Lee et al. who successfully validated the developed aptasensor for the detection of
malaria by Plasmodium lactate dehydrogenase (pLDH) with infected blood samples
from human patients [31] and Shi et al. who used serum samples of cancer patients to
validate the biosensor for ATP detection [152]. We found 47 other impedimetric
aptasensors (most covered in this chapter) that could detect the analyte in serum
samples, which show the tendency of real sample validation. However, more clinical
sample validation experiments are needed. Key parameters that should be tested in
real samples are repeatability, non-specific binding, and sample to sample variation.
The performance of aptasensors in real sample settings could be improved by
adjusting the buffer during SELEX to mimic real samples.
The SELEX procedure has been developed substantially in the last decade. Still,
the intrinsic capability to engineer an aptamer adapted to the specific needs of the
application has not been fully utilized. During the SELEX process several steps can
be included to reduce non-specific binding [153, 154] and to include a regeneration
technique [155]. For example, Wehbe et al. included an EDTA wash step to obtain
switchable aptamers (SwAps) that were used to recover vesicular stomatitis virus
after capture to allow further analytics of the sample with recovery rates of ~33%.
This technique would allow performing several analyses on the same sample for,
e.g., lab-on-a-chip developments.
Further strategies for the regeneration of the aptamer-modified sensors were
developed, mostly based on chemicals changing the ionic strength or pH of the
media, e.g. 2 M NaCl [156], 6 M guanidine hydrochloride [157], 6 M urea [32], pH
4 [158], 0.2 M glycine-HCl [112], or imidazole [159]. Others use more complex
elution buffers, e.g. Tris buffer containing 20% of methanol [107] or mixtures of
different chemicals, e.g. 0.05 M NaOH and 1 M NaCl [160] or variations of several
properties, like 1 M NaOH at 4
C [45]. But also more complex mechanisms for the
regeneration of the aptamer-target-bond were developed, e.g. the abovementioned
SwAps or the usage of mixed aptamers (MBA) that are oligonucleotides containing
more than one aptamer sequence directed against different targets. Du et al. used a
combination of ATP and thrombin aptamer, whereas the MBA was immobilized by
a complementary DNA probe immobilized on the electrode surface [161]. Part of the
ATP aptamer was complement to the DNA probe leading to a release of the MBA
when it binds to ATP. The binding of thrombin did not lead to a release but to an
increase in impedance. Thus, this aptasensor can be used to detect thrombin and can
be regenerated with ATP. A similar technique used by Liu et al. to detect Mucin
1 protein and carcinoembryonic antigen [51] is hybridization of the aptamer to an
immobilized complementary oligonucleotide, where upon binding to its target the
aptamer is released and the biosensor can be easily regenerated.
Detection of only one marker is not enough to establish a reliable diagnosis, but
biosensors targeting multiple analytes would enable early detection and help doctors
to provide a personalized therapy [162]. Although several publications report about
micro- and nanoarrays of electrodes [163–166] that are suitable for multiplexing
including repetitions and controls, unfortunately, no report on impedimetric
aptasensor used for the simultaneous detection of multiple analytes was found.
Multiplex impedimetric aptasensors would significantly enhance the chance for
market penetration and thus more research in this direction is needed.
Impedimetric Aptamer-Based Biosensors: Applications
79
malaria by Plasmodium lactate dehydrogenase (pLDH) with infected blood samples
from human patients [31] and Shi et al. who used serum samples of cancer patients to
validate the biosensor for ATP detection [152]. We found 47 other impedimetric
aptasensors (most covered in this chapter) that could detect the analyte in serum
samples, which show the tendency of real sample validation. However, more clinical
sample validation experiments are needed. Key parameters that should be tested in
real samples are repeatability, non-specific binding, and sample to sample variation.
The performance of aptasensors in real sample settings could be improved by
adjusting the buffer during SELEX to mimic real samples.
The SELEX procedure has been developed substantially in the last decade. Still,
the intrinsic capability to engineer an aptamer adapted to the specific needs of the
application has not been fully utilized. During the SELEX process several steps can
be included to reduce non-specific binding [153, 154] and to include a regeneration
technique [155]. For example, Wehbe et al. included an EDTA wash step to obtain
switchable aptamers (SwAps) that were used to recover vesicular stomatitis virus
after capture to allow further analytics of the sample with recovery rates of ~33%.
This technique would allow performing several analyses on the same sample for,
e.g., lab-on-a-chip developments.
Further strategies for the regeneration of the aptamer-modified sensors were
developed, mostly based on chemicals changing the ionic strength or pH of the
media, e.g. 2 M NaCl [156], 6 M guanidine hydrochloride [157], 6 M urea [32], pH
4 [158], 0.2 M glycine-HCl [112], or imidazole [159]. Others use more complex
elution buffers, e.g. Tris buffer containing 20% of methanol [107] or mixtures of
different chemicals, e.g. 0.05 M NaOH and 1 M NaCl [160] or variations of several
properties, like 1 M NaOH at 4
C [45]. But also more complex mechanisms for the
regeneration of the aptamer-target-bond were developed, e.g. the abovementioned
SwAps or the usage of mixed aptamers (MBA) that are oligonucleotides containing
more than one aptamer sequence directed against different targets. Du et al. used a
combination of ATP and thrombin aptamer, whereas the MBA was immobilized by
a complementary DNA probe immobilized on the electrode surface [161]. Part of the
ATP aptamer was complement to the DNA probe leading to a release of the MBA
when it binds to ATP. The binding of thrombin did not lead to a release but to an
increase in impedance. Thus, this aptasensor can be used to detect thrombin and can
be regenerated with ATP. A similar technique used by Liu et al. to detect Mucin
1 protein and carcinoembryonic antigen [51] is hybridization of the aptamer to an
immobilized complementary oligonucleotide, where upon binding to its target the
aptamer is released and the biosensor can be easily regenerated.
Detection of only one marker is not enough to establish a reliable diagnosis, but
biosensors targeting multiple analytes would enable early detection and help doctors
to provide a personalized therapy [162]. Although several publications report about
micro- and nanoarrays of electrodes [163–166] that are suitable for multiplexing
including repetitions and controls, unfortunately, no report on impedimetric
aptasensor used for the simultaneous detection of multiple analytes was found.
Multiplex impedimetric aptasensors would significantly enhance the chance for
market penetration and thus more research in this direction is needed.
Impedimetric Aptamer-Based Biosensors: Applications
79