Surface modification of poly(methyl methacrylate) used in the fabrication of microanalytical devices

We report here the chemical modification of poly(methyl methacrylate) (PMMA) surfaces by their reaction with the monoanion of alpha,omega-diaminoalkanes (aminolysis reaction) to yield amine-terminated PMMA surfaces. It is found that the amine functionalities are tethered to the PMMA backbone through an alkane bridge to amide bonds formed during the aminolysis of the surface ester functionalities. The distribution of the amine termini is quite uniform as judged by fluorescence micrographs. It is found that the electroosmotic flow in aminated PMMA microchannels is reversed when compared to that in unmodified channels. In addition, it is demonstrated that enzymes can be immobilized onto the amine-terminated PMMA surfaces and are effective in the restriction digestion of dsDNAs. Finally, the availability of the surface amine groups is further demonstrated by their reaction with n-octadecane-1-isocyanate to form PMMA surfaces terminated with well-ordered and highly crystalline octadecane chains.     

Construction of a biomimetic surface on microfluidic chips for biofouling resistance

A biomimetic surface has been formed on the poly(methyl methacrylate) (PMMA) microfluidic chips for biofouling resistance on the basis of a simple modification. Accordingly, an amphiphilic phospholipid copolymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate (PMB) was developed to introduce the phosphorylcholine functional groups onto the PMMA surface via the anchoring of hydrophobic n-butyl methacrylate units. The 2-methacryloyloxyethyl phosphorylcholine segments could form hydrophilic domains, considered to be located on the surface, to provide a biocompatible surface. X-ray photoelectron spectroscopy and Fourier transform infrared spectra confirmed the success of surface functionalization. The PMB-modified microchips containing phosphorylcholine moieties exhibited more stable electroosmotic mobility compared with the untreated one. In addition to being characterized for minimized nonspecific adhesion of serum proteins and plasma platelets, the PMB-functionalized microchannels have been exemplified by electrophoresis of proteins. This one-step procedure offers an effective approach for a biomimetic surface design on microfluidic chips, which is promising in high-throughput and complex biological analysis.     

Fabrication of discontinuous surface patterns within microfluidic channels using photodefinable vapor-based polymer coatings

In this report, we introduce a surface modification method for the fabrication of discontinuous surface patterns within microfluidic systems. The method is based on chemical vapor deposition (CVD) of a photodefinable coating, poly(4-benzoyl-p-xylylene-co-p-xylylene), onto the luminal surface of a microfluidic device followed by a photopatterning step to initiate spatially controlled surface binding. During photopatterning, light-reactive groups of the CVD polymer spontaneously react with molecules adjunct to the surface, such as poly(ethylene oxide). We demonstrate the potential of these reactive polymers for surface modification by preventing nonspecific protein adsorption on different substrates including silicon and poly(dimethylsiloxane) as measured by fluorescence microscopy. More importantly, three-dimensional patterns have successfully been created within polymer-based microfluidic channels, establishing spatially controlled, bioinert surfaces. The herein reported surface modification method addresses a critical challenge with respect to surface engineering of microfluidic devices, namely, the fabrication of discontinuous patterns within microchannels.     

Generation of hydrophilic poly(dimethylsiloxane) for high-performance microchip electrophoresis

Poly(dimethylsiloxane) (PDMS) has become one of the most widely used materials for microchip capillary electrophoresis and microfluidics. The popularity of this material is the result of its low cost, simple fabrication, and rugged elastomeric properties. The hydrophobic nature of PDMS, however, limits its applicability for microchip CE, microfluidic patterning, and other nonelectrophoresis applications. The surface of PDMS can be made hydrophilic using a simple air plasma treatment; however, this property is quickly lost through hydrophobic recovery caused by diffusion of unreacted oligomer to the surface. Here, a simple approach for the generation of hydrophilic PDMS with long-term stability in air is presented. PDMS is rendered hydrophilic through a simple two-step extraction/oxidation process. First, PDMS is extracted in a series of solvents designed to remove unreacted oligomers from the bulk phase. Second, the oligomer-free PDMS is oxidized in a simple air plasma, generating a stable layer of hydrophilic SiO2. The conversion of surface-bound siloxane to SiO2 was followed with X-ray photoelectron spectroscopy. SiO2 on extracted-oxidized PDMS was stable for 7 days in air as compared to less than 3 h for native PDMS. Furthermore, the contact angle for modified PDMS was reduced to <40 degrees and remained low throughout the experiments. As a result of the decreased contact angle, capillary channels self-wet through capillary action, making the microchannels much easier to fill. Finally, the modification significantly improved the performance of the devices for microchip electrophoresis. The electroosmotic flow increased from 4.1 x 10(-4) to 6.8 x 10(-4) cm(2)/V.s for native compared to oxidized PDMS. Separation efficiencies for electrochemical detection also increased from 50 000 to 400 000 N/m for a 1.1-nL injection volume. The result of this modification is a significant improvement in the performance of PDMS for microchip electrophoresis and microfluidic applications.     

