pubs.acs.org/Biomac Article Functionalized Prion-Inspired Amyloids for Biosensor Applications Marta Díaz-Caballero,‡ Susanna Navarro,‡ and Salvador Ventura* Cite This: Biomacromolecules 2021, 22, 2822−2833 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Protein amyloid nanofibers provide a biocompatible platform for the development of functional nanomaterials. However, the functionalities generated up to date are still limited. Typical building blocks correspond to aggregation-prone proteins and peptides, which must be modified by complex and expensive reactions post-assembly. There is high interest in researching alternative strategies to tailor amyloid-based nanostructures’ functionality on demand. In the present study, the biotin- streptavidin system was exploited for this purpose. Prion-inspired heptapeptides (Ac-NYNYNYN-NH2, Ac-QYQYQYQ-NH2, and Ac-SYSYSYS-NH2) were doped with biotin-conjugated counter- parts and assembled into amyloid-like fibers under mild conditions. The scaffolds’ versatile functionalization was demonstrated by decorating them with different streptavidin conjugates, including gold nanoparticles, quantum dots, and enzymes. In particular, they were functionalized with peroxidase or phosphatase activities using streptavidin conjugated with horseradish peroxidase and alkaline phosphatase, respectively. Modification of amyloid-like nanostructures has generally been restricted to the addition of a single protein moiety. We functionalized the fibrils simultaneously with glucose oxidase and horseradish peroxidase, coupling these activities to build up a nanostructured glucose biosensor. Overall, we present a simple, modular, and multivalent approach for developing amyloid-based nanomaterials functionalized with any desired combination of chemical and biological moieties. 1. INTRODUCTION class of binary patterned heptapeptides whose compositions Molecular self-assembly is a leading bottom-up strategy for were inspired by those of prion-like domains (Ac-NYNYNYN- fabricating novel complex nanostructures.1−3 This approach NH2, Ac-QYQYQYQ-NH2, and Ac-SYSYSYS-NH2). These exploits the selective recognition between molecular building synthetic short prion-inspired peptides fully recapitulate the units to fabricate novel architectures with nanometric slow hierarchical amyloid self-assembly of intrinsically dimensions. Bionanomaterials, such as protein nanofibers, disordered prion-like domains.21 However, their synthesis is offer significant advantages over inorganic assemblies since much simpler and cheaper than the recombinant production they provide a cost-effective and environmentally friendly way and purification of the long natural sequences, and it is fully to produce versatile biocompatible nanomaterials.4,5 Among scalable. The peptides self-assemble spontaneously, forming them, amyloid fibrils have been gathering considerable regular steric zippers with an antiparallel β-sheet disposition in attention as biocompatible functional materials3,6−9 and for which polar residues are packed at the interface between β- their application as scaffolds in tissue engineering.10,11 They sheets, yielding a strong network of hydrogen bonds that are held by densely packed, hydrogen-bonded β-sheets,12 and stabilize the inner amyloid architecture, whereas in the surface this distinctive supramolecular configuration endows them of the steric zipper, Tyr residues form ladders of aromatic− with a rigid internal order that results in nanostructures with aromatic interactions.22 Thus, in the resulting nanofibers, every high strength, stability, and high-morphological aspect ratios,13 side chain contributes to the structure and stability. In contrast which, together with their insolubility in aqueous media, make to inorganic nanoparticles or carbon nanotubes, these prion- them optimal materials for bionanotechnological applica- inspired biocompatible nanostructures are formed under mild tions.3,14 Many proteins can form amyloids under appropriate experimental conditions.15,16 This sequences’ amyloidogenic Received: February 18, 2021 potential is concentrated in defined short segments,17−19 and Revised: June 18, 2021 peptides corresponding to these regions self-assemble auton- Published: July 1, 2021 omously into amyloids;20 thus, short peptides, rather than full- length proteins, are the preferred building blocks in nano- technology.4 In this context, we have recently described a new © 2021 American Chemical Society https://doi.org/10.1021/acs.biomac.1c00222 2822 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article experimental conditions, such as room temperature, neutral mM stock solution for NY7, QY7, and SY7 peptides and 1 mM stock pH, and ambient pressure. solution for biotinylated NY7, QY7, and SY7 peptides, aliquoted and The ability to decorate peptide nanofibers with functional frozen at −80 °C until their use. groups is of paramount importance to utilize them as sensors,5 2.2. Peptide Self-Assembly. Peptides were mixed in a 1:5 (biotinylated peptide:nonbiotinylated peptide) molar ratio to a final drug delivery systems,23,24 or cell scaffolds,25,26 among other concentration of 250 μM (accounting for both peptides) in 100 mM applications. Fiber post-assembly functionalization is challeng- K2HPO4/KH2PO4 at pH 7.0. The peptides were diluted directly from ing, especially when folded globular proteins should be HFIP stock solutions. HFIP was 2.5% (v/v) in the final solutions. chemically attached to preformed nanofibers because of the Self-assembling reactions were performed at room temperature for 7 limited availability and expensive polypeptide conjugation days under quiescent conditions. chemistry, cross-reactivity, the lack of stoichiometry control, 2.3. Quantification of Biotinylated and Nonbiotinylated and the unavoidable reduction in the proportion of conforma- NY7, QY7, and SY7 Assemblies. After 7 day incubation of the tionally active molecules in the assembly.27 Therefore, there is reactions under the described conditions, the soluble and insoluble fractions were separated by centrifugation at 12,000g for 30 min at a need for alternative methods that allow developing hybrid room temperature. Supernatant and pellets were preserved for their nanoarchitectures decorated with intact and active chemical quantification. Self-assembled peptides corresponding to the insoluble and biological moieties in a stoichiometrically controlled fraction were diluted in 1:10 final volume and incubated with 2.5 M manner. In this regard, the use of noncovalent interactions guanidinium thiocyanate (GITC) in 100 mM K2HPO4/KH2PO4 with high affinity and specificity to functionalize nanostructures buffer at pH 7.0 for a minimum of 12 h under soft agitation to is gaining momentum.27−29 equilibrate the reaction. Calibration curves were obtained using a The biotin-streptavidin system provides the strongest known concentration of biotinylated and nonbiotinylated peptides noncovalent interaction in nature (Kd ≈ 10−14−10−16 M), ranging from 0 to 50 μM incubated under the same conditions. being exceptionally stable against physical and chemical Tyrosine intrinsic fluorescence was recorded on a Jasco FP-8200 fluorescence spectrophotometer (Jasco Corporation, Japan) in the perturbations, including heating, the presence of denaturant range 280−400 nm, excited at a 268 nm wavelength, using an agents or detergents, or extensive washing steps.30 In this excitation and emission bandwidth of 5 nm at 25 °C to determine the system, the first component is biotin, also known as vitamin H, concentration of peptides present in each fraction. Fluorescence a small 245 Da molecule acting as a cofactor in many emission at 303 nm, which corresponds to tyrosine emission enzymatic reactions. The second molecule corresponds to maximum, was used to quantify the peptides. streptavidin, a 52 kDa β-barrel protein homotetramer that 2.4. Thioflavin-T binding. Thioflavin-T (Th-T) was employed to binds four biotins per monomer. The particular properties of determine the presence of amyloid structures in the reactions. this noncovalent interaction have been exploited for various Incubated peptides were diluted 1:10 in 100 mM K2HPO4/KH2PO4 applications such as immunodetection,31 affinity chromatog- at pH 7.0, and Th-T was added to a final concentration of 25 μM. Th- T emission fluorescence was detected on a Jasco FP-8200 raphy,32 imaging,33,34 synthetic chemistry,35 chemical biol- fluorescence spectrophotometer (Jasco Corporation, Japan) in the ogy,36 and therapeutics.37 This is facilitated by a large number range 460−600 nm using an excitation wavelength of 445 