(SPCE) covered with 4-carboxyphenyl diazonium salt (4-CP) and 5
0 -aminohexaethyleneglycol modified aptamers against AFM1 [114]. They reached in buffer
a LoD of 1.15 ng/l and in milk a linear range of 20–1,000 ng/kg. Measurements of
raw milk and pasteurized milk spiked with 500 ng/kg AFM1 resulted in recovery
rates of 99–111%, whereas measurements with a standard immunoassay resulted in
similar recoveries of 98–114%. The comparison to a standard method proves its
functionality, although interferences with other substances should be tested.
Hianik’s research group immobilized aptamers against AFM1 on the PAMAM
G4 modified electrodes and compared them to biotin-avidin surfaces [117]. The
results demonstrate that both immobilization methods show the same characteristics
with a dynamic range of 15–120 ng/l and a LoD of 8.5 ng/l in milk which is
sufficient for the legislative limits.
Ochratoxin is produced by fungi of the genera Aspergillus and Penicillium. It can
be found in cereals, coffee, dried fruits, cocoa, grapes, and spices as well as in
processed food like red wine, bread, beverages and it can be accumulated in animals,
e.g. pork meat. Ochratoxin A (OTA) is the most prevalent and relevant representative of the ochratoxins and has several toxicological effects such as neurotoxicity,
nephropathy, immunosuppression, and carcinogenicity [118]. The European Commission set regulatory limits of OTA in foodstuff depending on the process level
from 2 to 20 ng/g and for foodstuff intended for babies or infants to 0.5 ng/g [116].
Castillo et al. from Hianik’s research group developed an aptamer-based
impedimetric biosensor for the detection of OTA by simple chemisorption of the
aptamers on a gold electrode and measuring the impedance in buffer with 1 mM of
the redox mediator ferri-/ferrocyanide [Fe(CN) 6 ]
3À/4À [105]. They tested different
modifications of the aptamer for immobilization; interestingly, the simplest modification with a thiol on the 5
0 end showed the best results reaching a LoD of 0.12 nM
(¼0.05 ng/ml) and they notified that Ca
2+ ions were essential for OTA binding. They
also measured the surface density of aptamers and obtained 1.85 Â 10
13 aptamers/
cm
2 . Furthermore, OTA showed a six times higher signal than OTB and N-Acetyl-Lphenylalanine. The recoveries in coffee, flour, and wine spiked with 1, 5, and 10 ng/
g OTA were 78–108%. The sensor could also be regenerated by immersion into
1 mM HCl and no significant decrease of the signal was observed within 10 cycles of
regeneration.
Evtugyn et al. from the same research group used silver nanoparticles to enhance
the signal and obtained a LoD of 0.05 nM (¼0.02 ng/ml) for OTA in buffer and a
concentration range of 0.3–30 nM [106], although they observed instability of the
coating and used significant higher concentrations of [Fe(CN) 6 ]
3À/4À (0.1 M instead
of 1 mM).
For the direct detection in situ, biosensors with a wide concentration range are
favored. Hayat et al. developed an aptamer-based impedimetric biosensor for the
detection of OTA with a concentration range of 1.25–500 ng/l and a LoD of 0.25 ng/l
[107]. This low sensitivity was achieved by the modification of a SPCE with
diazonium salts and the immobilization of the aptamer via click chemistry. The
surface modification led to high impedances of about 150 kΩ and thus low frequencies of 10 mHz were required to measure the charge-transfer resistance in a buffer
Impedimetric Aptamer-Based Biosensors: Applications
71
0 -aminohexaethyleneglycol modified aptamers against AFM1 [114]. They reached in buffer
a LoD of 1.15 ng/l and in milk a linear range of 20–1,000 ng/kg. Measurements of
raw milk and pasteurized milk spiked with 500 ng/kg AFM1 resulted in recovery
rates of 99–111%, whereas measurements with a standard immunoassay resulted in
similar recoveries of 98–114%. The comparison to a standard method proves its
functionality, although interferences with other substances should be tested.
Hianik’s research group immobilized aptamers against AFM1 on the PAMAM
G4 modified electrodes and compared them to biotin-avidin surfaces [117]. The
results demonstrate that both immobilization methods show the same characteristics
with a dynamic range of 15–120 ng/l and a LoD of 8.5 ng/l in milk which is
sufficient for the legislative limits.
Ochratoxin is produced by fungi of the genera Aspergillus and Penicillium. It can
be found in cereals, coffee, dried fruits, cocoa, grapes, and spices as well as in
processed food like red wine, bread, beverages and it can be accumulated in animals,
e.g. pork meat. Ochratoxin A (OTA) is the most prevalent and relevant representative of the ochratoxins and has several toxicological effects such as neurotoxicity,
nephropathy, immunosuppression, and carcinogenicity [118]. The European Commission set regulatory limits of OTA in foodstuff depending on the process level
from 2 to 20 ng/g and for foodstuff intended for babies or infants to 0.5 ng/g [116].
Castillo et al. from Hianik’s research group developed an aptamer-based
impedimetric biosensor for the detection of OTA by simple chemisorption of the
aptamers on a gold electrode and measuring the impedance in buffer with 1 mM of
the redox mediator ferri-/ferrocyanide [Fe(CN) 6 ]
3À/4À [105]. They tested different
modifications of the aptamer for immobilization; interestingly, the simplest modification with a thiol on the 5
0 end showed the best results reaching a LoD of 0.12 nM
(¼0.05 ng/ml) and they notified that Ca
2+ ions were essential for OTA binding. They
also measured the surface density of aptamers and obtained 1.85 Â 10
13 aptamers/
cm
2 . Furthermore, OTA showed a six times higher signal than OTB and N-Acetyl-Lphenylalanine. The recoveries in coffee, flour, and wine spiked with 1, 5, and 10 ng/
g OTA were 78–108%. The sensor could also be regenerated by immersion into
1 mM HCl and no significant decrease of the signal was observed within 10 cycles of
regeneration.
Evtugyn et al. from the same research group used silver nanoparticles to enhance
the signal and obtained a LoD of 0.05 nM (¼0.02 ng/ml) for OTA in buffer and a
concentration range of 0.3–30 nM [106], although they observed instability of the
coating and used significant higher concentrations of [Fe(CN) 6 ]
3À/4À (0.1 M instead
of 1 mM).
For the direct detection in situ, biosensors with a wide concentration range are
favored. Hayat et al. developed an aptamer-based impedimetric biosensor for the
detection of OTA with a concentration range of 1.25–500 ng/l and a LoD of 0.25 ng/l
[107]. This low sensitivity was achieved by the modification of a SPCE with
diazonium salts and the immobilization of the aptamer via click chemistry. The
surface modification led to high impedances of about 150 kΩ and thus low frequencies of 10 mHz were required to measure the charge-transfer resistance in a buffer
Impedimetric Aptamer-Based Biosensors: Applications
71