Direct Bottom-Up In Situ Growth: A Paradigm Shift for Studies in Wet-Chemical Synthesis of Gold Nanoparticles.

Plasmonic gold nanoparticles have been used increasingly in solid-state systems because of their applicability in fabricating novel sensors, heterogeneous catalysts, metamaterials, and thermoplasmonic substrates. While bottom-up colloidal syntheses take advantage of the chemical environment to control size, shape, composition, surface chemistry, and crystallography of the nanostructures precisely, it can be challenging to assemble nanoparticles rationally from suspension onto solid supports or within devices. In this Review, we discuss a powerful recent synthetic methodology, bottom-up in situ substrate growth, which circumvents time-consuming batch presynthesis, ligand exchange, and self-assembly steps by applying wet-chemical synthesis to form morphologically controlled nanostructures on supporting materials. First, we briefly introduce the properties of plasmonic nanostructures. Then we comprehensively summarize recent work that adds to the synthetic understanding of in situ geometrical and spatial control (patterning). Next, we briefly discuss applications of plasmonic hybrid materials prepared by in situ growth. Overall, despite the vast potential advantages of in situ growth, the mechanistic understanding of these methodologies remains far from established, providing opportunities and challenges for future research.

Engineered Hemostatic Biomaterials for Sealing Wounds.

Hemostatic biomaterials show great promise in wound control for the treatment of uncontrolled bleeding associated with damaged tissues, traumatic wounds, and surgical incisions. A surge of interest has been directed at boosting hemostatic properties of bioactive materials via mechanisms triggering the coagulation cascade. A wide variety of biocompatible and biodegradable materials has been applied to the design of hemostatic platforms for rapid blood coagulation. Recent trends in the design of hemostatic agents emphasize chemical conjugation of charged moieties to biomacromolecules, physical incorporation of blood-coagulating agents in biomaterials systems, and superabsorbing materials in either dry (foams) or wet (hydrogel) states. In addition, tough bioadhesives are emerging for efficient and physical sealing of incisions. In this Review, we highlight the biomacromolecular design approaches adopted to develop hemostatic bioactive materials. We discuss the mechanistic pathways of hemostasis along with the current standard experimental procedures for characterization of the hemostasis efficacy. Finally, we discuss the potential for clinical translation of hemostatic technologies, future trends, and research opportunities for the development of next-generation surgical materials with hemostatic properties for wound management.

Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells.

Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.

Bio-macromolecular design roadmap towards tough bioadhesives.

Emerging sutureless wound-closure techniques have led to paradigm shifts in wound management. State-of-the-art biomaterials offer biocompatible and biodegradable platforms enabling high cohesion (toughness) and adhesion for rapid bleeding control as well as robust attachment of implantable devices. Tough bioadhesion stems from the synergistic contributions of cohesive and adhesive interactions. This Review provides a biomacromolecular design roadmap for the development of tough adhesive surgical sealants. We discuss a library of materials and methods to introduce toughness and adhesion to biomaterials. Intrinsically tough and elastic polymers are leveraged primarily by introducing strong but dynamic inter- and intramolecular interactions either through polymer chain design or using crosslink regulating additives. In addition, many efforts have been made to promote underwater adhesion via covalent/noncovalent bonds, or through micro/macro-interlock mechanisms at the tissue interfaces. The materials settings and functional additives for this purpose and the related characterization methods are reviewed. Measurements and reporting needs for fair comparisons of different materials and their properties are discussed. Finally, future directions and further research opportunities for developing tough bioadhesive surgical sealants are highlighted.

An absence of lamin B1 in migrating neurons causes nuclear membrane ruptures and cell death.

Deficiencies in either lamin B1 or lamin B2 cause both defective migration of cortical neurons in the developing brain and reduced neuronal survival. The neuronal migration abnormality is explained by a weakened nuclear lamina that interferes with nucleokinesis, a nuclear translocation process required for neuronal migration. In contrast, the explanation for impaired neuronal survival is poorly understood. We hypothesized that the forces imparted on the nucleus during neuronal migration result in nuclear membrane (NM) ruptures, causing interspersion of nuclear and cytoplasmic contents-and ultimately cell death. To test this hypothesis, we bred Lmnb1-deficient mice that express a nuclear-localized fluorescent Cre reporter. Migrating neurons within the cortical plate of E18.5 Lmnb1-deficient embryos exhibited NM ruptures, evident by the escape of the nuclear-localized reporter into the cytoplasm and NM discontinuities by electron microscopy. The NM ruptures were accompanied by DNA damage and cell death. The NM ruptures were not observed in nonmigrating cells within the ventricular zone. NM ruptures, DNA damage, and cell death were also observed in cultured Lmnb1-/- and Lmnb2-/- neurons as they migrated away from neurospheres. To test whether mechanical forces on the cell nucleus are relevant to NM ruptures in migrating neurons, we examined cultured Lmnb1-/- neurons when exposed to external constrictive forces (migration into a field of tightly spaced silicon pillars). As the cells entered the field of pillars, there were frequent NM ruptures, accompanied by DNA damage and cell death.

