Yeast Surface Display Platform for Rapid Selection of an Antibody Library via Sequential Counter Antigen Flow Cytometry.

Yeast surface display techniques have been increasingly employed as a tool for both the discovery and affinity maturation of antibodies. In this study, we describe the use of yeast surface display for the selection and affinity maturation of antibodies targeted to small molecules (haptens). In this approach, we coupled 4 to 15 sequential cycles of error-prone PCR to introduce heterogeneity into the sequence of an 12F6 scFv antibody that binds to chelated uranium; the resulting full-length constructs were combined to create a yeast-displayed scFv-library with high diversity. We also developed a stringent selection technique utilizing fluorescence-activated cell sorting; this was based on sequentially dropping the target antigen concentration, while concomitantly increasing the concentration of potential cross-reactive haptens in subsequent selection cycles. As a proof of the efficacy this approach, we confirmed that the antibodies identified via this approach retained binding to the target antigen (UO22+ complexed to a chelator), while binding with lesser affinity than the parental scFv to a structurally related haptens (the same chelator complexed to other metal ions). As will be described in this report, these scFv variants perform more efficiently in sensor-based assay than the parental 12F6 antibody. Combining the generation of scFv libraries via error-prone PCR with selection of yeast-displayed antibodies by fluorescence activated cell sorting will provide an efficient new method for the isolation of scFvs and other binding proteins with high affinity and specificity.

Quantitative Analysis of Heavy Metals and Organic Compounds in Soil from Deir Kanoun Ras El Ain Dump, Lebanon.

Recently, there has been a worldwide concern regarding soil contamination by heavy metals and organic compounds, especially in the developing countries including Lebanon that has suffered from solid waste mismanagement for decades. Deir Kanoun Ras El Ain is a village in southern Lebanon that possesses one of the country's worst dumps, and its leachates influx into a running canal that irrigates surrounding agricultural lands. The aim of this study was to determine the levels of some toxic heavy metals and organic compounds in different soil samples collected from the dump and along the canal during winter and summer seasons. Six research sites (four from the dump and two along the canal) were selected, and the soil samples for analysis were collected from a depth of around 10 cm. Heavy metals (lead, cadmium, arsenic, and mercury) and organic compounds (phthalates, bisphenol A, and polyaromatic hydrocarbons) content were determined using atomic absorption and high pressure liquid chromatography, respectively. The conducted research confirmed high levels of contamination in the collected soil samples by both heavy metals and organic compounds. The present study provided evidence that different sampling sites accumulated heavy metals at concentrations that exceeded the average maximum permissible levels for sewage sludge and agricultural land. These findings suggest the need for mitigation measures by the Lebanese authorities and new waste management programs to resolve the problems associated with uncontrolled dumping of solid wastes in Lebanon.

Antimicrobial activity and biocompatibility of slow-release hyaluronic acid-antibiotic conjugated particles.

Here, the aim was to design and use a long-lasting antibiotic release system for prevention of postoperative infections in ophthalmic surgery. Ciprofloxacin and vancomycin-conjugated hyaluronic acid (HA) particles were prepared as drug carriers for sustained release of antibiotics. The antimicrobial effects of the released drugs were determined by disc-diffusion and macro-dilution tests at different times up to 2 weeks. Slow degradable HA particles were obtained with 35.2 wt% degradation within 21 days. The drug loading amount was increased by employing two sequential chemical linking (conjugation, 2C) and one physical absorption loading (A) procedures (2C + A processes) from 148 ± 8 to 355 ± 11 mg/g HA particles for vancomycin. The amounts of vancomycin and ciprofloxacin that were released linearly was estimated as 64.35 ± 7.35 and 25.00 ± 0.68 mg/g, respectively, from drug-conjugated HA particles in 100 h. Antimicrobial studies revealed that antibiotic-conjugated HA particles could inhibit the growth of microorganisms from 1 h to 1 week. The MBC values were measured as 0.25, 4.0, and 0.25 mg/mL against Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, respectively, after 72 h incubation time. Cytotoxicity studies showed no difference between fibroblast growth or corneal thickness after 5 days with or without HA-antibiotic particles. The drug release studies and antimicrobial activity of antibiotic-loaded HA particles with time against various bacteria further revealed that HA particles are very effective in preventing bacterial infections. Likewise, cytotoxicity studies suggest that these particles pose no toxicity to eukaryotic cells, including corneal endothelium.

