Rapid detection of φX-174 virus based on synchronous fluorescence of tryptophan

Abstract

The development of rapid methods for the detection of virus particles based on their intrinsic fluorescence (the native auto-fluorescence that originates from the non-labeled analyte) is challenging. Pure viruses may be detected in filtered solutions, based on the strong fluorescence of the amino acid tryptophan (Trp) in their proteins. Nevertheless, Trp also exists in high quantities in the hosts and host cultivation media. In this work, we developed a new method for the detection of the naked φX-174 virus . We show that a separation of φX-174 from its Escherichia coli host (grown on the standard cultivation medium nutrient agar) by simple extraction and filtration is not sufficient for its detection based on the intrinsic fluorescence since ~ 70% of the Trp fluorescence is derived from impurities. We formulate a new cultivation medium with a very low Trp concentration. We apply synchronous fluorescence measurements to show that no Trp fluorescence is detected in the extract solution upon incubation of this medium substrate with ammonium acetate extraction buffer. Finally, we apply synchronous fluorescence to detect φX-174 based on the spectral fingerprint of its native Trp content. Such a method is more rapid than usual traditional separation and detection methods which can take several hours and does not require any addition of labeling agents such as fluorescent dyes or antibodies for the detection. As other virus species contain Trp as one of the amino acids presents in their proteins, this method has the potential to apply to the detection of other viral species. Graphical Abstract

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“…5,22 To further characterize the ES of ARPE19 cells and study whether NAD(P)H can be released with and without application of an external potential, we measured the 2D-fluorescence spectra (λ Excitation = 260−400 nm, λ Emission = 300−550 nm) of the ES of intact cells and after 10 min of chronoamperometry treatment. The obtained spectra showed a peak with a maximum around (λ Excitation = 300 nm, λ Emission = 350 nm) that corresponds to the spectral fingerprint of the amino acids tyrosine and tryptophan 43,44 (Figure 2d), and a minor peak corresponding to the fingerprint of NAD(P)H (λ Excitation = 350 nm, λ Emission = 450 nm). 5 The intensity of both peaks increased after chronoamperometry treatment (Figure 2e).…”