Thermally Assisted Acoustofluidic Separation Based on Membrane Protein Content.

The over- and under-expression of certain proteins in extracellular vesicles has been observed in many physiological and pathological conditions; however, a simple method to sort vesicles based on contrast in protein content is yet to be developed. We herein present a nonaffinity-based method for rapid and inexpensive isolation of lipid vesicles based on their membrane protein content. Based on a composition-specific thermophysical property change of vesicles at different protein contents, an acoustic property change that enabled an acoustophoretic separation was observed. This change was demonstrated in a thermally modulated acoustofluidic device in the form of a shift in vesicle migration from the nodal plane to antinodal plane at a specific temperature known as the acoustic contrast temperature (TΦ). Using phosphatidylcholine vesicles containing the membrane proteins gramicidin D, alamethicin, and melittin at molar contents ranging from 0.001% to 10%, we observed that increasing the membrane protein content brought about conformational changes in the membrane which afforded the vesicles distinctive acoustic properties. Then, by establishing an acoustic contrast temperature window, vesicles with the same protein but different molar content were successfully separated. The efficiency of the separation was studied for various vesicle mixtures and a separation efficiency as high as 97% was accomplished. In order to confirm the technique's applicability for biological samples, sheep red blood cells with various melittin peptide contents similarly demonstrated the depressing effects of melittin on membrane bending modulus and depressed the TΦ of the cells. This method holds promise for a myriad of applications in the biomedical field, especially in bioanalytical research.

Perspectives on moving ionic liquid chemistry into the solid phase.

Ionic liquid (IL) chemistry has evolved over the past century, such that these organic salts have impacted virtually every area of science and engineering. In the area of chemistry, initial applications of these salts were primarily the domain of chemists or chemical engineers who desired to manipulate the properties of IL solvents for a variety of applications including tuning various chemical processes. Since then, the chemistry of these organic salts has progressed such that changing an important property of a solvent (e.g., melting point or hydrophobicity) often involves simply altering the counterion of the organic salt. It is with this simplicity in mind that we have recently embarked upon the use of such chemistry to manipulate important properties of solid-phase ionic organic materials. To differentiate this chemistry from ionic liquid chemistry, we have coined the acronym GUMBOS (group of uniform materials based on organic salts). In this perspective article, we describe and demonstrate how ionic liquid chemistry can provide distinct and sometimes unique chemistry for solid-phase applications. Solid phase properties which can be manipulated via this chemistry include, but are not limited to, magnetism, melting point, hydrophobicity, fluorescence quantum yields, nanoformulations, material aggregation, viscosity, viscoelasticity, and cytotoxicity. In addition, we discuss a few examples to demonstrate how GUMBOS chemistry, until now, has been beneficial to the general area of materials chemistry and, more broadly, to the field of analytical chemistry. We also project future applications of this technology.

In vitro activity studies of hyperthermal near-infrared nanoGUMBOS in MDA-MB-231 breast cancer cells.

A new kind of material called nanoGUMBOS, comprised entirely of cations and anions, has been developed by pairing various functional ions that exhibit fluorescence activity with biocompatible ions, in a process very much akin to that employed in ionic liquid chemistry. In the present study, spectral and biological properties of NIR absorbing nanoGUMBOS were evaluated using electron microscopy, dynamic light scattering, absorbance, thermal imaging, and live/dead fluorescence assays in conjunction with malignant MDA-MB-231 and non-malignant HS-578-BST epithelial human breast cells. The primary focus of this study was to maximize heat generation using NIR laser irradiation and minimize non-specific cytotoxicity using biocompatible constituent ions (e.g. amino acids, vitamins, or organic acids). Concurrently, in order to generate highly responsive nanomaterials for NIR-laser-triggered hyperthermia, optimization of the nanoparticle size, shape, and uniformity was carried out. Evaluation of data from hyperthermal studies of NIR absorbing nanoGUMBOS shows that these materials can achieve temperatures above the threshold for killing cancerous cells. Additionally, in vitro cell based assays demonstrated their promising hyperthermal effects on cancer derived epithelial cells.

Minimizing human infection from Escherichia coli O157:H7 using GUMBOS.

