Fabrication and characterization of a multiwell array SERS chip with biological applications

Uniform, large surface area substrates for surface-enhanced Raman spectroscopy (SERS) are fabricated by oblique angle deposition. The SERS-active substrates are patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-di(4-pyridyl)ethylene (BPE) ≥98%, we show that this device provides a uniform Raman signal enhancement from well to well with a detection limit of at least 10⁻⁸ M of the BPE solution or 10⁻¹⁸ mol of BPE. The SERS intensity is also demonstrated to vary logarithmically with the log of BPE concentration and the apparent sensitivity of the patterned substrate is compared to previous reports from our group on non-patterned substrates. Avian influenza is analyzed to demonstrate the utility of SERS multiwell patterned substrates for biosensing. The spectra acquired from patterned substrates show better reproducibility and less variation compared to the unpatterned substrates according to multivariate analysis. Our results highlight potential advantages of the patterned substrate.     

Urinary proteome profiling using microfluidic technology on a chip

Clinical diagnostics and biomarker discovery are the major focuses of current clinical proteomics. In the present study, we applied microfluidic technology on a chip for proteome profiling of human urine from 31 normal healthy individuals (15 males and 16 females), 6 patients with diabetic nephropathy (DN), and 4 patients with IgA nephropathy (IgAN). Using only 4 microL of untreated urine, automated separation of proteins/peptides was achieved, and 1-7 (3.8 +/- 0.3) spectra/bands of urinary proteins/peptides were observed in the normal urine, whereas 8-16 (11.3 +/- 1.2) and 9-14 (10.8 +/- 1.2) spectra were observed in urine samples of DN and IgAN, respectively. Coefficient of variations of amplitudes of lower marker (1.2 kDa), system spectra (6-8 kDa), and upper marker (260.0 kDa) were 22.84, 24.92, and 32.65%, respectively. ANOVA with Tukey post-hoc multiple comparisons revealed 9 spectra of which amplitudes significantly differed between normal and DN urine (DN/normal amplitude ratios ranged from 2.9 to 3102.7). Moreover, the results also showed that 3 spectra (with molecular masses of 12-15, 27-28, and 34-35 kDa) were significantly different between DN and IgAN urine (DN/IgAN amplitude ratios ranged from 3.9 to 7.4). In addition to the spectral amplitudes, frequencies of some spectra could differentiate the normal from the diseased urine but could not distinguish between DN and IgAN. There was no significant difference, regarding the spectral amplitude or frequency, observed between males and females. These data indicate that the microfluidic chip technology is applicable for urinary proteome profiling with potential uses in clinical diagnostics and biomarker discovery.     

Rapid self-assembly of DNA on a microfluidic chip

Background  

DNA self-assembly methods have played a major role in enabling methods for acquiring genetic information without having to resort to sequencing, a relatively slow and costly procedure. However, even self-assembly processes tend to be very slow when they rely upon diffusion on a large scale. Miniaturisation and integration therefore hold the promise of greatly increasing this speed of operation.     

Novel microbial photobioelectrochemical cell using an invasive ultramicroelectrode array and a microfluidic chamber

Objective

To fabricate a novel microbial photobioelectrochemical cell using silicon microfabrication techniques.

Results

High-density photosynthetic cells were immobilized in a microfluidic chamber, and ultra-microelectrodes in a microtip array were inserted into the cytosolic space of the cells to directly harvest photosynthetic electrons. In this way, the microbial photobioelectrochemical cell operated without the aid of electron mediators. Both short circuit current and open circuit voltage of the microbial photobioelectrochemical cell responded to light stimuli, and recorded as high as 250 pA and 45 mV, respectively.

Conclusion

A microbial photobioelectrochemical cell was fabricated with potential use in next-generation photosynthesis-based solar cells and sensors.

