Antifouling Performance of D6 and A6 Hexapeptides

Katharina E. Dvorak Author

08/03/2021 Added

4 Plays

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For sensors with potential applications in complex biological systems, preventing surface fouling (non-specific binding) is vital to ensure specific recognition of the target molecule and avoid false positive results. This project investigated the hexapeptide D6-K-MB as a candidate for antifouling applications in specific biosensors.

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[00:00:00.000]My name is Kate Dvorak and my summer research project
[00:00:04.759]is antifouling performance of D6 and A6
[00:00:07.999]hexapeptides.
[00:00:12.000]So for some background on this project,
[00:00:17.916]For sensors with potential applications in
[00:00:20.404]complex biological systems, preventing
[00:00:22.724]surface fouling (non-specific binding) is
[00:00:25.064]vital to ensure specific recognition
[00:00:27.174]of the target molecule and avoid false
[00:00:28.994]positive results. This can be achieved by
[00:00:31.534]integrating other antifouling molecules
[00:00:34.244]into the self-assembled monolayer (SAM)
[00:00:36.844]on the sensor surface. Antifouling
[00:00:40.984]peptides, which are just chains of amino
[00:00:43.404]acids, are commonly used in biosensors due
[00:00:46.348]to their ability to create a hydration
[00:00:48.584]layer that prevents nonspecific protein
[00:00:50.985]adsorption. Our goals for the project were
[00:00:57.050]to investigate the peptide C6-D6 as a
[00:00:59.819]candidate for antifouling applications in
[00:01:02.191]specific biosensors. D6 is a chain of
[00:01:04.792]six aspartic acid residues and was
[00:01:08.099]predicted to have high antifouling
[00:01:10.129]ability due to its highly polar nature
[00:01:12.324]and acidic side chain at neutral pH.
[00:01:14.344]Alternating current voltammetry
[00:01:16.425]was used to analyze adsorption of proteins,
[00:01:19.015]such as bovine serum albumin and proteins
[00:01:22.003]in fetal bovine serum, at different
[00:01:25.095]concentrations on gold electrodes
[00:01:27.345]modified with the D6 peptide probe and a
[00:01:29.885]control probe, A6. Reference data was
[00:01:32.205]also recorded for methylene-blue modified
[00:01:34.775]electrodes.
[00:01:35.685]We first fabricated the electrodes by
[00:01:38.015]dissolving the peptide (either D6 or A6)
[00:01:40.255]in buffer and then dropcasting
[00:01:42.215]it on the surface of the electrode,
[00:01:44.276]letting it sit for one hour, then placing
[00:01:46.511]it in diluent (which attaches the peptide
[00:01:48.911]to the gold electrode surface) for 17-18
[00:01:51.341]hours in the fridge. We then interrogated
[00:01:55.053]the electrodes using ACV by equilibrating
[00:01:59.053]at 10 Hz until the methylene blue current
[00:02:01.513]was stable, which took 40-60 minutes,
[00:02:03.941]then determining the capacitance with CV
[00:02:06.543]and collecting the probe coverage data,
[00:02:08.973]which determines how much of the
[00:02:11.223]probe covers the electrode surface, which
[00:02:13.832]we determined in molecules/cm^2 for both
[00:02:16.392]D6, A6, and KMB, which was our reference,
[00:02:18.952]and then finally collecting the ACV
[00:02:21.126]response at 50 and 200 Hz. And we did this
[00:02:25.259]in Phys 2- that was spiked with different
[00:02:27.901]concentrations of BSA (bovine serum
[00:02:30.211]albumin) and fetal bovine serum.
[00:02:37.660]So because the peptides we were
[00:02:39.560]investigating for their antifouling
[00:02:41.325]performance have a methylene blue
[00:02:42.938]at the end of the amino acid chain, we
[00:02:44.819]wanted to determine that the methylene blue,
[00:02:47.489]which is what causes the peak in the
[00:02:49.638]voltammograms we were obtaining, was not
[00:02:52.380]what was causing our observed signal
[00:02:54.630]suppression, if any. So this was our K-MB
[00:02:56.880]reference calibration: K-MB is a methylene
[00:02:59.350]blue attached to one lysine residue which
[00:03:01.870]is then put on the surface of the
[00:03:04.400]electrode using C6-OH diluent, just like
[00:03:06.740]the rest of our probes, and we only
[00:03:09.079]observed about a 5% signal enhancement
[00:03:13.139]in FBS concentrations of 10, 25, 50, 75,
[00:03:15.679]and 100%. Shown here is some raw data
[00:03:22.829]we obtained in terms of ACV overlays of
[00:03:25.309]our probes, D6 and A6, in different
[00:03:27.517]concentrations of FBS or BSA. So for an
[00:03:30.597]antifouling experiment, what we would be
[00:03:33.177]looking for as we ran the experiment is
[00:03:36.267]that the peaks would overlay perfectly, so
[00:03:38.721]the before peak would match up with the
[00:03:41.721]100 uM or 1000 uM peak for BSA or the 50%
[00:03:44.401]or 100% peak for FBS. For our control,
[00:03:51.171]so for A6, which was not intended to be a
[00:03:55.652]good antifouling, we would be looking for
[00:03:58.122]an increase in signal suppression. So
[00:04:00.195]what this would look like is decreased
[00:04:02.455]peak height with increasing concentration.
[00:04:05.040]And as you can see here with our D6
[00:04:07.270]peaks, which are on the top, for FBS they
[00:04:10.060]don't exactly overlay perfectly,
[00:04:12.166]indicating there is some amount of signal
[00:04:14.316]suppression, for BSA they overlay pretty