nm and of commercially available streptavidin conjugates, including with excitation and emission bandwidth of 5 nm. enzymes, antibodies, and different inorganic molecules.38 2.5. Light Scattering. Light scattering was measured in a Jasco The avidin/neutravidin/streptavidin-biotin interaction has FP-8200 fluorescence spectrophotometer (Jasco Corporation, Japan), been utilized for the decoration of peptide nanostructures with which was excited at 330 nm, and recorded in the range 320−340 nm fluorescent quantum dots,39 enzymes,31,40,41 and metallic using an excitation and emission bandwidth of 5 nm. nanoparticles42,43 and exploited for the generation of fibril- 2.6. Fourier Transform Infrared (FTIR) Spectroscopy. Non- metallic hybrid nanowires,44 the development of biosensors,45 biotinylated and biotinylated peptides were incubated for 7 days as or the design of immunodetection systems.31,46 In many of described above, centrifuged at 12,000g for 30 min and resuspended in MilliQ water. Samples were placed on the ATR crystal and dried these applications, the nanofibers require post-assembly with nitrogen flow. The experiments were carried out in a Bruker modifications to conjugate them to biotin and incorporate a Tensor 27 FTIR (Bruker Optics, United States) supplied with a single functional component. Here, we have exploited the slow Specac Golden Gate MKII ATR accessory. Each spectrum consists of hierarchical self-assembly of prion-inspired heptapeptides to 32 acquisitions measured at a resolution of 1 cm−1. Data were dope them with biotinylated counterparts, obtaining amyloid- acquired and normalized using the OPUS MIR Tensor 27 software like nanofibers that incorporate biotin moieties without a need (Bruker Optics, United States). IR spectra were fitted employing a for any additional manipulation. Using streptavidin conjugates, nonlinear peak-fitting equation using PeakFit package v4.12 (Systat these nanostructures were functionalized with quantum dots, Software, San Jose, CA). The area for each Gaussian curve was gold nanoparticles, and enzymes like horseradish peroxidase or calculated in the amide I region from 1700 to 1600 cm−1 using the second derivative deconvolution method in the PeakFit package v4.12 alkaline phosphatase. Importantly, this modular strategy allows (Systat Software, San Jose, CA). obtaining multivalent nanofibers, anchoring simultaneously 2.7. Determination of Critical Aggregation Concentration two different catalytic entities, and allowing them to perform (CAC) by Th-T Binding. Nonbiotinylated and biotinylated peptide coupled enzymatic reactions and act as solid biosensing reactions were prepared at different concentrations ranging from 0 to platforms. 100 μM in 100 mM K2HPO4/KH2PO4 at pH 7.0. After 7 day incubation at room temperature, assemblies were diluted to 10 μM in 100 mM K2HPO4/KH2PO4 at pH 7.0, and Th-T was added to a final 2. EXPERIMENTAL METHODS concentration of 5 μM. The absorbance values at 492 nm were 2.1. Peptide Preparation. Synthetic heptapeptides NY7 (Ac- employed to plot the Th-T fluorescence signal versus the peptide NYNYNYN-NH2), biotinylated NY7 (Biotin-APAANYNYNYN- concentration. Linear regressions of Th-T fluorescence intensities and NH2), QY7 (Ac-QYQYQYQ-NH2), biotinylated QY7 (Biotin- the basal Th-T signal were calculated using GraphPad Prism 6.0. Two APAAQYQYQYQ-NH2), SY7 (Ac-SYSYSYS-NH2), and biotinylated different regressions were calculated: the first one corresponding to SY7 (Biotin-APAASYSYSYS-NH2) were purchased from CASLO ApS low concentration reactions (between 0 and 10 μM) with similar (Scion Denmark Technical University). Lyophilized peptides were intensities to that of the Th-T basal signal and the second one dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to obtain a 10 corresponding to concentrations ranging from 25 to 100 μM with 2823 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article higher Th-T intensity. The intersection of the two linear regressions United States) in a VersaDoc Imaging device (Bio-Rad, United corresponds to the critical aggregation concentration (CAC) for each States) using the blotting application with Chemi Ultra sensitivity, reaction. Measurements were acquired in triplicates, and the mean clear 0.5× gain and 4 × 4 Bin. The exposure for the acquisition of and standard deviation were calculated. images was 10 s. 2.8. Streptavidin-Gold Nanoparticles (Gold NPs) Binding to 2.13. Dot Blotting Streptavidin-Alkaline Phosphatase Biotinylated Scaffolds. A total of 100 μL of NY7, QY7, and SY7 Coupling to Biotin Scaffolds. NY7, QY7, and SY7 fibers were biotinylated fibers was centrifuged at 12,000g for 30 min. The pellet centrifuged at 12,000g for 30 min. The pellet was resuspended in the was resuspended in 50 μL of MilliQ water. Streptavidin-gold NP same volume of 100 mM K2HPO4/KH2PO4 at pH 7.0. A total of 50 conjugate solution (Sigma Aldrich, Germany) was diluted 1:10 (v/v) μL of isolated biotinylated and nonbiotinylated fibers were deposited in MilliQ H2O and equilibrated at room temperature for 20 min. After onto a PVDF membrane (Immobilon-P Transfer Membranes, equilibration, 50 μL of biotin-containing fibers and 50 μL of diluted Millipore Corporation, United States), and drops were let dry at streptavidin-gold NP solution were mixed to a final dilution 1:20 of room temperature. A total of 50 μL of each biotinylated NY7, QY7, streptavidin-gold NPs, and the solutions were incubated overnight at and SY7 non-assembled peptide at 60 μM was used as controls. After room temperature with soft agitation. Samples were washed twice by sample deposition, the membrane was blocked with Blocking Solution centrifugation at 12,000g for 30 min and resuspension in MilliQ water (5% w/v Milk in 1× Tween20 Tris buffered saline (TTBS 1×) buffer) to remove the excess of NPs. Samples were deposited onto carbon- for 1 h at room temperature. After blocking, it was incubated with coated copper grids for 10 min and stained with 0.5% uranyl acetate streptavidin-AP (Invitrogen, United States) diluted 1:1000 in 5% (w/ solution for 30 s for their visualization by transmission electron v) Milk TTBS 1× solution overnight at 4 °C. The membrane was microscopy (TEM). washed three times with TTBS 1× buffer for 15 min every washing 2.9. Inductively Coupled Plasma-Atomic Optic Emission step. Development of the membrane was performed with a Check Spectrometry (ICP-OES) for Gold Content Quantification. BCIP/NBT Liquid Substrate System (Sigma Aldrich, Germany) for 3 Biotinylated NY7, QY7, and SY7 fibrils and control nonbiotinylated h. NY7, QY7, and SY7 fibers were washed and incubated in the presence 2.14. Streptavidin-Alkaline Phosphatase Binding to Bio- of streptavidin-gold NP conjugate solution (Sigma Aldrich, Germany) tinylated Scaffolds. After 7 days of incubation, NY7, QY7, and SY7 diluted 1:20 (v/v) in MilliQ water overnight at room temperature biotinylated peptide samples were centrifuged at 12,000g for 30 min under agitation. Samples were washed twice by centrifugation at to isolate fibrils and the pellet was resuspended in the same volume of 12,000g for 30 min and resuspension in MilliQ water to remove the 100 mM K2HPO4/KH2PO4 at pH 7.0. Streptavidin-AP solution (1 excess of NP. Since some NPs could still precipitate nonspecifically mg/mL, Invitrogen, United States) was added to the biotin- with the fibrils, incubated nonbiotinylated fibrils were used as negative containing scaffolds at a 1:120 ratio (biotin:streptavidin-AP) and controls. Pellets were stored for their subsequent analysis. Pelleted incubated overnight at room temperature with soft agitation. Samples fibers were resuspended in a HNO3 and HCl solution in a ratio 1:3 were then centrifuged at 12,000g for 30 min, and pellets were and heated in a DINKO D-65 heating block to promote peptide resuspended in four times the initial volume of 100 mM K2HPO4/ digestion. Digestion products were injected in an inductively coupled KH2PO4 at pH 7.0. plasma-optic emission Optima 4300DV mass spectrometer (Perki- 2.15. Alkaline Phosphatase Activity Assay. Kinetics of AP nElmer, United States) to quantify Au content. The amount of activity were performed using one-step pNPP (p-nitrophenyl oxidized Au was calculated relative to that of initial fibrils. All samples phosphate) solution (Thermo Fisher, United States), which was were measured in duplicates, and the standard deviation was transformed to p-nitrophenol (pNP), developing yellow color and an calculated. Significance was calculated with a one-way ANOVA absorbance maximum at 405 nm. This assay was performed in a 96- statistical test. well plate with