Impact of Zero-Valent Iron Nanoparticles on Fremyella diplosiphon Transesterified Lipids and Fatty Acid Methyl Esters.

Efforts to enhance the transformative potential of biofuels is an important step to achieving an environment-friendly and sustainable energy source. Fremyella diplosiphon is an ideal third-generation biofuel agent due to its ability to produce lipids and desirable essential fatty acids. In this study, the impact of Nanofer 25s nanoscale zero-valent iron nanoparticles (nZVIs) on total lipid content and fatty acid composition of F. diplosiphon strains SF33 and B481 was investigated. We observed significant increases (P < 0.05) in the growth of F. diplosiphon treated with 0.2-1.6 mg L-1 Nanofer 25s, indicating that trace concentrations of nZVIs were not toxic to the organism. Chlorophyll a, carotenoids, and phycobiliprotein levels were not altered in F. diplosiphon treated with nZVIs ranging from 0.4 to 1.6 mg L-1, confirming that these concentrations did not negatively impact photosynthetic efficacy. In addition, Nanofer 25s ranging from 0.2 to 1.6 mg L-1 had an optimal impact on SF33 and B481 total lipid content. We identified significant increases in unsaturated fatty acid methyl esters (FAMEs) from F. diplosiphon Nanofer 25s-treated transesterified lipids. Theoretical chemical and physical biofuel properties revealed a product with elevated cetane number and oxidative stability for both strains. Scanning electron microscopy and energy-dispersive X-ray spectroscopy validated the localization of nZVIs. Our findings indicate that Nanofer 25s nZVIs significantly enhance F. diplosiphon total lipid content and essential FAMEs, thus offering a promising approach to augment the potential of the cyanobacterium as a large-scale biofuel agent.

Rapid Sensing of Biological and Environmental Analytes Using Microwave-Accelerated Bioassays and a MATLAB Application.

We report a method for rapid detection and analysis of biological and environmental analytes by microwave-accelerated bioassays (MABs) and a novel MATLAB-based image processing of colorimetric signals. In this regard, colorimetric bioassays for histidine-rich protein 2 (HRP-2) and microcystin-leucine arginine (MC-LR) toxin were carried out using MABs and without microwave heating (i.e, gold standard bioassays). Our MATLAB-based detection method is based on the direct correlation of color intensity of a solution calculated from images captured with a smartphone with the concentration of the biomolecule of interest using a MATLAB code developed in-house. We demonstrated that our MATLAB-based detection method can yield bioassay sensitivity comparable to the colorimetric gold standard tool, i.e., UV-Visible spectroscopy. In addition, colorimetric bioassay time for the HRP-2 assay (used in malaria diagnosis) and colorimetric MC-LR bioassay (used in MCLR toxin diagnosis) was reduced from up to 2 hours at room temperature without microwave heating to 15 minutes using the MABs technique.

Metal-Assisted and Microwave-Accelerated Decrystallization of Pseudo-Tophus in Synthetic Human Joint Models.

In this paper, we tested a hypothesis that the metal-assisted and microwave-accelerated decrystallization (MAMAD) technique, based on the combined use of low-power medical microwave heating (MWH) and gold nanoparticles (Au NPs), can be used to decrystallize laboratory-prepared monosodium urate monohydrate crystal aggregate (pseudo-tophus) placed in three-dimensional (3D) synthetic human joint models. To simulate a potential treatment of chronic tophaceous gout using the MAMAD technique, we used three different 3D synthetic human joint models and assessed the percent mass reduction (PMR, i.e., decrystallization) of pseudo-tophus and microwave-induced synthetic skin patch damage after MAMAD sessions (a MAMAD session = 120 s of MWH in the presence of Au NPs). Our three synthetic joint models are: Model 1: Application of seven MAMAD sessions in a closed synthetic joint with a pseudo-bursa containing a pseudo-tophus submerged in a solution of 20 nm Au NPs followed by dehydration of pseudo-tophus after each MAMAD session to assess PMR. Model 2: Application of seven MAMAD sessions in a closed or open synthetic joint with a pseudo-bursa containing a pseudo-tophus submerged in a solution of Au NPs followed by intermittent dehydration of pseudo-tophus after seven MAMAD sessions to assess PMR. Model 3: Application of 18 MAMAD sessions in a rotated closed synthetic joint (three sides are heated separately) with a pseudo-bursa containing a pseudo-tophus submerged in a solution of Au NPs followed by dehydration after every three MAMAD sessions to assess PMR. After a single MAMAD session, pseudo-tophus exposed to MWH and Au NPs had an average PMR of 8.30% (up to an overall PMR of 15%), and microwave-induced damage to the synthetic skin can be controlled by the use of a sacrificial skin sample and by adjusting the duration and the number of the MAMAD sessions. Computational electromagnetic simulations predict a 10% absorption of electric field by the pseudo-tophus placed in the synthetic joint models, which led us to conclude that a medical microwave source with higher power than 20 W can potentially be used with the MAMAD technique.

