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3D printing of LFIA
to adapt to these changes and provide rapid solutions to 2.2. Conjugation of the gold nanoparticles
protect our community. To conjugate the gold nanoparticles (AuNP) with S1
The advantage of using 3D printing over the spike protein (40591-V08H, Fisher) and Rabbit antibody,
traditional methods, such as laser cutting, is that 3D as shown in Figure 1A, 1 ml of 1 OD AuNP solution was
printing offers a prototyping method to test multiple used and the pH was adjusted to 8.5-9 using potassium
designs in short period of time. In addition, ease of carbonate (K CO ). The protein was added to the AuNP
accessibility to 3D printers makes them suitable for suspension at a ratio of protein solution: AuNP solution
2
3
prototyping in a laboratory setting. Once the design is of 1:100 (v: v) with a final protein concentration of
tested and finalized using 3D printing technology, it can 10 μg/ml, and the suspension was incubated for 2 h while
be sent out for mass production. mixing. Then, 250 μl of 5% BSA in TRIS buffer was
Here, we developed a LFIA test using multiple added and mixed for an additional 15 min. Afterward,
technologies to efficiently optimize the testing strips. 100 μl of 1% TWEEN 20 in TRIS buffer was added to
Bioprinting and 3D printing techniques were used the suspension and centrifuged for 10 min at 8000×g and
during the development of a rapid test for the detection 4°C. The supernatant was removed, and the precipitate
of antibodies against COVID-19. A material extrusion- was resuspended in 1 ml of Tris buffer (pH 8.5) + 1%
based bioprinting setup utilizing a robotic arm was BSA + 1% TWEEN 20 and centrifuged as before. Finally,
used during the construction of the strips to aid the the precipitate was resuspended in 100 μl of Tris buffer
dispensing of the capturing materials. In addition, an (pH 8.5) + 1% BSA + 1% TWEEN 20 + 20% sucrose to
additive manufacturing technology was used to build a achieve a conjugated AuNP concentration of 10 OD. The
housing unit for the strip in a layer-by-layer manner using same process was used when conjugating the S1 spike
photopolymerization technique. The design of the cassette protein and rabbit antibodies to the AuNP.
was modified as needed to adapt with the rapid changes To confirm the conjugation of the protein to the
in the testing strip during the optimization process. Using gold nanoparticles, UV–Vis Spectrometer (PerkinElmer
finite element analysis (FEA), we were able to simulate Lambda 1050) was used to compare the UV–Vis spectra
the physical strains on the designed cassette to determine of the conjugated AuNP with ligand-free AuNP. We
its minimum thickness while ensuring the practicality of assessed the red shift in peak absorbance between the
use and durability when conducting the test. conjugated AuNP and ligand-free AuNP which can
be used to confirm the conjugation. The sample was
2. Materials and methods scanned from 800 nm to 250 nm with a data interval of
2.1. Materials 1 nm and a scanning speed of 266.75 nm/min. To assess

Forty nanometers Gold NanoSpheres 2 mM citrate (OD=1)
was purchased from nanohybrids. SARS-CoV-2 (2019- A
nCoV) Spike S1-His Recombinant Protein (HPLC-
verified) (Cat. No.: 40591-V08H) and Normal Rabbit
Control IgG (Cat. No.: CR1) were purchased from Sino
Biological. SARS-CoV-2 Spike Protein (S-ECD/RBD)
Monoclonal Antibody (bcb03) (Cat. No.: MA5-35950)
and Goat Anti-Rabbit IgG (H+L) Superclonal Secondary B
Antibody (Cat. No.: A27033) were purchased from Fisher
Scientific. Mouse monoclonal (JDC-10) Anti-Human
IgG Fc (HRP) (Ab99759) was purchased from Abcam.
1×Phosphate-buffered saline (PBS), sucrose, TWEEN 20,
and bovine serum albumin (BSA), potassium carbonate,
and Tris buffer were purchased from Sigma-Aldrich. All
chemicals were used as received, without purification
or modification. Cellulose fiber sample pads, glass fiber
conjugate pads, and high-flow nitrocellulose membrane
were purchased from EMD Millipore Corporation.
Sample pads, conjugate pads, and absorbent pads of
different sizes were purchased from Ahlstrom-Munksjo. Figure 1. (A) Schematic representation of the physical conjugation
Backing cards KN-2211 were purchased from Kenosha. process between the gold nanoparticles and the proteins of interest.
FormLabs Photopolymer Resin White (FLGPWH04) was (B) Detection of conjugated AuNP with antibodies and proteins by
purchased from FormLabs. UV/VIS spectroscopy.

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