Watching a Single Enzyme at Work Using Single-Molecule Surface-Enhanced Raman Scattering and DNA Origami-Based Plasmonic Antennas

The detection of a single-enzyme catalytic reaction by surfaced-enhanced Raman scattering (SERS) is presented by utilizing DNA origami-based plasmonic antennas. A single horseradish peroxidase (HRP) was accommodated on a DNA origami nanofork plasmonic antenna (DONA) containing gold nanoparticles, enabling the tracing of single-molecule SERS signals during the peroxide reduction reaction. This allows monitoring of the structure of a single enzymatic catalytic center and products under suitable liquid conditions. Herein, we demonstrate the chemical changes of HRP and the appearance of tetramethylbenzidine (TMB), which works as a hydrogen donor before and after the catalytic reaction. The results show that the iron in HRP adopts Fe4+ and low spin states with the introduction of H2O2, indicating compound-I formation. Density functional theory (DFT) calculations were performed for later catalytic steps to rationalize the experimental Raman/SERS spectra. The presented data provide several possibilities for tracking single biomolecules in situ during a chemical reaction and further developing plasmon-enhanced biocatalysis.


Activity test of modified HRP
The assays were performed by checking the color change of TMB when oxidized by HRP.We mixed a TMB solution (0.5 mM) with the modified HRP, and incubated it for 5 min.If the enzyme is active, a blue color should appear.Figure S-9 shows the result for the HRP modified with DNA with different concentrations of HRP in solution.
We can see that even at the 3 nM concentration of HRP, we could observe a faint blue color, showing that the enzyme is active.This HRP concentration experiment determined the minimum concentration of NF needed to observe a color change.The second experiment was done with the DNA origami nanofork: first containing the nanofork without the SH-strand in the bridge and the other situation where the SH is present.Without the SH, it is expected that the HRP will not bind to the nanofork and will be washed away during the purification steps.Therefore, no color is expected.From the photography in Figure S-9, we can see the blue color in the solution for the sample where HRP should be present, while for the sample without the SH, the solution is still transparent, showing that there is no or not detectable non-specific binding of HRP to the nanofork and that the bound HRP is still active during our experiments.

Figure S- 1 .
Figure S-1.Large scale AFM image showing several DNA origami nanoforks modified with HRP (NFHRP).Overview of Raman imaging and SEM correlation image for snapshot measurement.

Figure S- 2 .
Figure S-2.Overview of AFM and Raman correlation image for time series measurement.

Figure S- 3 .
Figure S-3.Scheme of the data analysis: first, spectra are collected over time; after this, spectra with high signals are individually collected, and then the collected signal is averaged.Different averaged individual single-molecule SERS signals of HRP during the catalytic reaction are shown in the boxes

Figure S- 4 .
Figure S-4.Simulation of theoretical TMB and diimine vibrational modes.Vibrational modes that contribute to most pronounced bands in experimental SERS spectrum were shown.

Figure S- 5 .
Figure S-5.Single-molecule SERS signals of HRP during the cyclic catalytic reaction.Spectra were extracted before (left) and after (right) adding TMB solution.

Figure S- 6 .
Figure S-6.Control experiment to investigate the interaction of AuNPs with TMB.Spectra were extracted after adding TMB solution to non-HRP-functionalized DONAs (blue), compared with TMB SERS spectrum.

Figure S- 7
Figure S-7.pH dependency of TMB SERS signal.TMB was mixed with AuNPs and SERS signal was detected in liquid.HCl was added during the time series measurement and spectra were extracted from each adding HCl.

Figure S- 8 .
Figure S-8.Normal Raman spectra of bulk HRP at different excitation wavelength.

Figure S- 9 .
Figure S-9.Activity test of modified HRP.