Detection of Acetoin and Diacetyl by a Tobacco Mosaic Virus-Assisted Field-Effect Biosensor

Abstract

Acetoin and diacetyl have a major impact on the flavor of alcoholic beverages such as wine or beer. Therefore, their measurement is important during the fermentation process. Until now, gas chromatographic techniques have typically been applied; however, these require expensive laboratory equipment and trained staff, and do not allow for online monitoring. In this work, a capacitive electrolyte–insulator–semiconductor sensor modified with tobacco mosaic virus (TMV) particles as enzyme nanocarriers for the detection of acetoin and diacetyl is presented. The enzyme acetoin reductase from Alkalihalobacillus clausii DSM 8716T is immobilized via biotin–streptavidin affinity, binding to the surface of the TMV particles. The TMV-assisted biosensor is electrochemically characterized by means of leakage–current, capacitance–voltage, and constant capacitance measurements. In this paper, the novel biosensor is studied regarding its sensitivity and long-term stability in buffer solution. Moreover, the TMV-assisted capacitive field-effect sensor is applied for the detection of diacetyl for the first time. The measurement of acetoin and diacetyl with the same sensor setup is demonstrated. Finally, the successive detection of acetoin and diacetyl in buffer and in diluted beer is studied by tuning the sensitivity of the biosensor using the pH value of the measurement solution.

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System

“…All these options are realized if conversion reactions by, e.g., enzymes immobilized on chip surfaces, are recorded in small or miniaturized devices, via detecting electrochemical changes arising from the generation or consumption of electrons or protons. Respective sensor chips equipped with TMV adapter coatings as enzyme-displaying nanomaterial have proven their beneficial properties repeatedly [ 41 , 42 , 44 , 48 , 49 ]. Hence, we sought to evaluate if TVCV PA immobilization on such chips can follow the protocols established for TMV before.…”

“…Evidently, TVCV PA adapter particles possess biomolecule-stabilizing properties similar to TMV in previous work [ 46 , 49 , 50 ], despite their deviant surface composition. This renders them promising candidates for use as adapter coatings on detector chips in biosensor setups applied for electrochemical, label-free real-time analytics, as established with TMV [ 41 , 42 , 44 , 48 , 49 ]. Recent methods for the electrostatic LbL adsorption of TMV on polyelectrolyte-modified Ta 2 O 5 chip surfaces could be adopted one-to-one and yield even, nearly closed TVCV PA layers.…”

“…This chip arrangement is applied commonly as an electrolyte-insulator-semiconductor capacitor (EISCAP) structure for electrochemical biosensors [ 89 ]. The preparation, cleaning, and immobilization of virus particles directly on the chips were described in previous studies [ 44 , 45 ]. The EISCAPs were divided into two groups.…”

“…Its easy production, physical and chemical stability, and options for generating TMV-derived designer particles of altered shapes offer versatile prospects for multi-functionalization with, e.g., peptides, enzymes, drugs, and/or synthetic and inorganic compounds, and corresponding uses [ 31 ]. Increasing numbers of studies have employed TMV-based adapters for the display of enzymes, and for installing enzymatic and other types of bioreceptors in different types of sensors [ 20 , 25 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ]. TMV, however, does not tolerate extensive genetic modifications.…”

Soft Sub‐Structured Multi‐Material Biosensor Hydrogels with Enzymes Retained by Plant Viral Scaffolds

“…With the spatially well-defined GM spot array and a known number of docking sites exposed on the TMV nanocarriers, it will be feasible to integrate a defined number of enzyme molecules per spot and to combine distinct enzymes on one biosensor platform via GM formulations with different nanoparticle-enzyme conjugates. TMV has already been successfully applied in various sensor layouts [39][40][41][62][63][64][65][66][67][68][69][70] and was shown to stabilize distinct enzymes including penicillinase in label-free electrochemical field-effect biosensors that retained their perfor-mance for at least a year of repetitive use. [41] This points to the future potential of the combination of plant viral bioscaffolds with hydrogel soft biosensors, for example, as sensor domains in 3D cell cultures grown for tissue replacement, in implanted online sensors, or for in situ detection purposes for process monitoring in biotechnological or food fabrication fermenters.…”