Immobilization of trypsin in the layer-by-layer coating of graphene oxide and chitosan on in-channel glass fiber for microfluidic proteolysis

In this report, trypsin was immobilized in the layer-by-layer (LBL) coating of graphene oxide (GO) and chitosan on a piece of glass fiber to fabricate microchip bioreactor for efficient proteolysis. LBL deposition driven by electrostatic forces easily took place on the surface of the glass fiber, providing mild environmental conditions so that the denaturation and autolysis of the immobilized trypsin was minimized. Prior to use, a piece of the prepared trypsin-immobilized glass fiber was inserted into the channel of a microchip to form a core-changeable bioreactor. The novel GO-based bioreactor can be regenerated by changing its fiber core. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin, myoglobin, cytochrome c, and hemoglobin and the digestion time was significantly reduced to less than 10 s. The obtained digests were identified by MALDI-TOF MS. The digestion performance of the core-changeable GO-based microchip bioreactor was comparable to that of 12-h in-solution tryptic digestion. The novel microchip bioreactor is simple and efficient, offering great promise for high-throughput protein identification.     

Immobilization of trypsin on silica-coated fiberglass core in microchip for highly efficient proteolysis

In this report, trypsin was immobilized on silica-coated fiberglass core in microchip to form a core-changeable bioreactor for highly efficient proteolysis. To prepare the fiber core, a layer of organic-inorganic hybrid silica coating was prepared on the surface of a piece of glass fiber by a sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, trypsin was immobilized on the coating with the aid of glutaraldehyde. Prior to use, the enzyme-immobilized fiber was inserted into the channel of a microchip to form an in-channel fiber bioreactor. The novel bioreactor can be regenerated by changing its fiber core. The scanning electron microscopy images of the cross-section of a trypsin-immobilized fiber indicated that a layer of ∼1 μm thick film formed on the glass substrate. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c) and the digestion time was significantly reduced to less than 10 s. The digests were identified by MALDI-TOF MS with sequence coverages of 45% (BSA) and 77% (Cyt-c) that were comparable to those obtained by 12-h conventional in-solution tryptic digestion. The fiber-based microchip bioreactor provides a promising platform for the high-throughput protein identification.     

Efficient sample proteolysis based on a microchip containing a glass fiber core with immobilized trypsin

Trypsin was immobilized on cellulose-coated glass fibers via a condensation reaction between the aldehyde groups of the oxidized cellulose and the primary amino groups of trypsin. A piece of the modified fiber was inserted into the main channel of a poly(methyl methacrylate) microchip to form a microfluidic proteolytic bioreactor. Scanning electron microscopy of the cross section of the fiber revealed a rough film on the surface of the fiber glass. The performance of the bioreactor was demonstrated by the tryptic digestion of hemoglobin and cytochrome c, where the time for digestion was reduced to <10?s. The digests were identified by MALDI-TOF-MS to obtain peptide mass fingerprint spectra. The results indicated that the digestion in the microfluidic bioreactor is comparable to that of a 12-h solution tryptic digest and thus provides a promising platform for the high throughput identification of proteins.

A sol-gel-modified poly(methyl methacrylate) electrophoresis microchip with a hydrophilic channel wall

A sol-gel method was employed to fabricate a poly(methyl methacrylate) (PMMA) electrophoresis microchip that contains a hydrophilic channel wall. To fabricate such a device, tetraethoxysilane (TEOS) was injected into the PMMA channel and was allowed to diffuse into the surface layer for 24 h. After removing the excess TEOS, the channel was filled with an acidic solution for 3 h. Subsequently, the channel was flushed with water and was pretreated in an oven to obtain a sol-gel-modified PMMA microchip. The water contact angle for the sol-gel-modified PMMA was approximately 27.4 degrees compared with approximately 66.3 degrees for the pure PMMA. In addition, the electro-osmotic flow increased from 2.13x10(-4) cm2 V(-1) s(-1) for the native-PMMA channel to 4.86x10(-4) cm2 V(-1) s(-1) for the modified one. The analytical performance of the sol-gel-modified PMMA microchip was demonstrated for the electrophoretic separation of several purines, coupled with amperometric detection. The separation efficiency of uric acid increased to 74,882.3 m(-1) compared with 14,730.5 m(-1) for native-PMMA microchips. The result of this simple modification is a significant improvement in the performance of PMMA for microchip electrophoresis and microfluidic applications.     

