Integrated label-free protein detection and separation in real time using confined surface plasmon resonance imaging

We demonstrate a label-free protein detection and separation technology for real-time monitoring of proteins in micro/nanofluidic channels, confined surface plasmon resonance imaging (confined-SPRi). This was achieved by fabricating ultrathin fluidic channels (500 nm high, 500 microm wide) directly on top of a specialized SPRi sensor surface. In this way, SPRi is uniquely used to detect proteins deep into the fluidic channel while maintaining high lateral accuracy of separated products. The channel fluid and proteins were driven electrokinetically under an external electric field. For this to occur, the metallic SPR sensor (46 nm of Au on 2 nm of Cr) was segmented into an array of squares (each 200 microm x 200 microm in size and spaced 8 microm apart) and coated with 30 nm of CYTOP polymer. In this work, we track label-free protein separation in real time through a simple cross-junction fluidic device with an 8-mm separation channel length under 30 V/cm electric field strength.     

Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules

An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.     

Electrochromatography in microchips: reversed-phase separation of peptides and amino acids using photopatterned rigid polymer monoliths

A microfabricated glass chip containing fluidic channels filled with polymer monolith has been developed for reversed-phase electrochromatography. Acrylate-based porous polymer monoliths were cast in the channels by photopolymerization to serve as a robust and uniform stationary phase. UV light-initiated polymerization allows for patterning of polymer stationary phase in the microchip, analogous to photolithography, using a mask and a UV lamp for optimal design of injection, separation, and detection manifolds. The monoliths are cast in situ in less than 10 min, are very reproducible with respect to separation characteristics, and allow easy manipulation of separation parameters such as charge, hydrophobicity, and pore size. Moreover, the solvent used to cast the polymer enables electroosmotic flow, allowing the separation channel to be conditioned without need for high-pressure pumps. The microchip was used for separation of bioactive peptides and amino acids labeled with a fluorogenic dye (naphthalene-2,3-dicarboxaldehyde) followed by laser-induced fluorescence detection using a Kr+ ion laser. The microchip-based separations were fast (six peptides in 45 s), efficient (up to 600,000 plates/m), and outperformed the capillary-based separations in both speed and efficiency. We have also developed a method for complete removal of polymer from the channels by thermal incineration to regenerate the glass chips.     

I-shaped microchannel array chip for parallel electrophoretic analyses

We demonstrate electrophoresis in I-shaped microchannels with a new design and operation principle. Unlike the conventional T- or cross-shaped microchannels, the simple I-shaped design makes it straightforward to integrate parallel microchannels with electrodes onto a microchip. The operation of the I-shaped microchannels has been enabled by the autonomous solution filling technique, which exploits the high gas solubility in poly(dimethylsiloxane) (PDMS). We fabricated an I-shaped microchannel array (IMA) chip by integrating 12 independent microchannels and 2 electrodes onto a 3 cm x 2 cm area in a PDMS-glass hybrid microchip. For autonomous regulation of stable sample plugs in all the microchannels, we discovered that O2 plasma treatment of the PDMS-made reservoirs is effective. On the IMA chip, size-dependent separation of double-stranded (ds) DNA and sequence-specific separation of single-stranded DNA were achieved. Specifically, 10 fragments in a 100-1000-bp dsDNA ladder were separated using hydroxyethylcellulose as a sieving matrix within a separation length of 2 mm, and polymerase chain reaction products of the wild-type K-ras gene and its point mutant were separated using a probe DNA-poly(dimethylacrylamide) conjugate on the basis of affinity capillary electrophoresis. The IMA chip presented here opens up a new possibility of large-scale integration of microchannels for high-throughput electrophoretic analyses.     

