Magnetic force-based multiplexed immunoassay using superparamagnetic nanoparticles in microfluidic channel

This paper describes a novel microfluidic immunoassay utilizing binding of superparamagnetic nanoparticles to beads and deflection of these beads in a magnetic field as the signal for measuring the presence of analyte. The superparamagnetic 50 nm nanoparticles and fluorescent 1 microm polystyrene beads are immobilized with specific antibodies. When target analytes react with the polystyrene beads and superparamagnetic nanoparticles simultaneously, the superparamagnetic nanoparticles can be attached onto the microbeads by the antigen-antibody complex. In the poly(dimethylsiloxane)(PDMS) microfluidic channel, only the microbeads conjugated with superparamagnetic nanoparticles by analytes consequently move to the high gradient magnetic fields under the specific applied magnetic field. In this study, the magnetic force-based microfluidic immunoassay is successfully applied to detect the rabbit IgG and mouse IgG as model analytes. The lowest concentration of rabbit IgG and mouse IgG measured over the background is 244 pg mL(-1) and 15.6 ng mL(-1), respectively. The velocities of microbeads conjugated with superparamagnetic nanoparticles are demonstrated by magnetic field gradients in microfluidic channels and compared with the calculated magnetic field gradients. Moreover, dual analyte detection in a single reaction is also performed by the fluorescent encoded microbeads in the microfluidic device. Detection range and lower detection limit can be controlled by the microbeads concentration and the higher magnetic field gradient.     

Dual magnetic-/temperature-responsive nanoparticles for microfluidic separations and assays

A stimuli-responsive magnetic nanoparticle system for diagnostic target capture and concentration has been developed for microfluidic lab card settings. Telechelic poly(N-isopropylacrylamide) (PNIPAAm) polymer chains were synthesized with dodecyl tails at one end and a reactive carboxylate at the opposite end by the reversible addition fragmentation transfer technique. These PNIPAAm chains self-associate into nanoscale micelles that were used as dimensional confinements to synthesize the magnetic nanoparticles. The resulting superparamagnetic nanoparticles exhibit a gamma-Fe2O3 core ( approximately 5 nm) with a layer of carboxylate-terminated PNIPAAm chains as a corona on the surface. The carboxylate group was used to functionalize the magnetic nanoparticles with biotin and subsequently with streptavidin. The functionalized magnetic nanoparticles can be reversibly aggregated in solution as the temperature is cycled through the PNIPAAm lower critical solution temperature (LCST). While the magnetophoretic mobility of the individual nanoparticles below the LCST is negligible, the aggregates formed above the LCST are large enough to respond to an applied magnetic field. The magnetic nanoparticles can associate with biotinylated targets as individual particles, and then subsequent application of a combined temperature increase and magnetic field can be used to magnetically separate the aggregated particles onto the poly(ethylene glycol)-modified polydimethylsiloxane channel walls of a microfluidic device. When the magnetic field is turned off and the temperature is reversed, the captured aggregates redisperse into the channel flow stream for further downstream processing. The dual magnetic- and temperature-responsive nanoparticles can thus be used as soluble reagents to capture diagnostic targets at a controlled time point and channel position. They can then be isolated and released after the nanoparticles have captured target molecules, overcoming the problem of low magnetophoretic mobility of the individual particle while retaining the advantages of a high surface to volume ratio and faster diffusive properties during target capture.     

Controlling the internal structure of giant unilamellar vesicles by means of reversible temperature dependent sol-gel transition of internalized poly(N-isopropyl acrylamide)

