Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis.

Nanosized extracellular vesicles (EVs) have been found to play a key role in intercellular communication, offering opportunities for both disease diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The study was performed with small EVs (sEVs) isolated from a non-small-cell lung cancer (NSCLC) cell line as well as from human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter > 70 nm), the microscopy based approach showed a better capacity for analyses of smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.

Characterization of molecularly imprinted polymer nanoparticles by photon correlation spectroscopy.

We follow template-binding induced aggregation of nanoparticles enantioselectively imprinted against (S)-propranolol, and the non-imprinted ones, using photon correlation spectroscopy (dynamic light scattering). The method requires no separation steps. We have characterized binding of (R,S)-propranolol to the imprinted polymers and determined the degree of non-specificity by comparing the specific binding with the results obtained using non-imprinted nanoparticles. Using (S)-propranolol as a template for binding to (S)-imprinted nanoparticle, and (R)-propranolol as a non-specific control, we have determined range of concentrations where chiral recognition can be observed. By studying aggregation induced by three analytes related to propranolol, atenolol, betaxolol, and 1-amino-3-(naphthalen-1-yloxy)propan-2-ol, we were able to determine which parts of the template are involved in the specific binding, discuss several details of specific adsorption, and the structure of the imprinted site.

Solid-state electrolyte sensors for rebreather applications: a preliminary investigation.

INTRODUCTION: Recently developed prototypes of zirconium dioxide and NASICON-based micro solid-state electrolyte oxygen (O2) and carbon dioxide (CO2) sensors were tested for their potential suitability in rebreathers. The O2 sensor has a quasi-indefinite lifetime, whilst that of the CO2 sensor is approximately 700 h. This is a preliminary report of a new technological application. METHODS: The O2 sensor was tested in a small pressure chamber to a partial pressure of oxygen (PO2) of 405 kPa (4 bar). The CO2 sensor was tested up to 10 kPa CO2. The response times to a step change of pressure were measured, and cross-sensitivity for helium tested using trimix. A rebreather mouthpiece was modified so that breath-by-breath gas recordings could be observed. Power consumption to heat the sensors was measured. RESULTS: The O2 sensor demonstrated non-linearity, particularly above 101.3 kPa (1 bar) PO2, whereas the output of the CO2 sensor showed an inverse logarithmic relationship. Cross-sensitivity to helium was observed. The mean t90 response times were 90 (SD 10) ms for the O2 sensor, and 100 (SD 10) ms for the CO2 sensor. Breath-by-breath recordings showed slight damping of the CO2 trace due to electronic filtering. Power consumption was 1.5-2 W per sensor. CONCLUSIONS: The fast response times would allow accurate breath-by-breath measurement. Even though the O2 sensor has a non-linear response, measurement is possible using multi-point calibration. Further design is necessary to allow trimix to be used as the diluent. A major disadvantage is the high power consumption needed to heat the sensors to high temperatures.

Towards an electrowetting-based digital microfluidic platform for magnetic immunoassays.

We demonstrate ElectroWetting-On-Dielectric (EWOD) transport and SQUID gradiometer detection of magnetic nanoparticles (MNPs) suspended in a 2 microl de-ionized water droplet. This proof-of-concept methodology constitutes the first development step towards a highly sensitive magnetic immunoassay platform with SQUID readout and droplet-based sample handling. Magnetic AC-susceptibility measurements were performed on MNPs with a hydrodynamic diameter of 100 nm using a high-Tc dc Superconducting Quantum Interference Device (SQUID) gradiometer as detector. We observed that the signal amplitude per unit volume is 2.5 times higher for a 2 microl sample droplet compared to a 30 microl sample volume.

Tailored magnetic nanoparticles for direct and sensitive detection of biomolecules in biological samples.

