Sample Pretreatment and Nucleic Acid-Based Detection for Fast Diagnosis Utilizing Microfluidic Systems

Recently, micro-electro-mechanical-systems (MEMS) technology and micromachining techniques have enabled miniaturization of biomedical devices and systems. Not only do these techniques facilitate the development of miniaturized instrumentation for biomedical analysis, but they also open a new era for integration of microdevices for performing accurate and sensitive diagnostic assays. A so-called "micro-total-analysis-system", which integrates sample pretreatment, transport, reaction, and detection on a small chip in an automatic format, can be realized by combining functional microfluidic components manufactured by specific MEMS technologies. Among the promising applications using microfluidic technologies, nucleic acid-based detection has shown considerable potential recently. For instance, micro-polymerase chain reaction chips for rapid DNA amplification have attracted considerable interest. In addition, microfluidic devices for rapid sample pretreatment prior to nucleic acid-based detection have also achieved significant progress in the recent years. In this review paper, microfluidic systems for sample preparation, nucleic acid amplification and detection for fast diagnosis will be reviewed. These microfluidic devices and systems have several advantages over their large-scale counterparts, including lower sample/reagent consumption, lower power consumption, compact size, faster analysis, and lower per unit cost. The development of these microfluidic devices and systems may provide a revolutionary platform technology for fast sample pretreatment and accurate, sensitive diagnosis.     

Paper-based analytical devices for point-of-care infectious disease testing

Paper-based devices provide an alternative technology for simple, low-cost, portable, and disposable or recyclable diagnostic tools for many applications, including clinical diagnosis, food quality control, and environmental monitoring. The present review focuses on new paper-based tests for point-of-care (POC) infectious disease testing. This review provides a brief presentation of the fabrication techniques and the main sample preparation procedures. Recent immunological and molecular testing formats based on new paper-based solutions which go beyond conventional lateral flow formats are also added. Emphasis is placed on how paper systems could be used for detecting whole and viable bacteria associated to infectious diseases. Paper has recently become attractive, since it is a ubiquitous and extremely cheap material. It is easy to store, easy to use, and is compatible with many (bio)chemical and (bio)medical applications. Paper absorbs and transports liquids by capillary force without additional mechanical assistance. Hence, paper-based analytical devices are promising and possibly game-changing, even if they still suffer from limitations, including accuracy and sensitivity. It is anticipated that, in the near future, with advances in fabrication procedures and associated analytical techniques, there will be a continuous flow of innovative paper-based diagnostics kits.     

Recombinant growth hormone delivery systems based on vinylpyrrolidone-hydroxyethyl methacrylate copolymer matrices: monitoring optimization by capillary zone electrophoresis

Experimental conditions for the fabrication of two new polymeric devices (i.e. films and slabs) useful for the controlled release of recombinant growth hormone (GH) are given. The release rate is controlled by the resorption profile of the vinylpyrrolidone-hydroxyethyl methacrylate (VP-HEMA) tested systems which is related to the copolymer composition. The suitability of capillary electrophoresis (CE) for following the complete preparation of the different VP-HEMA devices is shown. Moreover, CE allows simultaneous monitoring of the controlled release of GH and dissolved polymer during in vitro experiments. From these results, guidelines are given for the fabrication of polymeric devices containing protein as active drug as well as for the correct selection of conditions during in vitro experiments.     

Recombinant growth hormone delivery systems based on vinylpyrrolidone-hydroxyethyl methacrylate copolymer matrices: Monitoring optimization by capillary zone electrophoresis

Experimental conditions for the fabrication of two new polymeric devices (i.e. films and slabs) useful for the controlled release of recombinant growth hormone (GH) are given. The release rate is controlled by the resorption profile of the vinylpyrrolidone-hydroxyethyl methacrylate (VPHEMA) tested systems which is related to the copolymer composition. The suitability of capillary electrophoresis (CE) for following the complete preparation of the different VP-HEMA devices is shown. Moreover, CE allows simultaneous monitoring of the controlled release of GH and dissolved polymer during in vitro experiments. From these results, guidelines are given for the fabrication of polymeric devices containing protein as active drug as well as for the correct selection of conditions during in vitro experiments.     

Microfabrication Technology for the Production of Capillary Array Electrophoresis Chips

Improvements in the fabrication, sample handling and electrical addressing of capillary array electrophoresis (CAE) chips have permitted the development of high density, high-throughput devices capable of analyzing 48 samples in about 20 minutes. The fabrication of high density capillary arrays on 10 cm diameter substrates required the characterization of glasses that yield high quality etches and the development of improved sacrificial etch masks. Using these improved fabrication techniques, high-quality, deep channel etches are routinely obtained. Methods for bonding large area substrates and for drilling arrays of 100 or more access holes have also been developed. For easier sample introduction, we use an array of sample wells fabricated from an elastomeric sheet. The practicality of these technologies is demonstrated through the analysis of 12 DNA samples in parallel on a microfabricated CAE chip, the development of methods for injecting multiple samples onto a single capillary without cross contamination, and the operation of a microfabricated array of 12 capillaries with 4 sample injections per capillary that can analyze 48 samples.     

