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.

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.

Rapid and Sensitive Detection of p53 Based on DNA-Protein Binding Interactions Using Silver Nanoparticle Films and Microwave Heating.

Tumor detection can be carried out via the detection of proteins, such as p53, which is known to play vital role in more than 50% of all cancers affecting humans. Early diagnosis of tumor detection can be achieved by decreasing the lower detection limit of p53 bioassays. Microwave-accelerated bioassay (MAB) technique, which is based on the use of circular bioassay platforms in combination with microwave heating, is employed for the rapid and sensitive detection of p53 protein. Direct sandwich ELISA was constructed on our circular bioassay platforms based on DNA-protein binding interactions. Colorimetric and fluorescence based detection methods were used for room temperature bioassay (control bioassay; total bioassay time is 27 hours) and bioassay using microwave heating (i.e., the MAB technique; total bioassay time is 10 minutes). In the colorimetric based detection, a very high background signal due to the non-specific binding of proteins for the bioassay carried out at room temperature and a LLOD of 0.01 ng/mL for p53 was observed using the MAB technique. The LLOD for the fluorescence-based detection using the MAB technique was found to be 0.01 ng/mL. The use of circular bioassay platforms in the MAB technique results in microwave-induced temperature gradient, where the specific protein binding interactions are significantly accelerated; thereby reducing the background signal and the lower limit of detection of p53 protein.

Circular Bioassay Platforms for Applications in Microwave-Accelerated Techniques.

In this paper, we present the design of four different circular bioassay platforms, which are suitable for homogeneous microwave heating, using theoretical calculations (i.e., COMSOL™ multiphysics software). Circular bioassay platforms are constructed from poly(methyl methacrylate) (PMMA) for optical transparency between 400-800 nm, has multiple sample capacity (12, 16, 19 and 21 wells) and modified with silver nanoparticle films (SNFs) to be used in microwave-accelerated bioassays (MABs). In addition, a small monomode microwave cavity, which can be operated with an external microwave generator (100 W), for use with the bioassay platforms in MABs is also developed. Our design parameters for the circular bioassay platforms and monomode microwave cavity during microwave heating were: (i) temperature profiles, (ii) electric field distributions, (iii) location of the circular bioassay platforms inside the microwave cavity, and (iv) design and number of wells on the circular bioassay platforms. We have also carried out additional simulations to assess the use of circular bioassay platforms in a conventional kitchen microwave oven (e.g., 900 W). Our results show that the location of the circular bioassay platforms in the microwave cavity was predicted to have a significant effect on the homogeneous heating of these platforms. The 21-well circular bioassay platform design in our monomode microwave cavity was predicted to offer a homogeneous heating pattern, where inter-well temperature was observed to be in between 23.72-24.13°C and intra-well temperature difference was less than 0.21°C for 60 seconds of microwave heating, which was also verified experimentally.

De-crystallization of Uric Acid Crystals in Synovial Fluid Using Gold Colloids and Microwave Heating.

In this study, we demonstrated a unique application of our Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC) technique for the de-crystallization of uric acid crystals, which causes gout in humans when monosodium urate crystals accumulate in the synovial fluid found in the joints of bones. Given the shortcomings of the existing treatments for gout, we investigated whether the MA-MAEC technique can offer an alternative solution to the treatment of gout. Our technique is based on the use of metal nanoparticles (i.e., gold colloids) with low microwave heating to accelerate the de-crystallization process. In this regard, we employed a two-step process; (i) crystallization of uric acid on glass slides, which act as a solid platform to mimic a bone, (ii) de-crystallization of uric acid crystals on glass slides with the addition of gold colloids and low power microwave heating, which act as "nano-bullets" when microwave heated in a solution. We observed that the size and number of the uric acid crystals were reduced by >60% within 10 minutes of low power microwave heating. In addition, the use of gold colloids without microwave heating (i.e. control experiment) did not result in the de-crystallization of the uric acid crystals, which proves the utility of our MA-MAEC technique in the de-crystallization of uric acid.

Design and Proof-of-Concept Use of a Circular PMMA Platform with 16-Well Sample Capacity for Microwave-Accelerated Bioassays.

