A flexible polymer tube lab-chip integrated with microsensors for smart microcatheter

A flexible polymer tube lab-chip integrated with physical and biochemical sensor modules mounted on a flexible spiral structure for measuring physiological (temperature/flow rate) and metabolic data (glucose concentration) in a catheter application was designed, fabricated and characterized in this work. This new approach not only provides a unique way to assemble multiple sensors on both the inside and outside the flexible polymer tube using standard microfabrication methods while avoiding wiring and assembling problems associated with previous methods, but also maintains catheter inherent lumen potency for in situ drug delivery or insertion of medical tools. Three well-known sensors: temperature sensor (RTD), flow rate sensor (hot film anemometry) and glucose biosensor (amperometric sensor) have been successfully fabricated and fully integrated outside the spirally rolled polymer tube (ID = 500 μm, OD = 650 μm) of this demonstration device. The fabricated sensors showed good performances not only in a planar configuration but also in a spirally rolled configuration. This flexible micro tube lab-chip system provides a generic platform for developing patient-specific “smart” microcatheters that incorporate microsensors, microactuators, microfluidic devices and wireless signal communication modules that are tailored for the patients’ unique condition.     

Prospects of electrochemical immunosensors for early diagnosis of preeclampsia

Preeclampsia is a vascular multisystem disorder that accounts for varying degree of morbidity and mortality of mother and the fetus. This can be significantly averted if diagnosed at an early (18‐20 weeks) stage of gestation, as there is no known way to prevent preeclampsia. In spite of extensive work on biomarker discovery, the existing method for its detection is mostly based on colorimetric immunoassays whose sensitivity is ranging in nanomolar range. Further, it has also been observed that change in the expression of a single biomarker is not sufficient to diagnose this condition. So, for early diagnosis (by 18‐20 weeks), an immuno‐diagnostic platform with detection limits in picomolar range and beyond along with the ability to do simultaneous detection of multiple analyte would be of great importance. A nano‐immunosensors with an electrochemical readout system can be a potential alternative that promises for the ultrasensitive detection of analyte with high specificity as well as suitability for on‐site analysis. Coupling the lateral flow technology with immunosensors would make it feasible to detect more than one biomarker simultaneously on a microchip. This review intends to summarize the potential preeclampsia biomarkers, limitations of existing diagnostic methods along with the recent advancements, and prospects to develop electrochemical immunosensors for early clinical diagnosis.     

Electrochemical detection of high-sensitivity CRP inside a microfluidic device by numerical and experimental studies

The concentration of C-reactive protein (CRP), a classic acute phase plasma protein, increases rapidly in response to tissue infection or inflammation, especially in cases of cardiovascular disease and stroke. Thus, highly sensitive monitoring of the CRP concentration plays a pivotal role in detecting these diseases. Many researchers have studied methods for the detection of CRP concentrations such as optical, mechanical, and electrochemical techniques inside microfluidic devices. While significant progress has been made towards improving the resolution and sensitivity of detection, only a few studies have systematically analyzed the CRP concentration using both numerical and experimental approaches. Specifically, systematic analyses of the electrochemical detection of high-sensitivity CRP (hsCRP) using an enzyme-linked immunosorbant assay (ELISA) inside a microfluidic device have never been conducted. In this paper, we systematically analyzed the electrochemical detection of CRP modified through the attachment of an alkaline phosphatase (ALP-labeled CRP) using ELISA inside a chip. For this analysis, we developed a model based on antigen-antibody binding kinetics theory for the numerical quantification of the CRP concentration. We also experimentally measured the current value corresponding to the ALP-labeled CRP concentration inside the microfluidic chip. The measured value closely matched the calculated value obtained by numerical simulation using the developed model. Through this comparison, we validated the numerical simulation methods, and the calculated and measured values. Lastly, we examined the effects of various microfluidic parameters on electrochemical detection of the ALP-labeled CRP concentration using numerical simulations. The results of these simulations provide insight into the microfluidic electrochemical reactions used for protein detection. Furthermore, the results described in this study should be useful for the design and optimization of electrochemical immunoassay chips for the detection of target proteins.     

