ATR-FTIR is seen as a quick and simple technique for IR analysis. Its low depth of penetration into the sample makes the task of analysing undiluted and minimally prepared samples extremely easy: as long as there is firm contact between the ATR reflection element and the sample, good spectra can be obtained.
Of course, like all common IR spectroscopy techniques, samples must be applied, measured, and cleaned off the accessory one at a time. This is fine when we want to measure the sample in its as-provided state, but the ATR technique in particular affords the ability to work with more complex sampling procedures and with larger numbers of tested materials. Hydrated films that have been evaporated on the crystal, electrochemical experiments using surface enhanced infrared absorption spectroscopy (SEIRAS) and other experiments that involve pre-treatment or functionalisation of the ATR element.
ATR-FTIR analysis of biological samples, including bloods & serums, liposomes & cellular vesicles, and proteins, often involve an evaporation step to concentrate the components of the mixture under investigation [1-2]. Without this step these analytes would be below the limits of detection by ATR. The sample preparation step ties up the ATR, and forces experiments to be conducted sequentially one at a time owing.
Review of sampling procedures for ATR-FTIR of biological samples
Dubailleul et al. [3] have investigated the use of yeast cells to prepare amino acids in a batch scale bioreactor. In the authors method the sample to be analysed was first dropped onto the diamond internal reflection element of a Specac Golden Gate ATR and then dried under a flow of nitrogen. FTIR was then used to analyse the amino acid product and can be used as a quick tool to verify the amino acid has formed with the correct folding structure.
Khoshmanesh et al. [4] have reported the potential to use ATR as a method for rapid detection of malaria parasites in blood samples. In their method they first dispersed blood samples in methanol before dropping onto the diamond internal reflection element of a Specac Golden Gate ATR and allowing it to dry with assistance from a heat source. Spectra were then collected, reprocessed using a second derivative and then analysed by chemometric methods (principal component analysis and partial least squares analysis). Detection limits were less than 1 parasite per μL of blood demonstrating the potential for this technology to be deployed at point of care facilities.
Hermans et al. [5] have investigated the components of oil paint to investigate spectra recorded from historic oil paintings. In their investigation mixtures of ionomers (zinc or lead sorbate) and linseed oil was dried onto a glass slide, and after 7 weeks ATR spectra were obtained. In their work they were able to demonstrate that the metal carboxylates were likely to be distributed throughout the polymer phase of the paint rather than present at the pigment-polymer interface. This important work sheds light on the chemical processes that occur within works of art and ultimately gives conservators vital information to work to preserve these culturally important objects. Oil paints dry via a polymerisation crosslinking reaction in the oil (commonly Linseed oil).
Arrow to the future?
Arrow is the world’s first consumable ATR solution designed to innovative ATR workflows. Its low-cost nature opens the door to batch sample preparation in which hundreds of samples are prepared at once. In this section we will review the existing literature to demonstrate how Arrow could be used to speed up experiments with an involved sample preparation step.
In the study by Khoshmanesh et al, for example, Arrow would have offered two advantages over conventional ATR spectroscopy: decoupling the drying step from the ATR and secondly eliminating any sample cross contamination concerns.
Tying up an ATR crystal for months on end (as was the case for the linseed oil samples) is also clearly not viable. The authors were limited to studies of the paint after the sample had set. Arrow would enable these long-term reactions to be monitored in real time with multiple samples run concurrently as demonstrated in our Application note [6].
Arrow can also be used to functionalise the surface prior to deposition of a sample on the surface for analysis. For instance, Ausilli et al. have reported studies on Ge ATR where the surface of the crystal is pre-treated by washing the crystal with an alkaline detergent, rinsed with deionised water and then further cleaned with methanol and chloroform. This surface treatment ensures the crystal is hydrophilic and as a result when an aqueous protein is dried onto the crystal promotes the formation of a membrane film with high long-range order. Different films with α-helix or β-sheets were studied using polarised ATR with the differences linked to orientation of the films on the surface.
Another potential application for Arrow is through the construction of microfluidic “lab on a chip” structures on top of the silicon element. Chan et al. have reported one such study performed on a ZnSe crystal. In their study the authors mixed H2O and D2O samples in the microfluidic channels and then used an FPA imaging detector to monitor the mixing to understand the fluid dynamics of their system.
The above examples are just some of the potential areas where Arrow could be deployed to improve workflows by facilitating batch sample preparation of hydrated films. Arrow offers an attractive scalable alternative to conventional ATR experiments avoiding the need to lock down the spectrometer for complex sample prep routines.
References
[1] Mihály et al. Biochimica et Biophysica Acta 1859 (2017) 459-466 DOI: 10.1016/j.bbamem.2016.12.005
[2] Natalello et al. Journal of Biological Chemistry Vol. 291, No. 18, pp 9678-9689 (2016) DOI: 10.1074/jbc.M116.720573
[3] Debailleul et al. Microbial Cell Factories 2013, 12:129 DOI: 10.1186/1475-2859-12-129
[4] Khoshmanesh et al. Analytical Chemistry 2014, 86, 4379-4386 DOI: 10.1021/ac500199x
[5] Hernans et al. RSC Adv. 2016, 6, 93363 DOI: 10.1039/C6RA18267D
[6] Specac Application Note: AN21-05 “Watching paint dry”
[7] Ausili et al. Biomedical Spectroscopy and Imaging 4 (2015) 159—170, DOI: 10.3233/BSI-150104
[8] Chan et al. Lab on a Chip, 2009,9, 2909-2913. DOI: 10.1039/B909573J