Nanoparticle-mediated Impact on Growth and Fatty Acid Methyl Ester Composition in the Cyanobacterium Fremyella diplosiphon.

Insufficient light supply is a major limitation in cultivation of cyanobacteria for scaled up biofuel production and other biotechnological applications, which has driven interest in nanoparticle-mediated enhancement of cellular light capture. In the present study, Fremyella diplosiphon wild type (Fd33) and halotolerant (HSF33-2) strains were grown in solution with 20, 100, and 200 nm-diameter gold nanoparticles (AuNPs) to determine their impact on biomass accumulation, pigmentation, and fatty acid methyl ester (FAME) production. Results revealed a significant increase in growth of Fd33 (0.244 ± 0.006) and HSF33-2 (0.112 ± 0.003) when treated with 200 nm AuNPs. In addition, we observed a significant increase in chlorophyll a accumulation in 200 nm AuNP-treated Fd33 (25.7%) and HSF33-2 (36.3%) indicating that NPs enhanced photosynthetic pigmentation. We did not observe any alteration in FAME composition and biodiesel properties of transesterified F. diplosiphon lipids among all AuNP treatments. Interactions between F. diplosiphon and AuNPs were visualized using scanning electron microscopy. Energy dispersive X-ray spectroscopy confirmed the presence of AuNPs outside cells with aggregation in high cell density locales. Our findings indicate that nanotechnological approaches could significantly enhance growth of the organism with no negative effect on FAME-derived biodiesel properties, thus augmenting F. diplosiphon as a potential biofuel agent.

Individually grown cobalt nanowires as magnetic force microscopy probes.

AC electric fields were utilized in the growth of individual high-aspect ratio cobalt nanowires from simple salt solutions using the Directed Electrochemical Nanowire Assembly method. Nanowire diameters were tuned from the submicron scale to 40 nm by adjusting the AC voltage frequency and the growth solution concentration. The structural properties of the nanowires, including shape and crystallinity, were identified using electron microscopy. Hysteresis loops obtained along different directions of an individual nanowire using vibrating sample magnetometry showed that the magnetocrystalline anisotropy energy has the same order of magnitude as the shape anisotropy energy. Additionally, the saturation magnetization of an individual cobalt nanowire was estimated to be close to the bulk single crystal value. A small cobalt nanowire segment was grown from a conductive atomic force microscope cantilever tip that was utilized in magnetic force microscopy (MFM) imaging. The fabricated MFM tip provided moderate quality magnetic images of an iron-cobalt thin-film sample.

Microwave Heating of Crystals with Gold Nanoparticles and Synovial Fluid under Synthetic Skin Patches.

