Concentrations Of Phosphate-buffered Saline Research Articles

Stable Polymer-Lipid Hybrid Nanoparticles Based on mcl-Polyhydroxyalkanoate and Cationic Liposomes for mRNA Delivery.

Background/Objectives: The development of polymer-lipid hybrid nanoparticles (PLNs) is a promising area of research, as it can help increase the stability of cationic lipid carriers. Hybrid PLNs are core-shell nanoparticle structures that combine the advantages of both polymer nanoparticles and liposomes, especially in terms of their physical stability and biocompatibility. Natural polymers such as polyhydroxyalkanoate (PHA) can be used as a matrix for the PLNs' preparation. Methods: In this study, we first obtained stable cationic hybrid PLNs using a cationic liposome (CL) composed of a polycationic lipid 2X3 (1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the hydrophobic polymer mcl-PHA, which was produced by the soil bacterium Pseudomonas helmantisensis P1. Results: The new polymer-lipid carriers effectively encapsulated and delivered model mRNA-eGFP (enhanced green fluorescent protein mRNA) to BHK-21 cells. We then evaluated the role of mcl-PHA in increasing the stability of cationic PLNs in ionic solutions using dynamic light scattering data, electrophoretic mobility, and transmission electron microscopy techniques. Conclusions: The results showed that increasing the concentration of PBS (phosphate buffered saline) led to a decrease in the stability of the CLs. At high concentrations of PBS, the CLs aggregate. In contrast, the presence of isotonic PBS did not result in the aggregation of PLNs, and the particles remained stable for 120 h when stored at +4 °C. The obtained results show that PLNs hold promise for further in vivo studies on nucleic acid delivery.

Luminescence enhancement of carbon dots synthesized by intense CW laser at 450 nm irradiating biocompatible solutions

Carbon dots (CDs) were synthesized by a continuum wave (CW) laser system irradiating vegetable charcoal placed in a Phosphate-Buffered Saline (PBS) solution. The laser operates at 450 nm with an output power of 50 W and 1 mm2 spot size, irradiating the carbon target for 1–3 h. The used charcoal consists of amorphous carbon that gives rise to CDs in liquids revealing high luminescence in the blue region. The formation of CDs is due to photo-thermal and photo-chemical effects induced by the prolonged laser irradiation inducing carbon atom ejection, scissions, radical formation, chemical bond breaking, molecular detachment bounded by weakened bonds and van de Walls forces, and nanoparticle functionalization in the used solution. The measurements have been concerned with the laser ablation yield, the Fourier Transmission Infrared (FTIR) spectroscopy, the UV-Vis optical spectroscopy, and the luminescence yield induced by UV excitation lamp at 365 nm wavelength. The main luminescence peak is observed at about 480 nm, characteristic of the blue color, with less significant involvement of the near regions at about 450 nm and 520 nm. Results suggest that CDs luminescence can be enhanced by the laser irradiation time, the PBS concentration, and the addition of acetic acid in the PBS solution. The presented luminescence in biocompatible solutions found many applications in different scientific fields. Overall, it is discussed its application for bioimaging in the biomedical field.

Gold Nanoparticles in Porous Silicon Nanotubes for Glucose Detection

Silicon nanotubes (Si NTs) have a unique structure among the silicon nanostructure family, which is useful for diverse applications ranging from therapeutics to lithium storage/recycling. Their well-defined structure and high surface area make them ideal for sensing applications. In this work, we demonstrate the formation of Au nanoparticles (NPs) functionalized with 4-Mercaptophenylboronic acid (MPBA) on porous Si NTs (pSi NTs) fabricated using ZnO nanowires as a template. The system was characterized, and the proposed structure was confirmed using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). Varying glucose concentrations in phosphate-buffered saline (PBS) (0.5–80 mM) were introduced to the Si NT nanocomposite system. The glucose is detectable at low concentrations utilizing surface-enhanced Raman spectroscopy (SERS), which shows a concentration-dependent peak shift in the benzene ring breathing mode (~1071 cm−1) of MPBA. Complementing these measurements are simulations of the Raman hot spots associated with plasmonic enhancement of the Au NPs using COMSOL. This biocompatible system is envisioned to have applications in nanomedicine and microfluidic devices for real-time, non-invasive glucose sensing.

