Comparison of different infrared measurement techniques in the clinical analysis of biofluids

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[ lo] E. Sanchez and B.R. Kowalski, J. Chemom., 2 (1988) 265. [ 111 E. Sanchez and B.R. Kowalski, Anal. Chem., 58 ( 1986) 496. [ 121 A.K. Smilde, Chemom. Intell. Lab. Syst., 15 (1992) 143. [ 13 ] J. Ohman, P. Geladi and S. Wold, J. Chemom., 4 (1990) 79. [ 141 R. Tamer, A. Izquierdo-Ridorsa, R. Gargallo and E. Casassas, Chemom. Intel. Lab. Syst., 27 (1995) 163. [ 151 R. Tauler, B.R. Kowalski and S. Fleming, Anal. Chem., 65 ( 1993) 2040. [ 16 ] R. Tauler, A.K. Smilde, J.M. Henshaw, L.W. Burgess and B.R. Kowalski, Anal. Chem., 66 ( 1994) 3337. [ 171 S. Lacorte, D. Barcelo and R. Tauler, J. Chromatogr. A, 697 ( 1995) 345. [ 18 ] R. Tauler and D. Barcelo, Trends Anal. Chem., 12 (1993) 319. [ 191 H.R. Keller, D.L. Massart and J.O. De Beer, Anal. Chem., 65 (1993) 471.

[ 201 E.R. Malinowski, Factor Analysis in Chemistry, Wiley, New York, 2nd edn., 1991. [ 211 E.R. Malinowski, Anal. Chem., 49 (1977) 612. [ 221 S. Wold, Technometrics, 20 (1978) 397. [ 23 ] R. Tauler, E. Casassas and A. Izquierdo-Ridorsa, Anal. Chim. Acta, 248 ( 1991) 447. [ 241 R. Tauler and E. Casassas, Chemom. Intell. Lab. Syst., 14 (1992) 305. [ 25 ] F. Cuesta Sanchez and D.L. Massart, Anal. Chim. Acta, 298 (1994) 331. [ 261 W.W. Yau and J.J. Kirkland, J. Chromatogr., 556 (1991) 111.

R. Gargallo and R. Tauler are at the Departament de Quimica Analitica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona, Spain. F. Cuesta-Sgnchez and D.L. Massart are at ChemoAC, Vrije Universiteit Brussel, Laarbeeklaan 103, B- 1090 Brussels, Belgium.

Comparison of different infrared measurement techniques in the clinical analysis of biofluids J. Wang, M. Sowa, H.H. Mantsch Winnipeg,

Canada

A. Bittner, H.M. Heise* Dortmund,

Germany

An infrared spectroscopy-based multicomponent assay of biofluids such as whole blood, urine, and synovial fluid holds considerable promise for the clinical laboratory. The methodology is reagentless, fast and readily automated. Infrared spectroscopic analysis is not hindered by the physical state of the sample and measurements can be made in a variety of instrument configurations. In this review we present the spectra of physiologi*Corresponding author. 01659936/96/$15.00 PIISO165-9936(96)00037-4

cal samples measured as liquids and dried films using transmission-, attenuated total reflection-, photoacoustic- and diffuse reflectance infrared spectroscopy. The advantages and limitations of each of these techniques are discussed in the context of developing a routine clinical analysis method for biofluid samples.

1. Introduction Traditionally, infrared (IR) spectroscopy has been one of the most important physical methods in the chemical laboratory as it plays an important role in the elucidation of structures and the identification of organic and inorganic compounds. The quantitative analysis of samples is straightforward 01996 Elsevier Science B.V. All rights reserved

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and can be reliably made down to the picogram level. An example is the analysis of organometallic complexes of biological derivatives with bench-top instrumentation [ 11. This methodology now provides the basis for a new immunological test. Furthermore, the IR spectroscopic analysis is not affected by the physical state of the sample: gaseous, liquid and homogeneous or inhomogeneous solid samples all can be conveniently studied. An example of the latter is the IR microscopic analysis of gallstones [ 2 1. To date, applications of IR spectroscopy in clinical chemistry have been scarce. An early application was the analysis of urinary calculi which used the old KBr pellet technique [ 3 1; more advanced investigations were carried out using multivariate data analysis [ 41. Recently, there is a growing interest in medical applications of IR spectroscopy [ 5,6]. IR pathology is becoming increasingly important, since diseases are manifested by changes in the composition of body fluids and tissues which can be elucidated by the wealth of information contained in their IR spectra. Classification of biomedical samples is helpful to physicians, providing a non-subjective aid in the diagnosis of the disease state, and even the potential for staging the disease. This is a pattern recognition approach. Quantitative analysis, on the other hand, provides several clinically relevant parameters, e.g. in blood, that may be used to indicate the metabolic status of the patient. IR spectroscopy offers a number of advantages for the analysis of body fluids. Unlike most colorimetric or electrochemical /enzymatic clinical chemistry assays, the IR spectroscopic analysis is applicable to a variety of biofluids: whole blood, plasma or serum, liquor, synovial fluid, aqueous humour, saliva and urine. The methodology is fast and readily automated, making it suitable for use as a routine analyzer in the clinical chemistry laboratory. IR spectrometers can be made to be compact, rugged, relatively inexpensive and user independent making the technology capable of point-of-care operation in the emergency room, critical care unit, in the general practitioner’s office or potentially even as a home monitoring device. Since no reagents or other consumables such as electrodes are required, IR spectroscopy has a low operational cost. Yet, the most attractive advantage of the method is the potential for a rapid multicomponent analysis to be carried out from a single measurement (spectrum), once the methodology has been calibrated.

