Microcontact printing of proteins inside microstructures

Microfluidic devices are well suited for the miniaturization of biological assays, in particular when only small volumes of samples and reagents are available, short time to results is desirable, and multiple analytes are to be detected. Microfluidic networks (MFNs), which fill by means of capillary forces, have already been used to detect important biological analytes with high sensitivity and in a combinatorial fashion. These MFNs were coated with Au, onto which a hydrophilic, protein-repellent monolayer of thiolated poly(ethyleneglycol) (HS-PEG) was self-assembled, and the binding sites for analytes were present on a poly(dimethylsiloxane) (PDMS) sealing cover. We report here a set of simple methods to extend previous work on MFNs by integrating binding sites for analytes inside the microstructures of MFNs using microcontact printing (muCP). First, fluorescently labeled antibodies (Abs) were microcontact-printed from stamps onto planar model surfaces such as glass, Si, Si/SiO2, Au, and Au derivatized with HS-PEG to investigate how much candidate materials for MFNs would quench the fluorescence of printed, labeled Abs. Au coated with HS-PEG led to a fluorescence signal that was approximately 65% weaker than that of glass but provided a convenient surface for printing Abs and for rendering the microstructures of the MFNs wettable. Then, proteins were inked from solution onto the surface of PDMS (Sylgard 184) stamps having continuous or discontinuous micropatterns or locally inked onto planar stamps to investigate how the aspect ratio (depth:width) of microstructures and the printing conditions affected the transfer of protein and the accuracy of the resulting patterns. By applying a controlled pressure to the back of the stamp, Abs were accurately microcontact-printed into the recessed regions of MFNs if the aspect ratio of the MFN microstructures was lower than approximately 1:6. Finally, the realization of a simple assay between Abs (used as antigens) microcontact-printed in microchannels and Abs from solution suggests that this method could become useful to pattern proteins in microstructures for advanced bioanalytical purposes.     

微流控芯片流动注射气体扩散分离光度测定系统的研究

提出了纳升级进样量的微流控芯片流动注射气体扩散分离光度检测系统. 制作三层结构微流控芯片, 在玻璃片上加工微反应通道, 用聚二甲基硅氧烷[Poly(dimethylsiloxane), PDMS]加工气体渗透膜和具有接收气体微通道的底片, 实现了生成气体的化学反应、气-液分离和检测在同一微芯片上的集成化. 采用缝管阵列纳升流动注射进样系统连续进样, 用吸光度法测定NH+4以验证系统性能. 结果表明， 该系统对NH+4的检出限为140 μmol/L(3σ), 峰高精度为3.7%(n=9). 在进样时间12 s、注入载流48 s和每次进样消耗200 nL试样条件下, 系统分析通量可达60样/h. 若加大样品量到800 nL, 使接收溶液停流1 min, 该系统对NH+4的检出限可达到35 μmol/L(3σ), 但分析通量降低到20样/h.     

基于液芯波导原理的微流控芯片长光程光度检测系统

提出了一种基于液芯波导(Liquidcorewaveguide,LCW)原理的微流控芯片吸收光度检测系统.通过芯片与外界接口技术实现液芯波导管与芯片的耦合,建立了芯片上长光程(毫米至厘米级)吸收光度检测池.采用邻菲啉-铁(Ⅱ)显色体系验证系统分析性能,以5.5cm外覆TeflonAF液芯波导管作为检测池(检测池体积240nL)时,芯片系统的检测线性范围为0.03～50μmol/L,对邻菲啉-铁(Ⅱ)配合物的检出限为8nmol/L,检测池有效光程达1.7cm,分析精度RSD(n=5)为0.8%.     

