Team:Wageningen UR/MethodsDetection

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= Detcection of VLPs =
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= Detection of Virus Like Particles =
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<p align="justify">
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A key part of our project is the detection of [[Team:Team:Wageningen_UR/VLPs|VLPs]]. We need sufficient visualization to get conclusive evidence of VLP formation. Besides standard approaches, we will investigate alternative methods to detect the formation and stability of Virus-Like Particles. In this paragraph we explain in short which methods we used and how these methods work. We also describe the limitations of the techniques, so that other people have an idea about what is possible.
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The most challenging piece of science is the detection. When producing a new VLP you don't know if it forms. So we put a lot of effort in it, to detect and visualize the Virus-Like-Particles
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== Electron Microscopy  ==
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== Electron Microscopy (EM) ==
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[[File:EM.jpg|400px|left|thumb|''Figure 1: Electron microscopy versus Light Microscopy, instead a light as a source to illuminate the sample EM used electrons and instead of glass lenses, EM uses electro-magnetic lenses'']]
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The most straight forward method to detect VLPs is Electron Microscopy (EM) [1]. We prepared and investigated multiple samples ourselves. This work was done at the Virology department of the Wageningen University.
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<br><br>
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Electron microscopy works similar to light microscopy (figure 1), but instead of visible light being used to illuminate the sample, it is done by electrons [1]. The EM has multiple lenses to focus the beam to magnify and light the sample. The lenses are electromagnetic instead of glass and therefore allow a much higher resolution than conventional light microscopy. In theory it is possible to reach a resolution of around 0.005 nm, but in practice it is mostly around 1-2 nm [1]. Possible reasons are certain errors of the lenses, inexperience of the operator (for example our team members) or the sample not being thin enough. All these factors can reduce the resolution of the electron microscope [1]. We investigated various samples obtained in our experiments with the help of the EM. The wild types of the [[Team:Wageningen_UR/ModifyingtheCCMV|Cowpea chlorotic mottle virus]] (CCMV) and [[Team:Wageningen_UR/ModifyingtheCCMV|Hepatitis B]] (HepB) were detected.  Multiple variations of CCMV have been tested too, unfortunately with mixed results.
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===EM results===
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<img src="https://static.igem.org/mediawiki/2012/5/58/CCMV_WT.jpg" title="Wild-type CCMV VLPs" />
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<img src="https://static.igem.org/mediawiki/2012/2/2c/CCMV_WT40.jpg" title="CCMV VLPs after 24h heat treatment" />
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<img src="https://static.igem.org/mediawiki/2012/5/5d/CCMV_WT65.jpg" title="Aggregates of CCMV VLPs" />
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<img src="https://static.igem.org/mediawiki/2012/c/cf/CCMVWT1.jpg" title="Overview picture of purified wild type CCMV VLPs" />
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<img src="https://static.igem.org/mediawiki/2012/6/63/HepBEM1.jpg" title="Wild-type Hepatitis B core Antigen VLPs" />
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<img src="https://static.igem.org/mediawiki/2012/0/0f/VLP%27s_with_iron2.png" title="Electron Microscopy image with lots of contamination" />
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<!--
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[[File:CCMV_WT.jpg|500px|center|thumb|''Wild type CCMV VLPs'']]
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[[File:CCMV_WT40.jpg|500px|center|thumb|''CCMV VLPs after 24h heat treatment'']]
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[[File:CCMV_WT65.jpg|500px|center|thumb|''Aggragates of CCMV VLPs'']]
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[[File:CCMVWT1.jpg|500px|center|thumb|''Overview picture of purified wild type CCMV VLPs CCMV'']]
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</p>
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=== EM Course ===
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<p align="justify">