Amperometric fluidic microchip array sensing device for nitric oxide determination in solution

In this paper, we report on the first use of an amperometric fluidic microchip array for the examination of nitric oxide in solution. The array chip is composed of 36 working platinum electrodes on a glass substrate. The electrodes have a diameter of 50 μm and are separated by 500 μm. The array chip is integrated within a flowing cell to obtain a fluidic-type sensing device. Two preliminary tests were performed. The first one consisted in assessing the fluidic set-up by using potassium ferrocyanide as test analyte. The second test was aimed at achieving the modification of the surface of the working electrodes by electrodepositing nickel tetrasulfonated phthalocyanine and Nafion® layers to show that the fluidic sensing device can be adapted to the analysis of nitric oxide in solution.     

Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.     

Electroosmotic flow-based pump for liquid chromatography on a planar microchip

An electroosmotic flow (EOF)-based pump, integrated with a sol-gel stationary phase located in the electric field-free region of a microchip, enabled the separation of six nitroaromatic and nitramine explosives and their degradation products via liquid chromatography (LC). The integrated pump and LC system were fabricated within a single quartz substrate. The pump region consisted of a straight channel (3.0 cm x 230 microm x 100 microm) packed with 5-microm porous silica beads. The sol-gel stationary phase was derived from a precursor mixture of methyltrimethoxy- and phenethyltrimethoxysilanes and was synthesized in the downstream, field-free region of the microchip, resulting in a stationary-phase monolith with dimensions of 2.6 cm x 230 microm x 100 microm. Fluid dynamic design considerations are discussed, especially as they relate to integrating the EOF pump with the LC system. Pump and separation performance, as characterized by flow rate measurements, injection, elution, separation, and detection, point to a viable analytical chemistry platform that encompasses all of the benefits expected of portable, laboratory-on-chip systems, including reduced sample requirements and small packaging.     

Microfluidic electrochemical aptameric assay integrated on-chip: a potentially convenient sensing platform for the amplified and multiplex analysis of small molecules

Aptamers are artificial oligonucleotides that have been widely employed to design biosensors (i.e., aptasensors). In this work, we report a microfluidic electrochemical aptamer-based sensor (MECAS) by constructing Au-Ag dual-metal array three-electrode on-chip for multiplex detection of small molecules. In combination with the microfluidic channels covering on the glass chip, different targets are transported to the Au electrodes integrated on different positions of the chip. These electrodes are premodified by different kinds of aptamers, respectively, to fabricate different sensing interfaces which can selectively capture the corresponding target. It is an address-dependent sensing platform; thus, with the use of only one electrochemical probe, multitargets can be recognized and detected according to the readout on a corresponding aptamer-modified electrode. In the sensing strategy, the electrochemical probe, [Ru(NH(3))(6)](3+) (RuHex), which can quantitatively bind to surface-confined DNA via electrostatic interaction, was used to produce chronocoulometric signal; Au nanoparticles (AuNPs) were used to improve the sensitivity of the sensor by amplifying the detection signals. Moreover, the sensing interface fabrication, sample incubation, and electrochemical detection were all performed in microfluidic channels. By using this detection chip, we achieved the multianalysis of two model small molecules, ATP, and cocaine, in mixed samples within 40 min. The detection limit of ATP was 3 × 10(-10) M, whereas the detection limit of cocaine was 7 × 10(-8) M. This Au-Ag dual metal electrochemical chip detector integrated MECAS was simple, sensitive, and selective. Also it is similar to a dosimeter which accumulates signal upon exposure. It held promising potential for designing electrochemical devices with high throughput, high automation, and high integration in multianalysis.     

Direct determination of carbohydrates, amino acids, and antibiotics by microchip electrophoresis with pulsed amperometric detection

The separation and detection of underivatized carbohydrates, amino acids, and sulfur-containing antibiotics in an electrophoretic microchip with pulsed amperometric detection (PAD) is described. This report also describes the development of a new chip configuration for microchip electrophoresis with PAD. The configuration consists of a layer of poly(dimethylsiloxane) that contains the microfluidic channels, reservoirs, and a gold microwire, sealed to a second layer of poly(dimethylsiloxane). Example separations of carbohydrates, amino acids, and sulfur-containing antibiotics are shown. The effect of the separation and injection potentials, buffer pH and composition, injection time, and PAD parameters were studied in an effort to optimize separations and detection. Detection limits ranging from 6 fmol (5 microM) for penicillin and ampicillin to 455 fmol (350 microM) for histidine were obtained.     

