ABSTRACT
Stability issues in membrane-free coacervates have been addressed with coating strategies, but these approaches often compromise the permeability of the coacervate. Here we report a facile approach to maintain both stability and permeability using tannic acid and then demonstrate the value of this approach in enzyme-triggered drug release. First, we develop size-tunable coacervates via self-assembly of heparin glycosaminoglycan with tyrosine and arginine-based peptides. A thrombin-recognition site within the peptide building block results in heparin release upon thrombin proteolysis. Notably, polyphenols are integrated within the nano-coacervates to improve stability in biofluids. Phenolic crosslinking at the liquid-liquid interface enables nano-coacervates to maintain exceptional structural integrity across various environments. We discover a pivotal polyphenol threshold for preserving enzymatic activity alongside enhanced stability. The disassembly rate of the nano-coacervates increases as a function of thrombin activity, thus preventing a coagulation cascade. This polyphenol-based approach not only improves stability but also opens the way for applications in biomedicine, protease sensing, and bio-responsive drug delivery.
Subject(s)
Drug Delivery Systems , Polyphenols , Tannins , Thrombin , Polyphenols/chemistry , Thrombin/metabolism , Thrombin/chemistry , Humans , Tannins/chemistry , Heparin/chemistry , Drug Liberation , Peptides/chemistry , Peptides/metabolism , ProteolysisABSTRACT
Colorimetric sensing offers instant reporting via visible signals. Versus labor-intensive and instrument-dependent detection methods, colorimetric sensors present advantages including short acquisition time, high throughput screening, low cost, portability, and a user-friendly approach. These advantages have driven substantial growth in colorimetric sensors, particularly in point-of-care (POC) diagnostics. Rapid progress in nanotechnology, materials science, microfluidics technology, biomarker discovery, digital technology, and signal pattern analysis has led to a variety of colorimetric reagents and detection mechanisms, which are fundamental to advance colorimetric sensing applications. This review first summarizes the basic components (e.g., color reagents, recognition interactions, and sampling procedures) in the design of a colorimetric sensing system. It then presents the rationale design and typical examples of POC devices, e.g., lateral flow devices, microfluidic paper-based analytical devices, and wearable sensing devices. Two highlighted colorimetric formats are discussed: combinational and activatable systems based on the sensor-array and lock-and-key mechanisms, respectively. Case discussions in colorimetric assays are organized by the analyte identities. Finally, the review presents challenges and perspectives for the design and development of colorimetric detection schemes as well as applications. The goal of this review is to provide a foundational resource for developing colorimetric systems and underscoring the colorants and mechanisms that facilitate the continuing evolution of POC sensors.
Subject(s)
Colorimetry , Humans , Coloring Agents/chemistry , Biosensing Techniques , Point-of-Care SystemsABSTRACT
Polymeric spherulites are typically formed by melt crystallization: spherulitic growth in solution is rare and requires complex polymers and dilute solutions. Here, we report the mild and unique formation of luminescent spherulites at room temperature via the simple molecule benzene-1,4-dithiol (BDT). Specifically, BDT polymerized into oligomers (PBDT) via disulfide bonds and assembled into uniform supramolecular nanoparticles in aqueous buffer; these nanoparticles were then dissolved back into PBDT in a good solvent (i.e., dimethylformamide) and underwent chain elongation to form spherulites (rPBDT) in 10 min. The spherulite geometry was modulated by changing the PBDT concentration and reaction time. Due to the step-growth polymerization and reorganization of PBDT, these spherulites not only exhibited robust structure but also showed broad clusterization-triggered emission. The biocompatibility and efficient cellular uptake of the spherulites further underscore their value as traceable drug carriers. This system provides a new pathway for designing versatile superstructures with value for hierarchical assembly of small molecules into a complicated biological system.
Subject(s)
Nanoparticles , Polymers , Crystallization , Polymers/chemistry , FreezingABSTRACT
We report here a colorimetric method for rapid detection of norovirus based on the valence-driven peptide-AuNP interactions. We engineered a peptide sequence named K1 with a cleavage sequence in between two lysine residues. The positively charged lysine groups aggregated the negatively charged nanoparticles leading to a purple color change. There was a red color when the cleavage sequence was digested by the Southampton norovirus 3C-like protease (SV3CP)-a protease involved in the life cycle of Human norovirus (HNV). The limit of detection was determined to be 320 nM in Tris buffer. We further show that the sensor has good performance in exhaled breath condensate, urine, and faecal matter. This research provides a potential easy and quick way to selectively detect HNV.