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The goal of the course was to get familiar with the EM and to handle it safely. Dr. Jan van Lent of the Virology department of Wageningen UR gave us an introduction to EM with a small presentation. During this instruction he explained in detail the electron microscope itself, the preparation of good samples and how to operate the EM properly. Afterwards he showed the sample preparation in the wet lab. Preparation of the sample is critical to have a good resolution and during the course we learned how to do it. The sample must be thin, between 2 - 300nm, and stable in the electron microscope. This can be done by drying or cryo-freezing the sample. After drying or freezing, the sample is stained with a coating containing a heavy metal salt. This coating reflects the electron waves of the microscope, whereas the spots with the VLPs have no coating at all. The missing coating allows the electron waves to go through unhindered. The information of reflection and permeability is processed to a full image which shows the VLPs.
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One of the direct methods to detect our VLPs is with Electron Microscopy or EM. We prepared and viewed multiple samples, this all was done in the Virology department of the Wageningen UR.
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(PICTURE EM AND LM COMPARISON)
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<img src="https://static.igem.org/mediawiki/2012/thumb/5/52/Emcourse1.JPG/500px-Emcourse1.JPG" title="The EM" />
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<img src="https://static.igem.org/mediawiki/2012/thumb/8/85/EMcourse2.JPG/500px-EMcourse2.JPG" title="Learning how to prepare the samples" />
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<img src="https://static.igem.org/mediawiki/2012/thumb/6/64/EMcourse3.JPG/500px-EMcourse3.JPG" title="A copper grid on which the sample has to be fixated" />
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<img src="https://static.igem.org/mediawiki/2012/thumb/6/6d/EMcourse5.JPG/500px-EMcourse5.JPG" title="Preparing a grid with Virus-Like Particles" />
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<img src="https://static.igem.org/mediawiki/2012/thumb/1/13/EMcourse6.JPG/500px-EMcourse6.JPG" title="The copper grids are very delicate, therefore they should be handled with great care" />
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<img src="https://static.igem.org/mediawiki/2012/thumb/b/bd/EMcourse8.JPG/500px-EMcourse8.JPG" title="After the VLPs have been applied to the grid, they have to be fixed with a heavy metal salt solution." />
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<img src="https://static.igem.org/mediawiki/2012/thumb/0/0a/EMcourse12.JPG/500px-EMcourse12.JPG" title="Jan van Lent explaining how to operate the EM itself" />
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<img src="https://static.igem.org/mediawiki/2012/thumb/d/d9/EMcourse15.JPG/500px-EMcourse15.JPG" title="Operating the EM ourselves." />
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<img src="https://static.igem.org/mediawiki/2012/thumb/c/c3/EMcourse14.JPG/500px-EMcourse14.JPG" title="The result, a screen full of VLPs" />
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<!--
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[[File:Emcourse1.JPG|500px|center|thumb|''The EM''</p>]]
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[[File:EMcourse2.JPG|500px|center|thumb|<p align="justify">''Learning how to prepare the samples''</p>]]
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[[File:EMcourse3.JPG|500px|center|thumb|<p align="justify">''A copper grid on which the sample has to be fixated''</p>]]
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[[File:EMcourse5.JPG|500px|center|thumb|<p align="justify">''Preparing a grid with Virus-Like Particles''</p>]]
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[[File:EMcourse6.JPG|500px|center|thumb|<p align="justify">''The copper grids are very delicate, therefore they should be handled with great care''</p>]]
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[[File:EMcourse8.JPG|500px|center|thumb|<p align="justify">''After the VLPs have been applied to the grid, they have to be fixed with a heavy metal salt solution.''</p>]]
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[[File:EMcourse12.JPG|500px|center|thumb|<p align="justify">''Jan van Lent explaining how to operate the EM itself''</p>]]
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[[File:EMcourse15.JPG|500px|center|thumb|<p align="justify">''Operating the EM ourselves.''</p>]]
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[[File:EMcourse14.JPG|500px|center|thumb|<p align="justify">''The result, a screen full of VLPs''</p>]]
