Digital colloid-enhanced Raman spectroscopy by single-molecule counting.

Quantitative detection of various molecules at very low concentrations in complex mixtures has been the main objective in many fields of science and engineering, from the detection of cancer-causing mutagens and early disease markers to environmental pollutants and bioterror agents1-5. Moreover, technologies that can detect these analytes without external labels or modifications are extremely valuable and often preferred6. In this regard, surface-enhanced Raman spectroscopy can detect molecular species in complex mixtures on the basis only of their intrinsic and unique vibrational signatures7. However, the development of surface-enhanced Raman spectroscopy for this purpose has been challenging so far because of uncontrollable signal heterogeneity and poor reproducibility at low analyte concentrations8. Here, as a proof of concept, we show that, using digital (nano)colloid-enhanced Raman spectroscopy, reproducible quantification of a broad range of target molecules at very low concentrations can be routinely achieved with single-molecule counting, limited only by the Poisson noise of the measurement process. As metallic colloidal nanoparticles that enhance these vibrational signatures, including hydroxylamine-reduced-silver colloids, can be fabricated at large scale under routine conditions, we anticipate that digital (nano)colloid-enhanced Raman spectroscopy will become the technology of choice for the reliable and ultrasensitive detection of various analytes, including those of great importance for human health.

Rapid and simultaneous detection of five mycotoxins and their analogs with a gold nanoparticle-based multiplex immuno-strip sensor.

Mycotoxins, as secondary metabolites produced by fungi, have been the focus of researchers in various countries and are considered to be one of the major risk factors in agricultural products. There is an urgent need for a rapid, simple and high-performance method to detect residues of harmful mycotoxins in agricultural foods. We have developed a gold nanoparticle-based multiplexed immunochromatographic strip biosensor that can simultaneously detect fifteen mycotoxins in cereal samples. With this optimized procedure, five representative mycotoxins, deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), tenuazonic acid (TEA) and alternariol (AOH) were detected in the range of 0.91-4.77, 0.04-0.56, 0.11-0.68, 0.12-1.02 and 0.09-0.75 ng/mL, respectively. The accuracy and stability of these measurements were demonstrated by analysis of spiked samples with recoveries of 91.8%-115.3% and coefficients of variation <8.7%. In addition, commercially available samples of real cereals were tested using the strips and showed good agreement with the results verified by LC-MS/MS. Therefore, Our assembled ICA strips can be used for the simultaneous detection of 5 mycotoxins and their analogs (15 mycotoxins in total) in grain samples, and the results were consistent between different types of cereal foods, this multiplexed immunochromatographic strip biosensor can be used as an effective tool for the primary screening of mycotoxin residues in agricultural products.

Silver nanoparticle-functionalized melamine-formaldehyde aerogel for online in-tube solid-phase microextraction of polycyclic aromatic hydrocarbons followed by HPLC-DAD analysis.

Based on the π-metal interaction between silver nanoparticles (AgNPs) and aromatic compounds, AgNPs were in-situ grown to melamine-formaldehyde (MF) aerogel for improving the extraction performance to polycyclic aromatic hydrocarbons (PAHs). The AgNPs/MF aerogel was regulated through varing the concentration of reactants, and characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and X-ray powder diffraction. As a new extraction coating, the AgNPs/MF aerogel was coated to stainless-steel wires for in-tube solid-phase microextraction (IT-SPME). The extraction effects of MF aerogels before and after the modification of AgNPs were compared, and the AgNPs greatly improved the extraction ability for PAHs reaching to 166.4 %. Combining IT-SPME with high performance liquid chromatographic detection, an online analytical system was constructed. Furthermore, the sampling volume and rate, concentration of organic solvent, and desorption time were optimized factor by factor. The online analytical method with low detection limits (0.003-0.010 µg L-1) and efficient enrichment factors (1998-3237) for PAHs was established, which fastly detected trace level of PAHs in drinking and environmental water samples. Compared with other methods, the method was comparable or better in the detection limit and linear range, indicating prospective application of the AgNPs/MF aerogel for sample preparation.

