An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications

This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.     

Performance of an integrated microoptical system for fluorescence detection in microfluidic systems

This article presents a new integrated microfluidic/microoptic device designed for basic biochemical analysis. The microfluidic network is wet-etched in a Borofloat 33 (Pyrex) glass wafer and sealed by means of a second wafer. Unlike other similar microfluidic systems, elements of the detection system are realized with the help of microfabrication techniques and directly deposited on both sides of the microchemical chip. The detection system is composed of the combination of refractive circular or elliptical microlens arrays and chromium aperture arrays. The microfluidic channels are 60 microm wide and 25 microm deep. The elliptical microlenses have a major axis of 400 microm and a minor axis of 350 microm. The circular microlens diameters range from 280 microm to 350 microm. The apertures deposited on the outer chip surfaces are etched in a 3000-A-thick chromium layer. The overall thickness of this microchemical system is < 1.6 mm. A limit of detection of 3.3 nM for a Cy5 solution in phosphate buffer (pH 7.4) was demonstrated. The cross-talk signal measured between two adjacent microchannels with 1 mm pitch was < 1:5600, meaning that < or = 1.8 x 10(-4)% of the fluorescence light power emitted from one microchannel filled with a 50 microM Cy5 solution reaches the photodetector at the adjacent microchannel. This performance compares very well with that obtainable in microchemical chips using confocal fluorescence systems, taking differences in parameters, such as excitation power into microchannels, data acquisition rates, and signal filtering into account.     

An integrated superconductive magnetic nanosensor for high-sensitivity nanoscale applications

An integrated magnetic nanosensor based on a niobium dc SQUID (superconducting quantum interference device) for nanoscale applications is presented. The sensor, having a washer shape with a hole of 200?nm and two Josephson-Dayem nanobridges of 80?nm × 100?nm, consists of a Nb(30?nm)/Al(30?nm) bilayer patterned by electron beam lithography (EBL) and shaped by lift-off and reactive ion etch (RIE) processes. The presence of the niobium coils, integrated on-chip and tightly coupled to the SQUID, allows us to easily excite the sensor in order to get the voltage-flux characteristics and to flux bias the SQUID at its optimal point. The measurements were performed at liquid helium temperature. A voltage swing of 75?μV and a maximum voltage-flux transfer coefficient (responsivity) as high as 1?mV/Φ(0) were directly measured from the voltage-flux characteristic. The noise measurements were performed in open loop mode, biasing the SQUID with a dc magnetic flux at its maximum responsivity point and using direct-coupled low-noise readout electronics. A white magnetic flux noise spectral density as low as 2.5?μΦ(0)?Hz(-1/2) was achieved, corresponding to a magnetization or spin sensitivity in units of the Bohr magneton of 100?spin?Hz(-1/2). Possible applications of this nanosensor can be envisaged in magnetic detection of nanoparticles and small clusters of atoms and molecules, in the measurement of nanoobject magnetization, and in quantum computing.     

Passively driven integrated microfluidic system for separation of motile sperm

This paper describes a self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques. The device isolates motile sperm from nonmotile sperm and other cellular debris, based on the ability of motile sperm to cross streamlines in a laminar fluid stream. The device is small, simple, and disposable yet is an integrated system complete with sample inlets, outlets, sorting channel, and a novel passively driven pumping system that provides a steady flow of liquid; it requires no external power source or controls. The device fulfills a need in clinical settings where small amounts of sperm need to be sorted. It also opens the way for convenient bioassays based on sperm motility including at-home motile sperm tests.     

Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.     