Conducting polymers for electrochemical DNA sensing

Conducting polymers (CPs) are a class of polymeric materials that have attracted considerable interest because of their unique electronic, chemical and biochemical properties, making them suitable for numerous applications such as energy storage, memory devices, chemical sensors, and in electrocatalysis. Conducting polymer-based electrochemical DNA sensors have shown applicability in a number of areas related to human health such as diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology due to their simplicity and high sensitivity. This review paper summarizes the advances in electrochemical DNA sensing based on conducting polymers as active substrates. The various conducting polymers used for DNA detection, along with different DNA immobilization and detection methodologies are presented. Current trends in this field and newly developed applications due to advances in nanotechnology are also discussed.     

A microfluidic immunomagnetic bead-based system for the rapid detection of influenza infections: from purified virus particles to clinical specimens

Seasonal and novel influenza infections have the potential to cause worldwide pandemics. In order to properly treat infected patients and to limit its spread, a rapid, accurate and automatic influenza diagnostic tool needs to be developed. This study therefore presents a new integrated microfluidic system for the rapid detection of influenza infections. It integrated a suction-type, pneumatic-driven microfluidic control module, a magnetic bead-based fluorescent immunoassay (FIA) and an end-point optical detection module. This new system can successfully distinguish between influenza A and B using a single chip test within 15 min automatically, which is faster than existing devices. By utilizing the micromixers to thoroughly wash out the sputum-like mucus, this microfluidic system could be used for the diagnosis of clinical specimens and reduced the required sample volume to 40 μL. Furthermore, the results of diagnostic assays from 86 patient specimens have demonstrated that this system has 84.8 % sensitivity and 75.0 % specificity. This developed system may provide a powerful platform for the fast screening of influenza infections.     

Opto-electronic DNA chip-based integrated card for clinical diagnostics

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip or lab-on-card systems. DNA chips, which provide multiparametric data, are privileged tools for genomic analysis. However, automation of molecular biology protocol and use of these DNA chips in fully integrated systems remains a great challenge. Simplicity of chip and/or card/instrument interfaces is amongst the most critical issues to be addressed. Indeed, current detection systems for DNA chip reading are often complex, expensive, bulky and even limited in terms of sensitivity or accuracy. Furthermore, for liquid handling in the lab-on-cards, many devices use complex and bulky systems, either to directly manipulate fluids, or to ensure pneumatic or mechanical control of integrated valves. All these drawbacks prevent or limit the use of DNA-chip-based integrated systems, for point-of-care testing or as a routine diagnostics tool. We present here a DNA-chip-based protocol integration on a plastic card for clinical diagnostics applications including: (1) an opto-electronic DNA-chip, (2) fluid handling using electrically activated embedded pyrotechnic microvalves with closing/opening functions. We demonstrate both fluidic and electric packaging of the optoelectronic DNA chip without major alteration of its electronical and biological functionalities, and fluid control using novel electrically activable pyrotechnic microvalves. Finally, we suggest a complete design of a card dedicated to automation of a complex biological protocol with a fully electrical fluid handling and DNA chip reading.     

A polymer microfluidic chip for quantitative detection of multiple water- and foodborne pathogens using real-time fluorogenic loop-mediated isothermal amplification

Inexpensive, portable, and easy-to-use devices for rapid detection of microbial pathogens are needed to ensure safety of water and food. In this study, a disposable polymer microfluidic chip for quantitative detection of multiple pathogens using isothermal nucleic acid amplification was developed. The chip contains an array of 15 interconnected reaction wells with dehydrated primers for loop-mediated isothermal amplification (LAMP), and requires only a single pipetting step for dispensing of sample. To improve robustness of loading and amplification, hydrophobic air vents and microvalves were monolithically integrated in the multi-layered structure of the chip using an inexpensive knife plotter. For quantification, LAMP was performed with a highly fluorescent DNA binding dye (SYTO-82) and the reactions monitored in real-time using a low-cost fluorescence imaging system previously developed by our group (Ahmad et al., Biomed. Microdevices 13(5), 929-937). Starting from genomic DNA mixtures, the chip was successfully evaluated for rapid analysis of multiple virulence and marker genes of Salmonella, Campylobacter jejuni, Shigella, and Vibrio cholerae, enabling detection and quantification of 10-100 genomes per μl in less than 20 min. It is anticipated that the microfluidic chip, along with the real-time imaging system, may be a key enabling technology for developing inexpensive and portable systems for on-site screening of multiple pathogens relevant to food and water safety.     

