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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 (D 10 ) and a cationic Arg-peptide (R 5 R 5 ) with a proteolytic cleavage site. The coacervates dissolve in the presence of the main protease (M pro ) 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 ( k cat /K M ) 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.

The transmission of SARS-CoV-2 coronavirus has led to the COVID-19 pandemic. Nucleic acid testing while specific has limitations for mass surveillance. One alternative is the main protease (M pro ) due to its functional importance in mediating the viral life cycle. Here, we describe a combination of modular substrate and gold colloids to detect M pro via visual readout. The strategy involves zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by M pro . Autolytic cleavage releases a positively charged moiety that assembles the nanoparticles with rapid color changes (t<10 min). We determine a limit of detection for M pro in breath condensate matrices <10 nM. We further assayed ten COVID-negative subjects and found no false-positive result. In the light of simplicity, our test for viral protease is not limited to an equipped laboratory, but also is amenable to integrating as portable point-of-care devices including those on face-coverings., (© 2021 Wiley-VCH GmbH.)

Gold nanorods possess optical properties that are tunable and highly sensitive to variations in their aspect ratio (length/width). Therefore, the development of a sensing platform where the gold nanorod morphology (i.e., aspect ratio) is modulated in response to an analyte holds promise in achieving ultralow detection limits. Here, we use a dithiol peptide as an enzyme substrate during nanorod growth. The sensing mechanism is enabled by the substrate design, where the dithiol peptide contains an enzyme cleavage site in-between cysteine amino acids. When cleaved, the peptide dramatically impacts gold nanorod growth and the resulting optical properties. We demonstrate that the optical response can be correlated with enzyme concentration and achieve a 45 pM limit of detection. Furthermore, we extend this sensing platform to colorimetrically detect tumor-associated inhibitors in a biologically relevant medium. Overall, these results present a subnanomolar method to detect proteases that are critical biomarkers found in cancers, infectious diseases, and inflammatory disorders.

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.

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.

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.

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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health and lacks an effective treatment. There is an urgent need for both real-time tracking and precise treatment of the SARS-CoV-2-infected cells to mitigate and ultimately prevent viral transmission. However, selective triggering and tracking of the therapeutic process in the infected cells remains challenging. Here, we report a main protease (Mpro)-responsive, mitochondrial-targeting, and modular-peptide-conjugated probe (PSGMR) for selective imaging and inhibition of SARS-CoV-2-infected cells via enzyme-instructed self-assembly and aggregation-induced emission (AIE) effect. The amphiphilic PSGMR was constructed with tunable structure and responsive efficiency and validated with recombinant proteins, cells transfected with Mpro plasmid or infected by SARS-CoV-2, and a Mpro inhibitor. By rational construction of AIE luminogen (AIEgen) with modular peptides and Mpro, we verified that the cleavage of PSGMR yielded gradual aggregation with bright fluorescence and enhanced cytotoxicity to induce mitochondrial interference of the infected cells. This strategy may have value for selective detection and treatment of SARS-CoV-2-infected cells.

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