Thursday, October 31, 2013

NMR Chemosensing

Welcome to my new NMR blog.  I hope to use this blog to discuss interesting new publications in the literature.

Today I'm going to review a recent JACs communication by Perrone et al.

The title of this paper is '"NMR Chemosensing" Using Monolayer-Protected Nanoparticles as Receptors.' 

A chemosensor is a "molecule of abiotic origin that signals the presence of matter or energy".  As a very general concept, a chemosensor can be imagined as a "supramolecular receptor" that selectively binds its target (the analyte), which causes some measurable change in a physical property (such as absorbance, redox potential, relaxivity, etc.)  It does not take a vivid imagination to dream up uses for chemosensors and thus, there are many scientists in the world making chemosensors.  To distinguish their chemosensor from all the other sensors out their, Perrone and co-workers make the following observation regarding the current state of chemosensor research:  for most chemosensors "the signal produced arise from a property of the chemosensor itself and therefore does not provide any direct information on the identity of the analyte detected."  These authors approach is the opposite.  In this publication they describe a chemosensor in which the analyte-sensor interaction alters a physical property of the analyte.  The measurement step queries physical properties of the analyte not the sensor.  OK.  It is novel.  Is it useful?  (I am not 100% convinced that the author's approach offers advantages over the more common receptor-centric approach, but I'm willing to give them the benefit of the doubt at this stage.)

How do they makes these sensors?  They use 2nm diameter gold nanoparticles coated with thiol monolayer, which seems to be the expertise of this group.  This monolayer is amphiphilic (for example, 7 carbon alkyl chain linked to a triethylene glycol head group via amide, 1).

Next, a mixture containing 70 uM chemosensor and 7 mM of 3-5 water soluble organic acids or sulfates (see the roster below) in D2O is prepared.   

A curious choice of molecules!  I wonder how the authors settled on these five.   

At any rate, let's get down to the experiment  It is expected that hydrophobic molecules will partition into the hydrophobic moiety of the monolayer, whereas hydrophilic molecules will interact with both the polar headgroup and polar solvent.  This mixture is probed using a NOE-pumping experiments, that works like STD NMR.  Briefly, the nuclear spins of aliphatic region of the monolayer are perturbed (inverted or saturated).  Some of this perturbation is transferred via NOE to small molecules partitioned into the hydrophobic monolayer.  Assuming these molecules are in fast exchange with free solvent, there will be signals in the resulting spectrum that arise from the analyte (not the sensor) as a direct consequence of interaction with the sensor.  The fact that the analyte has to be in fast exchange with the receptor has consequences for the detection limit, acquisition time and affinity towards the receptor.   

The first example presented is a mixture of 4, 7 and 8.  The chemosensor + NOE pumping experiment shows remarkable selectivity and signals from only one molecule are detected! 

The top spectrum is the aromatic region of the 1D 1H of a mixture of 4, 7 and 8 at a concentration of 7 mM in D2O.  The bottom spectrum is the aromatic region of the NOE-pumping experiment of the same mixture with 70 uM 1-coated gold nanoparticles.  I am impressed.  Two comments, though.  First, you can almost see a little bit of 7 peaking out of the noise.  Am I wrong?  Second, the authors do not mention the experimental conditions of the NOE-pumping experiment.  I understand that this publication is a JACs communication and the authors do not have a lot of space for extraneous information.  It is just that I'm curious about the sensitivity of the NOE-pumping experiment.  How many scans did they have to record to get the bottom spectrum?  (At a later point in the paper it is implied that the acquisition time is 4 hours using NOE-pumping.)

