Infrared spectroscopic analysis of human interstitial fluid in vitro and in vivo using FT-IR spectroscopy and pulsed quantum cascade lasers (QCL): Establishing a new approach to non invasive glucose measurement

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Abstract

Interstitial fluid, i.e. the liquid present in the outermost layer of living cells of the skin between the Stratum corneum and the Stratum spinosum, was analyzed by Fourier transform infrared spectroscopy and by infrared spectroscopy using pulsed quantum cascade infrared lasers with photoacoustic detection. IR spectra of simulated interstitial fluid samples and of real samples from volunteers in the 850–1800 cm−1 range revealed that the major components of interstitial fluid are albumin and glucose within the physiological range, with only traces of sodium lactate if at all. The IR absorbance of glucose in interstitial fluid in vivo was probed in healthy volunteers using a setup with quantum cascade lasers and photoacoustic detection previously described [11]. A variation of blood glucose between approx. 80 mg/dl and 250 mg/dl in the volunteers was obtained using the standard oral glucose tolerance test (OGT). At two IR wavelengths, 1054 cm−1 and 1084 cm−1, a reasonable correlation between the photoacoustic signal from the skin and the blood glucose value as determined by conventional glucose test sticks using blood from the finger tip was obtained. The infrared photoacoustic glucose signal (PAGS) may serve as the key for a non-invasive glucose measurement, since the glucose content in interstitial fluid closely follows blood glucose in the time course and in the level (a delay of some minutes and a level of approx. 80–90% of the glucose level in blood). Interstitial fluid is present in skin layers at a depth of only 15–50 μm and is thus within the reach of mid-IR energy in an absorbance measurement. A non-invasive glucose measurement for diabetes patients based on mid-infrared quantum cascade lasers and photoacoustic detection could replace the conventional measurement using enzymatic test stripes and a drop of blood from the finger tip, thus reducing pain and being a cost-efficient alternative for millions of diabetes patients.

Highlights

► The composition of interstitial fluid was studied by ATR-FT-IR spectroscopy. ► Interstitial fluid was simulated as a combination of glucose and albumin. ► Glucose was measured in interstitial fluid by photacoustic spectroscopy.

Introduction

Perhaps the most frequently performed bioanalytical measurement is the determination of glucose in blood determined by diabetes patients from a drop of blood taken from the finger tip; approx. 6 million type II diabetes patients perform around 10–15 million measurements per day only in Germany. This “invasive” measurement is considered painful by patients and is costly for the health insurances because of the enzymatic test stripes used. A true “non-invasive” device to measure spot blood glucose which could replace the finger prick is not available to the patient, in spite of the many different approaches by various research groups. None of the techniques has ever managed to successfully reach the market, presumably because of the low specificity of most of the physical or chemical principles used [1], [2]. Among these are near infrared (NIR) spectroscopy of the glucose overtones [3], [4], skin impedance spectroscopy [1] or optical coherence tomography, OCT [1], [5]. Semi-invasive technologies have been proposed and possibly offer more specific information on blood glucose [6], [7]. However, they require frequent surgical intervention, which may be the reason that they are not easily accepted by patients.

Mid-infrared spectroscopy (MIR) in the range of approx. 800–1300 cm−1 is very specific in the identification and quantification of glucose and other body fluids [8], [9]. The absorption of glucose in aqueous solution within this range exhibits maxima and shoulders near 1152, 1106, 1080, 1036 and 992 cm−1, all of them arising from coupled ring–C–O–H stretching and bending modes. This infrared glucose fingerprint can be used to quantify glucose concentration in blood or in blood plasma samples and has been successfully developed by us towards a reagent-free point-of-care measuring system for glucose and for several other blood parameters [9]. Infrared spectroscopy in this wavelength range, although highly specific for glucose, has the drawback that it reaches only the upper layers of skin, i.e. the Stratum corneum and the Stratum spinosum, with most of the IR energy deposited in a depth less than 50 μm. Within this range, blood vessels are not in reach.