287

It has already been demonstrated that the multicomponent analysis of several blood substrates in using attenuated total human EDTA-plasma, reflectance ( ATR) mid-infrared ( mid-IR) spectroscopy, can be achieved with a precision well within the clinical acceptance tolerance limits [ 7,8]. Other IR spectroscopic studies were concerned with the spectral range best suited for a quantitative analysis. Infrared bands in the nearIR region are combination and overtone vibrations arising from the C-H, O-H and N-H moieties of molecules whereas the bands which dominate the mid-IR region are fundamental molecular vibrations of all functional groups. Thus, the near-IR and the mid-IR regions carry a different information content. Nevertheless, near-IR assays of such substrates as total protein, total cholesterol and triglycerides, compare well in performance with those which use the mid-IR range [ 6,9]. A recent example of a successful near-IR multicomponent analysis of compounds is the assay of urea and creatine in urine [ 10 1. Quantitative IR spectroscopic analysis is based on the Lambert-Beer law. Liquid samples have generally been measured in fixed-pathlength transmission cells. Since a quantitative analysis usually achieves its best performance with the lowest noise over a rather small absorbance the transmission pathlength is usually dictated by the absorption characteristics of the liquid sample matrix. Due to the high water absorptivities in the mid-IR, aqueous samples are limited to pathlengths of less than 30 pm. Routinely, filling cells of such short pathlengths is difficult and the situation is exasperated by the highly viscous nature of certain biofluids. Another requirement for quantitative biofluid analysis in the mid-IR range is the use of an IR transparent window material that is compatible with the water matrix. Materials such as CaF2, BaF2 or ZnSe are necessary. These materials, however, possess a rather high refractive index compared to that of the aqueous biofluid, so that interference fringes in the baseline, due to multiple reflections within the cell, interfere with the analysis of compounds of low concentrations. In the mid-IR, the ATR technique is a very convenient technique for measuring aqueous fluids with the same high level of reproducibility that can be achieved with flow-through transmission microcells. However, the optical sample pathlength is wavelength dependent and can be varied either by using ATR crystals with a different refractive index, by changing the reflection angle, or by

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applying multiple reflections. Another technique, which is ideally suited to optically ‘thick’ solid samples, is photoacoustic spectroscopy (PAS). In PAS, the optical sample pathlength also depends on several measurement parameters and certain thermal properties of the sample. Measurement of aqueous fluids in the near-IR is almost exclusively carried out in transmission, whereas the diffuse reflectance technique is standard for studying highly scattering solids. Spectroscopy in the long-wavelength near-IR ( 1800-2500 nm) range usually requires a cell pathlength of less than 1 mm. For viscous fluids this can be problematic, but micro flow-through cells with volumes similar to that used in mid-IR studies ( = 25 ~1) can be constructed to address this problem. However, unlike in the mid-IR, near-IR transmission cells can be made from conventional glass or quartz window material. The cell pathlength in the short wavelength near-IR (800-1600 nm) range is about a factor of ten greater for physiological fluids than in the longer wavelength range, this is because the reduced water absorptivities observed in this range. The longer pathlength requirements are more convenient, but they also dictate a larger sample volume, which may not be readily available for certain biofluids. In addition, quantitative analysis in the short-wavelength near-IR region is usually less specific and less sensitive than comparable analyses in the longer wavelength near-IR regions. The IR spectrum of water shows a significant dependence on temperature and electrolyte concentration, which gives rise to additional variations in the spectra, if these parameters are not strictly controlled. This tightens the constraints for a reliable quantitative assay on aqueous samples by using IR spectroscopy. Improvements which would reduce these intrinsic limitations of aqueous liquid phase IR spectroscopy would be of value. The formation of films, dried from the physiological fluids, seems a promising alternative. Water, as well as other volatile components, is removed by exposing the sample to atmospheric air or placing it under a gentle vacuum. Removing the water from the fluid acts as an enrichment process that enables quantitation of compounds which previously would have been difficult to analyze due to their low concentration in the original fluid. In blood, for instance, uric acid which has a normal concentration range of around 0.25 mmol/l becomes an accessible analyte to IR spectroscopy, after removal of water from blood. In this report, we compare spectra of biofluids obtained by employing different IR measurement

techniques such as ATR, transmission, PAS and diffuse reflectance for the fluids as such and dried films derived from those fluids.

