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1.
In this contribution, we present a system for efficient preconcentration of pathogens without affecting their viability. Development of miniaturized molecular diagnostic kits requires concentration of the sample, molecule extraction, amplification, and detection. In consequence of low analyte concentrations in real-world samples, preconcentration is a critical step within this workflow. Bacteria and viruses exhibit a negative surface charge and thus can be electrophoretically captured from a continuous flow. The concept of phaseguides was applied to define gel membranes, which enable effective and reversible collection of the target species. E. coli of the strains XL1-blue and K12 were used to evaluate the performance of the device. By suppression of the electroosmotic flow both strains were captured with efficiencies of up to 99%. At a continuous flow of 15 μl/min concentration factors of 50.17 ± 2.23 and 47.36 ± 1.72 were achieved in less than 27 min for XL1-blue and K12, respectively. These results indicate that free flow electrophoresis enables efficient concentration of bacteria and the presented device can contribute to rapid analyses of swab-derived samples.  相似文献   

2.
Lin CH  Wang YN  Fu LM 《Biomicrofluidics》2012,6(1):12818-1281811
An integrated microfluidic chip is proposed for rapid DNA digestion and time-resolved capillary electrophoresis (CE) analysis. The chip comprises two gel-filled chambers for DNA enrichment and purification, respectively, a T-form micromixer for DNA/restriction enzyme mixing, a serpentine channel for DNA digestion reaction, and a CE channel for on-line capillary electrophoresis analysis. The DNA and restriction enzyme are mixed electroomostically using a pinched-switching DC field. The experimental and numerical results show that a mixing performance of 97% is achieved within a distance of 1 mm from the T-junction when a driving voltage of 90 V/cm and a switching frequency of 4 Hz are applied. Successive mixing digestion and capillary electrophoresis operation clearly present the changes on digesting φx-174 DNA in different CE runs. The time-resolved electropherograms show that the proposed device enables a φx-174 DNA sample comprising 11 fragments to be concentrated and analyzed within 24 min. Overall, the results presented in this study show that the proposed microfluidic chip provides a rapid and effective tool for DNA digestion and CE analysis applications.  相似文献   

3.
The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 μm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane  相似文献   

4.
Wei Hou H  Gan HY  Bhagat AA  Li LD  Lim CT  Han J 《Biomicrofluidics》2012,6(2):24115-2411513
Sepsis is an adverse systemic inflammatory response caused by microbial infection in blood. This paper reports a simple microfluidic approach for intrinsic, non-specific removal of both microbes and inflammatory cellular components (platelets and leukocytes) from whole blood, inspired by the invivo phenomenon of leukocyte margination. As blood flows through a narrow microchannel (20 × 20 µm), deformable red blood cells (RBCs) migrate axially to the channel centre, resulting in margination of other cell types (bacteria, platelets, and leukocytes) towards the channel sides. By using a simple cascaded channel design, the blood samples undergo a 2-stage bacteria removal in a single pass through the device, thereby allowing higher bacterial removal efficiency. As an application for sepsis treatment, we demonstrated separation of Escherichia coli and Saccharomyces cerevisiae spiked into whole blood, achieving high removal efficiencies of ∼80% and ∼90%, respectively. Inflammatory cellular components were also depleted by >80% in the filtered blood samples which could help to modulate the host inflammatory response and potentially serve as a blood cleansing method for sepsis treatment. The developed technique offers significant advantages including high throughput (∼1 ml/h per channel) and label-free separation which allows non-specific removal of any blood-borne pathogens (bacteria and fungi). The continuous processing and collection mode could potentially enable the return of filtered blood back to the patient directly, similar to a simple and complete dialysis circuit setup. Lastly, we designed and tested a larger filtration device consisting of 6 channels in parallel (∼6 ml/h) and obtained similar filtration performances. Further multiplexing is possible by increasing channel parallelization or device stacking to achieve higher throughput comparable to convectional blood dialysis systems used in clinical settings.  相似文献   

