Polystyrene-b-poly (acrylic acid) nanovesicles coated by modified chitosans for encapsulation of minoxidil

Abstract In this work, polystyrene-b-poly (acrylic acid) (PS-b-PAA) nanovesicles were coated by modified chitosans aiming at studying its physicochemical parameters. The chitosan (CS) was chemically modified to add hydrophilic and/or hydrophobic groups, obtaining three modified chitosans. The PS-b-PAA nanovesicles were obtained by organic (1,4-dioxane) cosolvent method in water, resulting in nanovesicles with less than 150 nm of diameter (polydispersibility index - PDI at 90° = 0.106), measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and negative zeta potential (-37.5 ± 3.2 mV), allowing the coating of its surface with oppositely charged polysaccharides, such as the CS and the modified chitosans. The coating process was made by mixing the colloidal suspensions with the CS and the modified chitosans at specific ENT#091;CS-xENT#093;/ENT#091;PS-b-PAAENT#093; ratios (0.001 to 1.0 wt %) and measuring the change in size and surface charge by DLS and zeta potential. Upon reaching maximum adsorption, the zeta potential parameter was positively stabilized (+26.7 ± 4.1 mV) with a hydrodynamic diameter slightly longer (< 200 nm of diameter). The encapsulation efficiency (EE) of minoxidil, quantified by capillary electrophoresis, was 50.7%, confirming their potential as drug delivery carriers and the coating process showed the possibility of controlling the surface charge nature of these nanovesicles

Emulsion-based isothermal nucleic acid amplification for rapid SARS-CoV-2 detection via angle-dependent light scatter analysis.

The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/µL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/µL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.

Estudo da eficácia de encapsulação da Cinarizina em cubossomos não-iônicos: caracterização estrutural e citotóxica / Study of the encapsulation efficacy of Cinnarizine in non-ionic cubosomes: structural and cytotoxic characterization

Os cubossomos são partículas nanoestruturadas em forma de bicamada lipídica, bicontínuas e altamente curvadas, as quais devem ser estabilizadas por um polímero não-iônico, neste caso o Pluronic® F-127. Podem ser compostos por alguns tipos de lipídios específicos que possuem a capacidade de se auto associar em estruturas cúbicas quando estão em excesso de água, como o fitantriol (PHY) e a monoleína (GMO). Devido a sua estrutura única, cubossomos possuem um grande potencial para serem considerados como sistemas drug delivery. Os sistemas drug delivery são amplamente utilizados na pesquisa farmacêutica e em contextos clínicos para aumentar a eficácia de compostos utilizados para diagnóstico e de fármacos. No caso da cinarizina (CNZ), fármaco já aprovado para o tratamento de náuseas, vômitos e vertigens causadas pela doença de Ménière, existem inúmeros efeitos colaterais associados a sua baixa solubilidade. Desta forma, a encapsulação em cubossomos se torna uma abordagem promissora para resolver os problemas de atividade farmacológica relacionados ao fármaco. Neste trabalho, realizamos uma caracterização biofísica da interação da CNZ em cubossomos (contendo PHY ou myverol, MYV, sendo este composto por 80% de GMO). As técnicas biofísicas utilizadas foram: espalhamento de raios-X em baixos ângulos (SAXS), espalhamento dinâmico de luz (DLS), microscopia eletrônica de transmissão (TEM), crio microscopia eletrônica de transmissão (Crio-TEM), análise de rastreamento de nanopartículas (NTA) e potencial zeta. A cromatografia líquida de alta eficiência (HPLC) foi realizada para verificar a porcentagem de eficiência de encapsulação (%EE) da CNZ nos cubossomos, enquanto que a citotoxicidade foi avaliada em eritrócitos através da análise da atividade hemolítica. Inicialmente, a influência de diferentes solventes (acetona, clorofórmio, etanol e octano) nas propriedades estruturais de cubossomos de PHY foi investigada, a fim de se minimizar os efeitos do solvente utilizados para a encapsulação da CNZ. Para amostras com acetona, descobriu-se que apenas altas concentrações tiveram influência na estrutura cristalográfica das nanopartículas, sendo o resultado foi totalmente reversível após 24h. O etanol fez com que o parâmetro de rede aumentasse de 10-15%. O clorofórmio e o octano tiveram efeitos diferentes sobre cubossomos de PHY em comparação com a acetona e o etanol; ambos induziram uma transição cúbico-hexagonal-micelar. Posteriormente, constatamos que as nanopartículas de PHY e MYV apresentaram diferentes estruturas cristalográficas, sendo elas Pn3m e Im3m, respectivamente. Devido a problemas com a baixa solubilidade de CNZ em PHY os estudos para esse lipídio foram suspensos. Nos testes para cubossomos de MYV ao incorporar a CNZ foi observado uma alteração da estrutura cúbica de Im3m para Pn3m e os valores dos parâmetros de rede se alteraram de acordo com a estrutura cristalina encontrada, porém os valores não apresentaram diferenças significativas de tamanho quando se trata da mesma estrutura, sugerindo que a CNZ não interferiu no parâmetro de rede. Os tamanhos das nanopartículas apresentaram uma população monodispersa com ~200 nm. DLS mostrou uma interferência da CNZ no tamanho dos cubossomos, variando de forma diretamente proporcional a concentração de CNZ na amostra, enquanto as técnicas de NTA e microscopia apresentaram nanopartículas de tamanhos bastante variados, mas independente da interferência da CNZ. A encapsulação de CNZ também foi dosada por HLPC em cubossomos de MYV, obtendo um limite superior de 0,6 mg/mL. A atividade citotóxica dos cubossomos foi testada em eritrócitos, revelando uma taxa de hemólise bastante inferior em cubossomos com CNZ em relação a cubossomos puros. Acreditamos que os cubossomos podem sim ser utilizados como sistemas carreadores de CNZ

