Preliminary scanning fluorescence detection of a minute particle running along a waveguide implemented microfluidic channel using a light switching mechanism

It has long been thought that an optical sensor, such as a light waveguide implemented total analysis system (TAS), is one of the functional components that will be needed to realize a “ubiquitous human healthcare system” in the near future. We have already proposed the fundamental structure for a light waveguide capable of illuminating a living cell or particle running along a microfluidic channel, as well as of detecting fluorescence even from the extremely weak power of such a minute particle. In order to develop novel functions to detect the internal structure of living cells quickly, an angular scanning method that sequentially changes the direction of illumination of the minute cell or particle may be crucial. In this paper, we investigate fluorescence detection from moving particles by switching the laser power delivery path of plural light waveguides as a preliminary experiment toward this novel method. To construct an experimental system able to incorporate a switching light source mechanism cost effectively, we utilized a conventional TAS chip with plural waveguide pairs arranged in parallel, and a forced vibration mechanism on an optical fiber tip by a piezoelectric actuator. With this system, we performed an experiment to detect extremely weak fluorescence using micro particles with a fluorescent substance attached and an optical TAS chip that incorporated a microfluidic channel and three pairs of laser-power-delivering light waveguide cores. We successfully obtained clear, quasi-triangular-shaped pulses in fluorescent signals from resin particles running across the intersection under three different conditions: (1) a particle with approximately the same velocity as that of a forced-vibrated optical fiber tip of approximately 700 mm/s, (2) a particle with velocity 1 digit smaller than that of an optical fiber tip, and (3) a particle with velocity approximately 1/20 that of an optical fiber tip.     

AC detection of directivity of fluorescence from a minute particle using TAS chip incorporated with radially arranged light-waveguides

Microsystem Technologies - In this paper, we attempt to drastically improve the S/N ratio of detected fluorescence signals from the resin-based optical TAS chip by applying AC-modulated laser power...     

Illumination and scattered light detection using a light source consisting of sub-micrometer defect arrays formed on a extremely flat light waveguide core

We have previously argued that an optical sensor combined total analysis system (TAS) is one of the indispensable functional components needed to realize a “ubiquitous human healthcare” system. To achieve this goal, we have proposed a fundamental structure for illuminating a minute cell or particle running along a microfluidic channel using a flat waveguide construction. It is desirable that the TAS light source should be arranged as close to the specimen flow as possible in order to acquire the necessary optical properties; hence, artificial defects formed on the surface of a flat light waveguide are considered to be a promising candidate for realizing the arbitrary-shaped light source for a highly functional optical TAS structure. Based on this idea, we fabricated a structure, constructing a flat and square light source consisting of rectangular solids, sub-micrometer in size, with a 1-μm thick and a 12-μm wide light waveguide core. We successfully trial-manufactured an optical TAS chip with a fluidic channel containing a 14 × 10-μm cross section, and an extremely flat light waveguide core. We repeatedly confirmed that the defect array could function as an approximately square light source when a 650-nm wavelength laser power was carefully introduced. Furthermore, we developed a hybrid numerical calculation method base on the finite-difference, time-domain method together with the beam propagation method. Utilizing this hybrid method, we evaluated the optical response when a particle runs across the light source while changing the aperture length of a shading mask to obtain signals with both higher intensity and shorter full width at half maximum. The numerical results were compared with experimental results obtained using an image acquisition system, and demonstrated good qualitative accord.     

Fabrication of microfluidic device channel using a photopolymer for colloidal particle separation

We have developed a method of fabricating microfluidic device channels for bio-nanoelectronics system by using high performance epoxy based dry photopolymer films or dry film resists (DFRs). The DFR used was with a trademark name Ordyl SY355 from Elga Europe. The developing and exposing processes as well as the time taken in making the channels are recorded. Finally from those recorded methods, the accurate procedures and time taken for DFR development and exposure have been found and ultimately been consistently used in fabricating our channels. These channels were patterned and sandwiched in between two glass substrates. In our advance, the channel was formed for the colloidal particle separation system. They can be used for handling continuous fluid flow and particle repositioning maneuver using dielectrophoresis that have showed successful results in the separation.     

