120
6 Fabrication of Robust Biomolecular Patterns …
successfully, as judged by FTIR spectroscopy (no data shown). Hence this methodology can be extended to activate chemically more stable protected carboxyl functionalities. Thereby the concept of using thin-activated polymer films to fabricate quasi3D bioreactive and bioactive matrices in a micropatterned format, using the reactive
microcontact printing approach described above, can be further expanded (Chap. 7).
Together with the flexibility of thin polymer films in topographic patterning, using
embossing or micromolding techniques (see also Chap. 5), applications of these platforms, not only for biosensor and microarray applications but also for the study of
cellular and sub-cellar behavior can be anticipated [29].
In summary, the development of a novel micropatterning strategy that relies
on reactive microcontact printing for the local passivation of thin substratesupported reactive polymer films to fabricate robust biomolecule microarrays has
been presented. The localized deposition and covalent coupling of amino endfunctionalized poly(ethylene glycol) was shown to the desired yield blocking layers,
while the high reactivity and high molecular loading of poly(N-hydroxysuccinimidyl
methacrylate) films was retained in the unfunctionalized areas. Using this methodology biomolecule microarrays can thus be conveniently fabricated in a two-step
procedure. As demonstrated by the immobilization of organic dyes, proteins, and
the hybridization of target DNA to immobilized probe DNA in micropatterns, the
concept of reactive μCP on activated polymer films appears to be very flexible for
obtaining robust platforms for biomolecule immobilization and screening. The extension of the pathogen detection work, discussed briefly in Chap. 4, to patterned sensor
formats is currently underway.
6.7 Experimental Section
Materials. The synthesis of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA)
(M n of 3650 g/mol; M w /M n of 1.3; T g of 140 °C) is described in Chap. 3. Reference
[25] 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 (amino end-functionalized probe DNA
(5
end): 25mer 5
-GGA ATG TGC CAT ACC GAA TCC GTG T-3
; Cy5-labeled
target DNA: 5
-CAC GGA TTC GGC ATG-3
; Cy5-labeled mismatch DNA: 5
-TGT
GCC TAA GCC ATA-3
(MWG BIOTEC AG, Ebersberg, Germany) were used as
received. The DNA samples were stored at −4 °C until use. Bovine serum albumin
(BSA) labeled with Alexa Fluor® 594 was bought from Molecular Probes Inc. and
was used as received.
Preparation of Thin Films. PNHSMA thin films were prepared by spin-coating
solutions of PNHSMA in DMSO (typical concentration between 10 and 20 mg/ml)
onto silicon wafers (111) or glass cover slides (Menzel-Glaser), which were previously cleaned by an oxygen plasma treatment using an Elektrotech PF 340 apparatus
(pressure of O 2 : 0.5 bar; current: 30 mA). All spin-coated samples were dried at
6 Fabrication of Robust Biomolecular Patterns …
successfully, as judged by FTIR spectroscopy (no data shown). Hence this methodology can be extended to activate chemically more stable protected carboxyl functionalities. Thereby the concept of using thin-activated polymer films to fabricate quasi3D bioreactive and bioactive matrices in a micropatterned format, using the reactive
microcontact printing approach described above, can be further expanded (Chap. 7).
Together with the flexibility of thin polymer films in topographic patterning, using
embossing or micromolding techniques (see also Chap. 5), applications of these platforms, not only for biosensor and microarray applications but also for the study of
cellular and sub-cellar behavior can be anticipated [29].
In summary, the development of a novel micropatterning strategy that relies
on reactive microcontact printing for the local passivation of thin substratesupported reactive polymer films to fabricate robust biomolecule microarrays has
been presented. The localized deposition and covalent coupling of amino endfunctionalized poly(ethylene glycol) was shown to the desired yield blocking layers,
while the high reactivity and high molecular loading of poly(N-hydroxysuccinimidyl
methacrylate) films was retained in the unfunctionalized areas. Using this methodology biomolecule microarrays can thus be conveniently fabricated in a two-step
procedure. As demonstrated by the immobilization of organic dyes, proteins, and
the hybridization of target DNA to immobilized probe DNA in micropatterns, the
concept of reactive μCP on activated polymer films appears to be very flexible for
obtaining robust platforms for biomolecule immobilization and screening. The extension of the pathogen detection work, discussed briefly in Chap. 4, to patterned sensor
formats is currently underway.
6.7 Experimental Section
Materials. The synthesis of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA)
(M n of 3650 g/mol; M w /M n of 1.3; T g of 140 °C) is described in Chap. 3. Reference
[25] 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 (amino end-functionalized probe DNA
(5
end): 25mer 5
-GGA ATG TGC CAT ACC GAA TCC GTG T-3
; Cy5-labeled
target DNA: 5
-CAC GGA TTC GGC ATG-3
; Cy5-labeled mismatch DNA: 5
-TGT
GCC TAA GCC ATA-3
(MWG BIOTEC AG, Ebersberg, Germany) were used as
received. The DNA samples were stored at −4 °C until use. Bovine serum albumin
(BSA) labeled with Alexa Fluor® 594 was bought from Molecular Probes Inc. and
was used as received.
Preparation of Thin Films. PNHSMA thin films were prepared by spin-coating
solutions of PNHSMA in DMSO (typical concentration between 10 and 20 mg/ml)
onto silicon wafers (111) or glass cover slides (Menzel-Glaser), which were previously cleaned by an oxygen plasma treatment using an Elektrotech PF 340 apparatus
(pressure of O 2 : 0.5 bar; current: 30 mA). All spin-coated samples were dried at