Screening of peptide specific to cholangiocarcinoma cancer cells using an integrated microfluidic system and phage display technology

Cholangiocarcinoma (CCA) is a cancer of the bile duct with high mortality rate and poor prognosis, owing to the difficulty in the early diagnosis and prognosis. The specific biomarkers or affinity reagents toward CCA cells could be great tools to assist in detection of CCA. However, screening of biomarkers/affinity reagents are generally labor-intensive, time-consuming and requiring large volume of samples and reagents. Therefore, we developed an integrated microfluidic system which could automatically perform selections of biomarkers and affinity reagents using phage display techniques. The experimental results showed that the selection of phage-displayed peptides bound to CCA cells was successfully demonstrated on the integrated microfluidic system using fewer reagents, samples and less time (5.25 h per biopanning round, and continuously performed for only 4 panning rounds). Three oligopeptides were screened, and their specificity and affinity toward CCA cells were characterized. Furthermore, comparing to conventional EpiEnrich beads for cancer cell capture, the screened CCA-specific peptides showed relatively low capture rate over control normal cells. It is envisioned that this microfluidic system may be a powerful tool for screening of biomarkers/affinity reagents in clinical diagnosis and target therapy for CCA.     

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.     

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.     

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.     

An integrated system for quantitation of chemotaxis using a 48-well millipore filter assay.

We have established a computerized system for quantification of human neutrophil motility using a 48-well chemotaxis assay. The software is primarily written in the Unix C-shell and is designed to integrate with standard statistical and graphics packages and permit analysis either under Unix or MS-DOS. We demonstrate how simple image analysis techniques may be used to count neutrophils that have traversed a polycarbonate filter. Methods of optical optimization, cell counting and integration with the Statistical Analysis System (SAS) are presented.     

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 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.     

A bead-based microfluidic system for joint detection in TORCH screening at point-of-care testing

Microsystem Technologies - A concise bead-based microfluidic system has been developed for joint detection in TORCH screening at point-of-care testing. Assisted by five functionalized polycarbonate...     

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 tissue P system and a DNA microfluidic device for solving the shortest common superstring problem

This paper describes a tissue P system for solving the Shortest Common Superstring Problem in linear time. This tissue P system is well suited for parallel and distributed implementation using a microfluidic device working with DNA strands. The approach is not based on the usual brute force generate/test technique applied in DNA computing, but it builds the space solution gradually. The possible solutions/superstrings are build step by step through the parallel distributed combination of strings using the overlapping concatenation operation. Moreover, the DNA microfluidic device solves the problem autonomously, without the need of external control or manipulation.An erratum to this article can be found at     

A simple method of constructing microfluidic solid-state quantum dot molecular beacon array for label-free DNA detection

Solid-state molecular beacons show great potential for label-free biomarker detection, however, it is challenging to construct robust and homogenous quantum dot molecular beacon microarrays in a microfluidic platform. Here, we report a simple and cost-effective method of constructing a mercaptopropionic acid quantum dot (MPA-QD) microarray in a microfluidic platform using a PDMS through-hole structure. This method combines soft lithography and surface functionalization to achieve uniform QD immobilization in a microfluidic network. With the simple fabricated quantum dot microarray sensor integrated in the microfluidic device, label-free biomarker detection was performed with high efficiency, specificity and sensitivity. We performed cardiovascular biomarker detection, and our microfluidic QD molecular beacon platform achieved target DNA sequence identification at a low concentration of 1 nM/L.     

Expert system for an INS/DGPS integrated navigator installed in a Bell 206 helicopter

This paper presents a novel real-time application of fuzzy logic for an integrated navigation avionics suite. A knowledge-based system, which uses a fuzzy rule-base for a real-time INS/DGPS integrated navigation system on-board a Bell 206 helicopter, has been designed and developed. This knowledge base is developed in such a way as to detect aircraft maneuvering and tune the integration algorithm (Kalman filter) of the INS/DGPS system accordingly. The signal processing method developed for the integration of INS and DGPS data provides accurate navigation even during dynamic maneuvering of the aircraft, while taking advantage of low-cost modular equipment rather than costly inertial navigation systems.     