Isoelectric focusing in a microfluidically defined electrophoresis channel

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.     

Microfabricated polymer analysis chip for optical detection

A coupling between multimode polymer waveguides and microfluidic channels on a polymethylmethacrylate (PMMA) capillary electrophoresis (CE)-chip for optical analytical applications has been successfully realised. This technology allows the integration of polymer optical waveguides together with hermetically sealed fluidic channels. The microchannels and waveguides are made in PMMA by the approved hot-embossing technology. The technology developed for the fabrication of polymer waveguides on the microfluidic chip offers the possibility of great flexibility in the choice of core materials, design and alignment of the polymer waveguides. The integration of polymer waveguides on an analysis chip enables highly spatially resolved optical detection without the large and expensive conventionally used apparatus. The optical properties of the analytical system developed are verified by transmission and propagation loss measurements. The results of measurements prove the suitability of the presented device for optical applications between 440 and 800 nm. This was shown with absorbance measurements of the dye Sulfanilazochromotrop (SPADNS) within 50 microm fluidic channels.     

Fully packed capillary electrochromatographic microchip with self-assembly colloidal silica beads

A fully packed capillary electrochromatographic (CEC) microchip showing improved solution and chip handling was developed. Microchannels for the CEC microchip were patterned on a cyclic olefin copolymer substrate by injection molding and packed fully with 0.8-microm monodisperse colloidal silica beads utilizing a self-assembly packing technique. The silica packed chip substrate was covered and thermally press-bonded. After fabrication, the chip was filled with buffer solution by self-priming capillary action. The self-assembly packing at each channel served as a built-in nanofilter allowing quick loading of samples and running buffer solution without filtration. Because of a large surface area-to-volume ratio of the silica packing, reproducible control of electroosmotic flow was possible without leveling of the solutions in the reservoirs resulting 1.3% rsd in migration rate. The capillary electrophoretic separation characteristics of the chip were studied using fluorescein isothiocyanate (FITC)-derivatized amino acids as probe molecules. A mixture of FITC and four FITC-derivatized amino acids was successfully separated with 2-mm separation channel length.     

Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).     

离体电生理检测微电极阵列传感器的设计与制备(英文)

针对动物离体组织电生理检测的实际需求,设计并制备了一种以载玻片为基底,以微电极阵列为敏感元件,并将灌流装置集成一体的传感器芯片.采用微电子机械系统(MEMS)技术中的薄膜工艺完成了微电极阵列的制备,其导电层和绝缘层分别是铂和氮化硅.采用聚二甲基硅烷(PDMS)浇铸制成埋有管道的方形灌流槽.该传感器可保持离体组织的生理活性,同时实现电生理信号的64通道同步记录.整个芯片结构紧凑,接口简单,使用方便.对芯片的电学性能进行了研究,结果表明,通过在微电极表面电镀修饰铂黑,可有效降低其交流阻抗,提高信噪比.     

Contactless conductivity detector for microchip capillary electrophoresis

A microfabricated electrophoresis chip with an integrated contactless conductivity detection system is described. The new contactless conductivity microchip detector is based on placing two planar sensing aluminum film electrodes on the outer side of a poly(methyl methacrylate) (PMMA) microchip (without contacting the solution) and measuring the impedance of the solution in the separation channel. The contactless route obviates problems (e.g., fouling, unwanted reactions) associated with the electrode-solution contact, offers isolation of the detection system from high separation fields, does not compromise the separation efficiency, and greatly simplifies the detector fabrication. Relevant experimental variables, such as the frequency and amplitude of the applied ac voltage or the separation voltage, were examined and optimized. The detector performance was illustrated by the separation of potassium, sodium, barium, and lithium cations and the chloride, sulfate, fluoride, acetate, and phosphate anions. The response was linear (over the 20 microM-7 mM range) and reproducible (RSD = 3.4-4.9%; n = 10), with detection limits of 2.8 and 6.4 microM (for potassium and chloride, respectively). The advantages associated with the contactless conductivity detection, along with the low cost of the integrated PMMA chip/detection system, should enhance the power and scope of microfluidic analytical devices.     

