Penicillin Detection by Tobacco Mosaic Virus-Assisted Colorimetric Biosensors

Abstract

The presentation of enzymes on viral scaffolds has beneficial effects such as an increased enzyme loading and a prolonged reusability in comparison to conventional immobilization platforms. Here, we used modified tobacco mosaic virus (TMV) nanorods as enzyme carriers in penicillin G detection for the first time. Penicillinase enzymes were conjugated with streptavidin and coupled to TMV rods by use of a bifunctional biotin-linker. Penicillinase-decorated TMV particles were characterized extensively in halochromic dye-based biosensing. Acidometric analyte detection was performed with bromcresol purple as pH indicator and spectrophotometry. The TMV-assisted sensors exhibited increased enzyme loading and strongly improved reusability, and higher analysis rates compared to layouts without viral adapters. They extended the half-life of the sensors from 4 - 6 days to 5 weeks and thus allowed an at least 8-fold longer use of the sensors. Using a commercial budget-priced penicillinase preparation, a detection limit of 100 µM penicillin was obtained. Initial experiments also indicate that the system may be transferred to label-free detection layouts.

Capacitive Field-Effect EIS Chemical Sensors and Biosensors: A Status Report

“…More recently, a novel promising approach for the development of EnEIS biosensors was described in [ 103 , 114 ], where a highly sensitive penicillin biosensor with a superior lifetime was realized by means of modification of a Ta 2 O 5 -gate EIS structure with tobacco mosaic virus (TMV) particles as scaffolds for the dense immobilization of enzymes. The TMV has a nanotube-like structure with an average length of 300 nm, an outer diameter of 18 nm, and an internal channel of 4 nm in diameter [ 115 , 116 ].…”

Immuno-Based Molecular Scaffolding of Glucose Dehydrogenase and Ferrocene Mediator on fd Viral Particles Yields Enhanced Bioelectrocatalysis

“…In pioneering works, Wege et al reported that adsorbing GOx-functionalized tobacco mosaic virus nanotubes onto a sensor electrode surface did not actually enhance O2-mediated catalysis, but significantly improved enzyme loading and sensor reusability. [30][31][32] The design of a bioanode bearing a random mesh of M13 bacteriophage particles decorated with GOx has also been reported, but in that case the viral particles were "mineralized" by being gold coated in order to promote direct electron transfer (DET). 33 Using an original immuno-based process, our group previously assembled, a non-integrated GOxbased system on fd bacteriophage particles adsorbed on gold electrodes.…”

Field-Effect Sensors for Virus Detection: From Ebola to SARS-CoV-2 and Plant Viral Enhancers

“…They can be applied to viral nanoparticles before or after their loading with target recognition elements to yield receptor layers of high surface densities, which may increase sensor sensitivity substantially in comparison to conventional layouts. Such "ultradense" presentation of efficiently immobilized capture units by way of plant VLP adapter templates has been demonstrated in several biosensor layouts for distinct systems with both indirect and label-free read-out (e.g., Szuchmacher Blum et al, 2011;Zang et al, 2014Zang et al, , 2017Fan et al, 2015;Koch et al, 2015Koch et al, , 2018aTinazzi et al, 2015;Bäcker et al, 2017;González-Gamboa et al, 2017;Poghossian et al, 2018;Yuste-Calvo et al, 2019b), as also detailed in the respective sections of recent reviews (Koch et al, 2016;Eiben et al, 2019;Benjamin et al, 2020).…”

“…Notwithstanding, we have found only two cases of electrostatic detection of plant virus particles with FEDs. The usability of capacitive field-effect EIS sensors for label-free electrical detection of plant virus particles was initially demonstrated by Koch et al (2018a) and Poghossian et al (2018) for tobacco mosaic virus (TMV). Here, EIS structures with adsorbed TMV particles were used for designing a penicillin biosensor, where the TMV particles served as nanocarriers for enzymes installed at high surface densities on the viral coat protein (CP) subunits.…”

“…Frequent outbreaks of TMV and related viruses in cultivated plants, namely in greenhouse crops such as tomato, pepper, cucurbits, and ornamentals thus demand for rapid identification to avoid substantial economic losses (Scholthof et al, 2011;Moriones and Verdin, 2020). This seems possible by way of FEDs: A single loading of TMV particles onto a Ta 2 O 5 -gate EIS sensor surface resulted in a large signal change of 230 mV (see Figure 1C; Koch et al, 2018a), which is associated with the negative charge of the TMV particles. The model study used biotinylated TMV, but should be valid also for native TMV particles exhibiting a similar charge.…”