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Blood Glucose Measurement by Multiple Attenuated Total Reflection and Infrared Absorption Spectroscopy YITZHAK MENDELSON, M E M B E R , I E E E , ALLEN C. CLERMONT, ROBERT A. PEURA, S E N I O R M E M B E R . IEEE. A N D BEEN-CHYUAN LIN

Abstract-The difficulty of measuring physiological concentration5 of glucose in blood by conventional infrared absorption 5pectroscopj is due to the intrinsic high background absorption of water. This limitation can he largely overcome by the use of a COz laser as an infrared source in combination with a multiple attenuated total reflection (ATR) technique. To demonstrate the applicability of this technique, w e compared in vitro measurements of glucose in blood obtained from an experimental infrared laser spectrometer with independent meawrements made by a standard YSI 23A laboratory gluco\e analyier. The capability of continuous measurement of blood gluco5e Concentration i s of primary importance in the future development of a glucose senwr for diabetic patients.

I. INTRODUCTION BSORPTION spectroscopy in the infrared (IR) region has been an important technique for the identification of unknown biological substances in aqueous solutions for many years [ l]-[3]. The technique is based on the phenomena that each molecule has specific resonance absorption peaks which are known as “fingerprints.” These characteristic peaks are caused by vibrational and rotational oscillations of the molecule. Biological molecules have a very complicated structure and, therefore, a large number of similar IR absorption peaks that are often overlaping. For example, as shown in Fig. 1, the characteristic IR spectrum of anhydrous D-glucose has, in the wavelength region of 2.5-10 pm, more than 20 absorption peaks [4]. Not all of these absorption peaks, however, are specific for only this molecule. Of particular significance is the absorption peak around 9.7 pm (1030 cm-I) which is the prominent absorption peak of glucose due to the carbon-oxygen-carbon bond in its pyrane ring. It is important to note also that this absorption peak is within the wavelength range emitted by a CO- 1aser. Besides the characteristic IR spectrum of glucose, the magnitude of its absorption peak is directly related to the concentration of glucose in the sample. In principle,

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therefore, the IR absorption intensity can provide, upon simple calculation using Beer-Lambert’s Law, a quantitative measure of the glucose concentration in a sample. In practice, however, two major practical difficulties must be overcome in measuring the concentration of glucose in an aqueous solution such as blood by means of conventional IR absorpjion spectroscopy: 1) pure water has an intrinsic high background absorption in the IR region, e.g., the absorption coefficient of water is approximately 640 cm-l at 9.7 pm [5], and 2) the normal concentration of glucose in human blood is relatively low (typically 90120 mg/dl or mg percent) [6]. The difficulty of measuring physiological concentrations of glucose by conventional IR spectrometers can be largely overcome by the use of a COz laser as an IR source [7]-[9]. Besides the much higher measuring sensitivity achieved with a powerful laser source, the monochromatic property of a laser (bandwidth ranging from IO-3 to cm-I) can provide an improvement in the measurement resolution. This is particularly important in multicomponent analysis of biological samples since two closely adjacent absorption peaks may be better separated. The capability of continuous measurement of glucose concentration in blood is of major importance in the treatment of diabetes [lo]. This paper describes an experimental IR spectrometer with a CO1 laser source based on a multiple attenuated total reflection (ATR) technique. To demonstrate the applicability of this laser spectrometer in medical diagnosis, we compared quantitative measurements of glucose in blood obtained from the prototype IR laser spectrometer with standard measurements made by a Yellow Springs Instruments (YSI) laboratory glucose analyzer. 11. PRINCIPLE OF MULTIPLE ATTENUATED TOTAL

REFLECTION Manuscript received June 15, 1988: revised October20, 1988. This work was supported in part by grants from W . M . Keck and Surdna Foundation5 and NIH Grant R15DK38845-01. The authors are with the Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester. M A 01609. IEEE Log Number 9034246.

