Binding Rate Research Articles

Conventional Receptor Radioligand Binding Techniques Applied to the Study of Monoamine Oxidase.

Designed to measure binding interactions between small molecules and receptor proteins, radioligand binding approaches may also be applied to interactions between monoamine oxidase (MAO) and its ligands. The technique may be used with tissue homogenates or with mitochondrial membranes and can provide information about binding site density, ligand affinity, binding rate constants, and binding events at sites that do not impact absorbance characteristics of the flavin cofactor and that may not be amenable to spectrophotometric studies. This overview describes the use of a cell harvester in a common filtration approach to measure binding to MAO of radiolabeled substrates, inhibitors, or allosteric ligands in saturation analyses and to take advantage of the principles of competition to obtain quantitative binding data for unlabeled ligands that may bind with much lower affinity. The quality and reproducibility of data are impacted by factors such as choice of ligand concentrations, pipetting technique, graphing and regression approaches, and scintillation counting parameters, and consideration is given to these and other factors that may influence the results.

Elucidation of the binding mechanism of astragaloside IV derivative with human serum albumin and its cardiotoxicity in zebrafish embryos.

LS-102 is a new derivative of astragaloside IV (AGS IV) that has been shown to possess potentially significant cardioprotective effects. However, there are no reports concerning its interaction with human serum albumin (HSA) and toxicology in vertebrates. The present investigation was undertaken to characterize the interaction of AGS IV and LS-102 with HSA using equilibrium dialysis and UHPLC-MS/MS methods, along with computational methods. Notably, the effects of AGS IV and LS-102 were studied in vivo using the zebrafish embryo model. Markers related to embryonic cardiotoxicity and thrombosis were evaluated. We showed that the plasma protein binding rate of AGS IV (94.04%–97.42%) was significantly higher than that of LS-102 (66.90%–69.35%). Through site marker competitive experiments and molecular docking, we found that AGS IV and LS-102 were located at the interface of subdomains IIA and IIIA, but the site I might be the primary binding site. Molecular dynamics revealed that AGS IV showed a higher binding free energy mainly due to the stronger hydrophobic and hydrogen bonding interactions. Moreover, the secondary structure implied no obvious effect on the protein structure and conformation during the binding of LS-102. LS-102 significantly ameliorated the astramizole-induced heart rate slowing, increased SV-BA spacing, and prevented arachidonic acid-induced thrombosis in zebrafish. To our knowledge, we are the first to reveal that LS-102 binds to HSA with reversible and moderate affinity, indicating its easy diffusion from the circulatory system to the target tissue, thereby providing significant insights into its pharmacokinetic and pharmacodynamic properties when spread in the human body. Our results also provide a reference for the rational clinical application of LS-102 in the cardiovascular field.

Comparison of soy protein isolate-(–)-epigallocatechin gallate complexes prepared by mixing, chemical polymerization, and ultrasound treatment

The effects of the preparation method (mixing, chemical polymerization, or ultrasound treatment) on the structure and functional properties of soy protein isolate-(–)-epigallocatechin-3-gallate (SPI-EGCG) complexes were examined. The mixing treated SPI-EGCG samples (M−SE) were non-covalently linked, while the chemical polymerization and ultrasound treated SPI-EGCG samples (C-SE and U-SE, respectively) were bound covalently. The covalent binding of EGCG with protein improved the molecular weight and changed the structures of the SPI by decreasing the α-helix content. Moreover, U-SE samples had the lowest particle size (188.70 ± 33.40 nm), the highest zeta potential (−27.82 ± 0.53 mV), and the highest polyphenol binding rate (59.84 ± 2.34 %) compared with mixing and chemical polymerization-treated samples. Furthermore, adding EGCG enhanced the antioxidant activity of SPI and U-SE revealed the highest DPPH (84.84 ± 1.34 %) and ABTS (88.89 ± 1.23 %) values. In conclusion, the SPI-EGCG complexes prepared by ultrasound formed a more stable composite system with stronger antioxidant capacity, indicating that ultrasound technology may have potential applications in the preparation of protein-polyphenol complexes.

Precise quantitative evaluation of pharmacokinetics of cisplatin using a radio-platinum tracer in tumor-bearing mice.

