Material-level countermeasures for securing microfluidic biochips.

Flow-based microfluidic biochips (FMBs) have been rapidly commercialized and deployed in recent years for biological computing, clinical diagnostics, and point-of-care-tests (POCTs). However, outsourcing FMBs makes them susceptible to material-level attacks by malicious actors for illegitimate monetary gain. The attacks involve deliberate material degradation of an FMB's polydimethylsiloxane (PDMS) components by either doping with reactive solvents or altering the PDMS curing ratio during fabrication. Such attacks are stealthy enough to evade detection and deteriorate the FMB's function. Furthermore, material-level attacks can become prevalent in attacks based on intellectual property (IP) theft, such as counterfeiting, overbuilding, etc., which involve unscrupulous third-party manufacturers. To address this problem, we present a dynamic material-level watermarking scheme for PDMS-based FMBs with microvalves using a perylene-labeled fluorescent dye. The dyed microvalves show a unique excimer intensity peak under 405 nm laser excitation. Moreover, when pneumatically actuated, the peak shows a predetermined downward shift in intensity as a function of mechanical strain. We validated this protection scheme experimentally using fluorescence microscopy, which showed a high correlation (R2 = 0.971) between the normalized excimer intensity change and the maximum principal strain of the actuated microvalves. To detect curing ratio-based attacks, we adapted machine learning (ML) models, which were trained on the force-displacement data obtained from a mechanical punch test method. Our ML models achieved more than 99% accuracy in detecting curing ratio anomalies. These countermeasures can be used to proactively safeguard FMBs against material-level attacks in the era of global pandemics and diagnostics based on POCTs.

Hierarchically Porous Calcium Carbonate Scaffolds for Bone Tissue Engineering.

Hierarchically porous CaCO3 scaffolds comprised of micro- (diameter = 2.0 ± 0.3 µm) and nano-sized (diameter = 50.4 ± 14.4 nm) pores were fabricated on silicon substrates using a supercritical CO2-based process. Differentiated human THP-1 monocytes exposed to the CaCO3 scaffolds produced negligible levels of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α), confirming the lack of immunogenicity of the scaffolds. Extracellular matrix (ECM) proteins, vitronectin and fibronectin, displayed enhanced adsorption to the scaffolds compared to the silicon controls. ECM protein-coated CaCO3 scaffolds promoted adhesion, growth, and proliferation of osteoblast MC3T3 cells. MC3T3 cells grown on the CaCO3 scaffolds produced substantially higher levels of transforming growth factor-beta and vascular endothelial growth factor A, which regulate osteoblast differentiation, and exhibited markedly increased alkaline phosphatase activity, a marker of early osteoblast differentiation, compared to controls. Moreover, the CaCO3 scaffolds stimulated matrix mineralization (calcium deposition), an end point of advanced osteoblast differentiation and an important biomarker for bone tissue formation. Taken together, these results demonstrate the significant potential of the hierarchically porous CaCO3 scaffolds for bone tissue engineering applications.

An integrative Raman microscopy-based workflow for rapid in situ analysis of microalgal lipid bodies.

BACKGROUND: Oils and bioproducts extracted from cultivated algae can be used as sustainable feedstock for fuels, nutritional supplements, and other bio-based products. Discovery and isolation of new algal species and their subsequent optimization are needed to achieve economical feasibility for industrial applications. Here we describe and validate a workflow for in situ analysis of algal lipids through confocal Raman microscopy. We demonstrate its effectiveness to characterize lipid content of algal strains isolated from the environment as well as algal cells screened for increased lipid accumulation through UV mutagenesis combined with Fluorescence Activated Cell Sorting (FACS). RESULTS: To establish and validate our workflow, we refined an existing Raman platform to obtain better discrimination in chain length and saturation of lipids through ratiometric analyses of mixed fatty acid lipid standards. Raman experiments were performed using two different excitation lasers (λ = 532 and 785 nm), with close agreement observed between values obtained using each laser. Liquid chromatography coupled with mass spectrometry (LC-MS) experiments validated the obtained Raman spectroscopic results. To demonstrate the utility and effectiveness of the improved Raman platform, we carried out bioprospecting for algal species from soil and marine environments in both temperate and subtropical geographies to obtain algal isolates from varied environments. Further, we carried out two rounds of mutagenesis screens on the green algal model species, Chlamydomonas reinhardtii, to obtain cells with increased lipid content. Analyses on both environmental isolates and screened cells were conducted which determined their respective lipids. Different saturation states among the isolates as well as the screened C. reinhardtii strains were observed. The latter indicated the presence of cell-to cell variations among cells grown under identical condition. In contrast, non-mutagenized C. reinhardtii cells showed no significant heterogeneity in lipid content. CONCLUSIONS: We demonstrate the utility of confocal Raman microscopy for lipid analysis on novel aquatic and soil microalgal isolates and for characterization of lipid-expressing cells obtained in a mutagenesis screen. Raman microscopy enables quantitative determination of the unsaturation level and chain lengths of microalgal lipids, which are key parameters in selection and engineering of microalgae for optimal production of biofuels.

