Gravity-Driven Separation for Enrichment of Rare Earth Elements Using Lanthanide Binding Peptide-Immobilized Resin.

Rare Earth Elements (REEs) constitute indispensable raw materials and are employed in a diverse range of devices, including but not limited to smartphones, electric vehicles, and clean energy technologies. While there is an increase in demand for these elements, there is a global supply challenge due to limited availability and geopolitical factors affecting their procurement. A crucial step in manufacturing these devices involves utilizing highly pure REEs, often obtained through complex and nonsustainable processes. These processes are vital in isolating individual REEs from mixtures containing non-REEs and other REEs. There exists an urgent requirement to explore alternative techniques that enable the selective recovery of REEs through more energy-efficient processes. To overcome the limitations mentioned above, we developed a microbead-based technology featuring immobilized lanthanide binding peptides (LBPs) for the selective adsorption of REEs. This technology does not require the utilization of external stimuli but uses gravity-based separation processes to separate the bound REE from the unbound REE. We demonstrate this technology's potential by enriching two relevant REEs (Europium and Terbium). Additionally, we propose a mechanism whereby REEs bind selectively to a particular LBP, leveraging the distinctive physicochemical characteristics of both the REE and the LBP. Moreover, these LBPs exhibit no binding affinity toward other frequently encountered industrial ions. Finally, we demonstrate the recovery of REEs through a change in system conditions and assess the reusability of the microbeads for subsequent adsorption cycles. We anticipate that this approach will address the challenges of REE recovery and demonstrate the potential of biomolecular strategies in advancing sustainable resource management.

Tuning Electrostatic Interactions To Control Orientation of GFP Protein Adsorption on Silica Surface.

The adsorption of green fluorescent protein (GFP) on silica surfaces has been the subject of growing interest due to its potential applications in various fields, including biotechnology and biomedicine. In this study, we used all-atom molecular dynamics simulations to investigate the charge-driven adsorption of wild type GFP and its supercharged variants on silica surfaces. The results showed that the positively charged variant of GFP adsorbed on the negatively charged silica surface with minimal loss in its secondary structure. Further studies were conducted to understand the role of surface charge distribution on two other positively charged variants of GFP, and the results showed that the orientation of GFP on silica can be easily tuned by careful mutations of the charged amino acid residues on the GFP. This study provides valuable molecular insights into the role of electrostatic-driven adsorption of GFP and highlights the importance of charge interactions in the adsorption process.

Self-Assembled Amino Acid Microstructures as Biocompatible Physically Unclonable Functions (BPUFs) for Authentication of Therapeutically Relevant Hydrogels.

Counterfeited biomedical products result in significant economic losses and pose a public health hazard for over a million people yearly. Hydrogels, a class of biomedical products, are being investigated as alternatives to conventional biomedical products and are equally susceptible to counterfeiting. Here, a biocompatible, physically unclonable function (BPUF) to verify the authenticity of therapeutically relevant hydrogels are developed. The principle of BPUF relies on the self-assembly of tyrosine into fibril-like structures which are incorporated into therapeutically relevant hydrogels resulting in their random dispersion. This unclonable arrangement leads to distinctive optical micrographs captured using an optical microscope. These optical micrographs are transformed into a unique security code through cryptographic techniques which are then used to authenticate the hydrogel. The temporal stability of the BPUFs are demonstrated and additionally, exploit the dissolution propensity of the structures upon exposure to an adulterant to identify the tampering of the hydrogel. Finally, a platform to demonstrate the translational potential of this technology in validating and detecting tampering of therapeutically relevant hydrogels is developed. The potential of BPUFs to combat hydrogel counterfeiting is exemplified by its simplicity in production, ease of use, biocompatibility, and cost-effectiveness.

Versatile Detection and Monitoring of Ionizing Radiation Treatment Using Radiation-Responsive Gel Nanosensors.

