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1.
Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)—transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.  相似文献   

2.
We report a 3D microfluidic device with 32 detection channels and 64 sheath flow channels and embedded microball lens array for high throughput multicolor fluorescence detection. A throughput of 358 400 cells/s has been accomplished. This device is realized by utilizing solid immersion micro ball lens arrays for high sensitivity and parallel fluorescence detection. High refractive index micro ball lenses (n = 2.1) are embedded underneath PDMS channels close to cell detection zones in channels. This design permits patterning high N.A. micro ball lenses in a compact fashion for parallel fluorescence detection on a small footprint device. This device also utilizes 3D microfluidic fabrication to address fluid routing issues in two-dimensional parallel sheath focusing and allows simultaneous pumping of 32 sample channels and 64 sheath flow channels with only two inlets.  相似文献   

3.
Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.  相似文献   

4.
This research presents a multiple enzyme-doped thread-based microfluidic system for blood urea nitrogen (BUN) and glucose detection in human whole blood. A novel enzyme-doped thread coated with a thin polyvinylchloride (PVC) membrane is produced for on-site electrochemical detection of urea and glucose in whole blood. Multiple enzymes can be directly applied to the thread without delicate pretreatment or a surface modification process prior to sealing the thread with PVC membrane. Results indicate that the developed device exhibits a good linear dynamic range for detecting urea and glucose in concentrations from 0.1 mM–10.0 mM (R2 = 0.9850) and 0.1 mM–13.0 mM (R2 = 0.9668), which is suitable for adoption in detecting the concentrations of blood urea nitrogen (BUN, 1.78–7.12 mM) and glucose (3.89–6.11 mM) in serum. The detection result also shows that the developed thread-based microfluidic system can successfully separate and detect the ions, BUN, and glucose in blood. The calculated concentrations of BUN and glucose ante cibum (glucose before meal) in the whole blood sample are 3.98 mM and 4.94 mM, respectively. The developed thread-based microfluidic system provides a simple yet high performance for clinical diagnostics.  相似文献   

5.
In this paper, we present a low cost and equipment-free blood filtration device capable of producing plasma from blood samples with mL-scale capacity and demonstrate its clinical application for hepatitis B diagnosis. We report the results of in-field testing of the device with 0.8–1 ml of undiluted, anticoagulated human whole blood samples from patients at the National Hospital for Tropical Diseases in Hanoi, Vietnam. Blood cell counts demonstrate that the device is capable of filtering out 99.9% of red and 96.9% of white blood cells, and the plasma collected from the device contains lower red blood cell counts than plasma obtained from a centrifuge. Biochemistry and immunology testing establish the suitability of the device as a sample preparation unit for testing alanine transaminase (ALT), aspartate transaminase (AST), urea, hepatitis B “e” antigen (HBeAg), hepatitis B “e” antibody (HBe Ab), and hepatitis B surface antibody (HBs Ab). The device provides a simple and practical front-end sample processing method for point-of-care microfluidic diagnostics, enabling sufficient volumes for multiplexed downstream tests.  相似文献   

6.
Inertial microfluidics is an emerging class of technologies developed to separate circulating tumor cells (CTCs). However, defining design parameters and flow conditions for optimal operation remains nondeterministic due to incomplete understanding of the mechanics, which has led to challenges in designing efficient systems. Here, we perform a parametric study of the inertial focusing effects observed in low aspect ratio curvilinear microchannels and utilize the results to demonstrate the isolation of CTCs with high purity. First, we systematically vary parameters including the channel height, width, and radius of curvature over a wide range of flow velocities to analyze its effect on size dependent differential focusing and migration behaviors of binary (10 μm and 20 μm) particles. Second, we use these results to identify optimal flow regimes to achieve maximum separation in various channel configurations and establish design guidelines to readily provide information for developing spiral channels tailored to potentially arbitrary flow conditions that yield a desired equilibrium position for optimal size based CTC separation. Finally, we describe a fully integrated, sheath-less cascaded spiral microfluidic device to continuously isolate CTCs. Human breast cancer epithelial cells were successfully extracted from leukocytes, achieving 86.76% recovery, 97.91% depletion rate, and sustaining high viability upon collection to demonstrate the versatility of the device. Importantly, this device was designed without the cumbersome trail-and-error optimization process that has hindered the development of designing such inertial microfluidic systems.  相似文献   

7.
Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 103 from 3.3 × 103 WBCs.  相似文献   

