The – 4 µM) as reviewed e.g. in

Theextensive worldwide use of antibiotics in human medicine and treatment offood-producing animals results in an alarming spread of antimicrobialresistance. To overcome this unfortunate consequence, it is of utmostimportance to monitor antibiotic residues in water, food and waste. Most antibioticsscreening methods used nowadays depend onappropriately equipped laboratories; they are based on enzyme-linkedimmunosorbent assays (ELISA), a combination of liquid chromatography (LC)and mass spectrometry (MS) 56, 57, microbiologicalinhibition assays 58, 59 or biosensors (see e.g.

reviews as 8, 11-13 and references therein). Manyof these sensors are label-free and often based on surface plasmon resonance (SPR)(see for original research e.g.

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60, 61 or for review e.g. 10, 62), quartz crystal microbalance (QCM)(reviewed in e.g. 10), or several electrochemicaltechniques (potentiometric, amperometric and conductometric) 8, 10-13. These biosensors allow highthroughput, short analysis times, automation and multiple repeated uses due to highregenerability.

The frequently utilized integration of biological recognitionelements such as enzymes entails a high specificity and great signalamplification. Enzyme-based biosensors have achieved LODs of 1.6 µg/kg –1.3 mg/kg (? 5 nM – 4 µM) as reviewed e.g.in 12. However, the biosensor design typicallysuffers from two main types of drawback: the first is the instability of thebiological sensing component (enzyme or antibody), which is susceptible totemperature, pH and other environmental stresses and may therefore quickly loseits activity.

The second is the elaborate sensor format with respect to the sizeof the transducer unit, the often complex and costly signal processing/readoutsetup, and the corresponding personnel training needed. Hence, long-term stablebiosensor units accessible to both official calibration and miniaturization dueto electronic readout might constitute an important step towards future on-sitemonitoring for antibiotic residues.Toour knowledge, this study is the first approach to use viral scaffolds as enzyme-carriersin antibiotic biosensing, in order to potentially abate at least limitationsdue to biomolecule degradation.

The integration of viral adapters into glucosesensors had revealed a remarkable stabilizing effect of TMV nanocarrier rods onboth GOx and HRP, extending the reusability and overall stability of thesensors over weeks, and enabling a high-density immobilization of activeenzymes on sensor surfaces 42, 43. Here, we sought to find out if TMV-assistedenzyme-based penicillin sensors (both colorimetric and potentiometric) wouldexhibit similarly improved characteristics. The application of TMV-derived nano-adaptersmediated increased enzyme loading compared to TMV-free layouts (factor: ? 1.6 x in microtiter plates (Figures 3)). The two-stepcoupling procedure of SA-conjugated enzymes to biotin-linkers was againsuitable to allow strong and specific immobilization of active enzymes (Figure 2A).The method made use of a commercial bulk penicillinase preparation that wasefficiently conjugated to streptavidin by a simple coupling reagent andretained its full activity.

Dense enzyme coverage of the TMV rods was confirmedby TEM (Figure 2B). As our study aimed at a first basic evaluation whetheror not enzymatic penG detection could be enhanced by the viral scaffolds, a standardreasonably-priced penicillinase was employed, not selected for maximumsensitivity in these sets of experiments, yet. Nethertheless,the enzyme used in this study allowed the detection of at least 3 differentantibiotics from seperate penicillin-derivate classes.Toour knowledge, this is the first work comparing six different pH indicators inthe same enzyme-based sensor system, including the two most frequently used halochromicdyes for acidometric penicillin detection: phenol red and bromcresol purple. Inour setup, bromcresol purple turned out to be best suited since it provoked thehighest pH-dependent absorption changes in a reasonable detection range (Figure S3;Table 2). The acidometric characterization of our first-generationTMV-assisted penicillin sensors with an input of 1 U SA-Pen showed a LODof 100 µM and a working range up to 20 mM penG (Figure 4).

ThisLOD is far beyond (factor: ? 8000 x) the regulation maxima (? 12 nM).However, a commercial enzyme, not particularly selected for maximumsensitivity, was used for first proof-of-principle experiments shown in thisstudy. The use of a high-performance enzyme with better specifity, higheractivity and further optimization of measuring conditions (such as pH, buffer,temperature and input amounts) could lead to a lowering of the detection limitobtained by these sensors. Integrationof modified TMV adapter sticks achieved increased reusability, betterstability, higher regenerability and higher analysis rates (Figure 3;Table 3). The use of TMV enzyme carriers extended the half-life of theacidometric sensors from 4?6 days to 5 weeks (Figure 3B).

Boththe GOx/HRP system tested on TMV carriers before 42 and the penicillinase applied hereexhibited a similar half-life of about four days under TMV-free controlconditions. However, compared to the GOx/HRP half-life prolonged aboutthreefold (to nearly two weeks) on TMV nanocarriers, that of SA-Pen was increasedto about five weeks, i.e. more than eightfold as obvious from Figure 3B.This impressive stabilization and preservation of the bioactive sensor elementsmight allow the applications of the sensors even over 2-3 months. Furthermore,a capacitive field-effect EIS structure equipped with TMV adapters, in order totest a different type of readout, allowed label-free analyte detection in penicillin-spikedsamples.