the corresponding streptavidin-AP enzyme controls. A 2.10. Transmission Electron Microscopy. Grids were exhaus- total of 10 μL of sample and 90 μL of pNPP solution were mixed. The tively scanned using a JEM 1400 transmission electron microscope reaction was stopped by adding 50 μL of 2 M NaOH at (JEOL Ltd., Japan) operating at 80 kV, and images were acquired corresponding time points. Selected time points were 0, 2, 5, 10, with a CCD GATAN ES1000W Erlangshen camera (Gatan Inc., 15, 20, 25, 30, 40, 50, and 60 min. Enzyme control was performed United States). with the same amount of streptavidin-AP added to the biotin- 2.11. Quantification of Gold NPs. TEM micrographs were containing scaffold solution (1:120 dilution) and diluted four times employed to calculate the number of gold NPs visualized in the just before the addition into the well. Absorbance was measured at samples. Image J Software (NIH, United States) was used to calculate 405 nm in a Victor3 (PerkinElmer, United States) plate reader. the number of gold NPs per μm2. We employed at least five Experiments were performed in triplicates, results correspond to the micrographs from two different experiments to analyze the retention mean of two independent experiments, and errors correspond to the capacity. Statistical analysis was performed by a one-way ANOVA test standard deviation. followed by a Bonferroni’s Multiple comparison test. 2.16. Glucose Oxidase and Horseradish Peroxidase 2.12. Dot Blotting Streptavidin-Horseradish Peroxidase Coupled Reaction Activity Assay. After 7 day incubation, 500 Coupling to Biotin Scaffolds. Biotinylated NY7, QY7, and SY7 μL of aggregated reactions at 250 μM was centrifuged at 12,000g for scaffolds were centrifuged at 12,000g for 30 min. The pellet was 30 min to isolate fibers and pellets were resuspended in the same resuspended in the same volume of 100 mM K2HPO4/KH2PO4 at pH volume of 100 mM K2HPO4/KH2PO4 at pH 7.0. Streptavidin-GOx 7.0. Isolated biotinylated scaffolds were deposited onto a PVDF solution (1 mg/mL, Stereospecific Detection Technologies GmbH, membrane (Immobilon-P Transfer Membranes, Millipore Corpo- Germany) and streptavidin-HRP solution (1 mg/mL, Abcam, United ration, United States) in different amounts (1 and 5 μL), and drops Kingdom) were added to the biotin-containing scaffolds in a 1:1 were let dry at room temperature. Nonbiotinylated NY7, QY7, and molar ratio (GOx:HRP) at a final streptavidin-enzyme concentration SY7 scaffolds were used as a negative control in the same amounts as of 50 nM. Samples were incubated overnight at room temperature biotin-containing fibers, and biotinylated NY7, QY7, and SY7 soluble with soft agitation. Then, samples were centrifuged at 12,000g for 30 peptides at 60 μM were used as positive controls, adding 5 μL. After min, and pellets were resuspended in eight times the initial volume of sample deposition, the membrane was blocked with Blocking Solution 100 mM K2HPO4/KH2PO4 at pH 7.0. For non-streptavidin-bound (5% w/v Milk in 1× Tween20 Tris buffered saline (TTBS 1×) buffer) GOx-containing reaction, biotinylated peptides were incubated in the for 1 h at room temperature. After blocking, it was incubated with presence of 50 nM streptavidin-HRP conjugate and washed twice. streptavidin-HRP (Abcam, United Kingdom) diluted 1:5000 in 5% Just before starting the reaction, 50 nM soluble glucose oxidase (w/v) Milk TTBS 1× solution overnight at 4 °C. The membrane was (GOx) (Sigma Aldrich, Germany) was added to the biotinylated self- washed three times with TTBS 1× buffer for 10 min every washing assembled samples. Kinetics of GOx-HRP coupled reaction were step. Development of the membrane was performed with an performed using D-glucose at a final concentration 1 mM (Sigma Immobilon Forte Western HRP substrate (Millipore Corporation, Aldrich, Germany) and ABTS reagent at a final concentration 2mM 2824 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article Figure 1. Biophysical characterization of biotinylated NY7, biotinylated QY7, and biotinylated SY7 amyloid scaffolds. Biotinylated NY7 (blue), biotinylated QY7 (red), and biotinylated SY7 (green) peptides were prepared at 250 μM to a 1:5 (biotinylated:nonbiotinylated peptide) ratio in buffer 100 mM K2HPO4/KH2PO4 at pH 7.0, and aggregation was analyzed after 7 days of incubation. (a) Synchronous light scattering. (b) Amyloid tinctorial properties of incubated peptides were assessed by Th-T in the absence (gray line) and in the presence of biotin-containing peptides. (c) FTIR absorbance spectra in the amide I region (solid line) of biotinylated NY7 (blue), biotinylated QY7 (red), and biotinylated SY7 (green) assembled peptides. Dashed black lines indicate the different signals contributing to the absorbance spectrum. (d) Morphology characterization by TEM. Scale bars correspond to 500 nm. (Sigma Aldrich, Germany), which was transformed to ABTS+, fibers at 250 μM was centrifuged at 12,000g for 30 min. Pellets were presenting a green-blue coloration. Reactions were performed in a resuspended in PBS-1% BSA and incubated with streptavidin-QD 96-well plate together with streptavidin-GOx and streptavidin-HRP (Thermo Fischer; United States) ranging from 150 to 2000 pM for 30 controls. Kinetics with soluble GOx and streptavidin-HRP and with min at room temperature. To discard the unbound streptavidin-QD, streptavidin-HRP alone were performed as a positive and negative reaction controls, respectively. For each reaction, 50 μL of sample was fibers were centrifuged at 12,000g for 30 min and the pellets were added to 50 μL of 4 mM D-glucose and 100 μL of 4 mM ABTS. The resuspended in PBS twice. QD fluorescence was measured using a absorbance of the product was measured at 405 nm in a Victor3 Victor3 Multilabel Reader (PerkinElmer Waltham, MA, USA) with an (PerkinElmer, United States) plate reader, and ABTS+ concentration excitation wavelength 300 nm, an emission wavelength 525 nm, and a was calculated using ε405nm = 3.65 × 104 M−1 cm−1. Experiments were bandwidth 20. Visualization of biotinylated NY7 fibers incubated with performed in triplicates, results correspond to the mean of three streptavidin-QD at 1000 pM was performed using a HCX PL APO 63 independent experiments, and error corresponds to standard deviation. × 1.4 oil immersion objective on a Leica TCS SP5 microscope (Leica 2.17. Streptavidin-Quantum Dots (QD) Binding to Biotiny- Microsystems, Germany) using excitation at 488 nm and the emission lated Scaffolds. A total of 100 μL of NY7 and biotinylated NY7 was collected at 515−560 nm with a line average 2. 2825 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article Figure 2. Characterization of biotinylated NY7, biotinylated QY7, and biotinylated SY7 amyloid scaffolds incubated with streptavidin-gold NP conjugates. (a) TEM micrographs of biotinylated NY7 (blue), biotinylated QY7 (red), and biotinylated SY7 (green) peptides were prepared at 250 μM to a 1:5 (biotinylated peptide:nonbiotinylated peptide) ratio, corresponding to the top-line images with continuous lines. Dashed lined images at the bottom correspond to nonbiotinylated scaffold controls. Streptavidin-gold NP conjugate control corresponds to the right image (black continuous line). Scale bars correspond to 500 nm. (b) Quantification of gold NPs. TEM micrographs acquired in panel (A) were used to quantify gold NPs, and the content was expressed as numbers of gold NPs/μm2. Data correspond to the average quantification from at least five micrographs from two independent experiments, and bars correspond to the SD of the mean. For all peptides, the one-way ANOVA statistical test was performed relative to the gold NP control (***P-value < 0.001, **P-value < 0.01, and *P-value < 0.05) followed by a Bonferroni’s multiple comparison test (***P-value < 0.001, **P-value < 0.01, and *P-value < 0.05) to compare biotinylated and nonbiotinylated samples. 