Modern radiation therapy workflow involves complex processes intended to maximize the radiation dose delivered to tumors while simultaneously minimizing excess radiation to normal tissues. Safe and accurate delivery of radiation doses is critical to the successful execution of these treatment plans and effective treatment outcomes. Given extensive differences in existing dosimeters, the choice of devices and technologies for detecting biologically relevant doses of radiation has to be made judiciously, taking into account anatomical considerations and modality of treatment (invasive, e.g., interstitial brachytherapy vs noninvasive, e.g., external-beam therapy radiotherapy). Rapid advances in versatile radiation delivery technologies necessitate new detection platforms and devices that are readily adaptable into a multitude of form factors in order to ensure precision and safety in dose delivery. Here, we demonstrate the adaptability of radiation-responsive gel nanosensors as a platform technology for detecting ionizing radiation using three different form factors with an eye toward versatile use in the clinic. In this approach, ionizing radiation results in the reduction of monovalent gold salts leading to the formation of gold nanoparticles within gels formulated in different morphologies including one-dimensional (1D) needles for interstitial brachytherapy, two-dimensional (2D) area inserts for skin brachytherapy, and three-dimensional (3D) volumetric dose distribution in tissue phantoms. The formation of gold nanoparticles can be detected using distinct but complementary modes of readout including optical (visual) and photothermal detection, which further enhances the versatility of this approach. A linear response in the readout was seen as a function of radiation dose, which enabled straightforward calibration of each of these devices for predicting unknown doses of therapeutic relevance. Taken together, these results indicate that the gel nanosensor technology can be used to detect ionizing radiation in different morphologies and using different detection methods for application in treatment planning, delivery, and verification in radiotherapy and in trauma care.

Radiation-Responsive Amino Acid Nanosensor Gel (RANG) for Radiotherapy Monitoring and Trauma Care.

Accurate detection of doses is critical for the development of effective countermeasures and patient stratification strategies in cases of accidental exposure to ionizing radiation. Existing detection devices are limited by high fabrication costs, long processing times, need for sophisticated detection systems, and/or loss of readout signal over time, particularly in complex environments. Here, we describe fundamental studies on amino acid-facilitated templating of gold nanoparticles following exposure to ionizing radiation as a new colorimetric approach for radiation detection. Tryptophan demonstrated spontaneous nanoparticle formation, and parallel screening of a library of amino acids and related compounds led to the identification of lead candidates, including phenylalanine, which demonstrated an increase in absorbance at wavelengths typical of gold nanoparticles in the presence of ionizing radiation (X-rays). Evaluation of screening, i.e., absorbance data, in concert with chemical informatics modeling led to the elucidation of physicochemical properties, particularly polarizable regions and partial charges, that governed nanoparticle formation propensities upon exposure of amino acids to ionizing radiation. NMR spectroscopy revealed key roles of amino and carboxy moieties in determining the nanoparticle formation propensity of phenylalanine, a lead amino acid from the screen. These findings were employed for fabricating radiation-responsive amino acid nanosensor gels (RANGs) based on phenylalanine and tryptophan, and efficacy of RANGs was demonstrated for predicting clinical doses of ionizing radiation in anthropomorphic thorax phantoms and in live canine patients undergoing radiotherapy. The use of biocompatible templating ligands (amino acids), rapid response, simplicity of fabrication, efficacy, ease of operation and detection, and long-lasting readout indicate several advantages of the RANG over existing detection systems for monitoring radiation in clinical radiotherapy, radiological emergencies, and trauma care.

Interrogating biomineralization one amino acid at a time: amplification of mutational effects in protein-aided titania morphogenesis through reaction-diffusion control.

