Learning and Experimenting with Dynamic Light Scattering

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Learning and Experimenting with Dynamic Light Scattering

This past week at my Ole Miss REU research program has been...interesting to say the least. Not in a bad way, just in a I'm-going-insane-because-all-my-data-is-wacky-no-matter-what-I-do kind of way.

The purpose of these experiments that I've been trying to do involves gaining data about the size and distribution of DNA/mercury complexes in solution. I outlined in my first post of this series how Dr. Wadkins has demonstrated that mercury complexes with DNA and induces it to form secondary structures. It's also postulated that these structures can be thought of as fully-fledged nanoparticles. 

Dynamic Light Scattering (DLS) is a common way to obtain data about nanoparticles. Therefore, getting usable data from the DLS instrument from my DNA/mercury samples was my first order of business in order to develop an idea of how increased mercury concentrations correlates with particle size in solution.

I thought it would be easy. I thought I could just throw in the DNA and the mercury and see what happened. There would be a clear pattern of increasing nanoparticle size as the concentration of mercury increased.

Well...I was wrong. And to understand why, I need to talk about how a DLS instrument actually garners this data for me.

(Disclaimer: At the time of this writing I still have not figured out the issue. I will go into how I think I can fix it, but I will give an update on that in a future post).

How a DLS Instrument Works (Sort of)

A DLS instrument works by shining a high-intensity laser through the sample, which is usually contained in a cuvette or a small well inside a well plate (I have been using a well plate).

When a sizeable particle in solution (on the scale of nanoparticles) enters the beam of light due to the random movements of Brownian motion, the light from the laser that hits the particle becomes scattered. 

The detector on the other side of the instrument receives the scattered light and makes some calculations. However, what's really important here is the fluctuations of the scattering of the laser because that's what really yields the data about the particle's size.

See, the fluctuations are a result of brownian motion of the molecules through solution. This creates phase shifts in the scattered light which can either constructively or destructively interfere. Therefore, the rate of this fluctuation is directly proportional to the rate of diffusion of the molecule through solution. The rate of diffusion is in turn related to the hydrodynamic radii of the molecules in solution scattering the light.

Smaller particles diffuse faster while larger particles diffuse slower. Therefore fast fluctuations indicate smaller particles and vice-versa (see the image at the top of this post).

The hydrodynamic radius in the contexts of DLS is the radius of a sphere with the same diffusion coefficient as the particles in the sample. It's not saying "this is the exact size of the particle" but it is quite close.

There's also something called the Polydispersity Index (PDI) which is a measure of the different kinds of particles/particle sizes that are present in the sample. For example, a PDI close to 0 or around 0.1 means that the sample is monodisperse (only one kind of particle) while higher values closer to 1 signal that the sample is polydisperse (the presence of different particles of different sizes.)
(Note: different instruments/software calculate PDI differently, so always check how yours defines it!)

A couple very last things to note are the amplitudes and the intensities of the signal. Amplitude is pretty much just the strength of the scattering from particles in the sample and is used to ascertain whether the data is good quality or not. Our target values are between 0.03 and 0.1 (there's still some details on that I'm a admittedly a bit fuzzy on). Intensity (or count rate) on the other hand is a measure of how many individual photons hit the sensor per second. Based on my research, anything between about 30,000 - 500,000 counts per second is decent while anything over one million is garbage data (way too many photons reaching the sensor). The problem I'm currently having is that I'm getting these one million or greater values......even when I run deionized water...High intensity doesn't mean good data, it means the detector’s getting blasted.

The Troubleshooting Part....What's Wrong?

Now that we've discussed Brownian motion, diffusion, hydrodynamic radii, polydispersity, amplitude, and intensity we're ready to get into what I currently think is wrong with my samples: There's nothing wrong with the samples (inherently). I think it has to do with the well plate I'm using (the plate that contains all of the divots that I pipette my samples into).

The reason I think this is the problem is because it accounts for the insanely low amplitudes I'm seeing and the insanely high intensities I'm seeing. 

Initially, I thought my very low amplitudes were due to the fact that the sample concentration was too low. After all, we were using a 6μM solution of single stranded DNA only 15 base pairs long. I thought that "there's not enough particles in solution and they're really small particles anyway. The instrument isn't even picking up on them!" So I nearly doubled the concentration to 10μM and compared with the 6μM solutions. No change. Still really wacky data that honestly made no sense. Still the same low amplitudes.

Then I thought, "maybe I'm using to steep of an addition of mercury and I'm oversaturating the samples with ionic mercury. I'll narrow it down to a smaller mercury concentration range to better 'resolve' what's happening in the samples." I tried that. Actually just did that right before writing this. The result? The same old garbage data.

Concentration isn't really changing much (unless I didn't increase it enough, but I also can't increase it too much) and the rate of mercury addition isn't really changing anything anyway.

The well plate I'm using has clear walls...this means the light coming up through solution could be bouncing and reflecting off of those clear reflective walls and creating more scattering than there actually is. This would explain the massive amounts of photons coming into the sensors. They're only designed to measure light from a specific angle, not from a plethora of other angles as a result of reflective light from within the well plate itself.

But does this also account for the really low amplitudes? I think it might. Amplitude is derived from the height (or y-intercept) of the correlation function that is based on the scattering of particles in solution. I think what could be happening is the high intensity noise is drowning out any actual scattering data that's able to come from the samples themselves thereby leading to a low amplitude.

We ordered the new plates with opaque well walls and we'll see if that makes a difference. Hopefully it does because I'm about to go insane over this DLS machine. In my next post I'll outline whether this fixed the issue or not!

Thanks for reading and I hope you enjoyed learning a little about Dynamic Light Scattering!

Where thoughts orbit stars and dreams power suns...













 

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