Enzyme Due to the electron accepting properties of

Enzyme activity is often affected upon immobilization on solid supports.
A carefully considered combination of an immobilization strategy with the
sensor surface of choice is therefore essential for the generation of
biosensors with good and reliable performance and high reusability. The
introduction of biological, charge-sensitive or conductive molecules, adapters
or films into the sensor layout may improve the immobilization efficiency,
analyte sensitivity or stability of the biological sensing molecule. Due to
their often unique and beneficial qualities a variety of organic and inorganic
materials (alone or as composite materials) are frequently used for enzyme
immobilization. Important examples are: gold nanoparticles (AuNP), magnetic
nanoparticles, graphene and carbon nanotubes (e.g. graphene oxide (GO), carbon
nanotubes (CNT), single-walled (SWNT), multiwalled nanotubes (MWNT)) 63-65. Remarkable examples
are nanocrystaline materials called quantum dots (QDs), which are luminescent
as well as semiconducting nanosized particles thus allowing spectroscopic
read-out in optical sensing systems 66. However, natural (e.g. gelatine,
starch, cellulose, chitin, collagen) or synthetic polymers (subclasses: e.g.
stimulus-responsive/smart polymer, conducting polymers), as well as inorganic
materials such as glass, quartz or silica are equally widespread 67, 68.

Recently, a cost effective colorimetric method for the detection of
cholesterol in aqueous solutions based on enzyme modified gold nanoparticles was
developed 69. Cholesterol oxidase immobilized
on AuNPs was used to produce H2O2 in proportion to the
level of cholesterol being detected in the solution. Due to the electron
accepting properties of H2O2, increasing levels lead to quenching
in optical properties of the AuNPs. Successful detection of urea was achieved
by layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced
GO 70. The integration of the weak
polyelectrolyte improved the pH response of the graphene-based FET due to its
transducing properties, thereby enabling a LOD of 1 ?M urea. To improve the water-dispersity
of reduced GO (rGO) and to allow efficient glucose detection, rGO particles
were integrated into a nanocomposite consisting of chitosan and AuNPs 71. The composite simultaneously provided
a beneficial microenvironment for the immobilization of GOx on a glassy carbon
electrode. Another group fabricated an electrochemical immunosensor based on
anti-penicillin G antibodies integrated in a supported bilayer lipid membrane
(s-BLM) modified with gold nanoparticles 72. Binding of penG to
anti-penicillin G resulted in an alteration of the impedance. The integration
of gold nanoparticles into the membrane provided a better microenvironment to
maintain the bioactivity of anti-penicillin G antibodies.

In addition to the viral adapters used in this study, penicillinase was
successfully immobilized on field-effect sensors functionalized with
layer-by-layer films containing SWNTs and polyamidoamine (PAMAM) dendrimers 52. The integration of nanostructured
PAMAM/SWNT LbL films resulted in higher biosensing abilities towards penicillin
covering the range between 5.0 µM to 25 mM penG. Compared to SWCNTs,
single-graphene nanosheets (SGNs) exhibit a significantly higher conductivity
and offer a larger surface area. Immobilization of penicillinase on electrodes
with layer-by-layer films containing SGNs preadsorbed with hematein
(SGN-hematein) and ionic liquids allowed high sensitivity detection of
penicillin in an amperometric setup 73. This electrochemical system was
also employed to analyze penicillin residues in milk samples. A similar
amperometric penicillin biosensor was developed by coimmobilization of MWCNTs,
hematein, and penicillinase on a glassy carbon electrode surface using a layer-by-layer
assembly technique 74. This sensor setup allowed the
detection of 50 nM penicillin V, a LOD more than two orders of magnitude
lower as the LOD reached by most of conventional pH change-based biosensors. More
recently, penicillinase was combined with carbon paste for the determination of
penG, allowing high sensitivity amperometric detection 75.

Nethertheless, the soft-mater surface of TMV, the obtained surface
enlargement due to the viral adapters and the strong stabilizing effect of the
virus microenviroment onto the enzyme activity are proving TMV particles as particularly
suitable, multivalent nanoscale platform for the site directed presentation of
bioactive molecules. So
far, the TMV-like enzyme carriers have now proved themselves worthwhile in two
different model sensing systems (for glucose and penicillin), and in four
different sensor setups. Therefore, the integration of TMV in further
biosensors and lab-on-a-chip devices with different setups and other analyte
targets is worth continuative testing. In the future, a toolbox of distinct viral adapter/enzyme-complexes may
be created, based on variable coupling methods and biomolecular partners
enabling substrate detection in numerous kinds of sensor layouts. The
enrichment of enzymes on sensor surfaces and the coinstantaneously occuring stabilization
of the enzyme activity over a prolonged time frame due to TMV-based nanocarriers,
in combination with more elaborated mesuring-setups or more sensitive
chip-materials, might enable a miniaturization of biosensor units to small future hand-held devices.
This might allow month-long detection in less developed and newly
industrialized countries without the need for fully equipped laboratories and
special training for operators.



The study has
demonstrated the general functionality of viral enzyme-nanocarriers in
penicillin detection. TMV nanotube adapters densely equipped with penicillinase
enzymes were integrated into setups allowing acidometric analyte detection. The
most important results are: (1) Compared to mere adsorption, the presence of
the TMV-based enzyme carriers increased selective enzyme loading of the sensor surfaces
with equal enzyme input by a factor of ? 1.6 x. (2) TMV-assisted sensors
exhibited superior performance along with a significantly increased half-life
(5 weeks instead of several days), thus allowing an at least 8-fold longer
use of the sensors. (3) Using a commercially available enzyme preparation, not particularly selected
for maximum sensitivity, a LOD of 100 µM penG was obtained.

The integration of TMV/enzyme
complexes in biosensors was shown to achieve superior working characteristics.
In the future, this could be used to promote detection of a wide range of
chemical and biological species in miniaturized detection devices.