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What Does a Nanocrystal Conjugate Look Like?

  
  
  
  
  

Hi, my name is Travis Jennings, PhD and I am a Materials Scientist here at eBioscience. I thought it might be useful to give a physical description of a Nanocrystal-Antibody (NC-Ab) conjugate and point out some of the implications that such a description may have for the end user.  It should be noted that standard users of our nanocrystal conjugates for Flow Cytometry or IHC/ICC can simply follow the instructions for use as a test size and expect to obtain outstanding results.  But for the researcher interested in using an eFluor® NC-conjugate in a custom application, this may mean that you need to think about your experiments differently. 

The figure below shows what a NC-Ab conjugate might look like (if antibodies were purple). Probably the most important aspect to realize is that our nanocrystals are NOT organic dyes – they’re fundamentally different.  Some differences are good, some are less good; it depends on the application. 

Nanocrystal Anatomy resized 600

eBioscience nanocrystals are:

    • Composed of semiconductor metals
    • Much bigger than traditional dye molecules
    • Similar in size to an antibody (see the Wiki page for more technical specifications)  

As you may imagine, solid semiconductor particles do not disperse naturally into water.  Thus, this is accomplished by encapsulating them into lipid polyethylene glycol (PEG) micelles, which may then be further functionalized and conjugated to an antibody of choice. 

The conjugate is exactly the reverse of Dye-Ab conjugates in that there are multiple Ab’s attached to each nanocrystal, instead of multiple dyes per Ab as is traditional, which may have several ramifications:

    1. This increases the avidity of the conjugate because multiple F’ab regions are present.
    2. This increases the size of the conjugate to ~20-30 nm hydrodynamic diameter.
    3. The lower fluorophore:protein (F:P) ratio does NOT imply that the conjugate is less bright.  Although this is a separate discussion on its own, suffice it to say that small NC’s (blue emission) may be similar or possibly less bright than a blue-emitting dye-Ab conjugate, while larger NC’s (red emission) may be several times brighter than a similarly red-emitting dye conjugate for a given application.
    4. Because the nanocrystal - and not the antibody - is the central unit in the conjugate, the concentration used for staining is determined by the NC concentration and NOT the Ab concentration as is typically done with organic dye conjugates.  In fact, NC’s absorb so much light that measuring Ab concentration at 260 nm is borderline impossible! 

The observations above will have implications to experiments involving kinetics, cell membrane diffusion, in vivo applications, etc....

Comments

Travis - thanks for the description which is very useful info. Two questions: 1) what chemistry underpins the Ab - PEG linkage? 2)If multiple ab molecules can bind is there an optimum? How can one determine if sufficient Abs are bound and stable. This would have implications for the NCs in blue emission? cheers, 
 
ian
Posted @ Wednesday, June 29, 2011 6:59 PM by ian beckman
Hello Ian, good questions! Unfortunately, I can't detail our methods for conjugating Ab's to the NC as we've spent lots of time and resources optimizing this. Generally, though, I can say that we use the same functionalized NC's that we sell normally (eg. 605NC-Amine, cat. 93-6366) and target either the ubiquitous amine groups or disulfide linkages found in the Ab hinge region. 
 
The Ab-NC conjugates are actually quite stable as long as they're stored properly about 4 C, and although it sounds circular, the best way to determine the optimal Ab:NC ratio is actually to use the conjugate in the desired application. We find that different Ab's have different optimal Ab:NC ratios. So, for the absolute best results on a particular Ab, we usually recommend performing titrated conjugations by varying the Ab:NC ratio and then testing the performance of each conjugate.  
 
I believe you're on the right track when you ask about how the ratio might affect performance of the blue-emitting NC's. High Ab:NC ratios will increase the avidity of the conjugate and lower the "off" kinetics theoretically, but will also limit the total number of conjugates that can bind per unit area. Low Ab:NC ratios will increase the density of conjugates on the cell surface (brighter), but will have a lower avidity and thus faster "off" kinetics (dimmer). So I believe that these are the competing factors dictating the performance of a NC-Ab conjugate. 
 
Best, 
Travis
Posted @ Thursday, June 30, 2011 2:07 PM by Travis Jennings
I am concerned that with the multiple aBs on the NC, I can no longer associate fluorescent signal to a single cell in flow cytometry because multiple cells can bind a single NC. Is this correct? 
Thanks
Posted @ Wednesday, December 05, 2012 7:21 AM by Wendy
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