A colorimetric immunoassay for the detection of human vascular endothelial growth factor 165 (VEGF165) based on anti-VEGF-iron oxide nanoparticle conjugation.

Vascular endothelial growth factor (VEGF) is an indispensable element in many physiological processes, while alterations in its level in the circulating system are signs of pathology-associated diseases. Therefore, its precise and selective detection is critical for clinical applications to monitor the progression of the pathology. In this study, an optical immunoassay biosensor was developed as a model study for detecting recombinant VEGF165. The VEGF165 sample was purified from recombinant Kluyveromyces lactis GG799 yeast cells. Indirect ELISA was used during the detection, wherein iron oxide nanoparticles (FeNPs) were utilized to obtain optical signals. The FeNPs were synthesized in the presence of lactose p-amino benzoic acid (LpAB). VEGF165 antibody was conjugated to the LpAB-FeNPs through EDC/NHS chemistry to convert the iron oxide nanoparticles into VEGF165 specific probes. The specificity of the prepared system was tested in the presence of potential serum-based interferents (i.e., glucose, urea, insulin, C-reactive protein, and serum amyloid A), and validation studies were performed in a simulated serum sample. The proposed immunoassay showed a wide detection range (0.5 to 100 ng/mL) with a detection limit of 0.29 ng/mL. These results show that the developed assay could offer a sensitive, simple, specific, reliable, and high-throughput detection platform that can be used in the clinical diagnostics of VEGF.

Identification of Marker Molecules in Aqueous Plant Extracts Affecting the Gold Nanostructures' Morphology and Size.

This work was performed as a comparative study using nine different aqueous pollen grain extracts from eight different genera (Juniperus, Biota, Cupressus, Abies, Pinus, Cedrus, Populus and Corylus) to synthesize gold nanostructures (AuNSs) to understand if there is any possible marker that helps to predict the final morphology and size of the AuNSs. Principal component analysis (PCA) revealed that Apigenin and Pinoresinol compounds are the marker molecules in determination of the AuNSs physical characteristics while total protein, reducing carbohydrate, flavonoid and phenol contents did not show any statistically meaningful outcome. The "dominancy hypothesis" was tested by paying attention to the most concentrated phenolic acids and flavonoids in the control of AuNSs morphology and size, for which correlation analysis were performed. The statistical findings were tested using two new more pollen extracts to validate the models. Three main findings of the study were (i) determination of Apigenin and Pinoresinol levels in pollen extract can give an insight into the AuNSs physical characters, (ii) the most concentrated phenolic acids and flavonoids don't need to be same to pose same dictative effect on AuNSs morphology and size, rather relatively abundant ones in the extract play the key role and (iii) differences in the polymeric structures (e. g. lignin, cellulosic compounds etc.) have minor effect on the final morphology and size of the AuNSs.

Sugar-Lectin Interactions for Direct and Selective Detection of Escherichia coli Bacteria Using QCM Biosensor.

Pathogenic Escherichia coli (E. coli) remains a safety concern in the preservation and quality of green leafy vegetables. Sugar-lectin interactions provide a reliable, specific, and effective sensing platform for the detection of bacteria as compared to the tedious conventional plate counting technique. Herein, we present the synthesis of 4-(N-mannosyl) benzoic acid (4-NMBA) and 4-thiophenyl-N-mannose (4-TNM) via a two-step reductive amination for the detection of E. coli using a quartz crystal microbalance (QCM) biosensor. The 4-NMBA was synthesized with mannose and para-aminobenzoic (4-PBA), while the 4-TNM was synthesized with mannose and 4-aminophenyl disulfide (4-AHP) using water and acetic acid in a 1:1 ratio. The resultant structure of mannose derivatives (4-NMBA and 4-TNM) was characterized and confirmed using analytical tools, such as Mass Spectrometer, SEM, and FTIR. The choice of ligands (mannose derivatives) is ascribed to the specific recognition of mannose to the FimH lectin of the type 1 pilus of E. coli. Furthermore, the 4-PBA and 4-AHP conjugated to mannose increase the ligand affinity to FimH lectins. The setup of the QCM biosensor was composed of modification of the crystal surface and the covalent attachment of ligands for the detection of E. coli. The piezoelectric effect (frequency shift of the quartz) was proportional to the change in mass added to the gold crystal surface. Both the 4-NMBA- and 4-TNM-coated QCM sensors had a limit of detection of 3.7 CFU/mL and 6.6 CFU/mL with a sensitivity of 2.56 × 103 ng/mL and 8.99 × 10-5 ng/mL, respectively, within the dynamic range of 103 to 106 CFU/mL. This study demonstrates the application of ligand-coated QCM biosensors as a cost-effective, simple, and label-free technology for monitoring pathogenic bacteria via molecular interactions on crystal surfaces.