ObjectiveThe platinum-based antineoplastic drug cisplatin is commonly used for chemotherapy in clinics. This work aims to demonstrate a radio-platinum tracer is useful for precisely quantifying small amounts of platinum in pharmacokinetics studies.MethodsA cisplatin radiotracer (radio-cisplatin) was synthesized, and a comprehensive evaluation of cisplatin over 7 days after its intravenous injection into nude mice bearing a subcutaneous lung tumor (H460) was conducted.ResultsA biphasic retention curve in the whole body and blood was observed [T1/2(α) = 1.14 h, T1/2(β) = 5.33 days for the whole body, and T1/2(α) = 23.9 min, T1/2(β) = 4.72 days for blood]. The blood concentration decreased within 1 day after injection. Most of the intact cisplatin was excreted via the kidneys in the early time points, and a small part was distributed in tissues including tumors. The plasma protein binding rate of cisplatin increased rapidly after injection, and the protein-bound cisplatin remained in the blood longer than intact cisplatin. The peak uptake in H460 tumors was 4.7% injected dose per gram at 15 min after injection, and the area under the curve (AUC0–7 days) was approximately one-half to one-third of the AUC0–7 days in the kidneys, liver, and bone, where some toxicity is observed in humans.ConclusionThe radio-platinum tracer revealed the highly quantitative biodistribution of cisplatin, providing insights into the properties of cisplatin, including its adverse effects. The tracer enables a precise evaluation of pharmacokinetics for platinum-based drugs with high sensitivity.

Dual-mode electrochemical immunoassay for Non-small cell lung cancer detection based on CoSe2-GO-Au and poly(MB)-Au

Combining the Potentiostatic Electrolysis i-t curve (i-t) and Differential Pulse Voltammetry (DPV), using CoSe2-GO-Au as the sensing substrate and poly(MB)-Au as the secondary antibody marker, the dual-mode electrochemical immunosensor was manufactured for the analysis and detection of Cytokeratin 19 Fragment Antigen (CYFRA 21-1). Cobalt selenide (CoSe2) had favourable catalytic activity of H2O2, which could provide an electrochemical signal through i-t. The huge specific surface area and excellent conductivity of graphene oxide (GO) provided a platform for the combination of CoSe2 and prevented the agglomeration of CoSe2. With the modification of Au nanoparticles, the conductivity of CoSe2-GO had been improved and the binding rates of Ab1 had been increased, which further expanded the electrochemical signal. Meanwhile, precious metal polymerized methylene blue (poly(MB)-Au) with good redox property was used as electrochemical probe, which could solve the shortcoming of MB falling off the electrode. Under the best experimental conditions, the sensitive detection of the CYFRA 21-1 was realized through the test methods of two modes. The dual-mode electrochemical immunosensor presented a linear current signal in 1 pg/mL–100 ng/mL with the detection limit of 0.36 pg/mL (i-t) and 0.24 pg/mL (DPV). To sum up, the prepared dual-mode electrochemical immunosensor provided a guarantee for the sensitive detection of CYFRA 21-1 in this experiment.

Plasma Protein Binding Rate and Pharmacokinetics of Lekethromycin in Rats.

Lekethromycin (LKMS), a novel macrolide lactone, is still unclear regarding its absorption. Thus, we conducted this study to investigate the characteristics of LKMS in rats. We chose the ultrafiltration method to measure the plasma protein binding rate of LKMS. As a result, LKMS was characterized by quick absorption, delayed elimination, and extensive distribution in rats following intramuscular (im) and subcutaneous (sc) administration. Moreover, LKMS has a high protein binding rate (78–91%) in rats at a concentration range of 10–800 ng/mL. LKMS bioavailability was found to be approximately 84–139% and 52–77% after im and sc administration, respectively; however, LKMS was found to have extremely poor bioavailability after oral administration (po) in rats. The pharmacokinetic parameters cannot be considered linearly correlated with the administered dose. Additionally, LKMS and its corresponding metabolites were shown to be metabolically stable in the liver microsomes of rats, dogs, pigs, and humans. Notably, only one phase I metabolite was identified during in vitro study, suggesting most of drug was not converted. Collectively, LKMS had quick absorption but poor absorption after oral administration, extensive tissue distribution, metabolic stability, and slow elimination in rats.

Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme

The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR–Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min−1 at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9–DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR–Cas9 technology.