[00:04:16.726]well. For our A6 probe, which was meant to
[00:04:19.620]be the control, again the FBS peaks
[00:04:22.800]exhibit a very slight amount of signal
[00:04:25.140]suppression, although not as much as D6,
[00:04:27.660]although we can't quantify this just
[00:04:29.797]by looking at the overlays. Interestingly
[00:04:32.217]for BSA it looks like they overlay pretty
[00:04:34.607]well, which indicates that A6 might
[00:04:36.631]actually be a good antifouling peptide.
[00:04:43.034]Shown here are our calibration curves,
[00:04:45.404]which quantify signal suppression
[00:04:47.514]based on the kind of raw data overlays we
[00:04:49.634]observed in the last side. So what we
[00:04:51.884]would be looking for is, ideally, for an
[00:04:53.814]antifouling peptide, which we expected D6
[00:04:56.030]to work pretty well, you would observe
[00:04:58.400]around 0% signal suppression hopefully.
[00:05:01.710]Anything under about 20% would be
[00:05:03.944]considered a good antifouling probe.
[00:05:06.534]Getting into the 30-40% signal suppression
[00:05:09.040]range would mean the sensor was actually
[00:05:11.433]sensing what we were putting on the
[00:05:13.583]surface, so in this case the proteins FBS
[00:05:15.873]and BSA. So for our D6 probe, the signal
[00:05:18.813]suppression was pretty good, it hovered
[00:05:21.217]under 15% for FBS and was really close to
[00:05:23.687]0% for BSA. But interestingly, for our A6
[00:05:26.927]probe, which was meant to be the control,
[00:05:29.432]we didn't expect it to work very well, we
[00:05:31.792]expected the signal suppression to be
[00:05:34.112]higher, for FBS the signal suppression
[00:05:36.212]was under 5% with increasing concentration
[00:05:38.822]and for BSA again, under 5%, which was
[00:05:41.252]unexpected. As shown in the last slide,
[00:05:47.097]the results we obtained for A6 were
[00:05:49.630]unexpected, in that we expected there to
[00:05:52.190]be a large amount of signal suppression
[00:05:54.430]and there really wasn't. A6 was seemingly
[00:05:56.750]performing a little bit better as an
[00:05:58.762]antifouling peptide than D6 was, which
[00:06:01.122]was unexpected because A6 is nonpolar
[00:06:04.012]and D6 is polar, meaning that it could
[00:06:06.391]probably bind better to proteins, which
[00:06:08.750]are typically very polar. So here we did
[00:06:11.957]an experiment where we put lower
[00:06:14.367]concentrations, lower coverage amounts, of
[00:06:16.987]A6 on the electrode. We thought that A6
[00:06:19.266]might have been performing so well because
[00:06:21.616]it had about 10^12 molecules/cm^2 on the
[00:06:24.021]electrode, where D6 dropcast at the same
[00:06:26.287]concentration had about 10^11 molecules/
[00:06:28.837]cm^2 on the electrode. So we did a lower
[00:06:32.227]concentration of the A6 that had about the
[00:06:34.407]same coverage, so 10^11 molecules/cm^2,
[00:06:37.572]and we obtained these results. So this is
[00:06:42.082]A6, about the same coverage as D6, in
[00:06:44.451]different concentrations of FBS. And
[00:06:48.553]we observed higher signal suppression in
[00:06:52.326]the lower-coverage A6 data than we did in
[00:06:54.706]the higher-coverage A6 data, but still
[00:06:57.697]under the amount we observed for D6 at
[00:07:03.341]the same coverage. So our control system,
[00:07:08.470]K-MB, exhibited just a slight signal
[00:07:11.188]enhancement (around 5%), but this did not
[00:07:13.428]significantly change with increasing
[00:07:15.578]concentration of FBS, indicating that the
[00:07:17.983]methylene blue label does not interfere
[00:07:20.147]with or alter the antifouling mechanism.
[00:07:22.176]A6 exhibited lower signal suppression than
[00:07:24.533]D6 in both BSA and FBS when both were
[00:07:27.063]fabricated using 3 uM peptide, which was
[00:07:30.533]unexpected as A6 (a six amino acid chain
[00:07:33.214]of alanine residues) is nonpolar and
[00:07:35.320]uncharged at neutral pH. Since A6 showed
[00:07:38.614]higher probe coverage when fabricated
[00:07:40.984]with the same peptide concentration of D6,
[00:07:43.244]a lower concentration of A6 was used to
[00:07:45.694]produce a coverage that was similar to
[00:07:47.844]D6. While D6 exhibited higher signal
[00:07:50.584]suppression with increasing concentration
[00:07:52.686]of FBS when compared to A6, the difference
[00:07:54.746]was not significant. Further antifouling
[00:07:56.996]experiments will be conducted using G6
[00:07:59.306](glycine hexapeptide)-modified electrodes
[00:08:01.736]as glycine is hydrophobic and uncharged
[00:08:03.706]at neutral pH, with the eventual goal of
[00:08:05.686]identifying a group of peptide-based
[00:08:07.546]antifouling diluents for use in biosensor
[00:08:09.926]fabrication. This work was funded by the
[00:08:14.359]UCARE grant program through the University
[00:08:16.479]of Nebraska-Lincoln with support from the
[00:08:18.499]Pepsi Quasi Endowment and Union Bank &
[00:08:20.403]Trust, and Nebraska MRSEC. I would like to
[00:08:22.403]thank my adviser, Dr. Lai, and my mentor,
[00:08:24.423]Kayleigh, and the rest of the Lai lab for
[00:08:26.472]their help and encouragement on this
[00:08:27.972]project.

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Antifouling Performance of D6 and A6 Hexapeptides

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