Carbon Quantum Dots Conjugated Rhodium Nanoparticles as Hybrid Multimodal Contrast Agents.

Nanoparticle (NP)-based contrast agents enabling different imaging modalities are sought for non-invasive bio-diagnostics. A hybrid material, combining optical and X-ray fluorescence is presented as a bioimaging contrast agent. Core NPs based on metallic rhodium (Rh) have been demonstrated to be potential X-ray Fluorescence Computed Tomography (XFCT) contrast agents. Microwave-assisted hydrothermal method is used for NP synthesis, yielding large-scale NPs within a significantly short reaction time. Rh NP synthesis is performed by using a custom designed sugar ligand (LODAN), constituting a strong reducing agent in aqueous solution, which yields NPs with primary amines as surface functional groups. The amino groups on Rh NPs are used to directly conjugate excitation-independent nitrogen-doped carbon quantum dots (CQDs), which are synthesized through citrate pyrolysis in ammonia solution. CQDs provided the Rh NPs with optical fluorescence properties and improved their biocompatibility, as demonstrated in vitro by Real-Time Cell Analysis (RTCA) on a macrophage cell line (RAW 264.7). The multimodal characteristics of the hybrid NPs are confirmed with confocal microscopy, and X-ray Fluorescence (XRF) phantom experiments.

On the Effect of Modified Carbohydrates on the Size and Shape of Gold and Silver Nanostructures.

Gold (Au) and silver (Ag) nanostructures have widespread utilization from biomedicine to materials science. Therefore, their synthesis with control of their morphology and surface chemistry have been among the hot topics over the last decades. Here, we introduce a new approach relying on sugar derivatives that work as reducing, stabilizing, and capping agents in the synthesis of Au and Ag nanostructures. These sugar derivatives are utilized alone and as mixture, resulting in spherical, spheroid, trigonal, polygonic, and star-like morphologies. The synthesis approach was further tested in the presence of acetate and dimethylamine as size- and shape-directing agents. With the use of transmission electron microscopy (TEM), selected area electron diffraction (SAED), x-ray diffraction (XRD), scanning electron microscopy (SEM), and ultraviolet-visible (UV-vis) absorption spectroscopy techniques, the particle size, shape, assembly, aggregation, and film formation characteristics were evaluated. NPs' attributes were shown to be tunable by manipulating the sugar ligand selection and sugar ligand/metal-ion ratio. For instance, with an imine side group and changing the sugar moiety from cellobiose to lactose, the morphology of the Ag nanoparticles (NPs) transformed from well dispersed cubic to rough and aggregated. The introduction of acetate and dimethylamine further extended the growth pattern and morphological properties of these NPs. As examples, L5 AS, G5AS, and S5AS ligands formed spherical or sheet-like structures when used alone, which upon the use of these additives transformed into larger multicore and rough NPs, revealing their significant effect on the NP morphology. Selected samples were tested for their stability against protein corona formation and ionic strength, where a high chemical stability and resistance to protein coating were observed. The findings show a promising, benign approach for the synthesis of shape- and size-directed Au and Ag nanostructures, along with a selection of the chemistry of carbohydrate-derivatives that can open new windows for their applications.