In this work, we present preparation and basic applications of lipid-bilayer-enclosed picoliter volumes (microcontainers) of solutions of poly(N-isopropylacrylamide) (PNIPAAm). Giant unilamellar vesicles (GUVs) were prepared from phospholipids using a standard swelling procedure and subsequently surface immobilized. Clear, slightly viscous solutions of PNIPAAm of varying concentration in aqueous buffer were directly pressure-microinjected into the GUVs, using a submicrometer-sized, pointed capillary. The GUV was subjected to changing temperature over a 21-40 degrees C range. The typical phase transition of the polymeric material upon heating and cooling across the lower critical solution temperature was followed using optical microscopy and shown to be reversible over multiple sequential heating/cooling cycles without compromising the integrity of the GUV membrane. Fluorescent, carboxylic acid modified 200 nm latex beads, co-injected with the PNIPAAm solution, were temperature-reversibly immobilized during the phase transition, practically freezing the Brownian motion of the entrapped particles in the volume. Furthermore, a co-injected water soluble fluorescent polysaccharide-dye conjugate was shown not to migrate from the aqueous phase into the hydrophobic polymer part upon heating, whereas the fluorescent beads were completely but reversibly immobilized in the hydrophobic domains of dense polymer agglomerates. The system reported here provides a feasible method for the reversible stabilization and solidification of GUV interior volumes, e.g., as a micrometer-sized model system for controlled drug release.     

Molded polyethylene glycol microstructures for capturing cells within microfluidic channels

The ability to control the deposition and location of adherent and non-adherent cells within microfluidic devices is beneficial for the development of micro-scale bioanalytical tools and high-throughput screening systems. Here, we introduce a simple technique to fabricate poly(ethylene glycol)(PEG) microstructures within microfluidic channels that can be used to dock cells within pre-defined locations. Microstructures of various shapes were used to capture and shear-protect cells despite medium flow in the channel. Using this approach, PEG microwells were fabricated either with exposed or non-exposed substrates. Proteins and cells adhered within microwells with exposed substrates, while non-exposed substrates prevented protein and cell adhesion (although the cells were captured inside the features). Furthermore, immobilized cells remained viable and were stained for cell surface receptors by sequential flow of antibodies and secondary fluorescent probes. With its unique strengths in utility and control, this approach is potentially beneficial for the development of cell-based analytical devices and microreactors that enable the capture and real-time analysis of cells within microchannels, irrespective of cell anchorage properties.     

Controlled pore functionalization of poly(ethylene terephthalate) track-etched membranes via surface-initiated atom transfer radical polymerization

A new method for surface-initiated atom transfer radical polymerization (ATRP) on the technical polymer poly(ethylene terephthalate) (PET) has been developed which allows controlling and estimating the layer thickness of the grafted polymer in the isocylindrical pores of track-etched membranes. After PET surface treatment by oxidative hydrolysis, the bromoalkyl initiator was immobilized on the PET surface in a two-step solid-phase reaction; the isoporous membrane structure was preserved, and the pore diameter was increased from 760 to 790 nm. Poly(N-isopropylacrylamide) (PNIPAAm) was grafted under ATRP conditions from a methanol/water mixture at room temperature. Both monomer concentration and reaction time could be used as parameters to adjust the degree of grafting. Effective grafted layer thickness and its response to temperature were estimated from pure water permeability. All data, especially the high polymer densities (0.37 g/cm3) in the swollen layers at 25 degrees C, indicate that grafted PNIPAAm with a "brush" structure has been achieved. For dry PNIPAAm layer thicknesses on the PET pore walls of up to 80 nm, a temperature-induced swelling/deswelling ratio of approximately 3 had been observed. Reduction of the brush grafting density, via composition of the reaction mixture used in solid-phase synthesis for initiator immobilization, led to an increase of that swelling/deswelling ratio. Further, density and temperature response of the grafted PNIPAAm layers synthesized via ATRP were compared with those obtained in the same membranes by less controlled photografting, leading to lower grafting density and larger gradients in grafted layer density and, consequently, much higher swelling/deswelling ratios (>15).     

一种新的压电免疫传感器中生物分子固定化方法的研究

生物分子固定化或传感界面设计技术是研制压电免疫传感器的关键之一。本文 结合自组装单分子膜（SAMs）和聚电解质静电吸附组装技术，提出了一种新的压电 免疫传感器中生物分子固定化方法，研制成一种检测补体C_3的压电免疫传感器。 先在石英晶振的金电极表面组装一层胱胺SAMs，再在膜上组装带相反电荷的聚苯磺 酸钠（PSS）单层膜，通过静电吸附作用固定抗体（抗原），实现对相应抗原（抗 体）的检测。利用扫描电镜技术，从形态上考察了晶振组装胱氨SAMs与PSS及固定 补体C_3抗体后的表面形貌。研究了抗体的固定化条件，探讨了传感器采用这种固 定化方法的响应与再生性能，并与戊二醛键合固定法进行比较。结果表明，这种固 定化方法不仅对蛋白质类生物分子的固定化具有普适性，而且对所固定的生物分子 的活性影响小，传感器的响应的频移值大，灵敏度高，选择性和再生性能均较好。     

Patterned self-assembled beads in silicon channels.