Investigation of the crystal formation from calcium silicate in human dentinal tubules and the effect of phosphate buffer saline concentration

Background/purposeBased on hydrodynamic theory, blocking the dentinal tubules can reduce discomfort caused by dentin hypersensitivity. This study identified the crystals formed in dentinal tubules from tricalcium silicate (TCS) in phosphate-buffered saline (PBS) and evaluated the effect of PBS concentration on crystal formation. Materials and methodsSixty-nine specimens were made by isolating the cervical part of extracted premolars. TCS was applied by brushing for 10,000 strokes on dentin surface simulating sensitive dentin. Specimens were stored in PBS or solutions with concentrations 1/100, 1/10, 10, and 100 times that of PBS for 1, 30, 60, or 90 days (n = 3). Another nine specimens applied TCS, were immersed in PBS for 3 months, and divided into three subgroups: no treatment, sonication for 10 min, and 1M acetic acid treatment for 3 min. Crystal formation was examined using a scanning electron microscope, assigned five grade scores (0–4) according to maturation, and analyzed by a nonparametric two-way ANOVA (α = 0.05). Crystal components were analyzed using X-ray diffraction (XRD) and energy dispersion X-ray spectroscopy (EDS). ResultsThe maturation of intratubular crystals was dependent on time and PBS concentration (P < 0.05). In all periods, the high-concentration group showed a higher maturation grade than the low-concentration group. Intratubular crystals were similar to hydroxyapatite according to XRD and EDS, and they withstood sonication and acid application. ConclusionTCS with nanosized particles formed hydroxyapatite-like crystals in the dentinal tubules, which were dependent on time and concentration of PBS and withstood sonication and acid application.

Sludge composting with self-produced carbon source by phosphate buffer coupled hyperthermophilic pretreatment realizing nitrogen retention

During sludge composting, ammonia emissions is the main pathway of nitrogen loss. In addition to regulating pH, increasing the carbon source is also important for controlling ammonia emissions. Previous studies have focused on additional carbon sources, while this study used phosphate-buffered saline (PBS) and hyperthermophilic pretreatment (HP) to release difficult-to-utilize carbon sources in extracellular polymeric substance (EPS) for composting. The PBS concentration was 0.22 mmol/g total matter (TM), PBS soaking days were 4.74, HP temperature was 149.46 °C, and the soluble chemical oxygen demand (SCOD) was 45.08 g/kg. Compared with the control check, the peak ammonia emission concentration in the pretreated group was reduced by 8.70 % and the total nitrogen loss was reduced by 18.10 %. Changes in EPS, ammonia assimilation activity, and microbial community composition demonstrated that PBS and HP released the carbon source from the sludge, promoted ammonia assimilation, and enriched bacteria related to ammonia assimilation such as Bacillus (16.52 %). HP treatment made Acinetobacter (12.5 %) the dominant genus in the calefactive phase, and the enrichment of Actinobacteriota (27.27 %) promoted the composting maturity. However, total pretreatment reduced the porosity and permeability of the sludge and affected the activity of microorganisms. Considering the cost of energy consumption, partial pretreatment has more practical application prospects.

Investigation of the Morphology and Electrical Properties of Graphene Used in the Development of Biosensors for Detection of Influenza Viruses.

