Name: computer numerical control micromilling of a microfluidic acrylic device with a staggered restriction for magnetic nanoparticle-based immunoassays
Uploaded: 2022-06-23T13:10:00
Description: Watch this Scientific Journal Video about Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays at JoVE.com

This work was supported by Conacyt, Mexico under grant 312231 of the &#34;Programa de Apoyos para Actividades Cient&#237;ficas, Tecnol&#243;gicas y de Innovaci&#243;n&#34;, and by AMEXCID and Mexican Foreign Relations Ministry (SRE) under grant &#34;Prueba serol&#243;gica r&#225;pida, barata y de alta sensibilidad para SARS-CoV-2&#34;. JAHO thanks Conacyt Mexico for their PhD scholarship.

1. Micromilling
<ol>
	<li>Surface grinding
	<ol>
		<li>Turn on the micromilling machine and the piezoelectric controller. Start their respective control software12.</li>
		<li>Select the required end mill bits (200 &#181;m and 800 &#181;m diameters). Place them in the appropriate compartment of the milling machine (Figure 1).</li>
		<li>Cut 9 mm x 25 mm rectangles of 1.3 mm thick PMMA with the 800 &#181;m end mill bit. Attach one of these rectangles...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-actuated+monolithic+acrylic+microfluidic+valves+and+pumps.">Pressure-actuated monolithic acrylic microfluidic valves and pumps.</a> Lab on a Chip. 18 (4), 662-669 (2018).</li><li>Guevara-Pantoja, P. E., Chavez-Pineda, O. G., Solis-Serrano, A. M., Garcia-Cordero, J. L., Caballero-Robledo, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+affordable+3D-printed+positioner+fixture+improves+the+resolution+of+conventional+milling+for+easy+prototyping+of+acrylic+microfluidic+devices.">An affordable 3D-printed positioner fixture improves the resolution of conventional milling for easy prototyping of acrylic microfluidic devices.</a> Lab on a Chip. 20 (17), 3179-3186 (2020).</li><li>Friedrich, C. R., Vasile, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+micromilling+process+for+high-aspect-ratio+microstructures.">Development of the micromilling process for high-aspect-ratio microstructures.</a> Journal of Microelectromechanical Systems. 5 (1), 33-38 (1996).</li><li>Malayath, G., Sidpara, A. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-pressure,+high-temperature+thermal+bonding+of+polymeric+microfluidic+devices+and+their+applications+for+electrophoretic+separation.">Low-pressure, high-temperature thermal bonding of polymeric microfluidic devices and their applications for electrophoretic separation.</a> Journal of Micromechanics and Microengineering. 16 (8), 1681-1688 (2006).</li><li>Tsao, C. W., Hromada, L., Liu, J., Kumar, P., DeVoe, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+bonding+of+PMMA+and+COC+microfluidic+substrates+using+UV/ozone+surface+treatment.">Low temperature bonding of PMMA and COC microfluidic substrates using UV/ozone surface treatment.</a> Lab on a Chip. 7 (4), 499-505 (2007).</li><li>Bamshad, A., Nikfarjam, A., Khaleghi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+simple+and+fast+thermally-solvent+assisted+method+to+bond+PMMA-PMMA+in+micro-fluidics+devices.">A new simple and fast thermally-solvent assisted method to bond PMMA-PMMA in micro-fluidics devices.</a> Journal of Micromechanics and Microengineering. 26 (6), 065017 (2016).</li><li>Ogilvie, I. R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+surface+roughness+for+optical+quality+microfluidic+devices+in+PMMA+and+COC.">Reduction of surface roughness for optical quality microfluidic devices in PMMA and COC.</a> Journal of Micromechanics and Microengineering. 20 (6), 065016 (2010).</li></ol>

An acrylic microfluidic device for immunoassays using nanoparticles as immunosupport was fabricated using a micromilling technique. The method of direct manufacturing on the substrate has the advantage of avoiding the use of a master mold and the time and costs that this implies. However, it is limited to rapid prototyping and high-volume device manufacturing.
Here, we used a previously reported accessory piezoelectric platform for the milling machine12. The platform wa...

