Successful Assembly Research Articles

Genome skimming herbarium specimens for DNA barcoding and phylogenomics

BackgroundThe world’s herbaria contain millions of specimens, collected and named by thousands of researchers, over hundreds of years. However, this treasure has remained largely inaccessible to genetic studies, because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today’s next-generation sequencing world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates.ResultsAs a practical test of routine recovery of rDNA and plastid genome sequences from herbarium specimens, we sequenced 25 herbarium specimens up to 80 years old from 16 different Angiosperm families. Paired-end reads were generated, yielding successful plastid genome assemblies for 23 species and nuclear rDNAs for 24 species, respectively. These data showed that genome skimming can be used to generate genomic information from herbarium specimens as old as 80 years and using as little as 500 pg of degraded starting DNA.ConclusionsThe routine plastome sequencing from herbarium specimens is feasible and cost-effective (compare with Sanger sequencing or plastome-enrichment approaches), and can be performed with limited sample destruction.

Land Assembly Without Eminent Domain: Laboratory Experiments of Two Tax Mechanisms

We use laboratory experiments to test the ability of two tax mechanisms to discourage seller holdout and facilitate land assembly. In one mechanism (“revealed assessment”), if a seller rejects a developer’s offer, then the property value is reassessed to be equal to the rejected offer and the taxes increase. In the second mechanism (“declared assessment”), a seller must declare a price at which he or she is willing to sell the property. The incentive to overstate the value is mitigated by using the declared price to assess a property tax. The incentive to understate the value is mitigated by allowing developers to buy the property at the declared price. We find that revealed assessment discourages the magnitude of seller holdout but not its frequency. Declared assessment has no effect on the magnitude of seller holdout but increases its frequency. Nevertheless, both tax mechanisms increase the rate of successful land assembly by more than 50 percent. Revealed assessment increases the gains from trade by 22.1 percent relative to the control treatment, but the difference is not statistically significant. Declared assessment increases the gains from trade by 124 percent, and the difference is highly statistically significant.

Fully Stretchable and Humidity-Resistant Quantum Dot Gas Sensors.

Stretchable gas sensors that accommodate the shape and motion characteristics of human body are indispensable to a wearable or attachable smart sensing system. However, these gas sensors usually have poor response and recovery kinetics when operated at room temperature, and especially suffer from humidity interference and mechanical robustness issues. Here, we demonstrate the first fully stretchable gas sensors which are operated at room temperature with enhanced stability against humidity. We created a crumpled quantum dot (QD) sensing layer on elastomeric substrate with flexible graphene as electrodes. Through the control over the prestrain of the flexible substrate, we achieved a 5.8 times improvement in NO2 response at room temperature with desirable stretchability even under 1000 stretch/relax cycles mechanism deformation. The uniformly wavy structural configuration of the crumpled QD gas-sensing layer enabled an improvement in the antihumidity interference. The sensor response shows a minor vibration of 15.9% at room temperature from relative humidity of 0 to 86.7% compared to that of the flat-film sensors with vibration of 84.2%. The successful assembly of QD solids into a crumpled gas-sensing layer enabled a body-attachable, mechanically robust, and humidity-resistant gas sensor, opening up a new pathway to room-temperature operable gas sensors which may be implemented in future smart sensing systems such as stretchable electronic nose and multipurpose electronic skin.

Assembly of Multifunctionalized Gold Nanoparticles with Chemiluminescent, Catalytic, and Immune Activity for Label-Free Immunoassays.