Bulk modification of polymeric microfluidic devices

The surface properties of microfluidic devices play an important role in their flow behavior. We report here on an effective control of the surface chemistry and performance of polymeric microchips through a bulk modification route during the fabrication process. The new protocol is based on modification of the bulk microchip material by tailored copolymerization of monomers during atmospheric-pressure molding. A judicious addition of a modifier to the primary monomer solution thus imparts attractive properties to the plastic microchip substrate, including significant enhancement and/or modulation of the EOF (with flow velocities comparable to those of glass), a strong pH sensitivity and high stability. Carboxy, sulfo, and amino moieties have thus been introduced (through the incorporation of methylacrylic acid, 2-sulfoethyl-methacrylate and 2-aminoethyl-methacrylate monomers, respectively). A strong increase in the electroosmotic pumping compared to the native poly(methylmethacrylate)(PMMA) microchip (ca. electroosmotic mobility increases from 2.12 to 4.30 x 10(-4) cm(2) V(-1) s(-1)) is observed using a 6% methylacrylate (MAA) modified PMMA microchip. A 3% aminoethyl modified PMMA microchip exhibits a reversal of the electroosmotic mobility (for example, -5.6 x 10(-4) cm(2) V(-1) s(-1) at pH 3.0). The effects of the modifier loading and the pH on the EOF have been investigated for the MAA-modified PMMA chips. The bulk-modified devices exhibit reproducible and stable EOF behavior. The one step fabrication/modification protocol should further facilitate the widespread production of high-performance plastic microchip devices.     

Rinse and evaporation coating of poly(methyl methacrylate) microchip for separation of sodium dodecyl sulfate-protein complex

We developed a novel channel wall coating on a poly(methyl methacrylate) (PMMA) microchip using methylcellulose (MC) as a coating reagent to suppress electroosmotic flow (EOF) following the strong analytes adsorption via hydrophobic interaction with channel walls of PMMA. Our coating was obtained by first rinsing channel walls with MC-containing aqueous solution followed by evaporation. The coating made the hydrophilic channel wall lowering EOF by two orders of magnitude (1.2 x 10(-5)cm(2)V(-1)s(-1)) as well as reducing the hydrophobic adsorption. On the coated channel walls, we successfully separated sodium dodecyl sulfate-protein complexes with high reproducibility and efficiency using dextran as a lower viscosity protein separation medium.     

注塑型聚甲基丙烯酸甲酯多通道微流控芯片的研制及其性能考察

微流控芯片技术因具有微量、快速、高效和高通量等特点,已成为分析化学领域中的研究热点之一．在微流控芯片中,最常见的可用作芯片的材料为玻璃、石英和各种塑料．玻璃和石英有很好的电渗性和光学性质,可采用标准的刻蚀工艺加工和用化学方法进行表面改性,但加工成本较高,封接难度较大．     

Channel wall coating on a poly-(methyl methacrylate) CE microchip by thermal immobilization of a cellulose derivative for size-based protein separation

We demonstrate channel wall coating using a cellulose derivative on a poly-(methyl methacrylate) (PMMA) CE microchip to eliminate EOF disturbing protein separation. The channel walls were modified by preconditioning with a solution containing the cellulose derivative and then thermally evaporating the solution to produce hydrophilic channel walls which prevent adsorption of analytes via a hydrophobic interaction. When the PMMA substrate was coated with the cellulose derivative hydroxypropylmethylcellulose (HPMC) 90SH, the water contact angle on the coated substrate was decreased (up to 15 degrees ) and EOF was significantly suppressed (up to 4.0 x 10(-6) cm2.V(-1)s(-1)). Three proteins (20.5, 68.0, and 114.6 kDa) were successfully separated on the 0.15% HPMC 90SH-coated channel walls with good reproducibility of migration time (RSD <1.75%) and high efficiency (theoretical plate number per meter: 2.62 x 10(5)).     