We developed nanoparticles with tailored magnetic properties for direct and sensitive detection of biomolecules in biological samples in a single step. Thermally blocked nanoparticles obtained by thermal hydrolysis, functionalized with specific ligands, are mixed with sample solutions, and the variation of the magnetic relaxation due to surface binding is used to detect the presence of biomolecules. The binding significantly increases the hydrodynamic volume of nanoparticles, thus changing their Brownian relaxation frequency which is measured by a specifically developed AC susceptometer. The system was tested for the presence of Brucella antibodies, a dangerous pathogen causing brucellosis with severe effects both on humans and animals, in serum samples from infected cows and the surface of the nanoparticles was functionalized with lipopolysaccharides (LPS) from Brucella abortus. The hydrodynamic volume of LPS-functionalized particles increased by 25-35% as a result of the binding of the antibodies, measured by changes in the susceptibility in an alternating magnetic field. The method has shown high sensitivity, with detection limit of 0.05 microg x mL(-1) of antibody in the biological samples without any pretreatment. This magnetic-based assay is very sensitive, cost-efficient, and versatile, giving a direct indication whether the animal is infected or not, making it suitable for point-of-care applications. The functionalization of tailored magnetic nanoparticles can be modified to suit numerous homogeneous assays for a wide range of applications.

QCM-D analysis of the performance of blocking agents on gold and polystyrene surfaces.

With today's developments of biosensors and medical implants comes the need for efficient reduction of nonspecific binding. We report on a comparison of the ability of traditionally used blocking agents and poly(ethylene glycol) (PEG) derivatives to prevent protein adsorption on both gold and polystyrene surfaces. The adsorption kinetics of blocking molecules and proteins was monitored gravimetrically using quartz crystal microbalance with dissipation (QCM-D). The resistance to nonspecific adsorption was evaluated on gold and polystyrene surfaces coated with bovine serum albumin (BSA) or casein, gold coated with three different 6-11 ethylene glycol (EG) long hydroxyl- or methoxy-terminated PEG-thiolates and polystyrene blocked with a PLL-g-PEG or three different 12 EG long benzyl-PEG-derivatives. The prevention of protein adsorption on the coated surfaces was evaluated by monitoring the mass uptake at the addition of both pure prostate specific antigen (PSA) and seminal plasma. We demonstrate that on pure gold the PEG-thiols are superior to the other blocking molecules tested, with the end group and length of the PEG-thiols used being of minor importance. On polystyrene surfaces blocking with PLL-g-PEG, BSA and casein gave the best results. These results have an impact on further development of an optimized immunoassay protocol.

Characterization of QCM sensor surfaces coated with molecularly imprinted nanoparticles.

Molecularly imprinted polymers (MIPs) are gaining great interest as tailor-made recognition materials for the development of biomimetic sensors. Various approaches have been adopted to interface MIPs with different transducers, including the use of pre-made imprinted particles and the in situ preparation of thin polymer layers directly on transducer surfaces. In this work we functionalized quartz crystal microbalance (QCM) sensor crystals by coating the sensing surfaces with pre-made molecularly imprinted nanoparticles. The nanoparticles were immobilized on the QCM transducers by physical entrapment in a thin poly(ethylene terephthalate) (PET) layer that was spin-coated on the transducer surface. By controlling the deposition conditions, it was possible to gain a high nanoparticle loading in a stable PET layer, allowing the recognition sites in nanoparticles to be easily accessed by the test analytes. In this work, different sensor surfaces were studied by micro-profilometry and atomic force microscopy and the functionality was evaluated using quartz crystal microbalance with dissipation (QCM-D). The molecular recognition capability of the sensors were also confirmed using radioligand binding analysis by testing their response to the presence of the test compounds, (R)- and (S)-propranolol in aqueous buffer.

Uniform molecularly imprinted microspheres and nanoparticles prepared by precipitation polymerization: the control of particle size suitable for different analytical applications.