In this study, we discuss the mechanisms behind changes in the conductivity, low-frequency noise, and surface morphology of biosensor chips based on graphene films on SiC substrates during the main stages of the creation of biosensors for detecting influenza viruses. The formation of phenylamine groups and a change in graphene nano-arrangement during functionalization causes an increase in defectiveness and conductivity. Functionalization leads to the formation of large hexagonal honeycomb-like defects up to 500 nm, the concentration of which is affected by the number of bilayer or multilayer inclusions in graphene. The chips fabricated allowed us to detect the influenza viruses in a concentration range of 10−16 g/mL to 10−10 g/mL in PBS (phosphate buffered saline). Atomic force microscopy (AFM) and scanning electron microscopy (SEM) revealed that these defects are responsible for the inhomogeneous aggregation of antibodies and influenza viruses over the functionalized graphene surface. Non-uniform aggregation is responsible for a weak non-linear logarithmic dependence of the biosensor response versus the virus concentration in PBS. This feature of graphene nano-arrangement affects the reliability of detection of extremely low virus concentrations at the early stages of disease.

Refractive index of phosphate-buffered saline in the telecom infrared C + L bands

The refractive index of phosphate-buffered saline (PBS) solutions, for concentrations ranging from 0 mol/L to 1.516 mol/L, was experimentally determined in the telecom C + L infrared bands, ranging from 1510 to 1620 nm, which is commonly used for silicon photonics biochemical sensors. The infrared measurements were performed with a silicon photonics arrayed waveguide sensor and compared with the optical visible range values measured with a handheld refractometer. An equation for the refractive index of PBS solutions as a function of wavelength and PBS concentration is presented. Moreover, the conductivity of the test solutions was determined and the relation between conductivity and refractive index was established, allowing for a fast, easy, cost-effective, indirect method of refractive index determination of PBS solutions. The data presented will enable more realistic simulation of optical biosensors and their precise experimental calibration.

Effect of rotating magnetic field on ferromagnetic structures used in hyperthermia

The study was aimed at investigating the heating efficiency of magnetically guided nanoparticles. The effect of graphene oxide (GO), magnetite (Fe3O4) and hybrid material (GO/Fe3O4) have been studied. The samples have been inductively heated at 42 °C in the presence of rotating magnetic field (RMF). The experiments have been conducted as a function of temperature different and samples concentrations in phosphate-buffered saline (PBS) medium. These measurements have been realized by means of the dynamic description of the obtained temperature changes. The field-dependent specific absorption rate (SAR) values of the applied systems with the nanoparticles were discussed. The results reveal that RMF may be successfully applied as a reinforcing factor in order to enhance the heating efficiency of the tested materials. The obtained results reveal the concentration dependent response to RMF and clear added value to study the hybrid materials in this fascinating field of interest.

Integrated Low Cost Optical Biosensor for High Resolution Sensing of Myeloperoxidase (MPO) Activity Through Carbon Nanotube Degradation

Myeloperoxidase (MPO) is an important biomarker for diseases associated with inflammation, and has been recognized as an early warning biomarker for cardiovascular diseases, with at-risk individuals typically exhibiting MPO concentration >350 ng/ml in blood. We present a high resolution and low cost optical biosensor capable of detecting MPO activity directly through degradation of single walled carbon nanotubes (SWCNTs), without requiring additional probe chemicals or reagents. The optical biosensor is assembled using commercially available semiconductor integrated circuit (IC) components. The change in optical contrast due to degradation of the SWCNTs is measured by a phase sensitive detection circuit as a voltage signal. The sensor system can be operated with merely 2 $\mu \text{l}$ of the test sample, and has sensitivity of 1.39 mV/$\mu \text{g}$ /ml with resolution of 26.8 ng/ml change in SWCNT concentration. The sensor is able to produce appreciable change in sensor output (>100 mV) and detect MPO activity for lyophilized MPO concentration in phosphate-buffered saline (PBS) and hydrogen peroxide (H2O2) as low as 327ng/ml.