5.
Here, we utilize microfluidic droplet technology to generate photopolymerizeable polyethylene glycol (PEG) hydrogel microbeads incorporating a fluorescence-based glucose bioassay. A microfluidic T-junction and multiphase flow of fluorescein isothiocyanate dextran, tetramethyl rhodamine isothiocyanate concanavalin A, and PEG in water were used to generate microdroplets in a continuous stream of hexadecane. The microdroplets were photopolymerized mid-stream with ultraviolet light exposure to form PEG microbeads and were collected at the outlet for further analysis. Devices were prototyped in PDMS and generated highly monodisperse 72 ± 2 μm sized microbeads (measured after transfer into aqueous phase) at a continuous flow rate between 0.04 ml/h—0.06 ml/h. Scanning electron microscopy analysis was conducted to analyze and confirm microbead integrity and surface morphology. Glucose sensing was carried out using a Förster resonance energy transfer (FRET) based assay. A proportional fluorescence intensity increase was measured within a 1–10 mM glucose concentration range. Microfluidically synthesized microbeads encapsulating sensing biomolecules offer a quick and low cost method to generate monodisperse biosensors for a variety of applications including cell cultures systems, tissue engineering, etc.  相似文献   

6.
Jena RK  Yue CY 《Biomicrofluidics》2012,6(1):12822-1282212
This report studies the surface modification of cyclic olefin copolymer (COC) by 2-methacryloyloxyethyl phosphorylcholine (MPC) monomer using photografting technique for the purpose of biointerface applications, which demonstrate resistance to both protein adsorption and cell adhesion in COC-based microfluidic devices. This is essential because the hydrophobic nature of COC can lead to adsorption of specific compounds from biological fluids in the microchannel, which can affect the results during fluidic analysis and cause clogging inside the microchannel. A correlation was found between the irradiation time and hydrophobicity of the modified substrate. Static water contact angle results show that the hydrophilicity property of the MPC-grafted substrate improves with increasing irradiation time. The contact angle of the modified surface decreased to 20 ± 5° from 88 ± 3° for the untreated substrate. The surface characterization of the modified surface was evaluated using x-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR spectroscopy). Attenuated total reflection-FTIR and XPS results show the presence of the phosphate group (P-O) on modified COC substrates, indicating that the hydrophilic MPC monomer has successfully grafted on COC. Finally, it was demonstrated that cell adhesion and protein adsorption on the MPC modified COC specimen has reduced significantly.  相似文献   

7.
Surface acoustic wave (SAW) devices with 64 μm wavelength were fabricated on a zinc oxide (ZnO) film deposited on top of an ultra-smooth nanocrystalline diamond (UNCD) layer. The smooth surface of the UNCD film allowed the growth of the ZnO film with excellent c-axis orientation and low surface roughness, suitable for SAW fabrication, and could restrain the wave from significantly dissipating into the substrate. The frequency response of the fabricated devices was characterized and a Rayleigh mode was observed at ∼65.4 MHz. This mode was utilised to demonstrate that the ZnO/UNCD SAW device can be successfully used for microfluidic applications. Streaming, pumping, and jetting using microdroplets of 0.5 and 20 μl were achieved and characterized under different powers applied to the SAW device, focusing more on the jetting behaviors induced by the ZnO SAW.  相似文献   

8.
Multiple sample DNA amplification was done by using a novel rotary-linear motion polymerase chain reaction (PCR) device. A simple compact disc was used to create the stationary sample chambers which are individually temperature controlled. The PCR was performed by shuttling the samples to different temperature zones by using a combined rotary-linear movement of the disc. The device was successfully used to amplify up to 12 samples in less than 30 min with a sample volume of 5 μl. A simple spring loaded heater mechanism was introduced to enable good thermal contact between the samples and the heaters. Each of the heater temperatures are controlled by using a simple proportional–integral–derivative pulse width modulation control system. The results show a good improvement in the amplification rate and duration of the samples. The reagent volume used was reduced to nearly 25% of that used in conventional method.  相似文献   

9.
Wang F  Li Y  Chen L  Chen D  Wu X  Wang H 《Biomicrofluidics》2012,6(1):14120-1412012
Hyperthermia can be used as an adjunctive method of chemotherapy, radiotherapy, and gene therapy to improve cancer treatment. In this study, we investigate the hyperthermic cell death of cervix cancer CaSki cells in a microchannel integrated with a directional heating scheme. Heat was applied from the inner end to the outer end of the channel and a temperature distribution from 60 °C to 30 °C was established. A three dimensional (3D) numerical model was conducted for the heat transfer simulation, based on which a simple fitting method was proposed to easily estimate the temperature distribution along the channel. Cell death along the channel was mapped 22 h after the heating treatment by dual fluorescent labeling and phase-contrast microscopy imaging. Upstream, where the temperature is higher than 42 °C, we observe necrotic death, late-stage and early stage apoptotic death in sequence along the channel. Downstream and in the middle of the channel, where the temperature is lower than 42 °C, significant cell detachment was noted. Vigorous detachment was observed even in the non-hyperthermic zone (temperature lower than 37 °C), which we believe is due to the direct effect of the hyperthermic zones (higher than 37 °C). The present work not only gives a vivid map of cell responses under a temperature gradient, but also reveals the potential interactions of the heated tumor cells and non-heated tumor cells, which are seldom investigated in conventional petri-dish experiments.  相似文献   

