Performance of an integrated microoptical system for fluorescence detection in microfluidic systems

This article presents a new integrated microfluidic/microoptic device designed for basic biochemical analysis. The microfluidic network is wet-etched in a Borofloat 33 (Pyrex) glass wafer and sealed by means of a second wafer. Unlike other similar microfluidic systems, elements of the detection system are realized with the help of microfabrication techniques and directly deposited on both sides of the microchemical chip. The detection system is composed of the combination of refractive circular or elliptical microlens arrays and chromium aperture arrays. The microfluidic channels are 60 microm wide and 25 microm deep. The elliptical microlenses have a major axis of 400 microm and a minor axis of 350 microm. The circular microlens diameters range from 280 microm to 350 microm. The apertures deposited on the outer chip surfaces are etched in a 3000-A-thick chromium layer. The overall thickness of this microchemical system is < 1.6 mm. A limit of detection of 3.3 nM for a Cy5 solution in phosphate buffer (pH 7.4) was demonstrated. The cross-talk signal measured between two adjacent microchannels with 1 mm pitch was < 1:5600, meaning that < or = 1.8 x 10(-4)% of the fluorescence light power emitted from one microchannel filled with a 50 microM Cy5 solution reaches the photodetector at the adjacent microchannel. This performance compares very well with that obtainable in microchemical chips using confocal fluorescence systems, taking differences in parameters, such as excitation power into microchannels, data acquisition rates, and signal filtering into account.     

集成一次性微流控芯片的便携式荧光检测系统

本文研究了一种新型的生物化学分析系统,该系统包括便携式荧光检测仪和带光纤的微流控芯片．采用基于MEMS技术的微泵将待测物与荧光试剂的混合物导入微流控芯片,采用PMT检测受激发产生的荧光,荧光强度与待测物浓度成一定比例．激发光则通过光纤将光源LED光信号导入微沟道中．随着液体在微沟道中的流动,可连续分析和检测不同的样品．该系统检测1～1000μg／L浓度的荧光素具有0．966的相关系数．基于荧光猝灭原理,该系统还可检测浓度为5ng／μL的硝基化合物．该生化分析系统除具有便携式和一次性微流控芯片优点外,还具有成本低．试剂、样品消耗量少,且分析时间短等优点该系统能实现现场检测,可应用于临床诊断、环境检测及生物战剂检测等领域     

High-efficiency single-cell entrapment and fluorescence in situ hybridization analysis using a poly(dimethylsiloxane) microfluidic device integrated with a black poly(ethylene terephthalate) micromesh

Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 microm in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. This microfluidic device has been employed for the detection of beta-actin mRNA expression in individual Raji cells. Differences in the levels of beta-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.     

Torque-actuated valves for microfluidics

This paper describes torque-actuated valves for controlling the flow of fluids in microfluidic channels. The valves consist of small machine screws (> or =500 microm) embedded in a layer of polyurethane cast above microfluidic channels fabricated in poly(dimethylsiloxane) (PDMS). The polyurethane is cured photochemically with the screws in place; on curing, it bonds to the surrounding layer of PDMS and forms a stiff layer that retains an impression of the threads of the screws. The valves were separated from the ceiling of microfluidic channels by a layer of PDMS and were integrated into channels using a simple procedure compatible with soft lithography and rapid prototyping. Turning the screws actuated the valves by collapsing the PDMS layer between the valve and channel, controlling the flow of fluids in the underlying channels. These valves have the useful characteristic that they do not require power to retain their setting (on/off). They also allow settings between "on" and "off" and can be integrated into portable, disposable microfluidic devices for carrying out sandwich immunoassays.     

Analysis of passive mixing behavior in a poly(dimethylsiloxane) microfluidic channel using confocal fluorescence and Raman microscopy

Confocal fluorescence microscopy (CFM) and confocal Raman microscopy (CRM) have been applied to monitor the laminar flow mixing behavior in a poly(dimethylsiloxane) (PDMS) microfluidic channel. Two passive PDMS micromixing devices were fabricated for this purpose: a two-dimensional round-wave channel and a three-dimensional serpentine channel. The microscale laminar flow mixing of ethanol and isopropanol was evaluated using the CFM and CRM at various flow rates. The mixing behavior of confluent streams in the microchannel was assessed by determining the degree of color change in Rhodamine 6G dye on mixing using the CFM. However, it was also possible to quantitatively evaluate the mixing process without employing a fluorescence label using the CRM. The results show a strong potential for CRM as a highly sensitive detection tool to measure fundamental fluid mixing processes and to provide detailed information on chemical changes of non-fluorescent reaction mixtures in a PDMS microfluidic channel.     

