Poly(dimethylsiloxane)-based protein preconcentration using a nanogap generated by junction gap breakdown

Simple and efficient sample concentration tools are the key to the application of proteomics in a biological system. In this paper, we developed a method to realize a nanofluidic preconcentrator on a poly(dimethylsiloxane) (PDMS)-based microfluidic channel. The originality of our preconcentration device is the simple nanogap formation using the junction gap breakdown phenomenon between two PDMS microchannels, without using any photolithography and etching techniques. From the dc current measurement, we confirm that the nanogap formed between two microchannel junctions with approximately 80 nm depth. Using this device, we achieve the concentration volume of beta-phycoerythrin protein as high as 70 pL, which is 120-fold larger than that from our previous reports, with a concentration factor as high as 10(4) within 1 h. Also we show the availability of protein preconcentration under several different buffers (phosphate, acetate) at several different pH values (pH 5 to pH 9).     

Increase of reaction rate and sensitivity of low-abundance enzyme assay using micro/nanofluidic preconcentration chip

We report a novel method of increasing both the reaction rate and the sensitivity of low-abundance enzyme assay using a micro/nanofluidic preconcentration chip. The disposable preconcentration device made out of PDMS with a surface-patterned ion-selective membrane increases local enzyme/substrate concentrations for rapid monitoring of enzyme activity. As a model system, we used trypsin as the enzyme and BODIPY FL casein as the fluorogenic substrate. We demonstrated that the reaction rate of trypsin-BODIPY FL was significantly enhanced by increasing the local concentrations of both trypsin and BODIPY FL casein in the preconcentration chip. The reaction time required to turn over substrates at 1 ng/mL was only approximately 10 min compared to approximately 1 h without preconcentration, which demonstrates a significantly higher reaction rate through the increase of the concentrations of both the enzyme and substrate. Furthermore, trypsin activity can be measured down to a concentration level of 10 pg/mL, which is a approximately 100 fold enhancement in sensitivity compared to the result without the preconcentration step. This micro/nanofluidic preconcentrator chip could be used as a generic micro reaction platform to study any enzyme-substrate systems, or other biochemical reaction systems in low concentration ranges.     

Flexible and Efficient Eletrokinetic Stacking of DNA and Proteins at an HF Etched Porous Junction on a Fused Silica Capillary

Better understanding of the mechanism is important for exploring the potentials of a preconcentration method. In this work, we show for the first time that the HF etched porous junction on a fused silica capillary behaves not only as a filter but also as an integrated nanofluidic interface. This junction exhibits an obvious ion concentration polarization (CP) effect, with which highly efficient electrokinetic stacking (ES) inside the capillary can be achieved without molecular size or charge type limitation. Two major types of CP based ES were proposed, and an autostop etching principle was presented for avoiding overetching. The ES can be performed in a broad range of pH and buffer concentration. Over a billion times of concentration was demonstrated by a fluorescein probe with laser induced fluorescent (LIF) detection. ES of fluorescently labeled and native DNA and protein were characterized by charge-coupled device (CCD) imaging and online capillary gel electrophoresis (CGE) with ultraviolet (UV) absorption detections, respectively. With this junction, highly efficient ES can be performed easily by voltage manipulation without any mechanical operation. We may foresee that the performance of capillary-based conventional and chip electrophoresis could be greatly enhanced with this junction in the analysis of low abundance biomolecules.     

Fabrication of porous polymer monoliths in polymeric microfluidic chips as an electrospray emitter for direct coupling to mass spectrometry

Coupling of polymeric microfluidic devices to mass spectrometry is reported using porous polymer monoliths (PPM) as nanoelectrospray emitters. Lauryl acrylate-co-ethylene dimethacrylate porous polymer monolith was photopatterned for 5 mm at the end of the channel of microfluidic devices fabricated from three different polymeric substrate materials, including the following: poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and cyclic olefin copolymer (COC). Spraying directly from the end of the chip removes any dead volume associated with inserted emitters or transfer lines, and the presence of multiple pathways in the PPM prevents the clogging of the channels, which is a common problem in conventional nanospray emitters. Spraying from a microfluidic channel having a PPM emitter produced a substantial increase in TIC stability and increased sensitivity by as much as 70x compared to spraying from an open end chip with no PPM. The performance of PPM emitter in COC, PMMA, and PDMS chips was compared in terms of stability and reproducibility of the electrospray. COC chips showed the highest reproducibility in terms of chip-to-chip performance, which can be attributed to the ease and reproducibility of the PPM formation due to the favorable optical and chemical properties of COC. We have further tested the performance of the COC chips by constant infusion of poly(propylene glycol) solution at organic content ranging from 10 to 90% methanol and at flow rates ranging from 50 to 1000 nL/min, showing optimum spraying conditions (RSD < 5%) at 50-70% organic content and at flow rates from 100 to 500 nL/min. The PPM sprayer was also used for protein preconcentration and desalting prior to mass spectrometric detection, and results were comparable with a chip spraying from an electrospray tip.     

