Thin-film electrode based droplet detection for microfluidic systems

We report on a droplet-producing microfluidic system with electrical impedance-based detection. The microfluidic devices are made of polydimethylsiloxane (PDMS) and glass with thin film electrodes connected to an impedance-monitoring circuit. Immiscible fluids containing the hydrophobic and hydrophilic phases are injected with syringe pumps and spontaneously break into water-in-oil droplet trains. When a droplet passes between a pair of electrodes in a medium having different electrical conductivity, the resulting impedance change signals the presence of the particle for closed-loop feedback during processing. The circuit produces a digital pulse for input into a computer control system. The droplet detector allows estimation of a droplet's arrival time at the microfluidic chip outlet for dispensing applications. Droplet detection is required in applications that count, sort, and direct microfluidic droplets. Because of their low cost and simplicity, microelectrode-based droplet detection techniques should find applications in digital microfluidics and in three-dimensional printing technology for rapid prototyping and biotechnology.     

Determination of dynamic contact angles within microfluidic devices

Here, we report a single-point detection method for the determination of dynamic surface conditions inside microfluidic channels. The proposed method is based on monitoring fluorescence amplitude as a function of the convolution of a laser beam with segmented flow consisting of two immiscible liquids, one containing fluorescent dye. The fluorescence amplitude is determined by the flow rate and the droplet shape, which is affected by the channel surface properties. We modeled the interaction of a droplet and a laser beam via computer-aided design software, using the laser beam location in relation to the droplet shape as a parameter. The method was applied to fused silica capillaries with both unmodified and modified surfaces, with segmented flow exhibiting water contact angles of ≈?30° and ≈?100°, respectively. The method allows discrimination between hydrophillic and hydrophobic surfaces, as well as the quality of the treatment. The results were verified using fluorescence imaging of the droplets via a stroboscopic technique. We also applied this method to the analysis of microfabricated channels with non-circular cross sections. We demonstrated that the technique enables the determination of the hydrophobicity of channel surfaces, a crucial property required for the generation of segmented flow or emulsions for applications such as digital PCR.     

Detection of microdroplet size and speed using capacitive sensors

Detection of the presence, size and speed of microdroplets in microfluidic devices is presented using commercially available capacitive sensors which make the droplet based microfluidic systems scalable and inexpensive. Cross-contamination between the droplets is eliminated by introducing a passivation layer between the sensing electrodes and droplets. A simple T-junction generator is used to generate droplets in microchannels. Coplanar electrodes are used to form a capacitance through the microfluidic channel. The change in capacitance due to the presence of a droplet in the sensing area is detected and used to determine the size and speed of the droplet. The design of a single pair of electrodes is used to detect the presence of a droplet and the interdigital finger design is used to detect the size and speed of the droplet. An analytical model is developed to predict the detection signal and guide the experimental optimization of the sensor geometry. The measured droplet information is displayed through a Labview interface in real-time. The use of capacitance sensors to monitor droplet sorting at a T-junction is also presented. The discussions in this paper can be generalized to any droplet detection application and can serve as a guideline in sensor selection.     

Droplet microfluidic preparation of au nanoparticles-coated chitosan microbeads for flow-through surface-enhanced Raman scattering detection

An integrated microfluidic device was fabricated to enable on-chip droplet forming, trapping, fusing, shrinking, reaction and producing functional microbeads for a flow-through single bead-based molecule detection. Dielectrophoresis (DEP) force was used to transport target polymer droplets into different predefined microwells, where the droplets were fused through electrocoalescence to form a new one with a desired diameter. In a continuous water loss process with water diffusion to oil phase, the polymer droplet was shrunken and solidified to form a polymer microbead. For a demonstration, Au nanoparticles-coated chitosan microbeads were in situ fabricated through droplet trapping, fusion and shrinking, followed by synthesis of Au nanoparticles on the microbead surface via a photoreduction process. The produced Au nanoparticle/chitosan microbead embedded in the microwell resulted in a highly sensitive, flow-through surface-enhanced Raman scattering (SERS) detection of Rhodamine 6G (R6G). This work successfully demonstrates an integrated droplet based lab-on-a chip and its application to fabricate an extremely high-throughput single bead based detection platform.     