The basic application of ATR was described by Fahrenfort [ l l ] and Harrick (121, [I31 and is illustrated in Fig. 2. The figure represents the path of a ray of light in the vicinity of a reflecting interface between two media of different refractive index n , and n2 where n l > n 2 . As

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internal reflections is directly proportional to the length of the plate and the angle of incidence and is inversely proportional to its thickness. The electric field at the interface and the depth of penetration into the sample medium depends on both the angle of incidence and the relative index of refractions at the interface. This arrangement allows the sample medium that must be placed against the surface of the ATR plate to be sampled several times. Weak absorptions can thus be enhanced considerably. Fig. 2. Schematic representation of the path o f a ray of light for total i n The essential advantage of the ATR method compared ternal reflection. The ray penetrates a fraction of a wavelength (d,,) beto conventional IR transmission measurements is that yond the reflecting interface into the rarer medium of refractive index n 2 . Note the displacement D of the ray upon reflection. heating of the sample inside the cell, which may ultimately destroy the sample, can be largely eliminated. In shown, the ray penetrates a distance df>beyond the reflec- addition, with layer thicknesses greater than three times tive interface into the rarer medium of refractive index n z . the test wavelength, the thickness of the sample medium This results in a certain displacement D upon reflection. no longer has any essential effect on the measured reflecThe depth of penetration d,, is a function of the incident tion by the ATR plate. This eliminates the need for filling and cleaning cells with very small path lengths. Another wavelength according to the following equation [ 121: advantage of ATR spectroscopy is the ability to perform noninvasive measurements as for example in surface analysis of biological media such as skin [ 151. LIGHT IN

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where d,, is the penetration depth (i.e., the distance from the interface where the electric field decreases to 1 / e of its initial value), 0 is the angle of incidence, h is the wavelength, and n21 = n 2 / n l . If the rarer medium is not transparent, the equilibrium between the incident and reflected energy is disturbed by radiation absorption due to an evanescent field near the boundary layer D.This property can be used for spectroscopic measurement. Kaiser [14] suggested that by combining a COz laser source with an ATR technique, it is possible to perform sensitive spectroscopic identification of biological substances. According to this approach, the incident beam is introduced into an ATR plate via the entrance aperture. The beam propagates down the length of the plate by multiple internal reflections from opposing flat surfaces and leaves the ATR plate via the exit aperture. The basic requirement is that the index of refraction of the ATR plate should be greater than the sample being analyzed. In addition, the ATR plate must have low intrinsic absorption at the test wavelength so that as little power as possible will be absorbed by the ATR plate itself and, therefore. strong heating will be largely eliminated. The number of

111. P R E L I M I N AEXPERIMENTS RY Freshly drawn whole human and pig blood, collected in heparinized syringes, were used in our experiments. The blood was first filtered through sterile gauze and then adjusted to a hematocrit of approximately 40%. The glucose concentration in the blood was varied by mixing the blood with different volumes of 5% isotonic glucose solutions. In order to determine if a conventional ATR technique utilizing Fourier transform IR (FTIR) spectrometry can be used to measure physiological concentration of glucose in blood, we first compared the ATR spectrum of freshly drawn whole human blood containing a normal concentration of 100 mg/dl glucose with the spectrum of the same blood sample after it was mixed with an abnormally high isotonic glucose concentration of 10 g/dl. The spectra, which were recorded with a Perkin-Elmer model 1700 FTIR equipped with a cylindrical ATR liquid cell attachment, are shown in Fig. 3. In order to enhance the absorption peaks due to glucose, the ATR spectrum of distilled water was automatically subtracted from the ATR

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spectrum of each blood sample. From this experiment it was found that despite the high (10 g / d l ) glucose concentration in the blood, a maximum decrease of only 3.34%in the transmittance was detected. This small decrease in transmittance indicates that in order to measure physiological concentrations of glucose in the blood with a resolution in the order of 10 mg/dl, a conventional FTIR spectrometer must be able to measure changes in transmittance corresponding to approximately 3.34 x lo-’ percent. This, however, is not within the presently attainable accuracy of conventional FTIR spectrometers. Next, in order to determine the optimal operating wavelength of the CO2 laser in our experimental IR spectrometer, we reexamined the expanded ATR absorption spectrum of a whole human blood sample containing 10 g/dl glucose. The spectrum, which is shown in Fig. 4 , was recorded by the same Perkin-Elmer FTIR spectrometer after the ATR spectrum of distilled water was automatically subtracted from the ATR spectrum of blood. Note the pronounced absorption peak around 1035 cm-I. This strong absorption peak, which corresponds to a CO2 laser wavelength of approximately 9.676 pm, was found to be in good agreement with the absorption peak of glucose shown in Fig. 1 . Therefore, this laser line was selected as the operating wavelength in our system.