2. Experimental 2.7. Attenuated total reflection, transmittance and diffuse reflectance spectroscopy ATR spectra of whole blood and plasma were recorded using a Bio-Rad FTS-40 spectrometer equipped with globar source, KBr beamsplitter, and a mercury cadmium telluride (MCT) detector. The spectra were collected at 4 cm-’ resolution between 500 and 4000 cm-’ with 128 coadded single-sided Fourier transformed interferograms. A circular ZnSe ATR crystal with sin le reflectance, Q flat surface area of about 4.84 cm and angle of incidence of 8 = 45”, was used for both film and liquid phase ATR measurements. Aliquots of whole blood or plasma were applied to the crystal as thin layers, and allowed to dry under atmospheric conditions. Thin dry films formed within a few minutes. ATR spectra of fluids were recorded by placing a drop of fluid on the active area of the crystal. An O-ring retainer and a CaF2 cover slip prevented fluid evaporation during the measurement. To ensure measurement reproducibility, the area of fluid on the crystal surface exceeded the active area from which spectra were recorded. For comparison, similar ATR spectra were recorded on a Perkin-Elmer FT-IR Model 1750 spectrometer equipped with a DTGS detector and a flow-through micro-CIRCLE cell (Spectra-Tech, Warrington, UK). Transmission spectra of dry films obtained from whole blood and/or plasma were prepared in a similar manner as described for the ATR measurements. A 3 yl volume of whole blood or plasma was deposited onto a 25 x 2 mm ZnSe window and spread over the entire window surface. The window was placed into a custom-built sample holder with holes of 3 mm inner diameter to obtain the transmission mid-IR spectra of dried whole blood or plasma films. In a similar way, diffuse reflectance spectra were recorded from samples spread onto a metal-coated rough substrate of 1000 grade sand paper. A sputtered layer of gold approximately 100 nm in thickness was used to coat the sand paper substrate. Diffuse reflectance measurements were made with a Bruker IFS 113~ spectrometer using a custom-built accessory consisting of a

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lightpipe (to illuminate the sample) and an ellipsoidal mirror (to collect the diffusely reflected radiation) [ 111. Near-IR measurements were carried out using a Bio-Rad FTS-175 spectrometer equipped with a quartz beamsplitter and an InSb detector. Spectra were recorded at 32 cm-’ over the 4000-10000 cm-’ range with 512 coadded single-side Fourier transformed interferograms. Transmission cells of 0.5 and 1.0 mm pathlength were employed for the measurement of the fluids. For dried film measurements, about 20 ul of each biofluid was deposited onto a CaF2 window and dried for 10 min at atmospheric pressure.

thermal diffusion length probed in a PA experiment is related to the thermal properties of the sample, as well as to the modulation frequency of the incident radiation. At increasingly higher modulation frequencies, the time interval monitored in which a photothermal wave can diffuse from the bulk of a sample to the sample surface decreases. At a modulation frequency of o Hz, a PA signal that takes longer than 1 /W s to reach the surface, will be aliased into the PA signals arising from shorter thermal diffusion times. The diffusion of the photothermal wave is described by the thermal diffusivity, a:=-

2.2. Photoacoustic

PC

spectroscopy

Photoacoustic (PA) spectra were collected on a Bio-Rad FTS-60A (Cambridge, MA) spectrometer using an MTEC Model 200 photoacoustic cell (MTEC, Ames, IA). A high surface area carbon black sample (MTEC) was used as the phase reference. Prior to recording a spectrum, the cell was purged with dry helium for at least 5 min. Phasemodulated depth profiling experiments at 400 Hz phase modulation freyency were collected at a step speed of 8.0 x 1O- m/s and a phase modulation amplitude of two He-Ne laser fringes. A Model 099- 1446 phase demodulator accessory (Bio-Rad, Cambridge, MA) was used to collect both the in-phase and quadrature signals. Double-sided interferograms consisting of 3950 data points were sampled at 2.5316 urn collection intervals and averaged for 16 scans. These interferograms correspond to a 8 cm-’ spectral resolution. The interferograms were then zero filled twice to give a spectral data point spacing of 4 cm-‘. The Mertz multiplicative phase correction parameters used for the in-phase and quadrature channels of the carbon black reference spectrum were used to separately phase correct the in-phase and quadrature channels of the whole blood and plasma spectra. Magnitude (or power) spectra, M(Vi), were calculated from the phase corrected in-phase (IP(Vi)) and quadrature (OP(Vi)) components of the photoacoustic response. M(4)=J/IP(z#

