Membrane assisted passive sampler for triazine compounds in water bodies--characterization of environmental conditions and field performance

This article reports the integration of the fiber optic-particle plasmon resonance (FO-PPR) biosensor with a microfluidic chip to reduce response time and improve detection limit. The microfluidic chip made of poly(methyl methacrylate) had a flow-channel of dimensions 4.0 cm × 900 μm × 900 μm. A partially unclad optical fiber with gold or silver nanoparticles on the core surface was placed within the flow-channel, where the volume of the flow space was about 14 μL. Results using sucrose solutions of various refractive indexes show that the refractive index resolution improves by 2.4-fold in the microfluidic system. The microfluidic chip is capable of delivering a precise amount of biological samples to the detection area without sample dilution. Several receptor/analyte pairs were chosen to examine the biosensing capability of the integrated platform: biotin/streptavidin, biotin/anti-biotin, DNP/anti-DNP, OVA/anti-OVA, and anti-MMP-3/MMP-3. Results show that the response time to achieve equilibrium can be shortened from several thousand seconds in a conventional liquid cell to several hundred seconds in a microfluidic flow-cell. In addition, the detection limit also improves by about one order of magnitude. Furthermore, the normalization by using the relative change of transmission response as the sensor output alleviate the demand on precise optical alignment, resulting in reasonably good chip-to-chip measurement reproducibility.     

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 polymer-based microfluidic device for immunosensing biochips

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.     

Fast immobilization of probe beads by dielectrophoresis-controlled adhesion in a versatile microfluidic platform for affinity assay

The use of probe beads for lab-on-chip affinity assays is very interesting from a practical point of view. It is easier to handle and trap beads than molecules in microfluidic systems. We present a method for the immobilization of probe beads at defined areas on a chip using dielectrophoresis (DEP)-controlled adhesion. The method is fast, i.e., it takes between 10 and 120 s--depending on the protocol--to functionalize a chip surface at defined areas. The method is versatile, i.e., it works for beads with different types of probe molecule coatings. The immobilization is irreversible, i.e., the retained beads are able to withstand high flow velocities in a flow-through device even after the DEP voltage is turned off, thus allowing the use of conventional high-conductivity analyte buffers in the following assay procedure. We demonstrate the on-chip immobilization of fluorescent beads coated with biotin, protein A, and goat-antimouse immunoglobulin G (IgG). The number of immobilized beads at an electrode array can be determined from their fluorescence signal. Further, we use this method to demonstrate the detection of streptavidin and mouse IgG. Finally, we demonstrate the feasibility of the parallel detection of different analyte molecules on the same chip.     

Surface plasmon resonance-based trace detection of small molecules by competitive and signal enhancement immunoreaction

A surface plasmon resonance (SPR)-immunosensor for detection of the low molecular weight compound 2,4-dinitorophenol (DNP) at ultra-low concentration has been developed. The sensor strategy is based on a competitive immunoreaction between DNP and a DNP-protein conjugate, namely DNP-bovine serum albumin conjugate (DNP-BSA). Anti-DNP monoclonal antibody was immobilized on a gold thin-film coated SPR-sensor chip by means of a chemical coupling process. DNP-BSA, on contact with the anti-DNP antibody immobilized SPR-immunosensor chip causes an increase in the resonance angle of the sensor chip. The optimum concentration of immobilized antibody on the SPR-sensor chip is 100 μg mL⁻¹. The SPR-immunosensor response for free DNP determination using the competitive immunoreaction had a response time of ca. 15 min. Using this method, DNP could be determined in the concentration range 1 ppt to 1 ppb. The SPR signal for ppt levels of DNP was enhanced by a factor of three by subsequently treating immuno-bound DNP-BSA with a secondary anti-DNP antibody.     

Integrated SPPS on continuous-flow radial microfluidic chip

A novel integrated continuous-flow microfluidic system was designed and fabricated for solid phase peptide synthesis (SPPS) using conventional reactants. The microfluidic system was composed of a glass-based radial reaction chip, a diffluent chip, amino acid feeding reservoirs and continuous-flow reagent pathways. A tri-row cofferdam-fence structure was designed for solid phase supports trapping. Highly cross-linked, porous and high-loading 4-(hydroxymethyl)phenoxymethyl polystyrene (HMP) beads were prepared for microfluidic SPPS. The transfer losses, hazardous handling and time-consuming processes in traditional peptide cleavage steps were avoided by being replaced with the on-chip cleavage treatment. Six peptides from an antibody affinity peptide library against β-endorphin with different lengths and sequences were obtained simultaneously on the constructed continuous-flow microfluidic system within a short time. This microfluidic system is automatic, integrated, effective, low-cost, recyclable and environment-friendly for not only SPPS but also other solid phase chemical syntheses.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman?, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.     

Microaffinity purification of proteins based on photolytic elution: toward an efficient microbead affinity chromatography on a chip

A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated.     

Rapid Analysis of Oligonucleotides on a Planar Microfluidic Chip

State-of-the-art microfluidic analytical systems are briefly surveyed. Attention is focused on the use of microchip capillary electrophoresis. The main results obtained in the development of a prototype analytical system with a laser-induced fluorescence detector for electrophoresis on a glass microfluidic chip are presented. Experimental data on electroosmotic flow and the distribution of sample fluorescence intensity over the cross section of a microchannel are analyzed. A procedure for the rapid analysis of oligonucleotides on a microfluidic chip is described.     