     

Dielectrophoretic separation of monocytes from cancer cells in a microfluidic chip using electrode pitch optimization

This study proposes a microfluidic device capable of separating monocytes from a type of cancer cell that is called T-cell acute lymphoblastic leukemia (RPMI-8402) in a continuous flow using negative and positive dielectrophoretic forces. The use of both the hydrodynamic and dielectrophoretic forces allows the separation of RPMI-8402 from monocytes based on differences in their intrinsic electrical properties and sizes. The specific crossover frequencies of monocytes and RPMI-8402 cells have been obtained experimentally. The optimum ranges of electrode pitch-to-channel height ratio at the cross sections with different electrode widths have been generally calculated by numerical simulations of the gradients of the electric field intensities and calculation their effective values (root-mean-square). In the device, the cell sorting has been conducted empirically, and then, the separation performance has been evaluated by analyzing the images before and after dielectrophoretic forces applied to the cells. In this work, the design of a chip with 77 μm gold–titanium electrode pitch was investigated to achieve high purity of monocytes of 95.2%. The proposed device can be used with relatively low applied voltages, as low as 16.5 V (peak to peak). Thus, the design can be used in biomedical diagnosis and chemical analysis applications as a lab-on-chip platform. Also, it can be used for the separation of biological cells such as bacteria, RNA, DNA, and blood cells.

     

Multianalyte immunoassay with self-assembled addressable microparticle array on a chip

This paper describes the random fluidic self-assembly of metallic particles into addressable two-dimensional microarrays and the use of these arrays as a platform for constructing a biochip useful for bioassays. The basic units in the assembly were the microfabricated particles carrying a straightforward visible code and the corresponding array template patterned on a glass substrate. The particles consisted of a hydrophobic and magnetic Ni-polytetrafluoroethylene (PTFE) composite layer on one face, and on the other face a gold layer that was modified for biomolecular attachment. An array template was photoresist-patterned with spatially discrete microwells in which an electrodeposited Ni-PTFE hydrophobic composite layer and a hydrophobic photo-adhesive coating were deposited. The particles, after biomaterial attachment and binding processes in bulk, were self-assembled randomly onto the lubricated bonding sites on the chip substrate, driven by a combination of magnetic, hydrophobic, and capillary interactions. The encoding symbol carried by the particles was used as the signature for the identification of each target/assay attached to the particle surface. We demonstrate here the utility of microfabricated-encoded particle arrays for conducting multianalyte immunoassays in a parallel fashion with the use of imaging detection.     

A prototype microfluidic chip using fluorescent yeast for detection of toxic compounds

A microfluidic chip has been developed to enable the screening of chemicals for environmental toxicity. The microfluidic approach offers several advantages over macro-scale systems for toxicity screening, including low cost and flexibility in design. This design flexibility means the chips can be produced with multiple channels or chambers which can be used to screen for different toxic compounds, or the same toxicant at different concentrations. Saccharomyces cerevisiae containing fluorescent markers are ideal candidates for the microfluidic screening system as fluorescence is emitted without the need of additional reagents. Microfluidic chips containing eight multi-parallel channels have been developed to retain yeast within the chip and allow exposure of them to toxic compounds. The recombinant yeast used was GreenScreentrade mark which expresses green fluorescent proteins when is exposed to genotoxins. After exposure of the yeast to target compounds, the fluorescence emission was detected using an inverted microscope. Qualitative and quantitative comparisons of the fluorescent emission were performed. Results indicated that fluorescent intensity per area significantly increases upon exposure to methyl-methanesulfonate, a well known genotoxic compound. The microfluidic approach reported here is an excellent tool for cell-based screening and detection of different toxicities. The device has the potential for use by industrial manufacturers to detect and reduce the production and discharge of toxic compounds, as well as to characterise already polluted environments.     

Electrophysiological recordings of single ion channels in planar lipid bilayers using a polymethyl methacrylate microfluidic chip

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented.     

Amperometric detection of the bacterial metabolic regulation with a microbial array chip

A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose.     