8.
Separation and sorting of biological entities (viruses, bacteria, and cells) is a critical step in any microfluidic lab-on-a-chip device. Acoustofluidics platforms have demonstrated their ability to use physical characteristics of cells to perform label-free separation. Bandpass-type sorting methods of medium-sized entities from a mixture have been presented using acoustic techniques; however, they require multiple transducers, lack support for various target populations, can be sensitive to flow variations, or have not been verified for continuous flow sorting of biological cells. To our knowledge, this paper presents the first acoustic bandpass method that overcomes all these limitations and presents an inherently reconfigurable technique with a single transducer pair for stable continuous flow sorting of blood cells. The sorting method is first demonstrated for polystyrene particles of sizes 6, 10, and 14.5 μm in diameter with measured purity and efficiency coefficients above 75 ± 6% and 85 ± 9%, respectively. The sorting strategy was further validated in the separation of red blood cells from white blood cells and 1 μm polystyrene particles with 78 ± 8% efficiency and 74 ± 6% purity, respectively, at a flow rate of at least 1 μl/min, enabling to process finger prick blood samples within minutes.  相似文献   

9.
Blood viscosity has been considered as one of important biophysical parameters for effectively monitoring variations in physiological and pathological conditions of circulatory disorders. Standard previous methods make it difficult to evaluate variations of blood viscosity under cardiopulmonary bypass procedures or hemodialysis. In this study, we proposed a unique microfluidic device for simultaneously measuring viscosity and flow rate of whole blood circulating in a complex fluidic network including a rat, a reservoir, a pinch valve, and a peristaltic pump. To demonstrate the proposed method, a twin-shaped microfluidic device, which is composed of two half-circular chambers, two side channels with multiple indicating channels, and one bridge channel, was carefully designed. Based on the microfluidic device, three sequential flow controls were applied to identify viscosity and flow rate of blood, with label-free and sensorless detection. The half-circular chamber was employed to achieve mechanical membrane compliance for flow stabilization in the microfluidic device. To quantify the effect of flow stabilization on flow fluctuations, a formula of pulsation index (PI) was analytically derived using a discrete fluidic circuit model. Using the PI formula, the time constant contributed by the half-circular chamber is estimated to be 8 s. Furthermore, flow fluctuations resulting from the peristaltic pumps are completely removed, especially under periodic flow conditions within short periods (T < 10 s). For performance demonstrations, the proposed method was applied to evaluate blood viscosity with respect to varying flow rate conditions [(a) known blood flow rate via a syringe pump, (b) unknown blood flow rate via a peristaltic pump]. As a result, the flow rate and viscosity of blood can be simultaneously measured with satisfactory accuracy. In addition, the proposed method was successfully applied to identify the viscosity of rat blood, which circulates in a complex fluidic network. These observations confirm that the proposed method can be used for simultaneous measurement of viscosity and flow rate of whole blood circulating in the complex fluid network, with sensorless and label-free detection. Furthermore, the proposed method will be used in evaluating variations in the viscosity of human blood during cardiopulmonary bypass procedures or hemodialysis.  相似文献   

10.
Optical based analysis in microfluidic and lab-on-a-chip systems are currently considered the gold standard methodology for the determination of end point reactions for various chemical and biological reaction processes. Typically, assays are performed using bulky ancillary apparatus such as microscopes and complex optical excitation and detection systems. Such instrumentation negates many of the advantages offered by device miniaturisation, particularly with respect to overall portability. In this article, we present a CO2 laser ablation technique for rapidly prototyping on-chip planar lenses, in conjunction with capillary action based autonomous microfluidics, to create a miniaturised and fully integrated optical biosensing platform. The presented self-aligned on-chip optical components offer an efficient means to direct excitation light within microfluidics and to directly couple light from a LED source. The device has been used in conjunction with a miniaturised and bespoke fluorescence detection platform to create a complete, palm sized system (≈60 × 80 × 60 mm) capable of performing fluoro-immunoassays. The system has been applied to the detection of cardiac Troponin I, one of the gold standard biomarkers for the diagnosis of acute myocardial infarction, achieving a lower detection limit of 0.08 ng/ml, which is at the threshold of clinically applicable concentrations. The portable nature of the complete system and the biomarker detection capabilities demonstrate the potential of the devised instrumentation for use as a medical diagnostics device at the point of care.  相似文献   

