eoe sub articles

EoE Sub Articles

1. Device preparation
<ol>
	<li>Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in an ultrasonic bath for 10 min to remove any remaining acrylic, adhesive, or unwanted material inside the channels.</li>
	<li>Empty the water inside the device channels. Use a syringe to introduce a blocking solution prepared with 5% (w/v) bovine serum albumin (BSA) diluted in 1x Tris-buffered saline (TBS) and previously filtered through a 0.2 &#181;m polyethersulfone (PES) syringe filter.</li>
	<li>Prepare a suspension of iron microparticles of 7.5 &#181;m diameter in 5% BSA. 
	NOTE: The microparticles are previously functionalized with a silica-polyethylene glycol (PEG) layer that confers resistance to protein absorption.</li>
	<li>Incubate the chip and the microparticle suspension with the blocking solution for at least 1 h at room temperature. If possible, allow blocking overnight at 4 &#176;C.</li>
</ol>
2. Microparticle trap formation
<ol>
	<li>Insert the microparticles into the chip with a syringe needle through the side channel outlet hose. Place the chip vertically and allow the microparticles to flow under the effect of gravity through the side channel. Rotate the chip 180&#176; in two steps of 90&#176; and allow the microparticles to target and compact at the 5 &#181;m restriction.</li>
	<li>Remove excess microparticles by gravity rotating 45&#176; toward the side channel.</li>
	<li>Keep the device upright to avoid undoing the microparticle trap. See Figure 1B for a summary of the microparticle trap formation process.</li>
</ol>
3. Immunoassay
<ol>
	<li>Nanoparticle preparation
	<ol>
		<li>Take 2 &#181;L of the suspension of 100 nm nanoparticles previously conjugated with lysozyme (antigen model). Add it to a 1.5 mL microcentrifuge tube with 100 &#181;L of blocking solution. Incubate overnight at 4 &#176;C.</li>
		<li>Add 150 &#181;L of wash buffer (1x TBS, 0.05% Tween 20).</li>
		<li>Place the 1.5 mL microcentrifuge tube in a magnetic separator. Keep for 15 min to allow the separation of the nanoparticles (Figure 2). 
		NOTE: The minimum volume for the magnetic separator is 200 &#181;L. Avoid using a smaller volume.</li>
		<li>Remove the liquid from the tube with a micropipette. Avoid contact with the wall of the tube where the nanoparticle pellet was formed.</li>
		<li>Add 250 &#181;L of fresh wash buffer. Keep the tube under agitation for 15 min.</li>
		<li>Repeat steps 3.1.3.-3.1.5. 2x more, shaking only for 5 min.</li>
		<li>Add the desired concentration of primary anti-lysozyme antibody (see the Table of Materials). Adjust to a final volume of 100 &#181;L in antibody diluent (1x TBS, 1% BSA, 0.05% Tween 20).</li>
		<li>Incubate for 15 min at 37 &#176;C. Keep shaking for an additional 15 min at room temperature.</li>
		<li>Repeat the washing steps 3.1.2.-3.1.6.</li>
		<li>Add 100 &#181;L of antibody diluent. Add the horseradish peroxidase-coupled secondary antibody (HRP-AbII) (see the Table of Materials) at a dilution of 1:500.</li>
		<li>Repeat the washing steps 3.1.2.-3.1.6.</li>
		<li>Keep the nanoparticles in a final volume of 50 &#181;L of antibody diluent.</li>
	</ol>
	</li>
</ol>
4. Experimental mounting
<ol>
	<li>Fill the two 100 &#181;L glass syringes with water, connect a 6.5 cm long hose to each syringe, insert a metal pin into the end of the hose, and place both syringes on the computer-controlled syringe pump.</li>
	<li>Seal all hoses of the acrylic device with heat.</li>
	<li>Cut the inlet hose and keep only a few millimeters. Fill the dispensing needle with wash buffer and insert it into the cut hose. Allow the solution to drip before connecting the needle to the device to prevent air access to the device.</li>
	<li>Cut the outlet hose from the lateral channel. Connect to the syringe pump. Next, do the same procedure for the main channel outlet hose. 
	&#8203;NOTE: It is key to perform steps 4.3.-4.4. in this order to avoid unpacking the microparticle trap. If possible, verify the state of the trap during these steps with the help of a magnifying glass.</li>
	<li>Place a glass slide on the microscope stage. Attach the magnet to the slide with double-sided tape and place a small piece of tape on each side to fix the chip edges to the glass.</li>
	<li>Set a flow rate of 50 &#181;L/h through the Flow Rate and Units tabs in the syringe pump controller. Select the Withdrawing mode and click on the Start button to activate the flow of the wash buffer.</li>
	<li>Carefully, approach the device toward the slide with the magnet in a horizontal manner so that the area of the chip containing the trap contacts the magnet.</li>
