Expert Insight: Bioanalytical methods for bacteria and protein characterization

Watch this on-demand webinar to expand your knowledge on particle characterization, Raman microscopy, fluorescence, and SPRi

03 Mar 2021



Nathalie Vollmer (left), Christelle Mégier (middle) and Thibault Brulé (right), from HORIBA Scientific

Molecular and cellular bioanalyses are increasingly moving towards faster and more sensitive approaches. Key techniques such as Raman spectroscopy, fluorescence spectroscopy, dynamic light scattering, nanoparticle tracking analysis and surface plasmon resonance imaging now allow for easy characterization of all kinds of analytes, from cells to small molecules.

In this webinar, Nathalie Vollmer, Christelle Mégier and Thibault Brulé, from HORIBA Scientific, discuss these different techniques and their application in life sciences and pharmaceutical research. The potential of these techniques is also illustrated through different application examples for bacteria and protein analysis. 
Read on for highlights from the live webinar’s Q&A session or register to watch the webinar on demand>>

Q: How does inner filter effect (IFE) lead to wavelength shift? 

NV: At high concentrations, reabsorption may occur and some amount of fluorescent light emerging from the shorter wavelength compounds is reabsorbed by the longer wavelength compounds, which in turn emits with a higher amplitude. Consequently, the maximum of the peak may see redshift.

Q: Can you comment on the impact of storage conditions on degradant formation in culture media?

CM: If culture media are not stored in the dark, light degradation catalyzes tryptophan degradation, which in turn reduces cell viability and growth. Several groups have reported looking at screening methods to detect various degradants, which are often byproducts of tryptophan, tyrosine, and riboflavin. 

Q: What is the main advantage of the absorbance, transmittance and a fluorescence excitation emission matrix (A-TEEM) method when applied to pharma solutions? 

NV: We showed that insulin sequences varying by only one or three amino acids can be differentiated by intrinsic fluorescent A-TEEM. In the study, we showed that the A-TEEM methods can be used to characterize protein therapeutic formulation and aggregation behavior, even under conditions where there are only small differences in the protein sequence. The A-TEEM method is a good way to characterize such proteins where traditional fluorescence methods may fail. For future applications, an A-TEEM fingerprinting technique could be used for the characterization of other protein therapeutics, such as vaccines, inhibitors, or antibodies. 

Q: What are the main differences between surface plasmon resonance (SPR) and SPR imaging (SPRi)?

NV: The main difference between these two techniques is the number of ligands we can immobilize on the sensor chip. With SPRi, we can immobilize hundreds of molecules, hundreds of ligands, on the same sensor chip. In this configuration, it is possible to follow, in real time, the interaction between these molecules and the different targets in parallel. Therefore, this saves time because you can immobilize many ligands on the biochip and can get the results very quickly compared to classical SPR. 

Q: Is it possible to reuse SPRi sensor chips? 

NV: In our configuration, once the ligands are immobilized on the sensor chip you cannot remove them, but you can regenerate the interaction between the ligands and the analytes. You can store and reuse the sensor chip with the ligands, but you cannot remove the ligands. 

Q: What are the benefits of EDF compared to standard DF elimination? 

NV: EDF is more interesting in the sense that it is composed of a specific condenser with a specific design that has been patented. Thanks to that, we managed to maximize the scattered signal from nanoparticles or nano-objects, and we can improve the detection limit and the signal ratio to get a better image contrast. This can be either used with a spectral imaging system or Raman imaging. 

Q: Is the Raman study of biological samples dependent on excitation regarding the source and slit width of the spectrometer?

TB: Most of the time, biological samples are fluorescence so the Raman signal will not depend on the laser source that is used. However, what will impact the spectrum will be the fluorescence coming from the samples. In that case, you have to choose your excitation source in order to reduce its fluorescence and so the fluorescence can be reduced by playing with the slit width of the spectrometer. 

Q: Would you consider dynamic light scattering (DLS) as an appropriate, complementary technique to size-exclusion chromatography (SEC) to determine soluble aggregates?

NV: Yes, you can have a DLS detector that can be coupled with SEC technology. In this case, with SEC, you will make a separation and then the DLS will allow you to evaluate the size of the monomer or dimer or trimer without any difficulty. However, when you use the DLS device alone, it will be impossible to get a separation of the different populations, because the technology itself will measure a global signal. SEC will allow you to separate the particles, but DLS alone will not have the capability to be resolutive on a sample with a mix of monomers, dimers, and trimers. 

Q: Gold nanoparticles can lead to very low, close enhancement effects. How reproducible are surface-enhanced Raman scattering (SERS) spectra of cells? 

TB: It depends on the protonation of the nanoparticles used to do the SERS spectra. The SERS spectra are highly dependent on the quality of the nanoparticles and their distribution in the cells, so to have reproducible SERS spectra of your cells, you need to have a good reproducibility of the distribution of the nanoparticles in the cells. When we do SERS analysis in cells, our interest is to investigate one spectrum from the inside of one cell. The idea is to target specific locations in the cell with the nanoparticles to see the interaction between the nanoparticles and these specific locations to help with characterization. 

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