Plastic microfluidic devices modified with polyelectrolyte multilayers

Control of the polymer surface chemistry is a crucial aspect of development of plastic microfluidic devices. When commercially available plastic substrates are used to fabricate microchannels, differences in the EOF mobility from plastic to plastic can be very high. Therefore, we have used polyelectrolyte multilayers (PEMs) to alter the surface of microchannels fabricated in plastics. Optimal modification of the microchannel surfaces was obtained by coating the channels with alternating layers of poly(allylamine hydrochloride) and poly(styrene sulfonate). Polystyrene (PS) and poly(ethylene terephthalate) glycol (PETG) were chosen as substrate materials because of the significant differences in the polymer chemistries and in the EOF of channels fabricated in these two plastic materials. The efficacy of the surface modification has been evaluated using XPS and by measuring the EOF mobility. When microchannels prepared in both PS and PETG are modified with PEMs, they demonstrate very similar electroosmotic mobilities. The PEMs are easily fabricated and provide a means for controlling the flow direction and the electroosmotic mobility in the channels. The PEM-coated microchannels have excellent wettability, allowing facile filling of the channels. In addition, the PEMs produce reproducible results and are robust enough to withstand long-term storage.     

Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.     

Patterning multiplex protein microarrays in a single microfluidic channel

The development of versatile biofunctional surfaces is a fundamental prerequisite in designing Lab on a Chip (LOC) devices for applications in biosensing interfaces and microbioreactors. The current paper presents a rapid combinatorial approach to create multiplex protein patterns in a single microfluidic channel. This approach consists of coupling microcontact printing with microfluidic patterning, where microcontact printing is employed for silanization using (3-Aminopropyl) triethoxysilane (APTES), followed by microfluidic patterning of multiple antibodies. As a result, the biomolecules of choice could be covalently attached to the microchannel surface, thus creating a durable and highly resistant functional interface. Moreover, the experimental procedure was designed to create a microfluidic platform that maintains functionality at high flow rates. The functionalized surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and monitored with fluorescence microscopy at each step of functionalization. To illustrate the possibility of patterning multiple biomolecules along the cross section of a single microfluidic channel, microarrays of five different primary antibodies were patterned onto a single channel and their functionality was evaluated accordingly through a multiplex immunoassay using secondary antibodies specific to each patterned primary antibody. The resulting patterns remained stable at shear stresses of up to 50 dyn/cm(2). The overall findings suggest that the developed multiplex functional interface on a single channel can successfully lead to highly resistant multiplex functional surfaces for high throughput biological assays.     

Protein nanopatterns and biosensors using gold binding polypeptide as a fusion partner

An efficient strategy for immobilizing proteins on a gold surface was developed by employing the gold binding polypeptide (GBP) as a fusion partner. Using the enhanced green fluorescent protein (EGFP), severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVme), and core streptavidin (cSA) of Streptomyces avidinii as model proteins, specific immobilization of the GBP-fusion proteins onto the gold nanoparticles and generation of protein nanopatterns on the bare gold surface were demonstrated. The GBP-fused SCVme bound to gold nanoparticles successfully interacted with its antibody and showed changes in absorbance and color, allowing efficient diagnosis of SARS-CoV. The fusion proteins could be successfully immobilized on the gold surface by nanopatterning and microcontact printing as examined by atomic force microscopy and surface plasmon resonance analysis. The poly(dimethylsiloxane) microfluidic channels were created on the gold surface and were used for antigen-antibody and DNA-DNA interaction studies. Specific immobilization of GBP-EGFP fusion protein and its interaction with the antibody in the microchannels could be demonstrated. By immobilizing the DNA probe through the use of GBP-fused cSA, specific hybridization of the target DNA prepared from Salmonella could also be achieved. The GBP-fusion method allows immobilization of proteins onto the gold surface without surface modification and in bioactive forms suitable for studying protein-protein, DNA-DNA, and other biomolecular interaction studies. Furthermore, these studies can be carried out in a microfluidic system, which allows high-throughput analysis of biomolecular interactions.     