Integrated Microfluidics/Electrochemical Sensor System for Monitoring of Environmental Exposures to Lead and Chlorophenols

A microanalytical system based on a microfluidics/electrochemical detection scheme was developed. The microfluidic platform was fabricated based on a multi-layer lamination method. Fluidic microchannels were produced by sandwiching laser-machined adhesive-backed polyimide gaskets between layers of the device. Individual components, such as microfabricated piezoelectrically actuated pumps and a microelectrochemical cell were designed and fabricated into plug-in modules which can be readily plugged into (or unplugged from) the microfluidic platform. This allowed rapid change-out and repair of individual components by incorporating plug and play concepts now standard in PC's. The detection of lead and chlorophenols were performed with the microanalytical system to demonstrate the capabilities of this new technology for on-site environmental characterization and for real-time non-invasive biomonitoring of toxic chemical mixtures.     

Magnetophoretic-based microfluidic device for DNA Concentration

Nucleic acids serve as biomarkers of disease and it is highly desirable to develop approaches to extract small number of such genomic extracts from human bodily fluids. Magnetic particles-based nucleic acid extraction is widely used for concentration of small amount of samples and is followed by DNA amplification in specific assays. However, approaches to integrate such magnetic particles based capture with micro and nanofluidic based assays are still lacking. In this report, we demonstrate a magnetophoretic-based approach for target-specific DNA extraction and concentration within a microfluidic device. This device features a large chamber for reducing flow velocity and an array of μ-magnets for enhancing magnetic flux density. With this strategy, the device is able to collect up to 95 % of the magnetic particles from the fluidic flow and to concentrate these magnetic particles in a collection region. Then an enzymatic reaction is used to detach the DNA from the magnetic particles within the microfluidic device, making the DNA available for subsequent analysis. Concentrations of over 1000-fold for 90 bp dsDNA molecules is demonstrated. This strategy can bridge the gap between detection of low concentration analytes from clinical samples and a range of micro and nanofluidic sensors and devices including nanopores, nano-cantilevers, and nanowires.     

A multilevel Lab on chip platform for DNA analysis

Lab-on-chips (LOCs) are critical systems that have been introduced to speed up and reduce the cost of traditional, laborious and extensive analyses in biological and biomedical fields. These ambitious and challenging issues ask for multi-disciplinary competences that range from engineering to biology. Starting from the aim to integrate microarray technology and microfluidic devices, a complex multilevel analysis platform has been designed, fabricated and tested (All rights reserved—IT Patent number TO2009A000915). This LOC successfully manages to interface microfluidic channels with standard DNA microarray glass slides, in order to implement a complete biological protocol. Typical Micro Electro Mechanical Systems (MEMS) materials and process technologies were employed. A silicon/glass microfluidic chip and a Polydimethylsiloxane (PDMS) reaction chamber were fabricated and interfaced with a standard microarray glass slide. In order to have a high disposable system all micro-elements were passive and an external apparatus provided fluidic driving and thermal control. The major microfluidic and handling problems were investigated and innovative solutions were found. Finally, an entirely automated DNA hybridization protocol was successfully tested with a significant reduction in analysis time and reagent consumption with respect to a conventional protocol.     

Design and integration of a generic disposable array-compatible sensor housing into an integrated disposable indirect microfluidic flow injection analysis system

We describe an integration strategy for arbitrary sensors intended to be used as biosensors in biomedical or bioanalytical applications. For such devices ease of handling (by a potential end user) as well as strict disposable usage are of importance. Firstly we describe a generic array compatible polymer sensor housing with an effective sample volume of 1.55 μl. This housing leaves the sensitive surface of the sensor accessible for the application of biosensing layers even after the embedding. In a second step we show how this sensor housing can be used in combination with a passive disposable microfluidic chip to set up arbitrary 8-fold sensor arrays and how such a system can be complemented with an indirect microfluidic flow injection analysis (FIA) system. This system is designed in a way that it strictly separates between disposable and reusable components- by introducing tetradecane as an intermediate liquid. This results in a sensor system compatible with the demands of most biomedical applications. Comparative measurements between a classical macroscopic FIA system and this integrated indirect microfluidic system are presented. We use a surface acoustic wave (SAW) sensor as an exemplary detector in this work.     