In this study, we report a universal label-free immunoassay to detect antigen based on multifunctionalized gold nanoparticles (MF-GNPs), which were obtained by successive assembly of N-aminobutyl- N-ethylisoluminol functionalized gold nanoparticles (ABEI-GNPs) with antibody, bovine serum albumin (BSA) and Co2+. MF-GNPs exhibited excellent chemiluminescent (CL), catalytic and immune activity. It was demonstrated that the CL signal of MF-GNPs decreased in the presence of antigens via antigen-antibody specific binding using human immunoglobulin G (hIgG) and corresponding antibody goat anti-human IgG (anti-hIgG) as a model system, due to that immunoreaction led to the aggregation of GNPs. According to the decreased CL intensity, hIgG could be determined in the range of 1.0 fM to 1.0 nM with a low detection limit of 0.13 fM. Furthermore, this CL strategy was also confirmed to be a general one by replacing hIgG with heart-type fatty acid-binding protein (H-FABP), which is a biomarker of early acute myocardial infarction (AMI). The CL strategy could be employed to detect H-FABP ranging from 10.0 fM to 10.0 nM, and the detection limit is 7.8 fM. The CL strategy also showed good selectivity. It might be extended to detect other antigens if their corresponding antibodies are available.

Modeling the Effect of the Relative Humidity on the Manipulation of Nanoparticles with an Atomic Force Microscope

In the manipulation of nanoparticles, different behaviors are typically observed including sliding, rolling and rotation. Most of investigations in this field have so far focused on describing the interaction forces under vacuum (dry air) environmental condition, while the effect of the relative humidity has been poorly considered. In this work we developed a model for simulating the dynamic nanoparticle motion (rolling and sliding) in an AFM-based manipulation of nanoparticles in a humid environment. In our method, the interaction forces include the adhesion force, mainly consisting of the capillary force and van der Waals force, the normal force and friction forces. We calculated the adhesion force by considering the contributions from the wet and dry portions of the particle. Our stimulations show that nanoparticles smaller than the AFM tip tend to slide before rolling, while in large nanoparticles the rolling occurs first. The particle motion is achieved if the applied force exceeds a critical value and the direction of the rolling movement depends on the applied force angle. Furthermore, small nanoparticles are more easily manipulated by the tip in low-humidity conditions while the manipulations with large nanoparticles need high-humidity conditions. Preliminary results can be used to adjust proper handling force for the accurate and successful assembly of particles.

The Membrane-Bound C Subunit of Reductive Dehalogenases: Topology Analysis and Reconstitution of the FMN-Binding Domain of PceC.

Organohalide respiration (OHR) is the energy metabolism of anaerobic bacteria able to use halogenated organic compounds as terminal electron acceptors. While the terminal enzymes in OHR, so-called reductive dehalogenases, are well-characterized, the identity of proteins potentially involved in electron transfer to the terminal enzymes remains elusive. Among the accessory genes identified in OHR gene clusters, the C subunit (rdhC) could well code for the missing redox protein between the quinol pool and the reductive dehalogenase, although it was initially proposed to act as transcriptional regulator. RdhC sequences are characterized by the presence of multiple transmembrane segments, a flavin mononucleotide (FMN) binding motif and two conserved CX3CP motifs. Based on these features, we propose a curated selection of RdhC proteins identified in general sequence databases. Beside the Firmicutes from which RdhC sequences were initially identified, the identified sequences belong to three additional phyla, the Chloroflexi, the Proteobacteria, and the Bacteriodetes. The diversity of RdhC sequences mostly respects the phylogenetic distribution, suggesting that rdhC genes emerged relatively early in the evolution of the OHR metabolism. PceC, the C subunit of the tetrachloroethene (PCE) reductive dehalogenase is encoded by the conserved pceABCT gene cluster identified in Dehalobacter restrictus PER-K23 and in several strains of Desulfitobacterium hafniense. Surfaceome analysis of D. restrictus cells confirmed the predicted topology of the FMN-binding domain (FBD) of PceC that is the exocytoplasmic face of the membrane. Starting from inclusion bodies of a recombinant FBD protein, strategies for successful assembly of the FMN cofactor and refolding were achieved with the use of the flavin-trafficking protein from D. hafniense TCE1. Mass spectrometry analysis and site-directed mutagenesis of rFBD revealed that threonine-168 of PceC is binding FMN covalently. Our results suggest that PceC, and more generally RdhC proteins, may play a role in electron transfer in the metabolism of OHR.

