Bipolar Electrochemiluminescence Imaging: A Way to Investigate the Passivation of Silicon Surfaces

This work depicts the original combination of electrochemiluminescence (ECL) and bipolar electrochemistry (BPE) to map in real-time the oxidation of silicon in microchannels. We fabricated model silicon-PDMS microfluidic chips, optionally containing a restriction, and monitored the evolution of the surface reactivity using ECL. BPE was used to remotely promote ECL at the silicon surface inside microfluidic channels. The effects of the fluidic design, the applied potential and the resistance of the channel (controlled by the fluidic configuration) on the silicon polarization and oxide formation were investigated. A potential difference down to 6 V was sufficient to induce ECL, which is two orders of magnitude less than in classical BPE configurations. Increasing the resistance of the channel led to an increase in the current passing through the silicon and boosted the intensity of ECL signals. Finally, the possibility of achieving electrochemical reactions at predetermined locations on the microfluidic chip was investigated using a patterning of the silicon oxide surface by etched micrometric squares. This ECL imaging approach opens exciting perspectives for the precise understanding and implementation of electrochemical functionalization on passivating materials. In addition, it may help the development and the design of fully integrated microfluidic biochips paving the way for development of original bioanalytical applications.     

Development of a microfluidic biosensor module for pathogen detection

The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 microL min(-1). The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.     

A pressure driven injection system for an ultra-flat chromatographic microchannel

A pressure-actuated on-chip injection system has been developed that is compatible with shallow microchannels with a very large aspect ratio, i.e. 1 microm deep and up to 1000 microm wide. Such channels offer potential advantages in the miniaturisation of liquid chromatography and other separation methods as they allow high loadability and low sample dispersion at the same time. Computational fluid dynamics simulations were performed to predict the flow profiles and the transport of a sample in the system and to justify the injection principle. Based on these simulations, a prototype integrated into a chip for hydrodynamic chromatography has been realised and tested experimentally. The performance of the device is satisfactory and the results are in qualitative agreement with the numerical models.     

Activity and lifetime of urease immobilized using layer-by-layer nano self-assembly on silicon microchannels

Urease has been immobilized and layered onto the walls of manufactured silicon microchannels. Enzyme immobilization was performed using layer-by-layer nano self-assembly. Alternating layers of oppositely charged polyelectrolytes, with enzyme layers “encased” between them, were deposited onto the walls of the silicon microchannels. The polycations used were polyethylenimine (PEI), polydiallyldimethylammonium (PDDA), and polyallylamine (PAH). The polyanions used were polystyrenesulfonate (PSS) and polyvinylsulfate (PVS). The activity of the immobilized enzyme was tested by pumping a 1 g/L urea solution through the microchannels at various flow rates. Effluent concentration was measured using an ultraviolet/visible spectrometer by monitoring the absorbance of a pH sensitive dye. The architecture of PEI/PSS/PEI/urease/PEI with single and multiple layers of enzyme demonstrated superior performance over the PDDA and PAH architectures. The precursor layer of PEI/PSS demonstrably improved the performance of the reactor. Conversion rates of 70% were achieved at a residence time of 26 s, on d 1 of operation, and >50% at 51 s, on d 15 with a six-layer PEI/urease architecture.     

Microfluidic chip accomplishing self-fluid replacement using only capillary force and its bioanalytical application

A polymer microfluidic chip accomplishing automated sample flow and replacement without external controls and an application of the chip for bioanalytical reaction were described. All the fluidic operations in the chip were achieved by only natural capillary flow in a time-planned sequence. For the control of the capillary flow, the geometry of the channels and chambers in the chip was designed based on theoretical considerations and numerical simulations. The microfluidic chip was made by using polymer replication techniques, which were suitable for fast and cheap fabrication. The test for a biochemical analysis, employing an enzyme (HRP)-catalyzed precipitation reaction, exhibited a good performance using the developed chip. The presented microfluidic method would be applicable to biochemical lab-on-a-chips with integrated fluid replacement steps, such as affinity elution and solution exchange during biosensor signaling.     

