The ActiPix™ TDA200 HT Nano-Sizing System
INTRODUCTION
The ActiPix™ TDA200 is a benchtop high throughput nano-sizing system which enables the rapid determination of size and purity for a range of molecules including proteins, antibodies, nanoparticles and quantum dots. It measures the hydrodynamic radius and diffusion coefficient for species in solution.
Key features and benefits include:
· Low sample requirement - between 10 to 100 nL of sample are injected into the system for each measurement. Low volume sample vials ensure no wastage of precious sample.
· Measures hydrodynamic radius for concentration range from 0.1 to 100 mg/mL. No dilution or sample preparation required.
· Fast - Most runs completed in less than five minutes, enabling faster decision making and less system downtime.
· Sample held in a temperature controlled environment from 4-40 oC during analysis. This ensures samples are held at optimum conditions for a variety of studies.
· Automated run lists allow multiple samples to be run overnight.
· Ideal for membrane proteins where surfactants are used. The TDA200 uses UV absorbance of the species to determine hydrodynamic radius. As detergents are typically invisible to UV light, difficult samples such as membrane proteins are easy to characterise without interference from surfactant micelles.
Overview of the ActiPix™ TDA200 Nano-Sizing System
DESCRIPTION OF TECHNIQUE
The TDA200 Nano-Sizing System uses a novel approach to measure the hydrodynamic radius for proteins, antibodies and nanoparticles. It utilises Paraytec’s award winning UV area imaging technology to capture high resolution absorbance data of the sample as it moves through a capillary . The technique allows analyses to be performed with up to 10 buffer samples in a sequence, enabling size measurements to be made for the samples in their native, aggregated or denatured forms.
Using a high precision injection system, a 10 to 100 nL slug of sample is introduced into the flowing stream contained in a looped capillary represented in Figure 1. The capillary, internal diameter typically 75 µm, has 2 detection windows along its length which enable absorbance measurements of the sample to be recorded at two different times using the ActiPix™ area imaging detector.
Figure 1. Illustration of an open ActiPix™ Nano-Sizing cartridge. The capillary loop can clearly be seen to the right of the picture. The two detection windows are labelled.
Whilst peak areas are the same, since area is proportional to the amount of sample injected, the widths of both peaks are different due to Taylor dispersion. The signal from the second window has a greater width and lower amplitude than the first window as shown in Figure 2. The data are automatically processed using Equation 1 below to provide absolute values of diffusion coefficient and hydrodynamic radius.
Figure 2. Illustration of the change in peak profile as due to Taylor Dispersion as a slug of sample is pushed through a capillary.
Taylor dispersion analysis (TDA) provides an absolute, simple and rapid method for determining diffusion coefficients and hydrodynamic radii.
In the TDA200 Nano-Sizing System, UV area imaging with two measurement points allow all data to be collected extremely quickly in a single experiment. The peaks are automatically fitted with a fitting function using Paraytec’s ActiPix™ TDA200 HT Nano-Sizing Software.
The area under the peak corresponds to the amount of sample injected. The times and variances of the two peaks are used to calculate the hydrodynamic radius of the sample. The ActiPix™ Nano-Sizing software presents the data in a convenient format.
This technique can also be used determining the radii of multiple components of different sizes in the same sample, as shown in Figure 3. Paraytec’s fitting functions deconvolute multiple Gaussian peak functions until the best quality of fit data is reported.
Example results from the TDA200
Table 1 shows typical results obtained from the TDA 200 Nano-Sizing System. The system can be used to precisely size a range of species with hydrodynamic radii from 0.3 nm to >20 nm.
* Multi-Component Fit, # Approximate diameter determined by TEM
SAMPLE REPORT
Powerful ActiPix™ software automatically generates detailed reports for each sample analysed. An example of a report is shown below. The software also enables the user to manually process data if required.
SYSTEM REPEATABILITY
The TDA200 Nano-Sizing System ensures data quality remains consistent from one day to the next.
SPECIFICATION
Controller
Light source Pulsed xenon lamp, nominal rate 20 Hz
Wavelengths 190 - 1100 nm, filter selectable
Filter bandpass 10 nm
Measurement range: caffeine 1 ppm (5 µM) to 5000 ppm (26 mM) at 254 nm
Measurement range: protein 0.1 mg/ml to 150 mg/ml at 214 nm
Baseline noise 10 µAU RMS
Imager properties
Spatial resolution 7 µm x 70 µm
Pixel size 7 x 7 µm
Imager area 9 x 7 mm (W x H)
Remote sensor head size 6 x 6.5 x 11 cm (H x W x L)
Sizing cartridge
Detection pathlength 75 µm
Capillary length 150 cm
Autosampler and pressure drive
Linear flow rate 1 mm/s to 20 mm/s selectable
Injection volume 10 - 100 nL selectable
Temperature Range 4 - 40 oC
Vials 300 µL or 4 mL glass
Adaptors for 250µl centrifuge tubes available
PC
High specification desktop PC with 22" monitor
Software for multi-component sizing analysis
Power
110 – 240 V AC 50/60 Hz
(4) power receptacles required as below:
(1) controller, (1) autosampler, (1) monitor, (1) PC
Bench space required
1 m x 1 m (Autosampler)
1 m x 1 m (PC)