3. RESULTS AND DISCUSSION capacity of the prion-inspired peptides21 was affected by the 3.1. Biotinylation of Prion-Inspired Heptapeptides. In addition of the biotin molecule and the spacer at the N- the design of an amyloid-based biotin-streptavidin system, it terminal edge. Once optimal self-assembly conditions were was important to avoid steric impairments that might interfere selected, the peptides were incubated in a 1:5 biotinylated- with the self-assembly or reduce the accessibility of peptide:nonbiotinylated peptide molar ratio. This doping streptavidin-conjugated partners to biotinylated peptides strategy provides a defined streptavidin binding stoichiometry once they become embedded into the amyloid fibers. and avoids possible steric constraints restricting incoming Therefore, a spacer was incorporated between the N-terminal molecules’ access to N-terminal biotins. In addition, if two conjugated biotin moiety and the prion-inspired heptapeptides. biotinylated peptides become adjacent in the fiber, the steric The short APAA sequence was selected since it has been zipper’s antiparallel nature22 would place their biotin moieties previously used as a connecting linker between amyloid prone at opposite ends of β-sheets, precluding steric clashing between peptides and biotin.47 Poly-Ala peptides have been described incoming streptavidin domains. Reactions were prepared at a to form α-helices,48 and Pro was incorporated in the second final peptide concentration of 250 μM in 100 mM K2HPO4/ position due to its dual α-helix and β-sheet disruption KH2PO4 buffer at pH 7.0 and incubated for 7 days at 25 °C capacity.49 The idea was that the linker would remain under quiescent conditions. From now on, assemblies formed disordered and would not interfere with the heptapeptide by a 1:5 biotinylated peptide:nonbiotinylated peptide molar prion-inspired core’s capability to assemble into a β-sheet ratio will be referred to as biotinylated assemblies, fibers, or structure. Additionally, to avoid the interference of repulsive scaffolds, unless otherwise indicated. charges in the self-assembling process, the C-termini of the The peptide mixture’s ability to self-assemble was first biotinylated peptides were blocked by amidation.21 The final monitored by synchronous light scattering (Figure 1a). A design of the peptide sequences corresponded to Biotin- significant scattering signal was recorded for the three APAANYNYNYN-NH2 (Biotin-NY7), Biotin-APAAQYQY- incubated peptides, reporting large assemblies in the solution. QYQ-NH2 (Biotin-QY7), and Biotin-APAASYSYSYS-NH2 Then, the amyloid-like nature of these peptide aggregates was (Biotin-SY7). assessed using the amyloid binding dye Thioflavin-T (Th-T). 3.2. Biotin-NY7, Biotin-QY7, and Biotin-SY7 Peptides All peptide solutions promoted a substantial increase in Th-T Self-Assemble into Amyloid Fibers. Initial experiments fluorescence intensity upon incubation with the dye, compared were addressed to determine if the amyloid self-assembling with Th-T alone (Figure 1b). 2826 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article The critical aggregation concentration (CAC) is a parameter to the fibers. The presence of some randomly distributed black often used to approach the concentration at which premicellar dots on the grids could be attributed to the gravitational aggregates are formed. The CAC values for biotinylated and deposition of NPs, as shown in the negative control nonbiotinylated assemblies were determined by measuring Th- micrograph in the absence of fibers (Figure 2a). T fluorescence, as an amyloid-like structure reporter, in the The number of gold NPs per μm2 was quantified in the 0.2−100 μM initial peptide concentration range (Figure S1 TEM micrographs (Figure 2b). As a reference, we employed and Table S1). The CACs of the different nonbiotinylated the negative control micrographs (Figure 2a), and they were peptides and of reactions containing a 1:5 ratio of compared with nonbiotinylated and biotinylated fibers. The biotinylated:nonbiotinylated peptides did not differ signifi- number of gold NPs detected in nonbiotinylated fibers was not cantly, lying in the 10−20 μM range in all cases. statistically different from that of the control for the three We used Fourier transform infrared (FTIR) to investigate peptides (Figure 2b). On the contrary, the number of gold NPs the secondary structure of NY7, QY7, and SY7 biotinylated in the three biotinylated fibers was higher than those in the assemblies, recording absorbance in the amide I region of the negative control and the corresponding nonbiotinylated fibers. spectrum (Figure 1c). The vibrational contributions were The difference in gold NP content for each pair of calculated from the Fourier transformed curves (Table S2). nonbiotinylated and biotinylated fibers was statistically The presence of the biotin molecule and the linker did not significant in all cases. However, the comparison between the significantly alter the secondary structure of the biotinylated gold NP content of biotinylated fibrils formed by different assemblies relative to the corresponding nonbiotinylated forms peptides is only qualitative since the number of bound (Figure S2) with a β-sheet content higher than 60% in all nanoparticles depends on the number of peptide monomers samples. However, there are important differences in the and the number of fibrils in the micrographs, which cannot be assemblies’ secondary structure depending on the constituent accurately quantified. peptide’s identity, with SY7 generating the more ordered To demonstrate orthogonally the ability of biotinylated assemblies (Figure 1, Figure S2, and Table S2). fibers to capture gold NPs, we used the inductively coupled The morphology of biotinylated and nonbiotinylated plasma-optic emission spectroscopy (ICP-OES) technique. assemblies was inspected by transmission electron microscopy The amount of gold NPs in biotinylated and nonbiotinylated (TEM). Incubated samples were deposited onto carbon-coated fibril samples was calculated relative to that of fibrils (Table copper grids and negatively stained using uranyl acetate S3). Nonspecific precipitation of gold NPs with fibrils during solution. The images’ visualization revealed the presence of the washing procedure resulted in the presence of Au in long fibers for the three biotin-containing heptapeptides nonbiotinylated samples. However, the quantity of Au in (Figure 1d), analogous in morphology to those observed for biotinylated fibril samples was significantly higher for all the the equivalent nonbiotinylated peptides21 (Figure S2b), in assayed peptides (P-value < 0.01). The specific binding of excellent agreement with their very similar secondary structure, streptavidin gold NPs to biotinylated scaffolds accounts for as analyzed by FTIR (Figure S2a). ∼20 ng gold NPs/μg fiber. Overall, the biophysical characterization confirmed that Gold nanoparticles and other metals have been combined neither biotin conjugation nor the linker at the N-terminus with different bionanostructures to build up conductive significantly influenced the peptides’ self-assembly into nanomaterials.44,51−53 In particular, metalized amyloids have amyloid-like fibrillar structures. been exploited for the generation of nanocables54 and A similar pre-assembly doping approach has been success- nanowires55,56 for molecular biosensing using plasmon fully used to build up biotin-decorated fibers of the resonance techniques and for imaging, among other Alzheimer’s-related Aβ40 peptide.50 Compared with the applications.43,44,51,57 In this context, the continuous coverage synthetic heptapeptides, Aβ40 fibrillation is more stochastic of the prion-inspired scaffold surface with inorganic metallic and challenging to reproduce lab to lab, the building unit is nanoparticles might allow the implementation of hybrid significantly more expensive, and the use of Aβ40 assemblies conductive nanomaterials. for biomedical applications is a subject of debate due to their 3.4. Functionalization of Nanofibers with Enzymatic potential cytotoxicity. In contrast, prion-inspired fibrils are Activities. Once the biotinylated scaffolds’ capacity to bind inert.21 Another successful pre-assembly strategy to decorate streptavidin was demonstrated, we exploited this property to amyloid-like fibers with biotin consists of genetically fusing an build up biocatalytic systems. The currently employed autobiotinylated peptide to the selected amyloidogenic protein strategies to generate enzymatically active matrices are often of interest.31,46 However, it requires the recombinant expensive and technically challenging. The traditional expression and posterior purification of a comparatively longer techniques for the immobilization of enzymes in solid supports protein fusion, being less straightforward, scalable, and rely on chemical modifications of the catalysts to promote their significantly more expensive than the approach that we use attachment, a process that might significantly impact their here. nativeness and activity. Protein engineering advances have 3.3. Functionalization of Nanofibers with Gold allowed the fusion of streptavidin to almost any protein of Nanoparticles. The binding of streptavidin-gold nanoparticle interest,34,38,58 allowing them to decorate biotin-modified (NP) conjugates to the