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Jung et al. Soft Sci 2024;4:15 https://dx.doi.org/10.20517/ss.2024.02 Page 9 of 44

adsorption may also occur. The key differentiation between ionic binding and physical adsorption pertains
to the strength of their interactions. Ionic binding demonstrates a distinctly stronger interaction, albeit not
as sturdy as that observed in covalent binding. Ionic binding offers the advantage of minimizing the
conformational alterations in the enzyme structure, thereby ensuring the preservation of proper enzymatic
activity. However, factors such as pH, temperature, and enzyme concentration can influence ionic
interactions. Consequently, maintaining optimal ionic strength becomes imperative to prevent leaching of
the immobilized enzyme in suboptimal conditions [154,155] .

Non-enzymatic electrochemical sensing
Antibodies
Immunological sensors, leveraging the intrinsic specificity of antibody-antigen interactions, are
instrumental in detecting biomolecules ranging from nanomolar to femtomolar concentrations [156-159] . These
sensors excel in detecting large biomolecules, such as proteins and cells, by harnessing the changes in
surface properties that occur upon antigen-antibody binding. Such interactions are pivotal for signal
transduction, primarily through electrochemical mechanisms within the domain of electrochemical
immunosensors. It is important to note that while antibodies are highly effective in detecting numerous
biomolecules such as some small molecules, the specific detection of certain analytes such as glucose,
lactate, potassium, sodium, and uric acid often relies on alternative sensing elements and mechanisms that
may not involve traditional antibody-antigen interactions. A diverse array of methodologies is employed in
[160]
this field, with the competitive and sandwich formats being particularly prominent . These methodologies
adeptly translate biochemical interactions into quantifiable electrical signals, thereby capitalizing on the
specificity and sensitivity inherent in antigen-antibody interactions for precise biomarker detection.

The competitive format of electrochemical immunosensors [Figure 3A (i)], primarily involves label-tagged
targets that compete with analytes for binding sites on specific antibodies. In this setup, the analyte and a
labeled analog are introduced to the system. The more analytes in the sample, the less labeled analog binds
to the antibody. The detection and quantification are negatively correlated with the analyte concentration,
as the signal decreases when the analyte concentration increases. This format is particularly useful for small
molecule detection where the epitope is limited, allowing for precise quantification even at low analyte
concentrations.

On the other hand, the sandwich format [Figure 3A (ii)], is characterized by its utilization of two antibodies:
a capture antibody and a labeled detector antibody. This method is tailored for larger molecules that possess
multiple epitopes. Initially, the target analyte is captured by the immobilized antibody on the sensor surface.
Subsequently, a second labeled antibody binds to a different epitope on the analyte. The sandwich format’s
signal amplification, resulting from multiple bindings, enhances the sensitivity and specificity of the sensor.
It is particularly effective for complex samples, providing robust detection even in the presence of
interfering substances.

However, these high-precision sensors necessitate meticulous bioreagent incubation and washing steps
before detection, underpinning the need for innovative solutions to streamline and adapt these processes for
wearable applications [161,162] .

Aptamers
Diverging from traditional antibody-based sensors, electrochemical aptamer-based (E-AB) sensors
represent a significant advancement and a paradigm shift in biosensing technology. Aptamers, single-
stranded DNA or RNA molecules, are discovered through the systematic evolution of ligands by