Jhony Orbulescu

Strong and Selective Adsorption of Lysozyme on Graphene Oxide

Biosensing methods and devices using graphene oxide (GO) have recently been explored for detection and quantification of specific biomolecules from body fluid samples, such as saliva, milk, urine, and serum. For a practical diagnostics application, any sensing system must show an absence of nonselective detection of abundant proteins in the fluid matrix. Because lysozyme is an abundant protein in these body fluids (e.g., around 21.4 and 7 μg/mL of lysozyme is found in human milk and saliva from healthy individuals, and more than 15 or even 100 μg/mL in patients suffering from leukemia, renal disease, and sarcoidosis), it may interfere with detections and quantification if it has strong interaction with GO. Therefore, one fundamental question that needs to be addressed before any development of GO based diagnostics method is how GO interacts with lysozyme. In this study, GO has demonstrated a strong interaction with lysozyme. This interaction is so strong that we are able to subsequently eliminate and separate lysozyme from aqueous solution onto the surface of GO. Furthermore, the strong electrostatic interaction also renders the selective adsorption of lysozyme on GO from a mixture of binary and ternary proteins. This selectivity is confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fluorescence spectroscopy, and UV–vis absorption spectroscopy.

Coumaryl crown ether based chemosensors: Selective detection of saxitoxin in the presence of sodium and potassium ions

Two novel fluorescent chemosensors in which an aza-crown is linked to 4-coumaryl fluorophores by a methylene spacer have been synthesized for sensing saxitoxin. Fluorescence enhancement was observed upon binding of the dicationic toxin molecule, whereas several metal ions produced no effect.

Photothermal ablation of amyloid aggregates by gold nanoparticles.

Amyloid peptide (Abeta) is found in the brain and blood of both healthy and diseased individuals alike. However, upon secondary structure transformation to a beta-sheet dominated conformation, the protein aggregates. These aggregates accumulate to form neuritic plaques that are implicated in the pathogenesis of Alzheimers disease. Gold nanoparticles are excellent photon-thermal energy converters. The extinction coefficient of the surface plasmon band of gold nanoparticles is very large when compared to typical organic dyes. In this study, gold nanoparticle-Abeta conjugates were prepared and the photothermal ablation of amyloid peptide aggregates by laser irradiation was studied. Monofunctional gold nanoparticles were prepared using a recently reported solid phase modification method and then coupled to fragments of Abeta peptide, namely Abeta(31-35) and Abeta(25-35). The conjugates were then mixed with Abeta fragments in solution. The aggregated peptide formation was studied by a series of spectroscopic and microscopic techniques. The peptide aggregates were then irradiated by a continuous laser. With gold nanoparticle-Abeta conjugates present the aggregates were destroyed by photothermal ablation. Gold nanoparticles without Abeta conjugation were not incorporated into the aggregates and when irradiated did not result in photothermal ablation. With gold nanoparticle-Abeta conjugates the ablation was selective to the site of irradiation and minimal damage was observed as a result of thermal diffusion. In addition to the application of photoablation to a protein-based sample the nanoparticles and the chemistry involved provide an easily monofunctionalized photothermal material for the biological conjugation.

Surface Chemistry and Spectroscopy of UG8 Asphaltene Langmuir Film, Part 1

While there has been much focus on asphaltenes in toluene, there has been much less focus on asphaltenes in other solvents. It is important to quantify characteristics of asphaltenes in solvents besides toluene in order to better assess their molecular architecture as well as their fundamental aggregation characteristics. The present work focuses on the investigation of UG8 asphaltene Langmuir films at the air-water interface using chloroform as spreading solvent. The results are compared to the results recently obtained using toluene as spreading solvent. Surface pressure-area isotherms and UV-vis spectroscopy indicate that asphaltenes form smaller nanoaggregates in chloroform than in toluene in similar concentration ranges. Still these nanoaggreates share common features with those in toluene. From the surface pressure-area and compression-decompression isotherms, Brewster angle microscopy, and p-polarized infrared reflection-absorption spectroscopy, it was concluded that small size aggregates are spread on the water surface and the compression of the film leads to formation of large aggregates. The films (Langmuir-Schaefer and Langmuir-Blodgett) studied by atomic force microscopy reveal the existence of nanoaggregates spread on the water surface that coexist with large aggregates formed during compression. In addition to these findings, the spreading solvent, chloroform, allows the determination of asphaltene absorption bands using in situ UV-vis spectroscopy at the air-water interface after 15 min waiting time period. The absorbance data carried out after waiting a time period of 1 h shows similar features with the ones carried out after only 15 min; therefore, there is no need to wait 1 h as in the case when toluene is used as spreading solvent. A comparison of the data obtained from chloroform and toluene shows that smaller aggregate sizes are obtained from chloroform as suggested from the surface pressure-area isotherm, in situ UV-vis spectroscopy, and atomic force microscopy. Nevertheless, the similarity of these nanoaggregates in different solvents suggests this formation is a fundamental property of asphaltenes. Moreover, the lack of the isolated absorption band for one-ring aromatics and only a small peak for two-ring aromatics in the UV spectrum of asphaltenes indicate that these groups are not present in asphaltenes in significant quantities.