Gout is a disease with elusive treatment options. Reduction of the size of l-alanine crystals as a model crystal for gouty tophi with the use of a monomode solid-state microwave was examined as a possible therapeutic aid. The effect of microwave heating on l-alanine crystals in the presence of gold nanoparticles (Au NPs) in solution and synovial fluid (SF) in a plastic pouch through a synthetic skin patch was investigated. In this regard, three experimental paradigms were employed: Paradigm 1 includes the effect of variable microwave power (5-10 W) and variable heating time (5-60 s) and Au NPs in water (20 nm size, volume of 10 µL) in a plastic pouch (1 × 2 cm2 in size). Paradigm 2 includes the effect of a variable volume of 20 nm Au NPs in a variable volume of SF up to 100 µL in a plastic pouch at a constant microwave power (10 W) for 30 s. Paradigm 3 includes the effect of constant microwave power (10 W) and microwave heating time (30 s), constant volume of Au NPs (100 µL), and variable size of Au NPs (20-200 nm) placed in a plastic pouch through a synthetic skin patch. In these experiments, an average of 60-100% reduction in the size of an l-alanine crystal (initial size = 450 µm) without damage to the synthetic skin or increasing the temperature of the samples beyond the physiological range was reported.

Metal oxide surfaces for enhanced colorimetric response in bioassays.

Physical stability of metal nanoparticle films on planar surfaces can be increased by employing surface modification techniques and/or type of metal nanoparticles. Subsequently, the enzymatic response of colorimetric bioassays can be increased for improved dynamic range for the detection of biomolecules. Using a model bioassay b-BSA, three planar platforms (1) poly (methyl methacrylate) (PMMA) with silver thin films (STFs), (2) silver nanowires (Ag NWs) on paper and (3) indium tin oxide (ITO) on polyethylene terephthalate (PET) were evaluated to investigate the extent of increase in the colorimetric signal. Bioassays for b-BSA and Ki-67 antigen (a real-life bioassay) in buffer were performed using microwave heating (total assay time is 25-30min) and at room temperature (a control experiment, total assay time is 3h). Model bioassays showed that STFs were removed from the surface during washing steps and the extent of ITO remained unchanged. The lowest level of detection (LLOD) for b-BSA bioassays were: 10-10M for 10nm STFs on PMMA and Ag NWs on paper and 10-11M for ITO. Bioassays for Ki-67 antigen yielded a LLOD of <10-9M on ITO platforms, while STFs platforms were deemed unusable due to significant loss of STFs from the surfaces.

Effect of Microwave Heating on the Crystallization of Glutathione Tripeptide on Silver Nanoparticle Films.

Effect of microwave heating on the crystallization of glutathione (GSH) tripeptide using the metal-assisted and microwave-accelerated evaporative crystallization (MA-MAEC) technique is reported. GSH crystals were grown from supersaturated solutions of GSH (300-500 mg/mL) on the iCrystal plates with silver nanoparticle films (SNFs) and without SNFs in three different microwave systems operating at 2.45 GHz: conventional (multimode, fixed power at 900W), industrial (monomode, variable power up to 1200 W), and the iCrystal system (monomode, variable power up to 100 W). The efficacy of the MA-MAEC technique, in terms of improvement in the crystallization time, crystal size and quality of GSH, was compared between the three microwave systems and the crystallization at room temperature (no microwave heating, a control experiment). Optical microscopy was used to visualize and quantify the growth of GSH crystals during and after microwave heating. Powder X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy data showed that GSH crystals had identical crystal structure to those grown at room temperature and microwave heating did not alter the chemical structure of GSH molecules during microwave heating, respectively. Using the MA-MAEC technique, the iCrystal system yielded high quality GSH crystals in a rapid manner.

Metal-Assisted and Microwave-Accelerated Decrystallization: An Alternative Approach to Potential Treatment of Crystal Deposition Diseases.