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Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, electrons are being used. The same as light microscopy, electron microscopy uses multiple lenses to focus the beam so that the sample is properly lighted. The only differences with lenses are that the lenses of electron microscopy are electromagnetic instead of glass. EM has also a much greater resolution than conventional light microscopy. It is theoretically possible of around 0.005 nm, but in practice it is around 1-2 nm, this is because the lenses has certain errors, the operator (us) is inexperienced and the sample must be as this as possible. All these factors reduces the resolution of the electron microscope.
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After the preparation of the samples Dr. van Lent explained the whole procedures of visualizing a sample. After this last introduction we were able to analyze and investigate multiple samples ourselves.
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At the end of the day we were able to use EM for our own experiments starting with sample preparation, handling of the microscope and proper visualization. After the course, the attending team members, were trained and allowed to use the EM without supervision.
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</p>
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We checked various samples in the electron microscope. The wild types of CCMV and HepB were detected. Multiple variations of CCMV were also tested with mixed results.
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=== Limits ===
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<p align="justify">
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Knowing the limit of a technique is required to interpret the raw data properly, this also applies to electron microscopy. One of the most troublesome limitations is that the preparation of the sample is laborious and can lead to potential debris, which can ruin the sample. Another limitation is that the preparation of the sample, operation of the EM and the analysis of the data requires special training. We were lucky enough to receive a course on how to use the EM. Also samples that are put into the EM must be resilient against the vacuum in the microscope [2].
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=== EM Cours ===
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== Dynamic Light Scattering ==
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[[File:Dynamic_light_scattering_beampath.jpg|500px|right|thumb|''Figure 2: DLS overview, laser is shooting light in the sample, scattered light is detected by a photon-detector and the data is processed by the computer'']]
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<p align="justify">
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Another method to detect VLPs is dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasi-elastic light scattering. This technique is used to measure the size of particles [3]. We applied this technique as a complimentary method to electron microscopy. To use this technique, first we must purify the sample  by [[Team:Wageningen_UR/MethodsPurification|FPLC]] to get good measurements. The standard setup of the DLS is seen in figure 2.
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When light travels through the sample and hits a particle, it gets scattered in all directions. This scattered light is detected by the DLS. Due to Brownian motion, the particles move randomly in the solution. These movements of the particles changes the distance of the particles with respect to the detector. This change of distance to the detector results in constructive or deconstructive interference of the scattered light. The change of intensity of the scattered light by the interference is related to the radius of the particle and can be calculated by the Stokes-Einstein equation [3].
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We received personally a course about the EM from Jan van Lent at the Virology department of the WageningenUR. In this course he explained how the EM works, how you prepare the samples and how to operate the EM.
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[[File:DLS_comb.jpg|800px|center|thumb|''Figure 3: A) Explanation on how the DLS works by Remco Fokkink, B) output screen of the DLS, C) Stokes-Einstein equation on white-board, D) DLS setup in real life'']]
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The goal of the course wa to get familiar with the EM and how to handle it. First there was a small presentation about how the EM works and how to prepare the samples. Next he showed us one time how to prepare the sample afterwards we can prepare our own samples.
 