Supramolecular Nanosubstrate-Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) is an efficient and precise gene-editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9•sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate-mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular-uptake of the RNP-laden nanoparticles are examined. Dose- and time-dependent CRISPR/Cas9-mediated gene disruption is further examined in a green fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). The utility of an optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells.

Detecting DNA and RNA and Differentiating Single-Nucleotide Variations via Field-Effect Transistors.

We detect short oligonucleotides and distinguish between sequences that differ by a single base, using label-free, electronic field-effect transistors (FETs). Our sensing platform utilizes ultrathin-film indium oxide FETs chemically functionalized with single-stranded DNA (ssDNA). The ssDNA-functionalized semiconducting channels in FETs detect fully complementary DNA sequences and differentiate these sequences from those having different types and locations of single base-pair mismatches. Changes in charge associated with surface-bound ssDNA vs double-stranded DNA (dsDNA) alter FET channel conductance to enable detection due to differences in DNA duplex stability. We illustrate the capability of ssDNA-FETs to detect complementary RNA sequences and to distinguish from RNA sequences with single nucleotide variations. The development and implementation of electronic biosensors that rapidly and sensitively detect and differentiate oligonucleotides present new opportunities in the fields of disease diagnostics and precision medicine.

Differential Charging in Photoemission from Mercurated DNA Monolayers on Ferromagnetic Films.

Spin-dependent and enantioselective electron-molecule scattering occurs in photoelectron transmission through chiral molecular films. This spin selectivity leads to electron spin filtering by molecular helices, with increasing magnitude concomitant with increasing numbers of helical turns. Using ultraviolet photoelectron spectroscopy, we measured spin-selective surface charging accompanying photoemission from ferromagnetic substrates functionalized with monolayers of mercurated DNA hairpins that constitute only one helical turn. Mercury ions bind specifically at thymine-thymine mismatches within self-hybridized single-stranded DNA, enabling precise control over the number and position of Hg2+ along the helical axis. Differential charging of the organic layers, manifested as substrate-magnetization-dependent photoionization energies, was observed for DNA hairpins containing Hg2+; no differences were measured for hairpin monolayers in the absence of Hg2+. Inversion of the DNA helical secondary structure at increased metal loading led to complementary inversion in spin selectivity. We attribute these results to increased scattering probabilities from relativistic enhancement of spin-orbit interactions in mercurated DNA.

Scalable Fabrication of Quasi-One-Dimensional Gold Nanoribbons for Plasmonic Sensing.

Plasmonic nanostructures have a wide range of applications, including chemical and biological sensing. However, the development of techniques to fabricate submicrometer-sized plasmonic structures over large scales remains challenging. We demonstrate a high-throughput, cost-effective approach to fabricate Au nanoribbons via chemical lift-off lithography (CLL). Commercial HD-DVDs were used as large-area templates for CLL. Transparent glass slides were coated with Au/Ti films and functionalized with self-assembled alkanethiolate monolayers. Monolayers were patterned with lines via CLL. The lifted-off, exposed regions of underlying Au were selectively etched into large-area grating-like patterns (200 nm line width; 400 nm pitch; 60 nm height). After removal of the remaining monolayers, a thin In2O3 layer was deposited and the resulting gratings were used as plasmonic sensors. Distinct features in the extinction spectra varied in their responses to refractive index changes in the solution environment with a maximum bulk sensitivity of ∼510 nm/refractive index unit. Sensitivity to local refractive index changes in the near-field was also achieved, as evidenced by real-time tracking of lipid vesicle or protein adsorption. These findings show how CLL provides a simple and economical means to pattern large-area plasmonic nanostructures for applications in optoelectronics and sensing.

