Characterization of nanoparticles and biomolecules

Zetasizer Ultra (Malvern Panalytical Ltd)

The Zetasizer Ultra uses the Dynamic Light Scattering (DLS) and the Electrophoretic Light Scattering (ELS) techniques to determine the particle size (from 1 nm to 10 μm) and the electrokinetic potential, the so-called Zeta potential, which can be used to determine the stability of the colloidal system. The Zetasizer Ultra is equipped with a He laser with a wavelength of 633 nm. The Non-Invasive Back-Scatter (NIBS) technique increases the sensitivity and versatility in the size determination. In addition to the back-scatter detector, operating at an angle of 173°, the instrument is equipped with an additional two detectors, a side-scatter 90°, and a forward-scatter 13°. This extension implements the Multi-Angle Dynamic Light Scattering (MADLS) technique. The MADLS allows the determination of the particle concentration, in a number of particles per volume, and further increases the sensitivity and resolution of measurements. The range of measurable concentrations varies, depending on the nature of the sample, from 1∙1013 to 1∙108 particles/ml. The minimum concentration for optimal MADLS analysis is 1 mg/ml, depending on the nature of the sample, for NIBS analysis, the minimum concentration is 0.1 mg/ml. Also, a fluorescent filter is available for NIBS which allows the measurement of the fluorescent samples and a capillary cuvette cell with a minimum measurable volume of 3 μl. For the Zeta potential measurement, the minimum sample concentration is 1 mg/ml, and a disposable cuvette with a minimum volume of 700 μl is available.

Zetasizer Nano ZSP (Malvern Panalytical Ltd)

The Zetasizer Nano ZSP, an older model from the series Zetasizer range uses the Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) technique to determine the particle or molecule size and to determine the electrokinetic potential, Zeta. The instrument is equipped with a He laser of 633 nm and with the Non-Invasive Back-Scatter (NIBS) technique at an angle of 173°. The measurement ranges in size from 1 nm to 10 µm and the molecular weight ranges from 980 Da to 20 MDa.

qNano (Izon Science Ltd)

The qNano uses the Tunable Resistive Pulse Sensing (TRPS) technique for the “particle by particle” analysis. The TRPS technique is based on measuring changes in the current flowing through the nanopore. Changes, referred to as resistive pulses, are due to the passage of particles (suspended in the electrolyte) through a given nanopore of an adjustable, defined size. By calibrating to a characterized polystyrene standard, this non-optical technique can be used to determine the size and the number of particles in volume. A wide range of the nanopore polyurethane membranes is available to cover the different analyte sizes from 40 nm to 1 μm, depending on the nature of the sample. The measurable sample concentration ranges from 1∙105 to 1∙1011 particles/ml.

Eclipse AF4 and accessories (Wyatt Technology Corporation)

The Eclipse is the control unit for Asymmetric Flow Field-Flow Fractionation (AF4). This technique belongs to the chromatographic methods enabling separation of the biomolecules or the particles based on the hydrodynamic behaviour. The sample is separated in the empty channel by a combination of laminar and perpendicular flow. Thanks to the adjustment of the separation force and the choice of both the separation profiles and the separation channels, the AF4 technique enables the separation of particles in the range from 1 nm to 1000 nm. The sample injection to the separation channel is automated (Agilent Technologies, Inc.) with a 100 μl injection loop. The separation takes place at a constant temperature in the adjustable range of 4 °C to 90 °C. The Analytical channel with a capacity of about 100 μg of the sample, the Semi-preparative channel with a capacity of about 1 mg of sample and the Hollow fiber channel (HF5) are available. Due to the small volume of the HF5 channel, the separation is processed in a small sample volume, high sensitivity and short analysis time. The separation is followed by a series of detectors: The Agilent 1200 Series Diode Array and Multiple Wavelength Detector UV-VIS DAD detector (Agilent Technologies, Inc.) with a flow cell allowing online determination of absorbance of up to 8 wavelengths ranging from 190 nm to 900 nm; The Agilent InfinityLab LC 1260 Series Infinity II Fluorescence Detector (Agilent Technologies, Inc.) for online measurements of multiple excitation or emission wavelengs. DAWN® HELEOS® (Wyatt Technology Corporation) Multi-Angle Light Scattering (MALS) detector measuring the angular dependence of the intensity of the scattered light to determine the particle size expressed as gyration radius (range 10 nm to 500 nm), molecular weight (200 Da to 1 GDa), and a second virial coefficient (describing the level of the interaction between the particles and the solvent). The sensitivity of the detector, depending on the nature of the sample, is about 0.2 μg/ml. The ambient detector has 18 angles between 15° and 160° and He laser with a wavelength of 658 nm. The 149° detector is equipped with a dynamic light scattering module allowing online measurement of the hydrodynamic radius (in the range of 1 nm to 300 nm). The Optilab® T-rEX ™ is an online detector for determining the differential refractive index (dRI) between the eluting sample and the reference liquid (pure mobile phase) and the absolute refractive index (aRI). As a light source, there is an LED with a wavelength of 660 nm. The AF4 allows the collection of individual fractions through a septum into tempered vessels using a 1260 Infinity II Analytical-Scale Fraction Collector (Agilent Technologies, Inc.).

Chronos DFD (ISS)

The Chronos DFD Fluorescence Spectrofluorometer characterizes the fluorescence properties of the analyte in solution. Characterization includes measurement of the stationary, polarized and time-resolved fluorescence. As a source of excitation light for the stationary and polarized fluorescence, the xenon lamp is used. A 370 nm laser diode and a 280 nm pulse LED are available for time-resolved fluorescence. A pair of PMT detectors are available to enable measurements in standard L-format and T-format geometry for fast and accurate polarization measurements. The instrument is capable of measuring in the wavelength range 200 nm to 900 nm and excited lifetime ranging from 10∙10−12 sec to 1 sec. in an adjustable temperature range from −30 °C to 105 °C (0.02 °C).

MicroCal PEAQ-DSC Automated (Malvern Panalytical Ltd)

The Differential scanning calorimeter MicroCal PEAQ-DSC is an automated analytical instrument for characterizing the thermal stability of biomolecules. Characterization involves the determination of the enthalpy, heat capacity, and transit temperature of thermally induced structural changes in the molecule in a solution. The instrument is equipped with a tempered (range 4 °C to 40 °C) sample storage in the form of 96-well disposable plates of 6 positions (96×6 in total, the measuring, and the reference cell (volume of 130 μl) are automated filled. The optimum sample volume is 325 μl over a concentration range of 0.01 mg/ml to 10 mg/ml, depending on the nature of the sample. The measurement is carried out in a temperature range of 2 °C to 130 °C with a maximum heating rate of 240 °C/h and a cooling rate of 60 °C/h.

MicroCal PEAQ-ITC (Malvern Panalytical Ltd)

The MicroCal PEAQ-ITC isothermal titration calorimeter determines binding affinity, stoichiometry, enthalpy and entropy by directly measuring the heat released or absorbed in a given biochemical binding reaction. The instrument is equipped with a 200 μl measuring cell with an automatic sample injection (injection volume ranges from 2 μl to 80 μl with an error below 1%) and an adjustable temperature between 2 °C and 80 °C.

Preparation of nanoparticles