biotinylated amyloid scaffolds was matrices. Importantly, in these matrices, a majority of the visualized by TEM. Micrographs clearly showed small black target protein keeps its native and active conformation. We dots corresponding to gold NPs distributed along the examined the functionalization of our biotinylated assemblies biotinylated fibers’ surface (Figure 2a). In contrast, when the with two of the most popular enzymes using either fibers formed by nonbiotinylated peptide counterparts were streptavidin-horse radish peroxidase (HRP) or streptavidin- incubated with streptavidin-gold NPs, such colocalization was alkaline phosphatase (AP) conjugates. not observed, indicating that the presence of biotin within 3.4.1. Functionalization of Nanofibers with Peroxidase these matrices was a requirement for the incorporation of NPs Activity. The first functionalization assay consisted of the 2827 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article decoration of the biotinylated prion-inspired assemblies with clear that biotinylated prion-inspired assemblies can be the streptavidin-HRP conjugate. HRP enzyme is employed in effectively functionalized with peroxidase activity. multiple detection techniques and commercial kits like 3.4.2. Functionalization of Nanofibers with Alkaline Western blotting or ELISA detection assays. It catalyzes a Phosphatase Activity. Further on, we addressed the reaction in which the luminol substrate and H2O2 are functionalization of prion-inspired amyloid scaffolds with a transformed into H2O, N2, and 3-aminophtalate (3-APA), streptavidin-AP conjugate. AP enzyme’s primary physiological emitting light (Figure 3a). To determine if streptavidin-HRP role corresponds to dephosphorylation. This activity has been for many applications, and in particular as a reporter in immunoassays. AP activity has been classically assessed using the p-nitrophenyl phosphate (pNPP) substrate, which is hydrolyzed into phosphoric acid and p-nitrophenol (pNP). This pNP product presents a yellow coloration at alkali pH (around pH 8.0) with a maximum absorbance peak at 405 nm (Figure 4a). Figure 3. Detection of streptavidin-HRP conjugate activity bound to biotinylated NY7, biotinylated QY7, and biotinylated SY7 amyloid scaffolds. (a) Schematic representation of the reaction catalyzed by HRP within the scaffold. (b) Dot blot of the biotinylated NY7, biotinylated QY7, and biotinylated SY7 scaffolds. Controls corre- spond to non-assembled biotinylated peptides and to scaffolds without biotin. Different volumes of scaffolds containing solutions were loaded on the membrane: 1 and 5 μL. conjugates could bind specifically to biotinylated fibers, we Figure 4. Biotinylated NY7, biotinylated QY7, and biotinylated SY7 performed a dot blot assay. The fibers were immobilized onto a amyloid scaffolds functionalized with the streptavidin-AP conjugate. membrane and incubated in the presence of streptavidin-HRP (a) Schematic representation of the reaction catalyzed by AP within conjugate; nonbiotinylated fibers were treated in the same the scaffolds. (b) Kinetics of the biotinylated NY7 (blue), biotinylated manner as a control. Samples containing biotin displayed a QY7 (red), and biotinylated SY7 (green) scaffolds bound to streptavidin-AP. Controls correspond to soluble streptavidin-AP quantity-dependent chemiluminescent signal after incubation (black) and to buffer (gray). Results correspond to the mean of with the HRP substrate (Figure 3b). Conversely, fibers without three independent experiments ± standard deviation. biotin did not exhibit any significant chemiluminescence. Non- assembled biotin-labeled peptides at 60 μM, dissolved in phosphate buffer immediately before their application to the Biotinylated scaffolds were incubated with streptavidin-AP, membrane, displayed faint signals. This concentration and the AP activity was detected by measuring the appearance corresponds to the maximum amount of biotinylated peptide of the pNP product. The experiment ratified the affinity of that can be potentially embedded in the scaffolds. Because in these assemblies for streptavidin conjugates (Figure 4b), with this control sample, all peptides bear a biotin moiety that can all biotinylated fibers displaying significant AP catalytic activity. bind to a streptavidin-HRP unit, a signal at least as strong as The binding of streptavidin-AP conjugates to biotinylated the one of the fibrils was expected. The significantly higher scaffolds was also assessed by a dot blot assay, as previously chemiluminescent signal in the biotinylated fibers indicates the described for HRP. Biotinylated fibers were immobilized onto advantage of concentrating the activity on a reduced surface to a membrane and incubated with the streptavidin-AP conjugate. increase the sensitivity for this kind of membrane-based assays. In this case, we detected the formation of visible NBT It could also be that the mild hydrophobic environment formazan. This product is insoluble and is deposited on the provided by the exposed tyrosine residues in the fibril might be membrane at the places where AP is present. In the presence of favorable for streptavidin binding.59 In any case, it becomes fibers, we observed dark blue precipitates (Figure S3), whereas 2828 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article Figure 5. Biotinylated NY7, biotinylated QY7, and biotinylated SY7 amyloid scaffolds functionalized with streptavidin-HRP and with streptavidin- GOx or soluble GOx. (a) Schematic representation of the reaction occurring in the graph below. (b) Biotinylated peptides in the presence of streptavidin-HRP and streptavidin-GOx. Reaction control was streptavidin-GOx and streptavidin-HRP (1 to 1 molar ratio). Results correspond to the mean of three independent experiments ± standard deviation. a 60 μM solution of non-assembled biotinylated peptides conditions for the cross-linking of one protein to the matrix in provided a faint blue background (Figure S3), indicating again a native conformation would not necessarily apply to the that the enzymatic activity concentrates on top of the discrete other/s. In this context, the use of the here described fibrillar aggregates. biocompatible amyloid scaffolds conjugated to biotin might The results in this and the previous section suggest that, in significantly reduce the time required for the conjugates’ principle, our scaffolds can be functionalized with any attachment and prevent the conformational damage and the enzymatic activity of interest, as long as a streptavidin associated loss of activity. At the same time, it might allow conjugate is available. The approach’s modularity presents modulating the quantity of enzyme retained in the surfaces advantages compared with previously explored strategies that according to the available biotin molecules within the matrix. rely on protein fusions to build up functionalized fibers, whose Despite these potential advantages, to the best of our activity is limited to the one encoded in the fused globular knowledge, coupled reactions of enzymes immobilized into domain.31,60,61 amyloid scaffolds employing the biotin-streptavidin interaction 3.5. Dual Functionalized Nanofibers Allow Glucose system have not been reported yet. Biosensing. Even when an enzyme reaction results in an We applied our prion-inspired biotinylated fibers to set up a undetectable product, it can still be possible to detect it using a dual-enzyme cascade inspired by glucose sensing.66 We coupled assay, where the product is used as the substrate of immobilized equimolar amounts of glucose oxidase (GOx) another easily detectable enzymatic reaction.62 Indeed, assays and horseradish peroxidase (HRP) simultaneously, exploiting in which two or more enzymes are coupled together are the biotin-streptavidin interaction. In this system, the first popular in clinical biochemistry. reaction corresponds to GOx, which catalyzes glucose’s A common approach to enhance operational performance of oxidation into D-gluconolactone and H2O2. Then, the biocatalytic systems is the immobilization of the enzymes in a secondary activity of HRP employs H2O2 to oxidize ABTS solid matrix. Up to date, the matrices and scaffolds employed into ABTS+, rendering a green-colored product (Figure 5a). for enzyme immobilization are mostly inorganic and demand Therefore, since there is no H2O2 in the initial reaction, the additional chemical modifications of the protein to attach it HRP activity is only detected in the presence of glucose when covalently to the matrix as well as long-time incubation the sugar is oxidized by GOx, generating H2O2 as an steps,63−65 