Seeded-Growth Experiment Demonstrating Size- and Shape-Dependence on Gold Nanoparticle-Light Interactions.

Gold nanoparticles are exciting materials in nanotechnology and nanoscience research and are being applied across a wide range of fields including imaging, chemical sensing, energy storage, and cancer therapies. In this experiment, students will synthesize two sizes of gold nanospheres (~20 nm and ~100 nm) and will create gold nanostars utilizing a seed-mediated growth synthetic approach. Students will compare how each sample interacts differently with light (absorption and scattering) based on the nanoparticles' size and shape. This experiment is ideal for high-school and early undergraduate students since all reagents are non-toxic, affordable, and no special characterization equipment is required.

Coupling Nanostructured Microchips with Covalent Chemistry Enables Purification of Sarcoma-Derived Extracellular Vesicles for Downstream Functional Studies.

Tumor-derived extracellular vesicles (EVs) play essential roles in intercellular communication during tumor growth and metastatic evolution. Currently, little is known about the possible roles of tumor-derived EVs in sarcoma because the lack of specific surface markers makes it technically challenging to purify sarcoma-derived EVs. In this study, a specific purification system is developed for Ewing sarcoma (ES)-derived EVs by coupling covalent chemistry-mediated EV capture/ release within a nanostructure-embedded microchip. The purification platform-ES-EV Click Chip-takes advantage of specific anti-LINGO-1 recognition and sensitive click chemistry-mediated EV capture, followed by disulfide cleavage-driven EV release. Since the device is capable of specific and efficient purification of intact ES EVs with high purity, ES-EV Click Chip is ideal for conducting downstream functional studies of ES EVs. Absolute quantification of the molecular hallmark of ES (i.e., EWS rearrangements) using reverse transcription Droplet Digital PCR enables specific quantification of ES EVs. The purified ES EVs can be internalized by recipient cells and transfer their mRNA cargoes, exhibiting their biological intactness and potential role as biological shuttles in intercellular communication.

Spin-Dependent Ionization of Chiral Molecular Films.

Spin selectivity in photo-emission from ferromagnetic substrates functionalized with chiral organic films was analyzed by ultraviolet photoelectron spectroscopy at room temperature. Using radiation with photon energy greater than the ionization potential of the adsorbed molecules, photoelectrons were collected that originated from both underlying ferromagnetic substrates and the organic films, with kinetic energies in the range of ca. 0-18 eV. We investigated chiral organic films composed of self-assembled monolayers of α-helical peptides and electrostatically adsorbed films of the protein, bovine serum albumin, with different α-helix and ß-sheet contents. Ultraviolet photoelectron spectral widths were found to depend on substrate magnetization orientation and polarization, which we attribute to helicity-dependent molecular ionization cross sections arising from photoelectron impact, possibly resulting in spin-polarized holes. These interactions between spin-polarized photoelectrons and chiral molecules are physically manifested as differences in the measured photoionization energies of the chiral molecular films. Substrate magnetization-dependent ionization energies and work function values were deconvoluted using surface charge neutralization techniques, permitting the measurement of relative spin-dependent energy barriers to transmission through chiral organic films.

Large-Area, Ultrathin Metal-Oxide Semiconductor Nanoribbon Arrays Fabricated by Chemical Lift-Off Lithography.

Nanoribbon- and nanowire-based field-effect transistors (FETs) have attracted significant attention due to their high surface-to-volume ratios, which make them effective as chemical and biological sensors. However, the conventional nanofabrication of these devices is challenging and costly, posing a major barrier to widespread use. We report a high-throughput approach for producing arrays of ultrathin (∼3 nm) In2O3 nanoribbon FETs at the wafer scale. Uniform films of semiconducting In2O3 were prepared on Si/SiO2 surfaces via a sol-gel process prior to depositing Au/Ti metal layers. Commercially available high-definition digital versatile discs were employed as low-cost, large-area templates to prepare polymeric stamps for chemical lift-off lithography, which selectively removed molecules from self-assembled monolayers functionalizing the outermost Au surfaces. Nanoscale chemical patterns, consisting of one-dimensional lines (200 nm wide and 400 nm pitch) extending over centimeter length scales, were etched into the metal layers using the remaining monolayer regions as resists. Subsequent etch processes transferred the patterns into the underlying In2O3 films before the removal of the protective organic and metal coatings, revealing large-area nanoribbon arrays. We employed nanoribbons in semiconducting FET channels, achieving current on-to-off ratios over 107 and carrier mobilities up to 13.7 cm2 V-1 s-1. Nanofabricated structures, such as In2O3 nanoribbons and others, will be useful in nanoelectronics and biosensors. The technique demonstrated here will enable these applications and expand low-cost, large-area patterning strategies to enable a variety of materials and design geometries in nanoelectronics.

