Enhanced FTIR Spectroscopy of Biological Liquid Samples Confined Between Ge Hemispherical ATR Element and Al Coated Glass Substrate

We demonstrated a modified SEIRA approach, which can be easily utilized for mea surements of various liquid samples, including biological specimens. In addition, the possibility of using Al, known as non-typical SERS metal, for enhancement of IR signal was demonstrated with enhancement factor varied from 10 to 1000 for different IR bands. This method is easily accessible and does not require deposition of metal on expensive internal reflection element. Instead metal can be deposited on cheap glass substrate, which is used in the suggested set up as top window during SEIRA measure -ments. Finally, the thickness of the analyte can be controlled by the position of the pressure applicator when using a grazing angle ATR attachment.


Introduction
It is well known that Infrared (IR) spectroscopy is a widely used as an effective analytical technique for investigation of molecular structure and composition as well as intermolecular interactions of specific and non-specific nature in various substances. However, this technique has some limitations in particular when applied to minute trace concentration of substances as well as for the aqueous solution of biological species and for single molecule detection.
Over last few decades different methods of Fourier Transform Infrared (FTIR) spectroscopy were developed to increase the sensitivity of this technique specifically for characterization of different biological samples (including proteins, DNA and RNA, cells and tissue specimens, etc.) as well as a single molecule detection [1][2][3][4][5]. Laboratory analysis of biological fluids is of great importance for medical diagnostics and health science in general.
Mid-infrared spectroscopy, using the Attenuated Total Reflection (ATR) technique, showed great potential for the quantitative multicomponent analysis of various body fluids [5][6][7][8][9]. There are many benefits in using Mid-infrared spectroscopy coupled to ATR including: trace samples can be measured; minimal sample preparation is required; no additional reagents or disposables are needed; continuous measurements and bulk measurements are easy to carry out; the concentrations of several analytes can be measured simultaneously from a single spectrum. optical internal reflection elements "(named ATR or IRE), such as hemispherical prism and trapezoidal elements, made from materials with high refractive index were developed for these methods. This resulted in significant enhancements of the infrared signal due to the grazing angle incidence (and reflection) and multiple reflections in IR Reflection Absorption Spectroscopy (IRRAS) techniques [1][2][3]. Starting from work of Hartstein et al. [10] a new modification of IRRAS method was proposed, namely by using a combination of IR ATR technique with a deposition of metallic underlayer (on the top of IRE) or overlayer on the top of the analyte in order to achieve surface enhancement of IR signal similar with Surface Enhanced Raman Spectroscopy (SERS).
Originally this technique was developed for polymer layer (or solid cast layer) investigation, therefore there was no problem to deposit a metallic layer on top of the ATR element or on top of the polymer layer [1,2]. As for the metal layer, firstly gold and silver (rarer copper) were used for the deposition similar to the initial developments of SERS technique. That is mainly due to a superior surface enhancement factor for Au and Ag [11]. However, later it was shown that some non-typical SERS metals (like Pt, Pd, Rh, Ni, Fe [1]) also demonstrate quite significant enhancement of the infrared signal. In analogy with SERS technique this new method was named as a SEIRA (surface enhanced infrared absorption).
The principles behind SEIRA [12,13] can be found elsewhere and will not be discussed here in details. A clear explanation for the mechanism of the absorption enhancement remains elusive but the most prominent theories include the electromagnetic [14] and chemisorption [15] theories. In reality it is probable that both mechanisms contribute to the absorption enhancement. It is also recognised that the morphology of the metal films affects the enhancement factor [16]. Similar approaches utilising SEIRA and ATR have been used to analyse biological fluids [17,18], environmental pollutants [19], organic semiconductor behaviour [20], as well as protein structures and interactions [21]. Therefore, there are numerous applications for this versatile method in many fields of science, medicine and engineering.
The main aim of this work is to develop a modified IR-ATR spectroscopy approach and to demonstrate the feasibility of the use of this technique for the qualitative analysis of biological molecules in solutions (i.e. in the liquid form). Therefore, in this report we demonstrate a modified SEIRA approach to enhance the infrared We used Al as a metal for deposition onto thick (~ 4 mm) glass substrate or in some cases onto widely available microscope glass slices (of ~ 1 mm thick). We believe that Al layer provides sufficient surface enhancement, is easy to deposit and is easy attainable. The low cost and simple experimental setup make our method very attractive for a prospective commercialization and a widespread use.