Bead-based microfluidic immunoassay for diagnosis of Johne's disease

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne's disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types of SAB, and types of magnetic beads) were optimized for a large degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD-negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested using commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads using laser-induced fluorescence. Antigen-coated magnetic beads treated with the bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples with the microfluidic system were compared to those analyzed with the flow cytometer, a high level of correlation (linear regression, r(2)=0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD.     

An integrated sensing system for detection of DNA using new parallel-motif DNA triplex system and graphene--mesoporous silica--gold nanoparticle hybrids

In this article, we demonstrate the use of graphene--mesoporous silica--gold NP hybrids (GSGHs) as an enhanced element of the integrated sensing platform for the ultra-sensitive and selective detection of DNA by using strand-displacement DNA polymerization and parallel-motif DNA triplex system as dual amplifications. We find that the present new sensing strategy based on GSGHs is able to detect target DNA with a fairly high detection sensitivity of 10 fm through the hybridization of duplex DNA to the acceptor DNA for the formation of parallel-motif DNA triplex on the multilayer film (containing GSGHs and redox probe) modified functional interface, and even has a good capability to investigate the single nucleotide polymorphisms (SNPs). The detection limit for target DNA is about two orders of magnitude lower than that of graphene-based DNA electrochemical impedance spectroscopy (EIS) sensor (6.6 pm), four orders of magnitude lower than those of graphene-based DNA sensors coupled with fluorescent assay (100 pm and 1 nm) and five orders of magnitude lower than those of field effect transistor (FET)-based assays (1 nm and 2 nm). Most importantly, our present sensing system can also be facilely achieved in the ITO electrode array, which is of paramount importance for possible multiplex analysis in lab-on-chip.     

A SU-8/PDMS Hybrid Microfluidic Device with Integrated Optical Fibers for Online Monitoring of Lactate

A microfluidic device with integrated optical fibres was developed for online monitoring of lactate. The device consists of a SU-8 waveguide, microfluidic channels and grooves for the insertion of optic fibres. It was fabricated by one-step photolithography of SU-8 polymer resist. Different channel widths (50–300 μm) were tested in terms of detection sensitivity. A wide range of flow rates were applied to investigate the influence of flow rate on signal fluctuations. The separation between optical fibre sensor and microfluidic channel and the width of fluidic channel have been optimized to maximize the detection sensitivity. It was revealed that 250 μm of channel width is the optimum light path length for a compromise between detection sensitivity and interference of ambient light. The independence of detection signals on flow rates was demonstrated within the range of flow rate (0.5–5 ml/hr) tested. Compared with conventional lactate detection, the device is proved to have high accuracy, relatively low limit of detection (50 mg/L) and reasonably fast response time (100 sec). The fabrication of device is simple and low cost. The present work has provided some fundamental data for further system optimization to meet specific detection requirements.     

A micro circulating PCR chip using a suction-type membrane for fluidic transport

A new micromachined circulating polymerase chain reaction (PCR) chip is reported in this study. A novel liquid transportation mechanism utilizing a suction-type membrane and three microvalves were used to create a new microfluidic control module to rapidly transport the DNA samples and PCR reagents around three bio-reactors operating at three different temperatures. When operating at a membrane actuation frequency of 14.29 Hz and a pressure of 5 psi, the sample flow rate in the microfluidic control module can be as high as 18 μL/s. In addition, an array-type microheater was adopted to improve the temperature uniformity in the reaction chambers. Open-type reaction chambers were designed to facilitate temperature calibration. Experimental data from infrared images showed that the percentage of area inside the reaction chamber with a thermal variation of less than 1°C was over 90% for a denaturing temperature of 94°C. Three array-type heaters and temperature sensors were integrated into this new circulating PCR chip to modulate three specific operating temperatures for the denaturing, annealing, and extension steps of a PCR process. With this approach, the cycle numbers and reaction times of the three separate reaction steps can be individually adjusted. To verify the performance of this circulating PCR chip, a PCR process to amplify a detection gene (150 base pairs) associated with the hepatitis C virus was performed. Experimental results showed that DNA samples with concentrations ranging from 10⁵ to 10²copies/μL can be successfully amplified. Therefore, this new circulating PCR chip may provide a useful platform for genetic identification and molecular diagnosis.     