We demonstrate the design and the proof-of-concept use of a new, circular poly(methyl methacrylate)-based bioassay platform (PMMA platform), which affords for the rapid processing of 16 samples at once. The circular PMMA platform (5 cm in diameter) was coated with a silver nanoparticle film to accelerate the bioassay steps by microwave heating. A model colorimetric bioassay for biotinylated albumin (using streptavidin-labeled horse radish peroxidase) was performed on the PMMA platform coated with and without silver nanoparticles (a control experiment), and at room temperature and using microwave heating. It was shown that the simulated temperature profile of the PMMA platform during microwave heating were comparable to the real-time temperature profile during actual microwave heating of the constructed PMMA platform in a commercial microwave oven. The model colorimetric bioassay for biotinylated albumin was successfully completed in ~2 min (total assay time) using microwave heating, as compared to 90 min at room temperature (total assay time), which indicates a ~45-fold decrease in assay time. Our PMMA platform design afforded for significant reduction in non-specific interactions and low background signal as compared to non-silvered PMMA surfaces when employed in a microwave-accelerated bioassay carried out in a conventional microwave cavity.

Crystallization of Amino Acids on a 21-well Circular PMMA Platform using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

We describe the design and the use of a circular poly(methyl methacrylate) (PMMA) crystallization platform capable of processing 21 samples in Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC). The PMMA platforms were modified with silver nanoparticle films (SNFs) to generate a microwave-induced temperature gradient between the solvent and the SNFs due to the marked differences in their physical properties. Since amino acids only chemisorb on to silver on the PMMA platform, SNFs served as selective and heterogeneous nucleation sites for amino acids. Theoretical simulations for electric field and temperature distributions inside a microwave cavity equipped with a PMMA platform were carried out to determine the optimum experimental conditions, i.e., temperature variations and placement of the PMMA platform inside a microwave cavity. In addition, the actual temperature profiles of the amino acid solutions were monitored for the duration of the crystallization experiments carried out at room temperature and during microwave heating. The crystallization of five amino acids (L-threonine, L-histidine, L-leucine, L-serine and L-valine HCl) at room temperature (control experiment) and using MA-MAEC were followed by optical microscopy. The induction time and crystal growth rates for all amino acids were determined. Using MA-MAEC, for all amino acids the induction times were significantly reduced (up to ~8-fold) and the crystal growth rates were increased (up to ~50-fold) as compared to room temperature crystallization, respectively. All crystals were characterized by Raman spectroscopy and powder x-ray diffraction, which demonstrated that the crystal structures of all amino acids grown at room temperature and using MA-MAEC were similar.

Rapid crystallization of L-arginine acetate on engineered surfaces using metal-assisted and microwave-accelerated evaporative crystallization().

We report the application of our newly described crystallization technique, which employs silver island films (SIFs) and microwave heating, to rapid crystallization of L-arginine acetate (LAA). Using our technique, LAA crystals (~ 1.2 mm in length) were grown from a 20 µl solution in 1 min on surface functionalized SIFs. In control experiments (glass slides and at room temperature) the growth of LAA crystals (0.1-0.3 mm) took ~ 55 min.

Rapid and Selective Crystallization of Acetaminophen using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

In this paper, we demonstrate the application of Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC) technique to rapid and selective crystallization of a small drug compound. i.e. acetaminophen. Subsequent characterization of the crystals by optical microscopy, powder X-ray diffraction (PXRD) and Raman spectroscopy showed quantitatively selective growth of different crystal forms at various experimental conditions. Acetaminophen crystals were grown predominantly as Form I (99%) on blank glass slides at room temperature. Form II crystals with 39% purity grown on SIFs using microwave energy.

Crystallization of l-alanine in the presence of additives on a circular PMMA platform designed for metal-assisted and microwave-accelerated evaporative crystallization.

Crystallization of l-alanine in the presence of l-valine and l-tryptophan additives on a circular poly(methyl) methacrylate (PMMA) platform designed for Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC) technique was investigated. Theoretical simulations predicted homogeneous temperature and electric field distributions across the circular PMMA platforms during microwave heating. Crystallization of l-alanine with and without additives on the blank and silver nanoparticle films (SNFs) modified sides of the circular PMMA platform occurred within 32-50 min using MA-MAEC technique, while the identical solutions crystallized within 161-194 min at room temperature. Optical microscopy studies revealed that l-alanine crystals without additives were found to be smaller in size and had several well-developed faces, whereas l-alanine crystals grown with additives appeared to be larger and had only one dominant highly-developed face. Raman spectroscopy and powder X-ray diffraction (XRD) measurements showed that all l-alanine crystals had identical peaks, despite the morphological differences between the l-alanine crystals with and without additives observed by optical microscope images.