Minute particle detection using a light-wave-guide incorporated optical total analysis system

An optical sensor combined total analysis system (TAS) is thought to be one of the most effective functional elements in realizing a ubiquitous human healthcare system. In accordance with this concept, we have proposed a fundamental structure of a light-wave-guide incorporated TAS and have developed a micro fluidic channel fabricated chip, where two light waveguides having approximately 10-??m cores were facing each other across a fluidic channel with a width of 12???m. By passing 5-??m-diameter polystyrene particles across the portion where a light-wave-guide and the fluidic channel intersect at a right angles, we confirmed that changes to transmitted light and scattered light were detectable even with an extremely low laser source power of 5???W, and they increased proportionally as the introduced laser power became higher. Increasing the introduced laser power clearly increased the correlation between the transmitted and scattered light change. Here we discuss in detail the two different correlation tendencies that appeared in the correlation maps.     

Magnetic particle dosing and size separation in a microfluidic channel

Separation of functional magnetic particles or magnetically labeled entities is a key feature for bioanalytical or biomedical applications and therefore also an important component of lab-on-a-chip devices for biological applications. We present a novel integrated microfluidic magnetic bead manipulation device, comprising dosing of magnetic particles, controlled release and subsequent magnetophoretic size separation with high resolution. The system is designed to meet the requirements of specific bioassays, in particular of on-chip agglutination assays for the detection of rare analytes by particle coupling as doublets. Integrated soft-magnetic microtips with different shapes provide the magnetic driving force of the bead manipulation protocol. The magnetic tips that serve as field concentrators of an external electromagnetic field, are positioned in close contact to a microfluidic channel in order to generate high magnetic actuation forces. Mixtures of 1.0 μm and 2.8 μm superparamagnetic beads have been used to characterize the system. Magnetophoretic size separation with high resolution was performed in static conditions and in continuous flow mode. In particular, we could demonstrate the separation of 1.0 μm single beads and doublets in a sample flow.     

On-demand particle enrichment in a microfluidic channel by a locally controlled floating electrode

A flexible strategy for the on-demand control of the particle enrichment and positioning in a microfluidic channel is proposed and demonstrated by the use of a locally controlled floating metal electrode attached to the channel bottom wall. The channel is subjected to an axially acting global DC electric field, but the degree of charge polarization of the floating electrode is governed largely by a local control of the voltage applied to two micron-sized control electrodes (CEs) on either side of the floating electrode (FE). This strategy allows an independent tuning of the electrokinetic phenomena engendered by the floating electrode regardless of the global electric field across the channel, thus making the method for particle manipulation far more versatile and flexible. In contrast to a dielectric microchannel wall possessing a nearly uniform surface charge (or zeta potential), the patterned metal strip (floating electrode) is polarized under electric field resulting in a non-uniform distribution of the induced surface charge with a zero net surface charge, and accordingly induced-charge electro-osmotic (ICEO) flow. The ICEO flow can be regulated by the control electric field through tuning the magnitude and polarity of the voltage applied to the CEs, which in turn affects both the hydrodynamic field as well as the particle motion. By controlling the control electric field, on-demand control of the particle enrichment and its position inside a microfluidic channel has been experimentally demonstrated.     

Cell separation in a microfluidic channel using magnetic microspheres

Magnetophoretic isolation of biological cells in a microfluidic environment has strong relevance in biomedicine and biotechnology. A numerical analysis of magnetophoretic cell separation using magnetic microspheres in a straight and a T-shaped microfluidic channel under the influence of a line dipole is presented. The effect of coupled particle–fluid interactions on the fluid flow and particle trajectories are investigated under different particle loading and dipole strengths. Microchannel flow and particle trajectories are simulated for different values of dipole strength and position, particle diameter and magnetic susceptibility, fluid viscosity and flow velocity in both the microchannel configurations. Residence times of the captured particles within the channel are also computed. The capture efficiency is found to be a function of two nondimensional parameters, α and β. The first parameter denotes the ratio of magnetic to viscous forces, while the second one represents the ratio of channel height to the distance of the dipole from the channel wall. Two additional nondimensional parameters γ (representing the inverse of normalized offset distance of the dipole from the line of symmetry) and σ (representing the inverse of normalized width of the outlet limbs) are found to influence the capture efficiency in the T-channel. Results of this investigation can be applied for the selection of a wide range of operating and design parameters for practical microfluidic cell separators.     

