An integrated microfluidic system for live bacteria detection from human joint fluid samples by using ethidium monoazide and loop-mediated isothermal amplification

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.     

A novel integrated microfluidic platform based on micro-magnetic sensor for magnetic bead manipulation and detection

We present a novel integrated microfluidic platform based on micro-magnetic sensor for manipulating and detecting magnetic beads (MB). A micro-spiral planar coil in MB manipulating system microfabricated by micro-electro-mechanical system technology is implemented to manipulate MB, and a giant magnetoimpedance (GMI) based micro-magnetic sensor is employed to detect the trapped MB. In our work, MB can be efficiently trapped by trapping force generated from micro-coil in microchannel. Next, trapped MB are detected by the changing ratio of impedance, as well as the variation of resistance and reactance in GMI sensor for trapped MB induce weak stray magnetic field under the magnetization by external magnetic field. The maximum difference of GMI ratio between with beads condition and without beads condition is 4.0% at the optimum driving frequency of 20 MHz under the external magnetic field of 15 Oe, and resistance ratio varies more significantly than reactance ratio. In comparison with traditional MB detecting methods by GMI sensor, the integrated microfluidic platform based on GMI sensor can not only manipulate and detect MB signal sensitively, but also enhance detection efficiency and decrease the experiment errors. Furthermore, this platform avoids contamination from the solutions in chemically reactive layers and reduces assay time in future biomarker detection. In our work, the microfluidic platform based on GMI sensor has potential applications in biomarker detection via MB manipulation and detection.     

An integrated microfluidic system for fast, automatic detection of C-reactive protein

C-reactive protein (CRP) is a well-known inflammation marker in human beings. This study reports a new microfluidic system for fast, automatic detection of CRP. It contains pneumatic micropumps, a vortex-type micromixer, a pneumatic micro-injector and several microvalves to automatically perform the entire protocol for CRP detection. This includes sample/reagent transportation, incubation between the target CRP and a CRP-specific aptamer, washing processes, and the chemiluminescence development process. In addition, the chemiluminescence signal is measured by using a custom-made optical system which consists of a photomultiplier tube, a portable air compressor and eight electronic magnet valves to quantify the concentration of CRP. When compared to previous works, not only can this new microfluidic system automatically perform the entire process via a new integrated micro-injector and new micropumps, but a new CRP-specific DNA aptamer with a higher affinity and specificity is also used for CRP measurement. Experimental data show that the developed system can automatically complete the entire protocol within 30 min with a detection limit of 0.0125 mg/L, which is superior to previous published results. Moreover, this study also measures CRP concentration from clinical samples to verify the performance of the developed microfluidic system. The results indicate that the measured CRP concentrations from human serums are consistent with those using a benchtop system. The developed system can also detect CRP concentrations from human whole blood without any external sample pretreatment process. This microfluidic system may be promising for point-of-care applications for CRP detection in the future.     

A microfluidic platform for electrical detection of DNA hybridization

Current methods used for detection of DNA hybridization involve the use of DNA microarrays which require overnight incubation times along with bulky and expensive fluorescent scanners. Here, we demonstrate electrical detection of DNA hybridization in an oligonucleotide functionalized microfluidic channel. We use microchannels functionalized with DNA probes integrated with electrodes for measuring conductance across the channel. As beads conjugated with the target DNA passing through the channel are captured on the surface, we are able to electrically detect changes in resistance due to bead capture. Our assay can be completed in less than an hour using less than a microliter of reagent, and has the potential for extensive multiplexing. Such a device can be useful as a handheld platform in a clinical setting where one would need to rapidly genotype a small number of genes rapidly.     

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.     

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD_15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.     

