Highly Sensitive Fluorometric Acetone Biosensor Using Hemi-Ellipsoidal Mirror Optics for Efficient Light Collection

Fiber-optic systems are fundamental optical platforms for constructing fluorometric biosensors. However, their light-collection efficiency at the fiber tip is inherently limited by the numerical aperture and is not particularly high. This poses a challenge for biosensors that require detecting trace molecules, such as in transcutaneous acetone gas sensing. Here, we propose a fluorescence light-collection approach that employs a hemi-ellipsoidal mirror that can be readily fabricated using a stereolithography-based 3D printer and a commercially available mirror-finish spray paint. Because an ellipse has two focal points, light emitted from one focus is reflected by the ellipsoidal surface and converges at the opposite focus. Based on this principle, we constructed and evaluated a hemi-ellipsoidal mirror-based fluorometric biosensor equipped with a flow cell and a photomultiplier tube (PMT) positioned at each focus. A secondary alcohol dehydrogenase (S-ADH) was employed in the fluorometric biosensor, which selectively reduces acetone and consumes the reduced form of nicotinamide adenine dinucleotide (NADH) with an autofluorescence property (ex 340 nm, fl 490 nm). The hemi-ellipsoidal mirror was fabricated by manually polishing the inner surface of a 3D-printed hemi-ellipsoidal shell, followed by coating with a mirror-finish spray to achieve a mirror surface. A collimated UV-LED light source, bandpass filters, a flow cell, and a PMT were aligned to assemble the system. Using this setup, NADH concentrations from 94 nM to 1 mM were quantified, achieving a limit of quantification nine times lower than that of a conventional fiber-optic system. Also, integrating an S-ADH-immobilized membrane enabled real-time monitoring of the acetone reduction reaction via fluorescence decrease. The dynamic range for acetone was 42 nM to 1 mM (14 times more sensitive than fiber-optics). These findings demonstrate that the hemi-ellipsoidal mirror enables highly sensitive fluorescence detection and holds strong potential for application in transcutaneous acetone gas sensing. Furthermore, the easily fabricable hemi-ellipsoidal mirror system showed high potential as a platform for fluorometric biosensors, capable of replacing fiber-optic systems.