2.1 Probe Design for Diagnostics
Effective tools to identify biomarkers are in high demand in medicine. Probe
development is one of the most frequently used strategies for diagnostics. Here, a
probe is considered using two components: the uptake component and a signal agent
to facilitate the measurement. In fact, a variety of diagnostic probes can be developed
using aptamers labeled via physical or chemical conjugation of both components, in
order to confer diverse functions.
There are several strategies to consider when creating a probe with high affinity
and specificity to the target [10]. Additional probe considerations relate to their
sensitivity, specific activity, signaling agent, and stability. The following describes
each consideration:
• High affinity to target. High uptake of the target usually requires nM-range Kd
values or less. This principally depends on the targeting component, but it is very
important that the recognition will not be affected by subsequent modifications.
Considering small molecules, any incorporations can affect charge and other
binding features.
• High specificity. This parameter permits the discrimination between the correct
target and others, because small molecular variations in specificity measure the
degree to which the probe differentiates between targets, isotypes, similar
domains, and conformational stages.
• Specific activity. This refers to the amount of signal component per mass unit.
This means how much signal component is available to be attached to the
molecular component and improve the detection.
• Signaling agent. For a probe to be used in quantification or qualification assays, it
needs a signaling agent, which can be a complementary dye, fluorophore, radionuclide, or a more complex system. Oligonucleotide composition exposes a
variety of chemical groups to attach signal agents to. Here, we must be very
careful in choosing the desired signal range which is directly dependent on the
application.
• Purity. The purity of the probe has direct consequences on the results. Free signal
agent increases the background noise affecting the real measurement. Free target
component and others decrease the site-specific signal.
• Stability. This is the most controversial point principally due to degradation of
biomolecule components. However, proper targeting and signal components
must be stable for as long as the assay requires. The pharmacokinetic parameters
should be also considered. Additionally, robust binding between the target and
signal agent is fundamentally related to the stability of the probe as a whole. A
covalent bond between both components is usually preferred. Extra links can be
added, but it is well documented that the addition has a profound impact on the
final physicochemical and biological characteristics [11].
• High sensitivity. Especially with small amounts of target, the probe must be
highly sensitive. The sensitivity is closely related to most of the features mentioned above but also is dependent on the detection equipment.
144
V. Calzada
Effective tools to identify biomarkers are in high demand in medicine. Probe
development is one of the most frequently used strategies for diagnostics. Here, a
probe is considered using two components: the uptake component and a signal agent
to facilitate the measurement. In fact, a variety of diagnostic probes can be developed
using aptamers labeled via physical or chemical conjugation of both components, in
order to confer diverse functions.
There are several strategies to consider when creating a probe with high affinity
and specificity to the target [10]. Additional probe considerations relate to their
sensitivity, specific activity, signaling agent, and stability. The following describes
each consideration:
• High affinity to target. High uptake of the target usually requires nM-range Kd
values or less. This principally depends on the targeting component, but it is very
important that the recognition will not be affected by subsequent modifications.
Considering small molecules, any incorporations can affect charge and other
binding features.
• High specificity. This parameter permits the discrimination between the correct
target and others, because small molecular variations in specificity measure the
degree to which the probe differentiates between targets, isotypes, similar
domains, and conformational stages.
• Specific activity. This refers to the amount of signal component per mass unit.
This means how much signal component is available to be attached to the
molecular component and improve the detection.
• Signaling agent. For a probe to be used in quantification or qualification assays, it
needs a signaling agent, which can be a complementary dye, fluorophore, radionuclide, or a more complex system. Oligonucleotide composition exposes a
variety of chemical groups to attach signal agents to. Here, we must be very
careful in choosing the desired signal range which is directly dependent on the
application.
• Purity. The purity of the probe has direct consequences on the results. Free signal
agent increases the background noise affecting the real measurement. Free target
component and others decrease the site-specific signal.
• Stability. This is the most controversial point principally due to degradation of
biomolecule components. However, proper targeting and signal components
must be stable for as long as the assay requires. The pharmacokinetic parameters
should be also considered. Additionally, robust binding between the target and
signal agent is fundamentally related to the stability of the probe as a whole. A
covalent bond between both components is usually preferred. Extra links can be
added, but it is well documented that the addition has a profound impact on the
final physicochemical and biological characteristics [11].
• High sensitivity. Especially with small amounts of target, the probe must be
highly sensitive. The sensitivity is closely related to most of the features mentioned above but also is dependent on the detection equipment.
144
V. Calzada