Low frequency electrochemical noise in AlGaN/GaN field effect transistor biosensors

Little has been studied on how the electrochemical noise impacts the limit of detection of field effect transistor (FET) biosensors. Herein, we investigate low frequency noise associated with phosphate-buffered saline (PBS) solutions at varying ionic strengths (Ni) under both weak and strong gate biases corresponding to saturation and sub-threshold regimes, respectively, in AlGaN/GaN heterojunction FET biosensors. We show that the electrochemical noise is strongly dependent on the ionic strength and gate biasing conditions. In the saturation regime (low bias), varying the ionic strength (a range of 10−6× PBS to PBS 1 × stock solutions used for testing) has little to no effect on the characteristic frequency exponent β(β=1), indicating a predominately diffusion-based process. Conversely, under higher biases (sub-threshold regime), the β parameter varies from 1 to 2 with ionic strength exhibiting both diffusion and drift characteristics, with a “cut point” at approximately 10−5× PBS (Ni≈9×1014/mL). Under a high bias, once the PBS concentration reaches 10−3×, the behavior is then drift dominant. This indicates that the higher bias likely triggers electrochemical reactions and by extension, faradaic effects at most physiologically relevant ionic strengths. The signal-to-noise ratio (SNR) of the device has an inverse linear relationship with the low frequency current noise. The device exhibits a higher SNR in the sub-threshold regime than in the saturation regime. Specifically, within the saturation regime, an inversely proportional relationship between the SNR and the ionic concentration is observed. The electrochemical noise induced from ionic activities is roughly proportional to Ni−1/2.

Electrochemical Detection of C-Reactive Protein in Human Serum Based on Self-Assembled Monolayer-Modified Interdigitated Wave-Shaped Electrode.

An electrochemical capacitance immunosensor based on an interdigitated wave-shaped micro electrode array (IDWµE) for direct and label-free detection of C-reactive protein (CRP) was reported. A self-assembled monolayer (SAM) of dithiobis (succinimidyl propionate) (DTSP) was used to modify the electrode array for antibody immobilization. The SAM functionalized electrode array was characterized morphologically by atomic force microscopy (AFM) and energy dispersive X-ray spectroscopy (EDX). The nature of gold-sulfur interactions on SAM-treated electrode array was probed by X-ray photoelectron spectroscopy (XPS). The covalent linking of anti-CRP-antibodies onto the SAM modified electrode array was characterized morphologically through AFM, and electrochemically through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The application of phosphate-buffered saline (PBS) and human serum (HS) samples containing different concentrations of CRP in the electrode array caused changes in the electrode interfacial capacitance upon CRP binding. CRP concentrations in PBS and HS were determined quantitatively by measuring the change in capacitance (ΔC) through EIS. The electrode immobilized with anti-CRP-antibodies showed an increase in ΔC with the addition of CRP concentrations over a range of 0.01–10,000 ng mL−1. The electrode showed detection limits of 0.025 ng mL−1 and 0.23 ng mL−1 (S/N = 3) in PBS and HS, respectively. The biosensor showed a good reproducibility (relative standard deviation (RSD), 1.70%), repeatability (RSD, 1.95%), and adequate selectivity in presence of interferents towards CRP detection. The sensor also exhibited a significant storage stability of 2 weeks at 4 °C in 1× PBS.

Combined effects of Marangoni, sedimentation and coffee-ring flows on evaporative deposits of superparamagnetic colloids

Evaporation of sessile colloidal droplets is a way to organize suspended particles. It is already known that the composition of the surrounding fluid modifies the dried deposit. For superparamagnetic particles, recent studies showed that external magnetic fields can act as remote controls for those deposits. In this paper, we study the configuration space given by the interplay of such fields and a modification of the fluid composition by considering various concentrations of phosphate buffered saline (PBS). We show that the magnetic field modifies the morphological properties of the deposit, while the composition (i.e. PBS concentration) modifies the density profile of the deposit. We then present an explanation of these influences considering the competition between (i) sedimentation, (ii) coffee-ring and (iii) Marangoni flows. From these considerations, we propose a master curve which should be able to model the deposit densities of any system where the above mechanisms compete with each other.