To emulate the control that biomineralizing organisms exert over reactant transport, we construct a countercurrent reaction-diffusion chamber in which an agarose hydrogel regulates the fluxes of inorganic precursor and precipitating solid-binding protein. We show that the morphology of the bioprecipitated titania can be changed from monolithic to interconnected particle networks and dispersed nanoparticles either by decreasing reaction time or by increasing agarose weight percentage at constant precursor and protein concentrations. More strikingly, protein variants with one or two substitutions in their metal oxide-binding domain yield unique peripheral morphologies (needles, threads, plates, and peapods) with distinct crystallography and photocatalytic activity. Our results suggest that diffusional control can magnify otherwise subtle mutational effects in biomineralizing proteins and provide a path for the green synthesis of morphologically and functionally diverse inorganic materials.

Solid-Binding Proteins: Bridging Synthesis, Assembly, and Function in Hybrid and Hierarchical Materials Fabrication.

There is considerable interest in the development of hybrid organic-inorganic materials because of the potential for harvesting the unique capabilities that each system has to offer. Proteins are an especially attractive organic component owing to the high amount of chemical information encoded in their amino acid sequence, their amenability to molecular and computational (re)design, and the many structures and functions they specify. Genetic installation of solid-binding peptides (SBPs) within protein frameworks affords control over the position and orientation of adhesive and morphogenetic segments, and a path toward predictive synthesis and assembly of functional materials and devices, all while harnessing the built-in properties of the host scaffold. Here, we review the current understanding of the mechanisms through which SBPs bind to technologically relevant interfaces, with an emphasis on the variables that influence the process, and highlight the last decade of progress in the use of solid-binding proteins for hybrid and hierarchical materials synthesis.

Phase Control of Nanocrystalline Inclusions in Bioprecipitated Titania with a Panel of Mutant Silica-Binding Proteins.

The biomimetic route to inorganic synthesis presents an opportunity to produce complex materials with superior properties under ambient conditions and from nontoxic precursors. While there has been significant progress in using solid-binding peptides (SBPs), proteins, and organisms to produce a variety of inorganic and hybrid structures, it has been more challenging to understand the interplay of solution conditions and solid-binding peptide (SBP) sequence, structure, and self-association on synthetic outcomes. Here, we show that fusing the Car9 silica-binding peptide-but not the silaffin-derived R5 peptide-to superfolder green fluorescent protein (sfGFP) enhances the ability of micromolar concentrations of protein to induce rapid titania (TiO2) precipitation from acidified solutions of tetrakis(di-lactato)-oxo-titanate (TiBALDH). TiO2 is produced stoichiometrically and although predominantly amorphous, contains nanosized anatase and monoclinic TiO2(B) inclusions. Remarkably, the phase of these nanocrystallites can be tuned from about 80% TiO2(B) to about 65% anatase by using Car9 mutants impaired in their ability to drive the formation of higher-order sfGFP-Car9 oligomers. Our results suggest that the presentation of multiple basic side chains in an extended plane formed by SBP self-association is critical to template the formation of monoclinic crystallites and underscore the subtle influence that single or dual substitutions in dodecameric SBPs can exert on the yield and crystallinity of biomineralized inorganics.

Flow-induced Shear Stress Confers Resistance to Carboplatin in an Adherent Three-Dimensional Model for Ovarian Cancer: A Role for EGFR-Targeted Photoimmunotherapy Informed by Physical Stress.

A key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.

Plasmonic gel nanocomposites for detection of high energy electrons.