IV. EXPERIMENTAL SYSTEM A . System Layout The schematic block diagram of the experimental IR spectrometer is shown in Fig. 5. A California Laser Model 82-7500-TG-T CW CO2 laser with a tunable wavelength range from 9.2 to 10.8 pm, maximum output power of about 8 W, and power fluctuations of less than 2 % , was chosen as an IR source. The laser, operating in the TEMm mode, is equipped with a grating device that can be manually adjusted by a micrometer for the accurate selection of a single rotational line. Temperature stability of the laser cavitv is achieved bv a built-in fan and a feedback

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controlled temperature cooling system. An optical engineering IR imaging plate was used to visualize the CO, laser beam during system alignment. An optical engineering COz spectrum analyzer was used to verify that the laser is tuned to the desired frequency. A photon technology, five-sector, aluminum mirrored, reflective surface chopper blade mounted on an EG&G Model 196 variable frequency optical chopper was used to deflect the COz laser beam in two perpendicular directions. The rotating chopper blade transmits the reference or sample beams alternatively at a chopping frequency of 193 Hz. The IR sample beam passes through the ATR plate which is in close contact with the sample medium. The IR reference beam is measured continuously by a Coherent Model 210 power detector in order to compensate for variations in the output power of the COz laser. It is important to note that a reflective chopper blade was used instead of a conventional zinc selenide (ZnSe) beam splitter since we found that temperature fluctuations caused significant variations in the splitting ratio of a ZnSe beam splitter. The power of the beam exiting the ATR plate is first measured by an Oriel Model 7084 pyroelectric detector

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and then amplified by an EG&G 5207 single phase lockin amplifier. An analog ratio circuit divides the outputs from the lock-in amplifier and the reference detector. An AT&T 6300 personal computer equipped with a MetraByte DASH-16, 12-b, A/D-D/A data acquisition board is used to acquire and process in real-time the output signals in the system. The optical set up was mounted on a stable vibrationfree table to minimize mechanical disturbances. The system was operated at room temperature.

B. ATR Plate A Harrick Scientific, 45 O bevel angle, trapezoidallyshaped ZnSe crystal (dimensions: 20 X 50 x 3 mm) was selected as the ATR plate. The angle of the entrance and exit ends with respect to the incident beam determines the interior angle of reflection. The ATR plate was aligned such that the laser beam enters at normal incidence. This resulted in a total of 17 internal reflections inside the ATR plate. A ZnSe ATR plate was selected based on its higher refractive index (2.42) relative to that of water (1.33) and its low intrinsic absorption at wavelengths between 9- 1 1 pm so that the power absorbed by the ATR plate is minimized. This reduces also the thermal loading on the test sample by preventing significant sample heating. ZnSe is also a suitable material for our application because it is water insoluable and relatively nontoxic. The penetration depth of the evanescent field into the absorbing medium depends on the angle of incident radiation and the relative index of refraction of the ATR plate with respect to the absorbing medium. For a waterZnSe interface and a CO2 laser operating at a wavelength of 9.6 pm, the penetration depth for each reflection was estimated to be approximately 1.3 pm. In order to increase the overall sensitivity of our system and avoid a temperature rise in the sample due to heating by the strong IR absorption of water, the sample solution was passed over both surfaces of the ATR plate. C. Sample Cell Holder A special sample cell holder was constructed to accommodate the single pass ATR plate and allow continuous pumping of the sample solution over both sides of the ATR plate. The ATR plate is clamped between the two parts of the grooved aluminum holder as shown in Fig. 6. A rubber O-ring provides a liquid tight seal. A steel rod mounts the sample cell holder on the optical table. A water pump was used to circulate the test medium through the cell so that the two largest surfaces of the ATR plate are continuously surrounded by the sample solution.

