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2.
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. 相似文献
3.
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. 相似文献
4.
This article reports a new miniature electrochemical detection system integrating a sample pretreatment device for fast detection
of glycosylated hemoglobin (HbA 1C), which is a common indicator for diabetes mellitus. In this system, circular micropumps, normally closed microvalves, dielectrophoretic
(DEP) electrodes, and electrochemical sensing electrode are integrated to perform several crucial processes. These processes
include separation of red blood cells (RBCs), sample/reagent transportation, mixing, cell lysis, and electrochemical sensing.
For the HbA 1C measurement, the RBCs are separated and are collected from whole human blood by using a positive DEP force generated by the
DEP electrodes. The collected RBCs are then lysed to release HbA 1C for the subsequent electrochemical detection processes. Experimental data show that the RBCs are successfully separated and
are collected using the developed system with a RBCs capture rate of 84.2%. The subsequent detection of HbA 1C is automatically completed by utilizing electrochemical sensing electrode. The microfluidic system only consumes a sample
volume of 200 μl. The entire process is automatically performed within a short period of time (10 min). The development of
this integrated microfluidic system may be promising for the clinical monitoring of diabetes mellitus. 相似文献
5.
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. 相似文献
8.
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. 相似文献
9.
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. 相似文献
10.
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. 相似文献
11.
Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA methylation. However, the standard detection assay utilizing a bisulfite treatment and HpaII/ MspI endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA methylation assay, including target DNA isolation, HpaII/ MspI endonuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experimentally determined to be 10 2 cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers. 相似文献
12.
A major problem when analyzing bionanoparticles such as influenza viruses (approximately 100 nm in size) is the low sample
concentrations. We developed a method for manipulating a single virus that employs optical tweezers in conjunction with dielectrophoretic
(DEP) concentration of viruses on a microfluidic chip. A polydimethylsiloxane microfluidic chip can be used to stably manipulate
a virus. The chip has separate sample and analysis chambers to enable quantitative analysis of the virus functions before
and after it has infected a target cell. The DEP force in the sample chamber concentrates the virus and prevents it from adhering
to the glass substrate. The concentrated virus is transported to the sample selection section where it is trapped by optical
tweezers. The trapped virus is transported to the analysis chamber and it is brought into contact with the target cell to
infect it. This paper describes the DEP virus concentration for single virus infection of a specific cell. We concentrated
the influenza virus using the DEP force, transported a single virus, and made it contact a specific H292 cell. 相似文献
14.
A micro flow cytometer with an integrated three-dimensional hydro-focusing unit and out-of-plane microlenses was successfully fabricated and tested. The entire system was fabricated with SU-8 ultra-violet lithography process. In the hydro-focusing unit, sheath flows pass through a trapezoid-shaped chamber with three 30° slopes to focus the sample flow in both horizontal and vertical directions. As an essential component in the on-chip optical detection system, integrated out-of-plane microlens was embedded in the sidewall of the fluid outlet channel in the detection area. A pre-aligned optical fiber holder was fabricated on chip to fix the output optical fiber in a position aligned to the microlens. Optical simulation and analysis were also conducted using commercial software Zemax. Numerical simulation results confirmed that the use of microlens substantially improved the detection efficiency by focusing the fluorescent light from the sample cells into the output optical fiber. Preliminary cell counting experiment was performed using the fabricated micro flow cytometer system and the experimental results proved the feasibility of the integrated micro flow cytometer design. 相似文献
15.
Colorimetric urinalysis is a commonly performed test for rapid and low-cost diagnosis. Conventional colorimetric urinalysis is manually conducted using dipsticks and suffers from difficulties in control of sample distribution and color interpretation. This paper reports a microfluidic platform for conducting automated colorimetric urinalysis. Centrifugal microfluidic technology was used for regulating the distribution of urine sample in designed volume and time sequence. The prototype of the microfluidic chip was fabricated using 3D printing technology. To test the feasibility of the prototype system, commercial urinalysis strips were integrated with the microfluidic system for detecting glucose, specific gravity, PH, and protein from simulated urine sample. The color change of the strips was recorded using a smartphone and analyzed to quantify the interested parameters. The H (hue), S (saturation) and V (value) coordinates of the HSV color space were extracted and related to the change of the four parameters. The intensity change of V channel showed good representation of the change of glucose concentration and specific gravity. The intensity change of S channel decreased as the increase of PH and protein concentration. The proposed Lab-on-CD platform has potential for automating colorimetric urinalysis to reduce the user errors, thus to made the testing results conducted by non-professionals more reliable. 相似文献
16.