Supercritical CO2 - assisted production of PLA and PLGA foams for controlled thymol release

Amorphous, medical grade poly(d,l-lactic acid) (PLA) and poly(d,l-lactic-co-glycolic acid) (PLGA) were used to develop systems for controlled release of a natural bioactive substance - thymol. Supercritical carbon dioxide (scCO2) was successfully used both as an impregnation medium for thymol incorporation into the polymer matrix and a foaming agent in a single-step batch process. Impregnation of samples using low to moderate scCO2 densities (273 kg/m3 and 630 kg/m3) and short processing times (2 h and 4 h) enabled thymol loading of 0.92%–6.62% and formation of microcellular foams upon system depressurization. Thymol effect on structural and thermal properties on foamed samples was proven by FTIR and DSC. The effect of CO2 under elevated pressure on the neat polymers was analysed by high pressure DSC. Foaming of polymers with lower molecular weight by CO2 of higher density yielded foams with smaller pores. All tested foams released thymol in a controlled manner in phosphate buffered saline (PBS) at 37 °C within 3 to 6 weeks. Higher loading and lower cell density favoured thymol release rate, while its concentration in PBS for the tested period depended on foam interaction with the medium. Representative PLGA foam sample with the highest thymol loading (6.62%) showed controlled thymol release within 72 h in mediums having pH values from 1.1 to 7.4.

Determination of the potentials of aqueous and ethanolic extracts of Buchholzia coriacea in preventing sodium metabisulphite-induced oxidation of some hemoglobin SS erythrocyte membrane proteins

BACKGROUND: The sustenance of the erythrocyte membrane integrity is crucial to ensuring a continuous lamina flow of erythrocytes through narrow blood vessels. So it is imperative to find a substance to attenuate the oxidation of erythrocytes that cause the membrane structure remodelling. OBJECTIVES: To investigate the hydrating strength and modulatory effect of Buchholzia coriacea seed extracts on erythrocyte membrane proteins degradation. MATERIALS AND METHODS: Aqueous and ethanolic extracts of B. coriacea were obtained following standard procedures. Blood samples (5.00 mL) from five known hemoglobin SS individuals in steady state were subjected to electrophoresis to establish the genotype of the individuals. The osmotic fragility assay was performed with 2.50, 5.00, 10.00 and 20.00 mg/mL of the extracts in the presence of different concentrations of phosphate-buffered saline. The graph of percentage hemolysis was plotted against saline concentration, and the mean corpuscular fragility (MCF) determined. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was also done using the same concentrations of extracts. RESULTS: There was no statistically significant difference (P > 0.01) between the average MCF in all the extract concentration and the control. Protein 4.1 and Ankyrin did not appear on the electrophoretic lanes of all the test samples when compared with the control. However, spectrin and ankyrin bands were visible with regular size and intensity. CONCLUSION: B. coriacea seed extracts possess antihemolytic character. However, it does not have the ability to maintain the erythrocyte membrane model despite its acclaimed antioxidant character.

A New Approach for Loading Anticancer Drugs Into Mesenchymal Stem Cell-Derived Exosome Mimetics for Cancer Therapy.

Exosomes derived from mesenchymal stem cells (MSCs) have been evaluated for their potential to be used as drug delivery vehicles. Synthetically personalized exosome mimetics (EMs) could be the alternative vesicles for drug delivery. In this study, we aimed to isolate EMs from human MSCs. Cells were mixed with paclitaxel (PTX) and PTX-loaded EMs (PTX-MSC-EMs) were isolated and evaluated for their anticancer effects against breast cancer. EMs were isolated from human bone marrow-derived MSCs. MSCs (4 × 106 cells/mL) were mixed with or without PTX at different concentrations in phosphate-buffered saline (PBS) and serially extruded through 10-, 5-, and 1-μm polycarbonate membrane filters using a mini-extruder. MSCs were centrifuged to remove debris and the supernatant was filtered through a 0.22-μm filter, followed by ultracentrifugation to isolate EMs and drug-loaded EMs. EMs without encapsulated drug (MSC-EMs) and those with encapsulated PTX (PTX-MSC-EMs) were characterized by western blotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The anticancer effects of MSC-EMs and PTX-MSC-EMs were assessed with breast cancer (MDA-MB-231) cells both in vitro and in vivo using optical imaging. EMs were isolated by the extrusion method and ultracentrifugation. The isolated vesicles were positive for membrane markers (ALIX and CD63) and negative for golgi (GM130) and endoplasmic (calnexin) marker proteins. NTA revealed the size of MSC-EM to be around 149 nm, while TEM confirmed its morphology. PTX-MSC-EMs significantly (p < 0.05) decreased the viability of MDA-MB-231 cells in vitro at increasing concentrations of EM. The in vivo tumor growth was significantly inhibited by PTX-MSC-EMs as compared to control and/or MSC-EMs. Thus, MSC-EMs were successfully isolated using simple procedures and drug-loaded MSC-EMs were shown to be therapeutically efficient for the treatment of breast cancer both in vitro and in vivo. MSC-EMs may be used as drug delivery vehicles for breast cancers.

