Empirical Optimization of Peptide Sequence and Nanoparticle Colloidal Stability: The Impact of Surface Ligands and Implications for Colorimetric Sensing.

Surface ligands play a critical role in controlling and defining the properties of colloidal nanocrystals. These aspects have been exploited to design nanoparticle aggregation-based colorimetric sensors. Here, we coated 13-nm gold nanoparticles (AuNPs) with a large library of ligands (e.g., from labile monodentate monomers to multicoordinating macromolecules) and evaluated their aggregation propensity in the presence of three peptides containing charged, thiolate, or aromatic amino acids. Our results show that AuNPs coated with the polyphenols and sulfonated phosphine ligands were good choices for electrostatic-based aggregation. AuNPs capped with citrate and labile-binding polymers worked well for dithiol-bridging and π-π stacking-induced aggregation. In the example of electrostatic-based assays, we stress that good sensing performance requires aggregating peptides of low charge valence paired with charged NPs with weak stability and vice versa. We then present a modular peptide containing versatile aggregating residues to agglomerate a variety of ligated AuNPs for colorimetric detection of the coronavirus main protease. Enzymatic cleavage liberates the peptide segment, which in turn triggers NP agglomeration and thus rapid color changes in <10 min. The protease detection limit is 2.5 nM.

Peptide valence-induced breaks in plasmonic coupling† † Electronic supplementary information (ESI) available: Materials, synthesis and instrumentations, characterization studies, computational simulations, one-pot assays, table of peptide sequences and molecular weights, table of operation windows of R2 and K2 peptides, structures of ligands and R2 peptide, HPLC and ESI-MS data, CCC, operation windows, LoD, and specificity measurements, initial structures for molecular dynamics simulation, and the colorimetric map of the sensing kit. See DOI: https://doi.org/10.1039/d2sc05837e

Electrostatic interactions are a key driving force that mediates colloidal assembly. The Schulze-Hardy rule states that nanoparticles have a higher tendency to coagulate in the presence of counterions with high charge valence. However, it is unclear how the Schulze–Hardy rule works when the simple electrolytes are replaced with more sophisticated charge carriers. Here, we designed cationic peptides of varying valencies and demonstrate that their charge screening behaviors on anionic gold nanoparticles (AuNPs) follow the six-power relationship in the Schulze–Hardy rule. This finding further inspires a simple yet effective strategy for measuring SARS-CoV-2 main protease (Mpro) via naked eyes. This work provides a unique avenue for fundamental NP disassembly based on the Schulze–Hardy rule and can help design versatile substrates for colorimetric sensing of other proteases. Electrostatic interactions are a key driving force that mediates colloidal assembly.

Peptide valence-induced breaks in plasmonic coupling.

Electrostatic interactions are a key driving force that mediates colloidal assembly. The Schulze-Hardy rule states that nanoparticles have a higher tendency to coagulate in the presence of counterions with high charge valence. However, it is unclear how the Schulze-Hardy rule works when the simple electrolytes are replaced with more sophisticated charge carriers. Here, we designed cationic peptides of varying valencies and demonstrate that their charge screening behaviors on anionic gold nanoparticles (AuNPs) follow the six-power relationship in the Schulze-Hardy rule. This finding further inspires a simple yet effective strategy for measuring SARS-CoV-2 main protease (Mpro) via naked eyes. This work provides a unique avenue for fundamental NP disassembly based on the Schulze-Hardy rule and can help design versatile substrates for colorimetric sensing of other proteases.

Peptide Amphiphile Mediated Co-assembly for Nanoplasmonic Sensing.

Aromatic interactions are commonly involved in the assembly of naturally occurring building blocks, and these interactions can be replicated in an artificial setting to produce functional materials. Here we describe a colorimetric biosensor using co-assembly experiments with plasmonic gold and surfactant-like peptides (SLPs) spanning a wide range of aromatic residues, polar stretches, and interfacial affinities. The SLPs programmed in DDD-(ZZ) x -FFPC self-assemble into higher-order structures in response to a protease and subsequently modulate the colloidal dispersity of gold leading to a colorimetric readout. Results show the strong aggregation propensity of the FFPC tail without polar DDD head. The SLPs were specific to the target protease, i.e., Mpro, a biomarker for SARS-CoV-2. This system is a simple and visual tool that senses Mpro in phosphate buffer, exhaled breath condensate, and saliva with detection limits of 15.7, 20.8, and 26.1 nM, respectively. These results may have value in designing other protease testing methods.

Spacer Matters: All-Peptide-Based Ligand for Promoting Interfacial Proteolysis and Plasmonic Coupling.

Plasmonic coupling via nanoparticle assembly is a popular signal-generation method in bioanalytical sensors. Here, we customized an all-peptide-based ligand that carries an anchoring group, polyproline spacer, biomolecular recognition, and zwitterionic domains for functionalizing gold nanoparticles (AuNPs) as a colorimetric enzyme sensor. Our results underscore the importance of the polyproline module, which enables the SARS-CoV-2 main protease (Mpro) to recognize the peptidic ligand on nanosurfaces for subsequent plasmonic coupling via Coulombic interactions. AuNP aggregation is favored by the lowered surface potential due to enzymatic unveiling of the zwitterionic module. Therefore, this system provides a naked-eye measure for Mpro. No proteolysis occurs on AuNPs modified with a control ligand lacking a spacer domain. Overall, this all-peptide-based ligand does not require complex molecular conjugations and hence offers a simple and promising route for plasmonic sensing other proteases.

Peptide-Induced Fractal Assembly of Silver Nanoparticles for Visual Detection of Disease Biomarkers.

We report the peptide-programmed fractal assembly of silver nanoparticles (AgNPs) in a diffusion-limited aggregation (DLA) mode, and this change in morphology generates a significant color change. We show that peptides with specific repetitions of defined amino acids (i.e., arginine, histidine, or phenylalanine) can induce assembly and coalescence of the AgNPs (20 nm) into a hyperbranched structure (AgFSs) (∼2 µm). The dynamic process of this assembly was systematically investigated, and the extinction of the nanostructures can be modulated from 400 to 600 nm by varying the peptide sequences and molar ratio. According to this rationale, two strategies of SARS-CoV-2 detection were investigated. The activity of the main protease (Mpro) involved in SARS-CoV-2 was validated with a peptide substrate that can bridge the AgNPs after the proteolytic cleavage. A sub-nanomolar limit of detection (0.5 nM) and the capacity to distinguish by the naked eye in a wide concentration range (1.25-30 nM) were achieved. Next, a multichannel sensor-array based on multiplex peptides that can visually distinguish SARS-CoV-2 proteases from influenza proteases in doped human samples was investigated.

A Charge-Switchable Zwitterionic Peptide for Rapid Detection of SARS-CoV-2 Main Protease.

The transmission of SARS-CoV-2 coronavirus has led to the COVID-19 pandemic. Nucleic acid testing while specific has limitations for mass surveillance. One alternative is the main protease (Mpro ) due to its functional importance in mediating the viral life cycle. Here, we describe a combination of modular substrate and gold colloids to detect Mpro via visual readout. The strategy involves zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by Mpro . Autolytic cleavage releases a positively charged moiety that assembles the nanoparticles with rapid color changes (t<10 min). We determine a limit of detection for Mpro in breath condensate matrices <10 nM. We further assayed ten COVID-negative subjects and found no false-positive result. In the light of simplicity, our test for viral protease is not limited to an equipped laboratory, but also is amenable to integrating as portable point-of-care devices including those on face-coverings.