Application of Heparin/Collagen-REDV Selective Active Interface on ePTFE Films to Enhance Endothelialization and Anticoagulation.

Expanded polytetrafluoroethylene (ePTFE) prosthetic valves have been widely used in clinical applications in Asian countries. However, these valves still have limits with regard to thrombosis, neointimal hyperplasia, restenosis, and valvar vegetation. The achievement of in situ endothelialization on implant materials is a promising way to overcome those limits. Here, heparin/collagen multilayers were fabricated on ePTFE films via a layer-by-layer (LBL) self-assembly technique, and then, the endothelial cell (EC) adhesive peptide sequence Arg-Glu-Asp-Val (REDV) was immobilized on the multilayers. After modification with the heparin/collagen multilayers with or without REDV peptide, less platelet activation and aggregation were observed, the blood coagulation time was increased, and the hemolysis rate was decreased comparedto that on pristine ePTFE films. The REDV-functionalized ePTFE films positively impacted early EC adhesion, later cell proliferation and cell activity. The EC barrier was confirmed to be successfully achieved on the functionalized ePTFE film surface in vitro. The successful assembly of the REDV-functionalized heparin/collagen multilayer on ePTFE films improved the blood compatibility, anticoagulant properties, and cell compatibility of the films in vitro, and thus, represents a candidate approach for applications requiring quick in situ endothelialization in vivo.

(Invited) Multifunctional Nano-Array Integrated Monolithic Devices: Toward Rational Engineered Nanomaterials Design and Scalable Nanomanufacturing

The scalable three-dimensional (3-D) integration of functional nanostructures into applicable platforms represents a promising technology to meet the ever-increasing demands of fabricating high performance devices featuring cost-effectiveness, structural sophistication and multi-functional enabling. Such an integration process generally involves a diverse array of nanostructural entities (nano-entities) consisting of dissimilar nanoscale building blocks such as nanoparticles, nanowires, and nanofilms made of metals, ceramics, or polymers. Various synthetic strategies and integration methods have enabled the successful assembly of both structurally and functionally tailored nano-arrays into a unique class of monolithic devices. The performance of nano-array based monolithic devices is dictated by a few important factors such as materials substrate selection, nanostructure composition and nano-architecture geometry. Therefore, the rational material selection and nano-entity manipulation during the nano-array integration process, aiming to exploit the advantageous characteristics of nanostructures and their ensembles, are critical steps towards bridging the design of nanostructure integrated monolithic devices with various practical applications. In this talk, we highlight the latest research progress of the two-dimensional (2-D) and 3-D metal and metal oxide based nanostructural integrations into prototype devices and systems applicable with ultrahigh efficiency, good robustness and improved functionality. Selective examples of nano-array integration, scalable nanomanufacturing and representative monolithic devices will be highlighted, including automotive catalytic converters, thermo-catalytic and photo-activated reactors for fuel/chemical production, and sensors, with practical nanotechnology implications ranging from energy, environmental, to chemical and biotechnology areas.

Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface of Lactobacillus plantarum, a Potent Member of the Gut Microbiome.

Heterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the "adaptor scaffoldin" strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia of Lactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all produced in vivo by the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.IMPORTANCE The multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering of Lactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System.

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

Smart Enough to Talk With Us? Foundations and Challenges for Dialogue Capable AI Systems

Smart Enough to Talk With Us? Foundations and Challenges for Dialogue Capable AI Systems

The influence of ligand charge and length on the assembly of Brome mosaic virus derived virus-like particles with magnetic core