Direct electrochemical bacterial sensor using ZnO nanorods disposable electrode

PurposeThis paper aims to report a prototype of a reliable method for rapid, sensitive bacterial detection by using a low-cost zinc oxide nanorods (ZnONRs)-based electrochemical sensor.Design/methodology/approachThe ZnONRs have been grown on the surface of a disposable, miniaturized working electrode (WE) using the low-temperature hydrothermal technique. Scanning electron microscopy and energy dispersion spectroscopy have been performed to characterize the distribution as well as the chemical composition of the ZnONRs on the surface, respectively. Moreover, the cyclic voltammetry test has been implemented to assess the effect of the ZnONRs on the signal conductivity between −1 V and 1 V with a scan rate of 0.01 V/s. Likewise, the effect of using different bacterial concentrations in phosphate-buffered saline has been investigated.FindingsThe morphological characterization has shown a highly distributed ZnONR on the WE with uneven alignment. Also, the achieved response time was about 12 minutes and the lower limit of detection was approximately 103 CFU abbreviation for Colony Forming Unit/mL.Originality/valueThis paper illustrates an outcome of an experimental work on a ZnONRs-based electrochemical biosensor for direct detection of bacteria.

Novel method for the detection of the facial nerve using electrical impedance spectroscopy during otologic surgery

Iatrogenic facial nerve injuries, which may occur during otologic surgery, can cause facial paralysis that eventually leads to devastating complications such as cosmetic problems, limitation to eye lubrication, and problems with eating, drinking, and speaking. We aimed to prevent iatrogenic nerve injury using an electrical impedance spectroscopy (EIS) technique and developed a facial nerve detector (FND) with coplanar bipolar electrodes on a flexible polyethylene terephthalate film using micro electro-mechanical systems technology. The discrimination capability of the FND was preliminarily evaluated by analyzing electrical impedances of solutions of various concentrations of phosphate-buffered saline and by extracting sample resistance using curve fitting with an electrical equivalent circuit. The impedances of bone, facial nerve covered by thin bone, and facial nerve of a cadaver were investigated using the optimal frequency range obtained based on the proposed discrimination index. To generalize the sensor output without regard to the shapes and dimensions of the EIS sensors, electrical conductivities of the samples were estimated by matching the extracted sample resistances with resistances obtained in the finite element method (FEM) simulation. Finally, in order to determine the location of the facial nerve before the facial nerve is exposed, resistance and conductivity changes were investigated using an FEM simulation based on the thickness of the bone covering the facial nerve. Thus, physicians can obtain information regarding the thickness of the bone covering the facial nerve while grinding the bone in otologic surgery. This will enable physicians to easily avoid iatrogenic facial nerve injury during otologic surgery.