Subject(s)
Metal Nanoparticles , Norovirus , Humans , Peptide Hydrolases , Colorimetry/methods , Norovirus/chemistry , Lysine , Peptides , Metal Nanoparticles/chemistry , Gold/chemistryABSTRACT
This study aims to restore grating lobe artifacts and improve the image resolution of sparse array ultrasonography via a deep learning predictive model. A deep learning assisted sparse array was developed using only 64 or 16 channels out of the 128 channels in which the pitch is two or eight times the original array. The deep learning assisted sparse array imaging system was demonstrated on ex vivo porcine teeth. 64- and 16-channel sparse array images were used as the input and corresponding 128-channel dense array images were used as the ground truth. The structural similarity index measure, mean squared error, and peak signal-to-noise ratio of predicted images improved significantly (p < 0.0001). The resolution of predicted images presented close values to ground truth images (0.18 mm and 0.15 mm versus 0.15 mm). The gingival thickness measurement showed a high level of agreement between the predicted sparse array images and the ground truth images, as indicated with a bias of -0.01 mm and 0.02 mm for the 64- and 16-channel predicted images, respectively, and a Pearson's r = 0.99 (p < 0.0001) for both. The gingival thickness bias measured by deep learning assisted sparse array imaging and clinical probing needle was found to be <0.05 mm. Additionally, the deep learning model showed capability of generalization. To conclude, the deep learning assisted sparse array can reconstruct high-resolution ultrasound image using only 16 channels of 128 channels. The deep learning model performed generalization capability for the 64-channel array, while the 16-channel array generalization would require further optimization.
Subject(s)
Deep Learning , Animals , Swine , Ultrasonography , Artifacts , Generalization, Psychological , Gingiva , Image Processing, Computer-AssistedABSTRACT
The surface of gold nanoparticles (AuNPs) can be conjugated with a wide range of highly functional biomolecules. A common pitfall when utilizing AuNPs is their tendency to aggregate, especially when their surface is functionalized with ligands of low molecular weight (no steric repulsion) or ligands of neutral charge (no electrostatic repulsion). For biomedical applications, AuNPs that are colloidally stable are desirable because they have a high surface area and thus reactivity, resist sedimentation, and exhibit uniform optical properties. Here, we engineer the surface of AuNPs so that they remain stable when decorated with coiled-coil (CC) peptides while preserving the native polypeptide properties. We achieve this by using a neutral, mixed ligand layer composed of lipoic acid poly(ethylene glycol) and lipoic acid poly(ethylene glycol) maleimide to attach the CCs. Tuning the surface fraction of each component within the mixed ligand layer also allowed us to control the degree of AuNP labeling with CCs. We demonstrate the dynamic surface properties of these CC-AuNPs by performing a place-exchange reaction and their utility by designing an energy-transfer-based caspase-3 sensor. Overall, this study optimizes the surface chemistry of AuNPs to quantitatively present functional biomolecules while maintaining colloid stability.
Subject(s)
Metal Nanoparticles , Thioctic Acid , Gold , Ligands , Peptides , Polyethylene GlycolsABSTRACT
Noroviruses are highly contagious and are one of the leading causes of acute gastroenteritis worldwide. Due to a lack of effective antiviral therapies, there is a need to diagnose and surveil norovirus infections to implement quarantine protocols and prevent large outbreaks. Currently, the gold standard of diagnosis uses reverse transcription polymerase chain reaction (RT-PCR), but PCR can have limited availability. Here, we propose a combination of a tunable peptide substrate and gold nanoparticles (AuNPs) to colorimetrically detect the Southampton norovirus 3C-like protease (SV3CP), a key protease in viral replication. Careful design of the substrate employs a zwitterionic peptide with opposite charged moieties on the C- and N- termini to induce a rapid color change visible to the naked eye; thus, this color change is indicative of SV3CP activity. This work expands on existing zwitterionic peptide strategies for protease detection by systematically evaluating the effects of lysine and arginine on nanoparticle charge screening. We also determine a limit of detection for SV3CP of 28.0 nM with comparable results in external breath condensate, urine, and fecal matter for 100 nM of SV3CP. The key advantage of this system is its simplicity and accessibility, thus making it an attractive tool for qualitative point-of-care diagnostics.
Subject(s)
Caliciviridae Infections , Metal Nanoparticles , Norovirus , Humans , Peptide Hydrolases , Norovirus/genetics , Gold , Colorimetry , Peptides , Endopeptidases , Feces , Caliciviridae Infections/diagnosis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Better insights into the fate of membraneless organelles could strengthen the understanding of the transition from prebiotic components to multicellular organisms. Compartmentalized enzyme reactions in a synthetic coacervate have been investigated, yet there remains a gap in understanding the enzyme interactions with coacervate as a substrate hub. Here, we study how the molecularly crowded nature of the coacervate affects the interactions of the embedded substrate with a protease. We design oligopeptide-based coacervates that comprise an anionic Asp-peptide (D10) and a cationic Arg-peptide (R5R5) with a proteolytic cleavage site. The coacervates dissolve in the presence of the main protease (Mpro) implicated in the coronavirus lifecycle. We capitalize on the condensed structure, introduce a self-quenching mechanism, and model the enzyme kinetics by using Cy5.5-labeled peptides. The determined specificity constant (kcat/KM) is 5817 M-1 s-1 and is similar to that of the free substrate. We further show that the enzyme kinetics depend on the type and quantity of dye incorporated into the coacervates. Our work presents a simple design for enzyme-responsive coacervates and provides insights into the interactions between the enzyme and coacervates as a whole.