Virus-like particles (VLPs) have sparked a great interest in the field of nanobiotechnology and nanomedicine. The introduction of superparamagnetic nanoparticles (SPIONs) as a core, provides potential use of VLPs in the hyperthermia therapy, MRI contrast agents and magnetically-powered delivery agents. Magnetite NPs also provide a significant improvement in terms of VLPs stability. Moreover employing viral structural proteins as self-assembling units has opened a new paths for targeted therapy, drug delivery systems, vaccines design, and many more. In many cases, the self-assembly of a virus strongly depends on electrostatic interactions between positively charged groups of the capsid proteins and negatively charged nucleic acid. This phenomenon imposes the negative net charge as a key requirement for the core nanoparticle. In our experiments, Brome mosaic virus (BMV) capsid proteins isolated from infected plants Hordeum vulgare were used. Superparamagnetic iron oxide nanoparticles (Fe3O4) with 15 nm in diameter were synthesized by thermal decomposition and functionalized with COOH-PEG-PL polymer or dihexadecylphosphate (DHP) in order to provide water solubility and negative charge required for the assembly. Nanoparticles were characterized by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Zeta Potential, Fourier Transformed Infrared Spectroscopy (FTIR) and Superconducting Quantum Interference Device (SQUID) magnetometry. TEM and DLS study were conducted to verify VLPs creation. This study demonstrates that the increase of negative surface charge is not a sufficient factor determining successful assembly. Additional steric interactions provided by longer ligands are crucial for the assembly of BMV SPION VLPs and may enhance the colloidal stability.

Multi-facet implications of PEGylated lysozyme stabilized-silver nanoclusters loaded recombinant PTEN cargo in cancer theranostics.

Amalgamation of delivery and tracking of therapeutically relevant moieties on a single platform is made possible by the application of metal nanoclusters, an innovative class of luminescent nanomaterials. Metal nanoclusters, possessing molecule-like attributes, display extraordinary size and shape tunable properties befitting theranostic applications. Herein, we report successful assembly of therapeutically significant phosphatase protein PTEN and fluorescent lysozyme-stabilized silver nanoclusters to accomplish delivery and tracking of the protein. Down-regulation of PTEN perturbs the cellular networking leading to copious pathological conditions. The integration of purified recombinant PTEN with silver nanoclusters was evaluated by fluorescence spectroscopy study. A key feature of this study is the use of polyethylene glycol coating that allows fabrication of the assembly into spherical nanocomposites as characterized by transmission electron microscope along with retention of both optical functionality of the cluster and biological activity of the protein. Prior to cellular application, the functional integrity of PTEN in the composite was determined in vitro, by enzymatic assay employing para-nitrophenylphosphate as substrate. Cellular internalization of the cargo was studied by confocal microscopy and flow cytometry analysis. The efficacy of the payload on modulation of cellular signaling was assessed on cell lines that expressed PTEN differentially. PTEN null U-87 MG and PTEN expressing MCF7 cell lines displayed successful alteration of AKT and FAK signaling proteins culminating in cell cycle arrest and reduced wound healing capacity. A dose dependent reduction in cell proliferation of MCF7 cells was achieved. For U-87 MG, treatment with the payload resulted in chemosensitization toward anti-cancer drug erlotinib. Thus, PEG coated GST-PTEN loaded silver nanoclusters serves as a comprehensive system encompassing cellular imaging and protein delivery with potential biomedical implications.

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.

Red algal Rubisco fails to accumulate in transplastomic tobacco expressing Griffithsiamonilis RbcL and RbcS genes.

In C3 plants, the carbon fixation step catalyzed by ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) represents a major rate‐limiting step due to the competing oxygenation reaction, which leads to the energy‐intensive photorespiration and lowers the overall photosynthetic efficiency. Hence, there is great biotechnological interest in replacing the Rubisco in C3 crops with a more efficient enzyme. The Rubisco enzymes from red algae are among the most attractive choices due to their remarkable preference for carboxylation over oxygenation reaction. However, the biogenesis of Rubisco is extremely complex. The Rubisco enzymes in plants, algae, and cyanobacteria are made up of eight large and eight small subunits. The folding of the large subunits and the assembly of the large subunits with the small subunits to form a functional holoenzyme require specific chaperonin complexes and assembly factors. As a result, previous success in expressing foreign Rubisco in plants has been limited to Rubisco large subunits from closely related plant species and simpler bacterial enzymes. In our previous work, we successfully replaced the Rubisco in tobacco with a cyanobacterial enzyme, which was able to support the phototrophic growth of the transgenic plants. In this work, we used the same approach to express the Rubisco subunits from the red alga Griffithsia monilis in tobacco chloroplasts in the absence of the tobacco Rubisco large subunit. Although the red algal Rubisco genes are being transcribed in tobacco chloroplasts, the transgenic plants lack functional Rubisco and can only grow in a medium containing sucrose. Our results suggest that co‐expression of compatible chaperones will be necessary for successful assembly of red algal Rubisco in plants.