Human health risk assessment and uncertainty analysis of silver nanoparticles in water.

Despite frequent detection in environmental waters, literature which quantifies the health risk of silver nanoparticles (Ag NPs) through oral ingestion is scarce. This study compiled literature data to find the removal of Ag NPs from different treatment schemes (i.e., natural, engineered, or hybrid). Ag NP concentrations were found either in surface water or in groundwater based on where the effluent of treatment schemes was discharged, i.e., either in surface water or in groundwater. Monte-Carlo simulation was carried out for probabilistic assessment of health risks for children for two hypothetical exposure scenarios: (a) ingesting river water while swimming and (b) drinking groundwater. Bio-accessible fraction, dietary metal adsorption factor, and concentrations of silver ions were incorporated to simulate realistic situations. Different treatment schemes were ranked for their nanoparticles' removal efficiency with respect to (i) exceedance probability from guideline value and (ii) health risk to children. Hybrid treatment combinations, i.e., conventional primary and secondary treatment units followed by nature-based units (constructed wetlands and soil aquifer treatment), were ranked the best. The health risk value was found to be less than 1, with the 99th percentile value less than 10-3 in all cases. The maximum allowable concentration of Ag NPs was found to be as low as 1.43 mg/L for groundwater, suggesting probable potential for risk. Uncertainty analysis revealed that the uncertainty of the influent NPs concentration in raw wastewater contributes > 99% to the variance of the hazard index. The results of this work indicate that the use of natural treatment technologies with existing engineered treatments provides higher nanoparticle removal from wastewater without the requirement of any tertiary treatment unit.

Investigating the behavior of ultratrace levels of nanoparticulate and ionic silver in a seawater mesocosm using single particle inductively coupled plasma - mass spectrometry.

Silver nanoparticles (AgNPs) nowadays appear in close to 24% of consumer products that contain engineered nanomaterials. Thus, they are expected to be released into the environment, where their fate and effect are still undetermined. Considering the evidenced efficacy of the single particle Inductively Coupled Plasma - Mass Spectrometry (sp ICP-MS) technique in the study of nanomaterials, this work reports on the use of sp ICP-MS along with an online dilution sample introduction system for the direct analysis of untreated and spiked seawater samples, as part of a larger scale experiment studying the fate of Ag (ionic and nanoparticles) in seawater mesocosm systems. Silver nanoparticles coated with branched polyethyleneimine (BPEI@AgNPs) or ionic silver (Ag+) were introduced gradually into the seawater mesocosm tanks at very low, environmentally relevant concentrations (50 ng Ag L-1 per day, for 10 consecutive days, up to a total of 500 ng Ag L-1), and samples were collected and analyzed daily, within a consistent time window. Using very low detector dwell time (75 µs) and specialized data treatment, information was obtained on the nanoparticles' size distribution and particle number concentration, as well as the ionic silver content, of both the AgNPs and the Ag+ treated seawater mesocosm tanks. The results for the AgNP treated samples indicated the rapid degradation of the added silver particles, and the subsequent increase of ionic silver, with recoveries close to 100% for the first days of the experiment. On the other hand, particle formation was observed in the Ag+ treated seawater tanks, and even though the number concentration of silver-containing nanoparticles increased throughout the experiment, the amount of silver per particle remained relatively constant from the early days of the experiment. In addition, the online dilution sample introduction system for the ICP-MS proved capable of handling the untreated seawater matrix without significant contamination issues and downtime, while the low dwell time and data treatment procedure developed were shown to be suitable for the analysis of nanomaterials at the low nm-scale, despite the complex and heavy matrix introduced into the ICP-MS.

Risk assessment of silver and microplastics release from antibacterial food containers under conventional use and microwave heating.