D. Data Processing The modulated signal from the pyroelectric detector was amplified and processed by the lock-in amplifier. The digital output signal from the chopper is connected to the reference input of the lock-in amplifier for synchroniza-

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tion. The incident laser power traversing the ATR plate is directly proportional to the glucose concentration in the blood. An analog ratio circuit based on a high-stability Analog Devices AD533 IC divider provides a continuous output voltage corresponding to the scaled ratio of the sample and reference channels. It is important to note that the output of the ratio circuit is independent of the input laser power leaving the measurement virtually unaffected by slow variations in the power of the COz laser. The normalized analog signal from the ratio circuit is first digitized at a rate of 10 samples per second and then averaged over a 10 s time interval by the computer.

V. RESULTS A . Calibration Experiments The CO, laser spectrometer was calibrated by comparing the relative IR absorption measured from different pig blood samples containing known glucose concentrations in the range between 90 and 270 mg/dl. The relative IR absorption for each blood sample was obtained by calculating the difference between the normalized IR power when the blood sample was pumped through the ATR plate and the corresponding attenuation by a 0.9% saline solution. The data obtained in this calibration study were analyzed using a linear regression statistical program. The result of this statistical analysis is plotted in Fig. 7. The solid line ( Y = 0.64 1.37 * lo-' X ) represents the linear regression line for a total of 15 paired data points. The correlation coefficient and the standard error of the estimate (SEE) were 0.98 and 0.015, respectively, ( p < 0.OOl). The 95% confidence interval is represented by the dashed lines. Note that the y-intercept of this regression line (offset absorption) is not equal to zero. This observation is important since it indicates that, besides water and glucose, other substances in the blood (e.g., proteins)

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can cause a noticeable IR absorption at the operating wavelength of the CO2 laser used in our system.

B. Glucose Prediction Experiments The slope and intercept of the linear regression line in Fig. 7 were used to establish a quantitative relationship between the IR spectrometer readings and unknown glucose concentrations in subsequent pig blood samples. Using these calibration coefficients, we performed another study in which we compared the concentrations of different glucose solutions as measured by the COz laser spectrometer with those measured simultaneously by the references YSI Model 23A glucose analyzer. The relationship between the actual and predicted values is shown in Fig. 8. A total of 98 data pairs, corresponding to glucose concentrations ranging from 48 to 270 mg/dl, were used in this regression analysis. The solid line represents the linear regression line. Linear regression analysis of this data revealed a slope and y-intercept of 1.008 and - 1.02 mg/dl, respectively. The correlation coefficient for actual versus predicted values was r = 0.969, ( p < 0.001), while the respective SEE, computed as the variation from the linear regression line, was found to be 20.2 mg/dl. The 95% confidence interval is shown by the dashed lines. In order to ascertain the measurement accuracy and precision of the IR spectrometer, we compared the mean and standard deviations for the difference between the measured and standard glucose concentrations for different glucose ranges. These comparisons are summarized in Fig. 9.

C. System Response To examine the response of the IR laser spectrometer to a transient step change in glucose concentration, we rapidly infused a bolus of 20 cc of the 5 % isotonic glucose solution into the 300 mL of whole pig blood that was pumped continuously through the ATR cell holder at a rate of 100 mL/min. After the glucose reached a steadystate equilibrium with the blood, an additional 300 mL of whole blood was added to the system in order to lower the glucose concentration in the blood. Periodic readings from the IR spectrometer were simultaneously compared against measurements of blood glucose concentrations obtained from the YSI glucose analyzer. The result of this experiment is shown in Fig. 10. VI. DISCUSSION

Several commercial products are presently available that can measure blood glucose concentration with acceptable accuracy. These measurements are performed on a sample of blood and require the use of a chemical reaction. The principle described in this paper has a distinct advantage since no chemical reactions are involved and, therefore, the measurement can be performed instantaneously and continuously. The work presented revealed that the use of a CO, laser source allows an improvement in measuring sensitivity compared with conventional ATR-based FTIR spectronieters. This improvement is believed to be due in part to the higher power and spectral resolution of the CO, laser

