Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

The overall goal of the following experiment is to construct an electronic tongue based on a combinatorial approach and perform surface plasma and resonance imaging for protein analysis. This is achieved by using lactose and sulfated lactose as building blocks and depositing their mixtures on the gold surface of a prism for self-assembly to create an array of combinatorial cross-reactive receptors. As a second step surface, plasma and resonance imaging is applied, which monitors binding events in real time and produces SPR images and a series of kinetic binding curves called sensor grams.

Next data processing is carried out based on the sensor grams using a math computing software in order to create a 2D continuous evolution profile and a 3D continuous evolution landscape for each sample, the results show the electronic tongue is able to generate distinct 3D continuous evolution landscapes for common purified proteins, and is efficient for their discrimination based on pattern recognition. This technique has several advantages over existing methods like classical, electronic gnosis and tongues. First, the preparation of sing receptors is largely simplified.

A great diversity of cross reactive receptors can be opting by mixing a small number of molecules. Second, thanks to the combinatory approach, the recognition patterns generated by our electronic tongue can be considered as continuous, so that abnormal signals can be more easily identified. Therefore, more reliable analysis can be obtained To prepare the combinatorial cross-reactive receptor array.

First, clean the gold surface of the prism 48 hours prior to use with a plasma cleaner for three minutes under 75%oxygen, 25%argon, 0.6 millibar, and 40 watts of power. Then prepare 100 milliliters of phosphate buffer solution containing 10%glycerol or PBSG. Adding glycerol to PBS is very important to reduce solvent evaporation and changes in the building block or BBB concentration after deposition.

Then prepare stock solutions of lactose or building block one and sulfated lactose, or building block two from the stock solutions. Prepare 11 pure and mixed solutions with ratios between zero and 100%in increments of 10%and a total building block concentration of 0.1 millimolar in PBSG deposit eight nanoliter droplets of these pure and mixed solutions on the prism surface using a non-contact spotter in quad duplicate for each ratio with 44 spots in total place the prism inside a Petri dish containing one milliliter of ultra pure water and leave it overnight at room temperature for self-assembly of BB one and BB two on the gold surface herein. It is assumed that the average surface composition in the mixed self-assembled monolayers reflects the composition of the deposition mixed solution.

The next day, wash the prism thoroughly with ultrapure water before drying it under a flow of Argonne. Set the incubator in which the surface plasma and resonance imaging apparatuses placed at 25 degrees Celsius to avoid refractive index changes induced by temperature variation during protein sensing. Insert a non functionalized rinsing prism in the 10 microliter polyether ether ketone flow cell connected to a computer controlled syringe pump, a desser and a six port medium pressure injection valve.

Fill the flow system with freshly filtered and Degas heaps running buffer. Remove the rinsing prism and insert the prism containing the combinational Cross-reactive receptor array in the flow cell run heaps at a flow rate of 100 microliters per minute. With the help of the CCD camera, remove any air bubbles present on the prism surface by passing running buffer.

Quickly define the study area for each spot by drawing a circle with the same diameter for the area of interest based on a well contrasted image of the array, and with the help of integrated software in the surface plasma and resonance imaging system, trace plasma curves, which represent reflectivity curves as a function of the incident angle for all the spots. Choose the working angle, which is the position at which kinetic curves will be recorded at the highest slope of the reflectivity curves. Rotate the scanning mirror to fix the selected working angle for kinetic measurements.

Continue to run heaps through the flow system until the reflectivity signal for all the spots is stable and constant. Then use a syringe to inject one milliliter of peanut lectin or a HL solution into the 500 microliter sample loop with the injection valve at the load position with the flow rate set to 100 microliters per minute, put the injection valve to the injection position. Start kinetic measurements by monitoring reflectivity variations against time simultaneously on all the spots at the end of protein injection.

Rinse the array with running buffer for eight minutes. Finally, inject one milliliter of 1%DS to regenerate the array. Repeat the same procedure for the other proteins following entry of protein solution into the flow cell.

Molecular binding occurs and induces a shift of the plasma curves and a variation of reflexivity. The image acquisition software converts the measured light intensity values to gray scale levels, giving SPR images and generates the variation of reflectivity versus time giving sensor grams. Next, use a math computing software to plot the reflectivity at the end of each protein injection versus the building block ratio to generate a 2D continuous evolution profile for each sample.

Finally, add the building block ratio into the sensor grams to generate a time dependent continuous recognition pattern called A 3D continuous evolution landscape for each protein to probe the ability of the electronic tongue for common protein analysis, three proteins were used a HL myoglobin and lysozyme for each protein. A distinct 2D continuous evolution profile was generated by the electronic tongue as shown 3D. Continuous evolution landscapes were also generated for a HL myoglobin and lysozyme.

The electronic tongue is sensitive to the surface characteristics of the proteins, such as the distribution of hydrophobic, neutral, and charged amino acid residues. The obtained continuous evolution profiles of landscapes can be used as fingerprints for efficient protein discrimination and prospective identification based on pattern recognition. While attempting this approach, it's important to remember to follow the optimized and standardized experimental procedures in order to ensure good reproducibility of the electronic tongue.

This procedures include clean and good surface of the prism, choosing the working angle for the surface plasma resonance imaging, and applying appropriate solution to regenerate the system completely for reuse. After watching this video, you should have a good understanding of how to construct the electronic tongue based on the COMBINATOR approach and SOFA plasma resonance imaging for protein analysis. Good luck for your experiments.