Antifouling Properties of Hexapeptides

Oliver Brauning Author

07/27/2021 Added

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Discussion of ongoing Lai Lab research

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[00:00:03.070]Hello, I'm Oliver Brauning,
[00:00:05.530]and I'm here to talk about the Lai Lab
[00:00:07.380]project of fabricating antifouling biosensors.
[00:00:11.600]One of the major problems with biosensors is surface fouling
[00:00:15.000]caused by non-specific absorption of proteins.
[00:00:18.910]So far antifouling research has been conducted on
[00:00:21.523]nucleic acid modified and ethylene glycol terminated probes,
[00:00:26.220]but not on short hydrophilic peptides.
[00:00:30.530]Our project is trying to fill this gap
[00:00:32.480]by testing two basic hexapeptides,
[00:00:35.470]lysine K six and arginine R six,
[00:00:38.810]and one acidic hexapeptide,
[00:00:41.360]glutamic acid
[00:00:42.700]E six.
[00:00:47.508]The experimental procedure starts out by dropcasting probe
[00:00:51.250]onto the surface of the gold disk working electrodes.
[00:00:56.051]A dropcasts occurs when you suck up with a pipette
[00:01:00.910]a solution and then use the pipette to drop
[00:01:05.100]the solution onto whatever your material is.
[00:01:08.830]In this case, a gold disk electrode.
[00:01:12.840]The probe concentrations used were 20 micromolar for K six,
[00:01:18.840]10 micromolar for R six,
[00:01:20.357]and 10 micromolar for E six as well.
[00:01:24.530]These sat overnight in two millimolar
[00:01:27.807]six mercapto one hexanol, or C6OH,
[00:01:32.560]which immobilizes the peptide by forming
[00:01:34.940]self-assembled monolayer.
[00:01:38.460]The coverage for K six on the electrodes is typically about
[00:01:42.020]0.2 to 0.5 TERRA molecules per centimeter squared,
[00:01:47.220]but the coverages for E six and R six are around
[00:01:51.100]one TERRA molecule per centimeter squared,
[00:01:54.010]with E six being a little more variable than R six.
[00:01:58.400]We place these modified electrodes into a cell
[00:02:01.140]with a silver reference electrode
[00:02:03.220]and a platinum counter electrode,
[00:02:05.390]so that we can apply the potential
[00:02:07.520]and monitor the current of the methylene blue tags,
[00:02:10.920]which are attached to the peptides.
[00:02:13.870]Our primary electro-chemical method used
[00:02:16.540]was alternating current voltammetry or ACV.
[00:02:21.790]Experiment propers begun by equilibrating
[00:02:25.190]the probes at 10 hertz
[00:02:27.360]and Fizz two at pH 7.6
[00:02:30.850]until they're stable enough to add a fouling agent.
[00:02:34.490]The equilibration time typically lasts an hour
[00:02:37.410]for K six and R six,
[00:02:39.390]but the E six probe is stable at 40 minutes.
[00:02:43.470]Once stable, the coverage is taken
[00:02:45.890]using ACV at one, two, four, and eight hertz.
[00:02:50.280]While the frequency response is also tested
[00:02:52.860]at 50 and 200 hertz from proximately
[00:02:56.170]negative 0.1 volts to negative 0.4 volts.
[00:03:00.870]Then capacitance data is taken using cyclical voltammetry
[00:03:05.570]at 51.2 volts per second,
[00:03:08.710]from negative 0.1 volts to positive 0.1 volts,
[00:03:12.550]as another other surface fouling monitoring technique.
[00:03:16.850]Finally after that we can add our fouling agent
[00:03:20.650]to the Fizz two solution,
[00:03:22.600]either bovine serum albumin, also known as BSA,
[00:03:26.970]or fetal bovine serum
[00:03:29.350]or FBS.
[00:03:32.410]The experiment proceeds by equilibrating the probes
[00:03:35.390]at a specified concentration for the same amount of time,
[00:03:38.640]and then taking 50 and 200 hertz ACV scans
[00:03:41.840]and a cyclical voltammetry capacitance scan.