Subject(s)
Peptides , Proteins , Peptides/chemistry , Oligopeptides , Peptide HydrolasesABSTRACT
We report the reversible aggregation of silver nanoparticle (AgNP) assemblies using the combination of a cationic arginine-based peptide and sulfur-capped polyethylene glycol (PEG). The formation and dissociation of the aggregates were studied by optical methods and electron microscopy. The dissociation of silver clusters depends on the peptide sequence and PEG size. A molecular weight of 1 kDa for PEG was optimal for the dissociation. The most important feature of this dissociation method is that it can operate in complex biofluids such as plasma, saliva, bile, urine, cell media, or even seawater without a significant decrease in performance. Moreover, the peptide-particle assemblies are highly stable and do not degrade (or express of loss of signal upon dissociation) when dried and resolubilized, frozen and thawed, or left in daylight for a month. Importantly, the dissociation capacity of PEG can be reduced via the conjugation of a peptide-cleavable substrate. The dissociation capacity is restored in the presence of an enzyme. Based on these findings, we designed a PEG-peptide hybrid molecule specific to the Porphyromonas gingivalis protease RgpB. Our motivation was that this bacterium is a key pathogen in periodontitis, and RgpB activity has been correlated with chronic diseases including Alzheimer's disease. The RgpB limit of detection was 100 pM RgpB in vitro. This system was used to measure RgpB in gingival crevicular fluid (GCF) samples with a detection rate of 40% with 0% false negatives versus PCR for P. gingivalis (n = 37). The combination of PEG-peptide and nanoparticles dissociation method allows the development of convenient protease sensing that can operate independently of the media composition.
Subject(s)
Metal Nanoparticles , Peptide Hydrolases , Porphyromonas gingivalis , Silver , Polymers , Endopeptidases , Peptides , Polyethylene GlycolsABSTRACT
OBJECTIVE: To develop a novel technique for localizing and reconstructing the greater palatine artery (GPA) using three-dimensional (3D) technology. METHODS: A miniaturized intraoral ultrasound transducer was used to imaging landmarks including the GPA, gingival margin (GM), and palatal masticatory mucosa (PMM). A 5-mm-thick solid hydrogel couplant was integrated to replace traditional ultrasound gel and avoid bubbles when moving the transducer. RESULTS: A panorama image provided the relative localization of landmarks including the GPA, PMM, and hard palate. Short- and long-axis imaging of GPA was performed in five subjects including 3D mapping of GPA branches and surrounding tissues in a volume of 10 mm × 8 mm × 10 mm. Full-mouth Doppler imaging was also demonstrated on both the dorsal and ventral tongue as well as buccal mucosa and sublingual region on two subjects. CONCLUSIONS: This study can measure the vertical distance from the GM to the GPA and depth from PMM to GPA and visualize the GPA localization in a 3D manner, which is critical to evaluate the available volume of palatal donor tissues and avoid sectioning of GPA during surgical harvesting of the tissues. Finally, the transducer's small size facilitates full-mouth Doppler imaging with the potential to improve the assessment, diagnosis, and management of oral mucosa.
Subject(s)
Arteries , Palate, Hard , Humans , Arteries/diagnostic imaging , Mouth Mucosa , FaceABSTRACT
Colorimetric biosensors based on gold nanoparticle (AuNP) aggregation are often challenged by matrix interference in biofluids, poor specificity, and limited utility with clinical samples. Here, we propose a peptide-driven nanoscale disassembly approach, where AuNP aggregates induced by electrostatic attractions are dissociated in response to proteolytic cleavage. Initially, citrate-coated AuNPs were assembled via a short cationic peptide (RRK) and characterized by experiments and simulations. The dissociation peptides were then used to reversibly dissociate the AuNP aggregates as a function of target protease detection, i.e., main protease (Mpro), a biomarker for severe acute respiratory syndrome coronavirus 2. The dissociation propensity depends on peptide length, hydrophilicity, charge, and ligand architecture. Finally, our dissociation strategy provides a rapid and distinct optical signal through Mpro cleavage with a detection limit of 12.3 nM in saliva. Our dissociation peptide effectively dissociates plasmonic assemblies in diverse matrices including 100% human saliva, urine, plasma, and seawater, as well as other types of plasmonic nanoparticles such as silver. Our peptide-enabled dissociation platform provides a simple, matrix-insensitive, and versatile method for protease sensing.