On-line chemiluminescence detection for isoelectric focusing of heme proteins on microchips

In this paper we present a sensitive chemiluminescence (CL) detection of heme proteins coupled with microchip IEF. The detection principle was based on the catalytic effects of the heme proteins on the CL reaction of luminol-H2O2 enhanced by para-iodophenol. The glass microchip and poly(dimethylsiloxane) (PDMS)/glass microchip for IEF were fabricated using micromachining technology in the laboratory. The modes of CL detection were investigated and two microchips (glass, PDMS/glass) were compared. Certain proteins, such as cytochrome c, myoglobin, and horseradish peroxidase, were focused by use of Pharmalyte pH 3-10 as ampholytes. Hydroxypropylmethylcellulose was added to the sample solution in order to easily reduce protein interactions with the channel wall as well as the EOF. The focused proteins were transported by salt mobilization to the CL detection window. Cytochrome c, myoglobin, and horseradish peroxidase were well separated within 10 min on a glass chip and the detection limits (S/N=3) were 1.2x10(-7), 1.6x10(-7), and 1.0x10(-10) M, respectively.     

Modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of DNA

Inexpensive and permanently modified poly(methyl methacrylate)(PMMA) microchips were fabricated by an injection-molding process. A novel sealing method for plastic microchips at room temperature was introduced. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values between 1-3% for the run-to-run and less than 2.1% for the chip-to-chip comparisons. Acrylonitrile-butadiene-styrene (ABS) was used as an additive in PMMA substrates. The proportions of PMMA and ABS were optimized. ABS may be considered as a modifier, which obviously improved some characteristics of the microchip, such as the hydrophilicity and the electro-osmotic flow (EOF). The detection limit of Rhodamine 6G dye for the modified microchip on the home-made microchip analyzer showed a dramatic 100-fold improvement over that for the unmodified PMMA chip. A detection limit of the order of 10(-20) mole has been achieved for each injected psiX-174/HaeIII DNA fragment with the baseline separation between 271 and 281 bp, and fast separation of 11 DNA restriction fragments within 180 seconds. Analysis of a PCR product from the tobacco ACT gene was performed on the modified microchip as an application example.     

EOF measurement by detection of a sampling zone with end-channel amperometry in microchip CE

A simple method for EOF measurement by detection of sampling zones with end-channel amperometry in microchip CE is developed. This method is based on the principle of the Kohlrausch regulating function (KRF). A dilute electroactive ionic species is added to the BGE as a continuously eluting electrophore which is used as a probe. When a BGE-like sample at a different concentration is injected, a peak of sampling zone appears and the migration time is related to EOF. In a microchip CE with hybrid PDMS/glass channel, a cathodic EOF of the hybrid glass/PDMS microchip was measured by end-channel amperometry; the effects of sample concentration and different probes on EOF rate were discussed. The present method was applied to monitor EOF rates in glass and in PDMS microchips. There was no significant difference between the values of EOF rates measured by the present method and the current-monitoring method. Detection of nonelectroactive analytes K(+), Na(+), and Li(+) can also be accomplished by the indirect amperometric method. Hence, the effective mobility of analyte can be accurately obtained.     

Improved Current-Monitoring Method for Low Electroosmotic Flow Measurement in Modified Microchip

Current-monitoring method is a widely used approach to measure electroosmotic flow (EOF) in microchip, but low and zero EOF is difficult to be measured. In this report, the mechanism of current-monitoring method was explained with Kohlraush regulation function principle, and an improved current-monitoring method was developed for low EOF measurement with tilting microchip. Fluid flow in the channel was accelerated with the help of hydrostatic pressure generated by tilting microchip, the time of dilute solution displacing the concentrated one in channel was shortened. EOF could be calculated according to the time difference between twice experiments under two different applied voltages by tilting microchip. Low even zero EOF could be measured by this improved current-monitoring method. Three modified microchips were characterized to verify the method. EOF in microchannels modified with poly(vinyl alcohol), bovine serum albumin and myoglobin were 0.27 ± 0.05, 0.16 ± 0.05 and ?0.45 ± 0.04 × 10^?4 cm² V^?1 s^?1, respectively.     

Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).     