A new optical platform for biosensing based on fluorescence anisotropy

A novel fluorescence-based optical platform for the interrogation of an optical biochip was designed and developed. The optical biochip was made of poly(methyl methacrylate) (PMMA) formed by two pieces of PMMA appropriately shaped in order to obtain four microchannels that are 500-μm wide and 400-μm high. The lower part includes the microchannels and the inlet and outlet for the fluidics, while the sensing biolayer was immobilized on the upper part. The optical signal comprised the fluorescence emitted by the biolayer, which was anisotropically coupled to the PMMA cover and suitably guided by the PMMA chip. The potentiality of the optical chip as a biosensor was investigated by means of a direct IgG/anti-IgG interaction carried out inside the flow channels. The mouse-IgG was covalently immobilized on the internal wall of the PMMA cover, and the Cy5-labelled anti-mouse IgG was used for the specific interaction. Several chemical treatments of the PMMA surface were investigated, poly(L-lactic acid), Eudragit L100 and NaOH, in order to obtain the most effective distribution of carboxylic groups useful for the covalent immobilisation of the mouse-IgG. The treatment with Eudragit L100 was found to be the most successful. Limits of detection and quantification of 0.05 μg mL⁻¹ and 0.2 μg mL⁻¹, respectively, were obtained with the configuration described.     

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.     

Obtaining calcium carbonate in a multiphase system by the use of new rotating disc precipitation reactor

Rotating disc reactor (RDR) was constructed to conduct gas–liquid–solid reactions with controlled reagent transfer from gaseous to liquid phase. The concept is based on continuous formation of thin liquid films at a surface of rotating discs where the mass transfer proceed in diffusion–convective way. The reactor was employed to run precipitation reaction of CaCO₃ via carbon dioxide absorption in lime slurry. During each reaction pH changes and Ca²⁺ concentration in time were measured. Disc rotations and gas flows were changed during the experiment and their influence on the obtained CaCO₃ powders has been examined and fully discussed.     

Silicon photonic sensors incorporated in a digital microfluidic system

Label-free biosensing with silicon nanophotonic microring resonator sensors has proven to be an excellent sensing technique for achieving high-throughput and high sensitivity, comparing favorably with other labeled and label-free sensing techniques. However, as in any biosensing platform, silicon nanophotonic microring resonator sensors require a fluidic component which allows the continuous delivery of the sample to the sensor surface. This component is typically based on microchannels in polydimethylsiloxane or other materials, which add cost and complexity to the system. The use of microdroplets in a digital microfluidic system, instead of continuous flows, is one of the recent trends in the field, where microliter- to picoliter-sized droplets are generated, transported, mixed, and split, thereby creating miniaturized reaction chambers which can be controlled individually in time and space. This avoids cross talk between samples or reagents and allows fluid plugs to be manipulated on reconfigurable paths, which cannot be achieved using the more established and more complex technology of microfluidic channels where droplets are controlled in series. It has great potential for high-throughput liquid handling, while avoiding on-chip cross-contamination. We present the integration of two miniaturized technologies: label-free silicon nanophotonic microring resonator sensors and digital microfluidics, providing an alternative to the typical microfluidic system based on microchannels. The performance of this combined system is demonstrated by performing proof-of-principle measurements of glucose, sodium chloride, and ethanol concentrations. These results show that multiplexed real-time detection and analysis, great flexibility, and portability make the combination of these technologies an ideal platform for easy and fast use in any laboratory.

Convenient formation of nanoparticle aggregates on microfluidic chips for highly sensitive SERS detection of biomolecules

Microfluidic chips combined with surface-enhanced Raman spectroscopy (SERS) offer an outstanding platform for rapid and high-sensitivity chemical analysis. However, it is nontrivial to conveniently form nanoparticle aggregrates (as SERS-active spots for SERS detection) in microchannels in a well-controlled manner. Here, we present a rapid, highly sensitive and label-free analytical technique for determining bovine serum albumin (BSA) on a poly(dimethylsiloxane) (PDMS) microfluidic chip using SERS. A modified PDMS pneumatic valve and nanopost arrays at the bottom of the fluidic microchannel are used for reversibly trapping gold nanoparticles to form gold aggregates, creating SERS-active spots for Raman detection. We fabricated a chip that consisted of a T-shaped fluidic channel and two modified pneumatic valves, which was suitable for fast loading of samples. Quantitative analysis of BSA is demonstrated with the measured peak intensity at 1,615 cm⁻¹ in the surface-enhanced Raman spectra. With our microfluidic chip, the detection limit of Raman can reach as low as the picomolar level, comparable to that of normal mass spectrometry.     