A microfluidic platform with integrated flow sensing for focal chemical stimulation of cells and tissue

A microfluidic platform for precise biochemical control of the extracellular microenvironment was developed. A chemical interface was established with cells or tissues through the precise and focal delivery of soluble chemical agents through a pore addressed by a polymer microchannel. Thermal flow sensors were integrated along the length of the microchannel and monitored internal flow rate. Sensor performance was characterized in anticipation of future studies with real-time feedback control of focal delivery. The microfluidic system was characterized by determining the fluid delivery rates through the pores and concentration profiles of agents delivered. Finally, focal delivery to rat retinal tissue was demonstrated.     

A droplet-based microfluidic platform for rapid immobilization of quantum dots on individual magnetic microbeads

Quantum dots (QDs) provide opportunities for the development of bioassays, biosensors, and drug delivery strategies. Decoration of the surface of QDs offers unique functions such as resistance to non-specific adsorption, selective binding to target molecules, and cellular uptake. The quality of decoration has substantial impact on the functionality of modified QDs. Single-phase microfluidic devices have been demonstrated for decorating QDs with biological molecules. The device substrate can serve as a solid-phase reaction platform, with a limitation being difficulty in the realization of reproducible decoration at high density of coverage of QDs. Magnetic beads (MBs) have been explored as an alternative form of solid-phase reaction platform for decorating QDs. As one example, controlled decoration to achieve unusually high density can be realized by first coating MBs with QDs, followed by the addition of molecules such as DNA oligonucleotides. Uniformity and high density of coatings on QDs have been obtained using MBs for solid-phase reactions in bulk solution, with the further advantage that the MBs offer simplification of procedural steps such as purification. This study explores the use of a droplet microfluidic platform to achieve solid-phase decoration of MBs with QDs, offering control of local reaction conditions beyond that available in bulk solution reactions. A microchannel network with a two-junction in-series configuration was designed and optimized to co-encapsulate one single 1 µm MB and many QDs into individual droplets. The microdroplet became the reaction vessel, and enhanced conjugation through the confined environment and fast mixing. A high density of QDs was coated onto the surface of single MB even when using a low concentration of QDs. This approach quickly produced decorated MBs, and significantly reduced QD waste, ameliorating the need to remove excess QDs. The methodology offers a degree of precision to control conjugation processes that cannot be attained in bulk synthesis methods. The proposed droplet microfluidic design can be widely adopted for nanomaterial synthesis using solid-phase assays.     

A novel integrated microfluidic platform to perform fluorescence in situ hybridization for chromosomal analysis

The fluorescence in situ hybridization (FISH) technique has been commonly employed to detect the chromosomal abnormalities. However, applications of this technique are limited due to its lengthy process and labor-intensive sample preparation. In this study, a novel integrated microfluidic chip capable of performing the entire FISH protocol automatically was reported. This novel technique can achieve several advantages, including reduce the consumption of bio-samples and reagents, automation and rapid analysis compared to the conventional method. In this study, several functional microfluidic devices were integrated on a single chip to perform automatic FISH on the microfluidic platform. Experimental data demonstrated that the developed microfluidic system successfully provided superior performance for probing the chromosomal abnormality of cells. Furthermore, the novel microfluidic system performed the entire process automatically within 3 h, where the conventional method required 10 h to perform the entire protocol manually. This data indicated superior performance of the novel method. Our findings conclude that the novel integrated FISH protocol is more convenient to perform large quantities of samples, which can be used in clinical trials.     

A paper-based microfluidic Dot-ELISA system with smartphone for the detection of influenza A

An automated, portable, and integrated paper-based microfluidic system has been developed for influenza A detection with smartphone at point-of-care (POC) settings. The low-cost paper-based microfluidic chip consists of a reagent storage and reaction modules. The storage module, which consists of a couple of reagent chambers with dispensation channels, is responsible for reagent storage and release. The reaction module consists of an absorbent pad and a nitrocellulose (NC) membrane which is functionalized with specific monoclonal antibodies on a test and control spots for immunoassay detection. Microfluidic Dot-ELISA is performed when the dispensed reagent flows through the NC membrane at a controllable speed and reaches the absorbent pad because of the gravity and capillary force without active pumping. A smartphone is used to capture image from the NC membrane with its own camera and process the image with an intelligent algorithm of custom application software which is developed with Java. With a smartphone, the detection result can be displayed and transmitted to other medical agencies if necessary. Experimental results show that, compared with the traditional methods, more convenient and efficient influenza A detection can be achieved with the developed paper-based POC microfluidic chip with the assistance of smartphone.     