Microfluidic blood filtration device

Rapid decentralized biomedical diagnostics have become increasingly necessary in a medical environment of growing costs and mounting demands on healthcare personnel and infrastructure. Such diagnostics require low-cost novel devices that can operate at bedside or in doctor offices using small amounts of sample that can be extracted and processed on the spot. Thus, point-of-care sample preparation is an important component of the necessary diagnostic paradigm shift. We therefore introduce a microfluidic device which produces plasma from whole blood. The device is inexpensive, reliable, easy to fabricate, and requires only 3.5 kPa pressure to operate. The device is fully compatible with microfluidic diagnostic chips. The output 23-gauge microtube of the former can be directly plugged into the input ports of the latter allowing immediate applicability in practice as a sample-prep pre-stage to a variety of emergent microfluidic diagnostic devices. In addition, the shown approach of filter encapsulation in elastomer has principle importance as it is compatible with and applicable to microfluidic sample-prep integration with analytical stages within the same elastomeric chip. This can eventually lead to finger-prick blood tests in point-of-care settings.     

Electrochemically Generated Conjugated Microporous Polymer Network Thin Films for Chemical Sensor Applications

Electrogenerated π‐conjugated microporous polymer network (CMPN) thin films are potential materials for highly sensitive detection of harmful chemical species as they show fast response, high signal amplification, and easy fabrication of devices. In the last few years, several CMPN thin films from carbazolyl and thienyl monomer building blocks having a variety of aromatic cores have been reported for different applications including organic electronics, electrochemical supercapacitors, chemical sensors. This article reviews the approaches toward the preparation of electrogenerated CMPN thin films and their application in electrochemical and fluorescence quenching–based sensing of nitroaromatics, metal ions, etc.     

Biosensors: a viable monitoring technology?

Biosensors for practical in vivo and in vitro applications are dependent on the effective integration of several biological and physical technologies. This review paper was stimulated by an IEE seminar. Some of the more recent advances aimed at taking techniques of fundamental and academic interest to various forms of practical reagentless biochemical analysis are highlighted, with associated clinical and commercial consequences. The paper describes some of the most recent developments in biosensor research, in particular those relating to material aspects of fabrication, including multilayer films for sensor applications, advances in ISFETs, conjugated polymers, new developments in quartz crystal based biosensors, as well as advances in amperometric enzyme electrodes and the application of devices for continuous monitoring.     

An automated micro-solid phase extraction device involving integrated \high-pressure microvalves for genetic sample preparation

This paper presents an automated micro-SPE device for DNA extraction using monolithically integrated high-pressure microvalves. The automated micro-SPE device was fabricated through glass-to-glass thermal bonding and microfluidic system interface technologies. To increase the DNA extraction efficiency, silica beads were packed in the extraction microchannel involving two weir structures. Experimental results show that the DNA extraction efficiency using the automated micro-SPE device containing bare silica beads was 75.87% in the first 8 μl of solution eluted by automated SPE procedure. In addition, the reproducibility of the DNA extraction was evaluated by ten successive measurements. Genomic DNA extracted from human WBCs had an absorbance ratio of DNA to protein (A₂₆₀/A₂₈₀) of 1.56. The applicability of this automated micro-SPE device to genetic sample preparation was verified by PCR amplification of a β-globulin gene using the genomic DNA extracted from WBCs. Consequently, we demonstrated that the proposed automatic micro-SPE device can extract nucleic acids from biological samples, thereby facilitating its integration with downstream genetic analyses in a micro format.     

Compact soft x-ray transmission microscopy with sub-50 nm spatial resolution

In this paper, the development of compact transmission soft x-ray microscopy (XM) with sub-50 nm spatial resolution for biomedical applications is described. The compact transmission soft x-ray microscope operates at lambda = 2.88 nm (430 eV) and is based on a tabletop regenerative x-ray source in combination with a tandem ellipsoidal condenser mirror for sample illumination, an objective micro zone plate and a thinned back-illuminated charge coupled device to record an x-ray image. The new, compact x-ray microscope system requires the fabrication of proper x-ray optical devices in order to obtain high-quality images. For an application-oriented microscope, the alignment procedure is fully automated via computer control through a graphic user interface. In imaging studies using our compact XM system, a gold mesh image was obtained with 45 nm resolution at x580 magnification and 1 min exposure. Images of a biological sample (Coscinodiscus oculoides) were recorded.     

