Engineering a chemical implementation device and an imaging device for detecting chemiluminescence with a Polaroid™ high‐speed detector film: application to influenza diagnostics with the ZstatFlu®‐II test

Abstract

We describe the engineering and product development of the chemiluminescent ZstatFlu-II Test kit for influenza diagnostics. The reaction vessel is a chemical implementation device with a polystyrene bottom chamber and a polypropylene top chamber that screw together. The patient's specimen is dispersed in a proprietary diluent and mixed inside the bottom chamber with the influenza viral neuraminidase-specific substrate, 1,2-dioxetane-4,7-dimethoxy-Neu5Ac. Neuraminidase catalysis releases the dioxetane. The top chamber contains 40% NaOH and is sealed at the top with an ABS plastic plug-crush pin assembly. The top chamber floor is 85% thinner at the centre, forming a frangible flap. An automated imaging device serves as an incubator for the chemical implementation devices and also facilitates the piercing of the flap by the crush pin. This action results in NaOH flushing into the bottom chamber, initiating chemiluminescence. The imaging device also exposes the Polaroid high-speed detector film to chemiluminescence. At the end of exposure, the film is automatically processed and ejected. Chemiluminescence from an influenza virus-positive specimen produces a "+"-shaped white image, archiving the diagnostic outcome. The modular ZstatFlu-II test kit components are easily adaptable for the chemiluminescent detection of a wide range of analytes.

Synthesis of novel N-acetylneuraminic acid derivatives as substrates for rapid detection of influenza virus neuraminidase

“…21–30 The α configuration of 1 and 2 were ascertained by the chemical shift ( δ 3.05 and 3.07) in the 1 H NMR spectrum for the signal of H-3eq which was diagnostic for the α configuration. 16, 31 It was also supported by the fact that 1 and 2 were cleavable by the neuraminidases from influenza virus as shown later. 32–33 …”

“…13–15 ZstatFlu and ZstatFlu-II tests exploit small molecular derivatives of N -acetylneuraminic acid coupling with a chromogenic or luminescent substance to directly detect influenza viral neuraminidase activity, exhibiting excellent specificity towards influenza virus A and B. 16–18 Bioluminescence-based assays have been used in many applications because of their comparatively high sensitivity. Among the biochemiluminescent substrates, luciferin in the firefly luciferin-luciferase system is best known and well characterized.…”

ZstatFlu®‐II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics

“…The non-specific chemiluminescent sialidase/neuraminidase (the two terms are used interchangeably) substrate, spiroadamantyl-1,2-dioxetane-Neu5Ac (Neuraminidase 'Star') chemiluminescent b-galactosidase assay kit (Galacton Plus and Galacton Star), and the specially formulated, neutral pH, 10Â light enhancer solutions, Emerald-II and Sapphire-II, were purchased from Applied Biosystems (Bedford, MA). The influenza viral neuraminidase-specific substrate, spiroadamantyl-1,2-dioxetane-4,7-dimethoxy-Neu5Ac was prepared in collaboration with Applied Biosystems according to the synthetic and purification procedures described previously (13,18,20). UV-inactivated influenza virus was obtained from Advanced Biotechnologies (Columbia, MD).…”

“…The ZstatFlu 1 -II test was carried out in a twinchambered reaction vessel called the Chemical Implementation Device, which was manufactured to our specifications (20). The top and bottom chambers screwed together to form a single-use, disposable unit.…”

“…The chemiluminescent influenza viral neuraminidase-specific substrate is composed of a chemiluminescent spiroadamantyl-1,2-dioxetane that is Oglycosidically linked to the recognition portion, the 4,7dimethoxy-N-acetylneuraminic acid. Enzymatically-released 1,2-dioxetane undergoes decomposition in the presence of the strong base, accompanied by light emission (5,8,(16)(17)(18)20). engineering and product development of the ZstatFlu 1 -II test kit components are described elsewhere (20).…”