Hi, my name is Francesco Lichi. I'm a postdoc researcher at the Bioanalytical Lab of Professor Palki at the University of Rome Tova Gata. Today, together with my colleagues, I'm going to show you preparation of an electrochemical CY for the detection of TCO mycotoxin in food samples.
Among the different methods used in immuno cell test, enzyme immuno cell is one of the most popular in this method. Either the antibody or antigen is level with an enzyme. Recently, the use of electrochemical methods for the detection of the enzyme duct as demonstrated to be particular advantages for certain applications.
Electrochemistry is, in fact, less prone to interferences, is suitable for miniaturization and for mass deduction at low cost. In this perspective, the use of screenprint electros is of great importance to ensure low cost and ness of procedure. Moreover, the use of magnetic beds could be coup with screenprinted electrodes in order to obtain the best sensitivity of method.
Today, we'll show you how to prepare an electrochemical mase based on the use of screen printed electrodes and magnetic bits for the detection of TCOs mycotoxin in food samples. Trics mycotoxins are commonly found in cereals, and for this reason represent a serious threat for the safety of cereal based food and feed stuff. The most important turtin within group A are T two and HT two toxins.
As we will see, this method can be easily performed by non-trained personnel and will low cost and portable instrumentation. This is one of the main objective of the integrated project bio funded by the European community in the frame of which this work has been carried out. Now let's see what is the principle of the method and the materials we'll use?
So let's get started. Materials magnetic beads will be used as support for the immunological chain reaction. A strip of eight screen printed electrodes will be used as sensing element.
Each sensor is comprised of a graphite, working electrode, a counter electrode, and a silver reference electrode. These sensors can be sequentially and individually operated in a very short time by a portable electrochemical instrument made by palm Sense principle of the method. The principle of the method is straightforward and is based on a competitive immunological reaction.
The conjugated toxin is immobilized on the magnetic beads, which are blocked afterwards to prevent non-specific absorption. The measurement itself starts with the competition step. In this phase, the coated beads are left to react with a fixed amount of monoclonal antibody and with the sample toxin present in the sample and the one immobilized on the magnetic beads will compete for the antibody binding sites.
The amount of antibody linked to the coded beads will be inversely proportional to the amount of toxin in a sample. The competition step is then followed by the labeling step. In this case, the magnetic beads are left to react with a secondary antibody conjugated with a labeling enzyme.
The secondary antibody will bind the monoclonal antibody and ensure efficient labeling. The labeling step is finally followed by the measurement step. In this case, the magnetic beads are placed in concentrated on the electrode surface with the aid of the magnetic platform, and we simply add the enzyme substrate one napt phosphate, and measure the production of one naptha.
The binding curve generated will show high current values at low toxin concentrations and low signals at high toxin content. The toxin concentration will be obtained by using a calibration curve generated in standard buffered solutions. To set up the actual assay, we prepare a rack of clean two mil fendor tubes, a separate one for each standard solution of the calibration curve, as well as for the samples.
Then 10 microliters of bead suspension is pipetted into each tube. This solution contains magnetic beads previously coated with the conjugate toxin. However, before running the assay, the storage liquid must be removed because the sodium azide used as a preservative would affect enzymatic activity of the label.
To do this, P-B-S-B-S-A buffer is added. The tubes are shaken, then the magnet is reinserted and the washing buffer is pipetted off. The first actual passage of the assay is called the blocking step.
During this passage, one mil of blocking solution is added to each of the tubes. Then they are put on the rotating sample mixer and left to incubate for half an hour at temperature. Then the blocking solution is removed for the next crucial step, the competition step, we add monoclonal specific antibody and toxin solutions to each tube.
To do this, 200 microliters of monoclonal antibody are injected at the optimized concentration. Then 200 microliters of each standard solution or sample is added to each tube. These reagents will be allowed to incubate for 30 minutes.
In order to optimize contact between the reagents immobilized on the magnetic particle surface and the molecules in solution to start this process, the tubes are put back on the rotating sample mixer to proceed to the labeling step. It is necessary to remove the solution from the previous step. Then 400 microliters of labeled secondary antibody is added to each tube, and they are returned to the rotating sample mixer and incubated for 30 minutes.
Then after removal of the labeling liquid, we performed washing procedures using various buffers in order to avoid any interference by unbound enzymatic reagents. Finally, to prepare for the actual measurements, the magnetic beads are resus suspended in 100 microliters of DEA buffer one that is suitable for optimizing the enzymatic activity of the label. The measurement is performed with this palm sends pot potentials stat that is shown connected to an eight electrode strip underneath.
There is a special support on which eight strong magnets are fixed exactly under the surface of each working electrode. The magnet serves to concentrate the beads on the working electrode surface. We pipette from each EOR a 20 microliter aliquot of the reactive magnetic bead dispersion and load it onto the corresponding working electrode surface.
To perform the actual measurement enzymatic substrate has to be added, but the sequence of steps also has to be carefully controlled. We add ad microliters of enzymatic substrate solution to the first electrode surface. Then we start a two minute countdown.
After 14 seconds, we add substrate solution onto the second electrode. After another 14 seconds, we add substrate solution to the third electrode. We proceed using the same time interval up to the last electrode.
In fact, the interval of 14 seconds is calculated as the time required for the potential stat to perform the measurement. Alright, After the two minute countdown from the first edition of enzymatic substrate solution, the electrochemical measurement protocol is started. The actual measurement of product formed is done using differential postal telemetry.
The curves for the successive electrodes begin to appear on the screen being a competitive assay. The current intensity of the signal obtained will be inversely proportional to the concentration of toxin that was present in the tube in the competition step. Current signal of standard solutions will be plotted in a sigmoidal curve and toxin content unknown samples will be obtained by extrapolating concentration value from the signal recorded.
Well, I hope this demonstration was clear and useful to understand the advantages of electrochemical immuno sensors. Thanks for watching and good luck with your experiments.