The evaluation of the migration of ionic silver and nanoparticulated silver (AgNPs) from antimicrobial plastic packaging to food is crucial to ensure its safety. Migration assays were performed on reusable silver-containing polypropylene (PP) food containers and a silicone baby bottle, using food simulants, under conventional or microwave heating and repeated use. The PP containers released significant amounts of silver, increasing with temperature, contact time, acidity and lower crystallinity. Silver migration in the silicone bottle was much lower. Risk assessment of released silver was done considering European authorities safety recommendations, with some containers far exceeding these levels. No significant AgNPs release was detected in the simulants by single particle-ICPMS. Silver-containing microplastics and silicone microparticles were detected by SEM in the food simulants after the migration assays. Consumers may be continuously exposed to the harmful effects of ionic silver and microplastics, which can potentially lead to health issues.

Development of a Pt-graphene nanocomposite-based solid-phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry for the determination of carbamate pesticides in fish.

In the present work, a potential solid-phase extraction (SPE) material based on graphene anchored with platinum nanoparticles (Pt-Graphene) was prepared and characterized by scanning electron micrographs and transmission electron micrograph. The carbamates residues in fish were enriched by SPE filled with Pt-Graphene and detected by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The proposed extraction protocol exhibited satisfactory recoveries (76.5-115.6%), low limit of quantitation values in µg kg-1 level, and good precision for the studied ten carbamates. These results demonstrated the feasibility of the proposed protocol. The developed Pt-Graphene nanoparticles showed excellent performance for extracting analytes at trace levels, indicating that it could be used as a potential SPE sorbent in food residue analysis.

Isolation of organophosphate pesticides from water using gold nanoparticles doped magnetic three-dimensional graphene oxide.

Pesticides are a large group of pristine organic contaminants, which are widely discharged into environmental water due to agricultural activities. Hence, extraction, determination, and removal of pesticides from water resources are necessary for human health. In this study, novel adsorbent was developed based on three-dimensional magnetic graphene coated with gold nanoparticles (3D-MG@AuNPs) for extraction of chlorpyrifos, dicrotophos, fenitrothion, and piperophos as four specific organophosphorus pesticides (OPPs) from wastewater and tap water samples. The proposed nanocomposite was characterized; FTIR and EDX are performed for the expected functional groups and elemental analysis, SEM showed the unique and spherical AuNPs are well dispersed over graphene sheets. In this investigation, the important parameters that have effect on the extraction efficiency, including the desorbing solvent, desorbing solvent volume, vortex time, the extraction time, adsorbent dosage, pH of sample solutions, and salt effect were evaluated. In conclusion, the measured amounts of the chosen OPPs were determined using the gas chromatography microelectron capture (µECD-GC) method. Limits of quantification (S/N ratio of 10) and detection (S/N ratio of 3) were attained at concentrations of 0.26-0.43 µg.L-1 and 0.08-0.14 µg.L-1, respectively. According to the results of the investigations, the synthesized 3D-MG@AuNPs did not require any complicated sample preparation methods; therefore, it is a very good choice for solid magnetic phase extraction studies.

Gallium Metal-Organic Nanoparticles with Albumin-Stabilized and Loaded Graphene for Enhanced Delivery to HCT116 Cells.

Background: Gallium (III) metal-organic complexes have been shown to have the ability to inhibit tumor growth, but the poor water solubility of many of the complexes precludes further application. The use of materials with high biocompatibility as drug delivery carriers for metal-organic complexes to enhance the bioavailability of the drug is a feasible approach. Methods: Here, we modified the ligands of gallium 8-hydroxyquinolinate complex with good clinical anticancer activity by replacing the 8-hydroxyquinoline ligands with 5-bromo-8-hydroxyquinoline (HBrQ), and the resulting Ga(III) + HBrQ complex had poor water solubility. Two biocompatible materials, bovine serum albumin (BSA) and graphene oxide (GO), were used to synthesize the corresponding Ga(III) + HBrQ complex nanoparticles (NPs) BSA/Ga/HBrQ NPs and GO/Ga/HBrQ NPs in different ways to enhance the drug delivery of the metal complex. Results: Both of BSA/Ga/HBrQ NPs and GO/Ga/HBrQ NPs can maintain stable existence in different solution states. In vitro cytotoxicity test showed that two nanomedicines had excellent anti-proliferation effect on HCT116 cells, which shown higher level of intracellular ROS and apoptosis ratio than that of cisplatin and oxaliplatin. In addition, the superior emissive properties of BSA/Ga/HBrQ NPs and GO/Ga/HBrQ NPs allow their use for in vivo imaging showing highly effective therapy in HCT116 tumor-bearing mouse models. Conclusion: The use of biocompatible materials for the preparation of NPs against poorly biocompatible metal-organic complexes to construct drug delivery systems is a promising strategy that can further improve drug delivery and therapeutic efficacy.

