A picoliter chamber array for cell-free protein synthesis

The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR.

The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify 'PCR friendly' surfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube.     

Macro/nao-structured silicon as solid support for antibody arrays

To facilitate high-throughput biomarker discovery and high-density protein-chip array analyses of complex biological samples, a novel macro-and nanoporous silicon surface for protein microarrays was developed. The surface offers three-dimensional surface enlarging properties and spot confinement, enabling both high sensitivity bioassays and design of high density arrays. Reproducible manufacturing of the protein chip surface was accomplished as demonstrated by the low imprecision when standard IgG bioassays were performed at 100 pM antigen level on a series of protein chips scanned at widely different locations within a silicon wafer, as well as between different wafers from two different manufacturers. The relative standard deviation (RSD) of fluorescence spot intensity within an array on a chip was less than 20%. Mean spot intensity RSD was 19% for all 25 microarray chips in the study. Within-manufacturer-lot RSDs in chips from either manufacturer were <15% of mean spot intensity. The detection limit and dynamic range of the novel protein chip surface were examined to evaluate whether they match criteria required in a search for novel biomarkers such as for prostate cancer. Monoclonal IgG against prostate-specific antigen (PSA) was arrayed on the porous silicon chips. These were subsequently incubated in serum samples containing widely different levels of fluorescence-labeled PSA. Detection of PSA in serum at concentrations from 0.7 ng/mL (26 pM) up to 10⁴-fold higher levels verified assay characteristics required in the search for prostate biomarkers (e.g., kallikrein gene products) at clinically relevant levels.     

A novel approach of protein immobilization for protein chips using an oligo-cysteine tag

Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamond-like, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.     

Fabrication of protein microarrays using the electrospray deposition (ESD) method: Application of microfluidic chips in immunoassay

In this study, antibody-based protein microarrays for high-throughput immunoassay were fabricated on an aldehydemodified indium-tin oxide glass plate using the electrospray deposition (ESD) method and their characteristics were evaluated immunochemically. The microarrays were also integrated into microfluidic chips with a polydimethylsiloxane (PDMS) micro-channel to detect human cytokines, which were quantitatively analyzed with a high resolution chargecoupled device. Simultaneous detection of various antigens was performed using the microarrays with considerable sensitivity (ca. 100 pg/mL). The results of this study indicate that microfluidic chip comprising a protein microarray formed by the ESD method and a PDMS micro-channel could be easy to handle, and offers high-throughput detection of molecular biomarkers.     

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.     

Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.     

聚合物微流控芯片消毒灭菌技术研究进展

聚合物微流控芯片成本低、易加工,目前在医药、生物检测和化学合成等领域得到了普遍应用。以热塑性聚合物聚甲基丙烯酸甲酯(polymethylmethacrylate,PMMA)和热固型聚合物聚二甲基硅氧烷(polydimethy lsiloxane,PDMS)为基材的高分子聚合物材料因具有较好的生物相容性和光学透明性,已逐渐成为聚合物微流控芯片加工的主导材料,被广泛应用于生物医药类微流控芯片的制备。鉴于该类芯片应用场景的特殊性,需在使用前进行消毒灭菌处理以避免微生物干扰。目前,针对PMMA和PDMS的消毒灭菌方法包括高压蒸汽灭菌、紫外线灭菌、电子束、60Co γ射线辐射灭菌、超临界二氧化碳灭菌、乙醇消毒、环氧乙烷灭菌、过氧化氢低温等离子体灭菌、绿原酸消毒、清洗剂消毒。本文从基本原理、消毒灭菌方法、应用场景等方面,回顾和总结了相关技术在PMMA和PDMS基体微流控芯片中的实现方法,并在芯片材质、适用范围等方面分析了所适用的消毒灭菌方法,为以聚合物为基材的生物医药类微流控芯片的消毒灭菌提供有益参考。     

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

Nanospheres lithographic (NSL) method has been used to fabricate nano-structured arrays (NAs) of hexagonally close-packed gold (Au) using polystyrene beads [PS, diameter ～300 nm] as mask. The developed NA was incorporated with a customized and cheap microfluidics system to demonstrate its applicability as an alternative easy and efficient platform for multiplex analysis and Lab-on-a-Chip applications. The chip functionality was demonstrated with horseradish peroxidase (HRP) and anti-HRP antibody as model for recognition system. The enzyme-linked immunosorbent assay (ELISA) performed on fabricated protein biochip had a detection limit 100 pg/mL for HRP. The antibody chip was also checked for the shelf-life and it was found that these chips could be stored for 50 days when stored at 4°C without any significant loss of activity. Therefore, NAs based protein biochip with the correct microfluidics could find huge potential application in diagnostics and biosensing technology.     

Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address

The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here, we present a unique method to attach a single DNA address to proteins in one step during the purification from the E. coli lysate by fusion to human O6-alkylguanine-DNA-alkyltransferase (SNAP-tag) and the Avitag. Use of the conjugates in converting a DNA chip into a protein chip by self assembly is demonstrated.     

Microfluidic chips controlled with elastomeric microvalve arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.     

Fabrication of disposable protein chip for simultaneous sample detection

In this study, we have described a method for the fabrication of a protein chip on silicon substrate using hydrophobic thin film and microfluidic channels, for the simultaneous detection of multiple targets in samples. The use of hydrophobic thin film provides for a physical, chemical, and biological barrier for protein patterning. The microfluidic channels create four protein patterned strips on the silicon surfaces with a high signal-to-noise ratio. The feasibility of the protein chips was determined in order to discriminate between each protein interaction in a mixture sample that included biotin, ovalbumin, hepatitis B antigen. In the fabrication of the multiplexed assay system, the utilization of the hydrophobic thin film and the microfluidic networks constitutes a more convenient method for the development of biosensors or biochips. This technique may be applicable to the simultaneous evaluation of multiple protein-protein interactions.     

Application of on-chip cell cultures for the detection of allergic response

In this report, the development of a microfluidic cell chip for monitoring allergic response is described. A rat basophilic leukemia cell line (RBL-2H3), a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus. The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods. The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods. In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine. The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE. When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope.     

The BARC biosensor applied to the detection of biological warfare agents

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.     

Neuronal circuits on a chip for biological network monitoring

Cultured neuronal networks (CNNs) are a robust model to closely investigate neuronal circuits’ formation and monitor their structural properties evolution. Typically, neurons are cultured in plastic plates or, more recently, in microfluidic platforms with potentially a wide variety of neuroscience applications. As a biological protocol, cell culture integration with a microfluidic system provides benefits such as accurate control of cell seeding area, culture medium renewal, or lower exposure to contamination. The objective of this report is to present a novel neuronal network on a chip device, including a chamber, fabricated from PDMS, vinyl and glass connected to a microfluidic platform to perfuse the continuous flow of culture medium. Network growth is compared in chips and traditional Petri dishes to validate the microfluidic chip performance. The network assessment is performed by computing relevant topological measures like the number of connected neurons, the clustering coefficient, and the shortest path between any pair of neurons throughout the culture's life. The results demonstrate that neuronal circuits on a chip have a more stable network structure and lifespan than developing in conventional settings, and therefore this setup is an advantageous alternative to current culture methods. This technology could lead to challenging applications such as batch drug testing of in vitro cell culture models. From the engineering perspective, a device's advantage is the chance to develop custom designs more efficiently than other microfluidic systems.     

An integrated chip for rapid, sensitive, and multiplexed detection of cardiac biomarkers from fingerprick blood

Cardiovascular diseases are the major cause of death among adults worldwide. Electrocardiogram (ECG) is a first test when a patient suffering from chest pain sees a doctor, however, it is lack of the required sensitivity. Standard assays to detect cardiac biomarkers, like enzyme-linked immunosorbent assay (ELISA) are sensitive, but suffer from important sample and reagent consumption in large-scale studies. Moreover they are performed in central laboratories of clinics and hospitals and take a long time, which is highly incompatible with the quick decisions needed to save a heart attack patient. Herein, we describe an integrated chip allowing rapid, sensitive, and simultaneous analysis of three cardiac biomarkers in fingerprick blood. The integrated chip is composed of a filtration chip for plasma separation from blood and a silicon nanowire (SiNW) array sensor chip for protein detection. These two chips are fabricated separately and bonded to form a single unit after alignment. The integrated chip is capable of reducing the dead volume of the sample by eliminating the tubing between the two chips. After the plasma is filtrated by the filtration chip, the SiNW sensor, spotted with three different antibodies, enabled us to detect three cardiac biomarkers, troponin T (cTnT), creatine kinase MM (CK-MM) and creatine kinase MB (CK-MB), simultaneously. The integrated chip is able to attain a low detection limit of 1 pg/ml for the three cardiac biomarkers from 2 μl blood in 45 min.     