Gout is a painful and prevalent crystal deposition disease caused by the overproduction of Uric Acid (UA) in the body and the atypical deposition in human synovial joints as Monosodium Urate Monohydrate (MSUM). Conventional treatments, such as NSAIDs, cyclooxygenase-2 inhibitors, and systemic glucocorticoids often present harmful side-effects and are short-lived. Long-term therapies including xanthine oxidase inhibitors and the use of uricosuric agents have been developed and aim to lower the UA serum levels in the body. As regards to post-crystals deposition, our research laboratory recently proposed and demonstrated the use of the Metal-Assisted and Microwave-Accelerated Decrystallization (MAMAD) technique for the breakdown of organic and biological crystals on planar surfaces. The MAMAD technique is based on the combined use of microwave heating and Au NPs in solution. The interactions of the Au NPs with microwave's electromagnetic field result in an increase in the kinetic energy of Au NPs, and subsequently, an increase in the collisions with target crystals placed on planar surfaces leading to rapid crystal breakdown. In this regard, our laboratory aims to develop the MAMAD technique as an alternative treatment for crystal deposition diseases, particularly gout, with minimal invasion and side-effects as compared to current treatments. In this review article, we will summarize our previous findings and provide additional data detailing the effectiveness of the MAMAD technique as a rapid and efficient method for the breakdown of gout related crystals and L-alanine crystals (a model crystal).

Ultra-Rapid Crystallization of L-alanine Using Monomode Microwaves, Indium Tin Oxide and Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

The use of indium tin oxide (ITO) and focused monomode microwave heating for the ultra-rapid crystallization of L-alanine (a model amino acid) is reported. Commercially available ITO dots (< 5 mm) attached to blank poly(methyl)methacrylate (PMMA, 5 cm in diameter with 21-well silicon isolators: referred to as the iCrystal plates) were found to withstand prolonged microwave heating during crystallization experiments. Crystallization of L-alanine was performed at room temperature (a control experiment), with the use of two microwave sources: a 2.45 GHz conventional microwave (900 W, power level 1, a control experiment) and 8 GHz (20 W) solid state, monomode microwave source with an applicator tip that focuses the microwave field to a 5-mm cavity. Initial appearance of L-alanine crystals and on iCrystal plates with ITO dots took 47 ± 2.9 min, 12 ± 7.6 min and 1.5 ± 0.5 min at room temperature, using a conventional microwave and focused monomode microwave heating, respectively. Complete evaporation of the solvent using the focused microwaves was achieved in 3.2 ± 0.5 min, which is ~52-fold and ~172-fold faster than that observed at room temperature and using conventional microwave heating, respectively. The size and number of L-alanine crystals was dependent on the type of the 21-well iCrystal plates and the microwave heating method: 33 crystals of 585 ± 137 µm in size at room temperature > 37 crystals of 542 ± 100 µm in size with conventional microwave heating > 331 crystals of 311 ± 190 µm in size with focused monomode microwave. FTIR, optical microscopy and powder X-ray diffraction analysis showed that the chemical composition and crystallinity of the L-alanine crystals did not change when exposed to microwave heating and ITO surfaces. In addition, theoretical simulations for the binding of L-alanine molecules to ITO and other metals showed the predicted nature of hydrogen bonds formed between L-alanine and these surfaces.

Microwave Heating of Synthetic Skin Samples for Potential Treatment of Gout Using the Metal-Assisted and Microwave-Accelerated Decrystallization Technique.

Physical stability of synthetic skin samples during their exposure to microwave heating was investigated to demonstrate the use of the metal-assisted and microwave-accelerated decrystallization (MAMAD) technique for potential biomedical applications. In this regard, optical microscopy and temperature measurements were employed for the qualitative and quantitative assessment of damage to synthetic skin samples during 20 s intermittent microwave heating using a monomode microwave source (at 8 GHz, 2-20 W) up to 120 s. The extent of damage to synthetic skin samples, assessed by the change in the surface area of skin samples, was negligible for microwave power of ≤7 W and more extensive damage (>50%) to skin samples occurred when exposed to >7 W at initial temperature range of 20-39 °C. The initial temperature of synthetic skin samples significantly affected the extent of change in temperature of synthetic skin samples during their exposure to microwave heating. The proof of principle use of the MAMAD technique was demonstrated for the decrystallization of a model biological crystal (l-alanine) placed under synthetic skin samples in the presence of gold nanoparticles. Our results showed that the size (initial size ∼850 µm) of l-alanine crystals can be reduced up to 60% in 120 s without damage to synthetic skin samples using the MAMAD technique. Finite-difference time-domain-based simulations of the electric field distribution of an 8 GHz monomode microwave radiation showed that synthetic skin samples are predicted to absorb ∼92.2% of the microwave radiation.