Aggregating-agent-assisted surface-enhanced Raman spectroscopy-based detection of acrylamide in fried foods: A case study with potato chips.

This study developed a simple, rapid, stable, and reliable technique for acrylamide (AAm) detection through surface-enhanced Raman scattering (SERS) on an AgNPs substrate with an aggregating agent. Specifically, the agglomeration effects of five types of salt solutions (NaCl, KCl, MgCl2, Na2SO4, and MgSO4) were investigated at different concentrations and optimized using an orthogonal experiment. The optimal amounts of the aggregating agent, analytes, and AgNPs were 4, 4, and 12 µL, respectively. A linear relationship (peak area I1449 = 7.4197x + 5984.8, R2 = 0.9971) between the characteristic peak area and AAm concentration was established in the range of 10 to 500 µg/L, and the LOD was 2.5 µg/L. The recoveries and relative standard deviations in the analysis of potato chips samples were 94.67 %-117.50 % and 8.43 %-12.29 %, respectively. The results of the proposed method were consistent with those obtained by LC-MS/MS method. This study demonstrated that SERS has excellent potential for application in the qualitative and quantitative analyses of AAm in fried foods.

Development of a fluorescent probe for cefazolin detection based on solvent-based de-emulsification dispersive liquid-liquid microextraction of silver nanoparticles.

A novel, simple, and rapid method has been developed for the fluorimetric determination of trace levels of cefazolin. The method is based on the synthesis of silver nanoparticles (AgNPs) as fluorescent probes using resorcinol as a reducing and capping agent and then their extraction into the 1-octanol by a highly efficient solvent-based de-emulsification dispersive liquid-liquid microextraction technique. The interaction of cefazolin with silver affected the fluorescence intensity of AgNPs in the organic phase that creates a micro-probe fluorimetric detection of this antibiotic at excitation/emission wavelengths of 410/527 nm. Under the established optimum conditions, the linear analytical range was from 0.80 to 12.00 ng mL-1 of cefazolin with a detection limit of 0.55 ng mL-1. The relative standard deviation for ten replicate measurements of 2 and 10 ng mL-1 of cefazolin was 4.18 and 1.88%, respectively. The suggested method was successfully applied to the determination of cefazolin in pharmaceutical formulation, human urine and plasma.

Characterization of titanium dioxide nanoparticles in confectionary products and estimation of dietary exposure level among the Chinese population.

Titanium dioxide (TiO2) is widely used in the food industry. Recently, European Commission has banned TiO2 as a food additive, raising public concern about its health risk, especially the nanoparticles (NPs) contained therein. This study aimed to reveal the existence of TiO2 NPs in food and further estimate the dietary exposure level among Chinese population by characterizing particle size distribution, determining Ti content and micro-distribution in food products, and calculating food consumption from the China Health and Nutrition Survey (CHNS). The results showed that TiO2 particle size in food additives and chewing gums was 53.5-230.3 nm and 56.8-267.7 nm respectively, where NPs accounted for 34.7% and 55.6% respectively. TiO2 was firstly in situ presented on the surface of confectionary products with hard shells. The content of TiO2 ranged from 3.2 to 3409.3 µg/g product. Besides, the mean dietary intake was 71.31 µg/kgbw/day for TiO2 and 7.75 µg/kgbw/day for TiO2 NPs among Chinese population, affected by people's dietary habits of different regions. Children's exposure levels was the highest due to their love of sweets. More attention should be paid to risk assessment and management of TiO2 NPs for children in China.

