部分支持这项工作是由Ishaya霍洛维茨基金。

IL - 8含量： <ol><li>反应混合物包括以下四个部分，在各自的浓度100μL缓冲液的最终体积。 10μL100珠/μL终浓度，2μL生物素IL - 8抗体nano-gram/μl终浓度为1，1μL和素荧光蛋白20nano-gram/μl终浓度与捕获抗体的磁珠， 0.48pico-gram/μl和1μl的IL - 8的目标。这些组件被添加缓冲液，然后30分钟动摇。 </li><li>一个控制反应是准备没有IL - 8的目标相同的方式。 </li><li>反应后放置的试管中，没有任何分离或洗涤步骤和检查使用MMB系统。 </li></ol> MMB的系统： <ol><li>将在其两者之间的电磁极点地位的比色皿。 </li><li>操作的调制电流，并等待30秒，让聚合和凝结珠</li><li>在锁定放大器使用示波器测量信号。 </li></ol>代表性的成果： 聚合珠的目视检查，提出了一个与目标之间的反应和没有目标的反应的明显差异。在所有的反应与目标，珠形成一个单一的，密集的聚集出的激光束在一个团结的方式提出。然而，在没有目标的反应，珠少汇总和它们的运动是不太团结（见图1）。 <img src="/files/ftp_upload/1935/1935fig1.jpg" alt="图1" /> 图1：目视检查三明治“免疫法（一）没有目标的IL - 8（二）随着IL - 8的目标。 极调制时钟（黄色）和光电倍增管的输出信号（洋红色），同时检测0.48微微克的IL - 8的目标是在图1（a）。调制频率为每极2赫兹。然而，因为这是理论上的预期，珠通过激光束时，光电倍增管检测荧光光有一个光电倍增管的输出电压的峰值。因此，解调频率为4赫兹。当光电倍增管的信号和一倍调制时钟（4赫兹）是美联储锁定放大器中，敏感的阶段检测器检测到的同步和高电压（见图2）的结果。 <img src="/files/ftp_upload/1935/1935fig2.jpg" alt="图2" /> 图2：（一）调制时钟（黄色）和光电倍增管的信号（洋红色）当检测0.48微微克的IL - 8的目标。 （b）在导致放大器的电压锁定在两个不同的扫描。 放大器的锁，没有检测到任何信号，当控制样品测试。这个事实，一起聚集在视觉上的差异表明，MMB系统可以清楚地识别存在的IL - 8的目标。

<ol>
	<li>Danielli, A., Arie, A., Porat, N., Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+fluorescent-labeled+probes+at+sub-picomolar+concentrations+by+magnetic+modulation.">Detection of fluorescent-labeled probes at sub-picomolar concentrations by magnetic modulation.</a> Optics Express. 16, 19253-19259 (2008).</li><li>Danielli, A., Porat, N., Arie, A., Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+homogenous+detection+of+the+Ibaraki+virus+NS3+cDNA+at+pico-molar+concentrations+by+magnetic+modulation.">Rapid homogenous detection of the Ibaraki virus NS3 cDNA at pico-molar concentrations by magnetic modulation.</a> Biosensors and Bioelectronics. 25, 858-863 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bio-Rad "Human angiogenesis assay panel". , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+Manual.">Instruction Manual.</a> Bio-Plex Precision Pro Cytokine Assay. , .</li></ol>

综上所述，我们发现，MMB系统可用于检测低浓度（0.5微微克的生物复杂精密临的细胞因子的检测[3,4]的检测限）的IL - 8的目标存在。该系统的能力并不局限于IL - 8和可用于检测其他蛋白质。 MMB系统的优点是能够迅速地检测到目标，并没有任何分离或洗涤步骤。因此，它有利于在检测过程中，允许系统在现场应用中使用。