A knowledge-based system for information management in an automated and integrated manufacturing system

In recent years there has been considerable interest in flexible automation of manufacturing processes in an effort to improve the productivity of manufacturing industry. Central to an integrated, flexible and intelligent manufacturing system is the information management function. The sharing of data is the basis of flexible and intelligent decision-making, the means of integrating the system components, the mode of synchronizing their processes and the method of maintaining the consistency of their states.

It appears that the data management and manipulation function in manufacturing systems is increasingly becoming a bottleneck for further performance improvement of manufacturing systems. New information management systems need to be developed that meet the needs of modern manufacturing systems in terms of efficiency, flexibility, reliability and intelligence. A new model for an integrated manufacturing information system is presented. A double structure of two parallel but independent system is proposed for manufacturing control and manufacturing information management. The objective is to improve service efficiency. representational adequacy, consistency of information and reliability in the information system.     

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.     

A microfluidic system for the study of the response of endothelial cells under pressure

Hydrostatic pressure can affect the structure and function of endothelial cells (ECs). A microfluidic system was built to study how ECs respond to applied pressure. The system included a syringe pump, a PDMS-glass microfluidic chip, and a digital manometer for pressure monitoring. The manometer was connected with the chip in two ways (one was before the inlet and the other after the outlet of the microchannel). The static control and flowing control systems were also set up. Human umbilical vein endothelial cells (HUVECs) were cultured in the 4 cm × 2 mm × 100 μm channel. Pressure of 12 ± 0.5 or 18 ± 0.5 kPa was applied on the cells for 8 h. The F-actin cytoskeleton and the nuclei of the cells were stained for examination and endothelin-1 (ET-1) released from the cells in the channel was assayed by ELISA. The results showed that the cell area and ET-1 concentration increased with the pressure and a higher pressure caused more damages to the cells. This microfluidic system provides a convenient and cost-effective platform for the studies of cell response to pressure.     

Structure overview of vegetation detection. A novel approach for efficient vegetation detection using an active lighting system

Fully autonomous navigation has been widely investigated for several decade of years; however, a safe and reliable navigation is still a daunting challenge in terrains containing vegetation. To improve the mobility capability of recent autonomous navigation systems, an additional vegetation detection function has been proposed. Since many proposals of generating vegetation classifier as well as suggestions of using different sensors to implement the function exist, a structured overview is required for vegetation detection in the context of outdoor navigation. Therefore, this paper studies and compares the accuracy and efficiency of existing vegetation detection approaches in a structured way. Furthermore, a new vision system set-up which combines CMOS sensor and Photo Mixer Device sensor with a near-infrared lighting system is also introduced to simultaneously provide depth, near-infrared and color images at high frame rate. Those near-infrared and color information are then used to compute vegetation index or train vegetation classifier to completely realize a real-time robust vegetation detection system. In this paper, a modification of the normalized difference vegetation index is devised, which is then defined as the new standard form of vegetation index for such vision system integrated with an additional lighting system. Finally, we will show the out-performance of the proposed approach in comparison with more conventional ones.     

Searching for optimal frame patterns in an integrated TDMAcommunication system using mean field annealing

In an integrated time-division multiple access (TDMA) communication system, voice and data are multiplexed in time to share a common transmission link in a frame format in which time is divided into slots. A certain number of time slots in a frame are allocated to voice and the rest are used to transmit data. Maximum data throughput can be achieved by searching for the optimal configuration(s) of relative positions of voice and data transmissions in a frame (frame pattern). When the problem size becomes large, the computational complexity in searching for the optimal patterns becomes intractable. In the paper, mean field annealing (MFA), which provides near-optimal solutions with reasonable complexity, is proposed to solve this problem. The determination of the related parameters are addressed. Comparison with the random search and simulated annealing algorithm is made in terms of solution optimality and computational complexity. Simulation results show that the MFA approach exhibits a good tradeoff between performance and computational complexity.