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

Summary

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.

Abstract

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 µm 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.

Introduction

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 movem....

Protocol

1. Micromilling

1. Surface grinding
   1. Turn on the micromilling machine and the piezoelectric controller. Start their respective control software¹².
   2. Select the required end mill bits (200 µm and 800 µm diameters). Place them in the appropriate compartment of the milling machine (Figure 1).
   3. Cut 9 mm x 25 mm rectangles of 1.3 mm thick PMMA with the 800 µm end mill bit. Attach one of these rectangles carefully with double-sided adhesive tape to the piezoelectric platform (Figure 2).
      NOTE: Make sure to always place the acrylic ....

Results

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 µm in height that operates as a staggered restriction in a 200 µm high channel is achieved. The simple design of the staggered restriction captures iron microparticles of 7.5 µm diameter which, when compacted in the microchannel, allow the creation of a magnetic trap when an external magnet approaches the device. Th.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.008 Endmill	KYOCERA SGS	2204	2FL 0.008x1/8x0.12x1-1/12
0.032 Endmill	KYOCERA SGS	2228	2FL 0.032x1/8x0.48x1-1/12
Carbonyl-iron microparticles	Sigma-Aldrich	44890	7 μm
Chloroform	Fermont	6201	Health Hazard: Moderate Flammability: None Reactivity: None Contact Hazard: Moderate
CMOS camera Moment	Teledyne Photometrics		Sensor Technology: CMOS Quantum Efficiency: 73% Pixel Size: 4.5 µm x 4.5 µm Supported Interfaces: USB 3.2 Gen 2
Dr Engrave Software	Roland DGA Corporation		Engraving software to design and create the engraving path on the surface
Extraction hood	Unknown	Unknown
Flexible Plastic Tubing	Tygon	AAD04103	ID = 0.020, OD = 0.060
Fluorescence microsope	ZEISS	Axio Vert.A1
High Precision Dispense Needle	Loctite	98612
Homemade piezoelectric controller application	LabView		See reference 12 for more details.
Loctite 495 instant adhesive	Henkel	49503	Apply with micropipette tip or dispensing needle
MagJET Separation Rack	thermoscientific		12 x 1.5 mL
Mechanic press	Home-made
Milling Machine	Roland	MDX-50
Piezoelectric platform	Home-made		See reference 12
Polymethylmethacrylate - Sheet - PMMA, Acrylic	Goodfellow	ME303018/1	Thickness: 1.3 mm, Transparency: Clear/Transparent
PVCamTest software	Teledyne Photometrics	Version 3.10.107	Image acquisition software
Stereo microscope	Nikon	SMZ 7457
SuperMag Carboxyl Beads	Ocean NanoTech	KSC0100	100 nm
Syringe pump	kd Scientific	KDS200	Can hold up to two syringes
Utrasonic bath	Branson	2800
VPanel software	Windows OS	Version 1.0.3.0	Software for controlling the micromilling machine