Surface-enhanced Raman spectroscopy combined with stable isotope probing to assess the metabolic activity of Escherichia coli cells in chicken carcass wash water.

Rapid evaluation of the metabolic activity of microorganisms is crucial in the assessment of the disinfection ability of various antimicrobial agents in the food industry. In this study, surface-enhanced Raman spectroscopy combined with isotope probing was employed for the analysis of the disinfection of single bacterial cells in the chicken carcass wash water. The Raman signals from single Escherichia coli O157:H7 cells were enhanced by in situ synthesis of silver nanoparticles. The ΔCD of the cells grown in presence of 0.5% hydrogen peroxide and 50 ppm chlorine was 5.86 ± 1.86% and 5.1 ± 2.3%, respectively, which showed significant reduction compared with cells grown in the absence of disinfecting agents (19.86 ± 2.51%) after 2 h of incubation. The study proved that the proposed method had the potential to assess the metabolic activity of microorganisms in other food products and optimize the disinfection process.

The Effect of Copper and Copper Oxide Nanoparticles on Rainbow Trout (Oncorhynchus mykiss W.) Spermatozoa Motility after Incubation with Contaminants.

The study aimed to analyse the effect of copper nanoparticles of similar particle size of Cu and CuO and copper ions (CuSO4) on the motility parameters of rainbow trout spermatozoa after long-term exposure and compare its harmful effect. Nanoproducts of Cu and CuO (Cu NPs, CuO NPs) of primary particle size around 50 nm and ionic solution of CuSO4 were used for the study. Suspension of concentrations 0, 1, 5, 10, 25, and 50 mg Cu·L-1 of Cu NPs, CuO NPs, and CuSO4 was dissolved in an artificial seminal plasma. Milt was mixed with the prepared solution and stored in a fridge, at 6 °C, for 96 h. At the defined incubation time, spermatozoa were activated for movement, and six motility parameters were evaluated using an automated system (CASA). Increasing concentrations of Cu NPs, CuO NPs, and CuSO4 in an incubation medium in parallel decreased the percentage of motile sperm (MOT). The effect of Cu NPs and ionic copper on MOT was more deleterious than that of CuO NPs. Copper products slightly increased the velocity (VCL) compared to the control, particularly up to 24 h of storage. Linearity (LIN) was improved by three tested products, particularly CuO NPs. Generally, the motility duration was prolonged when the sperm was incubated with copper products compared to the control. Nanoproducts made from different compounds of the same elements of similar particle size have a different effect on cells. Cu NPs were more harmful than CuO NPs. The effect of Cu NPs was similar to an ionic form of CuSO4. When incubated, the copper nanoproducts and ionic form exert a slightly positive effect on spermatozoa velocity, linearity, and motility duration, particularly up to 24 h of storage.

Comparative toxicity of silver nanoparticles and silver nitrate in freshwater fish Oreochromis mossambicus: A multi-biomarker approach.

Silver nanoparticles (AgNPs) in the aquatic environment affect ecological repercussions and have fatal impacts on aquatic animals. The current study examined and correlated the toxicity of silver nitrate (AgNO3) and silver nanoparticles (AgNPs) to the Mozambique tilapia, Oreochromis mossambicus. The comparative toxicity studies were done by exposing O. mossambicus to various doses of AgNO3 and AgNPs (0, 25, 50, 75, and 100 µg/L) over a 7-day subacute exposure period. AAS analysis was used to detect Ag accumulation, while the histological examination established gill tissue damage. Oxidative stress affects lipid peroxidation (LPO) and protein carbonyl activity (PCA) in the gill tissue. Antioxidant parameters such as glutathione-S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase activity (CAT), and non-enzymatic antioxidants such as metallothionein (MT) and reduced glutathione. The serum in the blood was used to determine non-specific immunological characteristics such as lysozyme (LYZ), myeloperoxidase (MPO), and respiratory burst activity (RBA). The neurotoxic impact of acetylcholine esterase activity (AChE) was investigated in brain tissues. The findings demonstrated that larger concentrations of AgNO3 than AgNPs improved enzymatic antioxidant activities in the gill tissue. Histological examination of fish gills demonstrated that both AgNPs and AgNO3 induced telangiectasia and epithelial cell hyperplasia. By increasing the concentration of AgNPs and AgNO3, the present research demonstrated that silver accumulation leads to inefficient oxidative stress and altered enzymatic and non-enzymatic parameters, leading to cellular damage.