Molecularly imprinted polymers (MIPs) are being increasingly used as selective adsorbents in different analytical applications. To satisfy the different application purposes, MIPs with well controlled physical forms in different size ranges are highly desirable. For examples, MIP nanoparticles are very suitable to be used to develop binding assays and for microfluidic separations, whereas MIP beads with diameter of 1.5-3 microm can be more appropriate to use in new analytical liquid chromatography systems. Previous studies have demonstrated that imprinted microspheres and nanoparticles can be synthesized using a simple precipitation polymerization method. Despite that the synthetic method is straightforward, the final particle size obtained has been difficult to adjust for a given template. In this work, we initiated to study new synthetic conditions to obtain MIP beads with controllable size in the nano- to micro-meter range, using racemic propranolol as a model template. Varying the composition of the cross-linking monomer allowed the particle size of the MIP beads to be altered in the range of 130 nm to 2.4 microm, whereas the favorable binding property of the imprinted beads remained intact. The chiral recognition sites were further characterized with equilibrium binding analysis using tritium-labeled (S)-propranolol as a tracer. In general, the imprinted sites displayed a high chiral selectivity: the apparent affinity of the (S)-imprinted sites for (S)-propranolol was 20 times that of for (R)-propranolol. Compared to previously reported irregular particles, the chiral selectivity of competitive radioligand binding assays developed from the present imprinted beads has been increased by six to seven folds in an optimized aqueous solvent.

Brownian motion of aggregating nanoparticles studied by photon correlation spectroscopy and measurements of dynamic magnetic properties.

We have investigated colloidal stability of magnetic nanoparticle suspensions in different buffer systems and NaCl concentrations commonly used for biological applications. We have also investigated how conjugation of proteins to magnetic nanoparticles affects colloidal stability. Two different techniques, giving complementary information on the state of the particle system studied, have been used and compared. We have monitored the rotational Brownian motion of particles using measurements of dynamic magnetic susceptibility in the frequency domain. The results were processed using an algorithm that enables us to quantify changes of particle size distribution for particle suspensions subjected to various buffer conditions. The measurements were compared to results obtained for the translational Brownian motion of the same nanoparticles using photon correlation spectroscopy (PCS). We demonstrate that the complementarity of the two techniques enables more precise characterization of particles in suspension, particularly for suspensions of particles with a wide distribution in size and shape, or systems that are close to the onset of agglomeration.

Long-term bone response to titanium implants coated with thin radiofrequent magnetron-sputtered hydroxyapatite in rabbits.

PURPOSE: The present study was designed to investigate the long-term bone response around machined screw-type uncoated and calcium phosphate (CaP) -coated commercially pure titanium implants. MATERIALS AND METHODS: Using a magnetron sputtering technique, implants with a CaP coating similar in composition and CaP ratio to hydroxyapatite were produced. Heat treatment was subsequently used to increase the crystallinity of the coatings. Four types of coatings (0.1 and 2.0 microm amorphous and 0.1 and 2.0 microm crystalline) were manufactured; uncoated implants served as a control. Three hundred twenty implants (64 of each type) were randomly placed in the tibial cortical and trabecular femoral bones of 40 rabbits. The rabbits were sacrificed 9 months after implant placement. RESULTS: Histomorphometric evaluation carried out on ground sections revealed that the crystalline CaP coatings achieved the highest bone-implant contact in both tibiae and femora compared with amorphous CaP-coated and uncoated titanium. DISCUSSION: The present study suggests that submicron crystalline hydroxyapatite coating adds bioactive properties to titanium oral implants. CONCLUSION: An ultra-thin, 0.1-microm crystalline CaP coating can elicit and maintain an improved long-term bone response compared to amorphous coated or uncoated Ti implants, without any adverse tissue reactions.

Biomolecular reactions studied using changes in Brownian rotation dynamics of magnetic particles.

We have used magnetic particles to study specific binding of prostate specific antigen (PSA) to the surfaces of the bioparticles. The used particles have a mean diameter of about 130 nm and are placed in phosphate buffer saline (PBS). Each particle is composed of clusters of magnetic single domains of magnetite, which are covered with dextran. Changes in surface chemistry of the particles give rise to a change in the hydrodynamic volume of the particles. The later is mirrored by the changed frequency response of the complex magnetic susceptibility of a fluid containing these particles. Using ordinary induction coils and the lock-in amplifier technique it is possible to measure the complex magnetic susceptibility of the particle solution in a frequency range from about 10 Hz up to 10 kHz. From the measurement of the complex susceptibility versus the excitation frequency (both at the excitation frequency as well as at higher harmonics) we have shown that it is possible to quantitatively study the binding of PSA to the surfaces of the magnetic particles and thus to determine the PSA concentration in solution containing known concentration of nanoparticles functionalised with a monoclonal PSA antibody. Our method allows to perform an immunoassay in a single step and is much faster and cheaper compared to conventional ELISA procedures.

