Electrochemical
The majority of electrochemical biosensors are based on a principle
called Electrochemical Impedance Spectroscopy, or EIS. Performing an EIS
requires electrodes be placed within the solution desired to be tested
and delivering a sinusoidal alternating current signal through the
solution, usually ranging from 2-10 mV. By varying the frequency of
these sinusoidal waves, an impedance spectrum can be created
81. The electrodes are coated in metal, to reduce
electric resistance. Proteins that are highly selective to endotoxin
components are then bound to these electrodes such that if the
endotoxins come in contact with the electrode-protein complex, they bind
to the proteins. These proteins are referred to as Endotoxin
Neutralizing Proteins, or ENPs 82,83. When endotoxins
bind to ENPs on the electrodes it increases the resistance of the
electrode. This was the case in an experiment run by Yeo et al.84 who constructed an electrode made of gold and a
complex of human recombinant toll-like receptor 4 (rhTLR4) and myeloid
differentiation-2 (MD-2) proteins (Figure 4) . They exposed
these electrodes to solutions of varying endotoxin concentrations and
created impedance spectrums for each of these concentrations. The
maximum current across all potential differences was lower at higher
concentrations of endotoxin 74. The study also reports
that this particular biosensor has high specificity towards endotoxins,
in order to prevent false positive results. It then goes on to state
that the sensor had a detection limit of 0.0002 EU/ml, or 2 x
10-5 ng/ml. This is drastically lower than the
standard LAL test limit of 0.003 ng/ml. Metal complexes immobilized upon
a gold electrode have been used and were able to detect endotoxins at
concentrations as low as 0.0001 ng/ml 85. Porous
silicon membranes (pSim) based electrochemical biosensors comprise of
array of nano-channels which are modified using Polymyxin-B, with strong
affinity to endotoxins. It shows very low limits of detection of 1.8
ng/ml. These sensors showed ability to detect endotoxins from various
bacterial strains like E. coli and S. typhimurium and all this is
done in a label free manner 86. Studies have also
reported highly sensitive peptide modified gold electrode based
electrochemical biosensors which are used for endotoxin detection with
very low limit of detection of 0.04 EU/ml 87. These
methods are faster, more accurate, and in most cases, more cost
effective than biological based techniques 88. Two
other electrochemical techniques are amperometric and potentiometric
methods. Amperometric transducers have been described as the most common
of the electrochemical sensors used for endotoxin detection
89. They work on the relying on the same principle of
EIS, wherein the concentration of the analyzed sample has a linear
relationship with the current measured. This method is able to use
premade, disposable testing strips, for fast, cost-efficient testing
90. Potentiometric methods are worth noting because
although their detection limits are relatively high, 1-5 EU/mL, they
were the first biosensor to be able to detect endotoxins in real time
81,91. The methods in which the electrodes are
created, as well as the ways in which they are measured and utilized,
are more complicated and labor-intensive than the biological methods
92. They require more sophisticated personnel and
equipment to be run effectively than RPT or LAL
tests93.