Evaluation of nano-silver concentrations using multi-media fate and transport models with different spatial resolutions.

Due to the complexity of the environmental matrix and the limitation of the detection level, it is important to use models to evaluate environmental exposure to nanomaterials. Based on this, this research established two multi-media fate and transport models of silver nanoparticles (AgNPs) with respective coarse and fine spatial resolutions. Using the case of the spatial distribution of AgNPs in the Xiangjiang River, China, we compared how the spatial resolution of a model impacts modeling results. The results show that the process of heterogeneous aggregation has the greatest impact on the modeled concentration of AgNPs in water, and the heterogeneous aggregation of free AgNPs with natural colloids and settling down to sediments is the dominant mechanism responsible for the loss of AgNPs. In addition, the use of different spatial resolutions gives similar trends in the modeled AgNPs concentration, despite the difference in absolute levels. Our work also shows that the reliability and accuracy of the rate constant of heterogeneous aggregation has a great influence on the modeled AgNPs concentrations.

Quantification of silver nanoparticle interactions with yeast Saccharomyces cerevisiae studied using single-cell ICP-MS.

Silver nanoparticles (AgNPs) have been used in many fields due to their anticancer, antimicrobial, and antiviral potential. Single-cell ICP-MS (SC-ICP-MS) is an emerging technology that allows for the rapid characterization and quantification of a metal analyte across a cell population in a single analysis. In this study, a new rapid and sensitive SC-ICP-MS method was developed to quantitatively study the interactions of AgNPs with yeast Saccharomyces cerevisiae. The method can quantify the cell concentration, silver concentration per cell, and profile the nanoparticle distribution in a yeast cell population. AgNP dosing time, concentration, and AgNP size were quantitatively evaluated for their effects on AgNP-yeast cell interactions. The results showed that the initial uptake of AgNPs was rapid and primarily driven by the mass of Ag per cell. The optimal dosing particle concentrations for highest uptake were approximately 1820, 1000, and 300 AgNPs/cell for 10, 20, and 40 nm AgNPs, respectively. Furthermore, this study also validated a washing method for the application to a microorganism for the first time and was used to quantitatively determine the amount of cell surface-adsorbed AgNPs and intracellular AgNPs. These results indicated that the mass (Ag in ag/cell) ratios of intracelluar vs cell surface-adsorbed AgNPs were similar for different AgNP sizes. This high throughput and ultrasensitive SC-ICP-MS method is expected to have many potential applications, such as optimization of methods for green synthesis of AgNPs, nanotoxicity studies, and drug delivery. This is the first quantification study on the interactions of AgNPs and S. cerevisiae using SC-ICP-MS.

Nanopharmaceuticals (Au-NPs) after use: Experiences with a complex higher tier test design simulating environmental fate and effect.