Rapamycin nanoparticles improves drug bioavailability in PLAM treatment by interstitial injection

BackgroundPulmonary lymphangiomyomatosis (PLAM) is a rare interstitial lung disease characterized by diffuse cystic changes caused by the destructive proliferation of smooth muscle-like cells or LAM cells. PLAM is more common in young women than other people, and a consensus is lacking regarding PLAM treatment. The clinical treatment of PLAM is currently dominated by rapamycin. By inhibiting the mTOR signaling pathway, rapamycin can inhibit and delay PLAM’s occurrence and development. However, the application of rapamycin also has shortcomings, including the drug’s low oral bioavailability and a high binding rate to hemoglobin, thus significantly decreasing the amount of drug distributed to the lungs.Methods and resultsHere, we developed a new mode of rapamycin administration in which the drug was injected into the intrathecal space after being nanosized; the directional flow characteristics of the liquid in the intrathecal space were exploited to increase the drug content in the interstitial fluid to the greatest extent possible. We studied the rapamycin content in the interstitial fluid and blood after intervaginal space injection (ISI). Compared with oral administration, ISI significantly increased the drug concentration in the lung interstitial fluid.ConclusionsThese results provided new ideas for treating PLAM and optimizing the dosing regimens of drugs with similar characteristics to rapamycin.

Mapping single-nephron filtration in the isolated, perfused rat kidney using magnetic resonance imaging.

The kidney has an extraordinary ability to maintain glomerular filtration despite natural fluctuations in blood pressure and nephron loss. This is partly due to local coordination between single-nephron filtration and vascular perfusion. An improved understanding of the three-dimensional (3-D) functional coordination between nephrons and the vasculature may provide a new perspective of the heterogeneity of kidney function and could inform targeted therapies and timed interventions to slow or prevent the progression of kidney disease. Here, we developed magnetic resonance imaging (MRI) tools to visualize single-nephron function in 3-D throughout the isolated perfused rat kidney. We used an intravenous slow perfusion of a glomerulus-targeted imaging tracer [cationized ferritin (CF)] to map macromolecular dynamics and to identify glomeruli in 3-D, followed by a bolus of a freely filtered tracer (gadolinium diethylenetriamine penta-acetic acid) to map filtration kinetics. There was a wide intrakidney distribution of CF binding rates and estimated single-nephron glomerular filtration rate (eSNGFR) between nephrons. eSNGFR and CF uptake rates did not vary significantly by distance from the kidney surface. eSNGFR varied from ∼10 to ∼100 nL/min throughout the kidney. Whole single-kidney GFR was similar across all kidneys, despite differences in the distributions eSNGFR of and glomerular number, indicating a robust adaptive regulation of individual nephrons to maintain constant single-kidney GFR in the presence of a natural variation in nephron number. This work provides a framework for future studies of single-nephron function in the whole isolated perfused kidney and experiments of single-nephron function in vivo using MRI.NEW & NOTEWORTHY We report MRI tools to measure and map single-nephron function in the isolated, perfused rat kidney. We used imaging tracers to identify nephrons throughout the kidney and to measure the delivery and filtration of the tracers at the location of the glomeruli. With this technique, we directly measured physiological parameters including estimated single-nephron glomerular filtration rate throughout the kidney. This work provides a foundation for new studies to simultaneously map the function of large numbers of nephrons.

Fucoidan-Modified Phase-Transitional Contrast Agent for Ultrasound Imaging and Targeting of Hepatoma Cells

To prepare a fucoidan-modified phase-transitional contrast agent (FPCA) and to evaluate its in vitro capabilities for ultrasound imaging and targeting of hepatoma cells. Nano-liposomes encapsulated with perfluoropentane were prepared using thin-film hydration and ultrasonic emulsification methods. Then, FPCA nanoparticles were prepared through chemical grafting of fucoidan and the characterization of their physical and chemical properties was performed. After applying external stimuli of heating with hot water bath and microwave irradiation, the phase-transition status of FPCA was observed with microscope. The imaging abilities of phase-transited FPCA on two-dimensional ultrasound and contrast-enhanced ultrasound were observed with ultrasonic diagnostic instrument. The ability of FPCA to target at hepatoma cells was evaluated and verified with fluorescence confocal observation and flow cytometry analysis. The FPCA prepared in the study had an average diameter of (222.1±32.5) nm, displaying spherical appearance, good dispersion, good stability, and good biocompatibility. The phase-transition of FPCA was induced by both heating with hot water bath and microwave irradiation. For phase transition, the optimal temperature was found to be 50 ℃ and the preferred microwave power was 1.5 W/cm 2. Moreover, after phase transition, FPCA showed significant imaging enhancement on both two-dimensional ultrasonography and contrast-enhanced ultrasonography. Through fluorescein isothiocyanate (FITC) labeling, FPCA could specifically bind with hepatoma cells at a high binding rate of (96.19±1.62)%, while it rarely bound with normal liver cells, showing a binding rate of less than 10%. A new type of phase-transitional ultrasound contrast agent with good stability and biocompatibility was successfully prepared. It not only could enhance ultrasound imaging through phase transition, but also had specific active hepatoma cell-targeting properties.