	<li>Stick the edges of the device to the glass with double-sided tape to prevent movement. Avoid obstructing the optical path for microscopy (Figure 1C).</li>
</ol>
5. Immunodetection
<ol>
	<li>Keep the wash buffer flowing for 10 min at 50 &#181;L/h to remove excess BSA.</li>
	<li>Remove the remaining wash buffer from the dispensing needle with a micropipette. Add 50 &#181;L of the nanoparticle suspension.</li>
	<li>Flow the suspension of nanoparticles for 7 min at a flow rate of 100 &#181;L/h. Subsequently, change the flow rate to 50 &#181;L/h and flow for another 15 min.</li>
	<li>Change the dispensing needle. Flow the wash buffer for 10 min at 50 &#181;L/h. Prepare the fluorogenic substrate according to the manufacturer&#39;s specifications during the washing step.</li>
	<li>Remove the remaining wash buffer from the dispensing needle with a micropipette. Add 100 &#181;L of the fluorogenic substrate (see the Table of Materials). Flow the fluorogenic substrate for 6 min at 50 &#181;L/h.</li>
	<li>Set the flow rate (1 &#181;L/h, 3 &#181;L/h, 5 &#181;L/h, and 10 &#181;L/h) and time (6 min) measurement parameters in the corresponding Flow Rate and Set Timer tabs of the interface that control the syringe pump. Be sure to select Withdrawing mode for each of the measurements to be performed.</li>
	<li>Set an additional Flow Rate tab at 50 &#181;L/h and Set Timer at 3 min for the wash step.</li>
	<li>Turn on the fluorescence of the microscope 15 s before the substrate at 50 &#181;L/h stops. Start the image capture with the software of the microscope camera 10 s before the substrate stops with an exposure time of 1,000 milliseconds. Perform imaging for 6 min at 1 frame/s (FPS).</li>
	<li>Click on the Start button of the desired flow rate parameter immediately after the substrate wash flow rate at 50 &#181;L/h stops. Click on the Start button of the wash flow (50 &#181;L/h) immediately after the selected measurement flow stops.</li>
	<li>Stop the image capture and turn off the fluorescence of the microscope to avoid photobleaching of the substrate.</li>
	<li>Repeat steps 5.8.-5.10. for each measurement flow rate used.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carbonyl-iron microparticles</td>
			<td>Sigma-Aldrich</td>
			<td>44890</td>
			<td>7 &#956;m</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fermont</td>
			<td>6201</td>
			<td>Health Hazard: Moderate 
			Flammability: None 
			Reactivity: None 
			Contact Hazard: Moderate</td>
		</tr>
		<tr>
			<td>CMOS camera Moment</td>
			<td>Teledyne Photometrics</td>
			<td></td>
			<td>Sensor Technology: CMOS 
			Quantum Efficiency: 73% 
			Pixel Size: 4.5 &#181;m x 4.5 &#181;m 
			Supported Interfaces: USB 3.2 Gen 2</td>
		</tr>
		<tr>
			<td>Dr Engrave Software</td>
			<td>Roland DGA Corporation</td>
			<td></td>
			<td>Engraving software to design and create the engraving path on the surface</td>
		</tr>
		<tr>
			<td>Extraction hood</td>
			<td>Unknown</td>
			<td>Unknown</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible Plastic Tubing</td>
			<td>Tygon</td>
			<td>AAD04103</td>
			<td>ID = 0.020, OD = 0.060</td>
		</tr>
		<tr>
			<td>Fluorescence microsope</td>
			<td>ZEISS</td>
			<td>Axio Vert.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>High Precision Dispense Needle</td>
			<td>Loctite</td>
			<td>98612</td>
			<td></td>
		</tr>
		<tr>
			<td>MagJET Separation Rack</td>
			<td>thermoscientific</td>
			<td></td>
			<td>12 x 1.5 mL</td>
		</tr>
		<tr>
			<td>Polymethylmethacrylate - Sheet - PMMA, Acrylic</td>
			<td>Goodfellow</td>
			<td>ME303018/1</td>
			<td>Thickness: 1.3 mm, Transparency: Clear/Transparent</td>
		</tr>
		<tr>
			<td>PVCamTest software</td>
			<td>Teledyne Photometrics</td>
			<td>Version 3.10.107</td>
			<td>Image acquisition software</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Nikon</td>
			<td>SMZ 7457</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperMag Carboxyl Beads</td>
			<td>Ocean NanoTech</td>
			<td>KSC0100</td>
			<td>100 nm</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>kd Scientific</td>
			<td>KDS200</td>
			<td>Can hold up to two syringes</td>
		</tr>
		<tr>
			<td>Utrasonic bath</td>
			<td>Branson</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>VPanel software</td>
			<td>Windows OS</td>
			<td>Version 1.0.3.0</td>
			<td>Software for controlling the micromilling machine</td>
		</tr>
	</tbody>
</table>

a magnetic nanoparticle-based immunoassay using a microfluidic device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://app.jove.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.