Targeted and Stimulus-Responsive Delivery of Surfactant to the Oil-Water Interface for Applications in Oil Spill Remediation.

The use of chemical dispersants is a well-established approach to oil spill remediation where surfactants in an appropriate solvent are contacted with the oil to reduce the oil-water interfacial tension and create small oil droplets capable of being sustained in the water column. Dispersant formulations typically include organic solvents, and to minimize environmental impacts of dispersant use and avoid surfactant wastage it is beneficial to use water-based systems and target the oil-water interface. The approach here involves the tubular clay minerals known as halloysite nanotubes (HNTs) that serve as nanosized reservoir for surfactants. Such particles generate Pickering emulsions with oil, and the release of surfactant reduces the interfacial tension to extremely low values allowing small droplets to be formed that are colloidally stable in the water column. We report new findings on engineering the surfactant-loaded halloysite nanotubes to be stimuli responsive such that the release of surfactant is triggered by contact with oil. This is achieved by forming a thin coating of wax to stopper the nanotubes to prevent the premature release of surfactant. Surfactant release only occurs when the wax dissolves upon contact with oil. The system thus represents an environmentally benign approach where the wax coated HNTs are dispersed in an aqueous solvent and delivered to an oil spill whereupon they release surfactant to the oil-water interface upon contact with oil.

Heavy metals and organic compounds contamination in leachates collected from Deir Kanoun Ras El Ain dump and its adjacent canal in South Lebanon.

Environmental pollution generated from uncontrolled dumping is a major problem in Lebanon due to the lack of proper waste management plans. Deir Kanoun Ras El Ain is the village that harbors the worst dumps in Lebanon. Wastewater leachates of this dump influx into an adjacent nearby canal used for irrigation and drinking purposes. The aim of this study is to assess the concentrations of heavy metals (Pb, Cd, As, and Hg) and the presence of organic compounds (phthalates, bisphenol A, and polycyclic aromatic hydrocarbons (PAHs)) in water samples collected from two different sites around the dump and two canal sites during winter and summer seasons. The concentrations of heavy metals were determined using atomic absorption spectrophotometry, while the identification of the extracted organic compounds was performed using High Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS). The carried analyses revealed that water samples collected from dump and canal were heavily polluted by Cd, As, Hg, phthalates, bisphenol A, and PAHs caused by pyrogenic and petrogenic sources. The concentrations of the found heavy metals were far above the maximum tolerable levels set by different guidelines. The findings suggest that the studied water sources are not safe for irrigation and drinking. The serious implications of dumping wastes on the health of inhabitants recall for an immediate employment of efficient waste management policies to resolve this problem.

Investigation of Amphiphilic Polypeptoid-Functionalized Halloysite Nanotubes as Emulsion Stabilizer for Oil Spill Remediation.

Halloysite nanotubes (HNTs), naturally occurring and environmental benign clay nanoparticles, have been successfully functionalized with amphiphilic polypeptoid polymers by surface-initiated polymerization methods and investigated as emulsion stabilizers toward oil spill remediation. The hydrophilicity and lipophilicity balance (HLB) of the grafted polypeptoids was shown to affect the wettability of functionalized HNTs and their performance as stabilizers for oil-in-water emulsions. The functionalized HNTs having relatively high hydrophobic content (HLB = 12.0-15.0) afforded the most stable oil-in-water emulsions containing the smallest oil droplet sizes. This has been attributed to the augmented interfacial activities of polypeptoid-functionalized HNTs, resulting in more effective reduction of interfacial tension, enhancement of thermodynamic propensity of the HNT particles to partition at the oil-water interface, and increased emulsion viscosity relative to the pristine HNTs. Cell culture studies have revealed that polypeptoid-functionalized HNTs are noncytotoxic toward Alcanivorax borkumensis, a dominant alkane degrading bacterium found in the ocean after oil spill. Notably, the functionalized HNTs with higher hydrophobic polypeptoid content (HLB = 12.0-14.3) were shown to induce more cell proliferation than either pristine HNTs or those functionalized with less hydrophobic polypeptoids. It was postulated that the functionalized HNTs with higher hydrophobic polypeptoid content may promote the bacterial proliferation by providing larger oil-water interfacial area and better anchoring of bacteria at the interface.