A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 microm wide and 50 microm deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 microm, has also been generated on the bottom of a 500 microm wide and 50 microm deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.     

Ultra-smooth glass channels for bioassay with motor proteins

Optically-flat glass channels were fabricated for the non-fluorescent observation of bio-molecules. Two bioassays of motor proteins were successfully performed, in which nanoscale beads and microtubules were clearly observed by a differential interference contrast (DIC) microscope and a dark-field microscope, respectively. The concentration of hydrofluoric acid (HF) was optimized to obtain an optically flat surface which was evaluated by AFM, SEM and dark-field microscopy. A glass channel was etched using a poly(dimethyl siloxane)(PDMS) microfluidic channel as a mask and sealed with a PDMS-coated coverslip. The channel volume of 2-3 microl realized the drastic reduction of proteins required per assay compared with a conventional flow cell method requiring 20 microl.     

Preparation of biotinylated glyconanoparticles via a photochemical process and study of their bioconjugation to streptavidin

We report here the preparation of novel biotinylated glyconanoparticles from well-defined biotinylated glycopolymers and poly(N-isopropylacrylamide) (PNIPAAm) synthesized via the reversible addition fragmentation chain transfer (RAFT) polymerization process. The in situ reduction of the biotinylated glycopolymers, PNIPAAm, poly(ethylene glycol), and HAuCl4 via a photochemical process resulted in the formation of biotinylated gold nanoparticles. The multifunctional biotinylated glyconanoparticles were then evaluated for their bioconjugation toward streptavidin using UV-vis spectroscopy and surface plasmon resonance (SPR). The biotinylated nanoparticles underwent aggregation in the presence of streptavidin as revealed by spectrophotometry, which indicates the accessibility of the biotin for conjugation. These results were further confirmed by surface plasmon resonance even in the case of surface-immobilized streptavidin.     

PET/PEN共混物的相容性与酯交换反应

通过用1H-NMR对聚对苯二甲酸乙二酯(PET)与聚2,6-萘甲酸乙二酯(PEN)、PET/PEN共聚物的共混物酯交换反应的研究,测得了反应速率常数、反应活化能和诱导期.根据酯交换反应程度和不同反应温度下的诱导期探讨了酯交换反应与相容性的关系,认为PET与PEN的相容导致或增强了酯交换反应,即相容性是酯交换的必要条件;同时酯交换的发生又促进了PET与PEN的相容.酯交换和相容是聚酯共混物熔融时相互关联的两个过程.     

Preparation of temperature‐responsive polymer gels physically immobilizing core‐shell type bioconjugates

Poly(N‐isopropylacrylamide) (PNIPAAm) gels are temperature‐responsive polymer gels; and were prepared by redox polymerization of N‐isopropylacrylamide in the presence of N,N′‐methylenebisacrylamide as a crosslinking reagent and core‐shell type bioconjugates, which were core‐crosslinked polyion complex micelles formed from the mixture of bovine pancreas trypsin and poly(ethylene glycol)‐block‐poly(α,β‐aspartic acid). The phase transition temperature of PNIPAAm gels was no change with physically immobilization of bioconjugates. Also, the enzymatic activity of bioconjugates was essentially maintained even in PNIPAAm gels, although enzymatic reaction rate was apparently controlled by temperature, i.e., by the degree of swelling of PNIPAAm gels. Further, the control of enzymatic reaction synchronizing the phase transition of PNIPAAm gels immobilized bioconjugates. PNIPAAm gels could immobilize core‐shell type bioconjugates, and were successfully prepared without interfering with the properties of temperature‐responsive polymer gels and the bionanoreactor. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5942–5948, 2007     

Fluorescence affinity sensing by using a self-contained fluid manoeuvring microfluidic chip

