Simplified Detection of Serotonin by FET-Based Sensor
31
2 Materials and Methods
2.1 Fabrication of ZnO FET
ZnO FET-based biosensor with lateral electrode configuration has been fabricated
using hydrothermal technique involving the ZnO seed layer preparation. Contacts
have been installed on one side of the glass substrate by photolithography. Here,
positive photoresist S1813 was spin-coated on top of glass surface by centrifugation
at 2500 rpm for 30 s. First, the ZnO precursor solution was prepared by mixing
50 mM zinc nitrate hexahydrate [Zn (NO 3 ) 2 ·6H 2 O] with 100 ml of deionized (DI)
water. The solution bath has been placed on a magnetic stirrer and was heated. Next,
4 ml of ammonium hydroxide [NH 4 OH] solution has been added in the solution as
soon as the bath temperature rises to 60 °C, followed by stirring up to 30 min. Then,
the solution was kept uninterrupted for 24 h. ZnO film is obtained from the precursor
solution and has been spin-coated on glass surface. After that, for evaporation of the
remaining solvent, ZnO sample has been kept on a hot plate at 250 °C for 10 min.
Next step involves the hydrothermal growth of ZnO nanorod on the seeded surface
area following the procedure as reported earlier. About 50 mM zinc nitrate hexahydrate [Zn (NO 3 ) 2 ·6H 2 O] with 50 ml DI water, whereas, on another beaker, 50 mM
hexamethylenetetramine [HMTA. C 6 H 12 N 4 ] has been blended with 50 ml DI water
and was stirred for 10 min respectively. Then both the solutions have been mixed
with one another and stirred at 260 rpm. The ZnO-seeded glass platform has been
suspended into the solution bath for 2 h. After this step, the glass platform has been
taken out from the solution and washed with DI water. To achieve better crystallization, the ZnO nanorod has been thereby annealed at 350 °C using the rapid thermal
annealing method for 5 min.
2.2 Functionalization of ZnO
For proper antibody immobilization, silanization process was followed by crosslinker attachment. The silane solution has been prepared by the addition of 0.4 ml
mercaptopropyltrimethoxysilane (MTS) and 9.258 ml ethanol with 0.341 ml DI
water and was pipetted on the sensor surface. The sensor was sealed and was
left in the nitrogen environment for 24 h. The sample was washed with ethanol
and then ultrasonicated for 5 min along with a drying session in the nitrogen
environment. Thereafter, the cross-linker attachment was achieved by 2 mM N-γmaleimidobutyryloxysuccinimide (GMBS) solution on the ZnO surface and was kept
for 24 h. The sample was then ultrasonicated in ethanol for 5 min and thereafter the
activated ZnO surface is now suitable for covalent attachment of antibody. Therefore, all the surface modification steps have been performed in room temperature
following the reported protocol (Corso et al. 2008).
31
2 Materials and Methods
2.1 Fabrication of ZnO FET
ZnO FET-based biosensor with lateral electrode configuration has been fabricated
using hydrothermal technique involving the ZnO seed layer preparation. Contacts
have been installed on one side of the glass substrate by photolithography. Here,
positive photoresist S1813 was spin-coated on top of glass surface by centrifugation
at 2500 rpm for 30 s. First, the ZnO precursor solution was prepared by mixing
50 mM zinc nitrate hexahydrate [Zn (NO 3 ) 2 ·6H 2 O] with 100 ml of deionized (DI)
water. The solution bath has been placed on a magnetic stirrer and was heated. Next,
4 ml of ammonium hydroxide [NH 4 OH] solution has been added in the solution as
soon as the bath temperature rises to 60 °C, followed by stirring up to 30 min. Then,
the solution was kept uninterrupted for 24 h. ZnO film is obtained from the precursor
solution and has been spin-coated on glass surface. After that, for evaporation of the
remaining solvent, ZnO sample has been kept on a hot plate at 250 °C for 10 min.
Next step involves the hydrothermal growth of ZnO nanorod on the seeded surface
area following the procedure as reported earlier. About 50 mM zinc nitrate hexahydrate [Zn (NO 3 ) 2 ·6H 2 O] with 50 ml DI water, whereas, on another beaker, 50 mM
hexamethylenetetramine [HMTA. C 6 H 12 N 4 ] has been blended with 50 ml DI water
and was stirred for 10 min respectively. Then both the solutions have been mixed
with one another and stirred at 260 rpm. The ZnO-seeded glass platform has been
suspended into the solution bath for 2 h. After this step, the glass platform has been
taken out from the solution and washed with DI water. To achieve better crystallization, the ZnO nanorod has been thereby annealed at 350 °C using the rapid thermal
annealing method for 5 min.
2.2 Functionalization of ZnO
For proper antibody immobilization, silanization process was followed by crosslinker attachment. The silane solution has been prepared by the addition of 0.4 ml
mercaptopropyltrimethoxysilane (MTS) and 9.258 ml ethanol with 0.341 ml DI
water and was pipetted on the sensor surface. The sensor was sealed and was
left in the nitrogen environment for 24 h. The sample was washed with ethanol
and then ultrasonicated for 5 min along with a drying session in the nitrogen
environment. Thereafter, the cross-linker attachment was achieved by 2 mM N-γmaleimidobutyryloxysuccinimide (GMBS) solution on the ZnO surface and was kept
for 24 h. The sample was then ultrasonicated in ethanol for 5 min and thereafter the
activated ZnO surface is now suitable for covalent attachment of antibody. Therefore, all the surface modification steps have been performed in room temperature
following the reported protocol (Corso et al. 2008).