Integration of a surface acoustic wave biosensor in a microfluidic polymer chip

SAW devices based on horizontally polarized surface shear waves (HPSSW) enable label-free, sensitive and cost-effective detection of biomolecules in real time. It is known that small sampling volumes with low inner surface areas and minimal mechanical stress arising from sealing elements of miniaturized sampling chambers are important in this field. Here, we present a new approach to integrate SAW devices with sampling chamber. The sensor device is encapsulated within a polymer chip containing fluid channel and contact points for fluidic and electric connections. The chip volume is only 0.9 microl. The polymeric encapsulation was performed tailor-made by Rapid Micro Product Development 3Dimensional Chip-Size-Packaging (RMPD 3D-CSP), a 3D photopolymerisation process. The polymer housing serves as tight and durable package for HPSSW biosensors and allows the use of the complete chips as disposables. Preliminary experiments with these microfluidic chips are shown to characterise the performance for their future applications as generic bioanalytical micro devices.     

Determination of antibiotic EC50 using a zero‐flow microfluidic chip based growth phenotype assay

Current existing assay systems for evaluating antimicrobial activity suffer from several limitations including excess reagent consumption and inaccurate concentration gradient preparation. Recently, microfluidic systems have been developed to provide miniaturized platforms for antimicrobial susceptibility assays. However, some of current microfluidic based assays require continuous flows of reagents or elaborate preparation steps during concentration preparation. In this study, we introduce a novel microfluidic chip based growth phenotype assay that automatically generates a logarithmic concentration gradient and allows observing the growth of pathogenic bacteria under different concentrations of antibiotics in nanoliter batch culture reactors. We chose pathogen bacterium Pseudomonas aeruginosa as a model strain and evaluated the inhibitory effects of gentamicin and ciprofloxacin. We determined the EC₅₀ values and confirmed the validity of the present system by comparing the EC₅₀ values obtained through conventional test tube method. We demonstrated that the EC₅₀ values acquired from present assay are comparable to those obtained from conventional test tube cultures. The potential application of present assay system for investigating combinatorial effects of antibiotics on multidrug resistant pathogenic bacteria is discussed and it can be further used for systematic evaluation of antifungal or antiviral agents.     

基于环介导等温扩增技术的微流控芯片在病原菌检测方面的研究进展

环介导等温扩增(LAMP)技术是一种新兴的核酸恒温扩增技术,与微流控芯片技术相结合,可实现对病原菌的快速检测,具有特异性强、灵敏度高、操作简单等优点。本文根据不同终产物的检测方法对目前检测病原菌的相关微流控LAMP芯片进行了分类与介绍,并对技术的改进和存在的问题进行了分析,以期为后续的相关研究提供参考。     

Fluorimetric urease inhibition assay on a multilayer microfluidic chip with immunoaffinity immobilized enzyme reactors

We fabricated a three-layer polydimethylsiloxane (PDMS)-based microfluidic chip for realizing urease inhibition assay with sensitive fluorescence detection. Procedures such as sample prehandling, enzyme reaction, reagent mixing, fluorescence derivatization, and detection can be readily carried out. Urease reactors were prepared by adsorption of rabbit immunoglobulin G (IgG) and immunoreaction with urease-conjugated goat anti-rabbit IgG. Acetohydroxamic acid (AHA) as a competitive inhibitor of urease was tested on the chip. Microfluidically generated gradient concentrations of AHA with substrate (urea) were loaded into urease reactors. After incubation, the produced ammonia was transported out of reactors and then reacted with o-phthalaldehyde (OPA) to generate fluorescent products. Urease inhibition was indicated by a decrease in fluorescence signal detected by microplate reader. The IC₅₀ value of AHA was determined and showed good agreement with that obtained in microplate. The presented device combines several steps of the analytical process with advantages of low reagent consumption, reduced analysis time, and ease of manipulation. This microfluidic approach can be extended to the screening of inhibitory compounds in drug discovery.     