Pharmacodynamic model of slow reversible binding and its applications in pharmacokinetic/pharmacodynamic modeling: review and tutorial.

Therapeutic responses of most drugs are initiated by the rate and degree of binding to their receptors or targets. The law of mass action describes the rate of drug-receptor complex association (kon) and dissociation (koff) where the ratio koff/kon is the equilibrium dissociation constant (Kd). Drugs with slow reversible binding (SRB) often demonstrate delayed onset and prolonged pharmacodynamic effects. This report reviews evidence for drugs with SRB features, describes previous pharmacokinetic/pharmacodynamic (PK/PD) modeling efforts of several such drugs, provides a tutorial on the mathematics and properties of SRB models, demonstrates applications of SRB models to additional compounds, and compares PK/PD fittings of SRB with other mechanistic models. We identified and summarized 52 drugs with in vitro-confirmed SRB from a PubMed literature search. Simulations with a SRB model and observed PK/PD profiles showed delayed and prolonged responses and that increasing doses/kon or decreasing koff led to greater expected maximum effects and a longer duration of effects. Recession slopes for return of responses to baseline after single doses were nearly linear with an inflection point that approaches a limiting value at larger doses. The SRB model newly captured literature data for the antihypertensive effects of candesartan and antiallergic effects of noberastine. Their PD profiles could also be fitted with indirect response and biophase models with minimal differences. The applicability of SRB models is probably commonplace, but underappreciated, owing to the need for in vitro confirmation of binding kinetics and the similarity of PK/PD profiles to models with other mechanistic determinants.

Understanding of promoting enzymatic hydrolysis of combined hydrothermal and deep eutectic solvent pretreated poplars by Tween 80

In this study, lignin blockers including non-catalytic protein and surfactants were employed to promote enzymatic digestibility of pretreated poplars. Among them, Tween 80 exhibited the most pronounced facilitation, improving the glucose yield from 26.6% to 99.6% at a low enzyme loading (10 FPU/g glucan), and readily reduced the required cellulase loading by 75%. The underlying mechanism for this remarkable improvement on glucose yields by Tween 80 was elucidated. The impacts of Tween 80 on the enzyme-lignin interaction were explored by quartz crystal microbalance analysis, revealing that the binding rate of Tween 80 on lignin surfaces was 3-fold higher than that of enzyme. More importantly, Tween 80 remarkably decreased the binding capacity and binding rate of enzyme on lignins. Furthermore, the substrate properties dominating the increase in glucose yields with Tween 80 were explored. The results facilitate to understand the underlying mechanism of the promotion of surfactants on enzymatic hydrolysis.

First-Principle Coarse-Graining Framework for Scale-Free Bell-Like Association and Dissociation Rates in Thermal and Active Systems

A new theory of ligand-receptor bonds in biological cells that accounts for membrane fluctuations reveals an orders-of-magnitude increase in binding rates and the range over which a receptor can recognize its ligand.

Binding and removal of polycyclic aromatic hydrocarbons in cold smoked sausage and beef using probiotic strains

The aim of this study is to bio-monitor the levels of 16 polycyclic aromatic hydrocarbons (PAHs) in cold smoked beef and sausages. The ability of probiotics to remove PAHs was also investigated as function of the cell viability (viable, non-viable and acid-treated cells), bacterial counts (107, 108, and 109 CFU/mL), pH (3, 5, and 7), and incubation time (6, 12, and 24 h). The results indicated a significant difference (p < 0.05) among the analyzed sausages and beef samples for the PAHs concentration. Non-viable probiotics achieved the highest PAHs reduction rates. Limosilactobacillus fermentum EMCC 1346 presented the lowest binding activity value (i.e. 41.10–56.80 %) for all PAHs, followed by Lacticaseibacillus rhamnosus EMCC 1105 with binding percentage of 50.40–65.80 %. On the other hand, the highest removal for all PAHs was achieved by Lactobacillus bulgaricus EMCC 1102 with binding rate of 60.50–76.80 %, at 109 CFU/mL, pH 7, after incubation for 24 h. The fortified sausages results revealed that L. bulgaricus EMCC 1102 cultures exhibited the maximum and significant reduction (p < 0.05) of PAHs with values of 44.71 µg/kg for the center part, compared to control non treated sausages (82.65 µg/kg). Regarding the sensorial profile, treated samples with probiotics led to a preference from the panelists, compared to control. Consequently, the results confirm that fermented probiotic suspension is a feasible future strategy to control PAHs levels in cold smoked meat stuffs.

