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Alrashoudi, et al.
the attachment of the proteins to the gold nanoparticles, membrane, and absorbent pad were cut into 5 mm wide
we performed UV–VIS absorption scan in which we strips.
compared AuNP-S1, AuNP-rabbit antibody conjugates to
ligand-free AuNP. The conjugated nanoparticles showed 2.4. Designing and 3D printing of the LFIA strip
a 3 nm shift compared to the ligand-free nanoparticles, cassettes
as shown in Figure 1B, indicating the success of the NX computer-aided design (CAD) software was used in
functionalization of the 1 mL gold nanoparticles to the 10 designing the housing units, along with SolidWorks as a
μg of the S1 protein, and rabbit antibody . The red shift supporting program. NX CAD was mainly used to design
[25]
in peak absorbance is an indication of the enlargement several iterations due to its capability in designing small
of the gold nanoparticle size from the attachment of the features. NX CAD provided the simulation tools needed
proteins to the nanoparticle . to test the assembly of the design before printing; the
[26]
2.3. Assembly of the strip simulation of the designed structure was needed to design
the locking mechanism as it requires a precise sizing to
To assemble the strip, first, the high-flow nitrocellulose ensure proper locking after printing.
membrane was mounted on the backing cards. Dispensing Multiple 3D printing technologies were used to
of the antibodies to the nitrocellulose membrane was ensure the design’s maximum potential in prototyping
done using a setup consisting of a robotic arm (Dobot these small housing units. Material extrusion 3D
Magician, Dobot, China) and a microfluidic pump (ExiGo, printing is the traditional 3D printing method, and vat
Cellix, Ireland), as depicted in Figure 2; this setup could photopolymerization 3D printing was used in prototyping
be considered as material extrusion-based bioprinting. the cassette designs. Using vat photopolymerization, 3D
Dispensing of the antibodies onto the nitrocellulose printing was about its ability to print a small housing unit
membrane was done using a 21 G needle attached to the with fine features in a short period of time. Furthermore,
robotic arm. The antibodies were dispensed at a rate of cassettes were printed with FormLabs white liquid resin
200 μl/min and the robotic arm was programmed to move and commercially available thermoplastic filament
at a speed of 17 mm/s; antibodies were diluted in 1×PBS to keep the housing unit price reasonable. The vat
to be dispensed at a concentration of 200 μg/ml. These photopolymerization 3D printer, FormLabs (Form 3),
parameters ensured that antibodies were dispensed in which was used in printing the housing unit, is capable of
a thick and continuous line. In particular, two antibody printing a hundred units a day.
lines were printed on the nitrocellulose membrane. A test We have devolved several design iterations to
line was printed with anti-human antibodies (Abcam, complement the rapid changes that we encounter during
Ab99759) and a control line was printed with anti-rabbit the test development. The first iteration of the designs
antibodies (Fisher, A27033). A video demonstrating the started with mimicking the commercially available rapid
printing process is provided as the supplementary file. tests . While modifying the test, the cassette design
[27]
The strip was then dried at room temperature for 1 h. was modified as well leading to the final iteration design.
Then, the absorption pad was added to the backing card Using different 3D printing technologies allowed us to try
with a 2-3 mm overlap with the nitrocellulose membrane. and change different features while developing the test.
Finally, the assembled backing card, nitrocellulose
2.5. FEA simulation for the final iteration of the
cassette
LFIA test strips are usually single-use tests, and the
housing units are there to protect the strip. Our aim during
the designing process was to reduce the amount of material
needed to build the cassette, thus reducing wasted and
discarded material from each cassette used. FEA simulation
was used to study the proposed design before printing to
ensure that the design can sustain handling forces without
breaking. The durability of the cassette is not essential in
single-use devices; however, a minimum thickness for the
proposed cassette is required to prevent it from breaking
during the test and handling when polyethylene is used as
a material for the designed cassette.
Figure 2. The printing setup consists of a robotic arm, microfluidics The FEA simulation was conducted using NX
pumps, and dispensing nozzle. software. As NX was the CAD software used to design the

International Journal of Bioprinting (2021)–Volume 7, Issue 4 79