Size and Shape-Dependent Antimicrobial Activities of Silver and Gold Nanoparticles: A Model Study as Potential Fungicides.

Plant-based pathogenic microbes hinder the yield and quality of food production. Plant diseases have caused an increase in food costs due to crop destruction. There is a need to develop novel methods that can target and mitigate pathogenic microbes. This study focuses on investigating the effects of luteolin tetraphosphate derived silver nanoparticles (LTP-AgNPs) and gold nanoparticles (LTP-AuNPs) as a therapeutic agent on the growth and expression of plant-based bacteria and fungi. In this study, the silver and gold nanoparticles were synthesized at room temperature using luteolin tetraphosphate (LTP) as the reducing and capping agents. The synthesis of LTP-AgNPs and LTP-AuNP was characterized by Transmission Electron Microscopy (TEM) and size distribution. The TEM images of both LTP-AgNPs and LTP-AuNPs showed different sizes and shapes (spherical, quasi-spherical, and cuboidal). The antimicrobial test was conducted using fungi: Aspergillus nidulans, Trichaptum biforme, Penicillium italicum, Fusarium oxysporum, and Colletotrichum gloeosporioides, while the class of bacteria employed include Pseudomonas aeruginosa, Aeromonas hydrophila, Escherichia coli, and Citrobacter freundii as Gram (-) bacteria, and Listeria monocytogenes and Staphylococcus epidermidis as Gram (+) bacterium. The antifungal study demonstrated the selective size and shape-dependent capabilities in which smaller sized spherical (9 nm) and quasi-spherical (21 nm) AgNPs exhibited 100% inhibition of the tested fungi and bacteria. The LTP-AgNPs exhibited a higher antimicrobial activity than LTP-AuNPs. We have demonstrated that smaller sized AgNPs showed excellent inhibition of A. nidulans growth compared to the larger size nanoparticles. These results suggest that LTP-AuNP and LTP-AgNPs could be used to address the detection and remediation of pathogenic fungi, respectively.

Antimicrobial Activity of a New Class of Phosphorylated and Modified Flavonoids.

The surge of resistant food pathogens is a major threat worldwide. Previous research conducted on phytochemicals has shown their antibacterial activity against pathogenic bacteria. The design of antimicrobial agents to curb pathogenic disease remains a challenge demanding critical attention. Flavonoids such as apigenin and quercetin were evaluated against Gram-positive and Gram-negative bacteria. The results indicated that the antibacterial activity of each flavonoid occurred at a different minimum inhibitory concentration. However, the antimicrobial activity results of the modified flavonoids were also reported, and it was observed that the Gram-positive bacteria were more susceptible in comparison to the Gram-negative bacteria. The cell wall structure of the Gram-positive and Gram-negative bacteria could be the main reason for the bacteria susceptibility. Modified flavonoids could be used as a suitable alternative antimicrobial agent for the treatment of infectious diseases. Our results indicated 100% inhibition of Listeria monocytogenes, Pseudomonas aeruginosa, and Aeromonas hydrophila with modified flavonoids.

Objective clinical pain analysis using serum cyclooxygenase-2 and inducible nitric oxide synthase in American patients.