OBJECTIVES: Reduction in faecal shedding of Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) in food-producing animals is a viable strategy to minimize human disease initiated by exposure to these microorganisms. To this end, an intervention strategy involving the electrostatic hybridization of two commonly used anti-infective agents for veterinary practice (i.e. chlorhexidine and ampicillin) was evaluated to curtail EHEC-transmitted disease from ruminant sources. Chlorhexidine di-ampicillin is a novel group of uniform material based on organic salts (GUMBOS) with inherent in vitro antibacterial activity that comes from its parent antimicrobial ions, chlorhexidine and ampicillin. METHODS: Antibacterial activities for chlorhexidine diacetate, sodium ampicillin, chlorhexidine di-ampicillin and stoichiometrically equivalent 1 : 2 chlorhexidine diacetate : sodium ampicillin were assessed using the serial 2-fold dilution method and time-kill studies against seven isolates of E. coli O157:H7 and one non-pathogenic E. coli 25922. Further studies to investigate synergistic interactions of reacted and stoichiometrically equivalent unreacted antimicrobial agents at MICs and possible mechanisms were also investigated. RESULTS: Synergism and in vitro antibacterial activities against EHEC were observed in this study, which suggests chlorhexidine di-ampicillin could be a useful reagent in reducing EHEC transmission and minimizing EHEC-associated infections. Likewise, chlorhexidine di-ampicillin reduced HeLa cell toxicity as compared with chlorhexidine diacetate or the stoichiometric combination of antimicrobial agents. Further results suggest that the mechanisms of action of chlorhexidine di-ampicillin and chlorhexidine diacetate against E. coli O157:H7 are similar. CONCLUSIONS: Reacting antimicrobial GUMBOS as indicated in this study may enhance the approach to current combination drug therapeutic strategies for EHEC disease control and prevention.

Irradiation induced fluorescence enhancement in PEGylated cyanine-based NIR nano- and mesoscale GUMBOS.

We report on the synthesis and characterization of a PEGylated IR786 GUMBOS (Group of Uniform Materials Based on Organic Salts). The synthesis of this material was accomplished using a three step protocol: (1) substitution of chloride on the cyclohexenyl ring in the heptamethine chain of IR786 by 6-aminohexanoic acid, (2) grafting of methoxy polyethylene glycol (MeOPEG) onto the 6-aminohexanoic acid via an esterification reaction, and (3) anion exchange between [PEG786][I] and lithium bis(trifluoromethylsulfonyl)imide (LiNTf(2)) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in order to obtain PEG786 GUMBOS. Examination of spectroscopic data for this PEG786 GUMBOS indicates a large stokes shift (122 nm). It was observed that this PEG786 GUMBOS associates in aqueous solution to form nano- and mesoscale self-assemblies with sizes ranging from 100 to 220 nm. These nano- and mesoscale GUMBOS are also able to resist nonspecific binding to proteins. PEGylation of the original IR786 leads to reduced cytotoxicity. In addition, it was noted that anions, such as NTf(2) and AOT, play a significant role in improving the photostability of PEG786 GUMBOS. Irradiation-induced J-aggregation in [PEG786][NTf(2)] and to some extent in [PEG786][AOT] produced enhanced photostability. This observation was supported by use of both steady state and time-resolved fluorescence measurements.

Tunable size and spectral properties of fluorescent nanoGUMBOS in modified sodium deoxycholate hydrogels.

Microstructures of sodium deoxycholate hydrogels were altered considerably in the presence of variable tris(hydroxymethyl)aminomethane (TRIS) concentrations. These observations were confirmed by use of X-ray diffraction, polarized optical microscopy, rheology, and differential scanning calorimetry measurements. Our studies reveal enhanced gel crystallinity and rigidity with increasing TRIS concentrations. The tunable hydrogel microstructures obtained under various conditions have been successfully utilized as templates to synthesize cyanine-based fluorescent nanoGUMBOS (nanoparticles from a group of uniform materials based on organic salts). A systematic variation in size (70-200 nm), with relatively low polydispersity and tunable spectral properties of [HMT][AOT] nanoGUMBOS, was achieved by use of these modified hydrogels. The gel microstructures are observed to direct the size as well as molecular self-assembly of the nanomaterials, thereby tuning their spectral properties. These modified hydrogels were also found to possess other interesting properties such as variable morphologies ranging from fibrous to spherulitic, variable degrees of crystallinity, rigidity, optical activity, and release profiles which can be exploited for a multitude of applications. Hence, this study demonstrates a novel method for modification of sodium deoxycholate hydrogels, their applications as templates for nanomaterials synthesis, as well as their potential applications in biotechnology and drug delivery.

Ratiometric coumarin-neutral red (CONER) nanoprobe for detection of hydroxyl radicals.