Rat osseous plate alkaline phosphatase as Langmuir monolayer : An infrared study at the air-water interface

A glycosylphosphatidylinositol (GPI)-anchored enzyme (rat osseous plate alkaline phosphatase-OAP) was studied as monolayer (pure and mixed with lipids) at the air-water interface. Surface pressure and surface potential-area isotherms showed that the enzyme forms a stable monolayer and exhibits a liquid-expanded state even at surface pressure as high as 30 mN m(-1). Isotherms for mixed dimyristoylphosphatidic acid (DMPA)-OAP monolayer showed the absence of a liquid-expanded/liquid-condensed phase transition as observed for pure DMPA monolayer. In both cases, pure or mixed monolayer, the enzyme preserves its native conformation under compression at the air-water interface as observed from in situ p-polarized light Fourier transform-infrared reflection-absorption spectroscopic (FT-IRRAS) measurements. Changes in orientation and conformation of the enzyme due to the presence or absence of DMPA, as well as due to the surface compression, are discussed.

Antibody–gold quantum dot–PAMAM dendrimer complex as an immunoglobulin immunoassay

Gold quantum dots (AuQDs) were synthesized and electrostatically conjugated to goat-derived anti-human IgG for the purpose of detecting human IgG in solution over a broad range of concentrations. The system is able to detect human IgG by linear fluorescence quenching over a micromolar to nanomolar concentration range. We have demonstrated the specificity and a wide dynamic range of the proposed immunoassay. The quenching is a result of competitive surface quenching of the AuQDs. Characterization, details of the immunoassay, and the quenching mechanism, are discussed.

Surface chemistry and spectroscopy of human insulin Langmuir monolayer.

The human insulin (HI) protein was examined to elucidate its structure at the air-water interface. Optimal experimental conditions were determined to prepare a homogeneous and stable human insulin (HI) Langmuir monolayer. HI insulin Langmuir monolayer can be used to study interactions of HI with a membrane as Langmuir monolayers are used as an in vitro model of biological membranes. Surface pressure and surface potential-area isotherms were used to characterize the HI Langmuir monolayer. The compression-decompression cycles and stability measurements showed a homogeneous and stable monolayer at the air-water interface. However, higher surface pressures resulted in a higher decrease in area and less stability. In situ UV-vis and fluorescence spectroscopy were used to verify the homogeneity of the HI monolayer and to identify the chromophore residues in the HI. Domain formation was examined through epifluorescence and Brewster angle microscopies. The conformation of HI was examined by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the aqueous phase and at the air-water interface by infrared reflection absorption spectroscopy (IRRAS). HI was found to exist as a monomer in 2-D.

Human insulin fibril-assisted synthesis of fluorescent gold nanoclusters in alkaline media under physiological temperature

Fluorescent insulin fibrils gold nanoclusters (Au NCs) have been synthesized through the reduction of gold by human insulin in fibrillated form. Likewise, nanocluster formation has been regulated by insulin, working as a protein-based template. Environment- and surface-controlled experiments have shown the optimized synthesis conditions is comprised of a pure aqueous alkaline solvent for insulin under constant heat at physiological temperature (37°C) prior to addition of the Au precursor (HAuCl4), followed by subsequent heating (37°C) and vigorous stirring after the addition of HAuCl4 until the completion of the synthetic approach. Microscopy experiments detected the presence of primordial fibril structures in samples of heated human insulin in the alkaline medium prior to addition of HAuCl4, while encountering more developed insulin fibrils in the terminal production of Au NCs. This investigation provides insight to the development of a novel synthesis of Au NCs in the alkaline medium, while providing a graphical description of the environmental and surface-dependent effects that were presented in the synthesis of human insulin nanoclusters. The study provides pertinent information for future synthetic procedures, as the protein state of several protein-nanoparticle systems may reflect on the results that were obtained herein.

Human cardiac troponin I: a Langmuir monolayer study.

Human cardiac troponin I (cTnI) is the preferred biomarker in the assessment of myocardial infarction. It is known to interact with troponin C and T to form a trimeric complex. Whereas small amounts are found in the cytoplasm, most of cTnI is in the form of a complex with actin located in myofilaments. To understand these interactions of cTnI better, we first investigated the surface chemistry of cTnI as a Langmuir monolayer spread at the air-water interface. We investigated the optimal conditions for obtaining a stable Langmuir monolayer in terms of changing the ionic strength of the subphase using different concentrations of potassium chloride. Monolayer stability was investigated by compressing the cTnI monolayer to a specific surface pressure and keeping the surface pressure constant while measuring the decrease in the molecular area as a function of time. Aggregation and/or domain formation was investigated by using compression-decompression cycles, in situ UV-vis spectroscopy, Brewster angle microscopy (BAM), and epifluorescence microscopy. To ensure that the secondary structure is maintained, we used infrared reflection-absorption spectroscopy (IRRAS) directly at the air-subphase interface. It was found that cTnI forms a very stable monolayer (after more that 5000 s) that does not aggregate at the air-subphase interface. The cTnI molecules maintain their secondary structure and, on the basis of the low reflectivity observed using BAM measurements and the low reflection-absorption intensities measured with IRRAS spectroscopy, lie flat on the subphase with the alpha-helices parallel to the air-subphase interface.

Surface Chemistry Studies of Quantum Dots (QDs) Modified with Surfactants

Since luminescent CdSe quantum dots (QDs) have shown great potential in biological labeling, the surface chemistry behavior of QDs at interfaces is of great research interest. In the present study, CdSe QDs with green luminescence were modified with hydrophobic chains of varying lengths [from C6 to C18]. These modified QDs can be utilized to form stable monolayers at the air/water interface. Surface pressure-area isotherms of modified QDs have been measured and limiting molecular areas have also been extrapolated in order to analyze the size of the QDs. UV absorption spectra of modified QDs at various surface pressures were also determined. Surface chemistry, as well as the topographic properties, of modified QDs in Langmuir and L-B films was discussed.