Enhancement of biocompatibility and carbohydrate absorption control potential of rosmarinic acid through crosslinking into microparticles.

Rosmarinic acid (RA), a bioflavonoid and antioxidant that exists in plants of the Lamiaceae family, was crosslinked into particles as poly(Rosmarinic Acid) (p(RA)) via an emulsion crosslinking method. The particles were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, solid state nuclear magnetic resonance 13C NMR spectroscopy, and thermal gravimetric analysis. The zeta potential values of p(RA) particles were determined at different pHs; the isoelectric point was estimated as pH 1.2. The release of monomeric RA from the particles at 37.5 °C was found to be similar at different pHs, 1.0, 7.4, and 11.0. The effects of p(RA) on hemolysis and coagulation were found to be minimal. The antioxidant activity of p(RA) particles and RA monomer were almost indistinguishable suggesting that p(RA) particles may be used as an antioxidant. On a per weight basis, p(RA) particles were ~66% less cytotoxic to mammalian cells that RA monomer, as assessed using COS-1 cells. In addition, p(RA) was an 8.6-fold stronger inhibitor of α-glycosidase than RA; the IC50s of the monomer and particles were 0.121 and 0.014 mg/mL, respectively. The strong inhibitory effect of p(RA) on α-glycosidase, coupled with its reduced cytotoxicity and antioxidant activity, provide new opportunities for the use of p(RA).

Uranium (VI) detection in groundwater using a gold nanoparticle/paper-based lateral flow device.

The contamination in groundwater due to the presence of uranium is nowadays a subject of concern due to the severe health problems associated with renal failure, genotoxicity and cancer. The standard methods to detect uranium require time-consuming processes and expensive non-portable equipment, so these measurements are rarely performed in-field, which increases the time until water samples are analysed. Furthermore, the few portable methods available do not allow quantitative analysis and the detection limit is often not low enough to reach the recommendations for drinking water (30 ppb or 126 nM of uranium). For the first time, we propose a portable, fast, inexpensive and sensitive paper-based biosensor able to detect in situ U(VI) in water samples: U(VI) selective gold nanoparticle-based lateral flow strips. Antibody-coated gold nanoparticles are used as labels in the proposed lateral flow system because of their biocompatibility; in addition, these nanoparticles provide high sensitivity due to their intense plasmonic effect. The antibody used in the assay recognizes soluble U(VI) complexed to the chelator, 2,9-dicarboxyl-1,10-phenanthroline (DCP). Because of the small size of the U(VI)-DCP complex, this assay employs a competitive format that reaches a limit of detection of 36.38 nM, lower than the action level (126 nM) established by the World Health Organization and the U.S. Environmental Protection Agency for drinking waters.

Polydopamine particles as nontoxic, blood compatible, antioxidant and drug delivery materials.