An application of a novel polymer microfluidic chip for sample exchange via natural capillary forces for immuno-analysis is described. The microfluidic device was designed to achieve sample replacement by capillary force only, which would therefore be suitable for point-of-care-testing. Complete and automatic replacement of the sample in the reaction chamber with another one makes the chip able to mimic affinity chromatography and immunoassay processes. The microfluidic chip was made using polymer replication techniques, which were suitable for fast and cheap fabrication. Micrometre-sized polystyrene beads were used for the functionalization of biomolecules. Dinitrophenyl (DNP) and anti-DNP antibody coordination was employed on the chip for fluorescence analysis. DNP was immobilized on the polymer beads via a pre-adsorbed dendrimer layer and the beads were placed in the reaction chamber. Fluorescein tagged anti-DNP was successfully observed by a fluorescence microscope after the completion of the entire flow sequence. A calibration curve was registered based on the anti-DNP concentration. A multiplex sensing was accomplished by adding biotin/streptavidin coordination to the system. DNP and biotin conjugated beads were placed in the reaction chamber in an ordered fashion and biospecific bindings of anti-DNP antibody and streptavidin were observed at their expected sites. A ratiometric analysis was carried out with different concentration ratios of anti-DNP/streptavidin. The microfluidic chip described in this work could be applied to various biological and chemical analyses using integrated washing steps or fluid replacement steps with minimum sample handling.     

蛋白质DNA混合微点阵和微流控芯片的整合

在活化的石英片上制作蛋白质和DNA微点阵, 并可逆地将其与含有通道的多聚二甲基硅氧烷弹性橡胶封接在一起, 使蛋白质和DNA微点阵组装在微通道列阵内; 实现在微通道列阵内同时检测和分析蛋白质与DNA的功能. 为了降低多聚二甲基硅氧烷弹性橡胶的疏水性, 增强其生物相容性, 实验通过多聚赖氨酸对多聚二甲基硅氧烷弹性橡胶的修饰, 提高了它的亲水性, 使溶液能够在微通道内顺畅地流通. 实验表明, 这种混合芯片能够提高检测速度和增加检测的信息量.     

Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel

A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 microg ml(-1)) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 microm width x 6 microm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 microM) from being transported to the electrode surface while nitric oxide (NO, 10 microM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 microM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.     

Fabrication of cell-containing hydrogel microstructures inside microfluidic devices that can be used as cell-based biosensors

This paper describes microfluidic systems containing immobilized hydrogel-encapsulated mammalian cells that can be used as cell-based biosensors. Mammalian cells were encapsulated in three-dimensional poly(ethylene glycol)(PEG) hydrogel microstructures which were photolithographically polymerized in microfluidic devices and grown under static culture conditions. The encapsulated cells remained viable for a week and were able to carry out enzymatic reactions inside the microfluidic devices. Cytotoxicity assays proved that small molecular weight toxins such as sodium azide could easily diffuse into the hydrogel microstructures and kill the encapsulated cells, which resulted in decreased viability. Furthermore, heterogeneous hydrogel microstructures encapsulating two different phenotypes in discrete spatial locations were also successfully fabricated inside microchannels.     

环氧基修饰周期性介孔有机氧化硅对漆酶的固定化作用

以三嵌段共聚物P123为模板剂, 在酸性条件下通过1,2-三乙氧基硅基乙烷(BTESE)和3-含氧缩水甘油基-丙基-三甲氧基硅烷(GPTMS)共水解缩聚合成环氧基修饰的周期性介孔氧化硅(PMOs), 以修饰后的PMOs为载体对漆酶进行固定化, 研究了环氧基修饰对固定化酶稳定性的影响. 通过XRD、TEM、固态NMR和N₂吸附等手段表征材料的修饰效果、孔结构以及漆酶的固定化. 结果表明, 修饰后的材料保持良好的孔结构, 环氧基的修饰有利于提高固定化酶的活力, 基于环氧基修饰PMOs的固定化酶具有较高稳定性.     

Multivalent binding of small guest molecules and proteins to molecular printboards inside microchannels

Beta-Cyclodextrin (beta-CD) monolayers have been immobilized in microchannels. The host-guest interactions on the beta-CD monolayers inside the channels were comparable to the interactions on beta-CD monolayers on planar surfaces, and a divalent fluorescent guest attached with a comparable binding strength. Proteins were attached to these monolayers inside microchannels in a selective manner by employing a strategy that uses streptavidin and orthogonal linker molecules. The design of the chip, which involved a large channel that splits into four smaller channels, allowed the channels to be addressed separately and led to the selective immobilization of antibodies. Experiments with labeled antibodies showed the selective immobilization of these antibodies in the separate channels.     