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Preparation of the sample is critical to have a good resolution, during the course we learned how to do it properly. The sample must be thin, between 2 - 300nm, and must be stable in the electron microscope, this can be done by drying or cryo-freezing the sample. After drying or freezing we stain the sample with a coating. The places where there is no coating (VLPs), electron wave can go through unhindered, while the places where there are coating, electron waves break apart. This result in a picture where you can see the particles.
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<br><br>
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We investigated various samples with the help of DLS. The main issue was that the sample was not pure or not concentrated enough. After optimization of the production and purification protocol we succeeded to detect VLPs. The main improvement was the implementation of FPLC, which filters out all aggregates and subunits, yielding pure VLP solutions.  
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</p>
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After the preperation of the samples we were shown where the EM is and how to operate it.  
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=== Limits ===
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<p align="justify">
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The same as electron microscopy, dynamic light scattering has its limitations. To detect particles with the DLS, the particle has to have a different refractive index than the solvent. If not, the particle doesn't scatter light. Also the sample must be very pure. Any dust particle or aggregate can ruin the data output. This is because large particles scatter way more light than small particles. The data analysis requires vast amounts of knowledge and time [5].  
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</p>
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== Nanoparticle Tracking Analysis ==
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<p align="justify">
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== Dynamic Light Scattering (DLS) ==
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[[File:Nanosight.png|200px|right|thumb|''Figure 4: Scematic overview of the NTA and the sample]]
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</p>
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<p align="justify">
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The nanoparticle tracking analysis (NTA) is been developed by Nanosight. This is a new method for a direct and real-time visualization of the virus-like-particles. On Tuesday 24th of October 3 samples were tested with varied results, but showed a promising technique that can be used for uncovering parameters of the assembly model.
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A focussed laser is passed through the sample and hits the particles in the detection volume. The light of the laser scatters in all directions making the particles detectable for a camera. These particles are seen moving due to Brownian motion. The NTA program identifies the particles and track the centre of each particle individually. The average distance each particles make is automatically calculated. From this value the diffusion coefficient can be obtained. With the diffusion coefficient we are able to get the radius by the Stokes-Einstein equation [6].
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In the slideshow below is our HepB wildtype VLPs visualized by the NTA program. We also tested two other samples, but there was no detection. In the slide show you can see aggregates, the VLps it self are too small to be seen on the slideshow, however they are being tracked by the program. Although no estimation of size was yet possible due to the wrong type of laser used during the measurement.
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<div class="detection_slider" style="width: 500px; height: 400px; margin: 0 auto; overflow: hidden;">
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<img src="https://static.igem.org/mediawiki/2012/e/e9/ScrshHepB1.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/a/a3/ScrshHepB2.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/7/72/ScrshHepB3.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/c/c3/ScrshHepB4.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/d/da/ScrshHepB5.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/c/c4/ScrshHepB6.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/8/88/ScrshHepB7.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/1/1d/ScrshHepB8.png" title="" />
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<img src="https://static.igem.org/mediawiki/2012/7/7a/ScrshHepB9.png" title="" />
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Thanks to the realtime and direct way of visualizing the particle sizes by the NTA program, it becomes possible to do studies about VLP assembly and aggregation [7]. With this technique it becomes possible to find parameters for the [[Team:Wageningen_UR/FormationModel|assembly]] and the [[Team:Wageningen_UR/HumanBody|human body]] model.
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Another method to detect virus-like-particles is with dynamic light scattering also known as photon correlation spectroscopy or quasi-elastic light scattering. This technique is used to measure the size of particles and is thus an indirect way to detect particles. We used this technique as a complimentary method next to the electron microscopy.
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</p>
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When light travels through the sample and hits a particle, light will scatter in all directions. The dynamic light scattering machine detect the scattered light under a fixed angle (picture formation). The particles in the solution will move randomly due to Brownian motion. As the particles move , the intensity will change. This is because the scattered light can undergo constructive or deconstructive interference with the scattered light of other particles. The larger the particle, the more slower it is in Brownian motion, the slower the change of intensity is. The change in intensity over time relates to the diffusion coefficient and this diffusion coefficient is related to the particle radius using the Stokes-Einstein equation (picture).
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== Comparison between the EM and DLS ==
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<p align="justify">
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We tested multiple samples with DLS. The main issue was that the sample was not pure enough or not concentrated enough. After optimizing the production and purification protocol we succeeded to detect VLPs.
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The electron microscope has been used for years to detect VLPs, but is rather expensive to use. So we searched for alternatives. One of the alternatives we found was the dynamic light scattering technique. Dynamic light scattering is sometimes mentioned as a way to detect VLPs, but is never explained thoroughly. We didn't know any possibilities or limitations of the DLS, so we wanted to see if the output of the DLS is similar to the output of the EM. If the results are positive we can use the DLS as a cheap and easy way to detect VLPs.
