A fluorometer is a device that measures the fluorescence, or the amount of visible light that fluoresces from an object. Fluorescence occurs as a result of exciting electrons in a material by one wavelength of light, such that they immediately emit light of a different wavelength. The mNeonGreen and mCherry pair has good overlap in their spectras, has a great intensity, is bright, and importantly has an extended dynamic linear range when compared to a GFP-RFP pair. Exisiting fluorometers are often used in a laboratory setting, where the device can be set up on a benchtop or lab desk. For this project, the aim was to make the device handheld and accessible for patients to be able to test the levels of key metabolites at home or in a doctor’s office.
Device Design Considerations
Excitation and Emmision
To make the biosensor portable and accessible, the size and cost of many of the precise components of traditional fluorometers could theoretically be reduced or eliminated. These include optical filters, collimating lenses, and high-powered lasers. This prototype included only a coloured LED and a light sensor to excite and detect the emission from the fluorophores. The LED must match the excitation wavelength of the specific fluorophore being examined. For the purposes of this project a GFP-RFP pair was used. GFP can be excited by a wavelength of 488nm, while RFP can be excited by a wavelength of 488 nm or 532 nm, giving overlap for both at 488 nm. This means that the optimal LED to use to excite these fluorophores would be a 488nm LED. The overlap in spectra for the mNeonGreen and mCherry pair can be shown in the figures below.
For a prototype, a blue LED would be the simplest option for excitation, as it is readily available with an appropriate wavelength range of between 450 and 490nm. For further development of the prototype, polarizers would be added to filter out the excitation light and allow only the emission signal to pass through to the photodiode. A laser at the precise wavelength would be the most accurate option.
The device itself is comprised of a microneedle array collection component which is placed onto the patient's forearm to capture interstitial fluid (or ISF) by capillary action. After our minimally-invasive approach of collecting interstitial fluid via a microneedle patch, the fluid is discharged into the fluorescent binding protein rich gold-plated chambers of our fluorometer device. The light source then replaces the microneedle patch to measure fluorescent intensity with a detector housed in the device body and communicates the metabolite concentration to the user through the app.
3D modelling was done using Solidworks. The device design was iterated to conclude with our unique revolver-like design for multiple chambers, one for each analyte detected, enabling convenience and cost-effectiveness by only requiring one detection channel.
The microneedle patches must be one-time use to comply with FDA regulations, but cost-effective manufacturing processes existing for glucose detection and insulin delivery applications could be used, including silicone molding or 3D printing. The casing would be made of metal such as stainless steel, but the patch could use polymers to be more cost effective as a disposable component.
A fluid distribution mechanism is important to get the sample to the antibody solution and buffer I the chambers, and then deal with the waste effectively. The major challenge was considering how to deal with the very low volumes involved. Various options were considered:
1. Extracting the ISF from a membrane on the back of the microneedle patch, after it has migrated throw the needles, directly into the chambers using a syringe.
2. Dispensing the ISF from the microneedle patch into the chambers from each array simultaneously by pressing a button or depressing a soft back on the patch to create pressure forcing the fluid out.
3. The viability of diffusion from the hollow needles into the buffer solution via capillary action. That is, the ISF readily flowing from the needles to the chambers full of buffer.
To further develop the system, an attempt would be made to make microfluidic channels for the ISF to flow into the device chambers, using capillary action to drive the fluid out of the needles.