Radiation therapy is a common treatment modality employed in the treatment of cancer. High energy photons are the primary source of radiation but when administered, they leave an exit dose resulting in radiation damage to the adjacent healthy tissues. To overcome this, high energy electrons are employed in cases of skin cancer to minimize radiation induced toxicity. Despite these advances, measurement of delivered radiation remains a challenge due to limitations with existing dosimeters including labor intensive fabrication, complex read-out techniques and post-irradiation instability. To overcome these limitations, we have developed a novel colorimetric plasmonic gel nanocomposite for the detection of therapeutic levels of radiation delivered in electron beam therapy. The plasmonic nanocomposite consists of an agarose gel matrix encapsulating precursor gold ions, which are reduced to gold nanoparticles as a result of exposure to high energy electrons. The formation of gold nanoparticles renders a change in color to the agarose matrix, resulting in the formation of plasmonic gel nanocomposites. The intensity of the color formed exhibits a linear relation with the delivered electron dose, which can be quantified using absorbance spectroscopy. The plasmonic gel nanocomposites were able to detect doses employed in fractionated electron therapy, including in an anthropomorphic phantom used for planning radiation treatments in the clinic. Furthermore, the use of glutathione as a quenching agent facilitated qualitative and quantitative spatial mapping of the delivered dose. Our results indicate that the ease of fabrication, simplicity of detection and quantification using absorbance spectroscopy, determination of spatial dose profiles, and relatively low cost make the plasmonic gel nanocomposite technology attractive for detecting electron doses in the clinic.

Determination of topographical radiation dose profiles using gel nanosensors.

Despite the emergence of sophisticated technologies in treatment planning and administration, routine determination of delivered radiation doses remains a challenge due to limitations associated with conventional dosimeters. Here, we describe a gel-based nanosensor for the colorimetric detection and quantification of topographical radiation dose profiles in radiotherapy. Exposure to ionizing radiation results in the conversion of gold ions in the gel to gold nanoparticles, which render a visual change in color in the gel due to their plasmonic properties. The intensity of color formed in the gel was used as a quantitative reporter of ionizing radiation. The gel nanosensor was used to detect complex topographical dose patterns including those administered to an anthropomorphic phantom and live canine patients undergoing clinical radiotherapy. The ease of fabrication, operation, rapid readout, colorimetric detection, and relatively low cost illustrate the translational potential of this technology for topographical dose mapping in radiotherapy applications in the clinic.

Protein-facilitated gold nanoparticle formation as indicators of ionizing radiation.

The use of X-ray radiation in radiotherapy is a common treatment for many cancers. Despite several scientific advances, determination of radiation delivered to the patient remains a challenge due to the inherent limitations of existing dosimeters including fabrication and operation. Here, we describe a colorimetric nanosensor that exhibits unique changes in color as a function of therapeutically relevant radiation dose (3-15 Gy). The nanosensor is formulated using a gold salt and maltose-binding protein as a templating agent, which upon exposure to ionizing radiation is converted to gold nanoparticles. The formation of gold nanoparticles from colorless precursor salts renders a change in color that can be observed visually. The dose-dependent multicolored response was quantified through a simple ultraviolet-visible spectrophotometer and the peak shift associated with the different colored dispersions was used as a quantitative indicator of therapeutically relevant radiation doses. The ease of fabrication, visual color changes upon exposure to ionizing radiation, and quantitative read-out demonstrates the potential of protein-facilitated biomineralization approaches to promote the development of next-generation detectors for ionizing radiation.

Hydrogel Nanosensors for Colorimetric Detection and Dosimetry in Proton Beam Radiotherapy.

Proton beam therapy (PBT) is a state-of-the-art radiotherapy treatment approach that uses focused proton beams for tumor ablation. A key advantage of this approach over conventional photon radiotherapy (XRT) is the unique dose deposition characteristic of protons, which results in superior healthy tissue sparing. This results in fewer unwanted side effects and improved outcomes for patients. Currently available dosimeters are intrinsic, complex, and expensive and are not routinely used to determine the dose delivered to the tumor. Here, we report a hydrogel-based plasmonic nanosensor for detecting clinical doses used in conventional and hyperfractionated proton beam radiotherapy. In this nanosensor, gold ions, encapsulated in a hydrogel, are reduced to gold nanoparticles following irradiation with proton beams. Formation of gold nanoparticles renders a color change to the originally colorless hydrogel. The intensity of the color can be used to calibrate the hydrogel nanosensor in order to quantify different radiation doses employed during proton treatment. The potential of this nanosensor for clinical translation was demonstrated using an anthropomorphic phantom mimicking a clinical radiotherapy session. The simplicity of fabrication, detection range in the fractionated radiotherapy regime, and ease of detection with translational potential makes this a first-in-kind plasmonic colorimetric nanosensor for applications in clinical proton beam therapy.