The current environmental hazard assessment is based on the testing of the pristine substance. However, it cannot be excluded that (nano)pharmaceuticals are excreted into sewage during the use phase followed by entry into wastewater treatment plants (WWTPs). Sorption to sewage sludge or release via effluent can result in modified ecotoxicological effects which possibly can only be detected with a modified test approach. The objective of our study was to investigate a realistic exposure scenario for metallic nanoparticles (NPs) in pharmaceutical products, excreted into effluent, and released into the environment after treatment in WWTPs. The test approach was illustrated by using gold (Au) NPs. Effluent from model WWTPs were investigated in aquatic tests (Daphnia magna, fish cell lines). Sewage sludge was used as a sole food source (Eisenia fetida) or mixed with soil and used as test medium (soil microorganisms, Folsomia candida, Enchytraeus crypticus). To cover the aspect of regulation, the test systems described in OECD-test guidelines (OECD TG 201, 211, 220, 232, 249, 317) were applied. Modifications and additional test approaches were included to meet the needs arising out of the testing of nanomaterials and of the exposure scenarios. The results were assessed regarding the suitability of the test design and the toxicity of Au-NPs. Except for activated sludge as a sole food source for E.fetida, the selected test approach is suitable for the testing of nanomaterials. Additional information can be gained when compared to the common testing of the pristine nanomaterials in the standardized test systems. Effects of Au-NPs were observed in concentrations exceeding the modeled environmental.

Analyzing the surface of functional nanomaterials-how to quantify the total and derivatizable number of functional groups and ligands.

Functional nanomaterials (NM) of different size, shape, chemical composition, and surface chemistry are of increasing relevance for many key technologies of the twenty-first century. This includes polymer and silica or silica-coated nanoparticles (NP) with covalently bound surface groups, semiconductor quantum dots (QD), metal and metal oxide NP, and lanthanide-based NP with coordinatively or electrostatically bound ligands, as well as surface-coated nanostructures like micellar encapsulated NP. The surface chemistry can significantly affect the physicochemical properties of NM, their charge, their processability and performance, as well as their impact on human health and the environment. Thus, analytical methods for the characterization of NM surface chemistry regarding chemical identification, quantification, and accessibility of functional groups (FG) and surface ligands bearing such FG are of increasing importance for quality control of NM synthesis up to nanosafety. Here, we provide an overview of analytical methods for FG analysis and quantification with special emphasis on bioanalytically relevant FG broadly utilized for the covalent attachment of biomolecules like proteins, peptides, and oligonucleotides and address method- and material-related challenges and limitations. Analytical techniques reviewed include electrochemical titration methods, optical assays, nuclear magnetic resonance and vibrational spectroscopy, as well as X-ray based and thermal analysis methods, covering the last 5-10 years. Criteria for method classification and evaluation include the need for a signal-generating label, provision of either the total or derivatizable number of FG, need for expensive instrumentation, and suitability for process and production control during NM synthesis and functionalization.

Protein-Protein Interactions: Insight from Molecular Dynamics Simulations and Nanoparticle Tracking Analysis.

Protein-protein interaction plays an essential role in almost all cellular processes and biological functions. Coupling molecular dynamics (MD) simulations and nanoparticle tracking analysis (NTA) assay offered a simple, rapid, and direct approach in monitoring the protein-protein binding process and predicting the binding affinity. Our case study of designed ankyrin repeats proteins (DARPins)-AnkGAG1D4 and the single point mutated AnkGAG1D4-Y56A for HIV-1 capsid protein (CA) were investigated. As reported, AnkGAG1D4 bound with CA for inhibitory activity; however, it lost its inhibitory strength when tyrosine at residue 56 AnkGAG1D4, the most key residue was replaced by alanine (AnkGAG1D4-Y56A). Through NTA, the binding of DARPins and CA was measured by monitoring the increment of the hydrodynamic radius of the AnkGAG1D4-gold conjugated nanoparticles (AnkGAG1D4-GNP) and AnkGAG1D4-Y56A-GNP upon interaction with CA in buffer solution. The size of the AnkGAG1D4-GNP increased when it interacted with CA but not AnkGAG1D4-Y56A-GNP. In addition, a much higher binding free energy (∆GB) of AnkGAG1D4-Y56A (-31 kcal/mol) obtained from MD further suggested affinity for CA completely reduced compared to AnkGAG1D4 (-60 kcal/mol). The possible mechanism of the protein-protein binding was explored in detail by decomposing the binding free energy for crucial residues identification and hydrogen bond analysis.