Solution-phase surface modification in intact poly(dimethylsiloxane) microfluidic channels

An improved approach composed of an oxidation reaction in acidic H2O2 solution and a sequential silanization reaction using neat silane reagents for surface modification of poly(dimethylsiloxane) (PDMS) substrates was developed. This solution-phase approach is simple and convenient for some routine analytical applications in chemistry and biology laboratories and is designed for intact PDMS-based microfluidic devices, with no device postassembly required. Using this improved approach, two different functional groups, poly(ethylene glycol) (PEG) and amine (NH2), were introduced onto PDMS surfaces for passivation of nonspecific protein absorption and attachment of biomolecules, respectively. X-ray electron spectroscopy and temporal contact angle experiments were employed to monitor functional group transformation and dynamic characteristics of the PEG-grafted PDMS substrates; fluorescent protein solutions were introduced into the PEG-grafted PDMS microchannels to test their protein repelling characteristics. These analytical data indicate that the PEG-grafted PDMS surfaces exhibit improved short-term surface dynamics and robust long-term stability. The amino-grafted PDMS microchannels are also relatively stable and can be further activated for modifications with peptide, DNA, and protein on the surfaces of microfluidic channels. The resulting biomolecule-grafted PDMS microchannels can be utilized for cell immobilization and incubation, semiquantitative DNA hybridization, and immunoassay.     

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.     

Surface modification method of microchannels for gas-liquid two-phase flow in microchips

A capillarity restricted modification method for microchannel surfaces was developed for gas--liquid microchemical operations in microchips. In this method, a microstructure combining shallow and deep microchannels and the principle of capillarity were utilized for chemical modification of a restricted area of a microchannel. A hydrophobic--hydrophilic patterning in microchannels was prepared as an example for guiding gas and liquid flows along the respective microchannels. Validity of the patterning was confirmed by measuring aqueous flow leak pressure from the hydrophilic microchannel to the hydrophobic one. The leak pressure of 7.7-1.1 kPa agreed well with that predicted theoretically from the Young-Laplace equation for the microchannel depth of 8.6-39 microm. In an experiment to demonstrate usefulness and effectiveness of the method, an air bubble was first introduced into the hydrophilic microchannel and purged from the hydrophobic-hydrophilic patterned microchannels. Next, the patterning structure was applied to remove dissolved oxygen by contacting the aqueous flow with a nitrogen flow. The concentration of dissolved oxygen decreased with contact time, and its time course agreed well with numerical simulation. These demonstrations showed that the proposed patterning method can be used in general microfluidic gas-liquid operations.     

A microfluidic vesicle screening platform: monitoring the lipid membrane permeability of tetracyclines

For many drugs including antibiotics such as tetracyclines it is crucial that the molecule has the ability to quickly and passively permeate lipid membranes. Hence, the understanding of the permeability in relation to the molecular structure is an important aspect to rationally design novel pharmaceutically active compounds with high bioavailability. Here, we present a versatile method to study the kinetics of tetracycline permeation across liposome membranes on a microchip. Liposomes are immobilized onto the glass surface in a stripe pattern via an avidin-biotin bond and covered by microchannels to allow continuous delivery of tetracycline and buffer. The fluid flow provides a constant concentration profile and thereby resembles the drug transport via blood in the human body. Total internal reflection fluorescence (TIRF) microscopy was used to image the formation of a fluorescent drug-europium complex inside the liposomes. The permeation rates of various tetracyclines were investigated and the results compared to a conventional method (water-octanol partitioning). The findings largely confirm the correlation between membrane permeability and lipophilicity of the permeating molecules (Overton's rule). However, slight deviations reveal that lipophilicity is an important but not the exclusive parameter for the prediction of permeation. The method is fast enough to study the permeation of unstable tetracyclines such as rolitetracycline. Additionally, with the use of different cholesterol concentrations, the influence of membrane composition on the permeation rate can be investigated conveniently. The microfluidic approach can be easily applied to investigate the kinetics of other processes such as ligand-membrane receptor association and dissociation, provided that the process can be visualized by means of fluorescence spectroscopy.     