We report a new two-axis active optical fiber manipulator for on-chip optical manipulation and detection in microfluidic environment. The system comprising of air chambers, fiber channels, controllable moving walls, and membrane structures were fabricated by using microelectromechanical systems technology. By adjusting air pressures to control the deflection of the pneumatic chambers placed orthogonal to and underneath the fiber channels, accurate alignment of a pair of approximately coaxial optical fibers, which was indicated by maximizing fiber-to-fiber optical-coupling measured in real time, has been achieved. A maximum displacement of a buried fiber as large as 13 mum at an applied pressure of 40 lb/in 2 for one air chamber has been demonstrated. It was sufficient to accurately align two approximately coaxial optical fibers to maximize the optical coupling efficiency. The maximum coupling efficiency for two single-mode optical fibers facing each other at a distance of 200 mum was measured to be 4.1%. The following features have been successfully demonstrated with this system: 1) stable optical trapping and stretching of a single red blood cell; 2) stable optical trapping of multiple microparticles; 3) optically driven controlled motion of single and multiple microparticles; and 4) integration of a counterpropagating dual-beam trap with single-beam optical tweezers. In addition to optical trapping and manipulation, the proposed device is promising for applications requiring coaxial input/output fibers for in-line optical analysis. Furthermore, it can be easily integrated with other microfluidic devices such as microcapillary electrophoresis channels or microflow cytometers for DNA, protein, and cell analysis. 相似文献
17.
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. 相似文献
19.
It has long been thought that an optical sensor, such as a light waveguide implemented total analysis system (TAS), is one of the functional components that will be needed to realize a “ubiquitous human healthcare system” in the near future. We have already proposed the fundamental structure for a light waveguide capable of illuminating a living cell or particle running along a microfluidic channel, as well as of detecting fluorescence even from the extremely weak power of such a minute particle. In order to develop novel functions to detect the internal structure of living cells quickly, an angular scanning method that sequentially changes the direction of illumination of the minute cell or particle may be crucial. In this paper, we investigate fluorescence detection from moving particles by switching the laser power delivery path of plural light waveguides as a preliminary experiment toward this novel method. To construct an experimental system able to incorporate a switching light source mechanism cost effectively, we utilized a conventional TAS chip with plural waveguide pairs arranged in parallel, and a forced vibration mechanism on an optical fiber tip by a piezoelectric actuator. With this system, we performed an experiment to detect extremely weak fluorescence using micro particles with a fluorescent substance attached and an optical TAS chip that incorporated a microfluidic channel and three pairs of laser-power-delivering light waveguide cores. We successfully obtained clear, quasi-triangular-shaped pulses in fluorescent signals from resin particles running across the intersection under three different conditions: (1) a particle with approximately the same velocity as that of a forced-vibrated optical fiber tip of approximately 700 mm/s, (2) a particle with velocity 1 digit smaller than that of an optical fiber tip, and (3) a particle with velocity approximately 1/20 that of an optical fiber tip. 相似文献
20.
The chemical stability of the vanadomolybdophosphoric acid method for phosphate determination in a microfluidic manifold is described. The reagent lifetime has been shown to extend to more than 1 year. A stopped flow regime has been implemented, which enabled a very simple microfluidic manifold design to be employed, and has the added advantages of low reagent consumption coupled with less waste generation and access to the complete reaction profile in the optical cuvette on the microfluidic chip. Optical detection was achieved with an UV-LED, integrated into the microfluidic chip holder, coupled to a portable spectrometer via an optical fibre. The manifold includes integration of reagent and sample introduction inlets, a mixing channel and an optical cuvette of 400 μm path length. Two reagent batches were prepared (December 1999 and April 2001) and were shown to still be highly comparable after 1 year in storage. Multiple calibrations have been performed on the microfluidic system over a 12-month period showing only minimal loss in performance and a standard orthophosphate-containing sample was analysed in the microfluidic manifold on a weekly basis with a relative standard deviation of <2.3%. 相似文献
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