Fabrication of plastic microchips by hot embossing

Plastic microchips with microchannels (100 microm wide, 40 microm deep) of varying designs have been fabricated in polymethylmethacrylate by a hot embossing process using an electroform tool produced starting with silicon chip masters. Hot-embossed chips were capped with a polymethylmethacrylate top using a proprietary solvent bonding process. Holes were drilled through the top of the chip to allow access to the channels. The chips were tested with fluid and shown to fill easily. The seal between the top of the chip and the hot embossed base was effective, and there was no leakage from the channels when fluid was pumped through the microchannels. The chips were also tested with a semen sample and the plastic chip performed identically to the previous silicon-glass and glass versions of the chip. This microfabrication technique offers a viable and potentially high-volume low cost production method for fabricating transparent microchips for analytical applications.     

Velocity measurement of particulate flow in microfluidic channels using single point confocal fluorescence detection.

This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (0.93 microm diameter) passing through a focused laser beam at a variety of flow rates (50 nL min(-1)-8 microL min(-1)). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate velocity data for the flowing particles. Fluid manipulation is achieved by hydrodynamically pumping fluid through microchannels (150 microm wide and 50 microm deep) structured in a polydimethylsiloxane (PDMS) substrate. The mean fluorescence burst frequency is shown to be directly proportional to flow speed. Furthermore, the Poisson recurrence time and width of recovered autocorrelation curves is demonstrated to be inversely proportional to flow speed. The component-based confocal fluorescence detection system is simple and can be applied to a diversity of planar chip systems. In addition, velocity measurement only involves interrogation of the fluidic system at a single point along the flow stream, as opposed to more normal multiple-point measurements.     

Simultaneous sample washing and concentration using a "trapping-and-releasing" mechanism of magnetic beads on a microfluidic chip

Simultaneous washing and concentration of functionalized magnetic beads in a complex sample solution were demonstrated by applying a rotational magnetic actuation system to a microfluidic chip under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to the fluidic channel carrying a magnetic bead suspension allows trapping and releasing of the beads along the fluidic channel in a periodical manner. Each trapping and releasing event resembles one washing cycle. A purification efficiency of magnetic beads out of a mixed magnetic and non-magnetic bead sample solution of 83±4% at a flow rate of 0.5 μL min(-1), and a magnetic bead recovery or concentration efficiency of 91±5% were achieved using a flow rate of 0.2 μL min(-1). The detection performance of the device was experimentally evaluated with two different bioassays, using either streptavidin-coated magnetic beads in combination with biotinylated fluorescent isothiocyanate (FITC), or a mouse antigen (Ag)-antibody (Ab) system.     

TiO2-ZrO2 affinity chromatography polymeric microchip for phosphopeptide enrichment and separation

We fabricated a TiO(2)-ZrO(2) affinity chromatography micro-column on 2 mm PMMA plates, and demonstrated the enrichment and separation of (a) a standard mono- and tetra-phosphopeptide, and (b) phosphopeptides contained in a tryptic digest of β-Casein. The chromatography column consisted of 32 parallel microchannels with common input and output ports and was fabricated by lithography directly on the polymeric substrate followed by plasma etching (i.e. standard MEMS processing) and sealed with lamination. The liquid deposited TiO(2)-ZrO(2) stationary phase was characterized by X-ray diffraction and was found to be mostly TiO(2) and ZrO(2) in crystalline phases. Off-chip UV detection and MALDI MS identification of the separated effluents were used. The chip had a capacity of >1.4 μg (0.7 nmol) of a prototype mono-phosphopeptide and a recovery of 94 ± 3%, and can be used with small samples (less than 0.1 μL depending on the syringe pump used). The chip design allows an expansion of its capacity by means of increasing the number of parallel microchannels at a constant sample volume. Our approach provided an alternative to off-line extraction tips (with typical capacities of 1-2 μg and sample volumes of 1-10 μL), and to on-chip efforts based on packed bed and frit formats.     