Orthogonal supramolecular protein assembly on patterned bifunctional surfaces.

We report successful and selective dual protein assembly on patterned bifunctional βCD-Ni(ii)NTA surfaces, using red fluorescent protein variants with hexahistidine-tags and teal fluorescent protein variants conjugated with a peptide containing three adamantyl groups. We show that dual protein patterns can only be assembled, when opposing supramolecular interactions have been optimized and nonspecific interactions have been sufficiently suppressed.

Intracellularly Actuated Quantum Dot-Peptide-Doxorubicin Nanobioconjugates for Controlled Drug Delivery via the Endocytic Pathway.

Nanoparticle (NP)-mediated drug delivery (NMDD) has emerged as a novel method to overcome the limitations of traditional systemic delivery of therapeutics, including the controlled release of the NP-associated drug cargo. Currently, our most advanced understanding of how to control NP-associated cargos is in the context of soft nanoparticles (e.g., liposomes), but less is known about controlling the release of cargos from the surface of hard NPs (e.g., gold NPs). Here we employ a semiconductor quantum dot (QD) as a prototypical hard NP platform and use intracellularly triggered actuation to achieve spatiotemporal control of drug release and modulation of drug efficacy. Conjugated to the QD are two peptides: (1) a cell-penetrating peptide (CPP) that facilitates uptake of the conjugate into the endocytic pathway and (2) a display peptide conjugated to doxorubicin (DOX) via three different linkages (ester, disulfide, and hydrazone) that are responsive to enzymatic cleavage, reducing conditions, and low pH, respectively. Formation of the QD-[peptide-DOX]-CPP complex is driven by self-assembly that allows control over both the ratio of each peptide species conjugated to the QD and the eventual drug dose delivered to cells. Förster resonance energy transfer assays confirmed successful assembly of the QD-peptide complexes and functionality of the linkages. Confocal microscopy was employed to visualize residence of the QD-[peptide-DOX]-CPP complexes in the endocytic pathway, and distinct differences in DOX localization were noted for the ester linkage, which showed clear signs of nuclear delivery versus the hydrazone, disulfide, and amide control. Finally, delivery of the QD-[peptide-DOX]-CPP conjugate resulted in cytotoxicity for the ester linkage that was comparable to free DOX. Attachment of DOX via the hydrazone linkage facilitated intermediary toxicity, while the disulfide and amide control linkages showed minimal toxicity. Our data demonstrate the utility of hard NP-peptide bioconjugates to function as multifunctional scaffolds for simultaneous control over cellular drug uptake and toxicity and the vital role played by the nature of the chemical linkage that appends the drug to the NP carrier.