Electrokinetic protein preconcentration using a simple glass/poly(dimethylsiloxane) microfluidic chip

We discovered that a protein concentration device can be constructed using a simple one-layer fabrication process. Microfluidic half-channels are molded using standard procedures in PDMS; the PDMS layer is reversibly bonded to a glass base such as a microscope slide. The microfluidic channels are chevron-shaped, in mirror image orientation, with their apexes designed to pass within approximately 20 microm of each other, forming a thin-walled section between the channels. When an electric field is applied across this thin-walled section, negatively charged proteins are observed to concentrate on the anode side of it. About 10(3)-10(6)-fold protein concentration was achieved in 30 min. Subsequent separation of two different concentrated proteins is easily achieved by switching the direction of the electric field in the direction parallel to the thin-walled section. We hypothesize that a nanoscale channel forms between the PDMS and the glass due to the weak, reversible bonding method. This hypothesis is supported by the observation that, when the PDMS and glass are irreversibly bonded, this phenomenon is not observed until a very high E-field was applied and dielectric breakdown of the PDMS is observed. We therefore suspect that the ion exclusion-enrichment effect caused by electrical double layer overlapping induces cationic selectivity of this nanochannel. This simple on-chip protein preconcentration and separation device could be a useful component in practically any PDMS-on-glass microfluidic device used for protein assays.     

Wafer-scale fabrication of nanofluidic arrays and networks using nanoimprint lithography and lithographically patterned nanowire electrodeposition gold nanowire masters

Wafer scale (cm(2)) arrays and networks of nanochannels were created in polydimethylsiloxane (PDMS) from a surface pattern of electrodeposited gold nanowires in a master-replica process and characterized with scanning electron microscopy (SEM), atomic force microscopy (AFM), and fluorescence imaging measurements. Patterns of gold nanowires with cross-sectional dimensions as small as 50 nm in height and 100 nm in width were prepared on silica substrates using the process of lithographically patterned nanowire electrodeposition (LPNE). These nanowire patterns were then employed as masters for the fabrication of inverse replica nanochannels in a special formulation of PDMS. SEM and AFM measurements verified a linear correlation between the widths and heights of the nanowires and nanochannels over a range of 50 to 500 nm. The PDMS replica was then oxygen plasma-bonded to a glass substrate in order to create a linear array of nanofluidic channels (up to 1 mm in length) filled with solutions of either fluorescent dye or 20 nm diameter fluorescent polymer nanoparticles. Nanochannel continuity and a 99% fill success rate was determined from the fluorescence imaging measurements, and the electrophoretic injection of both dye and nanoparticles in the nanochannel arrays was also demonstrated. Employing a double LPNE fabrication method, this master-replica process was also used to create a large two-dimensional network of crossed nanofluidic channels.     

In situ curing of sliding SU-8 droplet over a microcontact printed pattern for tunable fabrication of a polydimethylsiloxane nanoslit

A tunable process for polydimethylsiloxane (PDMS) nanoslit fabrication is developed for nanofluidic applications. A microcontact printing (μCP) of a laterally spreading self-assembled hexadecanethiol (HDT) layer, combined with in situ curing of a sliding SU-8 droplet, enables precise and independent tuning of a nanoslit-mold width and height using a single μCP master mold. The SU-8 nanoslit-mold is replicated using a hard-soft composite PDMS to prevent channel collapse at low (<0.2) aspect ratio (height over width). The fluidic characteristics as well as dimensions of nanoslits fabricated with various conditions are analyzed using a fluorescein sample and AFM images. Finally, concentration polarization-based sample preconcentration is successfully demonstrated at the nanoslit boundary where an electric double-layer is overlapped.     

Covalent micropatterning of poly(dimethylsiloxane) by photografting through a mask

A new photografting method to micropattern a covalent surface modification on poly(dimethylsiloxane) (PDMS) provides advantages in simplicity and efficiency. To accomplish the entire process on the benchtop, the PDMS was initially treated with benzophenone dissolved in a water/acetone mixture. This process permitted limited diffusion of the photoinitiator into the PDMS surface. Polymerization of acrylic acid was initiated by exposure of the benzophenone-implanted PDMS to UV radiation through a photomask with a thin aqueous layer of acrylic acid sandwiched between the PDMS and photomask. This procedure resulted in patterned poly(acrylic acid) (PAA) on the PDMS surface. In the modified regions, PAA and PDMS formed an interpenetrating polymer network extending 50 microm into the PDMS with an X-Y spatial resolution of 5 microm. The carboxyl groups of the PAA graft could be derivatized to covalently bond other molecules to the patterned PAA. Two bioanalytical applications of this micropatterned surface were demonstrated: (1) a guide for cell attachment and growth and (2) a substrate for immunoassays. 3T3 cells were shown to selectively localize to modified surface regions where they could be cultured for up to 7 days. Additionally, the micropatterned surface was used to immobilize either protein A or antibody for heterogeneous immunoassays.     

