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Microstereolithography-fabricated microneedles for fluid sampling of histamine-contaminated tuna

inlet port for the sample diluent. incubated overnight with the histamine solutions in a
The devices were fabricated using a photosensitive 4°C refrigerator.
acrylate-based, class-IIa biocompatible polymer known In addition, tuna samples were prepared for a time
by the tradename eShell 200 (Envisiontec, Ferndale, course examination, which involved placing samples
USA) [30] . This material was polymerized into the de- in the refrigerator for up to seven days. Tuna steaks
sired component geometries based on STL files, which again were cut into approximately 100 g individual
were created using computer aided design software. A pieces, placed into vacuum bags, and zip sealed; in
Perfactory III SXGA+ visible light dynamic mask this study, the bags were not vacuumed to remove air.
microstereolithography system with an Enhanced Res- Five time points were used to measure the fish spoi-
olution Module (EnvisionTEC GmbH, Gladbeck, Ger- lage over the course of the seven day period. For the
many) was utilized to fabricate the devices in an addi- “Day 0” time point, a piece of tuna flesh was imme-
tive layer-by-layer manner. A z-direction step size of diately frozen. Tuna flesh from days 1, 3, 5, and 7
50 µm was used to build the devices; the dynamic were also removed from incubation in the refrigerator
mask was illuminated with visible light at a lamp and frozen for histamine testing at a later time.
power of 550 mW. Lastly, a piece of spoiled tuna was examined with
Following the microstereolithography step, the de- the microneedle sampling system procedure and the
vices were rinsed with isoproponal 2–3 times and manufacturer-described procedure to obtain a com-
dried with compressed air. The test strip holders were parison between the two procedures. The tuna flesh
immersed in isopropanol for 15 minutes and sonicated for this study was stored in a vacuum sealed bag con-
in an ultrasonic bath. The parts were then removed, taining 1 × PBS with the air removed by vacuum; the
dried with compressed air, and hand-rinsed with iso- sample was then placed in a refrigerator overnight.
propanol as needed to remove unpolymerized resin. The tuna flesh was originally designated as a negative
Both types of devices were dried in a heated chamber control during the calibration steps described above;
at 30˚C for at least 30 min utes before undergoing a however, it was determined that the tuna flesh was
post curing procedure. For post curing, the parts were spoiled upon acquisition from the source. This occur-
loaded into an Otoflash Post Curing Light Pulsing rence provided an opportunity to study a tuna flesh
Unit (EnvisionTEC GmbH, Gladbeck, Germany) and sample acquired from a commercial source in an au-
exposed to two sets of 2000 light pulses. This units thentic spoilage scenario.
utilizes light pulses in the 300–700 nm spectral range
at 10 Hz to polymerize residual unpolymerized ma- 2.3 Histamine Testing Procedure
terial within the devices [31] . The devices were imaged To conduct the histamine screening, colorimetric lat-
with a VHX-5000 optical microscope (Keyence, eral flow tests were acquired as components of Re-
Itaska, IL, USA). veal for Histamine screening test kits (Neogen Cor-
®
®
2.2 Fish Preparation poration, Lansing, MI, USA), which provide a detec-
tion threshold of 50 ppm. For the microneedle sam-
Tuna steaks that were cut to a 1-inch thickness were pling system procedure, the microneedle array was
used to perform histamine testing. The tuna samples pressed into the tuna sample for 5 seconds to acquire a
were acquired from a local fresh fish market. To fluid sample. The microneedle array was then placed
calibrate the microneedle testing procedure, tuna into the test strip holder as seen in Figure 1. With the
pieces were incubated overnight in histamine solu- microneedle array in place, 1000 µL of the sample
tions within vacuum seal bags in a refrigerator. Pieces diluent provided in the test kit was added to the input
of tuna were trimmed to ~100 g pieces and loaded into port of the test strip holder. This fluid flowed over the
individual vacuum seal bags. Histamine (Sigma-Aldrich microneedle array, washing the test fluid sample into
®
Co. LLC, St. Louis, MO, USA) solutions were prepared the reservoir, where one of the Reveal test strips was
in 1 × phosphate buffered saline (PBS) to 0.5 mg/mL placed to begin the screening. The test strip was al-
and 1.0 mg/mL concentrations; 1 × PBS alone was lowed to incubate in the sample fluid for 10 minutes
used as a negative control. A volume of 5 mL of each or less before the result was determined.
of these solutions was added to individual vacuum bags To more objectively compare positive test results
®
containing the tuna and zip-sealed; most of the air in and negative test results, an Accuscan Gold (Neo-
®
the bags was then removed. The tuna samples were gen Corporation, Lansing, MI, USA) test strip reader
74 International Journal of Bioprinting (2016)–Volume 2, Issue 1