Cubosomes are nanostructured particles in the form of a lipid bilayer, bicontinuous and highly curved, which must be stabilized by a non-ionic polymer, in this case Pluronic® F-127. They can be composed of some types of specific lipids that have the ability to self-associate in cubic structures when they are in excess of water, such as phytantriol (PHY) and monolein (GMO). Due to their unique structure, cubosomes have a great potential to be considered as drug delivery systems. Drug delivery systems are widely used in pharmaceutical research and clinical settings to increase the efficacy of compounds used for diagnostics and drugs. In the case of cinnarizine (CNZ), a drug already approved for the treatment of nausea, vomiting and vertigo caused by Ménière's disease, there are numerous side effects associated with its low solubility. Thus, cubosomal encapsulation becomes a promising approach to solve drug-related problems of pharmacological activity. In this work, we performed a biophysical characterization of the CNZ interaction in cubosomes (containing PHY or myverol, MYV, which is composed of 80% GMO). The biophysical techniques used were: low angle X-ray scattering (SAXS), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryo transmission electron microscopy (Crio-TEM), nanoparticle tracking analysis (NTA) and zeta potential. High performance liquid chromatography (HPLC) was performed to verify the percentage of encapsulation efficiency (%EE) of CNZ in cubosomes, while cytotoxicity was evaluated in erythrocytes by analyzing the hemolytic activity. Initially, the influence of different solvents (acetone, chloroform, ethanol and octane) on the structural properties of PHY cubosomes was investigated in order to minimize the effects of the solvent used for the encapsulation of CNZ. For samples with acetone, it was found that only high concentrations had an influence on the crystallographic structure of the nanoparticles, with the result being fully reversible after 24h. Ethanol caused the network parameter to increase by 10-15%. Chloroform and octane had different effects on PHY cubosomes compared to acetone and ethanol; both induced a cubic-hexagonal-micellar transition. Later, we found that PHY and MYV nanoparticles presented different crystallographic structures, being Pn3m and Im3m, respectively. Due to problems with the low solubility of CNZ in PHY, studies for this lipid were suspended. In the tests for MYV cubosomes when incorporating CNZ, a change in the cubic structure from Im3m to Pn3m was observed and t he lattice parameters changed according to the crystal structure found, but the differences observed were not significant when it comes to the same structure, suggesting that the CNZ did not interfere with the network parameter. The nanoparticle sizes showed a monodisperse population with ~200 nm. DLS showed an interference of CNZ in the size of the cubosomes, varying directly proportionally to the concentration of CNZ in the sample, while NTA and microscopy techniques showed nanoparticles of widely varying sizes, but independent of CNZ interference. CNZ encapsulation was also dosed by HLPC in MYV cubosomes, obtaining an upper limit of 0.6 mg/ml. The cytotoxic activity of cubosomes was tested in erythrocytes, revealing a much lower rate of hemolysis in cubosomes with CNZ compared to pure cubosomes. We believe that cubosomes can indeed be used as CNZ carrier systems

Structural Characterization of Biomaterials by Means of Small Angle X-rays and Neutron Scattering (SAXS and SANS), and Light Scattering Experiments.