[00:03:46.530]When the experiment is finished,
[00:03:48.000]a way we interpret its results
[00:03:49.430]is by looking at the voltammograms and examining the gap
[00:03:54.230]or the signal suppression between the peaks
[00:03:57.300]of the before signal and the peaks of the signal
[00:03:59.920]once a concentration of fouling agent is added.
[00:04:05.400]The voltammograms on the poster only have three different
[00:04:07.930]concentrations of fouling agent.
[00:04:10.240]That's for simplicity's sake,
[00:04:12.190]as we actually took data from six concentrations,
[00:04:17.430]five,
[00:04:18.300]10,
[00:04:19.133]50,
[00:04:19.984]100,
[00:04:20.817]500,
[00:04:21.650]and 1,000 micromolar BSA.
[00:04:24.880]The three voltammograms compare the potentials
[00:04:27.490]of the same probe at different concentrations.
[00:04:30.710]However, the bottom right graph compares the potential
[00:04:33.830]of different probes at the same concentration.
[00:04:38.490]K six and R six showed modest signals suppression.
[00:04:43.230]R six under 40% and K six under 30%.
[00:04:48.300]While E six provided almost no signal suppression,
[00:04:51.350]even at the very high concentration of one millimolar.
[00:04:55.780]All the data on these graphs was collected
[00:04:57.930]at a low frequency of 10 hertz.
[00:05:02.900]As we look at the results from the
[00:05:04.480]fetal bovine serum experiment,
[00:05:07.210]the first thing that jumps out is
[00:05:09.260]there's one fewer graph.
[00:05:11.790]That's because we had a little bit of trouble getting
[00:05:14.550]the R six probes stable enough
[00:05:16.590]to be able to interpret results.
[00:05:18.920]We also will have one fewer data point.
[00:05:21.160]We only tested the FBS concentrations of 10%, 25%,
[00:05:26.990]50%, 75%
[00:05:29.421]and 100% percent.
[00:05:33.420]The baselines of the curves on voltammograms
[00:05:36.440]observed a shift to the right
[00:05:38.580]because the cell solution became more alkaline
[00:05:41.790]as we had a greater concentration of FBS.
[00:05:47.360]Both K six and E six performed worse in FBS then in BSA.
[00:05:51.820]Although E six is still within an acceptable
[00:05:54.960]threshold of percent signal suppression.
[00:06:00.620]A general trend we saw within both experiments
[00:06:03.340]was that even a small amount of fouling agent
[00:06:05.670]would destabilize the probe and increase signal suppression.
[00:06:09.690]But once the probe stabilized again,
[00:06:11.920]adding more fouling agent would hardly cause
[00:06:14.230]any more signal suppression.
[00:06:17.220]FBS did more to degrade the probes then BSA.
[00:06:21.930]All the peptides performed reasonably well
[00:06:24.580]in the fouling agents,
[00:06:26.010]although E six behaved better than K six and R six.
[00:06:29.890]One reason for the difference between the performance
[00:06:32.620]of E six and the others
[00:06:34.550]might be because lysine and arginine have
[00:06:37.810]positive side chains,
[00:06:39.520]which could be attracted to the negatively charged BSA.
[00:06:44.620]However, because K six and R six performed reasonably well,
[00:06:49.350]future work should look further into
[00:06:51.130]the mechanism for antifouling,
[00:06:53.450]to see how much of the antifouling ability
[00:06:55.920]comes from the peptide
[00:06:57.710]and how much comes from the methylene blue, or C6OH.
[00:07:02.790]Additionally,
[00:07:03.623]the R six experimental data needs to be completed for FBS.
[00:07:10.030]Although some areas still remain to be investigated,
[00:07:14.070]E six has shown itself to be a capable antifouling species.

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Antifouling Properties of Hexapeptides

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