BACKGROUND: Pain is a multidimensional condition of multiple origins. Determining both intensity and underlying cause are critical for effective management. Utilization of painkillers does not follow any guidelines relying on biomarkers, which effectively eliminates objective treatment. The aim of this study was to evaluate the use of serum cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as pain biomarkers. This work could significantly advance the diagnosis and treatment of pain. METHODS: We assessed the potential utility of serum COX-2 and iNOS as objective measures of pain in a sample of American patients. Pain was scaled between level 0-5 in accordance with the level reported by the patients. Blood samples were collected from 102 patients in the emergency room. Sandwich ELISA was used to determine the COX-2 and iNOS levels in the blood serum while statistical analysis was performed using Pearson product-moment correlation coefficients, Regression and Receiver Operating Characteristics (ROC) analyses. The biomarker results were also compared with self-reports of pain by the patients using conventional pain ratings and patients were asked to report the cause of the pain. Pain levels were clustered into four groups as 0 [self-reported 0], 1 [self-reported as 1], 2 [self-reported as 2 and 3] and 3 [self-reported as 4 and 5]. Co-expression of COX-2 and iNOS could significantly alter pain development and its sensitization. Therefore, iNOS dependent COX-2 levels were employed as categorized level. RESULTS: Self-reported pain levels did not show a correlation with the serum level of COX-2 and iNOS. The lack of correlation is attributed to multiple reasons including patients' intake of painkillers prior to participation, painkiller intake habit, chronic diseases, and subjectivity of self-reported pain. Increased serum COX-2 levels were reported in relation to the subtypes of these health issues. Further, 83% of the patients who reported pain also showed the presence of COX-2 in serum, while only 53% of the patients showed the presence of iNOS in serum. Moderate relation was found between the clustered pain level and categorized COX-2 and iNOS- levels. CONCLUSIONS: The findings support the requirement of further studies to use COX-2 and iNOS as prognostic biomarkers for objective quantification of pain at the clinical level.

Selective Sensing and Imaging of Penicillium italicum Spores and Hyphae Using Carbohydrate-Lectin Interactions.

The blue-green mold Penicillium italicum is among the most problematic post-harvest plant infections limiting the integrity of citrus and many other crops during storage and transportation, but there is no sensor for its on-site or field detection. We hereby, for the first time, report the development of novel biomolecular sensor for assessing the presence of P. italicum spores and hyphae using carbohydrate-lectin recognitions. Two approaches were developed: (i) lateral tests using standalone poly(amic) acid (PAA) membranes and glass surfaces and (ii) quantitative tests on 96-well polystyrene plates and paper electrodes. In both cases, the surfaces were functionalized with novel derivatized sugar based ligands while staining was performed with gold nanoparticles. Both approaches provided strong signals for 104 spores/mL of P. italicum isolated from experimentally infected lemons as the lowest-reliable concentration. The 96-well plate-based gave the most sensitive detection with a 4 × 102 spores/mL limit of detection, a linear dynamic range between 2.9 × 103 and 6.02 × 104 spores/mL ( R2 = 0.9939) and standard deviation of less than 5% for five replicate measurements. The selectivity of the ligands was tested against Trichaptum biforme, Glomerulla cingulata ( Colletotrichum gloeosporioides), and Aspergillus nidulans fungi species. The highest selectivity was obtained using the sugar-based gold-nanoparticles toward both the spores and the hyphae of P. italicum. The advanced specificity was provided by the utilized sugar ligands employed in the synthesis of gold nanoparticles and was independent from size and shapes of the AuNPs. Accuracy of the sensor response showed dramatic dependence on the sample preparation. In the case of 5-10 min centrifugation at 600 rpm, the spores can be isolated free from hyphae and conidiophore, for which spiked recovery was up to 95% (std ±4). In contrast, for gravity-based precipitation of hyphae, the spiked recovery was 88% (std 11).

Flavonoid-derived anisotropic silver nanoparticles inhibit growth and change the expression of virulence genes in Escherichia coli SM10.