In recent years, microfluidics has played an important role in immunoassay techniques1. Miniaturization technology has many outstanding advantages compared to traditional immunoassays, such as reduced sample and reagent consumption, shorter incubation times, efficient solution exchange, and higher integration and automation2.
Furthermore, microfluidic systems in immunoassays, in association with magnetic nanoparticles as immunosupport, considerably reduce incubation times, achieving high detection sensitivity due to the increased surface-to-volume ratio3. Brownian movement of the particles improves the reaction kinetics during the formation of the antigen-antibody complex4,5. Moreover, the magnetic properties of nanoparticles provide the versatility to be integrated into different microfluidic device configurations, making them an ideal candidate for signaling and molecule capture in miniaturized on-chip biosensing systems5. However, magnetic forces are significantly weaker than drag forces at the nanometer scale due to the high surface-to-volume ratio6. Therefore, capturing nanoparticles for crucial immunoassay steps such as washing and detection can be challenging, and a conventional magnet is insufficient4.
An efficient way to manipulate the nanoparticles is the use of a microfluidic magnetic trap formed by iron microparticles, which are packed in a microfluidic structure3. Therefore, when an external magnet approaches, a complex interaction is created within the magnetized porous medium between the magnetic and flux forces. The magnetic force acting on the nanoparticles is strong enough to capture them and resist flow drag3,4,7. This approach requires microfabrication techniques that achieve resolutions in the order of a few micrometers to generate micrometric structures that retain the microparticles.
Current microfabrication techniques allow the high-resolution fabrication of structures from a few microns to hundreds of nanometers8. However, many of these techniques require specialized, expensive, or complicated equipment. One of the main difficulties is the requirement for a cleanroom for mold fabrication, which remains costly and time-consuming8,9. Recently, microfluidic engineers have overcome this drawback by developing a variety of alternative fabrication methods, with various advantages such as reduced costs, faster turnaround times, cheaper materials and tools, and increased functionality8. In this way, the development of new microfabrication techniques brought low-cost, non-cleanroom methods that achieve resolutions as low as 10 &#181;m8. Patterning can be used directly on a substrate without generating an expensive molding pattern, thus avoiding a time-consuming process. Direct fabrication methods include CNC milling, laser ablation, and direct lithography8. All these methods are suitable for producing high-aspect-ratio channels in a wide range of materials, regardless of their hardness9, enabling new and advantageous geometries, physical behaviors, and qualities in microfluidic devices8.
CNC micromilling creates microscale structures using cutting tools that remove bulk material from a substrate and is an effective fabrication method for microfluidic devices10,11. The micromilling technique can be useful in microfluidic applications to create microchannels and features directly on the work surface, offering a key advantage: a workpiece can be fabricated in a short time (less than 30 min), significantly reducing the turnaround time from design to prototype12. In addition, the wide availability of cutting accessories of different materials, sizes, and shapes makes CNC milling machines a suitable tool that has allowed the fabrication of different features in many types of low-cost disposable materials13.
Among all the materials commonly used in micromilling, thermoplastics remain a leading choice due to their many favorable properties and compatibility with biological applications10,14. Thermoplastics are an attractive substrate for microfluidic systems due to their significant advantages for developing low-cost, disposable analytical systems9. In addition, these materials are highly amenable to high-volume manufacturing processes, making them suitable for commercialization and mass production. For these reasons, thermoplastics such as PMMA have been considered reliable and robust materials since the early years of microfluidics10. Different protocols have been described to fabricate closed channels in thermoplastics, such as solvent bonding15, heat bonding16, and ultraviolet (UV)/ozone surface treatment bonding17.
In many cases, the positioning resolution achieved with conventional micromilling machines is not sufficient for some microfluidic applications that require structures smaller than 10 &#181;m. High-end micromilling has enough resolution. Unfortunately, due to high prices, its use is limited to a handful of users12. Previously, our research group reported the fabrication and manipulation of a low-cost tool that allows machining structures of less than 10 &#181;m, overcoming the resolution of conventional milling machines12. The fixture is a platform manufactured by 3D printing with simple electronics, containing three piezoelectric actuators. The surface contains hinge-shaped joints that allow it to be lifted when the piezoelectric elements act simultaneously. Z-axis displacement can be controlled with a resolution of 500 nm and an accuracy of &#177;1.5 &#181;m12.
This paper presents the steps of the manufacturing process of an acrylic device (PMMA) through a micromilling technique. The chip design consists of a main channel 200 &#181;m wide and 200 &#181;m high and a side channel with the same dimensions to purge the flow of the reagents. In the central region, the channel is interrupted by a physical restriction of only 5 &#181;m in height, fabricated with the 3D-printed piezoelectric platform made by this group12, to capture magnetic microparticles that make up a magnetic trap for nanoparticles by placing an external magnet. We show the operation of the microfluidic device by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device combines different features that make it unique4: the use of magnetic nanoparticles as immune support reduces the total test time from hours to minutes; using a fluorogenic enzyme for detection allows for limits of detection that are comparable to those of standard enzyme-linked immunosorbent assays (ELISAs); and the use of a thermoplastic as a fabrication material makes it compatible with mass production, which was not the case for previous microfluidic nanoparticles&#39; magnetic traps3, and makes it an excellent candidate to develop POCT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.008 Endmill</td>
			<td>KYOCERA SGS&#160;</td>
			<td>2204</td>
			<td>2FL 0.008x1/8x0.12x1-1/12</td>
		</tr>
		<tr>
			<td>0.032 Endmill</td>
			<td>KYOCERA SGS&#160;</td>
			<td>2228</td>
			<td>2FL 0.032x1/8x0.48x1-1/12</td>
		</tr>
		<tr>
			<td>Carbonyl-iron microparticles&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>44890</td>
			<td>7 &#956;m&#160;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fermont</td>
			<td>6201</td>
			<td>Health Hazard: Moderate 
			Flammability: None 
			Reactivity: None 
			Contact Hazard: Moderate&#160;</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&#160;</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>Homemade piezoelectric controller application</td>
			<td>LabView&#160;</td>
			<td></td>
			<td>See reference 12 for more details.</td>
		</tr>
		<tr>
			<td>Loctite 495 instant adhesive</td>
			<td>Henkel</td>
			<td>49503</td>
			<td>Apply with micropipette tip or dispensing needle&#160;</td>
		</tr>
		<tr>
			<td>MagJET Separation Rack</td>
			<td>thermoscientific</td>
			<td></td>
			<td>12 x 1.5 mL</td>
		</tr>
		<tr>
			<td>Mechanic press</td>
			<td>Home-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milling Machine</td>
			<td>Roland</td>
			<td>MDX-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric platform&#160;</td>
			<td>Home-made</td>
			<td></td>
			<td>See reference 12</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&#160;</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>&#160;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&#160;</td>
			<td>Windows OS</td>
			<td>Version 1.0.3.0</td>
			<td>Software for controlling the micromilling machine</td>
		</tr>
	</tbody>
</table>