Are microfluidics-based blood viscometers ready for point-of-care applications? A review

In recent years, the engineering of blood viscometers for the diagnosis, prognosis, and prevention of cardiovascular and other diseases has been the subject of significant research interest. Conventional blood viscometers such as rotational viscometers and capillary viscometers typically rely on mechanical techniques in quantifying whole-blood viscosity, a process in which resistance to blood flow is measured in response to an applied force. The direct applicability of conventional viscometers as point-of-care diagnostic and clinical tools is subject to several limitations mainly related to their macro-structural features that augment the sampling size and reduce portability. The development of new fabrication technologies to scale down experimental processes has opened up the reality of miniaturizing existing concepts of blood viscometers into microchips, and paves the road for future development of blood viscometers. These micro-blood viscometers are advantageous because they use very small sample volumes for quick, routine clinical purposes. The easy fabrication of microsystems and large-scale production not only result in a lower cost, but also render these devices portable and disposable, both of which are highly desirable for clinical applications. The underlying challenges of these devices are associated with red blood cell clogging, measurement stability, reliability, and reproducibility. The present review discusses the state-of-the-art and emerging trends in the field of microfluidics to provide elegant solutions for quantifying blood viscosity with vastly improved efficacy and with the potential for use at the patient's bedside.     

Rapid Biocompatible Micro Device Fabrication by Micro Electro-Discharge Machining

Fabrication of a biocompatible micro device is predominantly done by silicon micromachining techniques. The lithographic and etching techniques require preparation and the use of masks which are time consuming and costly. Since bio research involves highly complex mechanisms, the modeling and simulation is difficult and experimental study is inevitable. To incorporate frequent design changes and to realize the hardware quickly, fabrication processes, complementary to the silicon micromachining techniques are required. In the present work the feasibility of using micro electro-discharge machining (EDM) for the fabrication of biocompatible microdevice has been studied. Micro channels with feature size as small as 25 microm are realized. The process is further improved by the introduction of ultrasonic vibration of the workpiece and the total time taken for the hardware realization is about 4 hours. The effects of ultrasonic vibration on the roughness of the spark eroded surface has been studied and reported. The potential of using micro EDM for making biocompatible devices for bio experiments is demonstrated and the surface finish achieved is well within the recommended Rz and Ra values of 3.4 and 0.4 microm respectively for biological studies like implant abutment.     

Microfluidics Section: Design and Fabrication of Integrated Passive Valves and Pumps for Flexible Polymer 3-Dimensional Microfluidic Systems

This paper describes the fabrication of flexible, polymeric 3-dimensional microfluidic systems with integrated check valves (flap and diaphragm valves) and a pump by stacking patterned poly(dimethylsiloxane) (PDMS) layers containing microchannels and vias. We describe this procedure for fabricating, manipulating, and bonding of PDMS membranes and bas-relief plates into multilayer microfluidic devices. The fabrication and demonstration of integrated check valves and a pump in a prototype polymer 3-dimensional microfluidic system is a step toward practical realization of all-polymer, flexible, low-cost, disposable microfluidic devices for biochemical applications.     

Microfluidic DNA microarrays in PMMA chips: streamlined fabrication <Emphasis Type="Italic">via</Emphasis> simultaneous DNA immobilization and bonding activation by brief UV exposure

This report presents and describes a simple and scalable method for producing functional DNA microarrays within enclosed polymeric, PMMA, microfluidic devices. Brief (30 s) exposure to UV simultaneously immobilized poly(T)poly(C)-tagged DNA probes to the surface of unmodified PMMA and activated the surface for bonding below the glass transition temperature of the bulk PMMA. Functionality and validation of the enclosed PMMA microarrays was demonstrated as 18 patients were correctly genotyped for all eight mutation sites in the HBB gene interrogated. The fabrication process therefore produced probes with desired hybridization properties and sufficient bonding between PMMA layers to allow construction of microfluidic devices. The streamlined fabrication method is suited to the production of low-cost microfluidic microarray-based diagnostic devices and, as such, is equally applicable to the development of diagnostics for both resource rich and resource limited settings.     

Autonomous microfluidic multi-channel chip for real-time PCR with integrated liquid handling

We report on a novel, polymer-based, multi-channel device for polymerase chain reaction that combines, for the first time, rapid sample processing in less than 5 min with high throughput at low costs. This is achieved by sample shuttling, during which submicroliter sample plugs (∼100 nl) are oscillated rapidly over three constant-temperature zones by pneumatic actuation with integrated system. The accuracy and the speed of the liquid handling have been significantly increased, while the design of the device can be kept very simple and allows for mass production using conventional low-cost polymer fabrication processes. Massive parallelization can lead to a throughput up to 100 samples in 10 min including the preparation time. The amplification can be optically monitored by means of online fluorescence detection. Successful real-time PCR and the determination of the threshold cycle, C _t, using the developed device were demonstrated with plasmid DNA in a fluorescent real-time format.     

