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1.
Multiwell plate and pipette systems have revolutionized modern biological analysis; however, they have disadvantages because testing in the submicroliter range is challenging, and increasing the number of samples is expensive. We propose a new microfluidic methodology that delivers the functionality of multiwell plates and pipettes at the nanoliter scale by utilizing drop coalescence and confinement-guided breakup in microfluidic parking networks (MPNs). Highly monodisperse arrays of drops obtained using a hydrodynamic self-rectification process are parked at prescribed locations in the device, and our method allows subsequent drop manipulations such as fine-gradation dilutions, reactant addition, and fluid replacement while retaining microparticles contained in the sample. Our devices operate in a quasistatic regime where drop shapes are determined primarily by the channel geometry. Thus, the behavior of parked drops is insensitive to flow conditions. This insensitivity enables highly parallelized manipulation of drop arrays of different composition, without a need for fine-tuning the flow conditions and other system parameters. We also find that drop coalescence can be switched off above a critical capillary number, enabling individual addressability of drops in complex MPNs. The platform demonstrated here is a promising candidate for conducting multistep biological assays in a highly multiplexed manner, using thousands of submicroliter samples.  相似文献   

2.
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   

3.
Manipulation of magnetic beads plays an increasingly important role in molecular diagnostics. Magnetophoresis is a promising technique for selective transportation of magnetic beads in lab-on-a-chip systems. We investigate periodic arrays of exchange-biased permalloy microstripes fabricated using a single lithography step. Magnetic beads can be continuously moved across such arrays by combining the spatially periodic magnetic field from microstripes with a rotating external magnetic field. By measuring and modeling the magnetophoresis properties of thirteen different stripe designs, we study the effect of the stripe geometry on the magnetophoretic transport properties of the magnetic microbeads between the stripes. We show that a symmetric geometry with equal width of and spacing between the microstripes facilitates faster transportation and that the optimal period of the periodic stripe array is approximately three times the height of the bead center over the microstripes.  相似文献   

4.
The development of widely applicable point-of-care sensing and diagnostic devices can benefit from simple and inexpensive fabrication techniques that expedite the design, testing, and implementation of lab-on-a-chip devices. In particular, electrodes integrated within microfluidic devices enable the use of electrochemical techniques for the label-free detection of relevant analytes. This work presents a novel, simple, and cost-effective bench-top approach for the integration of high surface area three-dimensional structured electrodes fabricated on polystyrene (PS) within poly(dimethylsiloxane) (PDMS)-based microfluidics. Optimization of PS-PDMS bonding results in integrated devices that perform well under pressure and fluidic flow stress. Furthermore, the fabrication and bonding processes are shown to have no effect on sensing electrode performance. Finally, the on-chip sensing capabilities of a three-electrode electrochemical cell are demonstrated with a model redox compound, where the high surface area structured electrodes exhibit ultra-high sensitivity. We propose that the developed approach can significantly expedite and reduce the cost of fabrication of sensing devices where arrays of functionalized electrodes can be used for point-of-care analysis and diagnostics.  相似文献   

5.
Xu G  Lee DY  Xie H  Chiew D  Hsieh TM  Ali EM  Lun Looi X  Li MH  Ying JY 《Biomicrofluidics》2011,5(3):34107-341079
Sample preparation is one of the most crucial processes for nucleic acids based disease diagnosis. Several steps are required for nucleic acids extraction, impurity washes, and DNA/RNA elution. Careful sample preparation is vital to the obtaining of reliable diagnosis, especially with low copies of pathogens and cells. This paper describes a low-cost, disposable lab cartridge for automatic sample preparation, which is capable of handling flexible sample volumes of 10 μl to 1 ml. This plastic cartridge contains all the necessary reagents for pathogen and cell lysis, DNA/RNA extraction, impurity washes, DNA/RNA elution and waste processing in a completely sealed cartridge. The entire sample preparation processes are automatically conducted within the cartridge on a desktop unit using a pneumatic fluid manipulation approach. Reagents transportation is achieved with a combination of push and pull forces (with compressed air and vacuum, respectively), which are connected to the pneumatic inlets at the bottom of the cartridge. These pneumatic forces are regulated by pinch valve manifold and two pneumatic syringe pumps within the desktop unit. The performance of this pneumatic reagent delivery method was examined. We have demonstrated the capability of the on-cartridge RNA extraction and cancer-specific gene amplification from 10 copies of MCF-7 breast cancer cells. The on-cartridge DNA recovery efficiency was 54-63%, which was comparable to or better than the conventional manual approach using silica spin column. The lab cartridge would be suitable for integration with lab-chip real-time polymerase chain reaction devices in providing a portable system for decentralized disease diagnosis.  相似文献   