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.

It was possible to establish a highly reproducible fabrication protocol that improves the resolution of the conventional micromilling technique. Using this protocol, the fabrication of a channel as small as 5 &#181;m in height that operates as a staggered restriction in a 200 &#181;m high channel is achieved. The simple design of the staggered restriction captures iron microparticles of 7.5 &#181;m diameter which, when compacted in the microchannel, allow the creation of a magnetic trap when an external magnet approaches...

Watch this Scientific Journal Video about Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays at JoVE.com

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

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.

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

Our protocol describes a high-resolution micromilling method to fabricate an acrylic microfluidic device for quantitative immunodetection of analyte concentrations comparable to the gold standard ELISA technique. The highlight is the fabrication of a simple and low-cost thermoplastic microfluidic device without a clean room, which allows for shorter assay times and good detection limits in a quantitative manner. Begin by surface grinding.Cut 9 x 25 millimeter rectangles of 1.3 millimeter thick PMMA with the 800 micrometer end mill bit. Attach one of these rectangles carefully with double-sided adhesive tape to the piezoelectric platform. Connect and place the Z-sensor on the surface of the PMMA rectangle.Select the detection pin and move it over the sensor surface. Lower the pin manually without contacting the sensor. Activate the Z-zero sensing mode.Spin the 200 micrometer end mill bit at 14, 500 rpm. Slowly lower it to the origin coordinate on the z-axis. Then reset the z-axis 30 micrometers below the origin.Set this coordinate as the new Z-origin. Click on the Cut button in the micromilling machine software to activate the cut panel. Click on the Add button and select the TXT file with a previously created code for the grinding of the acrylic surface.Click on the Output button to start the process. For milling of the five micrometer restriction, first, set the speed of rotation of the end mill bit to 11, 000 rpm. Then, raise the platform by 6.5 micrometers with the interface of the piezoelectric platform.Move the end mill bit along the y-axis by 500 micrometers. Return the piezoelectric platform to its initial value on the z-axis with the control interface. Next, perform milling of microchannels by opening the previously created design file from the design software.Click on the Print button, access the properties menu and click on the color window corresponding to the layer containing the design to be machined. Set the fabrication parameters in the tool panel. For milling of holes, switch to the 800 micrometer end mill bit.Activate the design layer of the 1.2 millimeter diameter holes by clicking on the corresponding color window and selecting the corresponding fabrication parameters. Machine two additional holes on contralateral corners of the rectangle for the alignment of the acrylic in an inverted manner on a new platform. Flip the acrylic and tape it with double-sided adhesive tape over the adapter with the machined pillars.Open the file with the design of the holes for the opposite face from the design software. Mill the remaining half of the reagent inlet and outlet holes with a diameter of 1.5 millimeters and a depth of 0.7 millimeters. Clean both acrylic rectangle sheets with isopropyl alcohol and rinse with distilled water.Immerse the acrylic in an ultrasonic bath for 10 minutes. Dry both acrylic sheets and tape them to the inside of a glass Petri dish lid with double-sided tape. Then place the base of the glass Petri dish inside a bigger glass Petri dish.Pour one milliliter of chloroform into the base of the Petri dish and quickly place the lid with the acrylic sheets. Immediately add distilled water to the base of the bigger Petri dish up to the level of the Petri dish lid. Allow exposure of the acrylic to chloroform gas for one minute.Then tilt the Petri dish to break the water seal and immediately uncover the Petri dish. For bonding, align both acrylics with the chloroform exposed sides face-to-face and form a sandwich. Place the acrylics in the press for two intervals of two minutes, changing the alignment of the acrylic.Then attach two to three centimeters of hose to each of the holes of the device with instant drying liquid adhesive. Fill the channels with distilled water using a syringe. Immerse