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In a previous award, we built a device that measures urinary calcium concentration using a disposable optrode to sample small amounts of urine and a handheld reader. The optrode was coated internally with a solid reagent consisting of calcein and sodium hydroxide (NaOH) buffer. The calcein binds with calcium in the urine and can produce a fluorometric signal when excited by the blue light in the reader. Our prototype worked but in this new project, we are refining the system to elevate the device technology readiness level for eventual spaceflight use.
The project has achieved multiple milestones. 1) Variations on the reagent recipe were tested and finalized. 2) Several new optrode designs were explored, and one design was chosen. 3) Manufacturability of the redesigned optrode tubes was explored and finalized and three hundred tubes were produced for human urine testing.
Regarding each: 1) Our reagent recipe consists of calcein with a buffering agent and the optional inclusion of potassium citrate. We tested whether NaOH or KOH was the preferred buffering agent and whether the inclusion of potassium citrate improved our reagent recipe. After preparing these four different reagent recipes, we created calcium standards (calcium dissolved in DI water) at several concentrations between 0 and 800 mg/L. We plotted the output voltage from our reader for each concentration of calcium standard mixed with each reagent recipe. We favored the recipe with the widest linear response. Calcein with KOH and potassium citrate was the favored recipe.
2) Several optrode concepts were explored. The concept we ultimately chose was a syringe-like design that could be preloaded with 18 microliters of reagent and an additional 2 microliters of urine could be drawn into the optrode. Once the urine was drawn into the tube, it would diffuse and mix with the reagent, producing the fluorescent signal that was detected by our reader.
3) To create a repeatable syringe optrode, we flared a polystyrene rod to create a seal with an outer tube. This formed the syringe. A manufacturing jig was created to ensure repeatability in flaring.
With these three objectives accomplished, we will compare the performance of the prototype device in measuring urinary calcium concentration against the urine chemistry analyzer at Dartmouth Hitchcock Medical Center (DHMC). The evaluation will use both calcium standards as well as human urine collected from 100 volunteers per our Institutional Review Board/IRB-approved protocol.
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