Identification of Potential ACE2-Derived Peptide Mimetics in SARS-CoV-2 Omicron Variant Therapeutics using Computational Approaches.

The COVID-19 pandemic has become a global health challenge because of the emergence of distinct variants. Omicron, a new variant, is recognized as a variant of concern (VOC) by the World Health Organization (WHO) because of its higher mutations and accelerated human infection. The infection rate is strongly dependent on the binding rate of the receptor binding domain (RBD) against human angiotensin converting enzyme-2 (ACE2human) receptor. Inhibition of protein–protein (RBDs(SARS-CoV-2/omicron)-ACE2human) interaction has been already proven to inhibit viral infection. We have systematically designed ACE2human-derived peptides and peptide mimetics that have high binding affinity toward RBDomicron. Our peptide mutational analysis indicated the influence of canonical amino acids on the peptide binding process. Herein, efforts have been made to explore the atomistic details and events of RBDs(SARS-CoV-2/omicron)-ACE2human interactions by using molecular dynamics simulation. Our studies pave a path for developing therapeutic peptidomimetics against omicron.

Parameter estimation on multivalent ITC data sets

The Wiseman fitting can be used to extract binding parameters from ITC data sets, such as heat of binding, number of binding sites, and the overall dissociation rate. The classical Wiseman fitting assumes a direct binding process and neglects the possibility of intermediate binding steps. In principle, it only provides thermodynamic information and not the kinetics of the process. In this article we show that a concentration dependent dissociation constant could possibly stem from intermediate binding steps. The mathematical form of this dependency can be exploited with the aid of the Robust Perron Cluster Cluster Analysis method. Our proposed extension of the Wiseman fitting rationalizes the concentration dependency, and can probably also be used to determine the kinetic parameters of intermediate binding steps of a multivalent binding process. The novelty of this paper is to assume that the binding rate varies per titration step due to the change of the ligand concentration and to use this information in the Wiseman fitting. We do not claim to produce the most accurate values of the binding parameters, we rather present a novel method of how to approach multivalent bindings from a different angle.

Primitive nucleobases @ sodium 2-Ketooctanoate vesicles with high salt resistance

• PNB@SKO is the strongest salt resistance P-SCA vesicle system to the best of our knowledge. • The rigid planar PNB molecules orientate via hydrogen bonding in the tilted bilayers of SKO. • The morphology, size, and membrane thickness of SKO vesicles are not affected by the PNB combination significantly. Proto-single chain amphiphiles (P-SCAs) vesicles are considered to be a plausible model of the protocell on early Earth due to their primordial relevancy and membrane structure. In this work, we selected P-SCAs sodium 2-ketooctanate (SKO) and proto-nucleobases (PNBs, including cytosine, uracil, adenine, and guanine) as the building constituents of the vesicles. The PNB molecules incorporate into SKO bilayers to form PNB@SKO vesicles with a binding rate of 7% − 12%. The interactions between them increase the solubility of PNB in water obviously while decreasing the critical vesicle concentration of SKO. The morphology, size, and membrane thickness of SKO PRB@SKO vesicles are not affected by the PNB combination significantly. A binding mechanism is proposed based on the results of Fourier transform infrared and small angle X-ray scattering. The rigid planar PNB molecules orientate via hydrogen bonding in the tilted bilayers of SKO, without excessive disruption to the membrane structure. Both ribozyme-catalyzed and non-enzymatic RNA copying reactions require high concentrations of divalent ions, fortunately, the salt resistance of these PNB@SKO vesicles is surprisingly remarkable, one or two orders of magnitude greater than the other typical P-SCAs vesicles systems. We expect that this primitively relevant vesicle system can exert its potential advantages in the proto-membrane mimicry of the origin of life.