基于微流控芯片的72重单核苷酸多态性族群推断系统的构建

建立了常染色体单核苷酸多态性（SNPs）复合检测芯片体系,用于未知个体的族群来源推断。基于前期筛选的74-SNPs组合,采用竞争性等位基因特异性聚合酶链式反应（PCR）的原理构建SNPs的扩增体系,在微流控芯片的每个反应孔内完成一个SNP的检测,通过高通量PCR微流控芯片实现了其中72个SNPs的同步检测。芯片的扩增由平板PCR仪完成,反应孔的荧光信号通过激光共聚焦扫描仪检测,最终通过提取的荧光值进行结果分析。使用该芯片检测获得52份样本的SNPs分型,分型结果的准确率为100%。以57个人群的3628个样本为参考人群数据库,进行20份样本的族群来源推断,推断结果与样本的实际来源一致。本研究建立的常染色体72个SNPs微流控芯片体系可以有效地进行SNP多态性分析检测,基于参考数据库,20份检测样本族群推断的准确性为100%。     

A novel microfluidic concept for bioanalysis using freely moving beads trapped in recirculating flows

There are only a few examples in which beads are employed for heterogeneous assays on microfluidic devices, because of the difficulties associated with packing and handling these in etched microstructures. This contribution describes a microfluidic device that allows the capture, preconcentration, and controlled manipulation of small beads (<6 microm) in etched microchannels using fluid flows only. The chips feature planar diverging and converging channel elements connected by a narrow microchannel. Creation of bi-directional liquid movement by opposing electro-osmotic and pressure-driven flows can lead to the generation of controlled recirculating flow at these elements. Small polymer beads can actually be captured in the controlled rotating flow patterns. The clusters of freely moving beads that result can be perfused sequentially with different solutions. A preliminary binding curve was determined for the reaction of streptavidin-coated beads and fluorescein-labelled biotin, demonstrating the potential of this bead-handling approach for bioanalysis.     

Microfluidic biofunctionalisation protocols to form multi‐valent interactions for cell rolling and phenotype modification investigations

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro‐fluidic protocols. Many available processes either require expensive and time‐consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi‐valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin–biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC‐I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC‐I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC‐I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC‐I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.     

On-column capture of a specific protein in capillary electrophoresis using magnetic beads

A method for capturing specific molecules separated by CE has been explored. To demonstrate on-column capture of migrating analyte molecules, two detection windows were fabricated on a capillary. Magnetic beads containing immobilized molecules that react with the specific molecules under study were placed between the detection windows in the capillary using magnets. Molecules in a sample solution injected into the capillary were separated and detected at the first detection window. After passing through the first detection window, the separated molecules encountered the magnetic beads, where the specific analyte was captured. As a result, the peak area for those analyte molecules decreased or disappeared completely at the second detection window. Rabbit IgG and carbonic anhydrase were employed to demonstrate on-column capture of a specific molecule. For rabbit IgG, magnetic beads containing the immobilized antibody (anti-rabbit IgG) were used. Rabbit IgG molecules were captured on the magnetic beads during CE migration. Furthermore, the capture of carbonic anhydrase was demonstrated by the reaction between magnetic beads (containing immobilized anti-rabbit IgG) and anti-carbonic anhydrase (rabbit IgG), before the beads were packed in the capillary. After packing the magnetic beads in the capillary, a mixture of two proteins was injected into the capillary. Two proteins were detected at the first detection window, while the peak corresponding to carbonic anhydrase disappeared at the second detection window. The results show that using an appropriate antibody, the present technique would be applicable to any proteins.     

A microfluidic protease activity assay based on the detection of fluorescence polarization

This article describes a fluorescence polarization (FP)-based protease assay on a microfluidic device that is compatible with fast and reproducible analyses of protease activities. The optical systems were arranged for simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes, and the binding of tetramethylrhodamine (TMR) labeled-biotin with streptavidin was utilized for optimizing FP detection in continuously flowing solutions within 74-μm wide, 12-μm deep microchannels of a glass chip. In developing off-chip FP-based assays for proteinase K, trypsin, papain and elastase, TMR conjugated-casein protein (TMR-α-casein) was employed as a universal substrate. After optimization of the hydrodynamic flow control to allow complete mixing of TMR-α-casein and short proteolysis time as possible, and of buffer composition to minimize protein sticking problems, the developed assay was transferred to the microfluidic chip by monitoring FP changes of TMR-α-casein in the main microchannel. The results indicate that the proposed device would serve as an integrated microfluidic platform with automated injection of reacting species, diffusion-controlled mixing, reaction and detection for protease activities without the need to separate the products.     

Controllable Synthesis of Multicompartmental Particles Using 3D Microfluidics

A microfluidic assembly method based on a microfluidic chip and capillary device was developed to create multicompartmental particles. The microfluidic chip design endows the particles with regulable internal structure. By adjusting the microstructure of the chip, the diameter of the capillary, the gap length between the two microfluidic components, and the flow rates, the size of the particles and the number or the ratio of different regions within the particle could be widely varied. As a proof of concept, we have produced some complicated particles that even contain 20 compartments. Furthermore, the potential applications of the anisotropic particles are explored by encapsulating magnetic beads, fluorescent nanoparticles, and the cells into different compartments of the microparticles. We believe that this method will open new avenues for the design and application of multicompartmental particles.     

Biochemical analysis with microfluidic systems

Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.     

Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system

A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.