For the development of handheld point-of-care tests, microfluidic chips need to
be established for the usage with aptamer-based impedimetric biosensors. Some
preliminary work was done by [167–169] who used microelectrodes on glass
substrate and fixed a molded microfluidic channel from polydimethylsiloxane
(PDMS) on the electrodes. The use of microfluidics in impedimetric biosensors
reduces the amount of sample and enables low-cost disposable biosensors. Additionally, different mixing mechanisms can be included to accelerate the reactiondiffusion kinetics [170], as for example used by Feng et al. who reduced the response
time from 60 min to only 15 min by magnetic stirring [171].
As impedimetric biosensors are valued for their fast detection, several
impedimetric aptasensors with analysis times below 15 min were developed, as
e.g. for OTA in 15, 9, or 5 min [104, 110, 111], for E. coli within 12 min [135] and
for S. aureus in 10 min [140]. If, instead of a whole spectrum, the signal at a single
frequency is measured, the response time is below ms and real-time monitoring can
be performed. Liao et al. developed a non-faradaic impedance aptasensor measuring
at 5 kHz and thus, enabling in vivo monitoring of PDGF-BB [172].
Another issue is the stability of aptamers, as RNA and DNA aptamers are
sensitive to nucleases abundant in biological fluids leading to their degradation
within minutes [173]. Thus modified nucleotides were developed to increase
nuclease-resistance, such as 2
0 -fluoro- and 2
0 -amino-pyrimidines or 2
0 -hydroxylpurines, which led to the first clinically approved aptamer known as Macugen
®
[174]. González-Fernández et al. developed an impedimetric biosensor using a fully
2
0 -O-methylated aptamer and detected tobramycin in diluted human serum
[175]. Other possibilities to increase the stability of aptamers are 3
0 - or 5
0 -end
cappings, locked amino acids [LNA], or peptide nucleic acids [PNA]. The aptamer
can be modified post-SELEX, but this often leads to a significant decrease in affinity
[176]. However, any modification on the aptamer increases its production costs and
may imply complications within synthesis. Thus, more research is still needed.
All over, we found electrochemical sensors based on aptamers for about 100 different targets and the number is rising. The thrombin aptamer is best examined, but
little is known about the mechanism of aptamer-target binding and how it is
influenced by its surrounding, such as pH, ions, and temperature. Beside experiments, more mathematical calculation and simulation of electrical properties are
needed.
Impedimetric aptasensors will not supersede well-established methods like PCR,
ELISA, or HPLC, but rather fill an important gap as e.g. for the detection of small
non-immunogenic molecules. With the advances in nanotechnology, enabling the
creation of nanostructures, it is likely that we will soon be able to detect single
molecules, especially in combination with handling of nanoliter volumes. This will
require sensor miniaturization, as e.g. with integrated microelectronics, for what
impedimetric aptasensors are optimally suited. With the already established variety
of surfaces, impedimetric aptasensors can be customized for many applications in
biotechnology, especially for extreme conditions such as high temperatures, high
pH, and complex biological matrices. Nevertheless, it is important to advance the
80
J.-A. Preuß et al.
be established for the usage with aptamer-based impedimetric biosensors. Some
preliminary work was done by [167–169] who used microelectrodes on glass
substrate and fixed a molded microfluidic channel from polydimethylsiloxane
(PDMS) on the electrodes. The use of microfluidics in impedimetric biosensors
reduces the amount of sample and enables low-cost disposable biosensors. Additionally, different mixing mechanisms can be included to accelerate the reactiondiffusion kinetics [170], as for example used by Feng et al. who reduced the response
time from 60 min to only 15 min by magnetic stirring [171].
As impedimetric biosensors are valued for their fast detection, several
impedimetric aptasensors with analysis times below 15 min were developed, as
e.g. for OTA in 15, 9, or 5 min [104, 110, 111], for E. coli within 12 min [135] and
for S. aureus in 10 min [140]. If, instead of a whole spectrum, the signal at a single
frequency is measured, the response time is below ms and real-time monitoring can
be performed. Liao et al. developed a non-faradaic impedance aptasensor measuring
at 5 kHz and thus, enabling in vivo monitoring of PDGF-BB [172].
Another issue is the stability of aptamers, as RNA and DNA aptamers are
sensitive to nucleases abundant in biological fluids leading to their degradation
within minutes [173]. Thus modified nucleotides were developed to increase
nuclease-resistance, such as 2
0 -fluoro- and 2
0 -amino-pyrimidines or 2
0 -hydroxylpurines, which led to the first clinically approved aptamer known as Macugen
®
[174]. González-Fernández et al. developed an impedimetric biosensor using a fully
2
0 -O-methylated aptamer and detected tobramycin in diluted human serum
[175]. Other possibilities to increase the stability of aptamers are 3
0 - or 5
0 -end
cappings, locked amino acids [LNA], or peptide nucleic acids [PNA]. The aptamer
can be modified post-SELEX, but this often leads to a significant decrease in affinity
[176]. However, any modification on the aptamer increases its production costs and
may imply complications within synthesis. Thus, more research is still needed.
All over, we found electrochemical sensors based on aptamers for about 100 different targets and the number is rising. The thrombin aptamer is best examined, but
little is known about the mechanism of aptamer-target binding and how it is
influenced by its surrounding, such as pH, ions, and temperature. Beside experiments, more mathematical calculation and simulation of electrical properties are
needed.
Impedimetric aptasensors will not supersede well-established methods like PCR,
ELISA, or HPLC, but rather fill an important gap as e.g. for the detection of small
non-immunogenic molecules. With the advances in nanotechnology, enabling the
creation of nanostructures, it is likely that we will soon be able to detect single
molecules, especially in combination with handling of nanoliter volumes. This will
require sensor miniaturization, as e.g. with integrated microelectronics, for what
impedimetric aptasensors are optimally suited. With the already established variety
of surfaces, impedimetric aptasensors can be customized for many applications in
biotechnology, especially for extreme conditions such as high temperatures, high
pH, and complex biological matrices. Nevertheless, it is important to advance the
80
J.-A. Preuß et al.