+ 0P(fiJ2

k

(1)

Finally, single beam PA magnitude spectra were then ratioed against a carbon black background. As this technique is rather distinct, some details of PA spectroscopy are discussed. The maximum

(2)

where k is the thermal conductivity of the sample, p is the density, and c is the specific heat. The thermal diffusion length ( u) of a sample is defined as the distance across which a thermal wave dissipates to 1 /e of its initial value. This is generally considered to be the maximum depth below the surface from which absorbed radiation can evolve and still be detected as a PA signal. For a homogeneous medium, the thermal diffusion length is expressed by the Rosencwaig-Gersho equation [ 121: p=

2a /- W

(3)

where o is the angular frequency of the modulation of the incident radiation. Thus by changing the modulation frequency as well as by examining the phase behaviour of the PA response some depthdependent information on the sample can be achieved. This forms the basis behind PA depth profiling.

3. Practical considerations As already mentioned, the measurement of aqueous biofluid samples in the mid-IR with transmission cells is difficult due to the short pathlengths which are necessary because of strong water absorptivities. The situation is exemplified in Fig. 1, where the water spectrum is recorded using a 50 urn pathlength CaF2 cell. Although water compatible, CaF2 as an IR material has the disadvantage of being opaque below 900 cm-‘. As shown in Fig. 1A, there is only a small transmission window in the fingerprint region which can be used, for instance, in the quantitative analysis of the important metabolite glucose (see insert in Fig. 1B). The

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0.6

St:: ._ E, 0.4 ii C 0.:

3ioo

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5do

Fig. 2. ATR measurements acquired during the drying process of a film of whole blood. 0.75

Q) 0.50 ii 2 b 9 0.25

0.00

L-h

---..I I

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1600

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I

I

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Fig. 1. Infrared spectra of aqueous fluids measured with a 50 pm pathlength transmission cell with CaF2 windows. (A) Transmission spectrum of water; (B) absorbance spectrum of a reference serum (Seroquant-AU) sample as recorded against a water-filled cell; insert is the absorbance spectrum of an aqueous glucose solution with a concentration of 5 g/l (water spectrum was eliminated by scaled absorbance subtraction).

difference spectrum of serum from which the water absorption bands were subtracted is also shown in Fig. 1B. Thus, proper water subtraction widens the useable analysis window in the mid-IR fingerprint region from 1800 to 900 cm-‘. Only a small interval between 1700 and 1600 cm-‘, which contains the strong Hz0 bending vibration band at 1640 cm-‘, has to be omitted. The multiple reflections with which one is faced when using water-compatible transmission cells affect the quantitative evaluation of small absorbance signals [ 13 1. This can be avoided when using the ATR technique. ATR spectra are similar to transmission spectra except that penetration depth and therefore pathlength depends on wavelength, as well as on the reflection angle and the optical constants of the materials defining the interface at

which total reflection occurs. In this context, transmission measurements aid in defining an equivalent sample thickness derived from the absorbances seen in the ATR spectra. Fig. 2 illustrates the drying process of a blood sample as monitored by the ATR technique. In the beginning, the spectrum is dominated by the water absorption bands. However, spectral features arising from other blood constituents gradually appear as the water evaporates and the water band absorptions decrease in intensity. Fig. 3 shows ATR spectra of dried films from whole blood and plasma, whereby the plasma was obtained from the same whole blood specimen. For comparison, Fig. 4 shows the ATR spectra of whole blood and plasma where a scaled water absorption subtraction factor was applied to the original fluid spectra. Exact water compensation in ATR aqueous spectra is fraught with difficulties. This is exemplified in the comparison of Fig. 3 and Fig. 4 where the intensity ratios of the amide I and amide II bands at 1650 and 1540 cm-‘, respectively, differ markedly between the dried film spectra (Fig. 3 ) and the ATR difference spectra (Fig. 4).

1

T 0.1 A.U

L

I

Plasma i”W

I 3500

3000 2500 2000 1500 Wavenumber I cm-l

1000

Fig. 3. Whole blood and blood plasma film absorbance spectra as measured by single reflection ATR.

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3000

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2000

1500

1000

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Fig. 4. ATR spectra of whole blood and plasma and the corresponding difference spectrum as measured with a micro Circle-ceil.

