Research
Our research interest is in the area of interfacial chemical kinetics. Our investigations of interfacial kinetics utilize self-assembled monolayers (SAM) or polymer matrices coupled with gold and silver nanoparticles. The nanoparticles have unique optical properties that provide an optical signal for following reactions in thin film matrices or on SAM's attached to the nanoparticles in solution.
Investigations of the immobilization of proteins and other biologically relevant molecules to solid substrates has been of interest because of the possibility of forming biosensors from biomolecules by stably bonding them to suitable organic thin films through a strong covalent bond. However, this strategy is not without its problems and numerous studies have addressed some of the major issues inherent in covalent immobilization of large molecules to surfaces.
Many reaction schemes have been developed to attach biological recognition systems to suitable surfaces. All of these methods have their own advantages and disadvantages and very few of them are understood in any fundamental kinetic and mechanistic way. Both the development of general methods and the application of existing methods for protein immobilization are hampered by a lack of basic understanding of the reactions and interactions that occur at thin-film and matrix surfaces. To date, there have been very few comprehensive studies of the kinetics and mechanism of covalent reactions between proteins and thin films. That is where we come in.
Our research is aimed at studying the reaction of a tethered N-hydroxysuccinimide ester with an amine in solution in detail using surface enhanced Raman spectroscopy (SERS). The N-hydroxysuccinimide ester is formed using a carbodiimide coupling reaction shown in scheme 1. This reaction is a well known way to make amide (from a carboxylic acid and an amine) or ester (from a carboxylic acid and an alcohol) bonds. In our case the carboxylic acid is tethered to a silver nanoparticle (Ag-NP) surface so that the resulting ester using N-hydroxysuccinimide as the alcohol is also tethered to the Ag-NP surface. We use 4-mercaptobenzoic acid (4MBA) as our carboxylic acid since the soft-soft Lewis acid-base interaction between the thiol sulfur and the silver in the Ag-NP is comparable to the strength of a covalent bond. This means that the tethered ester that is produced will not readily desorb off the surface and will stay intact for subsequent chemical transformations.
Scheme 1: General carbodiimide coupling reaction with N-hydroxysuccinimide
Our previous research has shown that Ag-NP suspended in solution will react with histidine (the amino acid with an imidazole side chain) with kinetics that are consistent with an SN2 mechanism. However, reaction with a large protein such as glucose oxidase is more complicated. The reaction is pH dependent and the kinetics appear to be consistent with an SN1 mechanism. This has led us to believe that the steric bulk around the nucleophiles on a large protein is responsible for a switch from SN2 to SN1 mechanism. To test this hypothesis there are several projects that we aim to complete.
Project 1:
Investigate the effect on the kinetics of increasing steric bulk around the nucleophilic N on imidazole derivatives.
Project 2:
Investigate the effect on the kinetics of changing (increasing or decreasing) the ester density on the Ag-NP surface. We can accomplish this by using benzene thiol and 4MBA in varying mole ratios to cover the Ag-NP surfaces. The higher the percentage of benzene thiol the more spread out the carboxylic acids (4MBA) will be on the surface leading to a lower surface density and thus less steric bulk at the surface.
Project 3:
Investigate the pH dependence of the imidazole or histidine reaction with the active ester on the Ag-NP surface. The protonated imidazole (imidazonium) is not nucleophilic and will not react with the active ester. Thus we would expect that as the pH increases, the imidazole/imidizonium (conjugate acid/conjugate base) ratio will increase, causing a higher concentration of nucleophile and thus a larger value of kobs. If the relationship of kobs to pH can be fully accounted for by knowing the pKa and thus the true concentration of imidazole in the solution then we can say that the overall reaction kinetics are not pH dependent, but rather dependent on the concentration of nucleophile as for any SN2 reaction.
Project 4:
Investigate the kinetics of the reaction as a function of the strength of the nucleophile in solution. By going from a primary, to a secondary, to a tertiary amine we can adjust the electronic conditions such that we increase the basicity of the nucleophile. We would expect the stronger base to react at a higher rate and thus have a larger rate constant.
Project 5:
Find a way to investigate the strength of the leaving groups on the surface. Tether other SERS active molecules to the surface with different leaving groups (halogens, mesylates, tosylate, etc.) and observe the kinetics the substitution reaction with a nucleophile in solution.