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In order to quantify the DLS, we set up an experiment to compare the size distribution of the VLPs. We produced a large batch of wild-type CCMV monomers and assembled them in vitro into VLPs. After purification we split up the sample. One part is analyzed by the electron microscope and the other part is analyzed by the dynamic light scattering technique. To calculate the size distribution of the VLPs with the EM, we took multiple pictures of the VLPs. We printed out the pictures and measured by hand each particle individually. After these measurements, we calculated the actual radius of each particle and plotted it on a graph. To calculate the size distribution of the VLPs with the DLS, we took the sample and measured it multiple times, so that anomalies were reduced. After measurement we used the CONTIN method to get the size distribution and plotted it in excel.
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[[File:Compairison.jpg|750px|center|thumb|''Figure 5: Up) The EM raw measurements (red) and the fitted normal distribution of the raw data (Blue), Bottom) Multiple size distributions of the wild-type CCMV VLPs using DLS'']]
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As seen in figure 5, the size distribution calculated by both methods looks similar. Both the EM and DLS detect particles around 15 nm, precisely what you expect from the literature. The minor differences can be explained by the methods used to calculate the size distribution. With the EM we visualized the particles first and then calculated the size distribution. With the DLS, the raw output was fitted and can be wrong if the sample is not pure or not concentrated enough. We took that into account by measuring multiple times.
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Both the EM and DLS can be used to detect VLPs. This means we've confirmed that both techniques combined create a solid and secure method to detect and analyze VLPs. It is possible to discover many characteristics of VLPs. The part [http://partsregistry.org/Part:BBa_K883001 BBa_K883001] that represents the wild-type CCMV gene with the IPTG induced promoter has been extendedly tested on the stability.
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==Stability experiments==
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<p align="justify">
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CCMV is a virus that can be useful in nanotechnology, because it is a well-studied virus and certain physical properties of the CCMV-VLP are already known. We want to provide additional information about the conditions in which the CCMVs are stable. These conditions are important when it comes to site-specific drug delivery. A better understanding of what will happen when the VLPs are injected into the bloodstream, will result into a better treatment for patients.
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To test the stability of the CCMV VLPs, we treat the VLPs under different conditions. One parameter we want to test is the temperature stability of the CCMV VLPs. To test this we put the CCMV VLPs for 24 hours in waterbaths with different temperatures. These temperatures range from 5 degrees Celcius to 40 degrees representing the ill human being. After the treatment we tested the samples under the DLS and EM.
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[[Image:T_depend.jpg|500px|center|thumb|''Figure 6: The temperature stability measurements of the wildtype CCMV VLPs'']]
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[[Image:CCMVWT40.jpg|500px|center|thumb|''Figure 7: EM pictures of wild-type CCMV VLPs after 24h heat treatment of 40 degrees Celcius'']]
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As seen in figure 6 and 7, the particles are still detected after a 24 hour treatment with 40 degrees Celcius. This means that the particles will not break down immediately  when exposed to the temperature of an ill human being.
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CCMV VLPs are renowned for its wide pH stability. It is stable between a pH range of 4.7 and 6.5. At a pH of 4.7 the CCMV is in it's unswollen form  with a radius of around 14 nm. At a pH of 6.5 it is in its swollen form, the VLPs radius is around 15-16nm. It is possible to change form, but change the pH gradually with the use of dialysis. If you change the pH too sudden, the VLPs are falling apart and forms aggregates as seen in figure 8.
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[[Image:CCMV WT65.jpg|500px|center|thumb|''Figure 8: Wildtype CCMV VLPs aggregates after a sudden change of pH, taken with the EM'']]
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To test the stability of the VLPs during a long period of time, we designed an experiment. We produced multiple CCMV VLPs and measured them after 3, 6 and 24 hours after formation (figure 9).
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[[Image:Time_depend.jpg|500px|center|thumb|''Figure 9: The temperature stability measurements of the wildtype CCMV VLPs'']]
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It is shown in graph 3 that we still detect wild-type CCMV VLPs after 24 hours. In theory, under perfect conditions (pH at 4.7, T = 4 degrees Celcius) it is possbile to store the CCMV VLPs for a good few weeks. Be aware that aggregate formation may occur when the VLPs are exposed to changing pH or temperature.
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==References==
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<ol>
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<li>David B. Williams, et al., Transmission Electron Microscopy: A Textbook for Materials Science, 2009</li>
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<li>Leonard F. Pease, et al., Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy Biotechnol Bioeng, 2009 102 (3):845-55</li>
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<li>Wolfgang Schartl, et al., Light Scattering from Polymer Solutions and Nanoparticle Dispersions, 2007</li>
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<li>Chao Chen, et al., Nanoparticle-Templated Assembly of Viral Protein Cages Nano Letters 2006 6 (4), 611-615</li>
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<li>Bruce J. Berne,Robert Pecora, Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics - </li>
 +
<li>Carr, R.J.G., Hole, P and Malloy, MNanoparticle detection and analysis: tracking nanoparticles in a sample, directly and individually, to give high resolution particle size distributions, 2007, Abs. Int Conf NanoParticles for European Industry II, Olympia London</li>
 +
<li>Strzelec, M., Laboratory of Laser Applications, Inst. OptoElectronics, Military University Technology, 2007, Warsaw, MNT Bulletin, Vol.8, No.3, p8.</li>
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</ol>
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  <a href="https://2012.igem.org/Team:Wageningen_UR/InsideModification#Inside_Modifications" title="Inside Modifications">5. Inside Modifications</a>
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  <a href="https://2012.igem.org/Team:Wageningen_UR/Applications#Applications" title="Applications">7. Applications</a>
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Latest revision as of 16:45, 26 October 2012