Excessive production of reactive oxygen species can lead to alteration of cellular functions responsible for many diseases including cardiovascular diseases, neurodegenerative diseases, cancer, and aging. Hydroxyl radical is a short-lived radical which is considered very aggressive due to its high reactivity toward biological molecules. In this study, a COumarin-NEutral Red (CONER) nanoprobe was developed for detection of hydroxyl radical based on the ratiometric fluorescence signal between 7-hydroxy coumarin 3-carboxylic acid and neutral red dyes. Biocompatible poly lactide-co-glycolide (PLGA) nanoparticles containing encapsulated neutral red were produced using a coumarin 3-carboxylic acid conjugated poly(sodium N-undecylenyl-Nε-lysinate) (C3C-poly-Nε-SUK) as moiety reactive to hydroxyl radicals. The response of the CONER nanoprobe was dependent on various parameters such as reaction time and nanoparticle concentration. The probe was selective for hydroxyl radicals as compared with other reactive oxygen species including O(2)(•-), H(2)O(2), (1)O(2), and OCl(-). Furthermore, the CONER nanoprobe was used to detect hydroxyl radicals in vitro using viable breast cancer cells exposed to oxidative stress. The results suggest that this nanoprobe represents a promising approach for detection of hydroxyl radicals in biological systems.

Enzymatic synthesis of L-lactic acid from carbon dioxide and ethanol with an inherent cofactor regeneration cycle.

Efficient conversion of carbon dioxide is of great interests to today's endeavors in controlling greenhouse gas emission. A multienzyme catalytic system that uses carbon dioxide and ethanol to produce L-lactate was demonstrated in this work, thereby providing a novel reaction route to convert bio-based ethanol to an important building block for synthesis biodegradable polymers. The synthetic route has a unique internal cofactor regeneration cycle, eliminating the need of additional chemical or energy for cofactor regeneration. Lactate was successfully synthesized with 41% of ethanol converted in a batch reaction, while a turnover number of 2.2 day⁻¹ was reached for cofactor regeneration in a reaction with continuous feeding of ethanol. A kinetic model developed based on reaction kinetic parameters determined separately for each reaction step predicted well the reaction rates and yields of the multienzyme reaction system.

Ephedrinium-based protic chiral ionic liquids for enantiomeric recognition.

We report the synthesis of a series of novel structurally related protic chiral ionic liquids (PCILs) derived from ephedrines. Enantiopure norephedrine, ephedrine, and methylephedrine were neutralized by use of fluorinated acids, bis(trifluoromethanesulfonyl)imide, and bis(pentafluoroethanesulfonyl)imide to afford six PCILs with protonated primary, secondary, and tertiary amines. The goal of this study is to investigate the influence of structure on both chiral recognition abilities and physicochemical properties of these closely related PCILs. The newly synthesized PCILs were characterized by use of nuclear magnetic resonance (NMR), thermal gravimetric analysis, differential scanning calorimetry, circular dichroism (CD), mass spectrometry, and elemental analysis. The PCILs were thermally stable up to 220°C and had glass transition temperatures between -60 and -30°C. Both enantiomers of the PCILs retained chirality throughout the synthesis as demonstrated by use of CD measurements. More interestingly, these ephedrinium PCILs displayed strong chiral recognition capabilities as evidenced by peak splitting of the chemical shift of the trifluoro group of potassium Mosher's salt by use of (19)F-NMR. In addition, these PCILs demonstrated enantiomeric recognition capabilities toward a range of structurally diverse analytes using steady-state fluorescence spectroscopy.

Lysine-based zwitterionic molecular micelle for simultaneous separation of acidic and basic proteins using open tubular capillary electrochromatography.

In this work, a zwitterionic molecular micelle, poly-epsilon-sodium-undecanoyl lysinate (poly-epsilon-SUK), was synthesized and employed as a coating in open tubular capillary electrochromatography (OT-CEC) for protein separation. The zwitterionic poly-epsilon-SUK containing both carboxylic acid and amine groups can be either protonated or deprotonated depending on the pH of the background electrolyte; therefore, either an overall positively or negatively charged coating can be achieved. This zwitterionic coating allows protein separations in either normal or reverse polarity mode depending on the pH of the background electrolyte. The protein mixtures contained four basic proteins (lysozyme, cytochrome c, alpha-chymotrypsinogen A, and ribonuclease A) and six acidic proteins (myoglobin, deoxyribonuclease I, beta-lactoglobulin A, beta-lactoglobulin B, alpha-lactalbumin, and albumin). Protein separations were optimized specifically for acidic (reverse mode) and basic (normal mode) pH values. Varying the polymer thickness by changing the polymer and salt concentration had a great influence on protein resolution, while nearly all peaks were also baseline resolved in both modes using the optimized poly-epsilon-SUK coating concentration of 0.4% (w/v). Proteins in human sera were separated under optimized acidic and basic conditions in order to demonstrate the general utility of this coating. Nanoscale characterizations of the poly-epsilon-SUK micellar coatings on silicon surfaces were accomplished using atomic force microscopy (AFM) to gain insight into the morphology and thickness of the zwitterionic coating. The thickness of the polymer coating ranged from 0.9 to 2.4 nm based on local measurements using nanoshaving, an AFM-based method of nanolithography.