Self-Collapse Lithography.

We report a facile, high-throughput soft lithography process that utilizes nanoscale channels formed naturally at the edges of microscale relief features on soft, elastomeric stamps. Upon contact with self-assembled monolayer (SAM) functionalized substrates, the roof of the stamp collapses, resulting in the selective removal of SAM molecules via a chemical lift-off process. With this technique, which we call self-collapse lithography (SCL), sub-30 nm patterns were achieved readily using masters with microscale features prepared by conventional photolithography. The feature sizes of the chemical patterns can be varied continuously from ∼2 µm to below 30 nm by decreasing stamp relief heights from 1 µm to 50 nm. Likewise, for fixed relief heights, reducing the stamp Young's modulus from ∼2.0 to ∼0.8 MPa resulted in shrinking the features of resulting patterns from ∼400 to ∼100 nm. The self-collapse mechanism was studied using finite element simulation methods to model the competition between adhesion and restoring stresses during patterning. These results correlate well with the experimental data and reveal the relationship between the line widths, channel heights, and Young's moduli of the stamps. In addition, SCL was applied to pattern two-dimensional arrays of circles and squares. These chemical patterns served as resists during etching processes to transfer patterns to the underlying materials (e.g., gold nanostructures). This work provides new insights into the natural propensity of elastomeric stamps to self-collapse and demonstrates a means of exploiting this behavior to achieve patterning via nanoscale chemical lift-off lithography.

Polymer-Pen Chemical Lift-Off Lithography.

We designed and fabricated large arrays of polymer pens having sub-20 nm tips to perform chemical lift-off lithography (CLL). As such, we developed a hybrid patterning strategy called polymer-pen chemical lift-off lithography (PPCLL). We demonstrated PPCLL patterning using pyramidal and v-shaped polymer-pen arrays. Associated simulations revealed a nanometer-scale quadratic relationship between contact line widths of the polymer pens and two other variables: polymer-pen base line widths and vertical compression distances. We devised a stamp support system consisting of interspersed arrays of flat-tipped polymer pens that are taller than all other sharp-tipped polymer pens. These supports partially or fully offset stamp weights thereby also serving as a leveling system. We investigated a series of v-shaped polymer pens with known height differences to control relative vertical positions of each polymer pen precisely at the sub-20 nm scale mimicking a high-precision scanning stage. In doing so, we obtained linear-array patterns of alkanethiols with sub-50 nm to sub-500 nm line widths and minimum sub-20 nm line width tunable increments. The CLL pattern line widths were in agreement with those predicted by simulations. Our results suggest that through informed design of a stamp support system and tuning of polymer-pen base widths, throughput can be increased by eliminating the need for a scanning stage system in PPCLL without sacrificing precision. To demonstrate functional microarrays patterned by PPCLL, we inserted probe DNA into PPCLL patterns and observed hybridization by complementary target sequences.

Double-Sided Opportunities Using Chemical Lift-Off Lithography.