Decrystallization of Crystals Using Gold "Nano-Bullets" and the Metal-Assisted and Microwave-Accelerated Decrystallization Technique.

Gout is caused by the overproduction of uric acid and the inefficient metabolism of dietary purines in humans. Current treatments of gout, which include anti-inflammatory drugs, cyclooxygenase-2 inhibitors, and systemic glucocorticoids, have harmful side-effects. Our research laboratory has recently introduced an innovative approach for the decrystallization of biological and chemical crystals using the Metal-Assisted and Microwave-Accelerated Evaporative Decrystallization (MAMAD) technique. In the MAMAD technique, microwave energy is used to heat and activate gold nanoparticles that behave as "nano-bullets" to rapidly disrupt the crystal structure of biological crystals placed on planar surfaces. In this study, crystals of various sizes and compositions were studied as models for tophaceous gout at different stages (i.e., uric acid as small crystals (~10-100 µm) and l-alanine as medium (~300 µm) and large crystals (~4400 µm). Our results showed that the use of the MAMAD technique resulted in the reduction of the size and number of uric acid and l-alanine crystals up to >40% when exposed to intermittent microwave heating (up to 20 W power at 8 GHz) in the presence of 20 nm gold nanoparticles up to 120 s. This study demonstrates that the MAMAD technique can be potentially used as an alternative therapeutic method for the treatment of gout by effective decrystallization of large crystals, similar in size to those that often occur in gout.

Synthesis, characterization, and evaluation of (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate as a biological agent and an anion sensor.

An amino acid based and bidentate Schiff base, (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate (ligand), was synthesized from the reaction of glycine-methyl ester hydrochloride with 2-hydroxy-1-naphthaldehyde. Characterization of the ligand was carried out using theoretical quantum-mechanical calculations and experimental spectroscopic methods. The molecular structure of the compound was confirmed using X-ray single-crystal data, NMR, FTIR and UV-Visible spectroscopy, which were in good agreement with the structure predicted by the theoretical calculations using density functional theory (DFT). Antimicrobial activity of the ligand was investigated for its minimum inhibitory concentration (MIC) to several bacteria and yeast cultures. UV-Visible spectroscopy studies also shown that the ligand can bind calf thymus DNA (CT-DNA) electrostatic binding. In addition, DNA cleavage study showed that the ligand cleaved DNA without the need for external agents. Energetically most favorable docked structures were obtained from the rigid molecular docking of the compound with DNA. The compound binds at the active site of the DNA proteins by weak non-covalent interactions. The colorimetric response of the ligand in DMSO to the addition of equivalent amount of anions (F-, Br-, I-, CN-, SCN-, ClO4-, HSO4-, AcO-, H2PO4-, N3- and OH-) was investigated and the ligand was shown to be sensitive to CN- anion.

A Highly Selective Sensor for Cyanide in Organic Media and on Solid Surfaces.

The application of IR 786 perchlorate (IR-786) as a selective optical sensor for cyanide anion in both organic solution (acetonitrile (MeCN), 100%) and solvent-free solid surfaces was demonstrated. In MeCN, IR-786 was selective to two anions in the following order: CN(-) > OH(-). A significant change in the characteristic dark green color of IR-786 in MeCN to yellow was observed as a result of nucleophilic addition of CN(-) to the fluorophore, i.e., formation of IR 786-(CN), which was also verified by a blue shift in the 775 nm absorbance peak to 430 nm. A distinct green fluorescence emission from the IR-786-(CN) in MeCN was also observed, which demonstrated the selectivity of IR-786 towards CN(-) in MeCN. Fluorescence emission studies of IR-786 showed that the lower detection limit and the sensitivity of IR-786 for CN(-) in MeCN was 0.5 µM and 0.5 to 8 µM, respectively. The potential use of IR-786 as a solvent-free solid state sensor for the selective sensing and monitoring of CN(-) in the environment was also demonstrated. On solvent-free solid state surfaces, the sensitivity of the IR-786 to CN(-) in water samples was in the range of 50-300 µM with minimal interference by OH(-).

