EPSRC grant (GR/T08746) "Spectroscopic Imaging for High-Throughput Analysis" (PI: S. G. Kazarian)

BBSRC grant (BB/K011030/1)  "Application of ATR-FTIR imaging to industrial scale production of therapeutic antibodies" (PI:  B. Byrne, Life Sciences; co-PI: S. G. Kazarian)

ERC Advanced Grant (ERC grant 227950): "Enhancing microfabricated devices with chemical imaging for novel chemical technology" (PI: S. G. Kazarian)


In this research we have developed applicability of FTIR spectroscopic imaging to study many samples simultaneously for high-throughput analysis. We have demonstrated for teh first time that FTIR spectroscopic imaging approach with macro ATR capability can be successfully used as a very powerful novel tool for high-throughput analysis of pharmaceutical formulations and can provide guidance to the design of pharmaceutical formulations for controlled release.

PDMS grid for high-throughput ATR

We developed macro attenuated total reflection (ATR) -FTIR imaging approach for measurements of many samples simultaneously (e.g. see cover article in Applied Spectrsocopy below, which is open access paper (doi)). This approach was successfully achieved using drop-on-demand device by preparing arrays of micro-drop samples directly on the surface of the ATR crystal. The use of PDMS grids to serve as sample support plates has also been realized.  The developed macro ATR-FTIR imaging approach has allowed us to simultaneously study more than 100 samples under controlled environment. Using this approach, it was possible to obtain "chemical snapshots" from a spatially-defined array of many different polymer/drug formulations under identical conditions. This high-throughput approach provided information about a specific weight fraction of ibuprofen in PEG which should not be exceeded in the formulation to avoid dimerization of ibuprofen. This method provides direct measurement of materials properties for high-throughput formulation design and optimisation. Simultaneous response (water sorption, crystallization, etc.) of the array of formulations to the environmental parameters was studied. Furthermore, a novel ATR accessory with an expanded field of view has been designed and applied to simultaneously obtain infrared spectra of more than 150 miniature samples. This approach enables the measurement of up to 1024 samples. The introduction of this new accessory with enhanced field of view provided an opportunity to combine ATR-FTIR spectroscopic imaging with a multi-channel grid that allowed simultaneous imaging of dissolution of several different formulations. Different molecular weights of poly(ethylene glycol) (PEG), with or without the addition of ibuprofen, have been used as model pharmaceutical formulations and chemical imaging of the simultaneous dissolution of five different formulations of PEG/ibuprofen has been demonstrated. Direct comparison between these different formulations under identical conditions was possible due to this imaging approach. The demonstrated approach is also the first example of application of spectroscopic imaging to microfluidics and may broaden its future use in miniaturisised high-throughput devices.

Front cover of Applied Spectroscopy

We have combined macro ATR-FTIR imaging with an inverted prism-shape ATR crystal with a poly(dimethylsiloxane)-based microfluidic mixing device and demonstrated that ATR-FTIR imaging can be used to chemically image different substances flowing within microfluidic channels. Chemical specificity of the ATR-FTIR imaging enhances detection approaches in microfluidics because this method does not require the use of fluorescent or absorbing labels. Specific examples used to demonstrate our approach include imaging of the mixing of two liquids of different viscosities and the imaging and mixing of H2O and D2O with consecutive H/D isotope exchange. The demonstrated approach pioneered by us can impact studying many processes in microfluidics ranging from reactions to separations. Further details about this research can be found either from the bibliography below (subscriptions to some journals required) or from the open access articles published though "Lab on a Chip" and highlighted online by Spectroscopy.