Scattering techniques represent non-invasive experimental approaches and powerful tools for the investigation of structure and conformation of biomaterial systems in a wide range of distances, ranging from the nanometric to micrometric scale. More specifically, small-angle X-rays and neutron scattering and light scattering techniques represent well-established experimental techniques for the investigation of the structural properties of biomaterials and, through the use of suitable models, they allow to study and mimic various biological systems under physiologically relevant conditions. They provide the ensemble averaged (and then statistically relevant) information under in situ and operando conditions, and represent useful tools complementary to the various traditional imaging techniques that, on the contrary, reveal more local structural information. Together with the classical structure characterization approaches, we introduce the basic concepts that make it possible to examine inter-particles interactions, and to study the growth processes and conformational changes in nanostructures, which have become increasingly relevant for an accurate understanding and prediction of various mechanisms in the fields of biotechnology and nanotechnology. The upgrade of the various scattering techniques, such as the contrast variation or time resolved experiments, offers unique opportunities to study the nano- and mesoscopic structure and their evolution with time in a way not accessible by other techniques. For this reason, highly performant instruments are installed at most of the facility research centers worldwide. These new insights allow to largely ameliorate the control of (chemico-physical and biologic) processes of complex (bio-)materials at the molecular length scales, and open a full potential for the development and engineering of a variety of nano-scale biomaterials for advanced applications.

Preparation and in vitro characterization of monoclonal antibody ranibizumab conjugated magnetic nanoparticles for ocular drug delivery

Gold coated magnetite nanoparticles were prepared and coated with ranibizumab as an ocular drug delivery system. The surface morphologies of the nanoparticles were determined by Scanning Electron Microscopy (SEM). The size and surface charge were determined by using the dynamic light scattering (DLS) technique. Crystallographic properties of the gold coated Fe3O4 nanoparticles were recorded on X-ray diffractometer (XRD) the XRD pattern of nanoparticlees were shown to have uniqe Fe3O4 and gold peaks. Conjugation of ranibizumab onto nanoparticles was achieved using the physical adsorption method. The amount of ranibizumab on the surface of the nanoparticles was determined by thermogravimetric analysis (TGA). In the in vitro release studies performed using UV spectroscopy; it was found that almost 60% of antibodies were released within the first 30 minutes. Antibody activity after release studies was also proved with ELISA. Non-toxicity of gold coated Fe3O4 particles were proved with MTT. Results of the studies, showed that the antibody conjugated magnetic nanoparticle system could be a potential treatment system for ocular diseases.

Rapid antimicrobial susceptibility testing on positive blood cultures through an innovative light scattering technology: performances and turnaround time evaluation.

BACKGROUND: A bacteremia diagnosis with speeded-up identification and antimicrobial susceptibility testing (AST) is mandatory to adjust empirical broad-spectrum antibiotherapy and avoid the emergence of multi-resistant bacteria. Alfred 60AST (Alifax, Polverara, PD, Italy) is an innovative automated system based on light scattering measurements allowing direct AST from positive blood cultures with rapid results. In this study we aimed to evaluate the system's performances and turnaround time (TAT) compared to routine AST. METHODS: The study was conducted during 2 non-consecutive 3-month periods at the microbiology laboratory of the Cliniques universitaires Saint-Luc. All blood cultures detected positive in the 0 AM-10 AM time frame with a pure Gram-positive cocci or Gram-negative bacilli stain were included for Alfred 60AST testing. Two customized EUCAST antibiotic panels were set up composed of 1) a "Gram-negative" panel including cefuroxime, ceftazidime Enterobacteriaceae, piperacillin-tazobactam Enterobacteriaceae, ciprofloxacine, and ceftazidime Pseudomonas 2) a "Gram-positive" panel including cefoxitin Staphylococcus aureus, cefoxitin coagulase-negative (CNS) Staphylococci and ampicillin Enterococci. Categorical agreement (CA), very major errors (VME), major errors (ME), minor errors (mE) and TAT to Alfred 60AST results were calculated in comparison with AST results obtained from direct testing on positive blood cultures with the Phoenix system (Becton Dickinson, Franklin Lakes, NJ, USA). RESULTS: Five hundred seventy and one hundred nine antibiotics were evaluated on respectively 166 Gram-negative bacilli and 109 Gram-positive cocci included in the studied population. During the first study period regarding Gram-negative strains a CA of 89.5% was obtained with a high rate of VME (19 and 15.4% respectively) for cefuroxime and piperacillin-tazobactam Enterobacteriaceae. Considering this, Alifax reviewed these antibiotics' formulations improving Gram-negative bacilli total CA to 92.2% with no VME during the second study period. For Gram-positive cocci, total CA was 88.1% with 2.3% VME, 13.8% ME (mainly cefoxitin CNS) and 12% mE rates both study periods combined. Median TAT to AST results was 5 h with Alfred versus 12 h34 with Phoenix. CONCLUSION: The Alfred 60AST system shows correct yet improvable microbiological performances and a major TAT reduction compared to direct automated AST testing. Clinical studies measuring the impact of the approach on antibiotic management of patients with bacteremia are recommended.