<img alt="Figure 1" src="/files/ftp_upload/22135/22135_Figure1.jpg" /> 
Figure 1: Final device configuration. (A) Acrylic device with the hoses attached to the corresponding inputs and outputs. The scale shows the dimensions of the device in centimeters. (B) Protocol for the formation of the microparticle trap. Microparticles flow through the channel by gravity when the device is placed in a vertical position. Microparticles are conc...

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

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 ...

Take a microfluidic device affixed to a microscope stage, comprising an inlet for dispensing reagents, interconnected channels for fluid flow, and outlets for purging the fluids.
The main channel contains a central staggered restriction with trapped aggregated iron microparticles.
The channels are incubated with a blocking solution to prevent non-specific antibody binding.
Flow a wash buffer, removing excess blocking solution.
Introduce an immunocomplex suspension at a defined flow rate. The immunocomplex consists of magnetic nanoparticles, coated with antigens bound to primary antibodies, which are further bound to peroxidase-conjugated secondary antibodies.
Apply an external magnetic field, magnetizing the aggregated microparticles and transforming them into a magnetic trap for capturing the immunocomplexes.
Introduce a fluorogenic substrate that is converted by the peroxidase into a fluorescent product and flows through the trap.
Using the microscope, compare the fluorescence before and after the trap, confirming the capture of immunocomplexes.

a-magnetic-nanoparticle-based-immunoassay-using-a-microfluidic-device

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

75 Görüntüleme. Kaynak: Hernández-Ortiz, J. A., et al. Manyetik Nanopartikül Tabanlı İmmünoanalizler için Kademeli Kısıtlamalı Bir Mikroakışkan Akrilik Cihazın Bilgisayarlı Sayısal Kontrol Mikro frezelemesi. J. Vis. Uzm. (2022). Bu video, manyetik nanoparçacık tabanlı bir immünolojik test için bir mikroakışkan sistemi göstermektedir. Algılama sistemi, kanalları içinde manyetik bir tuzak içeren bir mikroakışkan cihaz kullanır. Primer ve peroksidaz konjuge sekonder antikorlara bağl?...

Video: Mikroakışkan Bir Cihaz Kullanan Manyetik Nanopartikül Bazlı Bir İmmünoassay - Kavram

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).

Otomatik Enzimbağlantılı İmmünosorbent Assay için Elektrowetting tabanlı Dijital Mikroakışkanplatformu

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

JoVE Journal

Biyomühendislik

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically delivered tumor antibody-targeted magnetic nanoparticles, at temperatures from 39 &#730;C (fever-level) to 1,000 &#730;C (electrocautery) and treatment times from seconds to hours. The temperature-time relationship (thermal dose) dictates the effect with high thermal doses resulting in the tissue ablation and lower thermal doses resulting in sublethal effects such as increased blood flow, accumulation of drugs and immune stimulation. One of the most promising current medical therapies is magnetic nanoparticle hyperthermia&#160; (mNPH). This technique involves activating magnetic nanoparticles, that can be delivered systemically or intratumorally, with a non-invasive, non-toxic alternating magnetic field. The size, construct and association of the magnetic nanoparticles and the frequency and field strength of the magnetic field are major heating determinants. We have developed sophisticated instrumentation and techniques for delivering reproducible magnetic nanoparticle hyperthermia in large and small animal models and cultured cells. This approach, using continuous, real time temperature monitoring in multiple locations, allows for the delivery of well-defined thermal doses to the target tissue (tumor) or cells while limiting non-target tissue heating. Precise control and monitoring of temperature, in multiple sites, and use of the industry standard algorithm (cumulative equivalent minutes at 43 &#730;C /CEM43), allows for an accurate determination and quantification of thermal dose. Our system, which allows for a wide variety of temperatures, thermal doses, and biological effects, was developed through a combination of commercial acquisitions and inhouse engineering and biology developments. This system has been optimized in a manner that allows for the rapid conversion between ex vivo, in vitro, and in vivo techniques. The goal of this protocol is to demonstrate how to design, develop and implement an effective technique and system for delivering reproducible and accurate magnetic nanoparticle therapy (mNP) hyperthermia.

This protocol presents techniques and methodology necessary for the accurate delivery of magnetic nanoparticle hyperthermia using a sophisticated delivery and monitoring system.

Özel Yapım Bir Dağıtım Sistemi Kullanarak Manyetik Nanopartikül Hipertermisinin In Vitro ve In Vivo Teslimatı

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically ...

Tıp

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

Yordam ve anahtar optimizasyon stratejileri için bir otomatik Kapiler elektroforetik tabanlı Immunoassay yöntemi

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

TRANSKRIPT

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device