Biomedical Engineering Reference
In-Depth Information
proportional to the concentration of the target species. Thus, by characterizing
the double layer capacitance, an accurate estimate of the concentration of the
target species can be measured. The changes to the double layer capacitance
can be represented as the measured impedance changes especially at low
frequencies (below 1 kHz). In the implemented sensor configuration, redox
probes are not used and it can be assumed that all the conduction occurring at
the interface is non-faradaic in nature, so the charge distribution dynamics at
the metal-solution interface characterizing the biomolecular interactions at the
surface can be modeled using the Helmholtz-Gouy-Chapman model with
Sterns correction. Since binding of antigens to the nanobodies is free of any
biochemical mediators, the impedance changes within the electrical double
layer (Figure 5.7(d)) is non-Faradaic and the electrical circuit model of the
sensor can be represented as a simple resistive-capacitive (RC) series circuit
whose values are extracted by a frequency response analyzer potentiostat. For
probing the impedance changes to the electrical double layer of the nanowell
electrodes, a very small amplitude sinusoidal voltage is applied to the electro-
chemical system and the output current response is sensed. The ratio of the
applied voltage phasor to the output current phasor is the resulting impedance,
which is characterized using a frequency response analyzer.
A commonly used electrochemical immunosensing technique is pulsed
amperometry. This method involves the immobilization of an immune-reagent
component on the electrode transducer and the use of an electrochemical active
substance produced by enzymatic reaction for signal generation. As simple as
this appears, there can be numerous problems associated such as inadequate
supply of enzyme inhibitors in the sample, instability of the enzyme over time,
irreproducibility of the electrode kinetics for the re-oxidizing reagent or
reducing oxidizing agent, redox active interferences which either react at the
electrode and/or couple with the reagent couple, and inadequate temperature
control. The electrochemical impedance spectroscopy (EIS) technique elim-
inates most of these problems since it doesn't rely on the redox properties of the
analyte and doesn't need an enzyme inhibitor. Another fundamental difference
between the two techniques is the sensing mechanism. Amperometry involves
detection of ions in the solution by applying a fixed voltage through electrodes
and measuring the current/change in current, whereas EIS involves char-
acterizing the electrical double layer at the electrode by sweeping a range of
frequencies and measuring the current.
Highly selective morphology specific reagents (isolated nanobodies) are
immobilized onto the electrode sensor surface using a chemical linker. The
electrode surface is first amine functionalized using 3-aminopropyl triethoxy-
silane (APTES, 2% in acetone buffer). A 100mL aliquot of 2% APTES is
applied on the electrode surface and incubated at room temperature for 30 s.
Excess APTES is then removed by flowing acetone over the surface. The
alumina membrane is then soldered to the silanized electrode surface. Then
3,3 0 -dithiobis succinimidyl propionate (DTSSP) dissolved in dimethyl sulfoxide
(DMSO) solvent (4mgmL 1 ) is used to cross-link the nanobodies to the
electrode surface.
d n 4 t 3 n g | 2
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