Techniques
Ag-NP surfaces:
We make Ag-NP surfaces by "decorating" hexagonal boron nitride nanosheets (h-BNNS) with Ag-NP's and capturing this assembly on a special filter paper. The h-BNNS are planar sheets of hexagonally arranged alternating boron and nitrogens similar to the structure of graphite. The B-N bonds are about 0.152 nm and our Ag-NP's are on the order of 30-40 nm in diameter. Thus, a very small portion of the surface, more or less to scale, would look like figure 1. Note the scale difference between the nanoparticle and the B/N hexagons!
These surfaces are formed by synthesizing the Ag-NP from AgNO3(aq) and NaBH4(aq) in a solution of BNNS. These nanostructures are filtered out of the reaction solution onto a filter membrane.
Figure 1: A more or less scale drawing of a silver nanoparticle on a hexagonal boron nitride sheet
The filter containing the Ag-NP/BNNS surface is then soaked in 4-mercaptobenzoic acid (4-MBA) to coat the Ag-NP's. Figure 2 is a cartoon visualization of a Ag-NP coated with 4-MBA.
Figure 2: Cartoon of Ag-NP coated with 4-MBA
Figure 3: SERS spectra of a surface with 4-MBA before reaction with NHS (blue) and after reaction with NHS (red). The large peaks at around 1090 cm-1 and 1580 cm-1 are ring modes of the benzene ring in 4MBA and the small peak that appears near 1770 cm-1 is due to the succinimide stretching motion of the NHS.
NHS active ester formation
These surfaces are then soaked in a mixture of 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form the NHS ester according to Scheme 1. Each step of the fabrication is monitored using Surface Enhanced Raman Spectroscopy (SERS).
Kinetic runs
These surfaces with the NHS ester tethered are then used to investigate the kinetics of reaction between the NHS surface group and a nucleophile in solution. Since the nucleophile in solution will always be in excess of the NHS on the surface, these reactions are pseudo first order in NHS with the [nucleophile] staying essentially constant over the course of the reaction. The reaction is monitored by monitoring the disappearance of the NHS peak in the SER spectrum as it is replaced with the nucleophile. A typical SERS spectrum of the surface with the NHS ester peak is shown in Figure 3.
SERS
Surface enhanced Raman spectroscopy (SERS) is a technique that allows us to obtain Raman spectra of a very small quantity of molecules like the approximately pmoles of material that is typically absorbed to a surface. This technique is an extension of Raman spectroscopy which relies on the inelastic scattering of photons (light) when they interact with molecules. When a photon hits a molecule it can scatter off the molecule elastically and not lose any energy (or momentum) to the molecule. This type of scattering is called Rayleigh scattering. But in some small number of collisions between a molecule and a photon, the photon loses energy to a molecular vibrational mode and then the scattered photon has its original energy minus the energy used to excite the vibration. Since vibrational frequencies are quantized, there are only certain energies that the photon can lose. If we build an instrument to detect the energy of the photons after interaction with the sample and subtract those energies from the initial energy of the photons before interaction (this difference is called the Raman shift) we can determine the energy lost to vibrations of the molecules in the sample. If we then count the number of photons that have lost each increment of energy, we can graph the intensity (number of photons) vs. the energy lost. This gives us a vibrational spectrum since the energy lost is equal to the energy of vibrations that were excited.
Unfortunately this is a very insensitive technique since most photons scatter elastically and the number of photons that undergo the inelastic Raman scattering is very small and hard to detect. This is where SERS comes into play.
Metals have a sea of valence electrons that are not bound to any particular nucleus making up the metal lattice. These electrons are free to move throughout the metal material. This sea of electrons will set up wave patterns much like the waves in an ocean. Since the electrons cannot get outside the metal structure, these wave patterns are quantized. If an electric field (like a light wave or photon) interacts with a metal structure (like a nanoparticle) the electric field created by the oscillating electrons in the nanoparticle and the electric field of the photon can couple to make an enhanced electric field right around the nanoparticle called the local field. Molecules that are near the nanoparticle surface feel this enhanced electric field. Higher intensity fields mean more photons and more photons mean a higher chance that some of them will undergo Raman scattering. Since the fields can often be enhanced by up to six orders of magnitude, this makes detecting the Raman scattered photons much easier.
To make it even better, we can put nanoparticle structures close to one another (like on the Ag-NP decorated BNNS surfaces). When the nanoparticles are close enough that their local fields overlap we get even more enhancement of the Raman signal. This is why we are able to get high resolution spectra of the molecules on our Ag-NP decorated BNNS surfaces so that we can follow the kinetics of the reactions on those surfaces.
Some examples of "recent" posters presented at ACS
ShawnBridgette ACS15.pdfsers.pdfMattEmma ACS15.pdf