which might compromise the enzyme’s conforma- intermediate substrate. tion and activities.65 This is especially critical when two or Enzyme kinetics revealed a time-dependent increase in more enzymes should be simultaneously bound to the surface absorbance due to ABTS+ production, indicating that the since they would differ in their stability, and the optimal incubated amyloid scaffolds bind both streptavidin-GOx and 2829 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article Figure 6. Quantum dots binding to biotinylated amyloid scaffolds. (a) Fluorescence emission intensity of streptavidin-QD bound to NY7 and biotinylated NY7 fibers. (b) Confocal microscopy images of biotinylated NY7 fibers decorated with QD. Left and right images correspond to fluorescence and bright-field microscopy, respectively. The scale bar corresponds to 10 μm. streptavidin-HRP conjugates (Figure 5b). A control with a Overall, we can conclude that the three biotinylated scaffolds soluble streptavidin-GOx and streptavidin-HRP mix at the allow the effective immobilization of two different streptavidin- same concentrations at which they were initially added to enzyme conjugates to create enzyme cascades. Additionally, a fibers was performed to know the maximum detectable single enzyme can be immobilized on top of the fibrils and the product. Under the assay conditions, the apparent product cascade initiated by the addition of the rate-limiting enzyme in concentration of the cascade reaction reaches ∼100 μM its soluble form. ABTS+ for all tested scaffolds, being comparable to the one The derivatization of protein fibers with the avidin-biotin attained by the free GOx and HRP enzyme mixture, although chemistry has been previously used to develop a glucose in this case, the reaction occurs faster. The distinct biotinylated biosensor by modifying the fibers post-assembly with thiol fibrils differed slightly in their reaction velocities. This might be groups and coupling them to a gold surface.45 However, only caused by a different amount of streptavidin conjugates in the GOx was used as a sensor, and therefore the system lacked the different fibrils or by small differences in the effective signal amplification characteristic of enzyme cascades that we concentration of fibrils in the final solution since we do not use here. expect that the identity of the fibrils or their morphology 3.6. Functionalization of Nanofibers with Quantum would impact per se the activity of the externally added Dots. Finally, we addressed the possibility of modifying the enzymes. heptapeptide fibers with optical probes. To this aim, biotinylated scaffolds were decorated with the inorganic It was recently shown that linking of the two enzymes fluorophores’ quantum dots (QD), whose particular semi- together or placing them in close proximity does not result in conducting properties make them suitable for a wide variety of activity enhancement in the GOx-HRP cascade because H2O2 applications. The binding of streptavidin-QD was confirmed by diffuses rapidly;67 therefore, we did not expect our fibrils to be incubating biotinylated NY7 fibers, used as a model system, more active than the mixture of the two free streptavidin- with QD at concentrations ranging from 150 to 2000 pM and enzyme conjugates alone. The higher velocity of this control measuring the fluorescence emission at 525 nm. The specificity reaction likely responds to the fact that the concentration of of the binding reaction was tested using nonbiotinylated NY7 one or both enzymes is higher than those in the fibril- fibers as controls. Under the assay conditions, the fluorescence containing solutions. Despite the fact that the concentration of signal in biotinylated NY7 fibers increased linearly with the the enzymes in the soluble mixture is the same as the one used concentration of QD up to 800 pM when the signal became in the incubations with the fibrils, not necessarily all the saturated (Figure 6a). A certain concentration-dependent available streptavidin-enzyme would bind to the fibrils. In nonspecific binding of streptavidin-QD to control fibers was addition, the control reaction was not subjected to washing observed. However, under all conditions, the fluorescence steps, which might result in the loss of a proportion of the emitted by modified fibers was significantly higher than those initially functionalized fibrils. on nonbiotinylated ones, with a fourfold difference in the GOx is the rate-limiting enzyme in this coupled reaction emitted signal at 800 pM. Biotinylated amyloid scaffolds since it displays the lowest reaction velocity.67 We set up an decorated with QD appeared as highly fluorescent when experiment incubating the biotinylated scaffolds with strepta- visualized by confocal microscopy (Figure 6b). vidin-HRP alone, washing them up to remove any non- The possibility to accommodate QD would allow endorsing attached HRP and adding soluble GOx to the solution at the biotinylated amyloid scaffolds with fluorescent and semi- same concentration as that in the mixture of the two free conductor properties. enzymes, that is, at the maximum concentration that can be potentially attached to the fibrils (Figure S4a,b). Under these 4. CONCLUSIONS conditions, all the reactions are very efficient and equally fast, The decoration of nanostructures with active proteins such as indicating that, in the double functionalized fibrils, the velocity enzymes, fluorescent proteins, or antibodies and chemical of the cascade reaction is determined by the concentration of entities, like metallic or fluorescent nanoparticles and other the slower GOx enzyme. Of course, in the absence of soluble ligands, is of major relevance for their nanotechnological use. GOx, streptavidin-HRP-conjugated fibrils exhibited a neglect- Here, we introduce a modular and simple method for able ability to convert glucose into ABTS+ (Figure S4c,d). functionalizing peptide nanofibers based on the extraordinarily 2830 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article Figure 7. Schematic representation of biotinylated functionalized heptapeptide scaffolds. In the scheme, there are some examples of functionalization shown using the biotin-streptavidin system. This strategy allows the functionalization of these scaffolds with a huge range of possibilities, including different enzymes (HRP, GOx, or PA), fluorescent proteins (GFP), quantum dots, antibodies, or gold NPs, among many others. high-affinity interaction between the streptavidin protein and that these assemblies might find application in a range of biotin. The incorporation of the biotin moieties to the different nanotechnologies. ■ nanofibers occurs during assembly and therefore does not require the post-assembly modification of fibrillar matrices reported in previous studies.43−45 Moreover, due to their ASSOCIATED CONTENT reduced size, both nonbiotinylated and biotinylated peptides * sı Supporting Information can be commercially acquired with high purity at low cost. The The Supporting Information is available free of charge at molecular architecture of prion-inspired fibrils,22 their slow and hierarchal assembly, and the doping strategy result in https://pubs.acs.org/doi/10.1021/acs.biomac.1c00222. functionalized fibrils with a defined biotin ratio, in contrast Critical aggregation concentration assessment for non- to the uncertain number of biotins attached in post-assembly biotinylated and biotinylated peptides, physicochemical modification strategies.44,45 In addition, it facilitates that characterization of nonbiotinylated self-assembled pep- consecutive biotinylated peptides become distanced enough tides, dot blot detection of streptavidin-AP conjugates to avoid steric interference between incoming tetrameric bound to biotinylated assemblies, activity of biotinylated streptavidin units, which, as shown, results in highly function- peptides in the presence and absence of soluble GOx, alized fibrils. CAC values calculated from the linear regression, FTIR A large number of hydrogen bonds at the interface of the secondary structure content of nonbiotinylated and steric zippers formed by prion-inspired peptides22 results in biotinylated scaffolds, and quantification of gold NP highly stable matrices whose chemical and physical resistance retention by nonbiotinylated and biotinylated peptides can be further improved, if required, through UV light-induced (PDF) Tyr cross-linking.21 This property, together with the ability to bind and retain metal nanoparticles, quantum dots, or enzymes like HRP, AP, or GOx and, potentially, any streptavidin- conjugate of interest (Figure 