An integrated framework for software vulnerability detection,analysis and mitigation: an autonomic system

Nowadays, the number of software vulnerabilities incidents and the loss due to occurrence of software vulnerabilities are growing exponentially. The current existing security strategies, the vulnerability detection and remediating approaches are not intelligent, automated, self-managed and not competent to combat against the vulnerabilities and security threats, and to provide secured self-managed software environment to the organizations. Hence, there is a strong need to devise an intelligent and automated approach to optimize security and prevent the occurrence of vulnerabilities or mitigate the vulnerabilities. The autonomic computing is a nature-inspired and self-management-based computational model. In this paper, an autonomic-computing-based integrated framework is proposed to detect, fire the trigger of alarm, assess, classify, prioritize, mitigate and manage the software vulnerability automatically. The proposed framework uses a knowledge base and inference engine, which automatically takes the remediating actions on future occurrence of software security vulnerabilities through self-configuration, self-healing, self-prevention and self-optimization as per the needs. The proposed framework is beneficial to industry and society in various aspects because it is an integrated, cross-concern and intelligent framework and provides more secured self-managed environment to the organizations. The proposed framework reduces the security risks and threats, and also monetary and reputational loss. It can be embedded easily in existing software and incorporated or implemented as an inbuilt integral component of the new software during software development.     

微流控LAMP芯片构建及在MRSA快速检测中的应用研究

构建一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP),集细菌在线裂解、核酸提取、目标基因扩增和产物检测一体化的用于病原菌快速检测的集成式微流控芯片。以耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus,MRSA)为模式菌,以mec A为靶基因,在优化条件下用芯片实现对病原菌的在线检测,完成对101~106cfu MRSA的在线裂解、LAMP扩增和产物测定,采用荧光原位检测可得101~105cfu的检测范围和101cfu的检出限。该微流控LAMP芯片结构简单,操作便捷,可在1 h内实现对MRSA mec A基因的快速检测,具有较高的灵敏度和特异性,为下一步临床生物样本病原菌快速检测微流控芯片系统的构建奠定前期研究基础。     

Emerging microfluidic devices for cancer cells/biomarkers manipulation and detection

Circulating tumour cells (CTCs) are active participants in the metastasis process and account for ∼90% of all cancer deaths. As CTCs are admixed with a very large amount of erythrocytes, leukocytes, and platelets in blood, CTCs are very rare, making their isolation, capture, and detection a major technological challenge. Microfluidic technologies have opened‐up new opportunities for the screening of blood samples and the detection of CTCs or other important cancer biomarker‐proteins. In this study, the authors have reviewed the most recent developments in microfluidic devices for cells/biomarkers manipulation and detection, focusing their attention on immunomagnetic‐affinity‐based devices, dielectrophoresis‐based devices, surface‐plasmon‐resonance microfluidic sensors, and quantum‐dots‐based sensors.Inspec keywords: microfluidics, bioMEMS, cancer, cellular biophysics, biomedical equipment, patient diagnosis, tumours, proteins, molecular biophysics, electrophoresis, surface plasmon resonance, quantum dotsOther keywords: quantum‐dot‐based sensors, surface‐plasmon‐resonance microfluidic sensors, dielectrophoresis‐based devices, immunomagnetic‐affinity‐based devices, cancer biomarker‐proteins, CTC detection, blood samples, microfluidic technology, platelets, leukocytes, leukocytes, erythrocytes, cancer deaths, metastasis process, circulating tumour cells, cancer cell‐biomarker detection, cancer cell‐biomarker manipulation, microfluidic devices     

An approach to multilayer microfluidic systems with integrated electrical, optical, and mechanical functionality

A general solution is presented for the realization of microfluidic systems containing multiple compartments with sequential functions. The approach utilizes multilayer plastic/glass/silicon microfluidic system configurations with integrated electrical, optical, and fluid control functionalities. The approach allows for the integration of on-column, on-chip electrical conductivity/impedance detectors and driving electrodes, as well as for the integration of on-column, off-chip optical detection using resonance light scattering. A process-compatible latex microvalve is demonstrated and characterized. The procedure utilizes a novel packaging technique for realizing integrated fluid and electrical interfaces between the microsystem and conventional tubing/instrumentation. The microfluidic system demonstrated in this work contains some of the most commonly used components in bioanalysis systems and can be used as a platform to fabricate more sophisticated microscale bioanalysis systems for a variety of applications.     

