Peptide Amphiphile Mediated Co-assembly for Nanoplasmonic Sensing

Abstract

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., M^pro , a biomarker for SARS-CoV-2. This system is a simple and visual tool that senses M^pro 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.

Keywords: Aromatic Interactions; Colorimetric Test; Main Protease; Peptide Amphiphile; Saliva.

Figure 1.

Library of surfactant-like peptides (SLPs) studied here for colorimetric assays. (a) Chemical structures of modular peptide amphiphiles have an aromatic amino acid sticker tethered by a polar head of increasing hydrophilicity; an M^pro cleavage sequence is in the center. The schematic illustrates the peptide self-assembly and subsequent co-assembly with plasmonic nanoparticle in the presence of M^pro. The intact peptides produce β-sheet structures rich in sulfhydryl groups after proteolysis, which favors plasmonic coupling via aromatic-stacking/hydrogen-bonding. (b) Synthetic peptide sequences spanning a wide range of aromatic residues, polar stretches, and interfacial affinities. The amino acids on the polar head, cleavage site, and sticker tail are color coded. M^pro cleaves the peptide at Q↓S (i.e., P1 and P1’ site). (c,d) HPLC and ESI-MS data confirm that M^pro cleaves the representative D₃F₂C₁ peptide between Q and S. Peaks with * are the intact peptide (blue); the fragments are in red. The specificity constant (k_cat/K_M) for D₃F₂C₁ substrate by M^pro is 4,178.6 M^−1.s⁻¹.

Figure 2.

(a) TEM micrographs of the stained SLP fragments prepared by incubating the corresponding intact peptide (600 μM) with M^pro (200 nM) and aging for 48 h. The SGFFPC and SGFFPFFPC form a network of ordered nanofibrils. (b) Hydrodynamic size (D_H) change of the D₃F_nC₁ peptide (n = 1, 2, 4, at 600 μM) when incubated with M^pro (200 nM) at 37 °C. The time of initializing macroscopic aggregation in D₃F₄C₁ and D₃F₂C₁ solutions is about 5 and 40 min, respectively. While the D₃F₁C₁ produced no secondary structure under the tested conditions. (c) ThT kinetic experiment showing fluorescence intensity at 485 nm for 50 μM ThT incubated with 300 μM peptides (e.g., D₃F₁C₁, D₃F₂C₁, D₃F₄C₁, and their aged proteolytic products), as recorded over 1 h. The negative control consisted of buffer only. Error bars = standard errors (n = 3). Inset shows the dye structure.

Figure 3.

M^pro-induced color change using the modular peptide amphiphiles and colloidal gold (citrate). (a) The time progression of optical absorption of AuNPs (3.4 nM, 100 μL) when incubated with D₃F₂C₁ intact (left) and its fragments (right, c = 1.5 μM). The curves were recorded every 1 min for 30 min. Arrows designate sizable optical changes at 520 and 600 nm. (b-c) The concentration- and time-dependent color evolution of AuNPs (3.4 nM, 100 μL) in the presence of intact D₃F₂C₁ and its pre-cleaved fragments. These are cropped images with a color bar where purple represents particle flocculation. See also TEM images of AuNPs (3.4 nM, 8 μL) when mixed with D₃F₂C₁ intact (d, monolayer) and its fragments (e, heterogeneous stacking). (f) DLS profiles of AuNPs (3.4 nM, 100 μL) incubated with D₃F₂C₁ intact (blue) and its fragments (red) of 0 – 100 μM. (g-h) View of MANTA^[22b] size measurement shows that AuNPs (c = 0.2 – 0.6 nM) scatter blue light with D₃F₂C₁ intact (2.0 μM), whereas the fragment-induced colloidal aggregates scatter red light. (i) Zeta potential of AuNPs (3.4 nM, 100 μL) when incubated with increasing concentrations of D₃F₂C₁ intact (blue, reduced from −26.0 to −40.4 mV) and its fragments (red, increased from −26.6 to −9.0 mV). Error bars represent triplicate measurements for one sample. (j) The agarose gel (0.7% w/v) electrophoresis image collected from citrate-AuNPs only, AuNPs incubated with HS-PEG_2k-OCH₃, and intact D₃F₂C₁ (from left to right). Samples were prepared using the AuNPs (~15 nM, 40 μL) mixed with glycerol (10 μL). Note that TBE buffer (1×) promotes instant aggregation of citrate-AuNPs. (k) Ca²⁺ cation (20 mM)-modulated dispersity of citrate (red), PEGylated (green), and D₃F₂C₁-capped AuNPs (blue). The colloidal dispersity is quantified by ratiometric signal, i.e., Abs₆₀₀/Abs₅₂₀. The DDD stretch negatively charges the surfaces and promotes colloidal stability via electrostatic double repulsion. (l-n) White-light image (top) and quantified reversal color change (bottom) of the gold pellet in different surfactant solutions (10 mM, 100 μL) or solvents (100 μL). Panel l indicates an aromatic stacking-driven co-assembly of D₃F₂C₁ fragments and AuNPs. The gold pellet is prepared by aggregating AuNPs (3.4 nM, 100 μL) with D₃F₂C₁/D₃Y₂C₁/D₃F₁C₁ fragments (at 600 μM, 30 μL). Error bars = standard deviations (n = 3).

Figure 4.

Dynamic range of peptide. Ratiometric signal (i.e., Abs₆₀₀/Abs₅₂₀ at 3 h readout time) recorded from DPPS-AuNPs (2.8 nM, 120 μL) incubated with various amount of D₃F₀C₁ intact (a), D₃F₁C₁ intact (b), D₃F₂C₁ intact (c), D₃F₄C₁ intact (d), D₃F₂C₀ intact (e), D₃Y₂C₁ intact (f), and their corresponding proteolytic fragments (red). The charged polar head, di-homo aromatic amino acid, and cysteine are indispensable modules for peptide amphiphiles to colorimetrically measure protease. The determined lower limit of dynamic range for D₃F₂C₁, D₃F₄C₁, and D₃Y₂C₁ SLP is 36.6, 16.5, and 31.1 μM, respectively. Error bars = standard deviations (n = 2).

Figure 5.

Sensitivity and specificity test. Ratiometric absorbance as a function of M^pro concentration: The D₃F₂C₁ substrate (100 μM), DPPS-AuNPs (2.4 nM), and a 6 h assay time are employed in (a), while citrate-AuNPs (2.4 nM) and a 3 h 10 min assay time are used in (b). The LoDs are shown and the linear region is provided in Figure S17.^[31] Error bar = standard deviation (n = 2). (c) Inhibition curve collected by titrating M^pro (50 nM) with varying amount of GC376 in the presence of D₃F₂C₁ substrate (100 μM): % active M^pro = [I]/[E] at the x-intercept (see dash green line, 72.6%).^[32] Inset is the chemical structure of inhibitor GC376. Error bar = standard deviation (n = 2). (d) Sensor performance interfered by other mammalian proteins (50 nM), including bovine serum albumin (BSA), hemoglobin, trypsin, thrombin, α-amylase (50 U/mL), and neuraminidase (5 U/mL). Samples with and without M^pro were the controls.