Bio-Doped Microbial Nanosilica as Optosensing Biomaterial for Visual Quantitation of Nitrite in Cured Meats

Abstract

A microbial optosensor for nitrite was constructed based on biomimetic silica nanoparticles, which were doped with R5, a polypeptide component of silaffin, as a robust biosilica immobilization matrix entrapped with Raoultella planticola and NAD(P)H cofactor during the in vitro biosilicification process of silica nanoparticles. Ruthenium(II)(bipy)2(phenanthroline-benzoylthiourea), the chromophoric pH probe, was physically adsorbed on the resulting biogenic nanosilica. Optical quantitation of the nitrite concentration was performed via reflectance transduction of the bio-doped microbial nanosilica at a maximum reflectance of 608 nm, due to the deprotonation of phen-BT ligands in the ruthenium complex, while the intracellular enzyme expression system catalyzed the enzymatic reduction of nitrite. Reflectance enhancement of the microbial optosensor was linearly proportional to the nitrite concentration from 1−100 mg L−1, with a 0.25 mg L−1 limit of detection and a rapid response time of 4 min. The proposed microbial optosensor showed good stability of >2 weeks, great repeatability for 5 repetitive assays (relative standard deviation, (RSD) = 0.2−1.4%), high reproducibility (RSD = 2.5%), and a negligible response to common interferents found in processed meats, such as NO3−, NH4+, K+, Ca2+, and Mg2+ ions, was observed. The microbial biosensor demonstrated an excellent capacity to provide an accurate estimation of nitrite in several cured meat samples via validation using a standard UV-vis spectrophotometric Griess assay.

Keywords: biosensor; nitrite; optic; silaffin; silica.

Figure 1

The FESEM micrograph, at a magnification of 50 k×, of the microbial R5-fusion nanosilica prepared by the biomimetic silicification process at ambient temperature and pressure. The inset shows that the photographic image of the corresponding R5-fusion microbial nanosilica appears to be orangish after immobilized with 0.1 mg mL⁻¹ ruthenium optical pH probe at the surface via physical adsorption.

Figure 2

The UV-vis spectra of absorption peak responses at 455 nm for (a) 0.01 mg mL⁻¹ Ru(II) complex solution, (b) free whole cell R. planticola and 0.048 mM NAD(P)H co-substrate in the 0.01 mg mL⁻¹ Ru(II) complex solution before and (c) after reaction with nitrite in 0.1 M K-phosphate buffer at pH 7.4.

Figure 3

The schematic diagram represents the biomimetic silicification process involving R5 peptide from the silaffin protein of C. fusiformis to initiate the formation of silica nanoparticles with the co-silicification of the microbial intracellular NNR enzyme and cofactor NAD(P)H. Ruthenium complex, the pH optical label, is simply adsorbed to the surface of the R5-fusion nanosilica. The microbial optosensor changes from orange to yellow hue in the presence of nitrite, which can be monitored by the optical reflectometric method.

Figure 4

Reflectance spectra of the Ru(II) complex-modified biomimetic nanosilica support (a) before and (b) after reaction with 100 mg L⁻¹ nitrite in 0.1 M K-phosphate buffer at pH 7.4. The bio-doped microbial nanosilica exhibited a maximum reflectance peak signal at 608 nm, and the reflectance peak signal significantly heightened due to the deprotonation of phen-BT ligands in the ruthenium complex, while the intracellular enzyme expression system catalyzed the enzymatic reduction of nitrite.

Figure 5

The nitrite concentration-dependent reflectometric microbial biosensor response at the nitrite concentration range of 1-1000 mg L⁻¹ in 0.1 M K-phosphate buffer at a neutral pH. The inset shows the dynamic linear response range of the reflectance nitrite biosensor from 1–100 mg L⁻¹ nitrite.

Figure 6

The repeatability performance of the reflectance microbial optosensor at 608 nm, which repeatedly reacted with 20 mg L⁻¹ nitrite for 4 min after washing the bacterial cells biosensor with 0.1 M K-phosphate buffer at pH 7.0.