An integrated silicon sensor with microfluidic chip for monitoring potassium and pH

We present ion-sensitive field effect transistor-based sensors, integrated with a microfluidic chip, for monitoring pH and potassium cations. The sensor is strategically located at the base of a well so that the response time of the device depends both on the mean flow through the device and the diffusion coefficient of the analyte being monitored. This would enable monitoring of ions in the presence of larger molecules. The dependence of the device response time on diffusive transport of analytes was examined through a numerical study of the flow field and the passive diffusion of a chemical species. The predicted device response time was compared with the experimental measurements and reasonable agreement found. The general dependence of device response time on geometry, flow rate, and analyte diffusion coefficient was derived. These devices can be used with biological fluids where monitoring of pH and cations provide vital information about the well-being of patients.     

Development of a biological detection platform utilizing a modular microfluidic stack

The goal of this project is to build a miniaturized, user-friendly cytometry setup (Datta et al. in Microfluidic platform for education and research. COMS, Baton Rouge, 2008; Frische et al. in Development of an miniaturized flow cytometry setup for visual cell inspection and sorting. Baton Rouge, Project Report, 2008) by combining a customized, microfluidic device with visual microscope inspection to detect and extract specific cells from a continuous sample flow. We developed a cytological tool, based on the Coulter particle counter principle, using a microelectrode array patterned on a borosilicate glass chip as electrical detection set-up which is fully embedded into a polymeric multi-layer microfluidic stack. The detection takes place between pairs of coplanar Cr/Au microelectrodes by sensing an impedance change caused by particles continuously carried within a microfluidic channel across the detection area under laminar flow conditions. A wide frequency range available for counting provides information on cell size, membrane capacitance, cytoplasm conductivity and is potentially of interest for in-depth cell diagnostic e.g. to detect damaged or cancerous cells and select them for extraction and further in-depth analysis.     

An integrated data exchange platform for Intelligent Transportation Systems

Intelligent Transportation Systems (ITSs) make use of advanced detection, communications, and computing technology to improve the safety and efficiency of surface transportation networks. An ITS incorporates a variety of equipment and devices all working in mutual harmony. However, each piece of equipment or device has its own data format and protocol so they cannot exchange data with each other directly. In this paper, a platform of data exchange in an ITS is proposed that can receive data from several types of equipment external to automobiles, repackage the received data, and then dispatch the data to different devices inside the vehicles.     

New production method of convex microlens arrays for integrated fluorescence microfluidic detection systems

A new method for producing microlens array with large sag heights is proposed for integrated fluorescence microfluidic detection systems. Three steps in this production technique are included for concave microlens array formations to be integrated into microfluidic systems. First, using the photoresist SU-8 to produce hexagonal microchannel array is required. Second, UV curable glue is injected into the hexagonal microchannel array. Third, the surplus glue is rotated by a spinner at high velocity and exposed to a UV lamp to harden the glue. The micro concave lens molds are then finished and ready to produce convex microlens in poly methsiloxane (PDMS) material. This convex microlens in PDMS can be used for detecting fluorescence in microfluidic channels because a convex microlens plays the light convergence role for optical fiber detection.     