the device in an ultrasonic bath for 10 minutes.Then empty the water inside the device channels and use a syringe to introduce 5%BSA solution. Prepare a suspension of iron microparticles of diameter at 7.5 micrometers in 5%BSA. Incubate the chip and the microparticle suspension with the blocking solution for at least one hour at room temperature.Next, insert the microparticles into the chip with a syringe needle through the side channel outlet hose. Place the chip vertically, then rotate the chip in two steps of 90 degrees such that the microparticles target and compact at the five micrometer restriction. Seal all hoses of the acrylic device with heat.Cut the inlet hose until only a few millimeters are left. Fill the dispensing needle with wash buffer and insert it into the cut hose. Allow the solution to drip and then connect the needle to the device.Cut the outlet hose from the lateral channel, then connect to the syringe pump. Next, repeat the same procedure for the main channel outlet hose. Then attach the chip to the magnet.For immunodetection, keep the wash buffer flowing for 10 minutes at 50 microliters per hour. Remove the remaining wash buffer from the dispensing needle with a micropipette and add 50 microliters of the nanoparticle suspension. Flow the nanoparticle suspension for seven minutes at a flow rate of 100 microliters per hour.Then change the flow rate to 50 microliters per hour and continue the flow for another 15 minutes. Change the dispensing needle and flow the wash buffer for 10 minutes at the same rate. Remove the remaining wash buffer from the dispensing needle with a micropipette and add 100 microliters of the fluorogenic substrate.Adjust the flow rate and time parameters for substrate input, fluorescence measurement, and wash step. Activate the input flow of the fluorogenic substrate for six minutes at 50 microliters per hour. 15 seconds before the substrate flow stops, turn on the fluorescence of the microscope.Start the image capture with the software of the microscope camera 10 seconds before the substrate stops with an exposure time of 1, 000 milliseconds. Click on the Start button of the desired flow rate parameter immediately after the substrate wash stops. Perform imaging for six minutes at one frame per second.Click on the Start button of the wash flow immediately after the selected measurement flow stops. Lysozyme conjugated nanoparticles and horse radish peroxidase conjugated secondary antibody were used for the immunoassay. An increase in fluorescence intensity for different concentrations of primary antibody was observed comparing regions before and after the trap, showing that the change in substrate fluorescence is directly proportional to the concentration of primary antibody.For a given concentration of primary antibody, the fluorescence intensity was plotted as a function of time at different flow rates of the fluorogenic substrate. The conversion capacity of the substrate by the HRP enzyme was inversely proportional to the flow rate, a maximum intensity was obtained for a flow rate of one microliter per hour. For the different flow rates for various primary antibody concentrations, curves of the fluorescence differences after and before immunoreaction showed that for a concentration of 1, 000 nanograms per milliliter, the fluorescence saturates for all the evaluated flow rates.A calibration curve was prepared using the maximum value of the differences in fluorescence intensity obtained with respect to the concentration of primary antibody for each flow rate. The high variability and high fluorescence levels at one microliter per hour suggested that the rate does not favor the flow of the reacting substrate and tends to accumulate just after the trap. Special attention should be paid to the sealing steps of the microchannels since exposure to chloroform is very sensitive to temperature.For reproducible results, the temperature must be always the same. Our system helps to understand where compactness and size of microparticles, nanoparticle size, antigens, detection antibody and substrate are determining factors for the limit of detection in nanoparticle-based microfluidic immunoassay.

Research

JoVE Journal

Engineering

Terahertz Microfluidic Sensing Using a Parallel-plate Waveguide Sensor

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In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging

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Aortic regurgitation (AR) refers to backward blood flow from the aorta into the left ventricle (LV) during ventricular diastole. The regurgitant jet ...