86
4 Reactive Thin Polymer Films as Platforms for the Immobilization …
presumably due to the absence of strong interactions with the anti-listeria antibody,
this result shows that the PNHSMA-based platform exhibits some selectivity. In
addition, these results suggest that protein G (or at least a large fraction of protein
G) has been coupled in an active state, i.e., in the correct orientation and that the
antibody attachment was also successful. Thus, PNHSMA films appear as a suitable
polymer substrate to be used in the biosensor field for detecting bacteria and likely
other species.
In summary, we have shown that spin-coated thin films of poly(Nhydroxysuccinimidyl methacrylate) on oxidized silicon, glass and gold substrates
are interesting coatings for obtaining robust reactive platforms for biomolecule
immobilization with tunable film thickness and high molecular loading capabilities.
Compared to SAMs, an increased loading was observed for the covalent coupling
of various low molar mass and polymeric primary amines, including 25mer probe
DNA, PEG, and proteins. This increased loading is attributed to reactions that take
place at the surface, as well as in the surface-near region, of the polymer films. The
results presented here also suggest that PNHSMA films possess the potential to be
applied in sensors that monitor biomolecular interactions, such as hybridization reactions between surface-attached probe oligonucleotides and complementary DNA in
solution using, e.g., SPFS measurements, and the detection of pathogens, such as
bacteria. In addition, these polymer films are amenable to chemical (and topographical) patterning via soft lithographic approaches to sub-micrometer length scales, as
will be discussed in Chap. 6, which increases the scope of the system to include also
array-based formats.
4.6 Experimental Section
Materials. The synthesis of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA)
(Scheme 1) (M n of 3650 g/mol; M w /M n of 1.3; T g of 140 °C) is described in Chap. 3.
Amino end-labeled PEG (denoted as PEG 500 -NH 2 ), purchased from Nektar UK
Company (M n = 500 g/mol, PDI = 1.1), fluoresceinamine (Molecular Probes, Inc.
The Netherlands) and the DNA samples (biotin-coupling group at 5
end): P: 25mer
5
-GGA ATG TGC CAT ACC GAA TCC GTG T-3
; Cy5-labeled target DNA: 5
-
CAC GGA TTC GGC ATG-3
-Cy5; Cy5-labeled mismatch DNA: 5
-TGT GCC TAA
GCC ATA-3
-Cy5 (MWG BIOTEC AG, Ebersberg, Germany) were used as received
(HPLC-purified >98%). The DNA samples were stored at −4 °C until use. Glycine
was from Sigma with 99% purity. Bovine serum albumin (BSA) labeled with Alexa
Fluor®594 was bought from Molecular Probes Inc. and was used as received.
Preparation of Thin Films. Polymer thin films were prepared by spin-coating
polymer solutions in DMSO (typical concentration between 10 and 20 mg/ml) onto
silicon wafers (111), Au, or glass cover slides (Menzel-Glaser), which were previously cleaned by oxygen plasma treatment using an Elektrotech PF 340 apparatus
(pressure of O 2 : 0.5 bar; current: 30 mA). The samples were spun at 3000 rpm for 30 s
4 Reactive Thin Polymer Films as Platforms for the Immobilization …
presumably due to the absence of strong interactions with the anti-listeria antibody,
this result shows that the PNHSMA-based platform exhibits some selectivity. In
addition, these results suggest that protein G (or at least a large fraction of protein
G) has been coupled in an active state, i.e., in the correct orientation and that the
antibody attachment was also successful. Thus, PNHSMA films appear as a suitable
polymer substrate to be used in the biosensor field for detecting bacteria and likely
other species.
In summary, we have shown that spin-coated thin films of poly(Nhydroxysuccinimidyl methacrylate) on oxidized silicon, glass and gold substrates
are interesting coatings for obtaining robust reactive platforms for biomolecule
immobilization with tunable film thickness and high molecular loading capabilities.
Compared to SAMs, an increased loading was observed for the covalent coupling
of various low molar mass and polymeric primary amines, including 25mer probe
DNA, PEG, and proteins. This increased loading is attributed to reactions that take
place at the surface, as well as in the surface-near region, of the polymer films. The
results presented here also suggest that PNHSMA films possess the potential to be
applied in sensors that monitor biomolecular interactions, such as hybridization reactions between surface-attached probe oligonucleotides and complementary DNA in
solution using, e.g., SPFS measurements, and the detection of pathogens, such as
bacteria. In addition, these polymer films are amenable to chemical (and topographical) patterning via soft lithographic approaches to sub-micrometer length scales, as
will be discussed in Chap. 6, which increases the scope of the system to include also
array-based formats.
4.6 Experimental Section
Materials. The synthesis of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA)
(Scheme 1) (M n of 3650 g/mol; M w /M n of 1.3; T g of 140 °C) is described in Chap. 3.
Amino end-labeled PEG (denoted as PEG 500 -NH 2 ), purchased from Nektar UK
Company (M n = 500 g/mol, PDI = 1.1), fluoresceinamine (Molecular Probes, Inc.
The Netherlands) and the DNA samples (biotin-coupling group at 5
end): P: 25mer
5
-GGA ATG TGC CAT ACC GAA TCC GTG T-3
; Cy5-labeled target DNA: 5
-
CAC GGA TTC GGC ATG-3
-Cy5; Cy5-labeled mismatch DNA: 5
-TGT GCC TAA
GCC ATA-3
-Cy5 (MWG BIOTEC AG, Ebersberg, Germany) were used as received
(HPLC-purified >98%). The DNA samples were stored at −4 °C until use. Glycine
was from Sigma with 99% purity. Bovine serum albumin (BSA) labeled with Alexa
Fluor®594 was bought from Molecular Probes Inc. and was used as received.
Preparation of Thin Films. Polymer thin films were prepared by spin-coating
polymer solutions in DMSO (typical concentration between 10 and 20 mg/ml) onto
silicon wafers (111), Au, or glass cover slides (Menzel-Glaser), which were previously cleaned by oxygen plasma treatment using an Elektrotech PF 340 apparatus
(pressure of O 2 : 0.5 bar; current: 30 mA). The samples were spun at 3000 rpm for 30 s