Oscillations in light-triggered logic microfluidic circuit

Control of droplets in microfluidic environments has numerous applications ranging from analysis and sample preparation for biomaterials synthesis (Mann and Ozin Nature 382:313–318, 1996) and medical diagnostics (Pipper et al. Nat Med 13:1259–1263, 2007) to photonics (Schmidt and Hawkins Nat Photonics 5:598–604, 2011). Here we study the oscillations present in a microfluidic circuit capable of sorting curable droplets on demand by triggering the circuit with UV-light. Prior to this paper we showed that a simple circuit can self-sort particles and produce a binary output, sorted or rejected stream of particles, based on the hydrodynamic resistance induced by the particles as they flow through the microfluidic channels. We showed that the cross-linking of droplets can modulate the resistance, and demonstrated particle switching by sorting of otherwise identical droplets of uncured and cured photocurable solution immersed in mineral oil solution. Before arriving at the sorting circuit, droplets made of a photocurable solution were illuminated by a UV-light from a mercury lamp, curing them. By tuning the outlet pressures, the switching threshold could be tuned so that uncured droplets were rejected while cured droplets were switched (Raafat et al. μTAS Proc 1826–1828, 2010; Cartas-Ayala et al. Small 9:375–381, 2013). Here we use this system to study the oscillations in this circuit due to particle–particle interactions in the circuit. The circuit oscillation can be used as a counter with a light ON/OFF switch. The circuit behavior agrees well with theoretical predictions of droplet oscillations. Furthermore, the circuit oscillations can be switched on or off by UV-light illumination. This experiment demonstrates switching of particles based on deformability, illustrates the switching of particles by using light, and the possibility of creating new managing schemes for droplets by combining light control with droplet generation-rate control.     

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.     

RNA amplification chip with parallel microchannels and droplet positioning using capillary valves

We present results from the MicroActive project which develops an instrument for molecular diagnostics. The instrument is first tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip are developed and will be inserted into the instrument for each patient sample analysis. The first chip will perform nucleic acid extraction from patient epithelial cervical cells, while mRNA amplification and fluorescent detection takes place in the second chip. This paper reports results on the amplification chip. Purified sample is inserted into the chip and split into ten smaller droplets for simultaneous amplification and detection of ten viruses. The droplets move in parallel channels, each with two chamber extensions containing dried reagents. Experimental results on parallel droplet movement using one external pump combined with hydrophobic restrictions show that the parallel droplet positions can be controlled. There are four valves with increasing burst pressures between 800 and 4,500 Pa in each parallel channel, positioning the droplets in metering zones and reaction chambers. The re-hydration times for the dried reagents in micro chambers have been monitored. After sample insertion, uniform concentration of the reagents in the droplet was reached after respectively 60 s and 10 min. These times are acceptable for successful amplification. Finally we show positive amplification of HPV type 16 viruses in a micro chamber.     

Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.     

Generation of uniform-size droplets by multistep hydrodynamic droplet division in microfluidic circuits

A microfluidic system is presented to generate multiple daughter droplets from a mother droplet, by the multistep hydrodynamic division of the mother droplet at multiple branch points in a microchannel. A microchannel network designed based on the resistive circuit model enables us to control the distribution ratio of the flow rate, which dominates the division ratios of the mother droplets. We successfully generated up to 15 daughter droplets from a mother droplet with a variation in diameter of less than 2%. In addition, we examined factors affecting the division ratio, including the average fluid velocity, interfacial tension, fluid viscosity, and the distribution ratio of volumetric flow rates at a branch point. Additionally, we actively controlled the volume of the mother droplets and examined its influence on the size of the daughter droplets, demonstrating that the size of the daughter droplets was not significantly influenced by the volume of the mother droplet when the distribution ratio was properly controlled. The presented system for controlling droplet division would be available as an innovative means for preparing monodisperse emulsions from polydisperse emulsions, as well as a technique for making a microfluidic dispenser for digital microfluidics to analyze the droplet compositions.     

