Robust and scalable single-molecule protein sequencing with fluorosequencing.

The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications.

Oxidative Degradation of Sequence-Defined Peptoid Oligomers.

Due to their N-substitution, peptoids are generally regarded as resistant to biological degradation, such as enzymatic and hydrolytic mechanisms. This stability is an especially attractive feature for therapeutic development and is a selling point of many previous biological studies. However, another key mode of degradation remains to be fully explored, namely oxidative degradation mediated by reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS are biologically relevant in numerous contexts where biomaterials may be present, thus, improving understanding of peptoid oxidative susceptibility is crucial to exploit their full potential in the biomaterials field, where an oxidatively-labile but enzymatically stable molecule can offer attractive properties. Toward this end, we demonstrate a fundamental characterization of sequence-defined peptoid chains in the presence of chemically generated ROS, as compared to ROS-susceptible peptides such as proline and lysine oligomers. Lysine oligomers showed the fastest degradation rates to ROS and the enzyme trypsin. Peptoids degraded in metal catalyzed oxidation conditions at rates on par with poly(prolines), while maintaining resistance to enzymatic degradation. Furthermore, lysine-containing peptide-peptoid hybrid molecules showed tunability in both ROS-mediated and enzyme-mediated degradation, with rates intermediate to lysine and peptoid oligomers. When lysine-mimetic side-chains were incorporated into a peptoid backbone, the rate of degradation matched that of the lysine peptide oligomers, but remained resistant to enzymatic degradation. These results expand understanding of peptoid degradation to oxidative and enzymatic mechanisms, and demonstrate the potential for peptoid incorporation into materials where selectivity towards oxidative degradation is necessary, or directed enzymatic susceptibility is desired.

Peptomer substrates for quantitative pattern-recognition sensing of proteases.

The utility of active proteases as biomarkers is often limited by overlapping substrate specificity. Here, this feature is leveraged to develop a quantitative pattern-recognition sensing system driven by the degradation patterns of peptide-peptoid hybrid substrates to classify proteases and estimate their concentration by multivariate data analysis.

Fluorescent Peptomer Substrates for Differential Degradation by Metalloproteases.

Proteases, especially MMPs, are attractive biomarkers given their central role in both physiological and pathological processes. Distinguishing MMP activity with degradable substrates, however, is a difficult task due to overlapping substrate specificity profiles. Here, we developed a system of peptomers (peptide-peptoid hybrids) to probe the impact of non-natural residues on MMP specificity for an MMP peptide consensus sequence. Peptoids are non-natural, N-substituted glycines with a large side-chain diversity. Given the presence of a hallmark proline residue in the P3 position of MMP consensus sequences, we hypothesized that peptoids may offer N-substituted alternatives to generate differential interactions with MMPs. To investigate this hypothesis, peptomer substrates were exposed to five different MMPs, as well as bacterial collagenase, and monitored by fluorescence resonance energy transfer and liquid chromatography-mass spectrometry to determine the rate of cleavage and the composition of degraded fragments, respectively. We found that peptoid residues are well tolerated in the P3 and P3' substrate sites and that the identity of the peptoid in these sites displays a moderate influence on the rate of cleavage. However, peptoid residues were even better tolerated in the P1 substrate site where activity was more strongly correlated with side-chain identity than side-chain position. All MMPs explored demonstrated similar trends in specificity for the peptomers but exhibited different degrees of variability in proteolytic rate. These kinetic profiles served as "fingerprints" for the proteases and yielded separation by multivariate data analysis. To further demonstrate the practical application of this tunability in degradation kinetics, peptomer substrates were tethered into hydrogels and released over distinct timescales. Overall, this work represents a significant step toward the design of probes that maximize differential MMP behavior and presents design rules to tune degradation kinetics with peptoid substitutions, which has promising implications for diagnostic and prognostic applications using array-based sensors.

Tuning hydrogel properties with sequence-defined, non-natural peptoid crosslinkers.

The native extracellular matrix (ECM) is composed of hierarchically structured biopolymers containing precise monomer sequences and chain shapes to yield bioactivity. Recapitulating this structure in synthetic hydrogels is of particular interest for tissue engineering and in vitro disease models to accurately mimic biological microenvironments. However, despite extensive research on hydrogels, it remains a challenge to recapitulate the hierarchical structure of native ECM with completely synthetic hydrogel platforms. Toward this end, this work presents a synthetic hydrogel system using commercially available poly(ethylene glycol) macromers with sequence-defined poly(N-substituted glycines) (peptoids) as crosslinkers. We demonstrate that bulk hydrogel mechanics, specifically as shear storage modulus, can be controlled by altering peptoid sequence and structure. Notably, the helical peptoid sequence investigated here increases the storage modulus of the resulting hydrogels with increasing helical content and chain length, in a fashion similar to helical peptide-crosslinked hydrogels. In addition, the resulting hydrogels are shown to be hydrolytically and enzymatically stable due to the N-substituted peptidomimetic backbone of the crosslinkers. We further demonstrate the potential utility of these peptoid-crosslinked hydrogels as a viable cell culture platform using seeded human dermal fibroblasts in comparison to peptide-crosslinked hydrogels as a control. Taken together, our system offers a strategy toward ECM mimics that replicate the hierarchy of biological matrices with completely synthetic, sequence-defined molecules.

Assessing the range of enzymatic and oxidative tunability for biosensor design.

