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3D printing of LFIA
cassette, it was also used to perform the FEA simulation gold nanoparticles to capture SARS-CoV-2 antibodies
to ensure accurate simulations results for the proposed in the sample tube. Then, the testing lines consisting of
design. Furthermore, before FEA simulation, the two secondary antibodies were printed on the nitrocellulose
sides of the cassette were assembled without restricting membrane to capture primary antibodies, and the strip
the locking mechanisms to mimic the printing assembly. was placed in a 3D-printed housing cassette after careful
This setting gave a better understanding of the behavior design and strain simulation.
of the dipsticks while exerting force (F). The type of
element applied during this simulation was tetrahedral, 3.1. LFIA cassette designs and 3D printing
with an element size equal to 1.43 mm. During the The housing unit design went through different iterations
simulation, one side of the cassette was fixed, while the for adapting the changes and requirements for the
other side was the location of the applied force to the test. These iterations were designed, fabricated, and
negative z-axis (Figure S1). assembled in the laboratory using specific requirements.
The first two iterations are illustrated in the supplemental
2.6. Testing of the assembled strip documents (Figures S2 and S3). These designs were
The assembled strip was added to the cassette. A solution made for testing using a full strip setup which includes
of AuNP-S1 and AuNP-rabbit antibody at a concentration the conjugate pad. Alternatively, the third iteration was a
of 10 OD was mixed together at a ratio of 1:1. The dipstick design which was made to house a strip that does
solution was then used by adding 10 μl of the mixture not contain a conjugate pad. This provides a faster way
into an Eppendorf tube, as shown in Figure 3. Afterward, to test the strip by removing an extra step that adds the
a 1 μl sample was added to the mixture and vortexed for 5 conjugated AuNP to the conjugate pad.
s. Here, we used SARS-CoV-2 Spike Protein Monoclonal In this study, we used two 3D printing technologies
Antibody (MA5-35950) at a concentration of 200 ug/ml to print the proposed cassettes. Material extrusion 3D
as a positive sample and 1×PBS was used as a negative printing technology using thermoplastic filament was
sample. Then, the strip was submerged into the AuNP developed in the early 1990’s . In the material extrusion
[28]
mixture until the strip fully absorbed the solution. Finally, 3D printing technique, a thermoplastic filament is fused
it was submerged into another tube that contains 1×PBS using a mounted motor, which heats and melts the
as a running buffer to wash strips from any remaining filament to be extruded during the printing process. On
particles. the other hand, vat photopolymerization technique uses
a liquid photopolymer resin as a printing material, which
3. Results and discussion is subjected to polymerization initiated by a projected
Here, we developed by assembling all the components laser. This process would selectively solidify the liquid
a successful LFIA test that detects IgG antibodies. resin against the platform, creating a 3D construct in a
[29]
First, we conjugated SARS-CoV-2 spike protein to layer-by-layer fashion . The advantages of using vat
photopolymerization over material extrusion 3D printing
are the higher printing resolution and smoother finish
surface . Table 1 provides a summary comparing the
[30]
two 3D printing technologies. Figure 4 demonstrates a
schematic of the process used to fabricate our cassettes
using these two printing methods.

3.2. Dipstick housing unit
The dipstick housing unit design consists of a smaller unit
with an opening from one side to be used for dipping the
strip in the test solution as demonstrated in Figure 5A.
This unit was engineered and designed by taking into
consideration the security of the testing strip, as shown in
Figure 5B. Since this design has a wide opening, it was
essential to secure the strip, especially during the testing
process. Therefore, a pressing locking mechanism was
designed inside the housing unit, as shown in Figure 5C.
Furthermore, the dipstick design was 3D printed using
Figure 3. Illustration of an assembled dipstick test using the Form 3 printer (FormLabs) while taking into consideration
conjugated gold nanoparticles. the resolution of the used material.
80 International Journal of Bioprinting (2021)–Volume 7, Issue 4