Electric field driven addressing of ATPS droplets in microfluidic chips

The possibility of controlled droplet motion (droplet addressing) mediated by DC electric field in aqueous two-phase systems (ATPS) is here reported for the first time. Three ATPS of polyethylene glycol (PEG)/salt type, namely PEG/phosphate, PEG/sulphate, and PEG/carbonate, were selected for this study. We observed fast motion of salty droplets dispersed in PEG continuous phase induced by electric field of relative low strength. Hence, three fluidic systems with separated electrode chambers for the evaluation of electrophoretic mobilities and for addressing experiments were fabricated. Electrophoretic mobilities of salty droplets always exceeded the value of \(1\times 10^{-7}\, \hbox {m}^2\hbox {V}^{-1}\hbox {s}^{-1}\), which is about by one magnitude higher value than those typically measured in water–oil droplet systems. The electrophoretic mobilities in systems with free surface are the same or even smaller than in closed microfluidic structures, which is accounted mainly to the fact that a significant part of salty droplets is exposed to air and does not contribute to droplet forcing. Series of addressing and merging experiments in a microfluidic chip shows that DC electric field can be used as a powerful tool for smart manipulation of droplets in microfluidic systems with PEG/salt ATPS.     

Controlled dispensing and mixing of pico- to nanoliter volumes using on-demand droplet-based microfluidics

We present an integrated droplet-on-demand microfluidic platform for dispensing, mixing, incubating, extracting and analyzing by mass spectrometry pico- to nanoliter-sized droplets. All of the functional components are successfully integrated for the first time into a monolithic microdevice. Droplet generation is accomplished using computer-controlled pneumatic valves. Controlled actuation of valves for different aqueous streams enables accurate dosing and rapid mixing of reagents within droplets in either the droplet generation area or in a region of widening channel cross-section. Following incubation, which takes place as droplets travel in the oil stream, the droplet contents are extracted to an aqueous channel for subsequent ionization at an integrated nanoelectrospray emitter. Using the integrated platform, rapid enzymatic digestions of a model protein were carried out in droplets and detected online by nanoelectrospray ionization mass spectrometry.     

荧光免疫层析芯片检测系统信号提取方法研究

基于智能手机硬件平台,开发了一种荧光免疫层析芯片检测系统,实现检测系统对荧光信号强度的准确提取和荧光芯片上免疫分析物的定量检测.提出了一种应用智能手机的荧光芯片图像特征提取和识别的方法,识别荧光芯片的信号区域并提取信号强度,对荧光免疫层析芯片检测系统的研发起到了指导性作用.以荧光层析芯片图像为基础,通过改进的Sobel算子检测出芯片图像中信号区域的特征,进行卷积运算,阈值分析,二值化,投影法获取检测区域边界,实现了对荧光信号强度准确提取.实验结果表明:图像特征识别方法实时性好、精度高,可应用于荧光免疫层析芯片检测系统中.临床应用结果表明:信号强度与免疫分析物浓度具有很好的相关性,检测系统能够很好地提取芯片的荧光信号强度.     

Optical measurement of flow field and concentration field inside a moving nanoliter droplet

Droplets-based method has been employed to enhance mixing in microfluidic systems. This paper presents experimental studies of the recirculating flow field inside a moving droplet and the characterization of the mixing of two aqueous droplets. In the first part, the velocity field inside the moving water droplet was measured using the micro-particle image velocimetry (micro-PIV) technique. The PIV measurements showed that recirculation flow exists inside the droplet. However, the findings suggested that the outer layer of droplets move at a faster velocity than the central part. The result is different from what is reported by other researchers. In the second part, two water droplets, a de-ionized (DI) water droplet and another DI water droplet with fluorescent dye, were brought together by the carrier fluid to form a bigger droplet. The mixing between the two aqueous droplets was characterized by the fluorescent dye concentration distribution.     