Development of multi-functional materials and biosensors that can achieve an in situ response designed by the user is a current need in the biomaterials field, especially in complex biological environments, such as inflammation, where multiple enzymatic and oxidative signals are present. In the past decade, there has been extensive research and development of materials chemistries for detecting and monitoring enzymatic activity, as well as for releasing therapeutic and diagnostic agents in regions undergoing oxidative stress. However, there has been limited development of materials in the context of enzymatic and oxidative triggers together, despite their closely tied and overlapping mechanisms. With research focusing on enzymatically and oxidatively triggered materials separately, these systems may be inadequate in monitoring the complexity of inflammatory environments, thus limiting in vivo translatability and diagnostic accuracy. The intention of this review is to highlight a variety of enzymatically and oxidatively triggered materials chemistries to draw attention to the range of synthetic tunability available for the construction of novel biosensors with a spectrum of programmed responses. We focus our discussion on several types of macromolecular sensors, generally classified by the causative material response driving ultimate signal detection. This includes sensing based on degradative processes, conformational changes, supramolecular assembly/disassembly, and nanomaterial interactions, among others. We see each of these classes providing valuable tools toward coalescing current gaps in the biosensing field regarding specificity, selectivity, sensitivity, and flexibility in application. Additionally, by considering the materials chemistry of enzymatically and oxidatively triggered biomaterials in tandem, we hope to encourage synthesis of new biosensors that capitalize on their synergistic roles and overlapping mechanisms in inflammatory environments for applications in disease diagnosis and monitoring.

Tunable biomaterials from synthetic, sequence-controlled polymers.

Polymeric biomaterials have many applications including therapeutic delivery vehicles, medical implants and devices, and tissue engineering scaffolds. Both naturally-derived and synthetic materials have successfully been used for these applications in the clinic. However, the increasing complexity of these applications requires materials with advanced properties, especially customizable or tunable materials with bioactivity. To address this issue, there have been recent efforts to better recapitulate the properties of natural materials using synthetic biomaterials composed of sequence-controlled polymers. Sequence control mimics the primary structure found in biopolymers, and in many cases, provides an extra handle for functionality in synthetic polymers. Here, we first review the advances in synthetic methods that have enabled sequence-controlled biomaterials on a relevant scale, and discuss strategies for choosing functional sequences from a biomaterials engineering context. Then, we highlight several recent studies that show strong impact of sequence control on biomaterial properties, including in vitro and in vivo behavior, in the areas of hydrogels, therapeutic materials, and novel applications such as molecular barcodes for medical devices. The role of sequence control in biomaterials properties is an emerging research area, and there remain many opportunities for investigation. Further study of this topic may significantly advance our understanding of bioactive or smart materials, as well as contribute design rules to guide the development of synthetic biomaterials for future applications in tissue engineering and regenerative medicine.

Formation of Metal Nanoparticles Directly from Bulk Sources Using Ultrasound and Application to E-Waste Upcycling.

A method for creating nanoparticles directly from bulk metal by applying ultrasound to the surface in the presence of a two-part surfactant system is presented. Implosive collapse of cavitation bubbles near the bulk metal surface generates powerful microjets, leading to material ejection. This liberated material is captured and stabilized by a surfactant bilayer in the form of nanoparticles. The method is characterized in detail using gold, but is also demonstrated on other metals and alloys, and is generally applicable. It is shown that nanoparticles can be produced regardless of the bulk metal form factor, and the method is extended to an environmentally important problem, the reclamation of gold from an electronic waste stream.

Non-volatile iron carbonyls as versatile precursors for the synthesis of iron-containing nanoparticles.

The most commonly used method for the formation of well-defined iron and iron-containing heterometallic nanoparticles is the thermal decomposition of iron pentacarbonyl (Fe(CO)5). However, iron pentacarbonyl is highly toxic and volatile, which introduces safety concerns and drastically diminishes control over the reaction stoichiometry. Here we alleviate these issues by beginning with an easy-to-handle solid, triiron dodecacarbonyl (Fe3(CO)12). The issue of poor solubility of this cluster is addressed by its reaction with amine, which renders the cluster fully soluble in common high boiling point solvents. This reaction generates non-volatile anionic iron carbonyl species in solution which are subsequently used as the nanoparticle precursor. We demonstrate that the thermolysis of this novel precursor solution yields well-defined Fe, Fe1-xCox, and Fe1-xPtx nanoparticles. In addition, the same approach overcomes the solubility issue of another poorly soluble iron carbonyl compound, diiron nonacarbonyl (Fe2(CO)9). By using these precursors in an array of nanoparticle-forming reactions, we demonstrate a convenient replacement for the commonly used Fe(CO)5, producing particles of similar quality, but without the drawbacks of the precursor volatility and high toxicity.

Thermally programmable pH buffers.

Many reactions in both chemistry and biology rely on the ability to precisely control and fix the solution concentrations of either protons or hydroxide ions. In this report, we describe the behavior of thermally programmable pH buffer systems based on the copolymerization of varying amounts of acrylic acid (AA) groups into N-isopropylacrylamide polymers. Because the copolymers undergo phase transitions upon heating and cooling, the local environment around the AA groups can be reversibly switched between hydrophobic and hydrophilic states affecting the ionization behavior of the acids. Results show that moderate temperature variations can be used to change the solution pH by two units. However, results also indicate that the nature of the transition and its impact on the pH values are highly dependent on the AA content and the degree of neutralization.