Room-temperature imprinting method for plastic microchannel fabrication

A new plastic imprinting method using a silicon template is demonstrated. This new approach obviates the necessity of heating the plastic substrate during the stamping process, thus improving the device yield from approximately 10 devices to above 100 devices per template. The dimensions of the imprinted microchannels were found to be very reproducible, with variations of less than 2%. The channel depths were dependent on the pressures applied and the materials used. Rather than bonding the open channels with another piece of plastic, a flexible and adhesive poly(dimethylsiloxane) film is used to seal the microchannels, which offers many advantages. As an application, isoelectric focusing of green fluorescence protein on these plastic microfluidic devices is illustrated.     

High-speed, whole-column fluorescence imaging detection for isoelectric focusing on a microchip using an organic light emitting diode as light source

An integrated and simplified microfluidic device using a 250 microm x 1-4 cm of organic light emitting diode (OLED) array as a two-dimensional light source for single-channel and multichannel whole-column imaging detection was developed. This fluorescence detection system was used for isoelectric focusing (IEF) of R-phycoerythrin in a microchip. The IEF conditions were optimized, and the total analysis time was extremely reduced to 30 s for 2-cm-long microchannels at 700 V/cm of electric field strength without the presence of electroosmotic flow. The compression of pH gradient caused by electrolytes drawing into the microchannels was efficiently restrained when 1% hydroxylpropylmethyl cellulose in 2% ampholyte was used as the carrier for IEF. Under optimized IEF conditions, the detection limit of this system was approximately 0.6 microg/mL or 45 pg at 75 nL/column injection of R-phycoerythrin. This OLED-induced fluorescence detection system for WCID provides a high-speed IEF technique with quantitative ability and the potential for high integration and throughput microchip systems.     

Performance of SU-8 microchips as separation devices and comparison with glass microchips

Effective analytical performance of native, all-SU-8 separation microdevices is addressed by comparing their performance to commercial glass microdevices in microchip zone electrophoresis accompanied by fluorescence detection. Surface chemistry and optical properties of SU-8 microdevices are also examined. SU-8 was shown to exhibit repeatable electroosmotic properties in a wide variety of buffers, and SU-8 microchannels were successfully utilized in peptide and protein analyses without any modification of the native polymer surface. Selected, fluorescent labeled, biologically active peptides were baseline resolved with migration time repeatability of 2.3-3.6% and plate numbers of 112,900-179,800 m(-1). Addition of SDS (0.1%) or SU-8 developer (1.0%) to the separation buffer also enabled protein analysis by capillary zone electrophoresis. Plate heights of 2.4-5.9 microm were obtained for fluorescent labeled bovine serum albumin. In addition, detection sensitivity through SU-8 microchannels was similar to that through BoroFloat glass, when fluorescence illumination was provided at visible wavelengths higher than 500 nm. On the whole, the analytical performance of SU-8 microchips was very good and fairly comparable to that of commercial glass chips as well as that of traditional capillary electrophoresis and chromatographic methods. Moreover, lithography-based patterning of SU-8 enables straightforward integration of multiple functions on a single chip and favors fully microfabricated lab-on-a-chip systems.     

Electrokinetically driven microfluidic chips with surface-modified chambers for heterogeneous immunoassays

This article presents the first example of a microfluidic chip for heterogeneous bioassays using a locally immobilized biospecific layer and operated electrokinetically. The reaction chamber has picoliter dimensions and is integrated into a network of microchannels etched in glass. The high affinity of protein A (PA) for rabbit immunoglobulin G (rIgG) was exploited for chip testing, with PA being immobilized on microchannel walls and fluorescently labeled (Cy5) rIgG serving as sample. It was possible to operate the chip in an immunoaffinity chromatographic manner, using electrokinetically pumped solutions. Concentration of antibody from dilute solution onto the solid phase was demonstrated, with signal gains of approximately 30 possible. A dose-response curve for Cy5-rIgG was obtained for concentrations down to 50 nM, for an incubation time of 200 s. The flexibility of chip layout was demonstrated for competitive immunoassay of rIgG, using both a combined sample/tracer incubation and sequential addition of these solutions. With assay times generally below 5 min for this unoptimized device, the microfluidic approach described shows great potential for many high-throughput screening applications.     