Digital microfluidic design and optimization of classic and new fluidic functions for lab on a chip systems

This paper deals with microfluidic studies for lab-on-a-chip development. The first goal was to develop microsystems immediately usable by biologists for complex protocol integrations. All fluid operations are performed on nano-liter droplet independently handled solely by electrowetting on dielectric (EWOD) actuation. A bottom-up architecture was used for chip design due to the development and validation of elementary fluidic designs, which are then assembled. This approach speeds up development and industrialization while minimizing the effort in designing and simplifying chip-fluidic programming. Dispensing reproducibility for 64 nl droplets obtained a CV below 3% and mixing time was only a few seconds. Ease of the integration was demonstrated by performing on chip serial dilutions of 2.8-folds, four times. The second part of this paper concerns the development of new innovative fluidic functions in order to extend EWOD-actuated digital fluidics’ capabilities. Experiments of particle dispensing by EWOD droplet handling are reported. Finally, work is shown concerning the coupling of EWOD actuation and magnetic fields for magnetic bead manipulation.     

Fiber Bragg grating sensor for two-phase flow in microchannels

A new non-intrusive measurement technique for two-phase flow in microchannels is presented. The development of an evanescent field-based optical fiber Bragg grating (FBG) sensor is described, and experiments coupled with flow visualization demonstrating the performance of this sensor are presented. Two adjacent 1-mm FBGs in etched D-shaped fiber are embedded into the surface of a PDMS microchannel. Experiments are conducted in both droplet and slug flow regimes and high-speed digital video is captured synchronously with the sensor data. The FBGs exhibit an on?Coff type response to the passage droplets which is shown to correlate precisely with the passage of the liquid phase. This correlation enables the measurement of droplet average velocity and size using only the sensor data. In addition to the use of both FBG signals for the purpose of measuring droplet speed and size, it is shown that for droplets larger than the FBG length, a single FBG can be used to estimate the convection velocity and size of fast moving droplets. This sensing method is potentially useful for monitoring two-phase flow in fuel cells and microfluidic applications such as micro-heat exchangers and lab-on-a-chip systems.     

Exploitation of a microfluidic device capable of generating size-tunable droplets for gene delivery

This study presents a new microfluidic chip that generates micro-scale emulsion droplets for gene delivery applications. Compared with conventional methods of droplet formation, the proposed chip can create uniform droplets (size variation <7.1%) and hence enhance the efficiency of the subsequent gene delivery. A new microfluidic chip was developed in this study, which used a new design with a pneumatic membrane chamber integrated into a T-junction microchannel. Traditionally, the size of droplets was controlled by the flow rate ratio of the continuous and disperse phase flows, which can be controlled by syringe pumps. In this study, a pneumatic chamber near the intersection of the T-junction channel was designed to locally change the flow velocity and the shear force. When the upper air chamber was filled with compressed air, the membrane was deflected and then the droplet size could be fine-tuned accordingly. Experimental data showed that using the new design, the higher the air pressure applied to the active tunable membrane, the smaller the droplet size. Finally, droplets were used as carriers for DNA to be transfected into the Cos-7 cells. It was also experimentally found that the size of the emulsion droplets plays an important role on the efficiency of the gene delivery. The preliminary results of this paper have been presented at the 2007 IEEE International Conference of Nano/Molecular Medicine and Engineering (IEEE NANOMED 2007), Macau, China, 6–9 August, 2007.     

Development of a biological detection platform utilizing a modular microfluidic stack

The goal of this project is to build a miniaturized, user-friendly cytometry setup (Datta et al. in Microfluidic platform for education and research. COMS, Baton Rouge, 2008; Frische et al. in Development of an miniaturized flow cytometry setup for visual cell inspection and sorting. Baton Rouge, Project Report, 2008) by combining a customized, microfluidic device with visual microscope inspection to detect and extract specific cells from a continuous sample flow. We developed a cytological tool, based on the Coulter particle counter principle, using a microelectrode array patterned on a borosilicate glass chip as electrical detection set-up which is fully embedded into a polymeric multi-layer microfluidic stack. The detection takes place between pairs of coplanar Cr/Au microelectrodes by sensing an impedance change caused by particles continuously carried within a microfluidic channel across the detection area under laminar flow conditions. A wide frequency range available for counting provides information on cell size, membrane capacitance, cytoplasm conductivity and is potentially of interest for in-depth cell diagnostic e.g. to detect damaged or cancerous cells and select them for extraction and further in-depth analysis.     