Flow cytometry of Escherichia coli on microfluidic devices.

Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%.     

Ion diffusion in channels containing random arrays of microspheres: an electrochemical time-of-flight method

Rates of chloride ion diffusion in narrow (ca. 3 microm thick), rectangular (ca. 0.1 x 1.0 mm(2)) channels partially filled with polystyrene microspheres are investigated by a potentiometric electrochemical time-of-flight (P-ETOF) method. Lithographically fabricated on glass slides, P-ETOF devices consist of a centrally positioned 10 microm wide, ca. 1 mm long generator microelectrode and two sensor microelectrodes of the same dimensions symmetrically positioned on both side of the generator at a distance of 50 microm. The electrodes are silver-plated and partially oxidized in a chloride electrolyte to form Ag/AgCl deposits. Constant current reduction of AgCl on the generator electrode is used to produce chloride ions at a constant rate. Ag/AgCl deposited on the sensor microelectrodes allows time-dependent potentiometric monitoring of the increasing concentration of chloride ions diffusing across the interelectrode gap. The device is enclosed with a parallel glass plate to form a narrow channel with the polystyrene microbeads serving as spacers. The packing density of the microspheres expressed in terms of the fractional void volume (rho) varied from ca. 0.6 to 1.0. Using rho, we modified a diffusion equation describing the change of chloride ion concentration at the sensor microelectrode to include the effect of the microspheres restricting the void volume. We rely on digital simulations as well as on direct P-ETOF experiments to show that the proposed equation does accurately account for the effect of rho on the diffusion processes. We thus demonstrate that P-ETOF can be used to measure the number of identical microspheres in the active region of a narrow channel device. In the latter context, a future application of P-ETOF as a signal transduction mechanism in biosensors is outlined.     

An integrated microfluidic device for rapid cell lysis and DNA purification of epithelial cell samples

In this paper, we describe the design and fabrication of a microfluidic device for cell lysis and DNA purification, and the results of device tests using a real sample of buccal cells. Cell lysis was thermally executed for two minutes at 80 degrees C in a serpentine type microreactor (20 microL) using an Au microheater with a microsensor. The DNA was then mixed with other residual products and purified by a new filtration process involving micropillars and 50-80 microm microbeads. The entire process of sample loading, cell lysis, DNA purification, and sample extraction was successfully completed in the microchip within five minutes. Sample preparation within the microchip was verified by performing a SY158 gene PCR analysis and gel electrophoresis on the products obtained from the chip. The new purification method enhanced DNA purity from 0.93 to 1.62 after purification.     

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.     

Compact microelectrode array system: tool for in situ monitoring of drug effects on neurotransmitter release from neural cells

This paper presents a compact microelectrode array (MEA) system, to study potassium ion-induced dopamine release from PC12 neural cells, without relying on a micromanipulator and a microscope. The MEA chip was integrated with a custom-made "test jig", which provides a robust electrical interfacing tool between the microchip and the macroenvironment, together with a potentiostat and a microfluidic syringe pump. This integrated system significantly simplifies the operation procedures, enhances sensing performance, and reduces fabrication costs. The achieved detection limit for dopamine is 3.8 x 10-2 muM (signal/noise, S/N = 3) and the dopamine linear calibration range is up to 7.39 +/- 0.06 muM (mean +/- SE). The effects of the extracelluar matrix collagen coating of the microelectrodes on dopamine sensing behaviors, as well as the influences of K+ and l-3,4-digydroxyphenylalanine concentrations and incubation times on dopamine release, were extensively studied. The results show that our system is well suited for biologists to study chemical release from living cells as well as drug effects on secreting cells. The current system also shows a potential for further improvements toward a multichip array system for drug screening applications.