The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface

Antibodies engage Fc γ receptors (FcγRs) to elicit healing cellular immune responses following binding to a target antigen. Fc γ receptor IIIa/CD16a triggers natural killer cells to destroy target tissues with cytotoxic proteins and enhances phagocytosis mediated by macrophages. Multiple variables affect CD16a antibody-binding strength and the resulting immune response, including a genetic polymorphism. The predominant CD16a F158 allotype binds antibodies with less affinity than the less common V158 allotype. This polymorphism likewise affects cellular immune responses and clinical efficacy of antibodies relying on CD16a engagement, though it remains unclear how V/F158 affects CD16a structure. Another relevant variable shown to affect affinity is composition of the CD16a asparagine-linked (N)-glycans. It is currently not known how N-glycan composition affects CD16a F158 affinity. Here, we determined N-glycan composition affects the V158 and F158 allotypes similarly, and N-glycan composition does not explain differences in V158 and F158 binding affinity. Our analysis of binding kinetics indicated the N162 glycan slows the binding event, and shortening the N-glycans or removing the N162 glycan increased the speed of binding. F158 displayed a slower binding rate than V158. Surprisingly, we found N-glycan composition had a smaller effect on the dissociation rate. We also identified conformational heterogeneity of CD16a F158 backbone amide and N162 glycan resonances using NMR spectroscopy. Residues exhibiting chemical shift perturbations between V158 and F158 mapped to the antibody-binding interface. These data support a model for CD16a F158 with increased interface conformational heterogeneity, reducing the population of binding-competent forms available and decreasing affinity.

Synthesis and in vitro proof-of-concept studies on bispecific iron oxide magnetic nanoparticles targeting PSMA and GRP receptors for PET/MR imaging of prostate cancer

Prostate cancer (PCa) is the most common malignancy worldwide in men. This is a proof-of-concept study describing the development of 68Ga-magnetic iron oxide nanoparticles (mNP) targeting prostate specific membrane antigen (PSMA) and gastrin releasing peptide (GRPR) receptors as potential tools for diagnosis of PCa with PET/MRI. Two pharmacophores targeting PSMA, 1, and GRPR, 2, were coupled to mNPs carrying -SH (mNP-S1/2) or –NH2 (mNP-N1/2) groups. The mNP-S1/2 and mNP-N1/2 were characterized for their size, zeta potential, structure, and efficiency of functionalization using dynamic light scattering (DLS), FT-IR and RP-HPLC. A direct 68Ga-labelling procedure was followed, where 68Ga-mNP-N1/2 proved superior to 68Ga-mNP-S1/2 regarding radiolabelling efficiency, and thus were further evaluated in vitro. Toxicity studies in PCa cells (LNCaP, PC-3) showed low toxicity, and minimal hemolysis of red blood cells. In vitro assays in cells expressing PSMA (LNCaP), and GRPR (PC-3), showed specific time-dependent binding (40 min to plateau), high avidity (PC-3: Kd = 28.27 nM, LNCaP: Kd = 11.49 nM) and high internalization rates for 68Ga-mNP-N1/2 in both cell lines.

The correlation of haptenation of gold nanoparticles and cysteine modified screen printed carbon electrode by impedance technique with local lymph node assay data

Skin sensitization occurs when a skin sensitizer binds covalently to skin proteins through the haptenation process. The objective of this study was to correlate the electrochemical impedance spectroscopy (EIS) data of a screen printed carbon electrode (SPCE) modified with cysteine and gold nanoparticles (AuNPs) with local lymph node assay (LLNA) data as a potential skin sensitizer biosensor. The EIS was used to quantify variations in charge transfer resistance of skin sensitizers (ΔRCTskin sensitizer) due to different binding rates of skin sensitizers to cysteine. SPCE was modified through electrodeposition of AuNPs/thiourea/self–assembly of AuNPs/cysteine (assigned as ETSC) for the detection of skin sensitizers. Surface analysis of modified SPCEs using FESEM and EDX revealed a smooth surface with an uneven distribution of cysteine with AuNPs molecules. The ETSC modified SPCE showed a significant skin sensitizer biosensor since the ΔRCTskin sensitizer readings were increased proportionally to the strength of the skin sensitizers, with strong/extreme skin sensitizers displaying higher ΔRCTskin sensitizer readings compared to moderate and weak/non–skin sensitizers. The skin sensitization analysis from this work was compared to LLNA (animal study), human cell line activation (h–CLAT), direct peptide reactivity assay (DPRA), and KeratinoSens™, surface plasmon resonance (SPR) matched the categorization of LLNA in the following descending order: 96%, 92%, 82%, 70%, 70%, and 12%. With just an 8% mismatch with LLNA data, the EIS approach used in this study could be used as a screening tool for skin sensitizers.