Silicon chip-based patch-clamp electrodes integrated with PDMS microfluidics

We report on a silicon wafer-based device that can be used for recording macroscopic ion channel protein activities across a diverse group of cell-types. Gigaohm seals were achieved for CHO-K1 and RIN m5F cells, and both cell-attached and whole-cell mode configurations were also demonstrated. Two distinct intrinsic potassium ion channels were recorded in whole-cell mode for HIT-T15 and RAW 264.7 cells. Polydimethylsiloxane (PDMS) microfluidics were also coupled with the micromachined silicon chips in order to demonstrate that a single cell could be selectively directed to a micropore, and membrane protein currents could subsequently be recorded. These silicon chip-based devices have significant advantages over traditional micropipette approaches, and may serve as combinatorial tools for investigating membrane biophysics, pharmaceutical screening, and other bio-sensing tasks.     

On-chip cell culture on a microarray of extracellular matrix with surface modification of poly(dimethylsiloxane)

Microfluidic cell culture chips allow to perform assays of small-volume samples rapidly and reproducibly. Most of these chips are made of poly(dimethylsiloxane) (PDMS), which is a flexible, durable, transparent and inexpensive polymer that can be easily applied to fabrication of microstructures by photolithography and replica molding. However, not many cells are able to grow on unmodified PDMS because the cells need appropriate scaffolds on the surface. Here we report surface modification of a PDMS substrate with a microarray of extracellular matrix (ECM) for on-chip cell culture. The ECM proteins collagen and fibronectin were covalently immobilized on an 8 x 8 microarray format by micropatterned UV-induced graft polymerization through a photomask and dehydration-condensation reaction through a microfabricated stencil. Identical spots of ECMs were successfully formed and the geometry of the spots accurately corresponded to the micropattern of the photomask and stencil. We demonstrate the culture of CHO-K1 cells on the ECM microarray chip. Cells proliferated on the fibronectin spots during the 2-day culture.     

Automatic bio-sampling chips integrated with micro-pumps and micro-valves for disease detection

The present study reports a microfluidic system using the concept of membrane-movement to design and fabricate micro-pneumatic valves and pumps to form a bio-sensing diagnostic chip. The automatic bio-sampling system includes a micro-diagnostic chip fabricated by using MEMS (micro-electro-mechanical systems) technology and an automatic platform comprising of a control circuit, a compressed air source and several electromagnetic valve switches. The control circuit is used to regulate the electromagnetic valve switches, causing thin PDMS membranes to deflect pneumatically by the compressed air and generate valving and pumping effects. The micro-diagnostic chip allows for the quick detection of diseases. Compared to large-scale systems, the new microfluidic system uses smaller amounts of samples and reagents and performs fast diagnosis in an automated format. Instead of using traditional pneumatic micro-pumps, the current study adopts a new design called "spider-web" micro-pumps to increase the pumping rate, and more importantly, improve the uniformity of flow rates inside multiple micro-channels. Experimental data show that for disease diagnosis, the bio-sensing chips integrated with the micro-pneumatic valves and the peristaltic micro-pumps could successfully perform diagnosis tests. Small amounts of samples and reagents could be injected into the diagnosis chips using the micro-pumps and the micro-pneumatic valves could effectively control the movement of the samples and reagents. In order to demonstrate the functionality of the developed device, detection of hepatitis C virus (HCV) and syphilis has been performed using the bio-sampling chips. Experimental data show that fluorescence signals from the microfluidic system were comparable to the ones using conventional testing methods. The developed chip could be easily extended for multiple disease detection. The automatic bio-sensing chips could provide a useful tool for fast disease detection and be crucial for a micro-total-analysis system.