Experimental: Sample Preparations
We analyzed a number of biological samples, such as various DNA, Glucose, Bilirubin, Creatine, etc. These substances are found in a various body fluid such as blood and urine. Monitoring of the concentrations of these substances is vital to the treatment and diagnostics of many pathological conditions such as cancer [23], liver cirrhosis, hepatitis [24], diabetes, renal disease [25], etc. However, in this report we included only four samples from the above list due to space limitations for a Short Communication   Figure 1a (see also [22] for details). A thin film of analyte was obtained by liquid compression between the Ge ATR prism and a ~ 4 mm thick Al coated glass substrate (see Figure 1a). In some cases, thick (~1 mm) mi-

Results and Discussion
The absorption spectra obtained for four biological samples, In addition to that, other vibrational bands are observed at 1396, 1339, 1303, 1242, 903 and 808 cm -1 in good correspondence with known IR bands of BSA from literature [26][27][28]. We note that the observed in this case a derivative-like shape of the enhanced IR spectrum is quite often seen in SEIRA spectra (see for example Refs. [1,29,30]). This is due to a dispersive effect which resulted in some molecular vibrations show up as negative absorption peaks, namely as reflectance maxima [1,29,31]. These spectral features can be simulated with Electromagnetic (EM) calculations based on Bergman model of Effective-Medium Approximation (EMA) [1,29].
The spectra registered for Salmon DNA (type of B-DNA) are shown in Figure 2b. In this case the IR spectrum appeared with all bands in absorption maxima and a significant enhancement (for some bands reached nearly of three orders of magnitude) is also obtained. The peak positions marked in Figure 2b agree with previously measured IR spectra in Refs [32][33][34]. We should mention here that some IR bands registered for Salmon DNA in KBr tablets Finally, the thickness of the analyte can be control by the position of the pressure applicator. As a preliminary interpretation we would like to note that few mechanisms can be responsible for the IR absorption enhancement in these experiments. This can be due to electromagnetic enhancement as a result of the local field effect, due to multiple reflection of the incident light between two elements with high refractive index and finally due to adsorbance of the investigated analyte's molecules to the porous Al film structure.
During our experiments we noticed that the enhancement of IR absorption (as well as relative intensity of different IR bands) depends on morphology of deposited Al film as well as on thickness of the liquid analyte. Obviously when film becomes thinner and thinner, most of the substance will be left inside the Al pores and chemisorbance effect will prevail over other mechanisms of the enhancement. Note: The black lines correspond to the measurements taken from free liquid drop deposited onto Ge IRE, while red lines correspond to the case of liquid confined between Ge IRE and Al coated glass substrate. The double band in the region 2300-2400 cm -1 labelled with asterisk belongs to uncompensated CO 2 from the air.
It is clear that more experimental and theoretical investigations using this approach are necessary in order to establish a complete quantitative analysis of biological samples (as well as any liquid sample) with weak IR bands. This short report presents only demonstration of the effect and a modified approach to SEIRA technique using grazing angle ATR technique and top substrate (made from dielectric or semiconducting material) coated by thin metal film. To conclude, we have developed a novel cheap and easy to use SEIRA method utilizing surface enhancement for the potential measurement of biochemical samples. This method overcomes some of the drawbacks of IR spectroscopy and provides an amplification of the analyte bands which allowed for detection of amido bands in the bulk water bands. However, further detailed studies will be necessary to understand mechanisms involved and optimize this technique. We believe that this method might find a range of important applications including analysis of very diluted biochemical and biomedical samples that will be of great value for medical diagnostics and forensic sciences.