Fabrication of a configurable, single-use microfluidic device.

This paper describes microfluidic devices that contain connections that can be opened by the user after fabrication. The devices are fabricated in poly(dimethylsiloxane) (PDMS) and comprise disconnected fluidic channels that are separated by 20 microm of PDMS. Applying voltages above the breakdown voltage of PDMS (21 V/microm) opened pathways between disconnected channels. Fluids could then be pumped through the openings. The voltage used and the ionic strength of the buffer in the channels determined the size of the opening. Opening connections in a specific order provides the means to control complex reactions on the device. A device for ELISA was fabricated to demonstrate the ability to store and deliver fluids on demand.     

Design of PDMS microlenses bonded to a lab-CD chip for ELISA applications

A novel device comprising polydimethyl-siloxane (PDMS) microlenses bonded to a microfluidic compact disk (CD) is proposed for enzyme-linked immunosorbent assay (ELISA) applications. The PDMS microlenses were fabricated using a simple soft replica molding method and were bonded to the microfluidic CD using oxygen plasma treatment. A commercial software tool (ZEMAX) has been used to analyze the focal length of the microlens. A laser-induced fluorescence bio-detection system, consisting of the integrated microfluidic CD/PDMS microlenses and an optical detection module, was constructed and used to examine the enzymatic reaction of 3-(4-hydroxy) phenly propionic acid. The experimental results show that the PDMS microlens focusing effect yields a significant improvement in the intensity of the detected fluorescence signals. As a result, the proposed device represents an ideal solution for ELISAs and other high-sensitivity bio-detection applications.     

Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation

We have developed a multipurpose microfluidic platform that allows for sensitive fluorescence detection on inexpensive disposable chips. The fabrication scheme involves rapid injection molding of thermoplastics, followed by silica deposition and covalent attachment of an unstructured flexible lid. This combines the virtues of elastomer technology with high-throughput compact disk injection molding. Using this technique, the time to produce 100 chips using a single master can be lowered from more than 1 week by standard PDMS technologies to only a couple hours. The optical properties of the fabricated chips were evaluated by studying individual fluorescence-labeled DNA molecules in a microchannel. Concatemeric DNA molecules were generated through rolling circle replication of circular DNA molecules, which were labeled by hybridization of fluorescence-tagged oligonucleotides. Rolling circle products (RCPs) were detected after as little as 5 min of DNA polymerization, and the RCPs in solution showed no tendency for aggregation. To illustrate the versatility of the platform, we demonstrate two additional applications: The flexible property of the lid was used to create a peristaltic pump generating a flow rate of 9 nL/s. Biocompatibility of the platform was illustrated by culturing Chinese hamster ovary cells for 7 days in the microfluidic channels.     

Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.     

Self-sealed vertical polymeric nanoporous-junctions for high-throughput nanofluidic applications

We developed a reliable but simple integration method of polymeric nanostructure in a poly(dimethylsiloxane) (PDMS)-based microfluidic channel, for nanofluidic applications. The Nafion polymer junction was creased by infiltrating polymer solution between the gaps created by mechanical cutting, without any photolithography or etching processes. The PDMS can seal itself with the heterogeneous polymeric nanoporous material between the PDMS/PDMS gap due to its flexibility without any (covalent) bonding between PDMS and the polymer materials. Thus, one can easily integrate the nanoporous-junction into a PDMS microchip in a leak-free manner with excellent repeatability. We demonstrated nanofluidic preconcentration of proteins (beta-phycoerythrin) using the device. Because the polymeric junction spans across the entire microchannel height, the preconcentration was achieved with high-pressure field or even in large channels, with the dimensions of 1000 microm width x 100 microm depth.     

Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.     

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.     