Gold Nanoflower-Enhanced Dynamic Light Scattering Immunosensor for the Ultrasensitive No-Wash Detection of Escherichia coli O157:H7 in Milk.

Gold nanoflowers (GNFs) exhibit stronger light scattering ability than gold nanospheres (GNSs) with the same diameter, thereby contributing to enhancing the sensitivity of the scattering-based sensing method. However, the application of GNFs in biosensors based on dynamic light scattering (DLS) has not been yet reported. Herein, we describe for the first time an improved no-wash immunosensor based on dynamic light scattering for the detection of Escherichia coli O157:H7 (E. coli O157:H7) in milk using GNFs for sensitive signal transduction. To achieve this goal, a thiolated amphiphilic carboxyl ligand was introduced to modify the GNF surface and improve solution stability and antibody functionalization. Several key factors that affect the detection sensitivity of our developed GNF_DLS immunosensor were systematically investigated. Under the optimal conditions, our proposed GNF_DLS immunosensor provided an excellent linear detection for E. coli O157:H7 within the range from 6 × 100 to 6 × 104 colony-forming units (CFU)/mL, with a limit of detection of 2.7 CFU/mL. Combined with our previously reported two-step large-volume immunomagnetic separation (IMS) method, the designed GNF_DLS immunosensor can sensitively, selectively, and accurately detect the presence of E. coli O157:H7 in pasteurized milk. The potential of our GNF_DLS method for monitoring the presence of a single bacterial cell in 1 mL of sample solution was also demonstrated. Overall, the developed GNF_DLS immunosensor can be used for the rapid and high-sensitivity determination of pathogenic bacteria and can be extended for the ultrasensitive no-wash detection of other trace analytes.

Portable System for Time-Domain Diffuse Correlation Spectroscopy.

We introduce a portable system for clinical studies based on time-domain diffuse correlation spectroscopy (DCS). After evaluating different lasers and detectors, the final system is based on a pulsed laser with about 550 ps pulsewidth, a coherence length of 38 mm, and two types of single-photon avalanche diodes (SPAD). The higher efficiency of the red-enhanced SPAD maximizes detection of the collected light, increasing the signal-to-noise ratio, while the better timing response of the CMOS SPAD optimizes the selection of late photons and increases spatial resolution. We discuss component selection and performance, and we present a full characterization of the system, measurement stability, a phantom-based validation study, and preliminary in vivo results collected from the forearms and the foreheads of four healthy subjects. With this system, we are able to resolve blood flow changes 1 cm below the skin surface with improved depth sensitivity and spatial resolution with respect to continuous wave DCS.

Employment of Iron-Binding Protein from Haemophilus influenzae in Functional Nanopipettes for Iron Monitoring.

Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, interest in the iron status of the central nervous system has increased significantly in the past decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington's, Parkinson's, and Alzheimer's. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the iron-binding protein (FBP) from Haemophilus influenzae, specific for ferrous ions. Subsequently, we employed this protein in our nanopipette platform and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 µM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.

Dynamic Light Scattering Analysis to Dissect Intermediates of SNARE-Mediated Membrane Fusion.

Dynamic light scattering (DLS) spectroscopy provides rapid information on the size distribution of a large number of particles in a mixture. Vesicle sizes change during the merger of lipid bilayers, and DLS analysis can provide rapid, accurate, and non-perturbative quantification of the size distribution of proteoliposomes in SNARE-dependent membrane fusion. In this chapter, we describe the methodologies and reagents used for DLS spectroscopy in a biochemical and biophysical study of SNARE-mediated membrane fusion.

Molecular fingerprinting of nanoparticles in complex media with non-contact photoacoustics: beyond the light scattering limit.