Contents

Detection of Virus Like Particles

A key part of our project is the detection of VLPs. We need sufficient visualization to get conclusive evidence of VLP formation. Besides standard approaches, we will investigate alternative methods to detect the formation and stability of Virus-Like Particles. In this paragraph we explain in short which methods we used and how these methods work. We also describe the limitations of the techniques, so that other people have an idea about what is possible.

Electron Microscopy

Figure 1: Electron microscopy versus Light Microscopy, instead a light as a source to illuminate the sample EM used electrons and instead of glass lenses, EM uses electro-magnetic lenses

The most straight forward method to detect VLPs is Electron Microscopy (EM) [1]. We prepared and investigated multiple samples ourselves. This work was done at the Virology department of the Wageningen University.

Electron microscopy works similar to light microscopy (figure 1), but instead of visible light being used to illuminate the sample, it is done by electrons [1]. The EM has multiple lenses to focus the beam to magnify and light the sample. The lenses are electromagnetic instead of glass and therefore allow a much higher resolution than conventional light microscopy. In theory it is possible to reach a resolution of around 0.005 nm, but in practice it is mostly around 1-2 nm [1]. Possible reasons are certain errors of the lenses, inexperience of the operator (for example our team members) or the sample not being thin enough. All these factors can reduce the resolution of the electron microscope [1]. We investigated various samples obtained in our experiments with the help of the EM. The wild types of the Cowpea chlorotic mottle virus (CCMV) and Hepatitis B (HepB) were detected. Multiple variations of CCMV have been tested too, unfortunately with mixed results.

















EM results



EM Course

The goal of the course was to get familiar with the EM and to handle it safely. Dr. Jan van Lent of the Virology department of Wageningen UR gave us an introduction to EM with a small presentation. During this instruction he explained in detail the electron microscope itself, the preparation of good samples and how to operate the EM properly. Afterwards he showed the sample preparation in the wet lab. Preparation of the sample is critical to have a good resolution and during the course we learned how to do it. The sample must be thin, between 2 - 300nm, and stable in the electron microscope. This can be done by drying or cryo-freezing the sample. After drying or freezing, the sample is stained with a coating containing a heavy metal salt. This coating reflects the electron waves of the microscope, whereas the spots with the VLPs have no coating at all. The missing coating allows the electron waves to go through unhindered. The information of reflection and permeability is processed to a full image which shows the VLPs.





After the preparation of the samples Dr. van Lent explained the whole procedures of visualizing a sample. After this last introduction we were able to analyze and investigate multiple samples ourselves. At the end of the day we were able to use EM for our own experiments starting with sample preparation, handling of the microscope and proper visualization. After the course, the attending team members, were trained and allowed to use the EM without supervision.


Limits

Knowing the limit of a technique is required to interpret the raw data properly, this also applies to electron microscopy. One of the most troublesome limitations is that the preparation of the sample is laborious and can lead to potential debris, which can ruin the sample. Another limitation is that the preparation of the sample, operation of the EM and the analysis of the data requires special training. We were lucky enough to receive a course on how to use the EM. Also samples that are put into the EM must be resilient against the vacuum in the microscope [2].