Focusing microparticles in a microfluidic channel with ferrofluids

We report a novel on-chip microparticles focusing technique using stable magnetic nanoparticles suspension (i.e., ferrofluids). The principle of focusing is based on magnetic buoyancy forces exerted on non-magnetic particles within ferrofluids under non-uniform magnetic field. The design, modeling, fabrication, and characterization of the focusing scheme are presented. Focusing of 4.8, 5.8, and 7.3 ??m microparticles at various flow rates are demonstrated in a microfluidic channel. Our scheme is simple, low-cost, and label-free compared to other existing techniques.     

A novel particle filter implementation for a multiple-vehicle detection and tracking system using tail light segmentation

This paper proposes a vision-based multiple vehicle automatic detection and tracking system which can be applied in different environments. To detect vehicles, tail light position is utilized for fast vehicle candidate localization. A back propagation neural network (BPNN) trained by a Gabor feature set is used. BPNN verifies vehicle candidates and ensures detection system robustness. In the vehicle tracking step, to overcome multiple vehicle tracking challenges, partial vehicle occlusion and temporarily missing vehicle problems, this paper propose a novel method implementing a particle filter. The color probability distribution function (CPDF) of detected vehicles is used twice in the vehicle tracking sub-system. Firstly, CPDF is adopted to seek potential target vehicle locations; secondly, CPDF is used to measure the similarity of each particle for target vehicle position estimation. Because of various illuminations or target vehicle distances, the same vehicle will generate different CPDFs; the initial CPDF cannot guarantee long-term different scale vehicle tracking. To overcome these problems, an accurate tracking result, which is chosen by a trained BPNN, is used to update target vehicle CPDF. In our experiments, the proposed algorithm showed 84% accuracy in vehicle detection. Videos collected from highways, urban roads and campuses are tested in our system. The system performance makes it appropriate for real applications.     

Improving particle detection sensitivity of a microfluidic resistive pulse sensor by a novel electrokinetic flow focusing method

A novel and simple method of improving the particle detection sensitivity of a microfluidic resistive pulse sensor was presented in this paper. This novel electrokinetic flow focusing method utilizes a focusing solution (with high resistivity) flowing from the upstream focusing channel to the downstream focusing channel. The focusing solution in the sensing gate works like a virtual insulation wall that greatly narrows the gate and thus improves the detection sensitivity. An equation was derived to relate the magnitude of the output signal to the resistivity and the width of the focusing solution. The width of the focused particle solution under different voltages was numerically predicted. The results show that the magnitude of output signal increases with the decrease in the width of the focused particle stream. More importantly, the detection sensitivity can be improved by decreasing the space occupied by the focusing solution in the upstream and downstream channels as much as possible. Detection of 1 μm particle with a sensing gate of 30 × 40 × 10 μm (width × length × height) was successfully achieved. The proposed method is simple and advantageous in detecting smaller particles without fabricating a small sensing gate.     

Simulation of advanced microfluidic systems with dissipative particle dynamics

Computational Fluid Dynamics (CFD) is widely and successfully used in standard design processes for microfluidic μTAS devices. But for an increasing number of advanced applications involving the dynamics of small groups of beads, blood cells or biopolymers in microcapillaries or sorting devices, novel simulation techniques are called for. Representing moving rigid or flexible extended dispersed objects poses serious difficulties for traditional CFD schemes. Meshless, particle-based simulation approaches, such as Dissipative Particle Dynamics (DPD) are suited for addressing these complicated flow problems with sufficient numerical efficiency. Objects can conveniently be represented as compound objects embedded seamlessly within an explicit model for the solvent. However, the application of DPD and related methods to realistic problems, in particular the design of microfluidics systems, is not well developed in general. With this work, we demonstrate how the method appears when used in practice, in the process of designing and simulating a specific microfluidic device, a microfluidic chamber representing a prototypical bead-based immunoassay developed in our laboratory (Glatzel et al. 2006a, b; Riegger et al. 2006). Thomas Steiner: born Glatzel.     