In-Plane Integration of Polymer Microfluidic Channels With Optical Waveguides– A Preliminary Investigation

The next major challenges for lab-on-a-chip (LoC) technology are 1) the integration of microfluidics with optical detection technologies and 2) the large-scale production of devices at a low cost. In this paper the fabrication and characterisation of a simple optical LoC platform comprising integrated multimode waveguides and microfluidic channels based on a photo-patternable acrylate based polymer is reported. The polymer can be patterned into both waveguides and microfluidic channels using photolithography. Devices are therefore both quick and cost-effective to fabricate, resulting in chips that are potentially disposable. The devices are designed to be highly sensitive, using an in-plane direct excitation configuration in which waveguides intersect the microfluidic channel orthogonally. The waveguides are used both to guide the excitation light and to collect the fluorescence signal from the analyte. The potential of the device to be used for fluorescence measurements is demonstrated using an aqueous solution of sodium fluorescein. A detection limit of 7 nM is achieved. The possibilities offered by such a device design, in providing a cost-effective and disposable measurement system based on the integration of optical waveguides with LoC technology is discussed.     

SPR imaging measurements of 1-D and 2-D DNA microarrays created from microfluidic channels on gold thin films

Microfluidic channels fabricated from poly(dimethylsiloxane) (PDMS) are employed in surface plasmon resonance imaging experiments for the detection of DNA and RNA adsorption onto chemically modified gold surfaces. The PDMS microchannels are used to (i) fabricate "1-D" single-stranded DNA (ssDNA) line arrays that are used in SPR imaging experiments of oligonucleotide hybridization adsorption and (ii) create "2-D" DNA hybridization arrays in which a second set of PDMS microchannels are placed perpendicular to a 1-D line array in order to deliver target oligonucleotide solutions. In the 1-D line array experiments, the total sample volume is 500 microL; in the 2-D DNA array experiments, this volume is reduced to 1 microL. As a demonstration of the utility of these microfluidic arrays, a 2-D DNA array is used to detect a 20-fmol sample of in vitro transcribed RNA from the uidA gene of a transgenic Arabidopsis thaliana plant. It is also shown that this array fabrication method can be used for fluorescence measurements on chemically modified gold surfaces.     

Microfluidic system for planar patch clamp electrode arrays

We present a microfluidic system integrated with disposable cell interface partitions for simultaneous patch clamp recordings. Glass-supported poly(dimethylsiloxane) (PDMS) partitions, having a 2 microm air-blown aperture, were reversibly sealed to a microfluidic system including PDMS channels with isolation valves and microfabricated Ag/AgCl electrodes. Gigaseal recordings from RBL-1 cells were obtained with a 24% success rate. Simultaneous whole cell recordings from valve-isolated electrodes were obtained.     

Deconvolution microscopy for flow visualization in microchannels

Quantitative visualization of microflows is often needed to evaluate the efficiency of fluid mixing, study flow properties, investigate unusual flow behavior, and verify computational fluid dynamic simulations. In this work, we explore the technique of coupling a conventional optical microscope with a computational deconvolution algorithm to produce images of three-dimensional flows in plastic microfluidic channels. The approach, called deconvolution microscopy, is achieved by (1) optically sectioning the flow in the microchannel by collecting a series of fluorescence images at different focal planes along the optical axis and (2) removing the out-of-focus fluorescence signal by a deconvolution method to reconstruct the corrected three-dimensional concentration image. We compare three different classes of deconvolution algorithms for a uniform concentration test case and then demonstrate how deconvolution microscopy is useful for flow visualization and analysis of mixing in microfluidic channels. In particular, we employ the method to confirm the presence of twisting flows in a microchannel containing microfabricated ridges.     

Polymer microfluidic chips with integrated waveguides for reading microarrays

A microfluidic chip with an integrated planar waveguide was fabricated in poly(methyl methacrylate), PMMA, using a single-step, double-sided hot-embossing approach. The waveguide was embedded in air on three sides, the solution being interrogated on the fourth. DNA probes were covalently attached to the waveguide surface by plasma activating the PMMA and the use of carbodiimide coupling chemistry. Successful hybridization events were read using evanescent excitation monitored by an imaging microscope, which offered high spatial resolution (2 microm) and a large field-of-view (20 mm diameter field-of-view), providing imaging of the entire array without scanning. The application of the microfluidic/waveguide assembly was demonstrated by detecting low abundant point mutations; insertion C mutations in BRCA1 genes associated with breast cancer were analyzed using a universal array coupled to an allele-specific ligation assay. DNA probes consisting of amine-terminated oligonucleotides were printed inside the microfluidic channel using a noncontact microspotter. Mutant and wild-type genomic DNAs of BRCA1 were PCR (polymerase chain reaction) amplified, with the amplicons subjected to ligation detection reactions (LDRs). LDR solutions were allowed to flow over the microarray positioned on the polymer waveguide with successful ligation events discerned through fluorescence signatures present at certain locations of the array. The microfluidic/waveguide assembly could detect polymorphisms present at <1% of the total DNA content.     

Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing

A solid-object printer was used to produce masters for the fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS). The printer provides an alternative to photolithography for applications where features of > 250 microm are needed. Solid-object printing is capable of delivering objects that have dimensions as large as 250 x 190 x 200 mm (x, y, z) with feature sizes that can range from 10 cm to 250 microm. The user designs a device in 3-D in a CAD program, and the CAD file is used by the printer to fabricate a master directly without the need for a mask. The printer can produce complex structures, including multilevel features, in one unattended printing. The masters are robust and inexpensive and can be fabricated rapidly. Once a master was obtained, a PDMS replica was fabricated by molding against it and used to fabricate a microfluidic device. The capabilities of this method are demonstrated by fabricating devices that contain multilevel and tall features, devices that cover a large area (approximately 150 cm2), and devices that contain nonintersecting, crossing channels.     

On-chip electric field driven electrochemical detection using a poly(dimethylsiloxane) microchannel with gold microband electrodes

An external electric field driven in-channel detection technique for on-chip electrochemical detection in micro fabricated devices is described based on a microfluidic system containing an array of 20 microband electrodes. It is shown that an external electric field induces a potential difference between two gold microband electrodes in a poly(dimethylsiloxane) (PDMS) microchannel, and that this enables the electrochemical detection of electroactive species such as ascorbic acid and Fe(CN) 6 (4-). The results, which are supported by simulations of the behavior of the microband electrodes in the microfluidic system, show that the induced potential difference between the electrodes can be controlled by altering the external electric field or by using different microbands in the microband array. As the obtained currents depend on the concentrations of electroactive species in the flowing solution and the detection can be carried out anywhere within the channel without interference of the external electric field, the present approach significantly facilitates electrochemical detection in capillary electrophoresis. This approach consequently holds great promise for application in inexpensive portable chip-based capillary electrophoresis (CE) devices.     

Microfluidic Three-Electrode Cell Array for Low-Current Electrochemical Detection

This paper reports the implementation and calibration of a microscopic three-electrode electrochemical sensor integrated with a polydimethylsiloxane (PDMS) microchannel to form a rapid prototype chip technology that is used to develop sensing modules for biomolecular signals. The microfluidic/microelectronic fabrication process yields identical, highly uniform, and geometrically well-defined microelectrodes embedded in a microchannel network. Each three-microelectrode system consists of a Au working electrode with a nominal surface area of 9 mum², a Cl₂ plasma-treated Ag/AgCl reference electrode, and a Au counter electrode. The patterned electrodes on the glass substrate are aligned and irreversibly bonded with a PDMS microchannel network giving a channel volume of 72 nL. The electrokinetic properties and the diffusion profile of the microchannels are investigated under electrokinetic flow and pressure-driven flow conditions. Cyclic voltammetry of 10 mM K₃ Fe(CN)₆ in 1 M KNO₃ demonstrates that the electrode responses in the cell are characterized by linear diffusion. The voltammograms show that the system is a quasi-reversible redox process, with heterogeneous rate constants ranging from 3.11 to 4.94times10^-3 cm/s for scan rates of 0.1-1 V/s. The current response in the cell is affected by the adsorption of the electroactive species on the electrode surface. In a low-current DNA hybridization detection experiment, the electrode cell is modified with single-stranded thiolated DNA. The electrocatalytic reduction of 27 muM Ru(NH₃)₆ ³⁺ in a solution containing 2 mM Fe(CN)₆ ^3- is measured before and after the exposure of the electrode cell to a 500-nM target DNA sample. The preliminary result showing an increase in the peak current response demonstrates the hybridization-based detection of a complementary target DNA sequence