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source. The improved resolution is of particular interest in biological measurements since most media are composed of multicomponent systems. It is important to recognize that besides glucose various other biological molecules, such as urea, cholesterol, alcohols, and other substances have very close absorption peaks in the same IR region used by our spectrometer. Therefore, future work in our laboratory includes the use

of multiple IR wavelengths in order to account for the presence of interfering IR absorbing substances in the blood. The output power of our COz laser at 9.676 pm was approximately 3 W. This power is higher than the typical power of conventional IR sources found in commercial IR spectrometers by a factor of 103-10s. The spectral resolution is determined by the width of the COz laser line

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that is typically between cm- . Spectrometers with conventional IR sources have spectral resolution typically in the order of 1-10 cm-'. The detection sensitivity of our system depends on several factors including: the fluctuations of the laser power, variations in sample temperature, and the relatively small active area of the photodetector (1 mm2). These limitations may be overcome by selecting a more stable CO2 laser and maintaining better temperature control of the sample cell. Furthermore, fluctuations of the spatial power distribution of the laser beam exiting the ATR plate was found to affect the measurement because the pyroelectric detector used in our system has a small sensitive active area. This work has demonstrated that a CO2 laser in combination with an ATR plate is suitable for measuring the concentration of glucose in human whole blood in the physiologic and diabetic range. The method described in this paper is obviously not restricted to the in vitro measurement of glucose in blood using an ATR plate configuration, although this was our primary application. The measurement of glucose and other organic molecules in various sample solutions including environmental, food, and industrial applications are of primary interest. Furthermore, the ATR principle is similar to that of an uncladded fiber optic sensor utilizing the evanescent wave phenomena. Therefore, the long term goal of this work is to replace the ATR prism by an 1R optical fiber with a partially uncladded tip. A specially designed fiber optic sensor may then be inserted into the blood stream in order to measure glucose continuously in diabetic patients. ACKNOWLEDGMENT The authors greatefully acknowledge the technical assistance provided by Prof. R. Quimby of our Department of Physics. REFERENCES [ I ] L. T . Rozelle, L. J . Hallgren, J. E. Bransford, and R. B. Kock, "The identification of major infrared absorbing components of human urine," Appl. Spectrosc., vol. 19, no. 4, 1965.

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121 N . K. Freeman. "Analysis of blood lipids by infrared spectroscopy." in Blood Lipids t i n t / Lipoprotciri c: Quonritufion. Corripo.siriori. r r ~ l Meruholism, G . J . Nelson. Ed. New York: Wiley. 1972. 131 F. S. Parker, Applic.tifion.\ of Irijrured. Raniuri. wid Rrsoiicrrice Rtrmuri Specrroxvpy iri Biochrmrsfry. New York: Plenum. 1983. [4] C . J . Pouchert, The Aldrich Lihrury of Irifrurecl SpcJctru. 3rd ed. Milwaukee, WI: Aldrich Chemcial Co., Inc., 1981. [ 5 ] G. M. Hale. and M. R. Querry. "Optical constants of water in the 200-nm to 200-pm wavelength region,'' Appl. Opr., vol. 12. no. 3. pp. 555-563. 1973. 161 A. C. Guyton. Texrbook of Medicul Physiology. 4th ed. Philadelphia, PA.: Saunders. 1971. [7] G. Kraus and M. Maier. "Detection of water pollutants by a CO, laser," Opt. Commun., vol. I I . pp. 174-177, June 1974. 181 N. Kaiser, "Access to metabolic processes in living matter made possible by the laser." in: Moderri Techniques in Physiologicd Scicv7c.c,.s. J . F . Gross, R. Kauffmann, and E. Wetterer. Eds. London. England; Academic, 1973. 191 G. Kraus and M. Maier, "Infrared absorption spectroscopy of aqueous solutions with a CO: laser," Appl. Pliys.. vol. 7. pp. 287-293. 1975. [IO] R. A. Peura and Y. Mendelson. "Blood glucose sensors: An overview." in Proc. IEEE Eng. Med. B i d . /NSF Syrrip. Biosen.sors. 1984. pp. 64-68. [ I I ] J . Fahrenfort, "Attenuated total reflection: A new principle for the production of useful infrared spectra of organic compounds." Spew rrochirn. A c f u , vol. 17, p. 689. 1961. [I21 N . J. Harrick, "Total internal reflection and its application to surface studies." Ann. N . Y. Acud. Sei.. vol. 101. p. 928. 1963. [ 131 N . J . Harrick. lrirerriul Relflecriori Specrroscop?. Ossining. NY: Harrick Scientific Co., 1979. [ 141 N. Kaiser, "Laser absorption spectroscopy with an ATR prism." IEEE Trans. Biomed. Eng.. vol. 26. no. IO, pp. 597-600. 1979. [ 151 R. E. Baier. "Noninvasive. rapid characterization of human skin chemistry in situ." J . Soc.. Cosmer. Chern.. vol. 29. pp. 283-306, 1978.