Subject(s)
COVID-19 , Metal Nanoparticles , Humans , Gold , Peptides , Peptide HydrolasesABSTRACT
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.
Subject(s)
Colorimetry , Metal Nanoparticles , Colorimetry/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Polymers , LigandsABSTRACT
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.
ABSTRACT
Transmembrane protease serine 2 (TMPRSS2) is a plasma membrane protease that activates both spike protein of coronaviruses for cell entry and oncogenic signaling pathways for tumor progression. TMPRSS2 inhibition can reduce cancer invasion and metastasis and partially prevent the entry of SARS-CoV-2 into host cells. Thus, there is an urgent need for both TMPRSS2-selective imaging and precise screening of TMPRSS2 inhibitors. Here, we report a TMPRSS2-responsive surface-potential-tunable peptide-conjugated probe (EGTP) with aggregation-induced emission (AIE) features for TMPRSS2 selective imaging and accurate inhibitor screening. The amphiphilic EGTP was constructed with tunable surface potential and responsive efficiency with TMPRSS2 and its inhibitor. The rational construction of AIE luminogens (AIEgens) with modular peptides indicated that the cleavage of EGTP led to a gradual aggregation with bright fluorescence in high TMPRSS2-expressing cells. This strategy may have value for selective detection of cancer cells, SARS-CoV-2-target cells, and screening of protease inhibitors.
Subject(s)
COVID-19 , Peptide Hydrolases , Humans , SARS-CoV-2 , Cell Membrane , Protease Inhibitors , Virus Internalization , Serine EndopeptidasesABSTRACT
OBJECTIVE: To customize a miniaturized ultrasound transducer to access full-mouth B-mode, color Doppler, and spectral Doppler imaging for monitoring oral health. METHODS: A customized periodontal ultrasound transducer SS-19-128 (19 MHz, 128 channels) 1.8-cm wide and 1-cm thick was developed and connected to a data acquisition (DAQ) system. B-mode, color Doppler, and spectral Doppler data could all be collected with SS-19-128. The imaging resolution and penetration capacity of SS-19-128 were characterized on phantoms. The gingival thickness was measured on 11 swine teeth by SS-19-128 for comparison with conventional transgingival probing via Bland-Altman analysis and Pearson correlation. Five human subjects were then recruited to demonstrate B-mode and Doppler imaging by SS-19-128. RESULTS: The axial and lateral spatial resolution at 5.5 mm depth is 102.1 µm and 142.9 µm, respectively. The penetration depth in a tissue-mimicking phantom is over 30 mm. In vivo B-mode imaging of all 28 teeth was demonstrated on one human subject, and imaging of tooth #18 was accessed on five human subjects. Gingival thickness measurement compared with transgingival probing showed a bias of -0.015 mm and SD of 0.031 mm, and a r = 0.9235 (p < 0.0001) correlation. In vivo color and spectral Doppler imaging of the supraperiosteal artery in human gingiva was performed to generate hemodynamic information. CONCLUSIONS: The small size of SS-19-128 offers important advantages over existing ultrasound technology-more specifically, whole-mouth scanning/charting reminiscent of radiography. This is nearly a two-fold increase in the number of teeth that can be assessed versus conventional transducers.
Subject(s)
Oral Health , Ultrasonography, Doppler , Humans , Animals , Swine , Ultrasonography , Transducers , MouthABSTRACT
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 26.1â nM, respectively. These results may have value in designing other protease testing methods.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , SARS-CoV-2 , Peptides/chemistry , Peptide Hydrolases , Surface-Active Agents , Endopeptidases , Gold/chemistryABSTRACT
3D-bioprinted skin-mimicking phantoms with skin colors ranging across the Fitzpatrick scale are reported. These tools can help understand the impact of skin phototypes on biomedical optics. Synthetic melanin nanoparticles of different sizes (70-500 nm) and clusters are fabricated to mimic the optical behavior of melanosome. The absorption coefficient and reduced scattering coefficient of the phantoms are comparable to real human skin. Further the melanin content and distribution in the phantoms versus real human skins are validated via photoacoustic (PA) imaging. The PA signal of the phantom can be improved by: 1) increasing melanin size (3-450-fold), 2) increasing clustering (2-10.5-fold), and 3) increasing concentration (1.3-8-fold). Then, multiple biomedical optics tools (e.g., PA, fluorescence imaging, and photothermal therapy) are used to understand the impact of skin tone on these modalities. These well-defined 3D-bioprinted phantoms may have value in translating biomedical optics and reducing racial bias.
Subject(s)
Melanins , Skin , Humans , Phantoms, Imaging , Optics and Photonics , Optical ImagingABSTRACT
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.