Lanthanide-based luminescent NanoGUMBOS.

Lanthanide photochemistry has been frequently studied for its high luminescence intensity, narrow emission band, and stable luminescent lifetime decay. In the work presented here, nanoparticles prepared using an aerosolization process were derived from europium-based GUMBOS (Group of Uniform Material Based on Organic Salts). These nanoparticles were characterized using electron microscopy, X-ray photoelectron spectroscopy (XPS), absorbance, and photoluminescence spectroscopy. An average diameter of 39.5 ± 8.4 nm for our nanoparticles was estimated by use of electron microscopy. Absorbance, luminescence, and luminescence lifetime decay measurements indicate intense and steady luminescence, which suggests a multitude of possible applications for lanthanide-based GUMBOS, especially in sensory devices, OLEDs, and photovoltaic devices.

Nontemplated approach to tuning the spectral properties of cyanine-based fluorescent nanoGUMBOS.

Template-free controlled aggregation and spectral properties in fluorescent organic nanoparticles (FONs) is highly desirable for various applications. Herein, we report a nontemplated method for controlling the aggregation in near-infrared (NIR) cyanine-based nanoparticles derived from a group of uniform materials based on organic salts (GUMBOS). Cationic heptamethine cyanine dye 1,1',3,3,3',3'-hexamethylindotricarbocyanine (HMT) was coupled with five different anions, viz., [NTf(2)(-)], [BETI(-)], [TFPB(-)], [AOT(-)], and [TFP4B(-)], by an ion-exchange method to obtain the respective GUMBOS. The nanoGUMBOS obtained via a reprecipitation method were primarily amorphous and spherical (30-100 nm) as suggested by selected area electron diffraction (SAED) and transmission electron microscopy (TEM). The formation of tunable self-assemblies within the nanoGUMBOS was characterized using absorption and fluorescence spectroscopy in conjunction with molecular dynamics simulations. Counterion-controlled spectral properties observed in the nanoGUMBOS were attributed to variations in J/H ratios with different anions. Association with the [AOT(-)] anion afforded predominant J aggregation enabling the highest fluorescence intensity, whereas [TFP4B(-)] disabled the fluorescence due to predominant H aggregation in the nanoparticles. Analyses of the stacking angle of the cations based on molecular dynamic simulation results in [HMT][NTf(2)], [HMT][BETI], and [HMT][AOT] dispersed in water and a visual analysis of the representative simulation snapshots also imply that the type of aggregation was controlled through the counterion associated with the dye cation.

Combinatorial approach to enantiomeric discrimination: synthesis and (19)F NMR screening of a chiral ionic liquid-modified silane library.

A parallel library of chiral ionic liquid (CIL)-modified silanes as potential chiral selectors was synthesized, and their enantiomeric discrimination abilities were screened by use of (19)F NMR spectroscopy. The screening method allows for rapid identification of the most enantioselective members of the library and simultaneous investigation of their chiral recognition mechanisms. The library compounds were synthesized using quaternization and anion-exchange reactions. Three major parameters (type of chiral cations, anions, and linker chain lengths) were included and investigated during the synthesis and screening. As expected, the structure of the chiral cation was found to play an important role in determining chiral recognition abilities. In addition, several types of intermolecular interactions including ion-pair, hydrogen bonding, pi-pi stacking, dipole stacking, and steric interactions were found to impact chiral discrimination.

Particle-tethered NADH for production of methanol from CO(2) catalyzed by coimmobilized enzymes.