We hereby present a novel greener and ecofriendly synthesis of anisotropic silver nanoparticles (AgNPs) using water soluble quercetin diphosphate (QDP). QDP was employed as a reducing, capping and stabilizing agent at room temperature without any extraneous reagents. The purpose of this study was to determine the effects of modified quercetin pentaphosphate silver nanoparticles (QPP-AgNPs) and quercetin diphosphate derived silver nanoparticles (QDP-AgNPs) on microbial growth and expressions of virulence-related genes in Escherichia coli SM10. The gene expression analysis was carried out for 12 genes which are related to virulence and stress in E. coli SM10, namely: RpoD, RpoS, ibpB, clpB, uspA, fliC, fimH, fimF, kdpE, artJ, hyaA, and gyrA. Results showed that QDP-AgNPs reduced the swarming motility by 98% which correlated with the reduction in the expression of FliC flagellar gene. A simultaneous increase in the expression of the fimbrial genes FimH and FimF that are related to motility was recorded. In contrast, treatment of the microbes with QPP-AgNPs resulted in 90% of the swarming motility at different patterns compared to QDP-AgNPs treatment for the gene expressions of motility elements. The study revealed that QDP-AgNPs up-regulated the stress related RpoD and ibpB expressions, while QPP-AgNPs up-regulated the stress related RpoS and uspA gene expressions. However, both QDP-AgNPs and QPP-AgNPs up-regulated kpdE, artJ and gry at different levels. QDP-AgNPs were also tested for their antibacterial and antifungal activities, which showed µmolar cidal activity. The growth kinetics of both Gram (-) and Gram (+) bacteria were strongly altered by QDP-AgNPs activity. Energy dispersive absorption spectroscopy (EDS) studies revealed that silver ions and/or the nanoparticles themselves transferred into bacterial cells. To the best of our knowledge, this is the first report ofstudying the genetic and kinetic response of bacteria to modified quercetin phosphate mediated silver nanoparticles and we hereby report that the molecules used to synthesize AgNPs bring about a strong effect on AgNPs manipulatory activity on the tested 12-genes.

Modification of chitosan-bead support materials with L-lysine and L-asparagine for α-amylase immobilization.

Maltose syrups have got wide-range utilizations in a variety of applications from bakery to drug-development. α-Amylases are among the most widely utilized industrial enzymes due to their high specificity in production of maltose syrup from starch. However, enzymes are not stable in ex vivo conditions towards alteration in pH, temperature, and such other parameters as high salt concentrations and impurities, where immobilization is required to advance the stability of the enzyme with which approach the requirement of isolation of the enzyme from media is eliminated as well. In this study, Termamyl® α-amylase was immobilized on the none-modified chitosan beads (NMCB), L-lysine-modified chitosan beads (LMCB), and L-asparagine-modified chitosan beads (AMCB) to assess effects of the support material on optimum conditions and kinetic parameters of the α-amylase activity in production of maltose from starch. Immobilization on NMCB, LMCB, and AMCB puts a strong influence on optimum pH, optimum temperature, stability, and kinetic parameters of α-amylase. Modification of chitosan beads with L-lysine and L-asparagine dramatically altered the overall immobilization yield, and enzyme's response to pH and temperature variations and the kinetic parameters. AMCB provided the best immobilization yield (49%), while LMCB only improved the yield by 2% from 22 to 24%.

Seedless synthesis and SERS characterization of multi-branched gold nanoflowers using water soluble polymers.

We report for the first time, the aqueous-based synthesis of multibranched, monodispersed gold nanoflowers (AuNFs) using pyromellitic dianhydride-p-phenylene diamine - PPDDs at room temperature. AuNF synthesis was achieved using PPDDs that converts Au precursor (Au3+) into AuNFs while serving as both the reducing and directional agent. The resulting branched AuNFs exhibited different degrees of anisotropy and protuberance lengths obtained by modulating the ratio of PPDDs and HAuCl4·3H2O. The surface roughness obtained ranged from small bud-like protuberances to elongated spikes, which enabled the tuning of the optical properties of the nanoparticles from ∼450 to 1100 nm. Systematic analysis revealed that the generation of urchin-like particles as well as their size depended on the PPDDs/HAuCl4·3H2O ratio. At a medium concentration of the precursor, spherical nanoparticles were formed. Whereas at lower precursor concentrations, urchin-like nanoparticles were obtained with their size and protuberances length increasing at even lower HAuCl4·3H2O concentration. Increasing the temperature to 100 °C resulted in the enhancement of the anisotropy of the AuNFs. The resulting gold nanoflowers exhibited an enhanced performance in surface-enhanced Raman scattering (SERS). This work provides a unique approach for anisotropic particle synthesis using water soluble polymer and greener approaches. The fabricated AuNFs exhibited variable UV-vis absorption and SERS enhancement as a function of branch morphology, indicating their potential application in biolabeling, biosensing, imaging, and therapeutic applications.