The difficulty in achieving a single satisfactory water absorption substraction scaling factor arises from the wavelength and refractive index dependence of the penetration of the evanescent wave into the fluid. A general equation to describe the penetration depth I, of the radiation electrical field within a transparent sample for an ATR experiment is given as: 1, =

x 27m,(sin2H - $)‘I2

(4)

where h is the radiation wavelength, 8 is the radiation incident angle, yipis the refractive index of the crystal and II, = ( n,lnp) is the ratio of the refractive index of the sample and that of the crystal. Since the refractive indices of water, blood and plasma differ and also have a slightly different wavelength dependence, the wavelength-dependent penetration depths for these samples will differ slightly. In addition, the refractivity index of a biofluid will depend on relative concentrations of fluid constituents. These relative concentration ranges vary considerably under normal physiological conditions. For instance, haematocrit has a wide clinical range in whole blood. This will impact upon the scattering and refractive index of the fluid and thus the ATR penetration depth. Therefore finding a reliable and general water absorbance subtraction factor which is applicable across the entire mid-IR range for ATR biofluid spectra is problematic. This is evident from the comparison between difference spectra (Fig. 4) and the spectra of dried films (Fig. 3). For the difference spectra in Fig. 4, the HZ0 absorption band at about 2100 cm-’

was used to determine the water subtraction scaling factor. However, despite the difficulties in achieving a rigorous water absorption subtraction, a reagent-free multicomponent assay for several blood substrates based on a multivariate partial least-squares (PLS) calibration of mid-IR spectra using a micro-CIRCLE ATR cell achieved comparable performance to current discrete test clinical analyzers [ 7,8]. The ATR method carries strict requirements on the thicknesses of biofluid films which can be reliably used in quantitative analysis. Two complications arise when thick dry films are used in conjunction with ATR. In addition, because of varying sedimentation and adherence characteristics of the biofluid components to the ATR substrate, particularly the protein components, a concentration gradient of these components can potentially arise in the film which is deposited on the ATR crystal. In thick films this concentration gradient may exceed beyond the penetration depth of the ATR measurement. If this situation arises, the ATR measurements on the dried film will not be representative of the component concentrations in the original biofluid. Since the sedimentation and adherence characteristics of biofluids and in particular whole blood and plasma vary markedly within the normal physiology range, ATR measurements on thick dried biofluid films might be expected to demonstrate a high variability due to this phenomenon. Robust quantitative analysis using ATR in conjunction with dried biofluid films seems most promising when film thicknesses on the order of the penetration depth of the measurement or less are used. The relationship between the thickness of the probed surface layer and absorbance in FTIR/ATR spectroscopy has recently been investigated [ 141. The thickness of the original biofluid layer applied to the substrate is also crucial and needs to be kept constant in the dried film transmission measurements. Compared to dried film ATR spectra (Fig. 3), the dried whole blood transmission spectrum (Fig. 5, top trace) displays different relative intensities. This discrepancy highlights the wavelength-dependent penetration depth and thus intensities of the ATR measurements (see also Eq. 4). The strongly sloping baseline which appears in the dried film transmission spectrum of whole blood primarily arises from scattering due to the red blood cells (haematocrit) in the sample. As expected, this scattering is wavelength dependent and much more prominent at the shorter mid-IR

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wavelengths (higher wavenumbers). In addition to the effect of scattering, Fresnel reflection might influence the transmission spectra of dried films with a noticeable change in dispersion in the vicinity of strong absorption bands. This effect can be avoided by embedding the dried fluid in an IRtransparent KBr matrix (Fig. 5, bottom trace). However, the hygroscopic nature of KBr tends to make it difficult to eliminate water pick-up by the KBr powder during the pellet preparation procedure. The broadened OH stretching region ( = 3500 cm-’ ) in the spectrum of dried whole blood embedded into a KBr pellet (Fig. 5, bottom trace) compared to the dried film spectrum of whole blood (Fig. 5, top trace) is indicative of this problem. The reflection-absorption technique when used in conjunction with dried films has similar practical concerns as dried film transmission measurements. In the reflection-absorption method, a dried film of fluid was prepared on a gold-coated, diffusely reflecting substrate. The accessory used for measuring the reflectance can discriminate quite well against the Fresnel reflection without the need for embedding the material in a KBr matrix [ 111. The reflectance-absorption spectra of dried films of whole blood and serum are shown in Figure 6. Because the radiation passes through the sample twice, sample absorption is twice as large as compared to the corresponding transmission measurement. Thus, the control of dried film thicknesses is similarly crucial when using the reflection-absorption configuration, as compared to using dried film transmission measurements for quantitative analysis. With the reflection-absorption technique,

I

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500

Fig. 5. Whole blood absorbance spectra measured in transmission as a thin film and embedded in a KBr matrix.