An additive manufacturing oriented design approach to mechanical assemblies

Abstract Firstly introduced as a prototyping process, additive manufacturing (AM) is being more and more considered as a fully-edged manufacturing process. The number of AM processes, along with the range of processed materials are expanding. AM has made manufacturable shapes that were too difficult (or even impossible) to manufacture with conventional technologies. This has promoted a shift in engineering design, from conventional design for manufacturing and assembly to design for additive manufacturing (DFAM). Research efforts into the DFAM field have been mostly dedicated to part's design, which is actually a requirement for a better industrial adoption. This has given rise to topologically optimized and/or latticed designs. However, since AM is also capable of manufacturing fully functional assemblies requiring a few or no assembly operations, there is a need for DFAM methodologies tackling product's development more holistically, and which are, therefore, dedicated to assembly design. Considering all the manufacturing issues related to AM of assembly-free mechanisms and available post-processing capabilities, this paper proposes a top-down assembly design methodology for AM in a proactive manner. Such an approach, can be seen as the beginning of a shift from conventional design for assembly (DFA) to a new paradigm. From a product's concept and a selected AM technology, the approach first provides assistance in the definition of the product architecture so that both functionality and successful manufacturing (including post-processing) are ensured. Particularly, build-orientation and downstream processes' characteristics are taken into account early in the design process. Secondly, for the functional flow (energy, material, signal) to be appropriately conveyed by the right amount of matter, the methodology provides guidance into how the components can be designed in a minimalism fashion leveraging the shape complexity afforded by AM. A mechanical assembly as case study is presented to illustrate the DFAM methodology. It is found that clearances and material (be it raw unprocessed material or support structures) within them plays a pivotal role in a successful assembly's design to be additively manufactured. In addition, the methodology for components' design proves to be an efficient alternative to topology optimization. Though, the approach can be extended by considering a strategy for part consolidation and the possibility to manufacture the assemblies with more than one AM process. As regards components' design, considering anisotropy can also improved the approach. Highlights Additive manufacturing is capable of printing fully functional assemblies without any assembly operations. It is found that Design For Additive Manufacturing is currently mainly focused on part's design. A process-independent, structured and systematic method for designing assembly-free mechanisms (for AM) is proposed. Build orientation and downstream processes (including post-processing capabilities) are taken into account early in the design process. A method - based on functional flows - for part's design in a minimalist fashion, is proposed.

How Surface Roughness Performance of Printed Parts Manufactured by Desktop FDM 3D Printer with PLA+ is Influenced by Measuring Direction

Design work related to the implementation of new elements requires the use of 3D CAD modelling techniques and rapid prototyping, which makes it possible to accelerate the deployment of new solutions significantly. In this paper, six successful assembly parts are 3D printed with advanced polylactic acid (PLA+) using the fused deposition modeling (FDM) method and are expressed by the arithmetic mean surface roughness, Ra. The surface roughness was measured in three different angular directions 0°, 45° and 90° during the investigation along with various independent process parameters of nozzle diameter (0.5, 0.3, 0.2 mm), layer height (0.3, 0.2, 0.1 mm) and other dependent variables, i.e., nozzle temperature (220°C), print speed (30 mm/s) and infill density (0%). Experimental results show that nozzle diameter and layer height play a major role in terms of part quality finish, build time and ultimately part cost. Nozzle diameter and a layer height of 0.3 mm and 0.1 mm, respectively, represent the optimal manufacturing process parameters that can be selected. The surface roughness behaviour does not change and remains relatively constant and follows a similar trend with minor variations for both 45° and 90° measuring angle. Whereas, the surface roughness values are susceptible to 0° measuring direction to the build orientation as compared to other angles.

Study on Low Warpage and High Reliability for Large Package Using TSV-Free Interposer Technology Through SMART Codesign Modeling

Through-silicon via (TSV)-free interposer (TFI) technology eliminates TSV fabrication and reduces manufacturing and material cost. In this paper, structure–material–assembly–reliability–thermal (SMART) codesign modeling methodology is established for a package using TFI technology by considering wafer process, package assembly and package/board-level temperature cycling reliability, and thermal performance to optimize structure design, assembly process, and material selection. Experimental results are used to validate wafer warpage modeling results first. Through wafer-level modeling, suitable carrier and molding compound materials are recommended to control wafer warpage less than 2 mm for 12-in molded wafer. Effects of coefficient of thermal expansion of package substrate and stiffener on package warpage induced by assembly reflow process are simulated and analyzed. The recommended materials and geometry design based on thermal cycling reliability simulation are aligned with that from wafer and package warpage simulation results. The final test vehicle design and material selection are determined based on SMART codesign modeling results for achieving successful TFI wafer process and package assembly and long-term board-level reliability.