10.
In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensemble-averaging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N = 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N = 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFP-labeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein γ-PKC and envisions a further feasible identification of more than one single protein in the future.  相似文献   

11.
Jen CP  Chen WF 《Biomicrofluidics》2011,5(4):44105-4410511
Manipulating and discriminating biological cells of interest using microfluidic and micro total analysis system (μTAS) devices have potential applications in clinical diagnosis and medicine. Cellular focusing in microfluidic devices is a prerequisite for medical applications, such as cell sorting, cell counting, or flow cytometry. In the present study, an insulator-based dielectrophoretic microdevice is designed for the simultaneous filtration and focusing of biological cells. The cells are introduced into the microchannel and hydrodynamically pre-confined by funnel-shaped insulating structures close to the inlet. There are ten sets of X-patterned insulating structures in the microfluidic channel. The main function of the first five sets of insulating structures is to guide the cells by negative dielectrophoretic responses (viable HeLa cells) into the center region of the microchannel. The positive dielectrophoretic cells (dead HeLa cells) are attracted to regions with a high electric-field gradient generated at the edges of the insulating structures. The remaining five sets of insulating structures are mainly used to focus negative dielectrophoretic cells that have escaped from the upstream region. Experiments employing a mixture of dead and viable HeLa cells are conducted to demonstrate the effectiveness of the proposed design. The results indicate that the performance of both filtration and focusing improves with the increasing strength of the applied electric field and a decreasing inlet sample flow rate, which agrees with the trend predicted by the numerical simulations. The filtration efficiency, which is quantitatively investigated, is up to 88% at an applied voltage of 50 V peak-to-peak (1 kHz) and a sample flow rate of 0.5 μl/min. The proposed device can focus viable cells into a single file using a voltage of 35 V peak-to-peak (1 kHz) at a sample flow rate of 1.0 μl/min.  相似文献   

12.
Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml−1), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.  相似文献   

13.
Clinical point of care testing often needs plasma instead of whole blood. As centrifugation is labor intensive and not always accessible, filtration is a more appropriate separation technique. The complexity of whole blood is such that there is still no commercially available filtration system capable of separating small sample volumes (10-100 μl) at the point of care. The microfluidics research in blood filtration is very active but to date nobody has validated a low cost device that simultaneously filtrates small samples of whole blood and reproducibly recovers clinically relevant biomarkers, and all this in a limited amount of time with undiluted raw samples. In this paper, we show first that plasma filtration from undiluted whole blood is feasible and reproducible in a low-cost microfluidic device. This novel microfluidic blood filtration element (BFE) extracts 12 μl of plasma from 100 μl of whole blood in less than 10 min. Then, we demonstrate that our device is valid for clinical studies by measuring the adsorption of interleukins through our system. This adsorption is reproducible for interleukins IL6, IL8, and IL10 but not for TNFα. Hence, our BFE is valid for clinical diagnostics with simple calibration prior to performing any measurement.  相似文献   

14.
In this paper, a poly(dimethylsiloxane) microchip with amperometric detector was developed for the electrophoretic separation and determination of neurotransmitters. For increasing the separation efficiency, the microchannel is modified by polystyrene sulphonate∕polystyrene nano-sphere self-assembly coating. A stable electro-osmotic flow (EOF) and higher separation efficiency are obtained in proposed modified microchannel. Under optimized conditions, dopamine, epinephrine, catechol, and serotonin are acceptably baseline separated in this 3.5 cm length separation channel with the theoretical plate number from 4.6 × 104 to 2.1 × 105 per meter and resolution from 1.29 to 12.5. The practicability of proposed microchip is validated by the recovery test with cerebrospinal fluid as real sample which resulted from 91.7% to 106.5%.  相似文献   