L

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Fig. 6. Reflection-absorption blood plasma.

spectra of serum and

slight time-dependent spectral changes arising from film crystallization were observed upon removal of water [ 6 1. The PA technique is quite different in that it does not rely on the direct detection of reflected, scattered or transmitted radiation. The absorption of modulated radiation gives rise to a periodic pressure fluctuation leading to an accompanying photoacoustic response which is proportional to the amount of radiation absorbed by the sample. Thus, sample absorption can be indirectly determined by monitoring the PA response of the sample. Although PA spectroscopy is not limited by the physical state of the sample, the technique is most sensitive when monitoring gaseous samples. However, the ability to non-destructively depth profile solid samples is a very powerful practical aspect of PA spectroscopy. Recently, PA spectroscopy has been used to non-destructively explore the depthdependent heterogeneity in soft and calcified tissues [ 15-17 1. The potential for non-invasive assays for blood compounds such as glucose or haemoglobin has also been investigated [ l&l9 1. Modulation of the intensity of the incident radiation beam is necessary in order to generate a PA response from the sample. There are two practical means of modulating the incident infrared beam with Fourier transform instrumentation. The scanning of the moving mirror in the interferometer leads to an intensity modulation at different frequencies for different wavenumbers (the modulation frequency,f, of the infrared radiation of wavenumber V, depends on the scan speed, v, of the Michelson interferometer, f= 2vV [ 201). Like ATR, PA measurements using scan modulation

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probe various depths across the spectrum. However, unlike ATR, the diffusion length probed in the sample can be modified by simply changing the modulation frequency. In step-scan interferometers, the moving mirror can be dithered about a single point in the mirror scan before proceeding to the next step in the mirror scan. This phase modulation modulates all wavelengths at the dither frequency, therefore phase-modulated PA spectroscopy will probe the same effective thermal diffusion length in the sample at all wavelengths. Fig. 7A compares PA spectra of a dried whole blood film using phase modulation (Fig. 7, upper trace) and rapid scanning FT-IR (Fig. 7, lower trace). A comparison of the relative intensities across the spectra clearly shows the wavelength dependence of the PA response in the rapid scan acquisition mode. Conversely, the intensity distri-

wpid

ScaJ

/ 3500

3000

2500

2000

1500

1000

(Blood -Plasma) x 2

1800

1600

1400 Wavenumber

1200

1000

/ cm-l

Fig. 7. Photoacoustic spectra of thin films of whole blood and plasma. (A) Whole blood films as measured by the phase-modulated step-scan PA method and the rapid-scan technique, respectively; (B) thinfilm PA spectra of match blood and plasma samples and the corresponding difference spectrum.

bution across the mid-IR phase-modulated PA spectrum is similar to that observed in reflectionabsorption spectra (Fig. 6). A comparison of the amide I to amide II ratios in the PA spectra also indicates an increased level of PA saturation in the rapid-scan spectra. This saturation is particularly evident in slow-scanning spectra as indicated by the near 1: 1 amide I to amide II ratio. PA saturation of the intense absorption bands like the amide I causes the intensities of the weaker bands, in the fingerprint region for instance, to be increased relative to the stronger absorption features in the spectrum. This accounts for the discrepancy in the relaintensities tive encountered between the transmission (Fig. 5) and the PA spectra of dried films. Fig. 7B compares the fingerprint region of dried whole blood with dried plasma derived from the same whole blood sample as well as the corresponding difference spectrum. This difference spectrum represents for a greater part the spectral contribution from the cellular blood components; similar spectral features can be seen in the ATR difference spectrum shown in Fig. 4. PA spectroscopy, like the reflection-absorption method described earlier, can be used to study highly scattering samples. Compared to the other methods used to measure dried films, the film deposition, drying and thickness is less critical with the PA technique. Indeed, one of the strengths of qualitative PA spectroscopy is that little, if any, sample preparation is required. However, the technique suffers from the disadvantages of being rather time consuming, particularly step-scan based acquisitions. PA spectra typically have a poorer signal-to-noise ratio than transmission or ATR measurements on solids and liquids, and the variability of PA saturation between sample types or in samples of widely varying compositions can be a major problem in the analysis. This lack of sensitivity coupled with the PA saturation problem severely limits the applicability of PA spectroscopy in the quantitative analysis of biotluids. 3.1. Near-/R transmission measurements with fluids and dry films Near-IR transmission spectroscopy has recently been used in quantitative measurements on biofluids [ 6,9,10]. Again, strong water absorptions limit the transmission pathlength which can be used for these measurements. Fig. 8A compares the near-IR transmission spectrum of whole blood and water as measured with a 0.5 mm pathlength

trends in analytical chemistry, vol. 15, no. 7, 1996

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9000

8000

10000

9000

8000

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6000

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4000

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Fig. 8. (A) Absorbance near-IR spectra of whole blood and plasma as measured in transmission using a 0.5 mm pathlength quartz cell; (B) near-R difference spectrum from the whole blood and water spectra as shown above; (C) near-IR difference spectrum of human blood plasma as measured against a water-filled cell with a pathlength of 1 mm.