6.
Liquid dielectrophoresis (L-DEP), when deployed at microscopic scales on top of hydrophobic surfaces, offers novel ways of rapid and automated manipulation of very small amounts of polar aqueous samples for microfluidic applications and development of laboratory-on-a-chip devices. In this article we highlight some of the more recent developments and applications of L-DEP in handling and processing of various types of aqueous samples and reagents of biological relevance including emulsions using such microchip based surface microfluidic (SMF) devices. We highlighted the utility of these devices for on-chip bioassays including nucleic acid analysis. Furthermore, the parallel sample processing capabilities of these SMF devices together with suitable on- or off-chip detection capabilities suggest numerous applications and utility in conducting automated multiplexed assays, a capability much sought after in the high throughput diagnostic and screening assays.  相似文献   

7.
Microfluidic technologies have several advantages in sample preparation for diagnostics but suffer from the need for an external operation system that hampers user-friendliness. To overcome this limitation in microfluidic technologies, a number of user-friendly methods utilizing capillary force, degassed poly(dimethylsiloxane), pushbutton-driven pressure, a syringe, or a pipette have been reported. Among these methods, the pushbutton-driven, pressure-based method has a great potential to be widely used as a user-friendly sample preparation tool for point-of-care testing or portable diagnostics. In this Perspective, we focus on the pushbutton-activated microfluidic technologies toward a user-friendly sample preparation tool. The working principle and recent advances in pushbutton-activated microfluidic technologies are briefly reviewed, and future perspectives for wide application are discussed in terms of integration with the signal analysis system, user-dependent variation, and universal and facile use.  相似文献   

8.
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   

9.
Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.  相似文献   

10.
We present an immunoaggregation assay chip for multiplexed biomarkers detection. This chip is based on immunoaggregation of antibody functionalized microparticles (Ab-MPs) to quantify concentrations of multiple biomarkers simultaneously. A mixture of multiple types of Ab-MPs probes with different sizes and magnetic properties, which were functionalized by different antibodies, was used for the multiplexed assay. The interactions between biomarkers and their specific Ab-MPs probes caused the immunoaggregation of Ab-MPs. A two-stage micro resistive pulse sensor was used to differentiate and count the Ab-MP aggregates triggered by different biomarkers via size and magnetic property for multiplexed detection. The volume fraction of each type of Ab-MP aggregates indicates the concentration of the corresponding target biomarker. In our study, we demonstrated multiplexed detection of two model biomarkers (human ferritin and mouse anti-rabbit IgG) in 10% fetal bovine serum, using anti-ferritin Ab and anti-mouse IgG Ab functionalized MPs. We found that the volume fraction of Ab-MP aggregates increased with the increased biomarker concentrations. The detection ranges from 5.2 ng/ml to 208 ng/ml and 3.1 ng/ml to 5.12 × 104 ng/ml were achieved for human ferritin and mouse anti-rabbit IgG. This bioassay chip is able to quantitatively detect multiple biomarkers in a single test without fluorescence or enzymatic labeling process and hence is promising to serve as a useful tool for rapid detection of multiple biomarkers in biomedical research and clinical applications.  相似文献   

11.
Techniques used to prepare clinical samples have been perfected for use in diagnostic testing in a variety of clinical situations, e.g., to extract, concentrate, and purify respiratory virus particles. These techniques offer a high level of purity and concentration of target samples but require significant equipment and highly trained personnel to conduct, which is difficult to achieve in resource-limited environments where rapid testing and diagnostics are crucial for proper handling of respiratory viruses. Microfluidics has popularly been utilized toward rapid virus detection in resource-limited environments, where most devices focused on detection rather than sample preparation. Initial microfluidic prototypes have been hindered by their reliance on several off-chip preprocessing steps and external laboratory equipment. Recently, sample preparation methods have also been incorporated into microfluidics to conduct the virus detection in an all-in-one, automated manner. Extraction, concentration, and purification of viruses have been demonstrated in smaller volumes of samples and reagents, with no need for specialized training or complex machinery. Recent devices show the ability to function independently and efficiently to provide rapid, automated sample preparation as well as the detection of viral samples with high efficiency. In this review, methods of microfluidic sample preparation for the isolation and purification of viral samples are discussed, limitations of current systems are summarized, and potential advances are identified.  相似文献   

12.
Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization.  相似文献   

13.
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   

14.
转化医学为基础医学研究和临床实践搭建了一个平台,其主要目的是把涉及的众多交叉基础学科理论转化为临床实践。如何在民族地区高职高专医学教育阶段将转化医学理念融入教学课堂已成为热门课题。文章通过在诊断学的课程整合中改进教学方法,阐述如何将转化医学理念融入高职高专诊断学教学中,以培养高素质基层医疗人才,让高职高专医学生尽快服务缺医少药的民族地区,为民族地区医疗卫生事业发展添砖加瓦。  相似文献   

15.
基于区域医疗共享的PACS用于将患者分布在各级医院中的诊疗信息、检验检查结果和医学影像进行基于国际IHE规范的共享交换和协同医疗。通过构造区域内部的医学影像信息交换平台,以实现区域内医院的医学影像资源的共享与整合。此平台是卫生主管部门推动双向转诊和远程医疗的关键技术辅助手段,可以有效地避免患者重复检查,远程诊断咨询或者远程会诊,远程教学和医学继续教育,区域内部医学影像资源共享或者医院内部PACS系统的互联互通,远程医学影像质量控制等,通过采用集中分布系统架,合理利用了已有的医院资源,同时又增加了数据的获取渠道,平衡利用有限网络带宽资源;影像数据中心系统的中心节点和医院节点自动互为备份,数据在医院和中心异地备份;较好的解决了共享的问题,在实际应用中取得了较好的效果。  相似文献   