Solution-gated graphene field effect transistors integrated in microfluidic systems and used for flow velocity detection

Solution-gated graphene field effect transistors (SGGT) were integrated in microfluidic systems. The transfer characteristics of a SGGT with an Ag/AgCl gate electrode shifted horizontally with the change of the ionic concentration of KCl solution in the microchannel and the relationship can be fitted with the Nernst equation, which was attributed to the change of the potential drop at the Ag/AgCl electrode. Therefore the gate electrode is one important factor for the ion sensitive property of the SGGT. Then SGGTs were used as flow velocity sensors, which were based on measuring the streaming potentials in microfluidic channels. A linear relationship between the shift of the transfer curve of the SGGT and the flow velocity was obtained, indicating that the SGGT is a promising transducer for measuring flow velocity in a microchip. Since the streaming potential is influenced by the three physical quantities, including the flow velocity, the ionic strength of the fluid and the zeta potential of the substrate, the device can be used for sensing any one of the three quantities when the other two were known. It is noteworthy that SGGTs have been used for various types of chemical and biological sensors. Array of the devices integrated in multichannel microchips are expected to find many important applications in the lab-on-a-chip systems in the future.     

An integrated microfluidic device for rapid cell lysis and DNA purification of epithelial cell samples

In this paper, we describe the design and fabrication of a microfluidic device for cell lysis and DNA purification, and the results of device tests using a real sample of buccal cells. Cell lysis was thermally executed for two minutes at 80 degrees C in a serpentine type microreactor (20 microL) using an Au microheater with a microsensor. The DNA was then mixed with other residual products and purified by a new filtration process involving micropillars and 50-80 microm microbeads. The entire process of sample loading, cell lysis, DNA purification, and sample extraction was successfully completed in the microchip within five minutes. Sample preparation within the microchip was verified by performing a SY158 gene PCR analysis and gel electrophoresis on the products obtained from the chip. The new purification method enhanced DNA purity from 0.93 to 1.62 after purification.     

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

An integrated software system for processing, analyzing, and classifying Raman spectra

Raman spectroscopy is a powerful technique for analyzing various substances, but a significant amount of processing is required to extract useful information from raw spectra. Many research groups develop custom software systems to process spectra, which leads to standard algorithms being repeatedly implemented and novel techniques being difficult to share. This paper presents an integrated software system that processes, analyzes, and classifies Raman spectra. The system is freely available (http://cares.wayne.edu/rp/), open, cross-platform, and extensible, enabling interested parties to utilize and contribute to a common implementation of Raman processing algorithms.A wide variety of useful features are included, such as noise filtering, automated background fluorescence subtraction, principal component analysis, linear discriminant analysis, artificial neural network classification, and support vector machine classification. The system supports customizable groupings of related spectra and is able to retrieve spectra using a searchable database. Everything is executed through a simple graphical user interface.The developed system was used to process two data sets containing different types of spectra. The results demonstrate that the system is capable of fast, accurate processing of Raman spectra. The open system will become even more powerful if other developers are willing to add new and improved features. In addition, it is intended that the freely available system will make Raman spectroscopy more accessible to all researchers and promote progress in the field.     

Self-regulated, droplet-based sample chopper for microfluidic absorbance detection

Akin to optical beam chopping, we demonstrate that formation and routing of aqueous droplets in oil can chop a fluidic sample to permit phase sensitive detection. This hand-operated microfluidic sample chopper (μChopper) greatly reduces the detection limit of molecular absorbance in a 27 μm optical path. With direct dependence on path length, absorbance is fundamentally incompatible with microfluidics. While other microfluidic absorbance approaches use complex additions to fabrication, such as fiber coupling and increased optical paths, this self-regulated μChopper uses opposing droplet generators to passively alternate sample and reference droplets at ~10 Hz each. Each droplet's identity is automatically locked-in to its generator, allowing downstream lock-in analysis to nearly eliminate large signal drift or 1/f noise. With a lock-in time constant of 1.9 s and total interrogated volume of 59 nL (122 droplets), a detection limit of 3.0 × 10(-4) absorbance units or 500 nM bromophenol blue (BPB) (29 fmol) was achieved using only an optical microscope and a standard, single-depth (27 μm) microfluidic device. The system was further applied to nanoliter pH sensing and validated with a spectrophotometer. The μChopper represents a fluidic analog to an optical beam chopper, and the self-regulated sample/reference droplet alternation promotes ease of use.     