Microwave-accelerated bioassay technique for rapid and quantitative detection of biological and environmental samples.

Quantitative detection of molecules of interest from biological and environmental samples in a rapid manner, particularly with a relevant concentration range, is imperative to the timely assessment of human diseases and environmental issues. In this work, we employed the microwave-accelerated bioassay (MAB) technique, which is based on the combined use of circular bioassay platforms and microwave heating, for rapid and quantitative detection of Glial Fibrillary Acidic Protein (GFAP) and Shiga like toxin (STX 1). The proof-of-principle use of the MAB technique with the circular bioassay platforms for the rapid detection of GFAP in buffer based on colorimetric and fluorescence readouts was demonstrated with a 900W kitchen microwave. We also employed the MAB technique with a new microwave system (called the iCrystal system) for the detection of GFAP from mice with brain injuries and STX 1 from a city water stream. Control bioassays included the commercially available gold standard bioassay kits run at room temperature. Our results show that the lower limit of detection (LLOD) of the colorimetric and fluorescence based bioassays for GFAP was decreased by ~1000 times using the MAB technique and our circular bioassay platforms as compared to the commercially available bioassay kits. The overall bioassay time for GFAP and STX 1 was reduced from 4h using commercially available bioassay kits to 10min using the MAB technique.

Enhancement of the Chemiluminescence Response of Enzymatic Reactions by Plasmonic Surfaces for Biosensing Applications.

We report the enhancement of chemiluminescence response of horseradish peroxidase (HRP) in bioassays by plasmonic surfaces, which are comprised of (i) silver island films (SIFs) and (ii) metal thin films (silver, gold, copper, and nickel, 1 nm thick) deposited onto glass slides. A model bioassay, based on the interactions of avidin-modified HRP with a monolayer of biotinylated poly(ethylene-glycol)-amine, was employed to evaluate the ability of plasmonic surfaces to enhance chemiluminescence response of HRP. Chemiluminescence response of HRP in model bioassays were increased up to ~3.7-fold as compared to the control samples (i.e. glass slides without plasmonic nanoparticles), where the largest enhancement of the chemiluminescence response was observed on SIFs with high loading. These findings allowed us to demonstrate the use of SIFs (high loading) for the detection of a biologically relevant target protein (glial fibrillary acidic protein or GFAP), where the chemiluminescence response of the standard bioassay for GFAP was enhanced up to ~50% as compared to bioassay on glass slides.

Enhancement of Colorimetric Response of Enzymatic Reactions by Thermally Evaporated Plasmonic Thin Films: Application to Glial Fibrillary Acidic Protein.

We report the enhancement of the colorimetric response of horseradish peroxidase (HRP) and alkaline phosphatase (AP) in bioassays by thermally evaporated silver, gold, copper and nickel thin films. In this regard, a model bioassay based on biotin-avidin interactions was employed. Biotin groups and enzymes were introduced to all surfaces using a biotinylated linker molecule and avidin, respectively. The colorimetric response of HRP in the model bioassay carried out on the plasmonic thin films were up to 4.4-fold larger as compared to control samples (i.e., no plasmonic thin films), where the largest enhancement of colorimetric response was observed on silver thin films. The colorimetric response of AP on plasmonic thin films was found to be similar to those observed on control samples, which was attributed to the loss of enzymes from the surface during the bioassay steps. The extent of enzymes immobilized on to plasmonic thin films was found to affect the colorimetric response of the model bioassay. These findings allowed us to demonstrate the use of silver thin films for the detection of glial fibrillary acidic protein (GFAP), where the colorimetric response of the standard bioassays for GFAP was enhanced up to 67% as compared to bioassays on glass slides.

Metal-Enhanced Fluorescence from Silver Nanowires with High Aspect Ratio on Glass Slides for Biosensing Applications.