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.     

PCR-free DNA detection using a magnetic bead-supported polymeric transducer and microelectromagnetic traps

A fluorescent polymeric hybridization transducer supported on magnetic microbeads was investigated for the rapid, ultrasensitive, and sequence-specific detection of DNA. We show that the polymer derivative can be used to detect target DNA directly on magnetic particles by preparing "target-ready" microbeads grafted with the polymer and suitable DNA probes. A detection limit of approximately 200 target copies in a probed volume of 150 muL (1.4 copies/muL) was obtained for a DNA sequence specific to Candida albicans This detection scheme does not require the release of the hybridized target DNA prior to its detection or the labeling or amplification of the nucleic acids. Furthermore, we show that the fluorescence from these biosensing magnetic beads can be read while magnetically confined in a small volume by a microelectromagnetic trap, which offers the possibility of performing both the preconcentration and detection steps simultaneously on the same support. The combination of the fluorescent polymer biosensor with magnetic particle-assisted DNA preconcentration extends the application of this ultrasensitive biosensor to biological samples with complex matrixes and to integrated lab-on-a-chip platforms, where it could be used for fast multitarget DNA detection in point-of-care diagnostics and field analysis.     

Fabrication of polymer waveguides by a replication method

We have developed a soft-lithography method to replicate polymer waveguides. In this method, the waveguides are produced by a two-step molding process where a master mold is first formed on a negative-tone photoresist and subsequently transferred to a polydimethylsiloxane (PDMS) mold; a PDMS silicone rubber mold is then used as a stamp to transfer the final waveguide pattern onto an UV cure epoxy. Initial results show good pattern transferring in physical shape. The optical performance is measured based on the propagation loss. In our design, the loss was measured at 0.28 dB/cm for 1.3 microm and 0.26 dB/cm for 1.55 microm.     

Determination of hydrocarbons using sapphire fibers coated with poly(dimethylsiloxane)

A poly(dimethylsiloxane) (PDMS) coated sapphire fiber has been investigated as a sensor for hydrocarbons (HCs) in the mid-infrared region around 3000 cm(-1). In order to optimize and predict sensor response, the diffusion behavior of the analytes into the PDMS preconcentration medium has been examined. A diffusion model based on Fickian diffusion was used to quantify diffusion. The model incorporated such factors as film thickness, refractive index of the polymer and the fiber core, and principal wavelength at which the analyte absorbs. A range of hydrocarbons, from hexane to pentadecane, was analyzed at 2930 cm(-1) using both fiber-coupled Fourier transform infrared spectroscopy and a modular prototype system. Diffusion coefficients were determined for these compounds and diffusion behavior examined and related to factors such as analyte polarity and molecular size. The diffusion coefficients were found to range from 6.41 x 10(-11) 5 x 10(-12) to 5.25 x 10(-11) +/- 9 x 10(-13) cm2 s(-1) for hexane and pentadecane into a 2.9 microm PDMS film, respectively. The diffusion model was also used to examine the effect of changing system parameters such as film thickness in order to characterize sensor response.     

A polydimethylsiloxane (PDMS) sponge for the selective absorption of oil from water

We present a sugar-templated polydimethylsiloxane (PDMS) sponge for the selective absorption of oil from water. The process for fabricating the PDMS sponge does not require any intricate synthesis processes or equipment and it is not environmentally hazardous, thus promoting potential in environmental applications. The proposed PDMS sponge can be elastically deformed into any shape, and it can be compressed repeatedly in air or liquids without collapsing. Therefore, absorbed oils and organic solvents can be readily removed and reused by simply squeezing the PDMS sponge, enabling excellent recyclability. Furthermore, through appropriately combining various sugar particles, the absorption capacity of the PDMS sponge is favorably optimized.     

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.     

Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels

The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE. The size exclusion membrane was polymerized in the injection channel using a shaped laser beam, and the sizing monolith was cast by photolithography using a mask and UV lamp. Proteins injected electrophoretically were trapped on the upstream side of the size exclusion membrane (MW cutoff approximately 10 kDa) and eluted off the membrane by reversing the electric field. Subsequently, the concentrated proteins were separated in a cross-linked polyacrylamide monolith that was patterned contiguous to the size exclusion membrane. The extent of protein preconcentration is easily tuned by varying the voltage during injection or by controlling the sample volume loaded. Electric fields applied across the nanoporous membrane resulted in a concentration polarization effect evidenced by decreasing current over time and irreproducible migration of proteins during sizing. To minimize the concentration polarization effect, sieving gels were polymerized only on the separation side of the membrane, and an alternate electrical current path was employed, bypassing the membrane, for most of the elution and separation steps. Electrophoretically sweeping a fixed sample volume against the membrane yields preconcentration factors that are independent of protein mobility. The volume sweeping method also avoids biased protein loading from concentration polarization and sample matrix variations. Mobilities of the concentrated proteins were log-linear with respect to molecular weight, demonstrating the suitability of this approach for protein sizing. Proteins were concentrated rapidly (<5 min) over 1000-fold followed by high-resolution separation in the sieving monolith. Proteins with concentrations as low as 50 fM were detectable with 30 min of preconcentration time. The integrated preconcentration-sizing approach facilitates analysis of low-abundant proteins that cannot be otherwise detected. Moreover, the integrated preconcentration-analysis approach employing in situ formation of photopatterned polymeric elements provides a generic, inexpensive, and versatile method to integrate functions at chip level and can be extended to lowering of detection limits for other applications such as DNA analysis and clinical diagnostics.     

利用水溶性纳米线制备微纳流体系统(英文)

在本课题的研究中,提出了一种利用水溶性钼酸钠铵纳米线和纳米探针系统来制备微纳流体系统的方法.借助这种方法可制备长度、直径以及横截面可控的的纳米通道.对于选用SU-8和聚二甲基硅氧烷(PDMS)两种材料组合而成的微纳流体系统而言,氧等离子处理可以很大程度上提高二者的键合强度并增加材料表面的亲水性.这种微纳流体器件在应用于离子输运、生物分子分离和其他相关研究上具有较大的发展前景.     

Micropatterning functional groups on polymer surfaces via masked vapor-phase photografting

Controlling cell behavior on biomaterial surface is the ultimate goal of cell and tissue engineering. Fabrication of biomaterials with alternatively hydrophilic/hydrophobic surface of parallel nanopatterned groves can provide biomaterial surfaces for the study of cell-surface interactions. In the present communication, masked vapor-phase photografting was used in patterning functional groups on flat polymeric substrates using poly (dimethylsiloxane) (PDMS) channels. Surface patterns were fabricated by UV-initiated photografting in the presence of a patterned PDMS mask. The approach is exemplified by patterning maleic anhydride (MAH) and acrylamide (AAm) onto poly (methyl methacrylate) (PMMA). The method offers another means to chemically functionalize and pattern polymer surface at the same time.     

Mechanically tunable three-dimensional elastomeric network/air structures via interference lithography

We show how to employ an interference lithographic template (ILT) as a facile mold for fabricating three-dimensional bicontinuous PDMS (poly(dimethylsiloxane)) elastomeric structures and demonstrate the use of such a structure as a mechanically tunable PDMS/air phononic crystal. A positive photoresist was used to make the ILT, and after infiltration with PDMS, the resist was removed in a water-based basic solution which avoided PDMS swelling or pattern collapse occurring during the ILT removal process. Since the period of the structure is approximately 1 microm, the density of states of gigahertz phonons are altered by the phononic PDMS/air crystal. Brillouin light scattering (BLS) was employed to measure phononic modes of the structure as a function of mechanical strain. The results demonstrate that the phononic band diagram of such structures can be tuned mechanically.     

Fabrication of a configurable, single-use microfluidic device.

This paper describes microfluidic devices that contain connections that can be opened by the user after fabrication. The devices are fabricated in poly(dimethylsiloxane) (PDMS) and comprise disconnected fluidic channels that are separated by 20 microm of PDMS. Applying voltages above the breakdown voltage of PDMS (21 V/microm) opened pathways between disconnected channels. Fluids could then be pumped through the openings. The voltage used and the ionic strength of the buffer in the channels determined the size of the opening. Opening connections in a specific order provides the means to control complex reactions on the device. A device for ELISA was fabricated to demonstrate the ability to store and deliver fluids on demand.     

Million-fold preconcentration of proteins and peptides by nanofluidic filter

We have developed a highly efficient microfluidic sample preconcentration device based on the electrokinetic trapping mechanism enabled by nanofluidic filters. The device, fabricated by standard photolithography and etching techniques, generates an extended space charge region within a microchannel, which was used to both collect and trap the molecules efficiently. The electrokinetic trapping and collection can be maintained for several hours, and concentration factors as high as 10(6)-10(8) have been demonstrated. This device could be useful in various bioanalysis microsystems, due to its simplicity, performance, robustness, and integrabilty to other separation and detection systems.