Herein, the potential biomedical application of poly(3,4-dihyroxyphenyl)ethylamine, (poly(dopamine)-p(DA)) particles is reported. P(DA) particles with the size about 100 nm, 18.05 m2/g specific surface area, and mesoporous structure (7.19 nm pore width) were prepared and shown to be chemically modifiable using chlorosulfonic acid (CSA) and 3-CHloro-2 hydroxypropyl) trimethylammonium chloride solution (CHPACl) to obtain sulfonic acid and quaternary amine group containing modified p(DA) particles, m-p(DA)-CSA and m-p(DA)-CHPACl particles, respectively. The hydrolytic degradation of p(DA) particles at different pHs, including 1, 7.4 and 11, was carried out at 37.5 °C. These degradation studies revealed that p(DA) is slightly degradable at pH 1 and pH 7.4 with weight losses of 13.01 ± 0.08% and 7.26 ± 0.23% in 11 days, respectively. At pH 11, a sustained degradation that is almost linear degradation with time was observed for up to 30 days, with a total weight loss of 21.42 ± 0.88%. Furthermore, p(DA) particles were tested for cell toxicity against COS-1 cells and found non-toxic up to 50 µg/mL with 95.6 ± 4.5% cell viability as compared to 37.5 ± 0.03% for DA molecules. The p(DA) particles and DA were also compared for their ability to inhibit α-glucosidase; both inhibited α-glucosidase inhibition activity a concentration-dependent fashion: at concentrations of 500-4000 µg/mL, p(DA) provided 8.52-27.67% inhibition while DA inhibited 42.8-67.7% over the same concentration range. Furthermore, p(DA) particles were found to be blood compatible e.g., non-hemolytic with 1.87 ± 0.97% hemolysis ratio up to 50 µg/mL concentration and with 86.7% blood clotting index. Interestingly, p(DA) particle can be considered as an effective antioxidant with 33.5 ± 3.9 µg/ mL total phenol content in terms of gallic acid equivalency and 0.89 ± 0. 30 µmol/g trolox equivalent antioxidant capacity (TEAC). Finally, p(DA) particles and their modified forms, m-p(DA)-CSA, and m-p(DA)-CHPACl, were shown to be useful as active agent/drug delivery devices by using acyclovir as a model drug that can be readily loaded into particles and released at longer times at higher amounts for the modified p(DA) particles at physiological conditions.

The Effects of Glaucoma Drainage Devices on Oxygen Tension, Glycolytic Metabolites, and Metabolomics Profile of Aqueous Humor in the Rabbit.

PURPOSE: Glaucoma drainage device (GDD) implantation can lead to corneal decompensation. We evaluated changes over time in oxygen tension and in the metabolic environment of the aqueous humor after GDD implantation in the rabbit eye. METHODS: Ahmed Glaucoma Valves were implanted in the left eyes of eight male New Zealand white rabbits. Right eyes were used as a control. Oxygen tension was measured immediately before surgery and at 1 and 2 months postoperation. Aqueous humor was collected from the surgical and control eyes at 1, 2, and 5 months postoperation. Aqueous humor samples collected at 1 and 5 months postoperation were selected for broad-spectrum metabolomics analysis using ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC TOF-MS). Multivariate analysis methods were used to identify metabolite profiles that separated the surgical and control eye at 1 and 5 months. RESULTS: There was a significant decrease in oxygen tension in aqueous humor of the surgical eyes (9 mm Hg, 95% confidence interval [CI]: -14.7 to -3.5). Differences in the metabolic profiles between the surgical and control eye at 1 and 5 months were observed, as were differences for the surgical eye at 1 and 5 months. In addition, a metabolite profile was identified that differentiated the surgical eyes at 1 and 5 months. CONCLUSION: Changes in the oxygen tension and metabolic intermediates occur within the aqueous humor as early as 1 month after GDD implantation. TRANSLATIONAL RELEVANCE: Corneal decompensation following GDD implantation could be secondary to disruption of the normal aqueous circulation, resulting in hypoxia and an altered metabolic profile. Alterations to the GDD design might minimize aqueous disruption and prevent corneal decompensation.

Bacterial proliferation on clay nanotube Pickering emulsions for oil spill bioremediation.

Halloysites (tubular aluminosilicate) are introduced as inexpensive natural nanoparticles that form and stabilize oil-water emulsions. Pickering emulsification can proceed with energies low enough to be afforded by ocean turbulence and the stability of droplets extends over more than a week. The oil/water interface is shown to be roughened and bacteria, which are added for oil degradation, are better attached to such oil droplets than to droplets without halloysites. The metabolic activity of Alcanivorax borkumensis, alkanotrophic bacteria widely distributed in marine environments, is enhanced by halloysite addition. A halloysite-based dispersant system is therefore environmentally friendly and promising for further optimization. The key elements of the described formulations are natural clay nanotubes, which are abundantly available in thousands of tons, thus making this technology scalable for environmental remediation.