A Colorimetric Plasmonic Nanosensor for Dosimetry of Therapeutic Levels of Ionizing Radiation.

Modern radiation therapy using highly automated linear accelerators is a complex process that maximizes doses to tumors and minimizes incident dose to normal tissues. Dosimeters can help determine the radiation dose delivered to target diseased tissue while minimizing damage to surrounding healthy tissue. However, existing dosimeters can be complex to fabricate, expensive, and cumbersome to operate. Here, we demonstrate studies of a liquid phase, visually evaluated plasmonic nanosensor that detects radiation doses commonly employed in fractionated radiotherapy (1-10 Gy) for tumor ablation. We accomplished this by employing ionizing radiation, in concert with templating lipid surfactant micelles, in order to convert colorless salt solutions of univalent gold ions (Au(1)) to maroon-colored dispersions of plasmonic gold nanoparticles. Differences in color intensities of nanoparticle dispersions were employed as quantitative indicators of the radiation dose. The nanoparticles thus formed were characterized using UV-vis absorbance spectroscopy, dynamic light scattering, and transmission electron microscopy. The role of lipid surfactants on nanoparticle formation was investigated by varying the chain lengths while maintaining the same headgroup and counterion; the effect of surfactant concentration on detection efficacy was also investigated. The plasmonic nanosensor was able to detect doses as low as 0.5 Gy and demonstrated a linear detection range of 0.5-2 Gy or 5-37 Gy depending on the concentration of the lipid surfactant employed. The plasmonic nanosensor was also able to detect radiation levels in anthropomorphic prostate phantoms when administered together with endorectal balloons, indicating its potential utility as a dosimeter in fractionated radiotherapy for prostate cancer. Taken together, our results indicate that this simple visible nanosensor has strong potential to be used as a dosimeter for validating delivered radiation doses in fractionated radiotherapies in a variety of clinical settings.

Biotemplating plasmonic nanoparticles using intact microfluidic vasculature of leaves.

Leaves are an abundant natural resource, and consist of a sophisticated microfluidic network of veins that transport nutrients and water, thereby enabling photosynthesis. Here, we simultaneously exploit the microfluidics as well as chemistry of processed leaf vasculature (venation) in order to template the in situ generation of plasmonic metal (gold and silver) nanoparticles under ambient conditions. This biotemplating approach involves capillary flow of metal salts through skeleton leaf vasculature, and does not require additional reducing agents for plasmonic nanoparticle formation. Gold nanoparticles, 30-40 nm in diameter, and silver nanoparticles, approximately 9 nm in diameter, were formed within the intact leaf vasculature using this method. Absorption spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and electron diffraction analyses were employed to ascertain the formation of nanoparticles in the leaf veins. Fourier transform infrared (FT-IR) spectroscopy was employed in order to obtain insights into functional groups responsible for formation of the plasmonic nanoparticles within the leaves. Gold nanoparticles, templated within leaves, demonstrated excellent catalytic properties, thereby imparting catalytic and plasmonic properties to the leaf itself. Furthermore, nanoparticles can be recovered from the leaves as soluble dispersions by simply combusting the organic leaf matter. Taken together, this is a simple yet powerful biotemplating approach for the generation of plasmonic nanoparticles and formation of biotic-abiotic structures for diverse, low-cost applications in sensing, catalysis, and medicine.

Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (C(n)ELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of C(n)ELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and C(n)ELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80-150 nm, while TEM imaging indicated the formation of gold cores 10-20 nm in diameter. Interestingly, C2ELPs formed 1-2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and C(n)ELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.