Single cell analysis in full body quartz glass chips with native UV laser-induced fluorescence detection

In order to investigate the individual and inhomogenous cellular response, e.g. to external stimuli, single cell analysis is mandatory and may provide new cognitions in proteomics as well as in other fields of systems biology in the future. Here, we report on novel chip architectures for single cell analysis based on full body quartz glass microfluidic chips (QG chips) that extend our previous studies in polydimethylsiloxane (PDMS) chips, and enhance the detection sensitivity of native UV laser-induced fluorescence (UV-LIF) detection. Detection of a 10nM tryptophan solution with an S/N ratio of 11.9, which gives a theoretical limit of detection of 2.5nM (with S/N=3), was possible. With these optimizations the three proteins alpha-chymotrypsinogen A, ovalbumin and catalase each at a concentration of 100mug/mL (equal to 4muM, 0.4muM and 2.2muM) were injected electrokinetically and could be separated with nearly baseline resolution. Furthermore, fluorescence spectra (excitation wavelength, lambda(ex)=266nm) clearly demonstrate the favourable properties like the very high UV transparency and the nearly vanishing background fluorescence of the QG chips as compared to PDMS chips and to PDMS quartz window (PQW) chips. Finally we exploit the improved sensitivity for single cell electropherograms of Spodoptera frugiperda (Sf9) insect cells.     

Bulk- and surface-modified combinable PDMS capillary sensor array as an easy-to-use sensing device with enhanced sensitivity to elevated concentrations of multiple serum sample components

To enhance sensitivity and facilitate easy sample introduction into a combinable poly(dimethylsiloxane) (PDMS) capillary (CPC) sensor array, PDMS was modified in bulk and on its surface to prepare "black" PDMS coated with a silver layer and self-assembled monolayer (SAM). India ink, a traditional Japanese black ink, was added to the PDMS pre-polymer for bulk modification. The surface was modified by a silver mirror reaction followed by SAM formation using cysteine. These modifications enhanced the fluorescence signals by reflecting them from the surface and reducing background interference. A decrease in the water contact angle led to enhanced sensitivity and easy sample introduction. Furthermore, a CPC sensor array for multiplex detection of serum sample components was prepared that could quantify the analytes glucose, potassium, and alkaline phosphatase (ALP). When serum samples were introduced by capillary action, the CPC sensor array showed fluorescence responses for each analyte and successfully identified the components with elevated concentrations in the serum samples.     

Determination of oxalate in urine by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel coupled to a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in urine was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (4.0) provided an adequate selectivity in the separation of oxalate from anionic urine constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 8 x 10(-8) mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5 x 10(-3) mol/L concentrations. Such a favorable analyte/matrix concentration ratio (in part, attributable to a transient isotachophoresis stacking in the initial phase of the separation) made possible accurate and reproducible (typically, 2-5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample) determination of oxalate in 500 nL volumes of 20-100-fold diluted urine samples. Short analysis times (about 280 s), no sample pretreatment (not considering urine dilution) and reproducible migration times of this analyte (0.5-1.0% RSD values) were characteristic for ZE on the chip. This work indicates general potentialities of the present chip design in rapid ZE analysis of samples containing the analyte(s) at high ionic matrix/analyte concentration ratios.     

Determination of oxalate in beer by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel combined with a 500 nL sample injection channel) and a pair of on‐chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in beer was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (3.8), implemented by aspartic acid and bis‐tris propane, provided an adequate selectivity in the separation of oxalate from anionic beer constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 0.5 μmol/L concentration also in samples containing chloride (a major anionic constituent of beer) at a 1800 higher concentration. Such a favorable analyte/matrix concentration ratio made possible accurate and reproducible [typically, 2–5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample] determination of oxalate in 500 nL volumes of 20–50‐fold diluted beer samples. Short analysis times (about 200 s), minimum sample preparation, and reproducible migration times of this analyte (0.5–1.0% RSD values) were characteristic for ZE on the chip.     

