Chemical imaging of microfluidic flows using ATR-FTIR spectroscopy K. L. Andrew Chan, a Shelly Gulati, bc Joshua B. Edel, bc Andrew J. de Mello b and Sergei G. Kazarian * a Received 14th May 2009, Accepted 9th July 2009 First published as an Advance Article on the web 22nd July 2009 DOI: 10.1039/b909573j Elucidating the chemical composition of microfluidic flows is crucial in both understanding and optimising reactive processes within small-volume environments. Herein we report the implementation of a novel detection methodology based on Attenuated Total Reflection (ATR)–Fourier Transform Infra-Red (FTIR) spectroscopic imaging using an infrared focal plane array detector for microfluidic applications. The method is based on the combination of an inverted prism-shape ATR crystal with a poly(dimethylsiloxane)-based microfluidic mixing device. To demonstrate the efficacy of this approach, we report the direct measurement and imaging of the mixing of two liquids of different viscosities and the imaging and mixing of H 2 O and D 2 O with consecutive H/D isotope exchange. This chemically specific imaging approach allows direct analysis of fluid composition as a function of spatial position without the use of added labels or dyes, and can be used to study many processes in microfluidics ranging from reactions to separations. Introduction As microfluidic technology continues to demonstrate its tremendous potential as a fundamental analytical tool in the chemical and biological sciences, the need to develop new robust, fast and sensitive detection techniques for chemical species detection intensifies. These detection methods must consider and account for the physical realities of microfluidic flows such as reduced diffusion lengths, confined geometries, and planar substrates 1 whilst preserving or enhancing the rapid, high- throughput, and small volume benefits and the ease of use of microfluidic systems. Small volume detection within microfluidic systems is most commonly performed using optical methods. Their widespread adoption is unsurprising, since glass and polymers (the substrate materials of choice) are normally trans- parent in the visible, near-UV and near-IR regions of the elec- tromagnetic spectrum and thus allow sample contained within to be probed via the absorption, emission or scattering of radiation. More specifically, fluorescence-based methods are the most prevalent due to the exquisite sensitivity and selectivity of emis- sion spectroscopy. 2,3 Furthermore, much of the early interest in microfluidic systems was driven by the need to create high- throughput tools for nucleic acid analysis. Importantly, estab- lished methods for fragment sizing, sequencing and DNA amplification all incorporate fluorescent chemistries to facilitate detection, making their transferral to planar chip formats facile. However, an obvious disadvantage of fluorescence detection methods relates to the need to label molecules not containing an appropriate fluorophore. As a result, there has been interest in integrating label-free detection techniques with microfluidics. Such techniques include infrared spectroscopic detection, 4–6 Raman spectroscopy, 7–9 and surface-enhanced Raman spectros- copy (SERS). 10,11 These techniques are advantageous for chem- ical analysis because they are label-free and have a broad applicability. However, these methods are usually less sensitive than fluorescence-based approaches because they are either absorption techniques or relay on inefficient Raman scattering and because several of them need detectors that are far less efficient than UV/vis detectors or because the absorptivity of the spectral features is low. Nevertheless, the inherent chemical specificity of vibrational spectroscopic methods and their quan- titative nature are often essential for studies of many systems, such as flows in microfluidic devices. Recently, the application of Fourier transform infrared (FTIR) spectroscopic imaging to microfluidics has been proposed. 12 The possibility of obtaining chemical and spatial information from dynamic systems in microfluidics offers new opportunities in this field. FTIR imaging using a highly sensitive multi-pixel detector or focal plane array (FPA) is recognised as a powerful material characterization method 13,14 and has been applied to a broad range of studies in the past decade. The main advantage of this imaging method is that while mid-IR spectra provide a wealth of chemical information, the thousands of detector pixels, each simultaneously measuring a spectrum from a specific location of the sample, provides the possibility of obtaining a chemical snapshot of a system in a matter of minutes or seconds. 15 We have recently developed a number of applications utilising inverted prism-shape crystals in macro Attenuated Total Reflection (ATR) mode with optimised spatial resolution and fields of view. 16,17 Measurement in an ATR mode also has the significant advantage of overcoming one of the primary diffi- culties in FTIR spectroscopy, namely the strong absorption of water in the infrared region obscuring other weaker spectral bands of interest. 18 The ATR mode employs a high refractive index infrared transparent crystal to create an internal reflection where the infrared light interacts, as an evanescent wave, in the lower refractive index medium (the sample). 19 The depth of this a Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, United Kingdom. E-mail: s. kazarian@imperial.ac.uk; Tel: +44 (0)20 7594 5574 b Department of Chemistry, Imperial College London, Exhibition Road, South Kensington, London, SW7 2AZ, United Kingdom c Institute of Biomedical Engineering, Imperial College London, Exhibition Road, South Kensington, London, SW7 2AZ, United Kingdom This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 2909–2913 | 2909 PAPER www.rsc.org/loc | Lab on a Chip