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.