Optofluidic differential spectroscopy for absorbance detection of sub-nanolitre liquid samples

We present a novel optofluidic differential method for carrying out absorbance spectroscopy of sub-nanolitre volumes of liquid samples on a microfluidic chip. Due to the reduction of liquid volume, the absorbance detection in microfluidics is often hindered by either low sensitivity or complex fabrication. To address this issue, we introduced an optofluidic modulator which can be easily integrated into a PDMS (polydimethylsiloxane) based microfluidic chip. The modulator was controlled by the fluid pressure and the absorbance spectrum of the analyte was obtained by taking differential measurements between the analyte and reference medium. An advantage is that this method doesn't need a complicated fabrication step. It is compatible with conventional microfluidic chips and measurements can be carried out on a normal transmission microscope. The performance of the device was tested by measuring solutions containing methylene blue, with concentrations as low as 13 μM.     

Development of an ultra-low volume flow cell for surface plasmon resonance detection in a miniaturized capillary electrophoresis system

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.     

High-sensitivity detection using isotachophoresis with variable cross-section geometry

We present a theoretical and experimental study on increasing the sensitivity of ITP assays by varying channel cross-section. We present a simple, unsteady, diffusion-free model for plateau mode ITP in channels with axially varying cross-section. Our model takes into account detailed chemical equilibrium calculations and handles arbitrary variations in channel cross-section. We have validated our model with numerical simulations of a more comprehensive model of ITP. We show that using strongly convergent channels can lead to a large increase in sensitivity and simultaneous reduction in assay time, compared to uniform cross-section channels. We have validated our theoretical predictions with detailed experiments by varying channel geometry and analyte concentrations. We show the effectiveness of using strongly convergent channels by demonstrating indirect fluorescence detection with a sensitivity of 100 nM. We also present simple analytical relations for dependence of zone length and assay time on geometric parameters of strongly convergent channels. Our theoretical analysis and experimental validations provide useful guidelines on optimizing chip geometry for maximum sensitivity under constraints of required assay time, chip area and power supply.     

Cholera toxin subunit B detection in microfluidic devices

Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM₁ was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL⁻¹ for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.     

Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection

A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300?nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.     

On-line preconcentration for capillary electrophoresis-atomic fluorescence spectrometric determination of arsenic compounds

An on-line preconcentration method was developed for capillary electrophoresis (CE) with hydride generation-atomic fluorescence spectrometric (HG-AFS) detection of arsenite, arsenate, dimethylarsenic acid, and monomethylarsenic acid. These arsenic species were negatively charged in the sample solution with high pH. When the potential was applied to the electrophoretic capillary, the negatively charged analyte ions moved faster and stacked at the boundary of sample and CE buffer with low pH. So, high sample pH in combination with low buffer pH allowed the injection of large sample volumes (approximately 1100 nL). Comparison of the preconcentration of analyte solution, prepared with doubly deionized water and that prepared with lake or river water, indicated that preconcentration was independent on the original matrix. With injection of approximately 1100 nL sample, an enrichment factor of 37-50-fold was achieved for the four species. Detection limits for the four arsenic species ranged from 5.0 to 9.3 microg.L(-1). Precisions (RSDs, n = 5) were in the range of 4.9-6.7% for migration time, 4.7-11% for peak area, and 4.3-7.1% for peak height, respectively. The recoveries of the four species in locally collected water solution spiked with 0.1 microg.mL(-1) (as As) ranged from 83 to 109%.     

Rapid fabrication of electrochemical enzyme sensor chip using polydimethylsiloxane microfluidic channel

Applicability of polydimethylsiloxane (PDMS) for easy and rapid fabrication of enzyme sensor chips, based on electrochemical detection, is examined. The sensor chip consists of PDMS substrate with a microfluidic channel fabricated in it, and a glass substrate with enzyme-modified microelectrodes. The two substrates are clamped together between plastic plates. The sensor chip has shown no leakage around the microelectrodes under continuous solution flow (34 μl/min). Amperometric response of the sensor chips developed in this work suggest that various types of enzyme sensors can be designed by using PDMS microfluidic channels.     