Optical instruments can probe physical systems even to the level of individual molecules. In particular, every molecule, solution, and structure such as a living cell has a unique absorption spectrum representing a molecular fingerprint. This spectrum can help identify a particular molecule from others or quantify its concentration; however, scattering limits molecular fingerprinting within a complex compound and must be overcome. Here, we present a new, non-contact photoacoustic (PA)-based method that can almost completely remove the influence of background light scattering on absorption measurements in heterogeneous highly scattering solutions and, furthermore, separate the intrinsic absorption of nanoscale objects from their scattering. In particular, we measure pure absorption spectra for solutions of gold nanorods (GNRs) as an example of a plasmonic agent and show that these spectra differ from the extinction measured with conventional UV-VIS spectrophotometry. Finally, we show how the original GNR absorption changes when nanoparticles are internalized by cells.

A sensitive resonance Rayleigh light scattering method for alpinetin using gold nanorods probes.

A sensitive resonance Rayleigh light scattering (RLS) assay for alpinetin was developed based on alpinetin-modified gold nanorods (AuNRs). Alpinetin could interact with AuNRs and formed a new assembly by electrostatic attraction. In pH 7.4 Tris-HCl buffer solution, the assembly of alpinetin-AuNRs showed a sensitive RLS signal. Under optimum conditions, the magnitude of enhanced RLS intensity (ΔIRLS ) was proportional to the concentration of alpinetin over the range 0.027-3.24 µg ml-1 , with a detection limit of 1.79 ng ml-1 (by 3σ). The developed RLS method was successfully applied to the detection of alpinetin in real or synthesized samples. Alpinetin recoveries were 90.4-108.7% with an RSD of 0.82-2.9% (n = 5) for Alpinia katsumadai samples, and 95.1-103.7% with an RSD of 0.28-3.9% (n = 5) for synthesized samples. The results showed that this new approach was convenient, reliable and sensitive.

Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches.

PURPOSE: Characterizing submicron protein particles (approximately 0.1-1µm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS). METHODS: Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment. RESULTS: A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4 × 107-1 × 1011 particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering. CONCLUSION: RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

Detection of nanoplastics in food by asymmetric flow field-flow fractionation coupled to multi-angle light scattering: possibilities, challenges and analytical limitations.

We tested the suitability of asymmetric flow field-flow fractionation (AF4) coupled to multi-angle light scattering (MALS) for detection of nanoplastics in fish. A homogenized fish sample was spiked with 100 nm polystyrene nanoparticles (PSNPs) (1.3 mg/g fish). Two sample preparation strategies were tested: acid digestion and enzymatic digestion with proteinase K. Both procedures were found suitable for degradation of the organic matrix. However, acid digestion resulted in large PSNPs aggregates/agglomerates (> 1 µm). The presence of large particulates was not observed after enzymatic digestion, and consequently it was chosen as a sample preparation method. The results demonstrated that it was possible to use AF4 for separating the PSNPs from the digested fish and to determine their size by MALS. The PSNPs could be easily detected by following their light scattering (LS) signal with a limit of detection of 52 µg/g fish. The AF4-MALS method could also be exploited for another type of nanoplastics in solution, namely polyethylene (PE). However, it was not possible to detect the PE particles in fish, due to the presence of an elevated LS background. Our results demonstrate that an analytical method developed for a certain type of nanoplastics may not be directly applicable to other types of nanoplastics and may require further adjustment. This work describes for the first time the detection of nanoplastics in a food matrix by AF4-MALS. Despite the current limitations, this is a promising methodology for detecting nanoplastics in food and in experimental studies (e.g., toxicity tests, uptake studies). Graphical abstract Basic concept for the detection of nanoplastics in fish by asymmetric flow field-flow fractionation coupled to multi-angle light scattering.

Optical and surface plasmonic approaches to characterize extracellular vesicles. A review.

Extracellular vesicles (EVs) have been recognized as messengers delivering various active molecules between cells. This feature of EVs drew the attention of clinicians as well as researchers from different fields. However, exciting ideas to employ EVs as means of drug delivery or to test them as biomarkers of cellular status require very thoughtful and attentive approaches to the selection of analytical techniques for EV characterization. Optical and surface plasmonic analytical methods offer a researcher an invaluable opportunity to use already sized and/or quantified EVs in further functional cell-based assays and in focused biochemical tests (nucleic acid and protein arrays, etc.). Moreover, a high sensitivity and relative flexibility of surface plasmonic sensors open a possibility to develop instruments performing quantitative, metrical and EV surface/content analysis in a single device. This review aims to consider the applicability of established and modern optical techniques as well as novel surface plasmonic approaches for different aspects of EV analysis.