Thread-based microfluidic system for detection of rapid blood urea nitrogen in whole blood

This study develops a thread-based microfluidic device with variable volume injection capability and 3-dimensional (3D) detection electrodes for capillary electrophoresis electrochemical (CE–EC) detection of blood urea nitrogen (BUN) in whole blood. A poly methyl methacrylate (PMMA) substrate with concave 3D electrodes produced by the hot embossing method is used to enhance the sensing performance of the CE–EC system. Results show that the chip with 3D sensing electrodes exhibits a measured current response nine times higher and signal-to-noise ratio five times higher when compared to the peak responses obtained using a chip with conventional 2D sensing electrodes. In addition, the developed thread-based microfluidic system is capable of injecting variable sample volumes into the separation thread simply by wrapping the injection thread different numbers of times around the separation thread. The peak S/N ratio can be further enhanced with this simple approach. Results also indicate that the CE–EC system exhibits good linear dynamic range for detecting a urea sample in concentrations from 0.1 to 10.0 mM (R ² = 0.9848), which is suitable for adoption in detecting the BUN concentration in human blood (1.78–7.12 mM). Separation and detection of the ammonia ions converted from BUN in whole blood is successfully demonstrated in the present study, with the developed thread-based microfluidic system providing a low-cost, high-performance method for detecting BUN in human blood.     

An automatic microfluidic system for rapid screening of cancer stem-like cell-specific aptamers

This study reports a microfluidic system which automatically performs the systematic evolution of ligands by exponential enrichment (SELEX) process for rapid screening of aptamers which are specific to cancer stem-like cells. The system utilizes magnetic bead-based techniques to select DNA aptamers and has several advantages including a rapid, automated screening process, and less consumption of cells and reagents. By integrating a microfluidic control module, a magnetic bead-based aptamer extraction module, and a temperature control module, the entire Cell-SELEX process can be performed in a shorter period of time. Compared with the traditional Cell-SELEX process, this microfluidic system is more efficient and consumes fewer sample volumes. It only takes approximately 3 days for an entire Cell-SELEX process with 15 screening runs, which is relatively faster than that of a traditional Cell-SELEX process (1 week for 15 rounds). The binding affinity of this resulting specific aptamer was measured by a flow cytometric analysis to have a dissociation constant (K _d) of 15.32 nM. The capture rate for cancer stem-like cells using the specific aptamer-conjugated bead is better than that using Ber-EP4 antibody-conjugated bead. This microfluidic system may provide a powerful platform for the rapid screening of cell-specific aptamers.     

A microfluidic platform for formation of double-emulsion droplets

A new method for actively controlling the number of internal droplets of water-in-oil-in-water (W/O/W) double-emulsion droplets was demonstrated. A new microfluidic platform for double-emulsion applications has been developed, which integrates T-junction channels, moving-wall structures, and a flow-focusing structure. Inner water-in-oil (W/O) single-emulsion droplets were first formed at a major T-junction. Then the droplets were sub-divided into smaller uniform droplets by passing through a series of secondary T-junctions (branches). The moving-wall structures beside the secondary T-junctions were used to control the number of the sub-divided droplets by selectively blocking the branches. Finally, double-emulsion droplets were formed by using a flow-focusing structure downstream. Experimental data demonstrate that the inner and outer droplets have narrow size distributions with coefficient of variation (CV) of less than 3.5% and 5.7%, respectively. Double-emulsion droplets with 1, 2, 3, and up to 10 inner droplets have been successfully formed using this approach. The size of the inner droplets and outer droplets could be also fine-tuned with this device. The development of this new platform was promising for drug delivery applications involving double emulsions.     

Replication and bonding techniques for integrated microfluidic systems

From the technical and economic points of view, systems integration, and packaging represent a crucial step in the production of microsystems. Compared to purely silicon- or glass-based systems, the variety of materials and geometries available for purely polymer microfluidic systems is much larger, due to the outstanding material properties. Moreover, polymers may be shaped and joined by comparably simple methods. Examples are polymer microreplication as well as various bonding methods. With them, complete polymer microsystems can be integrated. In addition, a number of established, compatible processes are available for the integration of functional elements that may also be made of other materials.