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.     

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.     

Capillary pumps for autonomous capillary systems

Autonomous capillary systems (CSs), where liquids are displaced by means of capillarity, are efficient, fast and convenient platforms for many bioanalytical applications. The proper functioning of these microfluidic devices requires displacing accurate volumes of liquids with precise flow rates. In this work, we show how to design capillary pumps for controlling the flow properties of CSs. The capillary pumps comprise microstructures of various shapes with dimensions from 15-250 microm, which are positioned in the capillary pumps to encode a desired capillary pressure. The capillary pumps are designed to have a small flow resistance and are preceded by a constricted microchannel, which acts as a flow resistance. Therefore, both the capillary pump and the flow resistance define the flow rate in the CS, and flow rates from 0.2-3.7 nL s(-1) were achieved. The placement and the shape of the microstructures in the capillary pumps are used to tailor the filling front of liquids in the capillary pumps to obtain a reliable filling behaviour and to minimize the risk of entrapping air. The filling front can, for example, be oriented vertically or tilted to the main axis of the capillary pump. We also show how capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in a CS.     

Combinable poly(dimethyl siloxane) capillary sensor array for single-step and multiple enzyme inhibitor assays

We describe a new method for fabricating a capillary-type sensor, called a combinable poly(dimethyl siloxane) (PDMS) capillary (CPC) sensor. The method for preparing the CPC simplifies enzyme inhibitor assays into a simple, single step assay. The sample inhibitor solution is introduced by capillary action. This triggers the spontaneous dissolution of physically adsorbed fluorescent substrates, and the substrate mixes with the inhibitor. This is followed by competitive reaction with insoluble enzyme to give a fluorescence response. CPC is composed of a convex-shaped PDMS stick containing reagents immobilized in an insoluble coating, and a concave-shaped PDMS stick containing reagents immobilized in a soluble coating. Since the concave-shaped PDMS has a deeper channel than the convex structure, combining these PDMS sticks is like closing the zipper of a "freezer bag". This allows easy fabrication of "thin and long" capillary structures containing different reagents inside the same capillary, without the need for precise alignment. This method allows the immobilization of two reactive reagents, such as enzyme and substrate required for a single step assay, which are typically very difficult to immobilize using commercially available conventional capillaries. Furthermore, by simply arraying various CPCs, the CPC sensor allows multiple assays. Here, we carried out a single-step enzyme inhibitor assay using the CPC. In addition, two independent CPCs were arrayed to demonstrate multiple assaying of a protease inhibitor.     

Silver nanoparticle-enhanced fluorescence in microtransponder-based immuno- and DNAhybridization assays

The aim of this study is to improve assay sensitivity in common solid-phase bioassay configurations as the result of using silver nanoparticles. The solid phase was provided by numerically indexed, silicon-based electronic chips, microtransponders (p-Chips) that have previously been used in multiplexed assays. Assay configurations investigated included an ELISA-type immunoassay and a DNA hybridization assay. The surface of p-Chips was derivatized with the silver island film (SIF) and a polymer, and then characterized with AFM and SEM. Silver nanoparticle sizes were in the range of 100 to 200 nm. Four fluorophores were tested for fluorescence enhancement; namely, green fluorescent protein, phycoerythrin, Cy3 and Alexa Fluor 555. We consistently observed significant fluorescence enhancement and sensitivity improvement in the p-Chip-based assays: the sensitivity in the cytokine IL-6 immunoassay was 4.3 pg/ml, which represented a 25-fold increase over the method not involving a SIF; and 50 pM in the hybridization assay, a 38-fold increase. The greatest enhancement was obtained for p-Chip surfaces derivatized first with the polymer and then coated with SIF. In conclusion, we show that the SIF-p-Chip-based platform is a highly sensitive method to quantify low-abundance biomolecules in nucleic acid-based assays and immunoassays.