High enhancement of fluorescence emission, improved fluorophore photostability, and significant reduction of fluorescence lifetimes have been obtained from high aspect ratio (>100) silver (Ag) nanowires. These quantities are found to depend on the surface loading of Ag nanowires on glass slides, where the enhancement of fluorescence emission increases with the density of nanowires. The surface loading dependence was attributed to the creation of intense electric fields around the network of Ag nanowires and to the coupling of fluorophore excited states that takes place efficiently at a distance of 10 nm from the surface of nanowires, which was confirmed by theoretical calculations. The enhancement of fluorescence emission of fluorescein isothiocyanate (FITC) was assessed by fluorescence spectroscopy and fluorescence-lifetime imaging microscopy (FLIM) to demonstrate the potential of high aspect ratio Ag nanowires. Fluorescence enhancement factors exceeding 14 were observed on Ag nanowires with high loading by FLIM. The photostability of FITC was the highest on nanowires with medium loading under continuous laser excitation for 10 min because of the significant reduction in the fluorescence lifetime of FITC on these surfaces. These results clearly demonstrate the potential of Ag nanowires in metal-enhanced fluorescence-based applications of biosensing on planar surfaces and cellular imaging.

Enhancement of enzymatic colorimetric response by silver island films on high throughput screening microplates.

In this study, we report the use of an enzyme-based hybrid platform, which is comprised of silver island films, enzymes (HRP and AP) and high-throughput screening (HTS) microplates, to enhance the colorimetric response of enzymatic reactions. The hybrid platform was designed in a two-step process: (i) deposition of SIFs onto HTS microplates with low, medium, and high loading (refers to the extent of the surface plasmon resonance peak of SIFs at 460 nm) using Tollen's reaction scheme; and (ii) attachment of b-BSA or BEA as linkers for the immobilization of enzymes. The presence of SIFs within the wells of the HTS microplates was confirmed using an optical spectrophotometer and real-color photography. Control experiments, where SIFs were omitted from the surfaces were carried out to confirm the effect of SIFs on the enzymatic colorimetric response. Significant colorimetric signal enhancement was observed for HRP or AP on SIFs (high loading) deposited HTS microplates using b-BSA (up to ~3-fold for AP and ~6-fold HRP) or BEA (up to ~7-fold for both HRP and AP), as compared to our control samples. The observed increase in colorimetric response can be attributed to the nature of BEA, which exposes surface-bound enzymes to the substrate present in bulk more efficiently than b-BSA. This study proves that SIFs can serve as a valuable tool to improve the signal output of existing bioassays carried out in HTS microplates, which can be applicable to the field biosensors and plasmonics.

Crystal Engineering of l-Alanine with l-Leucine Additive using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

In this work, we demonstrated that the change in the morphology of l-alanine crystals can be controlled with the addition of l-leucine using the metal-assisted and microwave accelerated evaporative crystallization (MA-MAEC) technique. Crystallization experiments, where an increasing stoichiometric amount of l-leucine is added to initial l-alanine solutions, were carried out on circular poly(methyl methacrylate) (PMMA) disks modified with a 21-well capacity silicon isolator and silver nanoparticle films using microwave heating (MA-MAEC) and at room temperature (control experiments). The use of the MA-MAEC technique afforded for the growth of l-alanine crystals with different morphologies up to ∼10-fold faster than those grown at room temperature. In addition, the length of l-alanine crystals was systematically increased from ∼380 to ∼2000 µm using the MA-MAEC technique. Optical microscope images revealed that the shape of l-alanine crystals was changed from tetragonal shape (without l-leucine additive) to more elongated and wire-like structures with the addition of the l-leucine additive. Further characterization of l-alanine crystals was undertaken by Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy and powder X-ray diffraction (PXRD) measurements. In order to elucidate the growth mechanism of l-alanine crystals, theoretical simulations of l-alanine's morphology with and without l-leucine additive were carried out using Materials Studio software in conjunction with our experimental data. Theoretical simulations revealed that the growth of l-alanine's {011} and {120} crystal faces were inhibited due to the incorporation of l-leucine into these crystal faces in selected positions.