Dynamic Light Scattering

Figure 2: DLS overview, laser is shooting light in the sample, scattered light is detected by a photon-detector and the data is processed by the computer

Another method to detect VLPs is dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasi-elastic light scattering. This technique is used to measure the size of particles [3]. We applied this technique as a complimentary method to electron microscopy. To use this technique, first we must purify the sample by FPLC to get good measurements. The standard setup of the DLS is seen in figure 2.

When light travels through the sample and hits a particle, it gets scattered in all directions. This scattered light is detected by the DLS. Due to Brownian motion, the particles move randomly in the solution. These movements of the particles changes the distance of the particles with respect to the detector. This change of distance to the detector results in constructive or deconstructive interference of the scattered light. The change of intensity of the scattered light by the interference is related to the radius of the particle and can be calculated by the Stokes-Einstein equation [3].

Figure 3: A) Explanation on how the DLS works by Remco Fokkink, B) output screen of the DLS, C) Stokes-Einstein equation on white-board, D) DLS setup in real life




We investigated various samples with the help of DLS. The main issue was that the sample was not pure or not concentrated enough. After optimization of the production and purification protocol we succeeded to detect VLPs. The main improvement was the implementation of FPLC, which filters out all aggregates and subunits, yielding pure VLP solutions.

Limits

The same as electron microscopy, dynamic light scattering has its limitations. To detect particles with the DLS, the particle has to have a different refractive index than the solvent. If not, the particle doesn't scatter light. Also the sample must be very pure. Any dust particle or aggregate can ruin the data output. This is because large particles scatter way more light than small particles. The data analysis requires vast amounts of knowledge and time [5].

Nanoparticle Tracking Analysis

Figure 4: Scematic overview of the NTA and the sample

The nanoparticle tracking analysis (NTA) is been developed by Nanosight. This is a new method for a direct and real-time visualization of the virus-like-particles. On Tuesday 24th of October 3 samples were tested with varied results, but showed a promising technique that can be used for uncovering parameters of the assembly model.

A focussed laser is passed through the sample and hits the particles in the detection volume. The light of the laser scatters in all directions making the particles detectable for a camera. These particles are seen moving due to Brownian motion. The NTA program identifies the particles and track the centre of each particle individually. The average distance each particles make is automatically calculated. From this value the diffusion coefficient can be obtained. With the diffusion coefficient we are able to get the radius by the Stokes-Einstein equation [6].

In the slideshow below is our HepB wildtype VLPs visualized by the NTA program. We also tested two other samples, but there was no detection. In the slide show you can see aggregates, the VLps it self are too small to be seen on the slideshow, however they are being tracked by the program. Although no estimation of size was yet possible due to the wrong type of laser used during the measurement.


Thanks to the realtime and direct way of visualizing the particle sizes by the NTA program, it becomes possible to do studies about VLP assembly and aggregation [7]. With this technique it becomes possible to find parameters for the assembly and the human body model.

Comparison between the EM and DLS

The electron microscope has been used for years to detect VLPs, but is rather expensive to use. So we searched for alternatives. One of the alternatives we found was the dynamic light scattering technique. Dynamic light scattering is sometimes mentioned as a way to detect VLPs, but is never explained thoroughly. We didn't know any possibilities or limitations of the DLS, so we wanted to see if the output of the DLS is similar to the output of the EM. If the results are positive we can use the DLS as a cheap and easy way to detect VLPs.
In order to quantify the DLS, we set up an experiment to compare the size distribution of the VLPs. We produced a large batch of wild-type CCMV monomers and assembled them in vitro into VLPs. After purification we split up the sample. One part is analyzed by the electron microscope and the other part is analyzed by the dynamic light scattering technique. To calculate the size distribution of the VLPs with the EM, we took multiple pictures of the VLPs. We printed out the pictures and measured by hand each particle individually. After these measurements, we calculated the actual radius of each particle and plotted it on a graph. To calculate the size distribution of the VLPs with the DLS, we took the sample and measured it multiple times, so that anomalies were reduced. After measurement we used the CONTIN method to get the size distribution and plotted it in excel.