7), suggests that they can find ■ AUTHOR INFORMATION Corresponding Author application in plasmon resonance techniques, buildup of Salvador Ventura − Institut de Biotecnologia i de Biomedicina hybrid protein−metal nanowires, imaging uses, or as recyclable and Departament de Bioquímica i Biologia Molecular, biocatalysts. All these applications might be implemented with Universitat Autònoma de Barcelona, 08193 Bellaterra the anchorage of a single type of chemical or biological entity. (Barcelona), Spain; orcid.org/0000-0002-9652-6351; To the best of our knowledge, we show for the first time a Phone: 34-93-5868956; Email: salvador.ventura@uab.es; streptavidin-biotin amyloid-like scaffold with simultaneous Fax: 34-93-5811264 decoration of its surface with two different sensing proteins, a property that allows sustaining coupled enzymatic reactions Authors to detect different substrates and ligands, as we show here Marta Díaz-Caballero − Institut de Biotecnologia i de implementing a glucose biosensor using commercially available Biomedicina and Departament de Bioquímica i Biologia streptavidin conjugates. Molecular, Universitat Autònoma de Barcelona, 08193 Multidecorated fibril assemblies, similar to the ones that we Bellaterra (Barcelona), Spain; Present Address: Present describe here, might find application as bio-nanoplatforms to ́ address: Insitut Quimic de Sarrià (IQS), Universitat develop tunable biosensors to detect a great diversity of Ramón Llull (URL), Grup d’Enginyeria de Materials molecules, just playing with the nature and ratio of the (GEMAT), Via Augusta 390, 08017, Barcelona, Spain enzymes fused to streptavidin. (M.D.-C.); orcid.org/0000-0001-9032-9819 Taken together, we describe here a simple and cost-effective Susanna Navarro − Institut de Biotecnologia i de Biomedicina method to generate a modular, biocompatible, biotin- and Departament de Bioquímica i Biologia Molecular, containing, amyloid-like nanostructured system that can be Universitat Autònoma de Barcelona, 08193 Bellaterra easily functionalized with any streptavidin conjugate of (Barcelona), Spain; orcid.org/0000-0001-8160-9536 interest. The diversity of commercially available streptavidin Complete contact information is available at: derivatives, from metals to antibodies or nucleic acids, suggests https://pubs.acs.org/10.1021/acs.biomac.1c00222 2831 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article Author Contributions via Tyrosines. Proc. Natl. Acad. Sci. U. S. A. 2021, 118, ‡ No. e2014139118. M.D.-C. and S.N. contributed equally. The manuscript was written through contributions of all authors. All authors have (10) Loo, Y.; Goktas, M.; Tekinay, A. B.; Guler, M. O.; Hauser, C. A. given approval to the final version of the manuscript. E.; Mitraki, A. Self-Assembled Proteins and Peptides as Scaffolds for Conceptualization was done by S.V.; methodology was done Tissue Regeneration. Adv. Healthcare Mater. 2015, 4, 2557−2586. (11) Deidda, G.; Jonnalagadda, S. V. R.; Spies, J. W.; Ranella, A.; by M.D.-C. and S.N.; formal analysis and validation were done Mossou, E.; Forsyth, V. T.; Mitchell, E. P.; Bowler, M. W.; Tamamis, by M.D.-C., S.N., and S.V.; writing (review and editing) was P.; Mitraki, A. Self-Assembled Amyloid Peptides with Arg-Gly-Asp done by M.D.-C., S.N., and S.V.; supervision was done by S.N (RGD) Motifs As Scaffolds for Tissue Engineering. ACS Biomater. Sci. and S.V.; and funding acquisition was done by S.V. Eng. 2017, 3, 1404−1416. Funding (12) Sunde, M.; Blake, C. The Structure of Amyloid Fibrils by This work was funded by the Spanish Ministry of Economy Electron Microscopy and X-Ray Diffraction. Adv. Protein Chem. 1997, and Competitiveness (BIO2016-78310-R) and the Spanish 50, 123−159. Ministry of Science and Innovation (PID2019-105017RB-I00) (13) Smith F, J.; Knowles, T. P. J.; Dobson, C. M.; MacPhee, C. E.; to S.V. and by ICREA, ICREA-Academia 2015 and 2020 to Welland, M. E. Characterization of the Nanoscale Properties of S.V. M.D.-C. was supported by the Spanish Ministry of Science Individual Amyloid Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, and Innovation via a doctoral grant (FPU14/05786). 15806−15811. (14) Díaz-Caballero, M.; Fernández, M. R.; Navarro, S.; Ventura, S. Notes Prion-Based Nanomaterials and Their Emerging Applications. Prion The authors declare no competing financial interest. 2018, 12, 266−272. ■ ACKNOWLEDGMENTS We acknowledge the technical support from the Servei de (15) Chiti, F.; Dobson, C. M. Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333− 366. (16) Chiti, F.; Dobson, C. M. Protein Misfolding, Amyloid Microscopia and from the Servei d’Anàlisi Química (SAQ) Formation, and Human Disease: A Summary of Progress Over the from Universitat Autònoma de Barcelona (UAB). ■ Last Decade. Annu. Rev. Biochem. 2017, 86, 27−68. (17) Ventura, S.; Zurdo, J.; Narayanan, S.; Parreño, M.; Mangues, ABBREVIATIONS R.; Reif, B.; Chiti, F.; Giannoni, E.; Dobson, C. M.; Aviles, F. X.; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); Serrano, L. Short Amino Acid Stretches Can Mediate Amyloid AP, alkaline phosphatase; ICP-OES, inductively coupled Formation in Globular Proteins: The Src Homology 3 (SH3) Case. plasma-atomic optic emission spectrometry; GOx, glucose Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 7258−7263. oxidase; HRP, horse radish peroxidase; NPs, nanoparticles; (18) Sant’Anna, R.; Fernández, M. R.; Batlle, C.; Navarro, S.; de pNP, p-nitrophenol; pNPP, p-nitrophenyl phosphate; PVDF, Groot, N. S.; Serpell, L.; Ventura, S. Characterization of Amyloid polyvinylidene difluoride; QD, quantum dots; TEM, trans- Cores in Prion Domains. Sci. Rep. 2016, 6, 34274. (19) Batlle, C.; De Groot, N. S.; Iglesias, V.; Navarro, S.; Ventura, S. mission electron microscopy; Th-T, thioflavin-T; TTBS, Characterization of Soft Amyloid Cores in Human Prion-Like Tween20 Tris buffered saline ■ Proteins. Sci. Rep. 2017, 7, 12134. (20) Wang, W.; Navarro, S.; Azizyan, R. A.; Baño-Polo, M.; REFERENCES Esperante, S. A.; Kajava, A. V.; Ventura, S. Prion Soft Amyloid Core (1) Wei, G.; Su, Z.; Reynolds, N. P.; Arosio, P.; Hamley, I. W.; Gazit, Driven Self-Assembly of Globular Proteins into Bioactive Nanofibrils. E.; Mezzenga, R. Self-Assembling Peptide and Protein Amyloids: Nanoscale 2019, 11, 12680−12694. From Structure to Tailored Function in Nanotechnology. Chem. Soc. (21) Díaz-Caballero, M.; Navarro, S.; Fuentes, I.; Teixidor, F.; Rev. 2017, 46, 4661−4708. Ventura, S. Minimalist Prion-Inspired Polar Self-Assembling Peptides. (2) Zhang, S. Fabrication of Novel Biomaterials through Molecular ACS Nano 2018, 12, 5394−5407. Self-Assembly. Nat. Biotechnol. 2003, 21, 1171−1178. (22) Peccati, F.; Díaz-Caballero, M.; Navarro, S.; Rodríguez- (3) Knowles, T. P. J.; Mezzenga, R. Amyloid Fibrils as Building Santiago, L.; Ventura, S.; Sodupe, M. Atomistic Fibrillar Architectures Blocks for Natural and Artificial Functional Materials. Adv. Mater. of Polar Prion-Inspired Heptapeptides. Chem. Sci. 2020, 11, 13143− 2016, 28, 6546−6561. 13151. (4) Gazit, E. Reductionist Approach in Peptide-Based Nano- (23) Maji, S. K.; Schubert, D.; Rivier, C.; Lee, S.; Rivier, J. E.; Riek, technology. Annu. Rev. Biochem. 2018, 87, 533−553. R. Amyloid as a Depot for the Formulation of Long-Acting Drugs. (5) Hauser, C. A. E.; Maurer-Stroh, S.; Martins, I. C. Amyloid-Based PLoS Biol. 2008, 6, No. e17. Nanosensors and Nanodevices. Chem. Soc. Rev. 2014, 43, 5326−5345. (24) Silva, R. F.; Araújo, D. R.; Silva, E. R.; Ando, R. A.; Alves, W. A. (6) Omosun, T. O.; Hsieh, M. C.; Childers, W. S.; Das, D.; Mehta, L -Diphenylalanine Microtubes as a Potential Drug-Delivery System: A. K.; Anthony, N. R.; Pan, T.; Grover, M. A.; Berland, K. M.; Lynn, Characterization, Release Kinetics, and Cytotoxicity. Langmuir 2013, D. G. Catalytic Diversity in Self-Propagating Peptide Assemblies. Nat. Chem. 2017, 9, 805−809. 29, 10205−10212. (7) Baruch-Leshem, A.; Chevallard, C.; Gobeaux, F.; Guenoun, P.; (25) Gras, S. L.; Tickler, A. K.; Squires, A. M.; Devlin, G. L.; Horton, Daillant, J.; Fontaine, P.; Goldmann, M.; Kushmaro, A.; Rapaport, H. M. A.; Dobson, C. M.; MacPhee, C. E. Functionalised Amyloid Fibrils Catalytically Active Peptides Affected by Self-Assembly and Residues for Roles in Cell Adhesion. Biomaterials 2008, 29, 1553−1562. Order. Colloids Surf. 2021, 203, 111751. (26) Martin, A. D.; Chua, S. W.; Au, C. G.; Stefen, H.; Przybyla, M.; (8) Lee, M.; Wang, T.; Makhlynets, O. V.; Wu, Y.; Polizzi, N. F.; Lin, Y.; Bertz, J.; Thordarson, P.; Fath, T.; Ke, Y. D.; Ittner, L. M. Wu, H.; Gosavi, P. M.; Stöhr, J.; Korendovych, I. V.; Degrado, W. F.; Peptide Nanofiber Substrates for Long-Term Culturing of Primary Hong, M. Zinc-Binding Structure of a Catalytic Amyloid from Solid- Neurons. ACS Appl. Mater. Interfaces 2018, 10, 25127−25134. State NMR. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 6191−6196. (27) Jia, F.; Narasimhan, B.; Mallapragada, S. Materials-Based (9) Shipps, C.; Ray Kelly, H.; Dahl, P. J.; Yi, S. M.; Vu, D.; Boyer, Strategies for Multi-Enzyme Immobilization and Co-Localization: A D.; Glynn, C.; Sawaya, M. R.; Eisenberg, D.; Batista, V. S.; Malvankar, Review. Biotechnol. Bioeng. 2014, 111, 209−222. N. S. Intrinsic Electronic Conductivity of Individual Atomically (28) Wang, Y.; Wu, C. Site-Specific Conjugation of Polymers to Resolved Amyloid Crystals Reveals Micrometer-Long Hole Hopping Proteins. Biomacromolecules 2018, 19, 1804−1825. 2832 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833 Biomacromolecules pubs.acs.org/Biomac Article (29) Pollino, J. M.; Weck, M. Non-Covalent Side-Chain Polymers: (47) Ganesan, A.; Debulpaep, M.; Wilkinson, H.; Van Durme, J.; De Design Principles, Functionalization Strategies, and Perspectives. Baets, G.; Jonckheere, W.; Ramakers, M.; Ivarsson, Y.; Zimmermann, Chem. Soc. Rev. 2005, 34, 193−207. P.; Van Eldere, J.; Schymkowitz, J.; Rousseau, F. Selectivity of (30) Wilchek, M.; Bayer, E. A.; Livnah, O. Essentials of Aggregation-Determining Interactions. J. Mol. Biol. 2015, 427, 236− Biorecognition: The (Strept)Avidin-Biotin System as a Model for 247. Protein-Protein and Protein-Ligand Interaction. Immunol. Lett. 2006, (48) Vanhalle, M.; Corneillie, S.; Smet, M.; Van Puyvelde, P.; 103, 27−32. Goderis, B. Poly(Alanine): Structure and Stability of the d and l- (31) Men, D.; Zhang, Z. P.; Guo, Y. C.; Zhu, D. H.; Bi, L. J.; Deng, J. Enantiomers. Biomacromolecules 2016, 17, 183−191. Y.; Cui, Z. Q.; Wei, H. P.; Zhang, X. E. An Auto-Biotinylated (49) Chou, P. Y.; Fasman, G. D. Empirical Predictions of Protein Bifunctional Protein Nanowire for Ultra-Sensitive Molecular Biosens- Conformation. Annu. Rev. Biochem. 1978, 47, 251−276. ing. Biosens. Bioelectron. 2010, 26, 1137−1141. (50) Chan, H. M.; Li, H. W. Multifunctional Encoded Self- (32) Diamandis, E. P.; Christopoulos, T. K. The Biotin-(Strept)- Assembling Protein Nanofibrils as Platform for High-Throughput and Avidin System: Principles and Applications in Biotechnology. Clin. Multiplexed Detection of Biomolecules. Anal. Chem. 2011, 83, 9370− Chem. 1991, 37, 625−636. 9377. (33) Chamma, I.; Letellier, M.; Butler, C.; Tessier, B.; Lim, K.-H.; (51) Gazit, E. Use of Biomolecular Templates for the Fabrication of Gauthereau, I.; Choquet, D.; Sibarita, J.-B.; Park, S.; Sainlos, M.; Metal Nanowires. FEBS J. 2007, 274, 317−322. Thoumine, O. Mapping the Dynamics and Nanoscale Organization of (52) Colby, R.; Hulleman, J.; Padalkar, S.; Rochet, J. C.; Stanciu, L. Synaptic Adhesion Proteins Using Monomeric Streptavidin. Nat. A. Biotemplated Synthesis of Metallic Nanoparticle Chains on an α- Commun. 2016, 7, 10773. Synuclein Fiber Scaffold. J. Nanosci. Nanotechnol. 2008, 8, 973−978. (34) Dundas, C. M.; Demonte, D.; Park, S. Streptavidin-Biotin (53) Feng, Y.; Wang, H.; Zhang, J.; Song, Y.; Meng, M.; Mi, J.; Yin, H.; Liu, L. Bioinspired Synthesis of Au Nanostructures Templated Technology: Improvements and Innovations in Chemical and from Amyloid β Peptide Assembly with Enhanced Catalytic Activity. Biological Applications. Appl. Microbiol. Biotechnol. 2013, 97, 9343− Biomacromolecules 2018, 19, 2432−2442. 9353. (54) Carny, O.; Shalev, D. E.; Gazit, E. Fabrication of Coaxial Metal (35) Schwizer, F.; Okamoto, Y.; Heinisch, T.; Gu, Y.; Pellizzoni, M. Nanocables Using a Self-Assembled Peptide Nanotube Scaffold. Nano M.; Lebrun, V.; Reuter, R.; Köhler, V.; Lewis, J. C.; Ward, T. R. Lett. 2006, 6, 1594−1597. Artificial Metalloenzymes: Reaction Scope and Optimization (55) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X. M.; Jaeger, Strategies. Chem. Rev. 2018, 118, 142−231. H.; Lindquist, S. L. Conducting Nanowires Built by Controlled Self- (36) Terai, T.; Kohno, M.; Boncompain, G.; Sugiyama, S.; Saito, N.; Assembly of Amyloid Fibers and Selective Metal Deposition. Proc. Fujikake, R.; Ueno, T.; Komatsu, T.; Hanaoka, K.; Okabe, T.; Urano, Natl. Acad. Sci. U. S. A. 2003, 100, 4527−4532. Y.; Perez, F.; Nagano, T. Artificial Ligands of Streptavidin (ALiS): (56) Reches, M.; Gazit, E. Casting Metal Nanowires within Discrete Discovery, Characterization, and Application for Reversible Control Self-Assembled Peptide Nanotubes. Science 2003, 300, 625−627. of Intracellular Protein Transport. J. Am. Chem. Soc. 2015, 137, (57) Wild, D.; Kodak, E. The Immunoassay Handbook; Elsevier: 10464−10467. 2013, DOI: 10.1016/C2010-0-66244-4. (37) Le, Q.; Nguyen, V.; Park, S. Recent Advances in the (58) Laitinen, O. H.; Nordlund, H. R.; Hytönen, V. P.; Kulomaa, M. Engineering and Application of Streptavidin-like Molecules. Appl. S. Brave New (Strept)Avidins in Biotechnology. Trends Biotechnol. Microbiol. Biotechnol. 2019, 103, 7355−7365. 2007, 25, 269−277. (38) Laitinen, O. H.; Hytönen, V. P.; Nordlund, H. R.; Kulomaa, M. (59) Aragón, P.; Noguera, P.; Bañuls, M. J.; Puchades, R.; Maquieira, S. Genetically Engineered Avidins and Streptavidins. Cell. Mol. Life Sci. Á .; González-Martínez, M. Á . Modulating Receptor-Ligand Binding in 2006, 63, 2992−3017. Biorecognition by Setting Surface Wettability. Anal. Bioanal. Chem. (39) Tang, M.; Pi, J.; Long, Y.; Huang, N.; Cheng, Y.; Zheng, H. 2018, 410, 5723−5730. Quantum Dots-Based Sandwich Immunoassay for Sensitive Detection (60) Men, D.; Guo, Y. C.; Zhang, Z. P.; Wei, H. P.; Zhou, Y. F.; Cui, of Alzheimer’s Disease-Related Aβ1−42. Spectrochim. Acta, Part A Z. Q.; Liang, X. S.; Li, K.; Leng, Y.; You, X. Y.; Zhang, X. E. Seeding- 2018, 201, 82−87. Induced Self-Assembling Protein Nanowires Dramatically Increase (40) Zhou, Y.; Yin, H.; Li, X.; Li, Z.; Ai, S.; Lin, H. Electrochemical the Sensitivity of Immunoassays. Nano Lett. 2009, 9, 2246−2250. Biosensor for Protein Kinase A Activity Assay Based on Gold (61) Faltova, L.; Küffner, A. M.; Hondele, M.; Weis, K.; Arosio, P. Nanoparticles-Carbon Nanospheres, Phos-Tag-Biotin and β-Galacto- Multifunctional Protein Materials and Microreactors Using Low sidase. Biosens. Bioelectron. 2016, 86, 508−515. Complexity Domains as Molecular Adhesives. ACS Nano 2018, 12, (41) Demircan, M. B.; Tohumeken, S.; Gunduz, N.; Khalily, M. A.; 9991−9999. Tekinay, T.; Guler, M. O.; Tekinay, A. B. Biotin Functionalized Self- (62) Sahoo, J. K.; Pappas, C. G.; Sasselli, I. R.; Abul-Haija, Y. M.; Assembled Peptide Nanofiber as an Adjuvant for Immunomodulatory Ulijn, R. V. Biocatalytic Self-Assembly Cascades. Angew. Chem., Int. Response. Biotechnol. J. 2020, 15, 2000100. Ed. 2017, 56, 6828−6832. (42) Wang, C.; Wang, K.; Wang, Z. Development of Gold (63) Yasutaka, K.; Takato, Y.; Takashi, K.; Kohsuke, M.; Hiromi, Y. Nanoparticle Based Colorimetric Method for Quantitatively Studying Enhancement in Adsorption and Catalytic Activity of Enzymes the Inhibitors of Cu2+/Zn2+ Induced β-Amyloid Peptide Assembly. Immobilized on Phosphorus- and Calcium-Modified MCM-41. J. Anal. Chim. Acta 2015, 858, 42−48. Phys. Chem. B 2011, 115, 10335−10345. (43) Povilonienė, S.; Č asaitė, V.; Bukauskas, V.; Š etkus, A.; Staniulis, (64) Qian, K.; Wan, J.; Liu, F.; Girault, H. H.; Liu, B.; Yu, C. A J.; Meškys, R. Functionalization of α-Synuclein Fibrils. Beilstein J. Phospho-Directed Macroporous Alumina - Silica Nanoreactor with Nanotechnol. 2015, 6, 124−133. Multi-Functions. ACS Nano 2009, 3, 3656−3662. (44) Reches, M.; Gazit, E. Biological and Chemical Decoration of (65) Sassolas, A.; Blum, L. J.; Leca-Bouvier, B. D. Immobilization Peptide Nanostructures via Biotin-Avidin Interactions. J. Nanosci. Strategies to Develop Enzymatic Biosensors. Biotechnol. Adv. 2012, Nanotechnol. 2007, 7, 2239−2245. 30, 489−511. (45) Sasso, L.; Suei, S.; Domigan, L.; Healy, J.; Nock, V.; Williams, (66) Ngo, T. T.; Lenhoff, H. M. A Sensitive and Versatile Chromogenic Assay for Peroxidase and Peroxidase-Coupled Reac- M. A. K.; Gerrard, J. A. Versatile Multi-Functionalization of Protein tions. Anal. Biochem. 1980, 105, 389−397. Nanofibrils for Biosensor Applications. Nanoscale 2014, 6, 1629− (67) Zhang, Y.; Tsitkov, S.; Hess, H. Proximity Does Not 1634. Contribute to Activity Enhancement in the Glucose Oxidase- (46) Lee, D. S.; Park, J. S.; Lee, E. J.; Kim, H. J.; Lee, J. A Protein Horseradish Peroxidase Cascade. Nat. Commun. 2016, 7, 13982. Nanofiber Hydrogel for Sensitive Immunoassays. Analyst 2013, 138, 4786−4794. 2833 https://doi.org/10.1021/acs.biomac.1c00222 Biomacromolecules 2021, 22, 2822−2833