<table><tbody><tr><td>Bio-Plex Pro Human Cytokine IL-8 set</td><td>Bio-Rad</td><td>171-B5008M</td><td></td></tr><tr><td>Recombinant human IL-8 target</td><td>Thermo Fisher Scientific, Inc.</td><td>R202625</td><td></td></tr><tr><td>Biotinylated monoclonal mouse anti human IL-8 antibody G265-8</td><td>BD Biosciences</td><td>554718</td><td></td></tr><tr><td>Streptavidin coupled fluorescent protein</td><td>Bio-Rad</td><td>Streptavidin PE</td><td></td></tr></tbody></table>

rapid homogeneous detection of biological assays using magnetic modulation biosensing system

磁调制生物传感系统（MMB）[1,2]迅速，并在没有任何清洗或分离步骤的低浓度均匀检测生物指标。当IL - 8的目标，目前，附有一个“sandwich&#39;为基础的检测IL - 8的捕获抗体的磁珠的IL - 8的目标和生物素标记的IL - 8抗体的链霉通过耦合荧光蛋白。磁珠操纵振荡运动，通过采用两个电磁两极交变磁场梯度。凝结成的荧光蛋白质，这是附着在磁珠检测区域和他们的运动和正交激光束产生一个周期性的使用同步检测荧光信号解调。以前使用的磁调制生物传感系统检测茨城病毒非结构蛋白3（NS3区）互补DNA（cDNA）的[2]的编码序列。外部操纵和颗粒凝结在这项工作中表现出的技术可用于其他应用程序，如提供磁耦合的药物<em&gt;在体内</em&gt;或提高对比度<em&gt;在体内</em&gt;成像应用。

Watch this Scientific Journal Video about Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System at JoVE.com

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

利用磁调制生物传感系统，迅速，灵敏，简单地检测生物检测的DNA分子和蛋白质，如。

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

rapid-homogeneous-detection-biological-assays-using-magnetic

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.6K 次观看.  Tel Aviv University. 利用磁调制生物传感系统，迅速，灵敏，简单地检测生物检测的DNA分子和蛋白质，如。

视频: Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

科研

生物工程

Frequency Mixing Magnetic Detection Scanner for Imaging Magnetic Particles in Planar Samples

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. It consists of two magnetic measurement heads on both sides of the sample mounted on the legs of a u-shaped support. The sample is locally exposed to a magnetic excitation field consisting of two distinct frequencies, a stronger component at about 77 kHz and a weaker field at 61 Hz. The nonlinear magnetization characteristics of superparamagnetic particles give rise to the generation of intermodulation products. A selected sum-frequency component of the high and low frequency magnetic field incident on the magnetically nonlinear particles is recorded by a demodulation electronics. In contrast to a conventional MPI scanner, p-FMMD does not require the application of a strong magnetic field to the whole sample because mixing of the two frequencies occurs locally. Thus, the lateral dimensions of the sample are just limited by the scanning range and the supports. However, the sample height determines the spatial resolution. In the current setup it is limited to 2 mm. As examples, we present two 20 mm &#215; 25 mm p-FMMD images acquired from samples with 1 &#181;m diameter maghemite particles in silanol matrix and with 50 nm magnetite particles in aminosilane matrix. The results show that the novel MPI scanner can be applied for analysis of thin biological samples and for medical diagnostic purposes.

A scanner for imaging magnetic particles in planar samples was developed using the planar frequency mixing magnetic detection technique. The magnetic intermodulation product response from the nonlinear nonhysteretic magnetization of the particles is recorded upon a two-frequency excitation. It can be used to take 2D images of thin biological samples.

混频磁探测仪在平面样品成像磁性微粒

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. ...

工程学

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

生物化学

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

基于磁性纳米颗粒免疫分析的交错限制微流控亚克力装置的计算机数控微铣削

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

基于分子印迹的电容生物传感器超灵敏检测生物标志物

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

免疫与感染

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

使用磁悬浮定量细胞密度和抗原特性

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

生物学

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63899">Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays.</a> J. Vis. Exp. (2022).
This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic substrate emitting fluorescence upon catalysis by the peroxidases, the captured immunocomplexes are detected under a microscope.

使用微流控设备的基于磁性纳米粒子的免疫测定

1. Device preparation

	Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in ...