Combining Yeast Display and Competitive FACS to Select Rare Hapten-Specific Clones from Recombinant Antibody Libraries.

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10(6)) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used first to enrich the library between 20- and 100-fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. This selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.

Effect of Two Novel Sustained-Release Drug Delivery Systems on Bleb Fibrosis: An In Vivo Glaucoma Drainage Device Study in a Rabbit Model.

PURPOSE: To evaluate two drug delivery systems, a nonbiodegradable poly(2-hydroxyethyl methacrylate) (P[HEMA]) system with mitomycin C (MMC) and a biodegradable poly(lactic-co-glycolic acid) (PLGA) system with 5-fluorouracil (5-FU) with and without MMC for their ability to reduce fibrosis when attached to an Ahmed glaucoma valve (AGV) and implanted in a rabbit model. METHODS: New Zealand albino rabbits (48) were divided into six equal groups, and AGVs, modified as described below, were implanted in the right eye of each rabbit. The groups included (1) PLGA alone; (2) P(HEMA) plus MMC (6.5 µg); (3) PLGA plus 5-FU (0.45 mg); (4) PLGA plus 5-FU (1.35 mg); (5) PLGA plus 5-FU and MMC (0.45 mg and 0.65 µg, respectively); (6) PLGA plus 5-FU and MMC (1.35 mg and 0.65 µg, respectively). The rabbits were followed for 3 months prior to euthanasia. RESULTS: The bleb wall thickness was significantly less in groups 2, 5, and 6 compared to the rest. At 3 months, the PLGA polymer had completely disappeared, while the P(HEMA) polymer remained intact. There were no statistical differences in the degree of clinically graded conjunctival injection, histologic inflammation, or histologic fibrosis among the six groups. CONCLUSIONS: We successfully created a sustained-release drug delivery system that decreased the postoperative fibrosis using both a nonbiodegradable P(HEMA) polymer and a biodegradable (PLGA) polymer. Both systems appear to work equally well with no side effects. TRANSLATIONAL RELEVANCE: These results are supportive of the antifibrotic effect of the slow-release drug delivery system following glaucoma drainage device implantation, thus paving the way for human pilot studies.

The RPTEC/TERT1 Cell Line as an Improved Tool for In Vitro Nephrotoxicity Assessments.

In earlier studies, we have characterized a newly developed cell line derived from the renal proximal tubule epithelial cells (RPTEC) of a healthy human male donor in order to provide an improved in vitro model with which to investigate human diseases, such as cancer, that may be promoted by toxicant exposure. The RPTEC/TERT1 cell line has been immortalized using the human telomerase reverse transcriptase (hTERT) catalytic subunit and does not exhibit chromosomal abnormalities (Evercyte Laboratories). We have previously conducted single-compound and binary mixture experiments with the common environmental carcinogens, cadmium (Cd), and benzo[a]pyrene (B[a]P). Cells exhibited cytotoxic and compound-specific responses to low concentrations of B[a]P and Cd. We detected responses after exposure consistent with what is known regarding these cells in a normal, healthy kidney including significant gene expression changes, BPDE-DNA adducts in the presence of B[a]P, and indications of oxidative stress in the presence of Cd. The RPTEC/TERT1 cell line was also amenable to co-exposure studies due to its sensitivity and compound-specific properties. Here, we review our earlier work, compare our findings with commonly used renal cell lines, and suggest directions for future experiments. We conclude that the RPTEC/TERT1 cell line can provide a useful tool for future toxicological and mixture studies.

Cadmium alters the formation of benzo[a]pyrene DNA adducts in the RPTEC/TERT1 human renal proximal tubule epithelial cell line.