Silica-on-silicon waveguide integrated polydimethylsiloxane lab-on-a-chip for quantum dot fluorescence bio-detection

Integration of microfluidics and optical components is an essential requirement for the realization of optical detection in lab-on-a-chip (LOC). In this work, a novel hybrid integration of silica-on-silicon (SOS) waveguide and polydymethylsiloxane (PDMS) microfluidics for realizing optical detection based biochip is demonstrated. SOS is a commonly used platform for integrated photonic circuits due to its lower absorption coefficient of silica and the availability of advanced microfabrication technologies for fabricating complicated optical components. However, the fabrication of complex microfluidics circuits on SOS is an expensive process. On the other hand, any complex 3D and high-aspect-ratio microstructures for the microfluidic applications can be easily patterned on PDMS using soft lithography. By exploring the advantages of these two materials, the proposed hybrid integration method greatly simplifies the fabrication of optical LOC. Two simple technologies--namely, diamond machining and soft lithography--were employed for the integration of an optical microfluidic system. Use of PDMS for the fabrication of any complex 3D microfluidics structures, together with the integration of low loss silica-on-silicon photonic waveguides with a straight microfluidic channel, opens up new possibilities to produce low-cost biochips. The performance of SOS-PDMS-integrated hybrid biochip is demonstrated with the detection of laser induced fluorescence of quantum dots. As quantum dots have immense application potential for biodetection, they are used for the demonstration of biodetection.     

Microchip module for blood sample preparation and nucleic acid amplification reactions

A computer numerical control-machined plexiglas-based microchip module was designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module is comprised of a custom-made heater-cooler for thermal cycling, a series of 254 microm x 254 microm microchannels for transporting human whole blood and reagents in and out of an 8--9 microL dual-purpose (cell isolation and PCR) glass-silicon microchip. White blood cells were first isolated from a small volume of human whole blood (<3 microL) in an integrated cell isolation--PCR microchip containing a series of 3.5-microm feature-sized "weir-type" filters, formed by an etched silicon dam spanning the flow chamber. A genomic target, a region in the human coagulation Factor V gene (226-bp), was subsequently directly amplified by microchip-based PCR on DNA released from white blood cells isolated on the filter section of the microchip mounted onto the microchip module. The microchip module provides a convenient means to simplify nucleic acid analyses by integrating two key steps in genetic testing procedures, cell isolation and PCR and promises to be adaptable for additional types of integrated assays.     

Fabrication of self-expandable NiTi thin film devices with micro-electrode array for bioelectric sensing,stimulation and ablation

Self-expandable medical devices provide mechanical functionality at a specific location of the human body and are viable for minimal invasive procedures. Besides radiopaque markers and drug-eluting coatings, next generation self-expandable devices can be equipped with additional functionality, such as conductive and flexible electrodes, which enables chronic recording of bioelectrical signals, stimulating or ablating tissue. This promises new therapeutic options in various medical fields, among them in particular neuromodulation (e.g. deep brain stimulation), BioMEMS, radio frequency ablation, mapping or denervation. However, the fabrication of such multi-functional devices is challenging. For this study we have realized a 35 μm thick, superelastic NiTi thin film stent structure with six isolated electrodes on the outer circumference, each electrode connected to a contact pad at the end of the stent structure, using magnetron sputtering, UV lithography and wet chemical etching. Mechanical and electrical properties of the device during typical loading conditions, i.e. crimping, simulated pulsatile and electrochemical testing, were characterized and reveal promising results. For the fabrication of future multifunctional, minimal invasive medical devices, such as electroceuticals or other intelligent implants, NiTi thin film technology is therefore a versatile alternative to conventional fabrication routes.     

A hybrid poly(dimethylsiloxane) microsystem for on-chip whole blood filtration optimized for steroid screening

Miniaturized biochemical devices in glass, silicon and polymer materials are starting to find their way from the academic laboratories to real-life applications. However, most attention has been given to miniaturize the downstream functions of various microfluidic systems, leaving the sample introduction and preparation steps to more conventional, bulkier solutions. For point-of-care diagnostics in particular, it becomes crucial to be able to handle complex human samples in a miniaturized format. In this work, we report on a microsystem for on-chip sample preparation that is able to remove blood cells from whole blood. The hybrid system consists of a commercially available membrane filter incorporated into a poly(dimethylsiloxane) (PDMS) casted device. Membrane materials were evaluated on the bases of low nonspecific adsorption of free and protein-bound testosterone as analyte substance. The hybrid system including a hydrophilic polypropylene filter successfully removed blood cells from diluted human whole blood. Surface oxidation was sufficient to make the plasma filtrate flow through the membrane filter and the channel system by capillary force alone and thus no external pumping source was needed.