11.
Blood plasma contains biomarkers and substances that indicate the physiological state of an organism, and it can be used to diagnose various diseases or body condition. To improve the accuracy of diagnostic test, it is required to obtain the high purity of blood plasma. This paper presents a low-cost, disposable microfluidics device for blood plasma extraction using magnetophoretic behaviors of blood cells. This device uses alternating magnetophoretic capture modes to trap and separate paramagnetic and diamagnetic cells away from blood plasma. The device system is composed of two parts, a disposable microfluidics chip and a non-disposable (reusable) magnetic field source. Such modularized device helps the structure of the disposable part dramatically simplified, which is beneficial for low-cost mass production. A series of numerical simulation and parametric study have been performed to describe the mechanism of blood cell separation in the microchannel, and the results are discussed. Furthermore, experimental feasibility test has been carried out in order to demonstrate the blood plasma extraction process of the proposed device. In this experiment, pure blood plasma has been successfully extracted with yield of 21.933% from 75 μl 1:10 dilution of deoxygenated blood.  相似文献   

12.
A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm in radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units.  相似文献   

13.
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   

14.
Cell culture in microfluidic systems has primarily been conducted in devices comprised of polydimethylsiloxane (PDMS) or other elastomers. As polystyrene (PS) is the most characterized and commonly used substrate material for cell culture, microfluidic cell culture would ideally be conducted in PS-based microsystems that also enable tight control of perfusion and hydrodynamic conditions, which are especially important for culture of vascular cell types. Here, we report a simple method to prototype perfusable PS microfluidics for endothelial cell culture under flow that can be fabricated using standard lithography and wet laboratory equipment to enable stable perfusion at shear stresses up to 300 dyn/cm2 and pumping pressures up to 26 kPa for at least 100 h. This technique can also be extended to fabricate perfusable hybrid PS-PDMS microfluidics of which one application is for increased efficiency of viral transduction in non-adherent suspension cells by leveraging the high surface area to volume ratio of microfluidics and adhesion molecules that are optimized for PS substrates. These biologically compatible microfluidic devices can be made more accessible to biological-based laboratories through the outsourcing of lithography to various available microfluidic foundries.  相似文献   

15.
We have developed a microfluidics based sampling system for tissue analytics. The proof-of-concept of the sampling system was demonstrated by extracting lipid samples from tissue biopsies. The sample collection system consists of a disposable silicon based multiport microneedle integrated with polymer microfluidics. The polymethyl methacrylate polymer microfluidic chip has a 10 μl sample reservoir and actuation membranes for liquid pumping. A special automated robotic system was developed to control the positioning of the needle and the sampling procedure on preselected spots on the tissue. Real breast cancer tissue samples were used to test the feasibility of the sampling system. We successfully measured indicative cancer biomarkers from the tissue surface. Phosphatidylcholine and phosphoethanolamine were extracted from the tissue membrane with methyl tert-butyl ether solvent and detected by mass spectrometry. In the future, this tool could be used in characterization of preoperative biopsies and tumour tissues removed during surgery.  相似文献   

16.
Accurate measurement of blood viscoelasticity including viscosity and elasticity is essential in estimating blood flows in arteries, arterials, and capillaries and in investigating sub-lethal damage of RBCs. Furthermore, the blood viscoelasticity could be clinically used as key indices in monitoring patients with cardiovascular diseases. In this study, we propose a new method to simultaneously measure the viscosity and elasticity of blood by simply controlling the steady and transient blood flows in a microfluidic analogue of Wheastone-bridge channel, without fully integrated sensors and labelling operations. The microfluidic device is designed to have two inlets and outlets, two side channels, and one bridge channel connecting the two side channels. Blood and PBS solution are simultaneously delivered into the microfluidic device as test fluid and reference fluid, respectively. Using a fluidic-circuit model for the microfluidic device, the analytical formula is derived by applying the linear viscoelasticity model for rheological representation of blood. First, in the steady blood flow, the relationship between the viscosity of blood and that of PBS solution (μBlood/μPBS) is obtained by monitoring the reverse flows in the bridge channel at a specific flow-rate rate (QPBSSS/QBloodL). Next, in the transient blood flow, a sudden increase in the blood flow-rate induces the transient behaviors of the blood flow in the bridge channel. Here, the elasticity (or characteristic time) of blood can be quantitatively measured by analyzing the dynamic movement of blood in the bridge channel. The regression formula (ABlood (t) = Aα + Aβ exp [−(t − t0)/λBlood]) is selected based on the pressure difference (ΔP = PA − PB) at each junction (A, B) of both side channels. The characteristic time of blood (λBlood) is measured by analyzing the area (ABlood) filled with blood in the bridge channel by selecting an appropriate detection window in the microscopic images captured by a high-speed camera (frame rate = 200 Hz, total measurement time = 7 s). The elasticity of blood (GBlood) is identified using the relationship between the characteristic time and the viscosity of blood. For practical demonstrations, the proposed method is successfully applied to evaluate the variations in viscosity and elasticity of various blood samples: (a) various hematocrits form 20% to 50%, (b) thermal-induced treatment (50 °C for 30 min), (c) flow-induced shear stress (53 ± 0.5 mL/h for 120 min), and (d) normal rat versus spontaneously hypertensive rat. Based on these experimental demonstrations, the proposed method can be effectively used to monitor variations in viscosity and elasticity of bloods, even with the absence of fully integrated sensors, tedious labeling and calibrations.  相似文献   

