College Papers

The – 4 µM) as reviewed e.g. in

The
extensive worldwide use of antibiotics in human medicine and treatment of
food-producing animals results in an alarming spread of antimicrobial
resistance. To overcome this unfortunate consequence, it is of utmost
importance to monitor antibiotic residues in water, food and waste. Most antibiotics
screening methods used nowadays depend on
appropriately equipped laboratories; they are based on enzyme-linked
immunosorbent assays (ELISA), a combination of liquid chromatography (LC)
and mass spectrometry (MS) 56, 57, microbiological
inhibition assays 58, 59 or biosensors (see e.g. reviews as 8, 11-13 and references therein).

Many
of these sensors are label-free and often based on surface plasmon resonance (SPR)
(see for original research e.g. 60, 61 or for review e.g. 10, 62), quartz crystal microbalance (QCM)
(reviewed in e.g. 10), or several electrochemical
techniques (potentiometric, amperometric and conductometric) 8, 10-13. These biosensors allow high
throughput, short analysis times, automation and multiple repeated uses due to high
regenerability. The frequently utilized integration of biological recognition
elements such as enzymes entails a high specificity and great signal
amplification. 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 typically
suffers from two main types of drawback: the first is the instability of the
biological sensing component (enzyme or antibody), which is susceptible to
temperature, pH and other environmental stresses and may therefore quickly lose
its activity. The second is the elaborate sensor format with respect to the size
of the transducer unit, the often complex and costly signal processing/readout
setup, and the corresponding personnel training needed. Hence, long-term stable
biosensor units accessible to both official calibration and miniaturization due
to electronic readout might constitute an important step towards future on-site
monitoring for antibiotic residues.

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To
our knowledge, this study is the first approach to use viral scaffolds as enzyme-carriers
in antibiotic biosensing, in order to potentially abate at least limitations
due to biomolecule degradation. The integration of viral adapters into glucose
sensors had revealed a remarkable stabilizing effect of TMV nanocarrier rods on
both GOx and HRP, extending the reusability and overall stability of the
sensors over weeks, and enabling a high-density immobilization of active
enzymes on sensor surfaces 42, 43. Here, we sought to find out if TMV-assisted
enzyme-based penicillin sensors (both colorimetric and potentiometric) would
exhibit similarly improved characteristics. The application of TMV-derived nano-adapters
mediated increased enzyme loading compared to TMV-free layouts (factor: ? 1.6 x in microtiter plates (Figures 3)). The two-step
coupling procedure of SA-conjugated enzymes to biotin-linkers was again
suitable to allow strong and specific immobilization of active enzymes (Figure 2A).
The method made use of a commercial bulk penicillinase preparation that was
efficiently conjugated to streptavidin by a simple coupling reagent and
retained its full activity. Dense enzyme coverage of the TMV rods was confirmed
by TEM (Figure 2B). As our study aimed at a first basic evaluation whether
or not enzymatic penG detection could be enhanced by the viral scaffolds, a standard
reasonably-priced penicillinase was employed, not selected for maximum
sensitivity in these sets of experiments, yet. Nethertheless,
the enzyme used in this study allowed the detection of at least 3 different
antibiotics from seperate penicillin-derivate classes.

To
our knowledge, this is the first work comparing six different pH indicators in
the same enzyme-based sensor system, including the two most frequently used halochromic
dyes for acidometric penicillin detection: phenol red and bromcresol purple. In
our setup, bromcresol purple turned out to be best suited since it provoked the
highest pH-dependent absorption changes in a reasonable detection range (Figure S3;
Table 2). The acidometric characterization of our first-generation
TMV-assisted penicillin sensors with an input of 1 U SA-Pen showed a LOD
of 100 µM and a working range up to 20 mM penG (Figure 4). This
LOD is far beyond (factor: ? 8000 x) the regulation maxima (? 12 nM).
However, a commercial enzyme, not particularly selected for maximum
sensitivity, was used for first proof-of-principle experiments shown in this
study. The use of a high-performance enzyme with better specifity, higher
activity and further optimization of measuring conditions (such as pH, buffer,
temperature and input amounts) could lead to a lowering of the detection limit
obtained by these sensors.

Integration
of modified TMV adapter sticks achieved increased reusability, better
stability, higher regenerability and higher analysis rates (Figure 3;
Table 3). The use of TMV enzyme carriers extended the half-life of the
acidometric sensors from 4?6 days to 5 weeks (Figure 3B). Both
the GOx/HRP system tested on TMV carriers before 42 and the penicillinase applied here
exhibited a similar half-life of about four days under TMV-free control
conditions. However, compared to the GOx/HRP half-life prolonged about
threefold (to nearly two weeks) on TMV nanocarriers, that of SA-Pen was increased
to about five weeks, i.e. more than eightfold as obvious from Figure 3B.
This impressive stabilization and preservation of the bioactive sensor elements
might allow the applications of the sensors even over 2-3 months. Furthermore,
a capacitive field-effect EIS structure equipped with TMV adapters, in order to
test a different type of readout, allowed label-free analyte detection in penicillin-spiked
samples.