16.
[目的/意义]突发紧急状况下将科研力量迅速有效地协同整合,对于实现高质量的应急研发以及有效遏制突发公共卫生危机的蔓延或扩大态势至关重要。[方法/过程]通过对具有代表性突发公共卫生事件的研究,提出科研应急力量需要具备的三个特点,在此基础上提出科研应急力量协同整合的流程及相应的整合方法;最后以突发公共卫生危机中诊断试剂的科研应急攻关作为假设背景,以诊断试剂应急研发中的科研力量整合作为实证研究。[结果/结论]研究发现可以将单磷酸、神经管和误诊率为主和以单核酸、抗原受体、精神分裂症为主的主题聚类下的机构整合成两股科研应急力量,从应急力量构成、应急地域、应急能力上看两股科研应急力量各具特点。  相似文献   

17.
This paper presents a low-cost, power-free, and easy-to-use spotter system for fabrication of microarrays. The spotter system uses embedded dispensing microchannels combined with a polydimethylsiloxane (PDMS) membrane containing regular arrays of well-defined thru-holes to produce precise, uniform DNA or protein microarrays for disease diagnosis or drug screening. Powered by pre-evacuation of its PDMS substrate, the spotter system does not require any additional components or external equipment for its operation, which can potentially allow low-cost, high-quality microarray fabrication by minimally trained individuals. Polyvinylpyrrolidone was used to modify the PDMS surface to prevent protein adsorption by the microchannels. Experimental results indicate that the PDMS spotter shows excellent printing performance for immobilizing proteins. The measured coefficient of variation (CV) of the diameter of 48 spots was 2.63% and that of the intensity within one array was 2.87%. Concentration gradient experiments revealed the superiority of the immobilization density of the PDMS spotter over the conventional pin-printing method. Overall, this low-cost, power-free, and easy-to-use spotting system provides an attractive new method to fabricate microarrays.  相似文献   

18.
The development of monoclonal antibodies combined with flow cytometry has revolutionized the analysis of lymphocyte subsets. These newer methods using the Q-prep leucocyte preparation system require only 1–2 ml of blood as compared to 10 ml required traditionally. One of the main impediments in the use of this superior technology in Indian laboratories has been the high cost of reagents. This study evaluated methods to reduce the cost of assays. In the first experiment from 26 healthy subjects, 2ml venous blood samples in EDTA (ethylenediamine tetra-acetate) were obtained. Each sample was divided into two equal portions, one portion was stained using diluted monoclonal antibody, whereas the other portion was stained using standard concentrations of antibodies. In the second experiment, blood samples from 12 subjects were again divided into 2 portions; one portion of each pair was processed using commercial Q-prep reagents while the other portion was processed using our own reagents. In the first experiment, which evaluated use of a diluted antibody against the standard recommended concentrations, a 5-tube panel that estimated CD3, CD4, CD8, CD20 was used. In the second experiment CD3, CD4 and CD8 were estimated. The total cost per sample for a 5-panel estimation was however reduced from $39.11 to $1.10. Given the proven advantages of using a whole blood stain-lyse method for T cell subset estimations, its use should be encouraged in developing country settings. With the suggested methods the whole blood Q-prep could be performed at appreciably reduced costs, without loss in precision.  相似文献   

19.
Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.  相似文献   

20.
Serum thyroglobulin (Tg) and thyroid stimulating hormone (TSH) measurements have evolved as important analytes for monitoring the prognosis of patients with differentiated thyroid cancer, post-thyroidectomy. Individual analyte immunoassay is the current practice in clinical pathology, but the simultaneous assay for all relevant analytes for a given disease, can reduce assay costs, improve patient compliance and give the clinician more information for an unequivocal diagnosis. Microarray immunoassay (MI) can achieve this goal and, hence, we have developed and validated a immuno-radiometric MI for quantitation of serum TSH and Tg by using highly micro-porous polycarbonate (PC) track-etched membranes (TEM) to immobilize the monoclonal anti-TSH and polyclonal anti-Tg antibodies in ~1 mm diameter spots. Non-competitive immunoassays were performed using mixture of 125I labeled monoclonal anti-TSH and anti-Tg antibodies. Phosphorimager was used to quantify the bound radioactivity. TSH and Tg were detected with detection limit of 0.07 µIU/ml and 0.13 ng/ml respectively, which is lower than the clinically required cut-off level. The assay showed: acceptable intra-assay precision within 20 % and recovery in the range of 76–111.2 %. MI compared well with the established immunoradiometric assay (IRMA) with r = 0.98, p < 0.01 (n = 41). No cross-reactivity was seen between the immobilized antibodies. Although two hormones are addressed in this report, MI using PC TEM and isotopic/non-isotopic tracers has the potential for highly automated multiplexed analysis.  相似文献   

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