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. 2020 Jul 31;11(8):4800-4816.
doi: 10.1364/BOE.398038. eCollection 2020 Aug 1.

Waveguide-based surface-enhanced Raman spectroscopy detection of protease activity using non-natural aromatic amino acids

Nina Turk  1   2 , Ali Raza  1   2   3 , Pieter Wuytens  1   2   4 , Hans Demol  5   6 , Michiel Van Daele  7 , Christophe Detavernier  7 , Andre Skirtach  2   8 , Kris Gevaert  5   6 , Roel Baets  1   2
Affiliations

Waveguide-based surface-enhanced Raman spectroscopy detection of protease activity using non-natural aromatic amino acids

Nina Turk et al. Biomed Opt Express. .

Abstract

Surface enhanced Raman spectroscopy (SERS) is a selective and sensitive technique, which allows for the detection of protease activity by monitoring the cleavage of peptide substrates. Commonly used free-space based SERS substrates, however, require the use of bulky and expensive instrumentation, limiting their use to laboratory environments. An integrated photonics approach aims to implement various free-space optical components to a reliable, mass-reproducible and cheap photonic chip. We here demonstrate integrated SERS detection of trypsin activity using a nanoplasmonic slot waveguide as a waveguide-based SERS substrate. Despite the continuously improving SERS performance of the waveguide-based SERS substrates, they currently still do not reach the SERS enhancements of free-space substrates. To mitigate this, we developed an improved peptide substrate in which we incorporated the non-natural aromatic amino acid 4-cyano-phenylalanine, which provides a high intrinsic SERS signal. The use of non-natural aromatics is expected to extend the possibilities for multiplexing measurements, where the activity of several proteases can be detected simultaneously.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Fig. 1.
Fig. 1.
(a) The concept of protease activity detection via peptide bond cleavage using SERS, where the SERS signal originates from the aromatic amino acids phenylalanine (F) and 4-cyano-phenylalanine (CN-F). The peptide forms a monolayer on the gold nanostructures and is then cleaved by a protease (here trypsin). As the cleaved-off part of the peptide diffuses away from the gold surface, the corresponding SERS peak intensity decreases. (b) Peptide substrate for trypsin, written with using the single-letter amino acid code. White letters represent the aromatic amino acids that provide SERS signals, namely phenylalanine (F) and the non-natural 4-cyano-phenylalanine (CN-F).
Fig. 2.
Fig. 2.
(top) Gold nanodomes are free-space SERS substrate with an average gap size of 12 nm. (bottom) Waveguide-based nanoplasmonic slot waveguides have an average gap size of 43 nm.
Fig. 3.
Fig. 3.
SERS spectra of the peptide before and after trypsin addition, recorded on gold nanodomes. Addition of trypsin results in a decrease in the intensity of the SERS peak of phenylalanine (F) at 1003 cm−1.
Fig. 4.
Fig. 4.
F/CN-F peak intensity ratio for the trypsin cleavage experiments performed on gold nanodomes. The box plot shows that the F/CN-F peak intensity ratio decreases by 30% after trypsin addition. If no trypsin is added, or after the addition of inactive trypsin, there is no change in the F/CN-F peak intensity ratio.
Fig. 5.
Fig. 5.
Raw SERS spectrum of the trypsin peptide substrate acquired on the nanoplasmonic slot waveguide. The silicon nitride peak at 2330 cm−1 indicates that the light is guided in the waveguide, whereas the other two peaks originate from the peptide itself. The background originates predominantly from the silicon nitride waveguide.
Fig. 6.
Fig. 6.
An example of the F/CN-F peak intensity ratio plot as a function of time obtained on a reference nanoplasmonic slot waveguide. The vertical dashed line indicates the average value of the F/CN-F peak intensity.
Fig. 7.
Fig. 7.
SERS spectra of the peptide before and after trypsin addition acquired on nanoplasmonic slot waveguide. The decrease in the F peak at 1003 cm−1 indicates trypsin-mediated cleavage of the peptide. Each spectrum shown in the graph is the average of 10 background-subtracted measurements. For better visualization, the spectra were smoothed with the simple moving average with the window size of 3.
Fig. 8.
Fig. 8.
A box plot of F/CN-F peak intensities before and after trypsin addition recorded on nanoplasmonic slot waveguide. Individual measurements are presented as gray dots.
Fig. 9.
Fig. 9.
RP-HPLC chromatogram of the intact peptide (trypsin substrate). The measured mass (in Da) along with the identified peptide fraction is written above each HPLC peak.
Fig. 10.
Fig. 10.
RP-HPLC chromatogram of the trypsin-digested peptide. The measured mass (in Da) along with the identified peptide fraction is written above each HPLC peak.
Fig. 11.
Fig. 11.
SERS spectra of the RP-HPLC separated peptides from a bulk trypsin cleavage experiment. The spectra are normalized on the CN-F maximum peak intensity for easier comparison and offset on the y axis. The horizontal vertical lines represent the zero of each spectrum. The two vertical lines represent the positions of the characteristic SERS peaks of the phenylalanine (F) and 4-cyano-phenylalanine (CN-F). After trypsin digestion, a complete disappearance of the SERS peak of phenylalanine F is evident, as expected.
Fig. 12.
Fig. 12.
SERS spectra of the intact peptide (green) and of the cleavage solution after 1 h of incubating the peptide with trypsin (blue). The three spectra for each condition come from three different gold nanodome samples, all labelled under the same conditions. All spectra are normalized on the CN-F peak at 1180 cm−1.
Fig. 13.
Fig. 13.
A box plot of F/CN-F peak intensities before (Ref) and after (Try) trypsin addition for the spectra shown in Fig. 12. Each box plot corresponds to one SERS spectrum in the previous figure.
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