Since Alexander Fleming 1, antibiotics have revolutionized

the discovery of penicillin G (the world’s first antibiotic; a ?-lactam
antibiotic synonymously known as benzylpenicillin) in 1928 by Sir Alexander
Fleming 1, antibiotics have revolutionized
the world of modern medicine. The treatment of wounds and infections with
penicillin has been saving millions of people around the world. Moreover,
antibiotics are being used to a great extent in livestock production. Due to the
extensive use in animal husbandry and inappropriate prescribing of antibiotics
in human medicine, many types of bacteria have developed resistance; some have even
become resistant to more than one type of antibiotic (reviewed e.g. in 2, 3). The antibiotic resistance of disease-causing
bacteria is of particular concern since they can be transmitted to humans both
by animal products and by daily contact with other humans, or upon medical
therapy. The incidence and spread of different resistance mechanisms is
threatening our ability to treat even common infections, which can result in
prolonged illness, epidemic/pandemic spread of infections, use of more toxic
drugs and increasing mortality rates of infectious diseases 4, 5. Therefore, it is important to
support efforts to minimize the inappropriate use of antibiotics and to monitor
the antibiotic residues in wastewater, offal and animal products, especially when
entering the human food chain via meat, milk and eggs. In order to control this
threat, the European Union (EU) has issued strict regulations including
specific maximum residue limits (MRLs) for each veterinary drug in animal
husbandry (Council Regulation 2377/90/EEC).
For instance, the MRL of benzylpenicillin (penicillin G) in milk is
4 µg/kg (? 12 nM) 6, 7. A multitude of analytical
methodologies has been developed (as e.g. comprehensively reviewed in 8-13). Some
methods were designed to easily yield clinically relevant information on
whether or not bacteria from hospitals can degrade antibacterial drugs and thus
are resistant to those. Important detection methods are based on the cleavage
of the ?-lactam ring of antibiotics such as penicillin and cephalosporin by the
bacterial enzyme ?-lactamase (Figure 1A). The activity of ?-lactamases, like
penicillinase, has been assayed by a variety of chemical methods such as
manometric techniques (detection of CO2 evolution in bicarbonate
buffers) 14, 15, growth inhibition, and a diversity
of chromogenic methods, including acidometric methods with pH indicators (halochromic
dyes 16, 17; Figure 1C), or directly with chromogenic
substrates (e.g. chromogenic cephalosporin 18; Figure 1D), or iodometric
assays (compared e.g. in 18-20; Figure 1B) in combination
with spectrophotometry. Acidometric methods, as
used in this work, are based on the formation of penicilloic acid with a
pH change, monitored by a pH indicator in a low-buffered system
(Figure 1C). The halochromic indicators mostly applied are phenol red
(e.g. 16, 21-23, 20) and bromcresol purple (19, 24, 25, for review 12).


Virus particles such as tobacco
mosaic virus (TMV), cowpea mosaic
virus (CPMV), cowpea chlorotic mottle
virus (CCMV) or the bacteriophage M13 are often used biotemplates for
effizient dection in sensing devices (as e.g. reviewed in 26-29. These particles
offer well-defined, nanostructured and stable protein backbones with option for
multivalent presentation of functional groups. Moreover, the use of plant
viruses and bacteriophages is very attractive, due to their ease of production,
their high-yield and their lack of toxicity and pathogenicity to mammals. So
far, M13 has been used for colorimetric detection of volatile organic compounds,
as humidity sensor 30, and for the detection explosives
such as TNT 31 or for antibiotics 32. M13 was further employed for e.g.
detection of pathogens such as salmonella or bacillus anthracis (reviewed in 26). Dual-functionalized hepatitis B virus was successfully used
as tracer in high sensitivity detection of troponin, a marker for acute
myocardial infarction 33. CPMV was utilized as platform
for significant signal enhancement in direct and sandwich immunoassays 34,
35, for detection of staphylococcal
enterotoxin B 36 or as frame work in avidin
detection 37. TMV was successfully
integrated as viral carrier in sensor systems for TNT-detection 38, selective antibody-detection in
an optical micro-disc resonator 39 and further integrated in an thin
film sensor for detection of volatile organic compounds 40. In combination with inorganic
materials, TMV nanoparticles coated with palladium as a sensing material have
promoted the development of a surface acoustic wave (SAW) hydrogen sensor 41.

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this study, a biosensor employing tobacco mosaic virus (TMV)-based enzyme
carrier scaffolds was developed for acidometric detection of penicillin. The
incorporation of viral adapters was pursued, as it has been shown recently that
biosensors can profit from TMV nanotubes as enzyme immobilization supports 42, 43. TMV is a highly stable, rod-shaped
plant virus with a precisely structured protein capsid. It has a length of
300 nm, a diameter of 18 nm and an inner channel of 4 nm
diameter. The capsid, with 2130 coat protein (CP) subunits, can be conjugated with
functional molecules by appropriate chemistry, facilitated by CP variants with highly
reactive coupling sites. TMV has acted as a well-defined high-yield
nanotemplate in proof-of-concept experiments for a multitude of possible
applications, reviewed in 44. Promising results were obtained
with full TMV sticks serving as nanocarriers for enzymes 42. The two-enzyme cascade system of
glucose oxidase (GOx) and horseradish peroxidase (HRP) was successfully
installed on the viral adapter rods, enabling a colorimetric detection of
glucose in combination with a suitable chromogenic co-substrate. A TMV mutant
(TMVCys) exposing thiol groups on its surface was functionalized
with bifunctional maleimide-PEG11-biotin linkers, allowing a dense
decoration with active streptavidin (SA)-conjugated enzymes. Besides
enrichement of enzymes on sensor surfaces, additional beneficial effects of TMV
rods were found: GOx and HRP exhibited an increased reusability, a greater storage
stability and a higher regenerability 42. Thus, a great potential
application spectrum of virus-enzyme conjugates in biosensors and related
bioanalytical devices is emerging. TMV enzyme carriers were also evaluated as
adapters on platinum (Pt) electrode arrays on sensor chips in electrochemical
biosensors for amperometric glucose detection, which effectuated a superior
sensor performance 43.

main goal, now, was to test whether the TMV nanoadapters could be
functionalized with penicillinase similarly. The performance of the TMV-assisted
antibiotic sensing layouts was determined, primarily in a classic colorimetric

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