Hot embossing of electrophoresis microchannels in PMMA substrates using electric heating wires

A simple method based on electric heating wires has been developed for the rapid fabrication of poly(methyl methacrylate) (PMMA) electrophoresis microchips in ordinary laboratories without the need for microfabrication facilities. A piece of stretched electric heating wire placed across the length of a PMMA plate along its midline was sandwiched between two microscope slides under pressure. Subsequently, alternating current was allowed to pass through the wire to generate heat to emboss a separation microchannel on the PMMA separation channel plate at room temperature. The injection channel was fabricated using the same procedure on a PMMA sheet that was perpendicular to the separation channel. The complete microchip was obtained by bonding the separation channel plate to the injection channel sheet, sealing the channels inside. The electric heating wires used in this work not only generated heat; they also served as templates for embossing the microchannels. The prepared microfluidic microchips have been successfully employed in the electrophoresis separation and detection of ions in connection with contactless conductivity detection.     

Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance

A suite of polymers were evaluated for their suitability as viable substrate materials for microchip electrophoresis applications, which were fabricated via replication technology. The relevant physiochemical properties investigated included the glass transition temperature (T(g)), UV-vis absorption properties, autofluorescence levels, electroosmotic flow (EOF) and hydrophobicity/hydrophilicity as determined by sessile water contact angle measurements. These physiochemical properties were used as a guide to select the proper substrate material for the intended microchip electrophoretic application. The T(g) of these polymers provided a guide for optimizing embossing parameters to minimize replication errors (REs), which were evaluated from surface profilometer traces. RE values ranged from 0.4 to 13.6% for the polymers polycarbonate (PC) and low-density polyethylene (LDPE), respectively. The absorption spectra and autofluorescence levels of the polymers were also measured at several different wavelengths. In terms of optical clarity (low absorption losses and small autofluorescence levels), poly(methyl methacrylate), PMMA (clear acrylic), provided ideal characteristics with autofluorescence levels comparable to glass at excitation wavelengths that ranged from 488-780 nm. Contact angle measurements showed a maximum (i.e., high degree of hydrophobicity) for polypropylene (PP), with an average contact angle of 104 degrees +/-3 degrees and a minimum exhibited by gray acrylic, G-PMMA, with an average contact angle of 27 degrees +/-2 degrees. The EOF was also measured for thermally assembled chips both before and after treatment with bovine serum albumin (BSA). The electrophoretic separation of a mixture of dye-labeled proteins including; carbonic anhydrase, phosphorylase B, beta-galactosidase, and myosin, was performed on four different polymer microchips using laser-induced fluorescence (LIF) excitation at 632.8 nm. A maximum average resolution of 5.04 for several peak pairs was found with an efficiency of 6.68 x 10(4) plates for myosin obtained using a BSA-treated PETG microchip.     

Poly(methyl methacrylate) CE microchips replicated from poly(dimethylsiloxane) templates for the determination of cations

A novel method for the rapid fabrication of poly(methyl methacrylate) (PMMA) microfluidic chips using poly(dimethylsiloxane) (PDMS) templates has been demonstrated. The PDMS molds were fabricated by soft lithography. The dense prepolymerized solution of methyl methacrylate containing thermal and UV initiators was allowed to polymerized between a PDMS template and a piece of a 1 mm thick commercial PMMA plate under a UV lamp. The images of microchannels on the PDMS template were precisely replicated into the synthesized PMMA substrates during the UV-initiated polymerization of the prepolymerized solution on the surface of the PMMA plate at room temperature. The polymerization could be completed within 10 min under ambient temperature. The chips were subsequently assembled by thermal bonding of the channel plate and the cover sheet. The new fabrication method obviates the need for specialized replication equipment and reduces the complexity of prototyping and manufacturing. Nearly 20 PMMA chips were replicated using a single PDMS mold. The attractive performance of the new microfluidic chips has been demonstrated by separating and detecting cations in connection with contactless conductivity detection. The fabricated PMMA microchip has also been successfully employed for the determination of potassium and sodium in environmental and biological samples.     