Previously, we demonstrated the sensitivity of RPTEC/TERT1 cells, an immortalized human renal proximal tubule epithelial cell line, to two common environmental carcinogens, cadmium (Cd) and benzo[a]pyrene (B[a]P). Here, we measured BPDE-DNA adducts using a competitive ELISA method after cells were exposed to 0.01, 0.1, and 1 µM B[a]P to determine if these cells, which appear metabolically competent, produce BPDE metabolites that react with DNA. BPDE-DNA adducts were most significantly elevated at 1 µM B[a]P after 18 and 24 hours with 36.34 +/- 9.14 (n = 3) and 59.75 +/- 17.03 (n = 3) adducts/108 nucleotides respectively. For mixture studies, cells were exposed to a non-cytotoxic concentration of Cd, 1 µM, for 24 hours and subsequently exposed to concentrations of B[a]P for 24 hours. Under these conditions, adducts detected at 1 µM B[a]P after 24 hours were significantly reduced, 17.28 +/- 1.30 (n = 3) adducts/108 nucleotides, in comparison to the same concentration at previous time points without Cd pre-treatment. We explored the NRF2 antioxidant pathway and total glutathione levels in cells as possible mechanisms reducing adduct formation under co-exposure. Results showed a significant increase in the expression of NRF2-responsive genes, GCLC, HMOX1, NQO1, after 1 µM Cd × 1 µM B[a]P co-exposure. Additionally, total glutathione levels were significantly increased in cells exposed to 1 µM Cd alone and 1 µM Cd × 1 µM B[a]P. Together, these results suggest that Cd may antagonize the formation of BPDE-DNA adducts in the RPTEC/TERT1 cell line under these conditions. We hypothesize that this occurs through priming of the antioxidant response pathway resulting in an increased capacity to detoxify BPDE prior to BPDE-DNA adduct formation.

Methylated phenanthrenes are more potent than phenanthrene in a bioassay of human aryl hydrocarbon receptor (AhR) signaling.

Alkylated polycyclic aromatic hydrocarbons (APAHs) are abundant in petroleum, but data regarding their toxicological properties are limited. A survey of all monomethylated phenanthrene structures revealed that they were 2 times to 5 times more potent than phenanthrene for activation of human aryl hydrocarbon receptor in a yeast bioassay. Phenanthrenes with equatorial methyl groups had the greatest potency. The greater potency of the methylated phenanthrenes highlights the need for more toxicological data on APAHs.

A novel antiproliferative drug coating for glaucoma drainage devices.

PURPOSE: The implantation of a glaucoma drainage device (GDD) is often necessary for intractable cases of glaucoma. Currently, the success rate of GDD implants is relatively low because fibrosis that develops during the wound-healing process ultimately blocks fluid drainage. We describe herein a novel porous coating for Ahmed glaucoma valves based on biodegradable poly(lactic-co-glycolic acid) (PLGA). MATERIALS AND METHODS: Thin films of PLGA were fabricated using a spin-coating technique. The procedure led to an asymmetric pore structure that was exploited to control the rate of dissolution. Double-layered porous films were constructed to achieve continuous drug release. A cell culture system was used to test the efficacy of these coatings. RESULTS: Double-layered films were manufactured to provide a burst of mitomycin C (MMC) release followed by a slow release of 5-fluorouracil (5-FU), which together prevented fibrosis over the most active period of postoperative wound healing (0 to 28 d). Double-layered films containing 5-FU only in the bottom layer showed a 3- to 5-day delay in drug release, followed by a sharp increase that continued for ~28 days. MMC was stable only when surface-loaded, and this drug was therefore surface-loaded onto the top PLGA layer to provide a continuous release of antifibrotics over the wound-healing period. CONCLUSIONS: The combined use of both MMC and 5-FU in a biodegradable device inhibits cell proliferation in a tissue culture model and has the potential to reduce fibrosis and increase the success rate of GDD implants. The design is simple and can be scaled for commercial production.

High sensitive gold-nanoparticle based lateral flow Immunodevice for Cd2+ detection in drinking waters.