Rapid prototyping of microfluidic devices for integrating with FTIR spectroscopic imaging

Chemical imaging of microfluidic flows using ATR-FTIR spectroscopy

 In our current BBSRC-funded project we investigate the issues related to protein aggregation with view to improving isolation protocols using ATR-FTIR spectroscopic imaging. This allows us to chemically image the distribution of the target protein molecules under different conditions both in static droplets and in dynamic systems similar to those used for large-scale isolation.  Our first studies were a proof of principle that we can use this method to study proteins under a range of conditions that would normally be experienced during the isolation process using purified protein samples in solution. The findings of this research have been published in Analytical Chemistry 2014. In addition we have developed methodologies to allow us to study the changes in the chromatographic resin during cleaning processes key to extending the lifetime of the resin. Our ATR-FTIR spectroscopic methods allow us for the first time to explore changes in the chemistry of the resin under harsh cleaning protocols. Our findings could help to optimise cleaning protocols and extend resin lifetime and reduce mAb production costs. These findings have been published in Analytical and Bioanalytical Chemistry 2015 (Open Access, see below)  and discussed in a recent article on spectroscopic imaging for the analysis of biopharmaceuticals in SAA  (doi) that has been featured in GEN (Genetic Engineering & Biotechnology News).  

Key references:

  • Chan K. L. A., Shalygin A. S., Martyuanov O. N., Welton T., Kazarian S. G. High throughput study of ionic liquids in controlled environments with FTIR spectroscopic imaging  Journal of Molecular Liquids (2021)  accepted and in press. 
  • Tiernan H., Byrne B., Kazarian S. G. Insight into heterogeneous distribution of protein aggregates at surface layer using ATR - FTIR spectroscopic imaging. Analytical Chemistry (2020) 92 (7) 4760-4764 (doi).
  • Boulet-Audet, M., Byrne B., Kazarian S. G.  High-throughput thermal stability analysis of a monoclonal antibody by ATR-FTIR spectroscopic imaging  Analytical Chemistry (2014) 86(19), 9786–9793. (Open Access)
  • Boulet-Audet M., Byrne B., Kazarian S. G.  Cleaning-in-place for immunoaffinity resin monitored by in situ ATR-FTIR spectroscopy Analytical and Bioanalytical Chemistry (2015) 407, 7111-7122. (Open Access
  • Chan K. L. A., Kazarian S. G. FTIR spectroscopic imaging of reactions in multiphase flow in microfluidic channels Analytical Chemistry 84(9) (2012) 4052-4056 (doi)
  • Glassford S., Chan K. L. A., Byrne B., Kazarian S. G. Chemical imaging of protein adsorption and crystallisation on a wettability gradient surface Langmuir   28 (2012) 3174-3179
  • Chan K. L. A., Gulati S., Edel J. B., de Mello,A. J., Kazarian, S. G. "Chemical imaging of microfluidic flows using ATR-FTIR spectroscopy" Lab on a Chip 9(2009) 2909-2913.
  • Kazarian, S. G., Chan K. L. A. "Micro- and macro-attenuated total reflection Fourier transform infrared spectroscopic imaging (Focal Point article)" Applied Spectroscopy 64 (2010) 135A-152A (doi) (open access)
  • Chan K. L. A., Niu X,, de Mello,A. J., Kazarian, S. G. "Rapid prototyping of microfluidic devices for integrating with FTIR spectroscopic imaging" Lab on a Chip 10(2010) 2170-2174.
  • Chan K. L. A., Govada L., Bill, R. M., Chayen, N. E., Kazarian S. G. ATR-FTIR spectroscopic imaging of protein crystallization Analytical Chemistry 81 (2009) 3769-3775.
  • Kazarian S. G. "Enhancing high-throughput technology and microfluidics with FTIR spectroscopic imaging" Anal. Bioanal. Chem. 388 (2007) 529-532. 
  • Chan K. L. A., Kazarian S. G., Vassou D., Gionis V., Chryssikos G. D. "In situ high-throughput study of drug polymorphism under controlled temperature and humidity using FTIR spectroscopic imaging" Vib. Spectrosc. 43 (2007) 221-226. 
  • Chan K. L. A., Kazarian S. G. "ATR-FTIR spectroscopic imaging with expanded field of view to study formulations and dissolution" Lab on a Chip 6 (2006) 864-870.
  • Chan K. L. A., Kazarian S. G. "High Throughput Study of PEG/ibuprofen formulations under controlled environment using FTIR imaging" J. Comb. Chem. 8 (2006) 26-31.
  • Chan K. L. A., Kazarian S. G. "FTIR Imaging for High-Throughput Analysis of Pharmaceutical Formulations" J. Comb. Chem., 7 (2005) 185-189.