The authors follow up on this experiment by demonstrating the selectivity of their chemosensor.  For instance, in a mixture of three isomers of salicylate (4, 5 and 6) with 1-coated nanoparticles, the NOE-pumping experiment only show signals for 4.  (This data can be found in the SI.  I have no idea why the author's buried it there.)  Once again I am impressed, even if a bit of 5 and 6 seem to peak out of the noise.  The authors measure the apparent association constant, as well, to demonstrate that quantitative analyte detection with their chemosensor.  The detection limit for salicylate is 2.5 mM (OK, now I am a bit less impressed.  By the way, at the end of the paper the author's mention STD-NMR can be used in lieu of NOE-pumping.  Using STD-NMR the detection limit is 250 uM and the acquistion time is 30 minutes.  Why did they bother with NOE-pumping?)  At this stage the authors have convinced the reader that their 1-coated nanoparticle in combination with the NOE-pumping experiment is selective for sodium salicylate, even in the presence of isomers. The authors do not report results for the detection of salicylate (4) in a solution enriched in 5 and/or 6.  One might accuse them of dodging the issue of false positives, but I'll cut them some slack because this article is a JACs communication.  The authors are excellent communicators and do a nice job of leading their audience away from the short-comings by highlighting the positives.  I wish I were this skillful at this technique!

Since the authors have convinced us of the "what", they turn the "how" and "why".  Why do 1-coated nanoparticles and the NOE-pumping experiment detect 4 and not 5?  It goes back my introduction of their technique.  Hydrophobic molecules partition themselves into the aliphatic moiety of 1.  The authors note that the elution order of a mixture of 4-8 using reverse-phase C18 HPLC is 8 << 7 < 6 < 5 << 4.  The interpretation of this result is that 4 is the most hydrophobic.  Likewise the computationally predicted logD at pH 7.4 concurs with the elution order.  In other words, it seems salicylate really is more hydrophobic than the isomers and the chemosensing technique described in this publication relies of this fact.  Of course, this highlights a potential weakness in selectivity.  Will you ever be able to distinguish between two hydrophobic compounds (with equal C18 retention time and/or logD)?  Can we modify the chemistry of the coating thiols to distinguish molecules using a property other than hydrophobicity?

The authors put a positive spin on these questions.  Let me quote the paper.  "Since there are no limitations on the chemical structures of the analytes and the nano particle-coating thiols, the NOE pumping experiment has the advantage of very general applicability."  Really?  No limitations?  The authors make 2 and 3-coated nanoparticles and return to their mixture of 4, 7 and 8 at a concentration of 7 mM in D2O.  The aromatic region of the NOE-pumping experiment with 2-coated nanoparticles as the receptor shows signals from 4, 7, 8 and an impurity of 8 (middle spectrum, below).  The authors refer to this result as "different selectivity."  I would call this NO selectivity myself, but hey you say PO-TAY-TOE, I say PO-TOT-TOE.  (  The aromatic region of the NOE-pumping experiment with 3-coated nanoparticles as the receptor shows signals from 4 and 7 (bottom spectrum, below).

Finally, to demonstrate applicability the authors spike 5 mM salicylate into a sample of human urine, mimicking the concentrations found in urine after a medium-dose administration of acetylsalicylic acid (aspirin).  The aromatic region of the 1D 1H spectrum is complicated due to the myriad of metabolites in urine.  The aromatic region of the NOE-pumping spectrum of the same sample in the presence of 1-coated nanoparticles show only signals for salicylate.   

Let me conclude by stating that I do not want my criticisms to overshadow the excellent science in this communication by Perrone et al.  I am a nobody, whereas the authors are doing the hard, creative work of producing scientific knowledge.  I am genuinely impressed by their approach and I appreciate that this publication is the first step in a long journey.  Having said that I will be more impressed when they design a sensor to detect something a bit more interesting than 5 mM salicylate spiked into urine!  One example that springs to mind is enantiomeric impurities.  There are pharmaceuticals (for examaple, penicillamine - that have a single chiral carbon.  One enantiomer is a powerful therapeutic agent; the other is a deadly poison!  In 2004 Tsourkas and co-workers published a communication ( describing an antibody-based magnetic relaxation switch capable of detecting an impurity of 0.1 uM D-Phenylalanine in the presence of 10 mM L-Phenylalanine.  (That is 99.998% ee if you are keeping score at home!)  It is difficult for me to see how the monolayer-protected nanoparticles introduced by Perrone et al. will approach this level of sensitivity and discrimination.