In this work for first time a lateral flow immunosensor device (LFID) for Cd(2+) determination in drinking and tap waters using the Cd-EDTA-BSA-AuNP conjugate as signal producer tool is introduced. The principle of working is based on competitive reaction between the Cd-EDTA-BSA-AuNP conjugate deposited on the conjugation pad strip and the Cd-EDTA complex formed in the analysis sample for the same binding sites of the 2A81G5 monoclonal antibody, specific to Cd-EDTA but not Cd(2+) free, which is immobilized onto the test line. The device has a large response range within 0.4-2000ppb, being the linear response between 0.4 and 10ppb. The quantification and detection limits of 0.4 and 0.1ppb, respectively, represent the lowest ones reported so far for paper based metal sensors. The obtained detection limit is 50 times lower than the maximum contamination level required for drinking water. Here we also show a new option for increasing the sensibility in the LFDs with competitive format, through the decreasing in concentrations of the Cd-EDTA-BSA-AuNP conjugate deposited in the conjugation strip and the mAbs deposited in the test and control zones until to reach optimized concentrations. It is an important result take into account that the increase in sensibility is one of the challenges in the field of LFD sensors, where are focused many of the ongoing researches. In addition, a specificity study of the device for several metal interferences, where potential metal interferences are masked with the use of the EDTA and OVA optimized concentrations, is presented too.

All-integrated and highly sensitive paper based device with sample treatment platform for Cd2+ immunodetection in drinking/tap waters.

Nowadays, the development of systems, devices, or methods that integrate several process steps into one multifunctional step for clinical, environmental, or industrial purposes constitutes a challenge for many ongoing research projects. Here, we present a new integrated paper based cadmium (Cd(2+)) immunosensing system in lateral flow format, which integrates the sample treatment process with the analyte detection process. The principle of Cd(2+) detection is based on competitive reaction between the cadmium-ethylenediaminetetraacetic acid-bovine serum albumin-gold nanoparticles (Cd-EDTA-BSA-AuNP) conjugate deposited on the conjugation pad strip and the Cd-EDTA complex formed in the analysis sample for the same binding sites of the 2A81G5 monoclonal antibody (mAb), specific to Cd-EDTA but not Cd(2+) free, which is immobilized onto the test line. This platform operates without any sample pretreatment step for Cd(2+) detection thanks to an extra conjugation pad that ensures Cd(2+) complexation with EDTA and interference masking through ovalbumin (OVA). The detection and quantification limits found for the device were 0.1 and 0.4 ppb, respectively, these being the lowest limits reported up to now for metal sensors based on paper. The accuracy of the device was evaluated by addition of known quantities of Cd(2+) to different drinking water samples and subsequent Cd(2+) content analysis. Sample recoveries ranged from 95 to 105% and the coefficient of variation for the intermediate precision assay was less than 10%. In addition, the results obtained here were compared with those obtained with the well-established inductively coupled plasma emission spectroscopy (ICPES) and the analysis of certificate standard samples.

Electrospray ionization-ion mobility spectrometry identified monoclonal antibodies that bind exclusively to either the monomeric or a dimeric form of prostate specific antigen.

Macroion mobility spectrometry was used to distinguish between a monoclonal antibody (clone M612165) that bound exclusively to monomeric prostate specific antigen and a different monoclonal antibody (clone M612166) that bound exclusively to a dimeric form of the antigen that only comprised 6.8% of the total protein. In the presence of excess antigen, the mobility spectrum of M612165 was replaced by a composite spectrum that represented a mixture of antibodies that included either one or two equivalents of the protein antigen. In similar circumstances, the mobility spectrum of M612166 was replaced by a composite spectrum that represented a mixture of antibodies that included either two or four equivalents of the protein antigen. When exposed to either of the two antibodies, the mobility spectrum of the prostate specific antigen showed a concomitant decrease in the monomeric antigen in one case and in the dimeric antigen in the other case. While sensitive kinetic exclusion assays demonstrated large differences in the antigen binding behavior of the two antibodies, these functional studies alone were insufficient to reveal the likely structural origins of the observed differences. Macroion mobility measurements were shown to be a useful and informative complement to functional studies in understanding complex macromolecular interactions.