by Dr Bob Carr
There are many situations in which an accurate count and characterisation of preparations of small particles such as viruses or phages is vital. For example, the development of viral purification procedures relies on accurate counts. Likewise, to be validated, viral vaccines must be shown to contain a specified amount of active ingredient. The aggregation status or polydispersity profile of virus preparations is also frequently required. Yet another need for accurate viral counting is in the novel use of bacteriophages to inactivate MRSA. This article describes a system of particle counting that is based on the tracking of the individual Brownian motion of each virion, gives examples of typical applications and describes its advantages with respect to standard assays.
NANOPARTICLE TRACKING ANALYSIS (NTA)
The process known as nanoparticle tracking analysis, (NTA) is based on a novel application of laser light scattering in which populations of suspended particles are visualised and sized dynamically on an individual basis. Depending on the actual virus involved, NTA functions over a broad range of virus sizes, down to a particle size of approximately 20nm; the system can thus handle (i.e. track, size and count) most viruses and bacteriophages. The system directly tracks the Brownian motion of each and every particle separately and simultaneously using a CCD camera. A high resolution plot of the particle size distribution profile as a function of time is then produced so that aggregation or flocculation is immediately apparent. This particle-by-particle approach avoids the intrinsic assumptions of dynamic light scattering (DLS). By providing a unique image, that goes beyond light scattering to assess polydisperse systems, NTA gives key insights into potentially important aggregation phemomena. The LM analysers from the NanoSight company are based on the NTA principle [Figure 1].
VIRUS COUNTING
Using NTA, viruses in liquid preparations can be directly and individually visualised in real time. High resolution distribution profiles of particle size can then be obtained. Fast, robust and accurate, the technique is also cost-effective and provides an attractive alternative or complementary approach to expensive and more complex methods of nanoparticle analysis such as dynamic light scattering (e.g. based on photon correlation spectroscopy) or electron microscopy.
By simultaneously and directly measuring the diffusion coefficient of every particle, dedicated NTA software allows the automatic counting and sizing of the virions in a sample. The results are displayed graphically, showing the size of the particle as a function of the number of individual particles (or size versus relative brightness). This overcomes the limitations inherent in other particle analysis systems which generate only mean particle size distribution data.
The NTA approach has several advantages. For example, sample pre-treatment is minimal, requiring only dilution to approximately 107 – 1010/ mL. Accurate and reproducible analyses are automatically obtained from video clips that take only a few seconds to record. The data obtained then allow the immediate and automatic generation of graphs of particle size as a function of particle number. The technique is therefore effectively a real-time measurement system, so changes in particle size distribution caused, for example, by aggregation or dissolution can be followed automatically. A wide range of virus types have been successfully analysed to date [Table 1].
Since the technique is absolute no calibration is required. Uniquely, the system provides a simple and direct qualitative view of the sample being analysed (e.g. to validate data obtained from other techniques such as PCS); from this qualititive view an independent quantitative estimation of not only sample size but also size distribution and concentration can be immediately obtained.
The ability to clearly differentiate monodisperse and polydisperse systems in the sub micron region is a significant advantage of the use of NTA. A plot of particle-scattered intensity versus particle size is the usual way in which such data are presented. Figure 2 is an example of such a plot and shows data generated in a study of a partially purified influenza-type virus sample; the ordinate represents the numbers of virus particles of a given size (particle concentration). The graph clearly shows that there exist several populations of aggregated particles of larger size and scattering intensity. This is in contrast with the plot shown in Figure 3. Here the data from the NTA analysis of a purified suspension of adenovirus show a clean monodisperse distribution profile with a mode diameter centred at 82nm; this was independently confirmed using electron microscopy.
VACCINE PRODUCTION
To be validated for use in humans, viral vaccine preparations must be proven to be not only stable but also to contain precisely known quantities of active ingredient. NTA provides an immediate and direct estimation of such product purity and concentration. Similarly, the degree and rate of formation of aggregates in a virus preparation can be easily estimated allowing the manufacturer to evaluate and develop improved product manufacturing processes that could result in optimised product shelf-life. Because NTA allows all the particles in the preparation to be visualised and sized, more information about nanoparticle content is made available to the user. The data could, for example show the presence of larger particles (which can both be sized and counted using NTA) which could represent either non-viral cell debris from the cell culture process or aggregates of virus particles containing many individual virions. Such aggregates and possible contaminants represent a potential problem for the manufacturers that should be immediately identified. In addition, the NTA system generates results much more quickly than could be achieved using conventional bioassays, such as plaque assays.
BACTERIOPHAGE-BASED MRSA PROTECTION
Virus particle detection and counting using NTA is providing essential information for a team of researchers at the University of Strathclyde’s Institute of Pharmacy and Biomedical Sciences (IPBS) in Scotland. The team has developed methods to employ naturally-occurring phages to combat Methicillin- resistant Staphylococcus aureus (MRSA) [Figure 4]. Because of the resistance to antibiotics that the microorgansim has developed, infections caused by the bacterium are extremely difficult to treat and can be potentially lethal. It is therefore important, particularly in hospitals, to ensure that possible sites of MRSA contamination such as surgical instruments, sutures and even the external surfaces of wounds, be disinfected as thoroughly as possible. In principle, this can be achieved through the use of solutions such as detergents. In practice, however the preparation of the detergent solution to the appropriate concentration is often inconsistent, as is its application to the potentially contaminated surfaces, so the detergent route of disinfection is often ineffective. It is for this reason that the team at IPBS have been investigating the use of bacteriophages. A key factor in this approach is the characterisation of bacteriophage cultures using NTA, prior to the use as a dry-coat on vulnerable surfaces. NTA enables the rapid evaluation of the bacteriophage size profile in real time and at low cost. Such characterisation requires assessment of aggregation in the 20nm – 1000nm range where quantitative sample characterisation by other methods is much more difficult and therefore more expensive.
THE AUTHOR
Dr Bob Carr
Founder and CTO,
Nanosight Ltd.,
Salisbury,
UK
email: bob.carr@nanosight.com