15.
Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).  相似文献   

16.
In microcirculation, red blood cells (RBCs) flowing through bifurcations may deform considerably due to combination of different phenomena that happen at the micro-scale level, such as: attraction effect, high shear, and extensional stress, all of which may influence the rheological properties and flow behavior of blood. Thus, it is important to investigate in detail the behavior of blood flow occurring at both bifurcations and confluences. In the present paper, by using a micro-PTV system, we investigated the variations of velocity profiles of two working fluids flowing through diverging and converging bifurcations, human red blood cells suspended in dextran 40 with about 14% of hematocrit level (14 Hct) and pure water seeded with fluorescent trace particles. All the measurements were performed in the center plane of rectangular microchannels using a constant flow rate of about 3.0 × 10−12 m3/s. Moreover, the experimental data was compared with numerical results obtained for Newtonian incompressible fluid. The behavior of RBCs was asymmetric at the divergent and convergent side of the geometry, whereas the velocities of tracer particles suspended in pure water were symmetric and well described by numerical simulation. The formation of a red cell-depleted zone immediately downstream of the apex of the converging bifurcation was observed and its effect on velocity profiles of RBCs flow has been investigated. Conversely, a cell-depleted region was not formed around the apex of the diverging bifurcation and as a result the adhesion of RBCs to the wall surface was enhanced in this region.  相似文献   

17.
We present a 91 MHz surface acoustic wave resonator with integrated microfluidics that includes a flow focus, an expansion region, and a binning region in order to manipulate particle trajectories. We demonstrate the ability to change the position of the acoustic nodes by varying the electronic phase of one of the transducers relative to the other in a pseudo-static manner. The measurements were performed at room temperature with 3 μm diameter latex beads dispersed in a water-based solution. We demonstrate the dependence of nodal position on pseudo-static phase and show simultaneous control of 9 bead streams with spatial control of −0.058 μm/deg ± 0.001 μm/deg. As a consequence of changing the position of bead streams perpendicular to their flow direction, we also show that the integrated acoustic-microfluidic device can be used to change the trajectory of a bead stream towards a selected bin with an angular control of 0.008 deg/deg ± 0.000(2) deg/deg.  相似文献   

18.
Recent studies show that reduction in cross-sectional area can be used to improve the concentration factor in microscale bioseparations. Due to simplicity in fabrication process, a step reduction in cross-sectional area is generally implemented in microchip to increase the concentration factor. But the sudden change in cross-sectional area can introduce significant band dispersion and distortion. This paper reports a new fabrication technique to form a gradual reduction in cross-sectional area in polymethylmethacrylate (PMMA) microchannel for both anionic and cationic isotachophoresis (ITP). The fabrication technique is based on hot embossing and surface modification assisted bonding method. Both one-dimensional and two-dimensional gradual reduction in cross-sectional area microchannels were formed on PMMA with high fidelity using proposed techniques. ITP experiments were conducted to separate and preconcentrate fluorescent proteins in these microchips. Thousand fold and ten thousand fold increase in concentrations were obtained when 10 × and 100 × gradual reduction in cross-sectional area microchannels were used for ITP.  相似文献   

19.
Increasingly, invitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% ± 13.5%, and enrichment ratio of 12.2 ± 8.4 (n = 8), while preserving cell viability, differentiation potential, and long-term function.  相似文献   

20.
Ma Q  Chen C  Wei S  Chen C  Wu LF  Song T 《Biomicrofluidics》2012,6(2):24107-2410712
Magnetotactic bacteria (MTB) are capable of swimming along magnetic field lines. This unique feature renders them suitable in the development of magnetic-guided, auto-propelled microrobots to serve in target molecule separation and detection, drug delivery, or target cell screening in a microfluidic chip. The biotechnology to couple these bacteria with functional loads to form microrobots is the critical point in its application. Although an immunoreaction approach to attach functional loads to intact MTB was suggested, details on its realization were hardly mentioned. In the current paper, MTB-microrobots were constructed by attaching 2 μm diameter microbeads to marine magnetotactic ovoid MO-1 cells through immunoreactions. These microrobots were controlled using a special control and tracking system. Experimental results prove that the attachment efficiency can be improved to ∼30% via an immunoreaction. The motility of the bacteria attached with different number of loads was also assessed. The results show that MTB can transport one load at a velocity of ∼21 μm/s and still move and survive for over 30 min. The control and tracking system is fully capable of directing and monitoring the movement of the MTB-microrobots. The rotating magnetic fields can stop the microrobots by trapping them as they swim within a circular field with a controllable size. The system has potential use in chemical analyses and medical diagnoses using biochips as well as in nano/microscale transport.  相似文献   

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