Surface modification of poly(dimethylsiloxane) microfluidic devices and its application in simultaneous analysis of uric acid and ascorbic acid in human urine

A novel and simple method based on layer-by-layer (LBL) technique has been developed for the modification of the channel in PDMS electrophoresis microchip to create a hydrophilic surface with a stable EOF. The functional surface was obtained by sequentially immobilizing chitosan and deoxyribonucleic acid (DNA) onto the microfluidic channel surface using the LBL assembly technique. Compared to the native PDMS microchips, the contact angle of the chitosan-DNA modified PDMS microchips decreased and the EOF increased. Experimental conditions were optimized in detail. The chitosan-DNA modified PDMS microchips exhibited good reproducibility and long-term stability. Separation of uric acid (UA) and ascorbic acid (AA) performed on the modified PDMS microchip generated 43,450 and 46,790 N/m theoretical plates compared with 4048 and 19,847 N/m with the native PDMS microchip. In addition, this method has been successfully applied to real human urine samples, without SPE, with recoveries of 97-105% for UA and AA.     

Low electroosmotic flow measurement by tilting microchip

A novel method for low electroosmotic flow (EOF) rates measurement by tilting microchip which based upon the hydrostatic pressure conception and sampling zone method is described. Sampling zone could be detected in the tilting microchip but not in non-tilting one due to the hydrostatic pressure driven. The method is fulfilled to calculate low EOF rates by detecting the liquid flow velocity driven by hydrostatic pressure, and difference between the apparent mobility of the migrating analyte in two modes is caused by the effect of hydrostatic pressure. And then the EOF rates in unknown low EOF microchip can be calculated. Different microchannels modified with bovine serum albumin (BSA), myoglobin (MB) and polyvinyl alcohol (PVA) were used to verify the method, the EOF rate value was 1.73+/-0.03, 1.21+/-0.05, 0.34+/-0.04 x 10(-4)cm(2) V(-1)s(-1), respectively. The results obtained by the proposed method were agreed well with conventional methods.     

A polymeric master replication technology for mass fabrication of poly(dimethylsiloxane) microfluidic devices

A protocol of producing multiple polymeric masters from an original glass master mold has been developed, which enables the production of multiple poly(dimethylsiloxane) (PDMS)-based microfluidic devices in a low-cost and efficient manner. Standard wet-etching techniques were used to fabricate an original glass master with negative features, from which more than 50 polymethylmethacrylate (PMMA) positive replica masters were rapidly created using the thermal printing technique. The time to replicate each PMMA master was as short as 20 min. The PMMA replica masters have excellent structural features and could be used to cast PDMS devices for many times. An integration geometry designed for laser-induced fluorescence (LIF) detection, which contains normal deep microfluidic channels and a much deeper optical fiber channel, was successfully transferred into PDMS devices. The positive relief on seven PMMA replica masters is replicated with regard to the negative original glass master, with a depth average variation of 0.89% for 26-microm deep microfluidic channels and 1.16% for the 90 mum deep fiber channel. The imprinted positive relief in PMMA from master-to-master is reproducible with relative standard deviations (RSDs) of 1.06% for the maximum width and 0.46% for depth in terms of the separation channel. The PDMS devices fabricated from the PMMA replica masters were characterized and applied to the separation of a fluorescein isothiocyanate (FITC)-labeled epinephrine sample.     

Fabrication and performance of fiber electrophoresis microchips

A method based on the in situ polymerization of methyl methacrylate (MMA) has been developed for the rapid fabrication of a novel separation platform, fiber electrophoresis microchip. To demonstrate the concept, prepolymerized MMA molding solution containing a UV initiator was sandwiched between a poly(methyl methacrylate) (PMMA) cover plate and a PMMA base plate bearing glycerol-permeated fiberglass bundles and was exposed to UV light. During the UV-initiated polymerization, the fiberglass bundles were embedded in the PMMA substrate to form fiberglass-packed microchannels. When the glycerol in the fiberglass bundles was flushed away with water, the obtained porous fiberglass-packed channels could be employed to perform electrophoresis separation. Scanning electron micrographs (SEMs) and microscopic images offered insights into the fiber electrophoresis microchip. The analytical performance of the novel microchip has been demonstrated by separating and detecting dopamine and catechol in connection with end-column amperometric detection. The fiber-based microchips can be fabricated by the new approach without the need for complicated and expensive lithography-based microfabrication techniques, indicating great promise for the low-cost production of microchips, and should find a wide range of applications.