transmission cell. For the near-IR spectral range also the wavelength for the spectrum abscissa is still frequently used as for UV-VIS spectroscopy ( 1000 nm is equivalent to 10 000 cm- , and 2 500 nm to 4000 cm-’ ). However, we recommend the use of wavenumbers, which are directly proportional to photon frequency or energy. Each spectrum is similarly dominated by two water absorption bands, the OH stretch-HOH bend combination ( = 5 100 cm-’ ) and the first OH stretching overtone ( = 6900 cm-‘) vibrations. Apart from the increased sloping baseline at short wavelengths due to the increased scattering of the sample, the hint of a band in the combination region (42004900 cm-’ ), and the broader water absorption features there is little difference between the whole blood and water spectrum. Scaled water absorption subtraction (Fig. 8B ) reveals additional features in

the combination region and in the region of the first overtone of the CH stretching vibration ( = 5800 cm-’ ) of the whole blood spectrum. Again, complete compensation of the water absorption is impossible due to the major interactions between water and other components of the plasma or whole blood. These interactions primarily involve the water hydrogen bonding network and lead to the observed changes in the shapes of the water overtone and combination bands. Inadequate water compensation is exemplified in Fig. 8C which compares near-IR transmission measurements of blood plasma samples which were directly ratioed to a water-filled transmission cell. Typically, the water absorption regions are not included in a quantitative analysis because of the large spectroscopic variations encountered in those regions. A recent study compared the assay performances for various blood substrates in plasma using ATR mid-IR and transmission near-IR spectroscopy [ 61. For optimal multivariate PLS calibration models, water absorption regions had to be excluded from both the nearIR and ATR mid-IR quantitative analysis. Analysis of dried biofluid films using near-IR transmission spectroscopy again offers the potential of extending the analytical range of the quantitation by eliminating the masking water absorptions. Fig. 9 compares near-IR transmission spectra of dried films of whole blood and plasma. The strongly sloping baseline for whole blood arises from the enhanced sample scattering due to the cellular components. However, compared with the near-IR transmission spectrum of the raw fluid (Fig. 8A), or with the water compensated spectrum (Fig. 8B), there is considerably more structure in

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Fig. 9. Absorbance near-R spectra of whole blood and blood plasma thin films as measured in transmission.

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the spectrum of the dried whole blood film. Similarly, diffuse reflectance near-IR spectroscopy of dried fluid films might also be an appropriate technique to extend the quantitation range of near-IR analysis of biofluids.

(HMH) is grateful to the Deutsche Forschungsgemeinschaft and the National Research Council of Canada for a travel grant to visit the Institute for Biodiagnostics in Winnipeg.

References 4. Future prospects of infrared spectroscopy in the clinical laboratory

[1 M. Salmain, A. Vessibes, G. Jaouen and I.S. Butler, Anal. Chem., 63 ( 1991) 2323-2329.

IR multicomponent assays on easily accessible biofluids have already been described by various groups. The reagentless determination of clinical parameters has some convincing arguments and holds considerable clinical promise. Instrumentation is available at relatively low cost and requires only minimal operator training. Current multivariate analysis software can be seamlessly integrated into the data acquisition and instrument control software making the results of the assay immediately available. Conversely, the analysis can be made post-hoc after spectral data acquisition and archived spectra can be reanalysed at any time. Sample volumes required by IR analysis are at the microliter scale, so that minimally invasive biofluid sampling techniques can be applied. Near-IR analysis holds the added promise that inexpensive glass, or in some cases plastic disposable, substrates and transmission cells can be used. The use of flexible fiber-optic probes is certainly another advantage. Dried biofluid film preparations have the potential of extended detection limits over raw-fluid-based analysis. Dried films have the added advantage of being able to be stabilized for long-term archival thus enabling clinical IR fingerprinting of the patient. Despite these attractive features, IR spectroscopy has yet to emerge as an important tool in the clinical analysis of biofluids, and chemometrics will play an increasing role in establishing such assays. The challenge a number of groups have undertaken is to lay the ground work that will allow extending the scope of this powerful physical method from the chemistry laboratory into the clinical chemistry setting.

Acknowledgements The Dortmund component of the work was supported by the Ministerium fiir Wissenschaft und Forschung des Landes Nordrhein-Westfalen and the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie. One of the authors