Fabrication of bienzyme nanobiocomposite electrode using functionalized carbon nanotubes for biosensing applications

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wider applicability. This work presents a novel approach to fabricate nanobiocomposite bienzymatic biosensor based on functionalized multiwalled carbon nanotubes (MWNTs) with the aim of evaluating their ability as sensing elements in amperometric transducers. Electrochemical behavior of the bienzymatic nanobiocomposite biosensor is investigated by Faradaic impedance spectroscopy and cyclic voltammetry. The results indicate that glucose oxidase (GOD) and horseradish peroxidase (HRP) are strongly adsorbed on the surface of the thionin (TH) functionalized MWNTs and demonstrate a facile electron transfer between immobilized GOD/HRP and the electrode via the functionalized MWNTs in a Nafion film. The functionalized carbon nanotubes act as molecular wires to allow efficient electron transfer between the underlying electrode and the redox centres of enzymes through TH. Linear ranges for these electrodes are from 10 nM to 10 mM for glucose and 17 nM to 56 mM for hydrogen peroxide with the detection limit of 3 and 6 nM, respectively. A remarkable feature of the bienzyme electrode is the possibility to determine glucose and hydrogen peroxide at a very low applied potential where the noise level and interferences from other electroactive compounds are minimal. Performance of the biosensor is evaluated with respect to response time, detection limit, selectivity, temperature and pH as well as operating and storage stability.     

Numerical design of a microfluidic chip for probing mechanical properties of cells

Microfluidic chips have been widely used to probe the mechanical properties of cells, which are recognized as a promising label-free biomarker for some diseases. In our previous work (Ye et al., 2018), we have studied the relationships between the transit time and the mechanical properties of a cell flowing through a microchannel with a single constriction, which potentially forms a basis for a microfluidic chip to measure cell’s mechanical properties. Here, we investigate this microfluidic chip design and examine its potential in performances. We first develop the simultaneous dependence of the transit time on both the shear and bending moduli of a cell, and then examine the chip sensitivity with respect to the cell mechanical properties while serializing a single constriction along the flow direction. After that, we study the effect of the flow velocity on the transit time, and also test the chip’s ability to identify heterogeneous cells with different mechanical properties. The results show that the microfluidic chip designed is capable of identifying heterogeneous cells, even when only one unhealthy cell is included. The serialization of chip can greatly increase the chip sensitivity with respect to the mechanical properties of cells. The flow with a higher velocity helps in not only promoting the chip throughput, but also in providing more accurate transit time measurements, because the cell prefers a symmetric deformation under a high velocity.     

Application of microfluidic chip with integrated optics for electrophoretic separations of proteins

This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.     

High throughput gene expression measurement with real time PCR in a microfluidic dynamic array

We describe a high throughput gene expression platform based on microfluidic dynamic arrays. This system allows 2,304 simultaneous real time PCR gene expression measurements in a single chip, while requiring less pipetting than is required to set up a 96 well plate. We show that one can measure the expression of 45 different genes in 18 tissues with replicates in a single chip. The data have excellent concordance with conventional real time PCR and the microfluidic dynamic arrays show better reproducibility than commercial DNA microarrays.     

A method of binding kinetics of a ligand to micropatterned proteins on a microfluidic chip

A combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein-ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background. Selectively patterned protein patterns of various sizes were used for quantitative analysis of the kinetic parameters of immobilized proteins on the circular patterns. As a model system, a streptavidin-patterned array of the same-sized pattern, i.e. 150 microm diameter, was used to capture FITC-BSA-biotin present in solution. The fluorescence intensity was well matched with the Langmuir isotherm model results, showing a dissociation constant of 2.43x10(-8)M. Similar streptavidin arrays with different-sized spots, ranging from 50 to 200 microm, showed a consistent dissociation constant of FITC-BSA-biotin with streptavidin pattern. Therefore, the reduction of pattern size of an immobilized protein did not change the dissociation rate of the ligand.     

三种细胞共培养微流控芯片的设计和制作

设计一种具有“微坝”和“微缝”结构的微流控芯片,能够物理隔离不同细胞,而且培养基中小分子营养物质可以自由流通。实验结果表明在芯片上可以共培养人肺腺癌细胞(A549)、人胚肺成纤维细胞(HLF-1)和人内皮细胞(HUVECs)三种细胞,在72 h培养后三种细胞生长状态良好,具有细胞图形化的特点和功能,为下一步开展多种细胞相互作用等相关研究提供重要的技术平台。