Synthesis and antibacterial characterization of sustainable nanosilver using naturally-derived macromolecules.

Greener nanosynthesis utilizes fewer amounts of materials, water, and energy; while reducing or replacing the need for organic solvents. A novel approach is presented using naturally-derived flavonoids including Quercetin pentaphosphate (QPP), Quercetin sulfonic acid (QSA) and Apigenin Triphosphate (ATRP). These water soluble, phosphorylated flavonoids were utilized both as reducing agent and stabilizer. The synthesis was achieved at room temperature using water as a solvent and it requires no capping agents. The efficiency of the resulting silver nanoparticle synthesis was compared with naturally-occurring flavonoid such as Quercetin (QCR). Results show that QCR reduced Ag(+) faster followed by QPP, QSA and ATRP respectively. This is the first evidence of direct utilization of QCR for synthesis of silver nanoparticles (AgNPs) in water. The percentage conversion of Ag(+) to Ag(0) was determined to be 96% after 35min. The synthesized nanoparticles were characterized using Transmission electron microscopy (TEM), Energy dispersive absorption spectroscopy (EDS), UV-vis spectroscopy, High resolution TEM (HR-TEM) with selected area electron diffraction (SAED). The particle sizes ranged from 2 to 80nm with an average size of 22nm and in the case of ATRP, the nanoparticle shapes varied from spherical to hexagonal with dispersed particle size ranging from 2 to 30nm. Crystallinity was confirmed by XRD and the SAED of (111), (200), and the fringes observed in HRTEM images. Results were in agreement with the UV resonance peaks of 369-440nm. The particles also exhibit excellent antibacterial activity against Staphylococcus epidermidis, Escherichia coli and Citrobacter freundii in water.

Biosensor for selective detection of E. coli in spinach using the strong affinity of derivatized mannose with fimbrial lectin.

Escherichia coli (E. coli) contamination in foods and water resources represents a major threat for human health and the environment. This work exploits the strong affinity of mannose-containing oligosaccharides with the fimbrial lectin of E. coli to design novel biosensors. Modified carbohydrate ligands were synthesized by introducing phenyl residues and aliphatic chains to mannose via reductive amination in order to increase both the affinity and selectivity to E. coli compared to other pathogenic bacteria. The synthesized ligands include p-thiolphenyl aminomannose (PTAM), p-carboxyphenyl aminomannose (PCAM), 1-deoxy-1-aminomannopyranoside (DAMP), glucosamine and low molecular weight chitosan bonded to mercapto undecanoic acid. The structures of the ligands were confirmed using (1)H NMR and 1H, (13)C, COZY NMR, and ESI/MS. The ligands were immobilized onto gold electrodes and SPR surfaces using-mercaptoundecanoic acid with glycine as deactivating agent. Two detection mechanisms were tested: (i) metal-enhanced electrochemical detection (MED) and (ii) label-free surface plasmon resonance (SPR) detection. The introduction of phenyl residues and aliphatic side groups to the mannose-containing oligosaccharides produced extremely high affinity for E. coli with detection limit of 1 cfu/mL. The relative selectivity of these ligands for E. coli, Citrobacter freundii, Staphylococcus epidermidis were 100%, 2.6% and 8.6% respectively. The biosensors were validated using spinach leaves at 3.0 cfu/mL. The work provides a generic biosensor for other pathogenic bacteria by enabling multivalent binding, immediate recognition for pathogens as well as inhibition of bacterial growth.