Continuous generation of hydrogel beads and encapsulation of biological materials using a microfluidic droplet-merging channel

In this paper, we describe a method for encapsulation of biomaterials in hydrogel beads using a microfluidic droplet-merging channel. We devised a double T-junction in a microfluidic channel for alternate injection of aqueous fluids inside a droplet unit carried within immiscible oil. With this device, hydrogel beads with diameter <100 μm are produced, and various solutions containing cells, proteins and reagents for gelation could merge with the gel droplets with high efficiency in the broad range of flow rates. Mixing of reagents and reactions inside the hydrogel beads are continuously observed in a microchannel through a microscope. By enabling serial injection of each liquid with the dispersed gel droplets after they are produced from the oil-focusing channel, the device simplifies the sample preparation process, and gel-bead fabrication can be coupled with further assay continuously in a single channel. Instantaneous reactions of enzyme inside hydrogel and in-situ formation of cell-containing beads with high viability are demonstrated in this report.     

Design of microfluidic channel networks with specified output flow rates using the CFD-based optimization method

We report an effective, easy-to-use, computational fluid dynamics-based optimization method for designing purely resistive microfluidic networks with desired flow rates at user-specified outlets. The detailed topology and shape of the microchannel networks are obtained by minimizing the fluidic resistance of channels under a fixed driving flow rate at the inlet. This proposed method allows flexibility in setting up the relative positions among the inlet and outlets so that the layout of channel networks can be compactly adjusted based on the specific design requirements.     

Size-selective separation of magnetic nanospheres in a microfluidic channel

This paper reported an efficient method to size-selective separate magnetic nanospheres using a self-focusing microfluidic channel equipped with a permanent magnet. Under external magnetic field, the magnetophoresis force exerted on particles leads to size-dependent deflections from their laminar flow paths and results in effective particles separation. By adjusting the distance between magnet and main path of channel, we obtained two monodisperse nanosphere samples (Ca. 90 nm, Ca. 160 nm) from polydispersing particles solution whose diameters varied from 40 to 280 nm. Based on the magnetostatic and laminar flow models, numerical simulations were also used to predict and optimize the nanospheres migrations. Two thresholds of particles diameters were obtained by the simulations and diverse at each position of magnet. Therefore, appropriate position of the magnet could be determined at a certain particle sizes’ range when the flow rate of the two inlets remains unchanged.     

Characterization of DNA hybridization kinetics in a microfluidic flow channel

In this investigation we report on the influence of volumetric flow rate, flow velocity, complementary DNA concentration, height of a microfluidic flow channel and time on DNA hybridization kinetics. A syringe pump was used to drive Cy3-labeled target DNA through a polydimethylsiloxane (PDMS) microfluidic flow channel to hybridize with immobilized DNA from the West Nile Virus. We demonstrate that a reduction of channel height, while keeping a fixed volumetric flow rate or a fixed flow velocity, enhances mass transport of target DNA to the capture probes. Compared to a passive hybridization, the DNA hybridization in the microfluidic flow channel generates higher fluorescence intensities for lower concentration of target DNA during the same fixed period of time. Within a fixed 2 min time period the fastest DNA hybridization at a 50 pM concentration of target DNA is achieved with a continuous flow of target DNA at the highest flow rate and the lowest channel height.     

Fabrication of microfluidic channel utilizing silicone rubber with vacuum casting

Microfluidic patterns of 100 μm in width and 50 μm height were replicated from a master using vacuum casting with silicone rubber. These silicone copies are subjected to thorough analysis for dimensional accuracy against the master pattern. Analysis of experimental results shows repeatability of the silicone rubber molds. To test the limits of vacuum casting with silicone rubber, an attempt was undertaken to replicate 8 μm microchannel and submicron features. The results show that casting of microfluidic channel via vacuum casting has high repeatability.