The liquid present in the compartment within the epidermal layers containing living cells, termed epidermal interstitial fluid, presents an alternative. This fluid compartment probably represents a significant proportion of 20% of the volume in the epidermal layer [1]. Although hardly characterized with respect to its constituents, it is known that the glucose content of interstitial fluid approximates that of blood (around 80–90% of blood glucose) [10]. Moreover, it is know that the glucose variation in interstitial fluid follows that of blood with only minimal delay (around 5–10 min, depending on the skin part probed), which is a necessary prerequisite for a reliable measurement required by the diabetes patient. Indeed, a long delay time between glucose concentrations appearing in a body fluid which may be easily accessible, such as tears, and blood glucose, presents a knock-out argument for a measuring method.

Interstitial fluid (ISF) thus provides a potential access for mid-infrared spectroscopy. It emerges from skin upon surface abrasions. When a lesion is produced in the skin by cooling, heating or by friction, the binding among the cells between the S. corneum and the S. spinosum is weakened provoking a local depression area around the lesion zone. The relatively high pressure of the surrounding causes accumulation of interstitial fluid under the lesion; then a bulla is produced on the skin.

In order to obtain a solid basis for a true non-invasive glucose measurement, we have analyzed here the composition of ISF using FT-IR spectroscopy of model interstitial fluid samples mixed from albumin, glucose and lactate, up to now considered the significant constituents of ISF. We have compared these model spectra with the IR spectra of real ISF samples obtained from volunteers. In a second attempt, we have studied the IR absorption of interstitial fluid in vivo at volunteers. Pulsed IR radiation at two wavelengths relevant for glucose, near the spectral maxima of glucose reported above, was obtained from quantum cascade lasers. The detection of IR absorbance in skin was performed with a photoacoustic cell in contact with the skin. In order to obtain variable glucose concentrations for the volunteers, standardized oral glucose tolerance tests (OGTs) were performed.

The results help to identify and quantify the constituents of interstitial fluid and demonstrate the glucose-dependent absorbance of skin for specific mid-IR wavelengths as a basis for a non-invasive yet specific glucose measurement.

Section snippets

Models for epidermal interstitial fluid

Model samples (63) for interstitial fluids were produced by mixing different potential components of interstitial fluid, such as glucose, albumin and sodium lactate in physiological buffer solution (PBS). For each component, three concentrations within the physiological range were chosen. The concentration values for each component are shown in Table 1. The 63 simulated interstitial samples are combinations of these concentration values for each component.

Real interstitial fluid samples

The real interstitial fluid samples

In vitro infrared spectroscopy of interstitial fluid models and of real interstitial fluid

The infrared absorption spectra of glucose, albumin, and sodium lactate within the fingerprint region of glucose (850–1250 cm−1) are shown in Fig. 1.

The absorption bands of these three components overlap in this spectral range and some interference is expected if glucose is to be quantitatively determined from infrared spectra of ISF. In the case of glucose, the peaks arise from coupled stretching and bending modes of the C–O–H group [8]. The interference due to the absorption peaks of sodium

Outlook

The advent of quantum cascade lasers probably starts a new era of infrared analysis in biomedicine. Spectroscopic analysis of body fluids, up to now, is based on laboratory instruments such as an FT-IR spectrometer. Future developments may be based on QCLs selectively tuned to specific wavelengths. In the case of glucose, the high pulse energy of QCLs opens the possibility for a non-invasive measurements through skin as proposed here. Finally, external cavity QCLs tunable over >200 cm−1 which

Acknowledgements

The authors would like to acknowledge valuable discussions with Dr. Georg Wille, Institut für Biophysik, on the protein analysis of ISF. The technical assistance of Hans Werner Müller for the polyacrylamide gel electrophoresis is greatly acknowledged. We are grateful for the help of Ernst Winter for sophisticated mechanical engineering.

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