[2 E. Wentrnp-Byrne, L. Rintou, J.L. Smith and P.M. Fredericks, Appl. Spectrosc., 49 (1995) 10281036. [31 A. Hesse, G. Schrnmpf and I. Schilling, Z. Urol., 67 (1974) 367-375. [41 M. Volmer, A. Bolck, B.G. Wolthers, A.J. de Rulter, D.A. Doombos and W. van der Slik, Clin. Chem., 39 (1993) 948-954. [51 M. Jackson and H.H. Man&h, in H.H. Mantsch and D. Chapman (Editors), Biomedical Infrared Spectroscopy in Infrared Spectroscopy of Biomolecules, Wiley-Liss, New York, 1996, pp. 31 l352. 161 H.M. Heise, Mikrochim. Acta, ( 1996) in press. [71 H.M. Heise, R. Marbach, T. Koschinsky and F.A. Gries, Appl. Spectrosc., 48 (1994) 85-95. [81 H.M. Heise and A. Bittner, J. Mol. Strnct., 348 (1995) 21-24. A. Bittner, R. Marbach and H.M. Heise, J. Mol. [91 Strllct., 349 (1995) 341-344. [ 101 R.A. Shaw, S. Kotowich, H.H. Mantsch and M. Leroux, Clin. Biochem., 29 (1996) 11-19. E.H. Korte and A. Otto, Appl. Spectrosc., 42 [Ill (1988) 38-43. [I21 A. Rosenzwaig, Photoacoustics and Photoacoustic Spectroscopy, Chemical Analysis, Vol. 57, Wiley, New York, 1980, Chap. 21. 1131 H.M. Heise, Fresenius’ J. Anal. Chem., 350 (1994) 505-513. [I41 K. Ohta and R. Iwamoto, Appl. Spectrosc., 39 (1985) 418-425. Cl51 M.G. Sowaand H.H. Mantsch, J. Mol. Strnct., 300 ( 1993 ) 239-244. [ 161 M.G. Sowa and H.H. Mantsch, Appl. Spectrosc., 48 (1994) 316-319. [I71 M.G. Sowa and H.H. Mantsch, Calcif. Tissue Int., 54 (1994) 481-485. 1181 K.M. Quan, G.B. Christison, H.A. MacKenzie and P. Hodgson, Phys. Med. Biol., 38 (1993) 191 I1922. 1191 G. Spanner and R. Niessner, Anal. Methods Instrnm., 1 (1993) 208-212. [201 P.R. Griffiths and J.A. de Haseth, Fourier Transform Infrared Spectrometry, Wiley, New York, 1986.

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trends in analyticalchemistry, vol.15, no.

J. Wang, M. Sowa and H.H. Mantsch are at the Institute for Biodiagnos tics, National Research Council Canada, 435 El/ice Avenue, Winnipeg, Manitoba, Canada R3B 7Y6.

7, 1996

A. Bittner and H.M. Heise are at the lnstitut fiir Spektrochemie und Angewandte Spektroskopie, Bunsen-Kirchhoff-Str. 11, D-44 139 Dortmund, Germany.

New materials for electrochemical sensing I. Rigid conducting composites F. Ckspedes, E. Martinez-Fhbregas, S. Alegret Bellaterra,

Spain

The development of composites based on conductive phases dispersed in polymeric matrices has led to important advances in analytical electrochemistry, particularly in sensor devices. These new materials combine the electrical properties of graphite with the ease of processing of plastics (epoxy, methacrylate, Teflon, etc.) and show attractive electrochemical, physical, mechanical and economical features compared to the classic conductors (gold, platinum, graphite, etc.). The properties of these composites are described, along with their application to the construction of conductometric, potentiometric and amperometric sensors. The chemical modification of the composites by blending fillers that improve the analytical characteristics of the resulting sensors is discussed, particularly for the case of amperometric devices.

1. Introduction Environmental, clinical and industrial samples show great diversity and analytical complexity. At the same time, competition in the industrial arena has heightened considerably in the 1990s. Under these circumstances, electrochemical sensors have enormous potential as reliable, sensitive and robust devices. Although a wide range of chemical sensors and biosensors has been reported in the literature, very few of them have found their way to the market. Instrumentation prototypes are designed and built in research laboratories, but 0165-9936/96/$15.00 fIISOl65-9936(96)00042-8

even when they show excellent analytical qualities, the devices and systems are often not suitable for industrial fabrication. Recent efforts are being made in the research of new electrochemical sensors in areas such as microelectrodes, chemically and biologically bulk- and surface-modified electrodes, and on devices based on new materials. In most instances, the frontiers between these research areas are blurred and they may be approached simultaneously to realize a common goal - the integration of the analytical processes into a simple device. In this article, the preparation of a new type of chemically bulk-modified electrodes (CBMEs ) is described. These electrodes are based on rigid and conducting composite materials of the carbonpolymer-modified kind. Biologically modified sensors based on these same composite materials will be dealt with in Part II.

2. Classification of conducting composite materials A composite results from the combination of two or more dissimilar materials. Each individual component keeps its original nature while giving the composite distinctive chemical, mechanical and physical qualities, different from those shown by the individual components [ 11. If composite are formed from a conductive and an insulating phase the first classification of the materials can be based logically on the arrangement of the conducting and insulating particles in the composite material. When an insulating polymer acts as a cementing agent the composite can be classified by the nature of the conducting material (platinum, gold, carbon, etc. ) and the arrangement of its particles (i.e. whether the conducting parti01996 Elsevier Science B.V. All rights reserved