Efficient cofactor regeneration and reuse are highly desired for many important biotransformation applications. Here we show for the first time that cofactor NAD(H) covalently attached to micro particles, which can be easily recovered and reused, effectively mediated multistep reactions catalyzed by enzymes that were also immobilized with the micro particles. Such an immobilized enzyme-cofactor catalytic system was examined for the production of methanol from CO(2) with in situ cofactor regeneration. Four enzymes including formate, formaldehyde, alcohol, and glutamate dehydrogenases were coimmobilized using the same particles as that used for cofactor immobilization (enzymes and cofactor were immobilized separately). Reactions were performed by bubbling CO(2) in a suspension solution of the particle-attached enzymes and cofactor. It appeared that the collision among the particles afforded sufficient interactions between the cofactor and enzymes, and thus enabled the sequential transformation of CO(2) to methanol along with cofactor regeneration. For a 30-min batch reaction, a productivity of 0.02 micromol methanol/h/g-enzyme was achieved. That was lower than but comparable to the 0.04 micromol methanol/h/g-enzyme observed for free enzymes and cofactor at the same reaction conditions. The immobilized system showed fairly good stabilities in reusing. Over 80% of their original productivity was retained after 11 reusing cycles, with a cumulative methanol yield based on the amount of cofactor reached 127%. That was a promising enhancement in cofactor utilization as compared to the single-batch yield of 12% observed with free enzymes and free cofactor.

An organic soluble lipase for water-free synthesis of biodiesel.

Lipase AK was modified with short alkyl chains to form a highly organic soluble enzyme and was used to catalyze the synthesis of biodiesel from soybean oil in organic media. The effects of several key factors including water content, temperature, and solvent were examined for the solubilized enzyme in comparison with several other commercially available lipases. Whereas native lipases showed no activity in the absence of water, the organic soluble lipase demonstrated reaction rates of up to 33 g-product/g-enzyme h. The biocatalyst remains soluble in the biodiesel product, and therefore, there is no need to be removed because it is expected to be burned along with the diesel in combustion engines. This provides a promising one-pot mix-and-use strategy for biodiesel production.

Thermo-acoustofluidic separation of vesicles based on cholesterol content.

Biomechanical properties of cells such as cellular stiffness have been increasingly considered as biomarkers for diseases. For instance, stiffness of cancer cells has been correlated to the malignant potential in certain cell lines. In cells, the cholesterol content plays a crucial role in determining stiffness. Changes in the cholesterol content in cellular membranes can be an indication of pathological disorders. Acoustophoresis as a separation and diagnostic tool is well positioned to help in the separation and diagnosis of cells taking advantage of its unique separation criteria of density and compressibility. However, under the same conditions, cells and vesicles secreted by these cells often have a positive contrast factor sign and thus do not yield simple separations. Thermally-assisted acoustophoresis, also referred to as thermo-acoustophoresis, solves this problem by adding a temperature dimension to the separation. In this work, we evaluate the acoustic contrast temperature (TΦ) of vesicles at different cholesterol molar ratios (Xchol) and develop a multi-stage lab-on-a-chip method to accomplish for the first time the separation of a three-vesicle mixture. Using Xchol = 0.1, 0.2, and 0.3 vesicles, we have obtained separation efficiencies exceeding 93%. The simplicity, rapidity, and label-free nature of this approach holds promise as a diagnostic and separation tool for cells and extracellular vesicles such as exosomes and microvesicles.

Composite Gel Polymer Electrolyte for Improved Cyclability in Lithium-Oxygen Batteries.

Gel polymer electrolytes (GPE) and composite GPE (cGPE) using one-dimensional glass microfillers have been developed for their use in lithium-oxygen batteries. Using glass microfillers, tetraglyme solvent, UV-curable polymer, and lithium salt at various concentrations, the preparation of cGPE yielded free-standing films. These cGPEs, with 1 wt % of microfillers, demonstrated increased ionic conductivity and lithium transference number over GPEs at various concentrations of lithium salt. Improvements as high as 50% and 28% in lithium transference number were observed for 0.1 and 1.0 mol kg-1 salt concentrations, respectively. Lithium-oxygen batteries containing cGPE similarly showed superior charge/discharge cycling for 500 mAh g-1 cycle capacity with as high as 86% and 400% increase in cycles for cGPE with 1.0 and 0.1 mol kg-1 over GPE. Results using electrochemical impedance spectroscopy, Raman spectroscopy, and scanning electron microscopy revealed that the source of the improvement was the reduction of the rate of lithium carbonates formation on the surface of the cathode. This reduction in formation rate afforded by cGPE-containing batteries was possible due to the reduction of the rate of electrolyte decomposition. The increase in solvated to paired Li+ ratio at the cathode, afforded by increased lithium transference number, helped reduce the probability of superoxide radicals reacting with the tetraglyme solvent. This stabilization during cycling helped prolong the cycling life of the batteries.

Thermally-assisted ultrasonic separation of giant vesicles.

We report on a newly-developed membrane stiffness-based separation of vesicles using a thermally-assisted acoustophoretic approach. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to the change in the acoustic compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature, corresponding to the temperature at which the acoustic contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop a separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. Our studies suggest that this method may be applied for the separation of cells affected by diseases that affect the cellular stiffness.