17.
Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream™ that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 × 106 peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% ± 3.1% (n = 12) and 71.2% ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4–2600 cells. The viability of MDA-MB-231 cancer cells captured with ApoStream was greater than 97.1% and there was no difference in cell growth up to 7 days in culture compared to controls. The ApoStream device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level. Isolation and enrichment of viable cancer cells from ApoStream enables molecular characterization of CTCs from a wide range of cancer types.  相似文献   

18.
Cryptosporidiosis has been reported to be associated with HIV/acquired immune deficiency syndrome, which greatly reduces the quality of life and shortens the life expectancy of HIV-infected patients. In order to properly treat the infected patients, accurate and automatic diagnostic tools need to be developed. In this study, a novel microfluidic immunochip system was presented for the surveillance and the rapid detection of Cryptosporidium infection in 190 HIV-infected patients from Guangxi, China, using the P23 antigen of Cryptosporidium. The procedure of detection can be completed within 10 min with 2 μl sample consumption. The system also was evaluated using the standard ELISA method. Among 190 HIV-infected individuals, the rate of P23 positivity was 13.7%. Seropositivity in HIV-infected individuals was higher in female patients. The seropositivity to P23 was higher in HIV-infected individuals with high viral load, although the difference was statistically insignificant. Significantly higher Cryptosporidium seropositivity was observed in HIV-infected individuals with a CD4+ T-cell count of <200 cells/μl than in those with ≥200 cells/μl. Our results also demonstrate that a lower CD4+ T-cell count may reflect an increased accumulated risk for cryptosporidiosis. The detection system was further validated using the standard ELISA method and good correlation between the two methods was found (r = 0.80). Under the same sensitivity, this new microfluidic chip device had a specificity of 98.2%. This developed system may provide a powerful platform for the fast screening of Cryptospordium infection in HIV-infected patients.  相似文献   

19.
This article describes a fabrication process for the generation of a leak proof paper based microfluidic device and a new design strategy for convenient incorporation of externally prepared test zones. Briefly, a negative photolithographic method was used to prepare the device with a partial photoresist layer on the rear of the device to block the leakage of sample. Microscopy and Field Emission Scanning Electron Microscopy data validated the formation of the photoresist layer. The partial layer of photoresist on the device channel limits sample volume to 7 ± 0.2 μl as compared to devices without the partial photoresist layer which requires a larger sample volume of 10 ± 0.1 μl. The design prototype with a customized external test zone exploits the channel protrusions on the UV exposed photoresist treated paper to bridge the externally applied test zone to the sample and absorbent zones. The partially laminated device with an external test zone has a comparatively low wicking speed of 1.8 ± 0.9 mm/min compared to the completely laminated device with an inbuilt test zone (3.3 ± 1.2 mm/min) which extends the reaction time between the analyte and reagents. The efficacy of the prepared device was studied with colorimetric assays for the non-specific detection of protein by tetrabromophenol blue, acid/base with phenolphthalein indicator, and specific detection of proteins using the HRP-DAB chemistry. The prepared device has the potential for leak proof detection of analyte, requires low sample volume, involves reduced cost of production (∼$0.03, excluding reagent and lamination cost), and enables the integration of customized test zones.  相似文献   

20.
Cancer biomarkers have significant potential as reliable tools for the early detection of the disease and for monitoring its recurrence. However, most current methods for biomarker detection have technical difficulties (such as sample preparation and specific detector requirements) which limit their application in point of care diagnostics. We developed an extremely simple, power-free microfluidic system for direct detection of cancer biomarkers in microliter volumes of whole blood. CEA and CYFRA21-1 were chosen as model cancer biomarkers. The system automatically extracted blood plasma from less than 3 μl of whole blood and performed a multiplex sample-to-answer assay (nano-ELISA (enzyme-linked immunosorbent assay) technique) without the use of external power or extra components. By taking advantage of the nano-ELISA technique, this microfluidic system detected CEA at a concentration of 50 pg/ml and CYFRA21-1 at a concentration of 60 pg/ml within 60 min. The combination of PnP polydimethylsiloxane (PDMS) pump and nano-ELISA technique in a single microchip system shows great promise for the detection of cancer biomarkers in a drop of blood.  相似文献   

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