利用CO2激光法快速制造PMMA基材的微流控分析芯片

提出一种广泛使用的CO2激光法,以直接读写烧蚀的方式,进行快速的聚甲基丙烯酸甲酯（PMMA）基材的微流控分析芯片的制造.利用此方法所制造的微流道,将以扫描电子显微镜（SEM）、原子力显微镜（AFM）及表面轮廓仪进行各项表面性质的分析.本文所发展的CO2激光烧蚀法,提供了一个可广泛使用及具有经济效应的PMMA基材的微流控分析芯片的制造方法.在此激光制程法中,微流控分析芯片的制造图案可由商业的套装软件绘制而成,再传输至激光系统中进行烧蚀微管道,结果显示利用离焦法的激光制程技术,在没有退火处理的情况下,就可以获得表面相当平滑的微流道,表面粗糙度小于4nm.     

Surface-directed, graft polymerization within microfluidic channels

We demonstrate a simple procedure to coat the surfaces of enclosed PDMS microchannels by UV-mediated graft polymerization. In prior applications, only disassembled channels could be coated by this method. This limited the utility of the method to coatings that could easily and tightly seal with themselves. By preadsorbing a photoinitiator onto the surface of PDMS microchannels, the rate of polymer formation at the surface was greatly accelerated compared to that in solution. Thus, a gel did not form in the lumen of enclosed microchannels. We demonstrate that the photoinitiator benzophenone remained on the surface of PDMS even after extensive washing. After addition of a variety of monomer solutions (acrylic acid, poly(ethylene glycol) monomethoxyl acrylate, or poly(ethylene glycol) diacrylate) and illumination with UV light, a stable, covalently attached surface coating formed in the microchannels. The electroosmotic mobility was stable in response to air exposure and to repeated cycles of hydration-dehydration of the coating. These surfaces also supported the electrophoretic separation of two model analytes. Placement of an opaque mask over a portion of the channel permitted photopatterning of the microchannels with a resolution of approximately 100 microm. By using an appropriate mixture of monomers combined with masks, it should be possible to fabricate PDMS microfluidic devices with distinct surface properties in different regions or channels.     

Analysis of lipoproteins by microchip electrophoresis with high speed and high reproducibility

A method for the fast analysis of lipoproteins by microchip electrophoresis with light-emitting diode confocal fluorescence detection has been developed. Lipoproteins labeled with BODIPY FL C(5)-ceramide are found to strongly adsorb on the bare surface of a poly(methyl methacrylate) (PMMA) microchip. Sodium dodecyl sulfate and cetyltrimethylammonium bromide were therefore utilized to alter lipoproteins and channel surface to make them bear the same type of charge. After modification, the peak shape of lipoproteins was greatly improved, demonstrating lipoprotein adsorption on a PMMA chip dramatically reduced due to electrostatic repulsion. In addition, polymers were added into the running buffer to suppress electroosmotic flow and to serve as a sieving matrix. As a result, lipoprotein separation was manipulated by both electrophoretic mobilities and particle sizes. Various separation parameters including surfactant concentration, buffer pH, and polymer concentration as well as on-line concentration were investigated systematically. Under optimal conditions, two baseline separations of standard lipoproteins including high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein were achieved with different selectivity. This method affords high separation speed (within 100 s) and high reproducibility. The intraassay and interassay RSDs of lipoprotein migration times were in the range of 0.90-1.9%, indicating this method is highly reliable.     

Phase-changing sacrificial materials for interfacing microfluidics with ion-permeable membranes to create on-chip preconcentrators and electric field gradient focusing microchips

We have developed a novel approach for interfacing ionically conductive membranes with microfluidic systems using phase-changing sacrificial layers. Imprinted microchannels in a polymer substrate are filled with a heated liquid that solidifies at room temperature, a monomer solution is placed over the protected channels and polymerized to form a rigid semipermeable copolymer, and then the protective layer is melted and removed, leaving an open microchannel interfaced with a polymer membrane. We have applied this method in miniaturizing electric field gradient focusing (EFGF) and carrying out on-chip protein preconcentration. A semipermeable copolymer in the EFGF microchips fills a region of changing cross-sectional area, which allows a gradient in electric field to be established when an electrical potential is applied. Our technique provides microchip EFGF devices that offer 3-fold improved resolution in protein focusing compared with capillary-based systems. In addition, these EFGF microchips can separate peptide samples with resolution similar to what is obtained in capillary electrophoresis microdevices, and the micro-EFGF systems enrich analytes by a factor of >150. Finally, we have fabricated membrane-integrated microfluidic devices that can concentrate protein samples (R-phycoerythrin) over 10 000-fold to facilitate microchip capillary electrophoresis. Interfacing microchannels with ion-permeable membranes has great potential to enhance microchip analysis of biomolecules.