Chemical and Biophysical Approaches for Complete Characterization of Lectin-Carbohydrate Interactions.

Lectins are carbohydrate-binding proteins unrelated to antibodies or enzymes. While carbohydrates are present on all cells and pathogens, lectins are also ubiquitous in nature and their interactions with glycans mediate countless biological and physical interactions. Due to the multivalency found in both lectins and their glycan-binding partners, complete characterization of these interactions can be complex and typically requires the use of multiple complimentary techniques. In this chapter, we provide a general strategy and protocols for chemical and biophysical approaches that can be used to characterize carbohydrate-mediated interactions in the context of individual oligosaccharides, as part of a glycoprotein, and ending with visualization of interactions with whole virions.

Liposome-Assisted Metabolic Glycan Labeling With Cell and Tissue Selectivity.

Metabolic labeling of glycans with sugar chemical reporters (i.e., unnatural sugars bearing a bioorthogonal group), followed by bioorthogonal reaction with imaging probes or affinity tags, has enabled visualization and proteomic analysis of glycosylation in live cells and in living animals. This two-step metabolic glycan labeling strategy has emerged as a powerful tool for probing glycosylation, but suffers from a lack of cell-type selectivity. Here we describe liposome-assisted bioorthogonal reporter (LABOR), a liposome-assisted format of metabolic glycan labeling that allows for cell-selective and tissue-specific glycan imaging and glycoproteomic profiling. After a brief introduction of the principles and applications of LABOR, we provide detailed protocols for performing LABOR in cell culture and in living mice.

Quantification of Scleral Biomechanics and Collagen Fiber Alignment.

The stiffness of the sclera is important in several ocular disorders, and there is hence a need to quantify the biomechanical properties of this tissue. Here, we present two methods for measuring the stiffness of scleral ocular tissues: ocular compliance testing and digital image correlation strain mapping. In tandem with these approaches, we provide two methods to spatially quantify the anisotropic alignment of collagen fibers making up the sclera, using second harmonic generation microscopy and small-angle light scattering. Together, these approaches allow specimen-specific measurement of tissue stiffness and collagen alignment, which are key factors in determining how the eye responds to mechanical loads.

Measuring proteins with greater speed and resolution while reducing sample size.

A multi-angle light scattering (MALS) system, combined with chromatographic separation, directly measures the absolute molar mass, size and concentration of the eluate species. The measurement of these crucial properties in solution is essential in basic macromolecular characterization and all research and production stages of bio-therapeutic products. We developed a new MALS methodology that has overcome the long-standing, stubborn barrier to microliter-scale peak volumes and achieved the highest resolution and signal-to-noise performance of any MALS measurement. The novel design simultaneously facilitates online dynamic light scattering (DLS) measurements. As National Institute of Standards and Technology (NIST) new protein standard reference material (SRM 8671) is becoming the benchmark molecule against which many biomolecular analytical techniques are assessed and evaluated, we present its measurement results as a demonstration of the unique capability of our system to swiftly resolve and measure sharp (20~25 µL full-width-half-maximum) chromatography peaks. Precise measurements of protein mass and size can be accomplished 10 times faster than before with improved resolution. In the meantime the sample amount required for such measurements is reduced commensurately. These abilities will have far-reaching impacts at every stage of the development and production of biologics and bio-therapeutic formulations.

Focal molography is a new method for the in situ analysis of molecular interactions in biological samples.

Focal molography is a next-generation biosensor that visualizes specific biomolecular interactions in real time. It transduces affinity modulation on the sensor surface into refractive index modulation caused by target molecules that are bound to a precisely assembled nanopattern of molecular recognition sites, termed the 'mologram'. The mologram is designed so that laser light is scattered at specifically bound molecules, generating a strong signal in the focus of the mologram via constructive interference, while scattering at nonspecifically bound molecules does not contribute to the effect. We present the realization of molograms on a chip by submicrometre near-field reactive immersion lithography on a light-sensitive monolithic graft copolymer layer. We demonstrate the selective and sensitive detection of biomolecules, which bind to the recognition sites of the mologram in various complex biological samples. This allows the label-free analysis of non-covalent interactions in complex biological samples, without a need for extensive sample preparation, and enables novel time- and cost-saving ways of performing and developing immunoassays for diagnostic tests.