A microfluidic device with integrated fluorimetric detection for flow injection analysis

This work describes the development of flow analysis microsystems with integrated fluorimetric detection cells. Channels (width of 300–540 μm and depth of 200–590 μm) were manufactured by deep-UV lithography in urethane–acrylate (UA) resin. Plastic optical fibers (diameter of 250 μm) were coupled to a 2.0-mm-long detection channel in order to guide the excitation radiation from an LED (470 nm) and collect the emitted radiation at a right angle towards a photomultiplier. A single-line miniaturized system, with a total internal volume of 10.4 μL, was evaluated by means of standard fluorescein solutions (0.53–2.66 μmol L⁻¹, pH 8.5). The analytical signals presented a linear relationship in the concentration range studied, with a relative standard deviation of 1.9% (n = 5), providing a detection limit of 0.37 μmol L⁻¹ and an analytical frequency of 60 samples/h, using a flow rate of 60 μL min⁻¹. Optical microscopy images and videos acquired in real time for the hydrodynamic injection of 130 and 320 nL of sample solutions indicated the good performance of the proposed sampling strategy. Another microsystem with a total internal volume of 38 μL was developed, incorporating a confluence point for two solutions. This device was applied to the determination of the total concentration of Ca²⁺ and Mg²⁺ in commercial mineral waters using the calcein method. Microscopy images and videos demonstrated the mixing efficiency of the solutions in the microchannels. A linear relationship was observed for the analytical signal in the Ca²⁺ concentration range from 25 to 125 μmol L⁻¹, with relative standard deviations of 3.5%. The analysis of mineral waters with the proposed system provided results that did not differ significantly from those obtained by the EDTA titration method at a confidence level of 95%. These results demonstrate the viability of developing micro flow injection systems with an integrated fluorimetric detection cell.      

Fast sol–gel technology: from fabrication to applications

A novel method to prepare crack-free sol–gel materials without shrinkage is reviewed. The method allows fabrication of a viscous sol–gel resin in a few minutes followed by either thermal-curing or UV-curing requiring several hours or several minutes, respectively. The method is distinguished by the short time required to achieve a solid monolith. The fast sol–gel method uses a combination of organically modified alkoxides with traditional alkoxides as precursors, to produce a final product which is an organic-inorganic hybrid with properties that vary from silicone rubbers to silica glass. Optical and physical properties, such as refractive index and thermal expansion, can be engineered by controlling the ratio between the precursors. This class of materials is a promising candidate for preparation of optical elements such as waveguides and submicron structured replicas and can also be used as an optical bonding material. This paper reviews the fast sol–gel technology, as well as methods to characterize the process and its final products. Various applications of fast sol–gel materials are presented.     

Design of a microfluidic device for comprehensive spatial two‐dimensional liquid chromatography

This study discusses the design aspects for the construction of a microfluidic device for comprehensive spatial two‐dimensional liquid chromatography. In spatial two‐dimensional liquid chromatography each peak is characterized by its coordinates in the plane. After completing the first‐dimension separation all fractions are analyzed in parallel second‐dimension separations. Hence, spatial two‐dimensional liquid chromatography potentially provides much higher peak‐production rates than a coupled column multi‐dimensional liquid chromatography approach in which the second‐dimension analyses are performed sequentially. A chip for spatial two‐dimensional liquid chromatography has been manufactured from cyclic olefin copolymer and features a first‐dimension separation channel and 21 parallel second‐dimension separation channels oriented perpendicularly to the former. Compartmentalization of first‐ and second‐dimension developments by physical barriers allowed for a preferential flow path with a minimal dispersion into the second‐dimension separation channels. To generate a homogenous flow across all the parallel second‐dimension channels, a radially interconnected flow distributor containing two zones of diamond‐shaped pillars was integrated on‐chip. A methacrylate ester based monolithic stationary phase with optimized macroporous structure was created in situ in the confines of the microfluidic chip. In addition, the use of a photomask was explored to localize monolith formation in the parallel second‐dimension channels. Finally, to connect the spatial chip to the liquid chromatography instrument, connector ports were integrated allowing the use of Viper fittings. As an alternative, a chip holder with adjustable clasp locks was designed that allows the clamping force to be adjusted.     

