. A detection limit (S/N = 3) of 10 nM FITC-labeled arginine was obtained in this work. The effects of modulation frequency on S/N level and on the rejection of noise caused by LED-driver current and detector were also studied.     

Microfluidic three-dimensional biomimetic tumor model for studying breast cancer cell migration and invasion in the presence of interstitial flow

Cell migration and invasion are critical steps in cancer metastasis, which are the major cause of death in cancer patients. Tumor-associated macrophages(TAMs) and interstitial flow(IF) are two important biochemical and biomechanical cues in tumor microenvironment, play essential roles in tumor progression. However, their combined effects on tumor cell migration and invasion as well as molecular mechanism remains largely unknown. In this work, we developed a microfluidic-based 3 D breast cancer model by co-culturing tumor aggregates, macrophages, monocytes and endothelial cells within 3 D extracellular matrix in the presence of IF to study tumor cell migration and invasion. On the established platform, we can precisely control the parameters related to tumor microenvironment and observe cellular responses and interactions in real-time. When co-culture of U937 with human umbilical vein endothelial cells(HUVECs) or MDA-MB-231 cells and tri-culture of U937 with HUVECs and MDA-MB-231 cells, we found that mesenchymal-like MDA-MB-231 aggregates activated the monocytes to TAM-like phenotype macrophages. MDA-MB-231 cells and IF simultaneously enhanced the macrophages activation by the stimulation of colony-stimulating factor 1(CSF-1). The activated macrophages and IF further promoted vascular sprouting via vascular endothelial growth factor(VEGFα) signal and tumor cell invasion. This is the first attempt to study the interaction between macrophages and breast cancer cells under IF condition. Taken together, our results provide a new insight to reveal the important physiological and pathological processes of macrophages-tumor communication. Moreover, our established platform with a more mimetic 3 D breast cancer model has the potential for drug screening with more accurate results.