Scale-up and control of droplet production in coupled microfluidic flow-focusing geometries

A single microfluidic chip consisting of six microfluidic flow-focusing devices operating in parallel was developed to investigate the feasibility of scaling microfluidic droplet generation up to production rates of hundreds of milliliters per hour. The design utilizes a single inlet channel for both the dispersed aqueous phase and the continuous oil phase from which the fluids were distributed to all six flow-focusing devices. The exit tubing for each of the six flow-focusing devices is separate and individually plumbed to each device. Within each flow-focusing device, the droplet size was monodisperse, but some droplet size variations were observed across devices. We show that by modifying the flow resistance in the outlet channel of an individual flow-focusing device it is possible to control both the droplet size and frequency of droplet production. This can be achieved through the use of valves or, as is done in this study, by changing the length of the exit tubing plumbed to the outlet of the each device. Longer exit tubing and larger flow resistance is found to lead to larger droplets and higher production frequencies. The devices can thus be individually tuned to create a monodisperse emulsion or an emulsion with a specific drop size distribution.     

Intra-droplet acoustic particle focusing: simulations and experimental observations

The aim of this paper is to study resonance conditions for acoustic particle focusing inside droplets in two-phase microfluidic systems. A bulk acoustic wave microfluidic chip was designed and fabricated for focusing microparticles inside aqueous droplets (plugs) surrounded by a continuous oil phase in a 380-μm-wide channel. The quality of the acoustic particle focusing was investigated by considering the influence of the acoustic properties of the continuous phase in relation to the dispersed phase. To simulate the system and study the acoustic radiation force on the particles inside droplets, a simplified 3D model was used. The resonance conditions and focusing quality were studied for two different cases: (1) the dispersed and continuous phases were acoustically mismatched (water droplets in fluorinated oil) and (2) the dispersed and continuous phases were acoustically matched (water droplets in olive oil). Experimentally, we observed poor acoustic particle focusing inside droplets surrounded by fluorinated oil while good focusing was observed in droplets surrounded by olive oil. The experimental results are supported qualitatively by our simulations. These show that the acoustic properties (density and compressibility) of the dispersed and continuous phases must be matched to generate a strong and homogeneous acoustic field inside the droplet that is suitable for high-quality intra-droplet acoustic particle focusing.     

Purification of a droplet using negative dielectrophoresis traps in digital microfluidics

The development of techniques for manipulating particles and integrating them into the digital microfluidic (DMF) devices has been the subject of several studies in recent years. This paper presents a dielectrophoretic-based method that uses triangular traps to manipulate particles and purify a droplet in DMF platforms. Numerical and experimental studies are conducted to show the effectiveness of the proposed trap geometry which is also compatible with the other operators in the DMF platform. The triangular trap geometry is used to move the polystyrene particles to one side of the trap using negative dielectrophoresis (nDEP). The droplet is then split into two smaller droplets with very low and high concentrations of particles using the electrowetting on dielectric technique. The average velocity of the particles (as they move along the trap) as a function of the vertex angle of the triangular trap and the gap between the top and bottom plate is examined. It is observed that the vertex angle of the trap plays more important role on the motion of the particles than the gap. Thus, to enhance the motion of the particles and minimize the effect of splitting on the purification process, the vertex angle and the slope of the side arms of the triangular trap are modified based on the results of the numerical model simulating the dielectrophoretic force on the particle. The enhanced geometry is fabricated and tested experimentally to show the effectiveness and ease-of-use of the proposed technique in purifying (or concentrating) a droplet in DMF. The results show that using the proposed nDEP electrode geometry purification (or concentration) can be performed with the efficiency of 90 %.