Preparation and trans–cis isomerization of azobenzene-containing TiO2/ormosils hybrid films derived at a low temperature sol–gel process for photonic applications

Azobenzene-containing TiO₂/γ-glycidoxypropyltrimethoxysilane and methyltrimethoxysilane hybrid films are prepared by combining a low temperature sol–gel process and a spin-coating technique. The trans–cis isomerization of azobenzene small molecules inside organic–inorganic hybrid films is induced by a photoirradiation under UV light. It is noted that below a baking temperature of 150 °C is necessary for the hybrid films in optical storage or optical switching applications. The change of the refractive index and thickness of the hybrid films with real-time heating temperature are observed by using a prism coupling technique. The structural properties of the hybrid films are also characterized and investigated by thermal gravimetric analysis, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The results indicate that the as-prepared hybrid films might allow directly integrating the optical storage or optical switching devices with the waveguide devices on the same chip.     

Integrated sorption–energy-dispersive X-ray fluorescence detection for automatic determination of lead and cadmium in low-concentration solutions

Sorbent material packed in a PTFE laboratory-made flow cell located in the specimen holder of an energy-dispersive X-ray fluorescence (EDXRF) detector has been used for in situ solid-phase extraction (SPE) preconcentration–detection of metals. The flow cell was connected to a single-channel flow-injection (FI) manifold (for full automation of the steps and proper development of the method) by two PTFE tubes of 0.5-mm inner diameter introduced into the spectrometer specimen holder by a small orifice without distortion or modification of the instrument. The optical window open in the PTFE flow cell was adjusted to the X-ray irradiation zone of the spectrometer and fixed to it. The approach was tested by using both Pb and Cd aqueous solutions and a Dowex 50 cation-exchange resin as a sorbent, and flushing the sample through the flow cell for EDXRF measurements after removal of the sample matrix. The limits of detection and the limits of quantification (LOQs) thus obtained were 0.15 and 0.5 μg for Pb and 0.3 and 0.8 μg for Cd, respectively, values that allow the approach to be used for the analysis of drinking water by injecting a 100-mL sample into the FI manifold, taking into account the EC drinking water directives. The linear dynamic ranges are between the LOQ and 600 μg for both analytes. The method was validated by the standard addition method using tap-water samples. In addition, the integrated SPE–EDXRF approach enables the study of the variables influencing the sorption step–namely the effects of the volume of sample flushed through the column, concentrations of the analytes in the sample, breakthrough volume of the resin, elution profiles, sample pH and retention and elution flow rates–in an automatic, cheap, fast and precise way.     

Second-order energy components in basis-set-superposition-error-free intermolecular perturbation theory

 Recently a basis-set-superposition-error-free second-order perturbation theory was introduced based on the “chemical Hamiltonian approach” providing the full antisymmetry of all wave functions by using second quantization. Subsequently, the “Heitler–London” interaction energy corresponding to the sum of the zero- and first-order perturbational energy terms was decomposed into different physically meaningful components, like electrostatics, exchange and overlap effects. The first-order wave function obtained in the framework of this perturbation theory also consists of terms having clear physical significance: intramolecular correlation, polarization, charge transfer, dispersion and combined polarization–charge transfer excitations. The second-order energy, however, does not represent a simple sum of the respective contributions, owing to the intermolecular overlap. Here we propose an approximate energy decomposition scheme by defining some “partial Hylleraas functionals” corresponding to the different physically meaningful terms of the first-order wave functions. The sample calculations show that at large and intermediate intermolecular distances the total second-order intermolecular interaction energy contribution is practically equal to the sum of these “physical” terms, while at shorter distances the overlap-caused interferences become of increasing importance. Received: 18 June 2001 / Accepted: 28 August 2001 / Published online: 16 November 2001