Direct observations of field-induced assemblies in magnetite ferrofluids.

Evolution of microstructures in magnetite-based ferrofluids with weak dipolar moments (particle size ≤ 10 nm) is studied with an emphasis on examining the effects of particle concentration (ϕ) and magnetic field strength (H) on the structures. Nanoparticles are dispersed in water at three different concentrations, ϕ = 0.15%, 0.48%, and 0.59% (w/v) [g/ml%] and exposed to uniform magnetic fields in the range of H = 0.05-0.42 T. Cryogenic transmission electron microscopy is employed to provide in-situ observations of the field-induced assemblies in such systems. As the magnetic field increases, the Brownian colloids are observed to form randomly distributed chains aligned in the field direction, followed by head-to-tail chain aggregation and then lateral aggregation of chains termed as zippering. By increasing the field in low concentration samples, the number of chains increases, though their length does not change dramatically. Increasing concentration increases the length of the linear particle assemblies in the presence of a fixed external magnetic field. Thickening of the chains due to zippering is observed at relatively high fields. Through a systematic variation of concentration and magnetic field strength, this study shows that both magnetic field strength and change in concentration can strongly influence formation of microstructures even in weak dipolar systems. Additionally, the results of two commonly used support films on electron microscopy grids, continuous carbon and holey carbon films, are compared. Holey carbon film allows us to create local regions of high concentrations that further assist the development of field-induced assemblies. The experimental observations provide a validation of the zippering effect and can be utilized in the development of models for thermophysical properties such as thermal conductivity.

Fabrication of hierarchically porous materials and nanowires through coffee ring effect.

We report a versatile method for the fabrication of nanowires and hierarchical porous materials from a wide variety of ceramic materials such as CaCO3, ZnO, CuO, Co3O4, Co-doped ZnO, and Ag2O. The method consists of evaporation of CO2-enriched water microdroplets (diameter ∼3 µm) deposited from an aerosol onto heated substrates (T = 120 °C). A variety of porous scaffolds with 1-3 µm sized pores can be generated by tuning the process conditions. Subsequent sintering of the scaffolds is shown to generate nanosized pores in the walls of the porous scaffold creating a dual hierarchy of pore sizes (∼50 nm and 1-3 µm). We propose a mechanism for the formation of scaffolds based on the coffee-ring effect during the evaporation of microdroplets. Ostwald-ripening of CaCO3 scaffolds prepared without sintering yields scaffold structures consisting of two-dimensional crystals of CaCO3 that are one unit cell thick. The favorable application of CaCO3 scaffolds for the enhancement of bone healing around titanium implants with improved biocompatibility is also demonstrated.

Long-term efficient gene delivery using polyethylenimine with modified Tat peptide.

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (≈5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.

Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis.

The HIV-1 Tat peptide has been successfully used for intracellular gene delivery. Likewise, various lipid-based methods have shown increased endocytosis and can influence endosomal escape. This study combines the favorable properties of Tat peptide with that of lipid systems for DNA delivery. We combined the lipid FuGENE HD (FH) with the Tat peptide sequence modified with histidine and cysteine residues (mTat). mTat/FH transfection was evaluated by luciferase expression plasmid in five cell types. mTat/FH produced significant improvement in transfection efficiency of all cell lines when compared to FH or mTat. Treatment with chloroquine, associated with energy-dependent endocytosis, significantly increased transfection efficiency with mTat/FH while incubation at low temperature decreased it. The zeta potential of mTat/FH/DNA was significantly higher compared to FH, mTat, or their DNA combination in the presence of serum, and it was correlated with transfection efficiency. The particle size of the FH/DNA complex was significantly reduced by addition of mTat. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/FH transfection, but transfection was increased by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. These findings demonstrated the feasibility of using a combination of mTat with lipids, utilizing temperature-dependent and caveolae-mediated endocytosis, as a potentially attractive non-viral gene vector.

Frozen cyclohexane-in-water emulsion as a sacrificial template for the synthesis of multilayered polyelectrolyte microcapsules.

This paper reports the application of frozen cyclohexane-in-water emulsions as sacrificial templates for the fabrication of hollow microcapsules through layer-by-layer assembly of polyelectrolytes, poly(styrenesulfonate sodium salt), and poly(allylamine hydrochloride). Extraction of the cyclohexane phase from frozen emulsions stabilized with 11 polyelectrolyte layers by compatibilization with 30% v/v ethanol leads to the formation of water-filled microcapsules while preserving the spherical geometry. The majority of microcapsules (>90%) are prepared with intact polyelectrolyte membranes as measured by their deformation induced by osmotic pressure. This work provides a new route for the synthesis of hollow multilayered microcapsules under mild operating conditions.