Experimental studies on soft contact lenses for controlled ocular delivery of pirfinedone: in vitro and in vivo

Context: Pirfinedone (PFD) is a novel agent which has the potential to prevent scarring in the eyes. The 0.5% PFD eye drops exhibits poor bioavailability. Whereas, the feasibility of using contact lens as ocular drug delivery device initiated novel possibilities.Objective: To evaluate the delivery of PFD by soft contact lens (SCL) in vivo, we screened the most suitable lens material for PFD among various commercially available SCL materials in vitro.Material and methods: Firstly, 11 different SCLs (−1.00 diopter) were respectively soaked in 2 ml of 0.05% PFD-loading solution for 24 h to fully absorb drug, and then placed in fresh phosphate buffered saline (PBS) to release the drug. PFD concentration in PBS was determined by ultraviolet absorbance at 310 nm. Secondly, by immersing in 2 ml of 0.5% PFD eye drops for 24 h, the polymacon lens (0.00 diopter) was then placed on the cornea of New Zealand rabbits. PFD concentrations were detected by high performance liquid chromatography (HPLC) in tears, aqueous humor, conjunctiva, cornea, and sclera at different time points.Results: PFD showed some affinity for pHEMA-based lenses and the polymacon lens more slowly released more amount of PFD than any other lens in vitro (p < 0.001). Compared with eye drops, drug-loaded SCLs greatly enhanced the retention time and concentrations of PFD in cornea and aqueous humor and consequently improved the bioavailability of PFD.Conclusion: Polymacon-based SCL is probably a promising vehicle to be an effective ophthalmic delivery system for PFD.

Development of an ELISA for nitrazepam based on a monoclonal antibody

We report the production of a specific monoclonal antibody against Nitrazepam (NZP). First, the hapten 7-aminonitrazepam (7-ANZP) was synthesized, then the hapten was coupled with bovine serum albumin and ovalbumin by the diazotization method, and used as immunogen and coating antigen. Factors affecting binding, ionic strength, pH, and methanol concentration in phosphate-buffered saline, were optimized. The monoclonal antibody had a satisfactory IC50 of 0.2 ng/mL for NZP. The cross-reactivities with other analogs were all lower than 0.1% except for 23% with 7-NZP, which suggested that the enzyme-linked immunosorbent assay method possessed high specificity. The recoveries were in the range of 84–95%, indicating that the method could be applied for the detection of NZP in urine.

Stable oligomeric clusters of gold nanoparticles: preparation, size distribution, derivatization, and physical and biological properties.

Reducing dilute aqueous HAuCl4 with NaSCN under alkaline conditions produces 2–3 nm diameter yellow nanoparticles without the addition of extraneous capping agents. We here describe two very simple methods for producing highly stable oligomeric grape-like clusters (oligoclusters) of these small nanoparticles. The oligoclusters have well-controlled diameters ranging from ∼5 to ∼30 nm, depending mainly on the number of subunits in the cluster. Our first [“delay-time”] method controls the size of the oligoclusters by varying from seconds to hours the delay time between making the HAuCl4 alkaline and adding the reducing agent, NaSCN. Our second [“add-on”] method controls size by using yellow nanoparticles as seeds onto which varying amounts of gold derived from “hydroxylated gold”, Na+[Au(OH4–x)Clx]−, are added-on catalytically in the presence of NaSCN. Possible reaction mechanisms and a simple kinetic model fitting the data are discussed. The crude oligocluster preparations have narrow size distributions, and for most purposes do not require fractionation. The oligoclusters do not aggregate after ∼300-fold centrifugal-filter concentration, and at this high concentration are easily derivatized with a variety of thiol-containing reagents. This allows rare or expensive derivatizing reagents to be used economically. Unlike conventional glutathione-capped nanoparticles of comparable gold content, large oligoclusters derivatized with glutathione do not aggregate at high concentrations in phosphate-buffered saline (PBS) or in the circulation when injected into mice. Mice receiving them intravenously show no visible signs of distress. Their sizes can be made small enough to allow their excretion in the urine or large enough to prevent them from crossing capillary basement membranes. They are directly visible in electron micrographs without enhancement, and can model the biological fate of protein-like macromolecules with controlled sizes and charges. The ease of derivatizing the oligoclusters makes them potentially useful for presenting pharmacological agents to different tissues while controlling escape of the reagents from the circulation.