A microfluidic device with an integrated waveguide beam splitter for velocity measurements of flowing particles by Fourier transformation

A microfabricated capillary electrophoresis device for velocity measurements of flowing particles is presented. It consists of a 1 x 128 planar waveguide beam splitter monolithically integrated with an electrically insulated fluidic channel network for fluorescence excitation at multiple points. Stray light rejection structures are included in order to suppress unwanted light between the detection regions. The emission pattern of particles passing the detection region was collected by a photomultiplier tube that was placed in close proximity to the channel, thereby avoiding the use of transfer optics. The integrated planar waveguide beam splitter was, furthermore, permanently connected to the light source by a glued-on optical fiber, to achieve a robust and alignment-free operation of the system. The velocity was measured using a Fourier transformation with a Shah function, since the response of the light array was designed to approximate a square profile. Deviations from this response were observed as a result of the multimode nature of the integrated waveguides.     

Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.     

Sample filtration,concentration, and separation integrated on microfluidic devices

Microfabricated devices integrating sample filtration, solid-phase extraction, and chromatographic separation with solvent programming were demonstrated. Filtering of the sample was accomplished at the sample inlet with an array of seven channels each 1 microm deep and 18 microm wide. Sample concentration and separation were performed on channels 5 microm deep and 25 microm wide coated with a C18 phase, and elution was achieved under isocratic, step, or linear gradient conditions. For the solid-phase extraction, signal enhancement factors of 400 over a standard injection of 1.0 s were observed for a 320-s injection. Four polycyclic aromatic compounds were resolved by open channel electrochromatography in under 50 s. Chip operation was unaffected by the presence of the 5-microm silica particles at the filter entrance.     

Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation

We have developed a multipurpose microfluidic platform that allows for sensitive fluorescence detection on inexpensive disposable chips. The fabrication scheme involves rapid injection molding of thermoplastics, followed by silica deposition and covalent attachment of an unstructured flexible lid. This combines the virtues of elastomer technology with high-throughput compact disk injection molding. Using this technique, the time to produce 100 chips using a single master can be lowered from more than 1 week by standard PDMS technologies to only a couple hours. The optical properties of the fabricated chips were evaluated by studying individual fluorescence-labeled DNA molecules in a microchannel. Concatemeric DNA molecules were generated through rolling circle replication of circular DNA molecules, which were labeled by hybridization of fluorescence-tagged oligonucleotides. Rolling circle products (RCPs) were detected after as little as 5 min of DNA polymerization, and the RCPs in solution showed no tendency for aggregation. To illustrate the versatility of the platform, we demonstrate two additional applications: The flexible property of the lid was used to create a peristaltic pump generating a flow rate of 9 nL/s. Biocompatibility of the platform was illustrated by culturing Chinese hamster ovary cells for 7 days in the microfluidic channels.     

Self-contained, biomolecular motor-driven protein sorting and concentrating in an ultrasensitive microfluidic chip

We developed a molecular sorter that operates without external power or control by integrating the microtubule-based, biological motor kinesin into a microfluidic channel network to sort, transport, and concentrate molecules. In our devices, functionalized microtubules that capture analyte molecules are steered along kinesin-coated microchannel tracks toward a collector structure, concentrated, and trapped. Using fluorescent analyte molecules and nanoliter sample volumes, we demonstrated 14 fM sensitivity, even in the presence of high concentrations of other proteins.     

On-chip high-speed sorting of micron-sized particles for high-throughput analysis

A new design of particle sorting chip is presented. The device employs a dielectrophoretic gate that deflects particles into one of two microfluidic channels at high speed. The device operates by focussing particles into the central streamline of the main flow channel using dielectrophoretic focussing. At the sorting junction (T- or Y-junction) two sets of electrodes produce a small dielectrophoretic force that pushes the particle into one or other of the outlet channels, where they are carried under the pressure-driven fluid flow to the outlet. For a 40 microm wide and high channel, it is shown that 6 microm diameter particles can be deflected at a rate of 300/s. The principle of a fully automated sorting device is demonstrated by separating fluorescent from non-fluorescent latex beads.