Yitzhak Mendelson (S'79-M'82) was born in Tel-Aviv, Israel. in 1949. He received the B.S. and M.S. degrees in electrical engineering from the State University of New York, Buffalo, in 1975 and 1976, respectively. and the Ph.D. degree in biomedical engineering from Case Western Reserve University, Cleveland, OH, in 1983. He is currently an Associate Professor of Biomedical Engineering at Worcester Polytechnic Institute. Worcester, MA. His research interests are in developing invasive and noninvasive techniques for blood gas and glucose measurements. biomedical sensors. m eroprocessor based medical instrumentation. and the study of light interaction with biological media. Dr. Mendelson is a member of SPIE. AAMI, and the Optical Society of America.

Allen C. Clermont received the B.S. degree in electrical engineering in 1985 and the M.S. degree in biomedical engineeriqg in 1988 from Worcester Polytechnic Institute, Worcester. MA, where his focus of study was on the design of noninvasive diagnostic devices. He is currently the senior research assistant of the William P. Beetham Eye Institute ofthe Joslin Diabetes Center. His major research interests i n clude the characterization of changes in retinal blood flow and the early detection of cataracts associated with diabetes and its ocular complications. Mr. Clermont has been an active member of the Engineering in Medicine and Biology Society since 1987.

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Robert A. Peura (S'62-M'68-SM'88) receivcd the B S degree i n electrical engineering troin Worcester Polytechnic Inqtitute. Worccjter, MA, in 1964, the M S degree in electrical engineering and the Ph D degree i n biomedical and electricdl engineering from Iowa State University. Arne\. in 1967 and 1969, respectively Since 1968 he ha\ been a\\ocidted with the Worcester Polytechnic Institute, where he is Pro fe\sor and Head of Biomedicdl Engineering dnd Professor of Electricdl Engineering He \erve\ ds Lecturer in Biomedical Engineering at the University of Mds\achu\ett\ Medical School He has served EMBS as Secretary/Trea\urer (1984-198s) and Vice President tor Financial& Long Range Planning (1985- 1989) H I \ research interest5 include the development ot biosen\ors to iiieribure the biochemistry of the body and noninvasive technique\ tor the evdludtion ot the peripheral circulation and cardiac function He hds received \upporl tor his work from the NIH, NSF. and several private founddtion\

465 Dr. Peura is a member of the FDA Circulatory System Devices Panel and is a recipient of NIH. NDEA. and NSF Science Faculty Fellowships. Dow Outstanding Young Faculty Award. and Western Electric Fund Award for Excellence in instruction of Engineering studenta.

Been-Chyuan Lin wa\ born i n Taiwan. R 0 . C , on October 2 I , 1958 He received the B S degree trom Chung-Yuan Univer\ity, Taiwan. R 0 C., i n 1980, and the M S degree from Worce\ter Polytechnic In\titute. Worce\ter. MA. in 1988. both in biomedical engineering He is currently d test engineer with Hottinger Baldwin Measurement\, Inc , working on various tran\ducer test automation projects Hi5 current interests include bio\en\or. medical in\trumentation, machine vision, and factory automation

Blood glucose measurement by multiple attenuated total reflection and infrared absorption spectroscopy.

The difficulty of measuring physiological concentrations of glucose in blood by conventional infrared absorption spectroscopy is due to the intrinsic ...
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