We discuss the origins, motivation, invention, development, applications, and future of chemical lift-off lithography, in which a specified pattern of a self-assembled monolayer is removed, i.e., lifted off, using a reactive, patterned stamp that is brought into contact with the monolayer. For Au substrates, this process produces a supported, patterned monolayer of Au on the stamp in addition to the negative pattern in the original molecular monolayer. Both the patterned molecular monolayer on the original substrate and the patterned supported metal monolayer on the stamp are useful as materials and for further applications in sensing and other areas. Chemical lift-off lithography effectively lowers the barriers to and costs of high-resolution, large-area nanopatterning. On the patterned monolayer side, features in the single-nanometer range can be produced across large (square millimeter or larger) areas. Patterns smaller than the original stamp feature sizes can be produced by controlling the degree of contact between the stamp and the lifted-off monolayer. We note that this process is different than conventional lift-off processes in lithography in that chemical lift-off lithography removes material, whereas conventional lift-off is a positive-tone patterning method. Chemical lift-off lithography is in some ways similar to microtransfer printing. Chemical lift-off lithography has critical advantages in the preparation of biocapture surfaces because the molecules left behind are exploited to space and to orient functional(ized) molecules. On the supported metal monolayer side, a new two-dimensional material has been produced. The useful important chemical properties of Au (vis-à-vis functionalization with thiols) are retained, but the electronic and optical properties of bulk Au or even Au nanoparticles are not. These metal monolayers do not quench excitation and may be useful in optical measurements, particularly in combination with selective binding due to attached molecular recognition elements. In contrast to materials such as graphene that have bonding confined to two dimensions, these metal monolayers can be straightforwardly patterned-by patterning the stamp, the initial monolayer, or the initial substrate. Well-developed thiol-Au and related chemistries can be used on the supported monolayers. As there is little quenching and photoabsorption, spectroscopic imaging methods can be used on these functionalized materials. We anticipate that the properties of the metal monolayers can be tuned by varying the chemical, physical, and electronic connections made by and to the supporting molecular layers. That is, the amount of charge in the layer can be determined by controlling the density of S-Au (or other) connections and the molecular backbone and functionality, which determine the strength with which the chemical contact withdraws charge from the metal. This process should work for other coinage-metal substrates and additional systems where the binding of the outermost layers to the substrate is weaker than the molecule-substrate attachment.

Understanding How Sterols Regulate Membrane Remodeling in Supported Lipid Bilayers.

The addition of single-chain lipid amphiphiles such as antimicrobial fatty acids and monoglycerides to confined, two-dimensional phospholipid bilayers can trigger the formation of three-dimensional membrane morphologies as a passive means to regulate stress. To date, relevant experimental studies have been conducted using pure phospholipid compositions, and extending such insights to more complex, biologically relevant lipid compositions that include phospholipids and sterols is warranted because sterols are important biological mediators of membrane stress relaxation. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we investigated membrane remodeling behaviors triggered by the addition of sodium dodecyl sulfate (SDS), lauric acid (LA), and glycerol monolaurate (GML) to supported lipid bilayers (SLBs) composed of phospholipid and cholesterol mixtures. The SLB platforms were prepared by the solvent-assisted lipid bilayer method in order to form cholesterol-rich SLBs with tunable cholesterol fractions (0-52 mol %). The addition of SDS or LA to fabricated SLBs induced tubule formation, and the extent of membrane remodeling was greater in SLBs with higher cholesterol fractions. In marked contrast, GML addition led to bud formation, and the extent of membrane remodeling was lower in SLBs with higher cholesterol fractions. To explain these empirical observations, we discuss how cholesterol influences the elastic (stiffness) and viscous (stress relaxation) properties of phospholipid/cholesterol lipid bilayers as well as how the membrane translocation properties of single-chain lipid amphiphiles affect the corresponding membrane morphological responses. Collectively, our findings demonstrate that single-chain lipid amphiphiles induce highly specific membrane morphological responses across both simplified and complex model membranes, and cholesterol can promote or inhibit membrane remodeling by a variety of molecular mechanisms.

The Impact of Prescription Drug Monitoring Programs and Prescribing Guidelines on Emergency Department Opioid Prescribing: A Multi-Center Survey.

Objective: Emergency department (ED) providers are high volume but low quantity prescribers of opioid analgesics (OA). Few studies have examined differences in opioid prescribing decisions specifically among ED providers. The aim of this study was to describe OA prescribing decisions of ED providers at geographically diverse centers, including utilization of prescribing guidelines and prescription drug monitoring programs (PDMP). Methods: This was a multi-center cross-sectional Web-based survey of ED providers who prescribe OA. Respondents were asked about their OA prescribing decisions, their use of PDMPs, and their use of prescribing guidelines. Data was analyzed using descriptive statistics and chi-square tests of association were used to assess the relationship between providers' opioid prescribing decisions and independent covariates. Results: The total survey population was 957 individuals and 515 responded to the survey for an overall response rate of 54%. The frequency respondents prescribed different types of pain medication was variable between centers. of respondents were registered to access a PDMP, and were not aware whether their state had a PDMP. Forty percent (172/426) of respondents used OA prescribing guidelines, while 24% (103/426) did not, and 35% (151/426) were unaware of prescribing guidelines. No significant differences in OA prescribing decisions were found between groups either by use of PDMP or by guideline adherence. Conclusions: In this multi-center survey study of ED clinicians, OA prescribing varied between centers The utilization of prescribing guidelines and PDMPs was not associated with differences in OA prescribing decisions.