Short-term bone response to titanium implants coated with thin radiofrequent magnetron-sputtered hydroxyapatite in rabbits.

BACKGROUND: It has been suggested that calcium phosphate (CaP) coatings initiate faster bone growth around implants. A major concern about the viable use of these coatings has been their biologic performance related to the coating characteristics. PURPOSE: The purpose of this study was to investigate the early bone response to micron- and submicron-thick hydroxyapatite (HA) coatings in cortical and trabecular bone. MATERIALS AND METHODS: CaP coatings were manufactured by magnetron sputtering. Heat treatment was subsequently used to increase the crystallinity of the coatings. Coatings were characterized by x-ray diffraction, Fourier transform infrared spectroscopy, inductively coupled plasma optical emission spectroscopy (ICP-OES), and stylus profilometry. Four types of CaP-coated implants were used (0.1 microm and 2.0 microm amorphous; 0.1 microm and 2.0 microm crystalline); uncoated machined commercially pure titanium implants served as controls. Four hundred eighty implants were randomly placed in 60 rabbits. Ten animals were followed up for 1 week, 10 for 3 weeks, and 40 for 6 weeks. The bone response was histomorphometrically evaluated. RESULTS: Coatings with a CaP ratio very close to that of HA were produced. Crystalline coatings significantly improved the early bone-implant contact whereas the amorphous-coated implants behaved similarly to uncoated titanium. CONCLUSIONS: Crystalline CaP coatings 100 nm thick on titanium implants elicited an improved early bone response compared with that of uncoated titanium implants. No further improvement in the bone response was observed with 2 microm coatings.

Characteristics of the surface oxides on turned and electrochemically oxidized pure titanium implants up to dielectric breakdown: the oxide thickness, micropore configurations, surface roughness, crystal structure and chemical composition.

Titanium implants have been used widely and successfully for various types of bone-anchored reconstructions. It is believed that properties of oxide films covering titanium implant surfaces are of crucial importance for a successful osseointegration, in particular at compromized bone sites. The aim of the present study is to investigate the surface properties of anodic oxides formed on commercially pure (c.p.) titanium screw implants as well as to study 'native' oxides on turned c.p. titanium implants. Anodic oxides were prepared by galvanostatic mode in CH3COOH up to the high forming voltage of dielectric breakdown and spark formation. The oxide thicknesses, measured with Auger electron spectroscopy (AES), were in the range of about 200-1000 nm. Barrier and porous structures dominated the surface morphology of the anodic film. Quantitative morphometric analyses of the micropore structures were performed using an image analysis system on scanning electron microscopy (SEM) negatives. The pore sizes were < or = 8 microm in diameter and had 1.27-2.1 microm2 opening area. The porosity was in the range of 12.7-24.4%. The surface roughness was in the range of 0.96-1.03 microm (Sa), measured with TopScan 3D. The crystal structures of the titanium oxide were amorphous, anatase, and a mixtures of anatase and rutile type, as analyzed with thin-film X-ray diffractometry (TF-XRD) and Raman spectroscopy. The chemical compositions consisted mainly of TiO2, characterized with X-ray photoelectron spectroscopy (XPS). The native (thermal) oxide on turned implants was 17.4 nm (+/- 6.2) thick and amorphous. Its chemical composition was TiO2. The surface roughness had an average height deviation of 0.83 microm (Sa). The present results are needed to elucidate the influence of the oxide properties on the biological reaction. The results of animal studies using the presently characterized surface oxides on titanium implants will be published separately.