Figure 5: Up) The EM raw measurements (red) and the fitted normal distribution of the raw data (Blue), Bottom) Multiple size distributions of the wild-type CCMV VLPs using DLS



As seen in figure 5, the size distribution calculated by both methods looks similar. Both the EM and DLS detect particles around 15 nm, precisely what you expect from the literature. The minor differences can be explained by the methods used to calculate the size distribution. With the EM we visualized the particles first and then calculated the size distribution. With the DLS, the raw output was fitted and can be wrong if the sample is not pure or not concentrated enough. We took that into account by measuring multiple times.
Both the EM and DLS can be used to detect VLPs. This means we've confirmed that both techniques combined create a solid and secure method to detect and analyze VLPs. It is possible to discover many characteristics of VLPs. The part [http://partsregistry.org/Part:BBa_K883001 BBa_K883001] that represents the wild-type CCMV gene with the IPTG induced promoter has been extendedly tested on the stability.

Stability experiments

CCMV is a virus that can be useful in nanotechnology, because it is a well-studied virus and certain physical properties of the CCMV-VLP are already known. We want to provide additional information about the conditions in which the CCMVs are stable. These conditions are important when it comes to site-specific drug delivery. A better understanding of what will happen when the VLPs are injected into the bloodstream, will result into a better treatment for patients. To test the stability of the CCMV VLPs, we treat the VLPs under different conditions. One parameter we want to test is the temperature stability of the CCMV VLPs. To test this we put the CCMV VLPs for 24 hours in waterbaths with different temperatures. These temperatures range from 5 degrees Celcius to 40 degrees representing the ill human being. After the treatment we tested the samples under the DLS and EM.

Figure 6: The temperature stability measurements of the wildtype CCMV VLPs
Figure 7: EM pictures of wild-type CCMV VLPs after 24h heat treatment of 40 degrees Celcius


As seen in figure 6 and 7, the particles are still detected after a 24 hour treatment with 40 degrees Celcius. This means that the particles will not break down immediately when exposed to the temperature of an ill human being.


CCMV VLPs are renowned for its wide pH stability. It is stable between a pH range of 4.7 and 6.5. At a pH of 4.7 the CCMV is in it's unswollen form with a radius of around 14 nm. At a pH of 6.5 it is in its swollen form, the VLPs radius is around 15-16nm. It is possible to change form, but change the pH gradually with the use of dialysis. If you change the pH too sudden, the VLPs are falling apart and forms aggregates as seen in figure 8.


Figure 8: Wildtype CCMV VLPs aggregates after a sudden change of pH, taken with the EM


To test the stability of the VLPs during a long period of time, we designed an experiment. We produced multiple CCMV VLPs and measured them after 3, 6 and 24 hours after formation (figure 9).


Figure 9: The temperature stability measurements of the wildtype CCMV VLPs


It is shown in graph 3 that we still detect wild-type CCMV VLPs after 24 hours. In theory, under perfect conditions (pH at 4.7, T = 4 degrees Celcius) it is possbile to store the CCMV VLPs for a good few weeks. Be aware that aggregate formation may occur when the VLPs are exposed to changing pH or temperature.

References

  1. David B. Williams, et al., Transmission Electron Microscopy: A Textbook for Materials Science, 2009
  2. Leonard F. Pease, et al., Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy Biotechnol Bioeng, 2009 102 (3):845-55
  3. Wolfgang Schartl, et al., Light Scattering from Polymer Solutions and Nanoparticle Dispersions, 2007
  4. Chao Chen, et al., Nanoparticle-Templated Assembly of Viral Protein Cages Nano Letters 2006 6 (4), 611-615
  5. Bruce J. Berne,Robert Pecora, Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics -
  6. Carr, R.J.G., Hole, P and Malloy, MNanoparticle detection and analysis: tracking nanoparticles in a sample, directly and individually, to give high resolution particle size distributions, 2007, Abs. Int Conf NanoParticles for European Industry II, Olympia London
  7. Strzelec, M., Laboratory of Laser Applications, Inst. OptoElectronics, Military University Technology, 2007, Warsaw, MNT Bulletin, Vol.8, No.3, p8.


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