82
4 Reactive Thin Polymer Films as Platforms for the Immobilization …
were carried out on samples patched with glycine (Fig. 4.11b). The discrimination
of different target DNA can be substantially improved using this procedure. Without
patching, a discrimination ratio of complementary to mismatch of 12 was observed,
which improved to 30 after patching with glycine.
The hybridization of target DNA was also investigated using fluorescence
microscopy. The corresponding micrographs are shown in Fig. 4.12. No fluorescence
emission was detected for PNHSMA films modified only with (unlabeled) probe
DNA, as well as neat PNHSMA films treated with the dye-labeled target DNA, which
is unreactive toward NHS ester groups (Figs. 4.12a, b). In contrast, if the complementary target DNA reacts with PNHSMA films modified with covalently attached
probe DNA, strong fluorescence emission was detected (Fig. 4.12c). For the corresponding blank experiment with fluorescently labeled mismatch DNA (Fig. 4.12d),
no fluorescence emission was detected, which suggests that the hybridization of
complementary target DNA was indeed successful. Discrimination ratio of complementary to mismatch of 25 was observed, which is a comparable value compared to
SPFS experiment to glycine-patched films surface.
Owing to the effects of fluorescence quenching and decaying coupling efficiency
between surface plasmons and surface-immobilized fluorophores, there is an optimized film thickness of ~30–50 nm for this particular detection method. Since the
film thicknesses of spin-coated films can be easily controlled through variations of
polymer solution concentration or spinning speed, reactive thin film systems that
show only limited swelling, such as PNHSMA, can be advantageous in this respect.
4.5 Toward the Detection of Pathogenic Bacteria
on PNHSMA Films
Recently, there has been an increased interest in methods for the detection of bacteria,
especially those species that are involved in food poisoning, water contamination,
clinical cases, and biological warfare [30]. This interest is largely a result of increased
incidences of Escherichia coli O157:H7, Salmonella, Clostridium, and Campylobacter being found in food and water. Hence, there is interest to monitor food and
water using suitable biosensors. As introduced also in Chap. 2, a chemical biosensor
is a device, which responds to an analyte selectively through a reversible chemical interaction and it can be used for quantitative or qualitative determinations.
All sensors are composed of two main regions: one where the selective chemistry
occurs and the second being the transducer. The detection of bacteria using sensors
is typically assessed via enzyme-tagged immuno-electrochemical assays.
To demonstrate the possibility to apply PNHSMA films in more complex biosensors, the detection of pathogenic bacteria (listeria) was investigated. Listeria is a
pathogenic (disease-causing) bacterium that is food-borne and causes an illness called
listeriosis. It is frequently overlooked as a possible cause of illness due to its unique
4 Reactive Thin Polymer Films as Platforms for the Immobilization …
were carried out on samples patched with glycine (Fig. 4.11b). The discrimination
of different target DNA can be substantially improved using this procedure. Without
patching, a discrimination ratio of complementary to mismatch of 12 was observed,
which improved to 30 after patching with glycine.
The hybridization of target DNA was also investigated using fluorescence
microscopy. The corresponding micrographs are shown in Fig. 4.12. No fluorescence
emission was detected for PNHSMA films modified only with (unlabeled) probe
DNA, as well as neat PNHSMA films treated with the dye-labeled target DNA, which
is unreactive toward NHS ester groups (Figs. 4.12a, b). In contrast, if the complementary target DNA reacts with PNHSMA films modified with covalently attached
probe DNA, strong fluorescence emission was detected (Fig. 4.12c). For the corresponding blank experiment with fluorescently labeled mismatch DNA (Fig. 4.12d),
no fluorescence emission was detected, which suggests that the hybridization of
complementary target DNA was indeed successful. Discrimination ratio of complementary to mismatch of 25 was observed, which is a comparable value compared to
SPFS experiment to glycine-patched films surface.
Owing to the effects of fluorescence quenching and decaying coupling efficiency
between surface plasmons and surface-immobilized fluorophores, there is an optimized film thickness of ~30–50 nm for this particular detection method. Since the
film thicknesses of spin-coated films can be easily controlled through variations of
polymer solution concentration or spinning speed, reactive thin film systems that
show only limited swelling, such as PNHSMA, can be advantageous in this respect.
4.5 Toward the Detection of Pathogenic Bacteria
on PNHSMA Films
Recently, there has been an increased interest in methods for the detection of bacteria,
especially those species that are involved in food poisoning, water contamination,
clinical cases, and biological warfare [30]. This interest is largely a result of increased
incidences of Escherichia coli O157:H7, Salmonella, Clostridium, and Campylobacter being found in food and water. Hence, there is interest to monitor food and
water using suitable biosensors. As introduced also in Chap. 2, a chemical biosensor
is a device, which responds to an analyte selectively through a reversible chemical interaction and it can be used for quantitative or qualitative determinations.
All sensors are composed of two main regions: one where the selective chemistry
occurs and the second being the transducer. The detection of bacteria using sensors
is typically assessed via enzyme-tagged immuno-electrochemical assays.
To demonstrate the possibility to apply PNHSMA films in more complex biosensors, the detection of pathogenic bacteria (listeria) was investigated. Listeria is a
pathogenic (disease-causing) bacterium that is food-borne and causes an illness called
listeriosis. It is frequently overlooked as a possible cause of illness due to its unique