Enhanced Microchip Electrophoresis of Neurotransmitters on Glucose Oxidase Modified Poly(dimethylsiloxane) Microfluidic Devices

In this paper, glucose oxidase (GOx) was employed to construct a functional film on the poly(dimethylsiloxane) (PDMS) microfluidic channel surface and apply to perform electrophoresis coupled with in‐channel electrochemical detection. The film was formed by sequentially immobilizing poly(diallyldimethylammonium chloride) (PDDA) and GOx to the microfluidic channel surface via layer‐by‐layer (LBL) assembly. A group of neurotransmitters (5‐hydroxytryptamine, 5‐HT; dopamine, DA; epinephrine, EP; dobuamine, DBA) as a group of separation model was used to evaluate the effect of the functional PDMS microfluidic devices. Electroosmotic flow (EOF) in the modified PDMS microchannel was well suppressed compared with that in the native one. Experimental conditions were optimized in detail. As expected, these analytes were efficiently separated within 110 s in a 3.7 cm long separation channel and successfully detected at a single carbon fiber electrode. Good performances were attributed to the decreased EOF and the interactions of analytes with the immobilized GOx on the PDMS surface. The theoretical plate numbers were 2.19×10⁵, 1.89×10⁵, 1.76×10⁵, and 1.51×10⁵ N/m at the separation voltage of 1000 V with the detection limits of 1.6, 2.0, 2.5 and 6.8 μM (S/N=3) for DA, 5‐HT, EP and DBA, respectively. In addition, the modified PDMS channels had long‐term stability and excellent reproducibility.     

Microfluidic immunoassay with plug-in liquid crystal for optical detection of antibody

Recent advance in liquid crystal (LqC) based immunoassays enables label-free detection of antibody, but manual preparation of LqC cells and injection of LqC are required. In this work, we developed a new format of LqC-based immunoassay which is hosted in a microfluidic device. In this format, the orientations of LqC are strongly influenced by four channel walls surrounding the LqC. When the aspect ratio (depth/width) of the channel is smaller than 0.38, LqC orients homeotropically inside the microchannel and appears dark. After antigens bind to immobilized antibodies on the channel walls, a shift of the LqC appearance from dark to bright (due to the disruption of LqC orientation) can be visualized directly. To streamline the immunoassay process, a tubing cartridge loaded with a sample solution, washing buffers and a plug of LqC is connected to the microfluidic device. By using pressure-driven flow, the cartridge allows antigen/antibody binding, washing and optical detection to be accomplished in a sequential order. We demonstrate that this microfluidic immunoassay is able to detect anti-rabbit IgG with a naked-eye detection limit down to 1 μg mL⁻¹. This new format of immunoassay provides a simple and robust approach to perform LqC-based label-free immunodetection in microfluidic devices.     

Determination of Ochratoxin A in apples contaminated with Aspergillus ochraceus by using a microfluidic competitive immunosensor with magnetic nanoparticles

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi of the genus Aspergillus and Penicillium that presents carcinogenic, teratogenic and nephrotoxic properties. In this work, we have developed, characterized and applied an immunoassay methodology comprised of magnetic nanoparticles (MNPs) as platform for immobilizing bioactive materials incorporated into a microfluidic system for rapid and sensitive quantification of Ochratoxin A (OTA) in apples (Red Delicious) contaminated with Aspergillus ochraceus. The sensor has the potential for automation and the detection of OTA was carried out using a competitive indirect immunoassay method based on the use of anti-OTA monoclonal antibodies immobilized on 3-aminopropyl-modified MNPs. The total assay time into the microfluidic competitive immunosensor was 16 min, and the calculated detection limit was 0.05 μg kg(-1). Moreover, the intra- and inter-assay coefficients of variation were below 6.5%. The proposed method can be a very promising analytical tool for the determination of OTA in apparently healthy fruits post-harvest and for its application in the agricultural industry.