Determination of single nucleotide polymorphisms in N-acetyltransferase2 gene using an electrochemical DNA chip and an automated DNA detection system

An electrochemical DNA chip using an electrochemically active intercalator and DNA probe immobilized on a gold electrode has been developed for genetic analysis. In this study, N-acetyltransferase2 (NAT2) gene polymorphisms (C481T G590A G857A) were determined by the electrochemical DNA chip and the automated DNA detection system that performs hybridization reaction, washing, detection, and data analysis. Human genomic DNAs were extracted from blood and DNA fragments containing the three polymorphisms were amplified by the polymerase chain reaction (PCR) method. Double-stranded PCR products were treated with T7 exonuclease and single-stranded target DNAs were obtained. A sample containing the single-stranded target DNAs was injected into a cassette including the electrochemical DNA chip and set in an automated system. The turnaround time for genotyping with this system was 90 min. A total of 38 samples were automatically genotyped by an SNP determination algorithm. The results of genotype were completely consistent with those determined by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Consequently, this method requires no labeling step and has the advantage of realizing a compact and automatic system, and so the system is expected to contribute to personalized medicine based on genotype.     

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.     

A fully microfabricated carbon nanotube three-electrode system on glass substrate for miniaturized electrochemical biosensors

We present an integration process to fabricate single-walled carbon nanotube (SWCNT) three-electrode systems on glass substrate for electrochemical biosensors. Key issues involve optimization of the SWCNT working electrode to achieve high sensitivity, developing an optimal Ag/AgCl reference electrode with good stability, and process development to integrate these electrodes. Multiple spray coatings of the SWCNT film on glass substrate enabled easier integration of the SWCNT film into an electrochemical three-electrode system. O? plasma etching and subsequent activation of spray-coated SWCNT films were needed to pattern and functionalize the SWCNT working electrode films without serious damage to the SWCNTs, and to remove organic residues. The microfabricated three-electrode systems were characterized by microscopic and spectroscopic techniques, and the electrochemical properties were investigated using cyclic voltammetry and chrono-amperometry. The fully-integrated CNT three-electrode system showed an effective working electrode area about three times larger than its geometric surface area and an improved electrochemical activity for hydrogen peroxide decomposition. Finally, the effectiveness of miniaturized pf-SWCNT electrodes as biointerfaces was examined by applying them to immunosensors to detect Legionella(L) pneumophila, based on a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with 3,3',5,5'-tetramethylbenzidine dihydrochloride/hydrogen peroxide(TMB/H?O?) as the substrate/mediator system. The lower detection limit of the pf-SWCNT-based immunosensors to L. pneumophila is about 1500 times lower than that of the standard ELISA assay.     

Integrated microfluidic system for electrochemical sensing of urinary proteins

This study reports a new microfluidic system integrated with a microfluidic control module and a micro electrochemical module for detection of urinary proteins. The integrated microsystem can automatically detect proteins in urine with a high sensitivity. The microfluidic control module consists of a new two-way, spiral-shape micropump which can transport the urine samples to the sensing regions. The net ionic charges of the protein samples can be detected while the samples flow through the sensing region of the micro electrochemical module. Two major urinary proteins including lysozyme and albumin are detected in a multiple-channel layout with little human intervention and are analyzed in a short period of time, while only consuming a 100-μl urine sample. The developed microfluidic system could lead to a convenient, yet crucial, platform for chemical and biological detection and diagnosis. Preliminary results of the current paper had been presented at the 1st Annual IEEE International Conference on Nano/Molecular Medicine and Engineering, August 6–9, 2007.