EP1277039A1

EP1277039A1 - Method for the long-term stable and well-reproducible spectrometric measurement of the concentrations of components of aqueous solutions, and device for carrying out said method

Info

Publication number: EP1277039A1
Application number: EP01940355A
Authority: EP
Inventors: Kai-Uwe Zirk; Harald Pötzschke
Original assignee: GlukoMediTech AG
Current assignee: Sanguibiotech GmbH
Priority date: 2000-04-27
Filing date: 2001-04-24
Publication date: 2003-01-22
Also published as: US20030147078A1; DE10020615C2; AU7396101A; DE10020615A1; WO2001084123A1

Abstract

The invention relates to a method for the long-term stable and well-reproducible spectrometric measurement of the concentrations of components of aqueous solutions, especially of dialysates of interstitial tissue fluids, especially the glucose concentration. The inventive method comprises splitting a measuring beam into two partial beams by means of a beam splitter (2), and guiding one partial beam through a measuring cuvette (3), that is separated from the solution to be measured for example by a diaphragm, or that is connected to a pump system with an exchange path that is separated from the solution to be measured by a diaphragm. The other partial beam is guided through a reference cuvette (4) that is filled with a reference solution. The light intensity of both partial beams is measured and the measured signals are subjected to a symmetrical signal processing, optionally after suitable amplification. The invention further relates to a device for carrying out the inventive method, which device can be especially miniaturized.

Description

Process for the long-term stable and reproducible spectrometric measurement of the concentrations of the components of aqueous solutions and device for carrying out this process

The invention relates to a method with the features of claim 1 and a device for carrying out this method with the features of claim 6. A preferred area of use is the measurement of the glucose concentration in the interstitial body fluid using a device according to the invention in a miniaturized design.

Usually 100 mL of human blood contains between 70 and 1 10 mg of glucose (glucose). With the common disease diabetes mellitus (the "diabetes"), from which only 3% of the adult population suffers in the industrialized countries alone, the average glucose content in the blood of the sufferers is usually significantly increased because these patients have an absolute or relative deficiency Insulin lowers the level of glucose in the blood by, among other things, promoting its absorption into the body cells. If the current blood glucose level of the diabetic can be determined continuously and instantaneously, this enables him to supply him with the precisely required missing amount of insulin at any time and thus normalizing his glucose metabolism to a large extent eliminates stress and late damage to the organism due to undesirable effects that occur due to unstable glucose levels, which - in addition to a general improvement in the quality of life of the patient - in particular also leads to a higher life expectancy Detectable glucose sensor is one way to continuously detect the glucose concentration in the body. This previously missing component, coupled with a known insulin pump, would contribute decisively to the realization of a so-called "artificial pancreas", ie a technical device for the fully automatic supply of the patient with the hormone insulin. Such an artificial pancreas could give many people with diabetes a life without insulin injections enable.

Research and development groups worldwide are trying to develop an implantable glucose sensor for the detection of the glucose concentration to market maturity. Different measuring principles are used. So far, mostly electrochemical glucose sensors have been designed, tried and developed. The application of such electrochemical sensors based on the use of suitable enzymes is experiencing enormous difficulties. In particular, this is the "poisoning" of the enzymes used (for example glucose oxidase) by the body's own substances, with subsequent long-term instability. To avoid this disadvantage, the invention described here relates exclusively to a purely physical measuring principle, spectrometry:

If a light beam propagates in an absorbing medium, the light intensity I decreases exponentially along the path s (within the scope of the linear optics). A cuvette of layer thickness d, which is filled with the solution of an absorbing substance of concentration c, is penetrated by a light beam. The ratio of the intensity of the light beam leaving the cuvette with light-absorbing substance in the cuvette (l (d)) to the corresponding value without this (lo) is the transmission T, which can be calculated in accordance with the LAMBERT-BEER law.

T = l (d) / lo = e ^{"dc ε}

The proportionality factor ε is the substance-specific absorption coefficient.

In order to achieve a resolution of, for example, 3 mg / dL of the concentration of an aqueous glucose solution in a wavelength range around 1.6 μm of the measuring radiation (this is infrared radiation of the NIR range, NIR: near infrared) over a measuring distance of d = 5 mm 7, 10 ^"4 [T] must be able to detect transmission changes. The stated accuracy of the detection corresponds, with an average glucose content in the blood of 100 mg / dL, to a relative error of about 3%. Greater inaccuracy should be , if this is technically feasible, will not be tolerated.

Some methods and devices for ex vivo measurement of blood glucose levels in the human body, which use the physical principle mentioned as a measurement method, are known from the patents WO 9510038, EP 0884970, US 5710630, US 5222496, US 5372135, US 5638816, US 5743262 and US 5772587 known. But in all of these methods, the "measuring beam" is radiated from the outside into or through tissue and the change in absorption or change in intensity of the scattered (reflected) radiation is used as a measure of the glucose concentration. That is, none of these inventions is concerned with the development of an implantable glucose sensor and all such measurements stand in the way of fundamental difficulties, such as sufficient specificity, due to the diverse structures of the tissue.

In the patents EP 0589191, EP 0561872 and US 5243983 it is disclosed, for example, to determine the glucose concentration spectrometrically in the eye wash. However, since the concentration in the eye wash adapts very slowly to the current blood glucose value, this method appears extremely unsuitable for a sufficiently delay-free determination of the blood glucose level.

A further method and a device for the quantitative determination of glucose are known from patent specification WO 9852469. IR heat radiation emitted by the eardrum then contains spectral information about the composition of the tissue and thus also the glucose concentration. However, due to the enormous complexity and number of substances and structures occurring in the body, this process should also not be considered unsuitable for a development to market maturity.

The object of the present invention is to provide a highly sensitive, simple and reproducible spectrometry method and corresponding devices for the quantitative determination, in particular, of glucose in interstitial body fluids - specifically in the context of an implantable detector. However, the method and device can also be used for other uses, for example for monitoring and controlling chemical processes.

If - as in the case of glucose in the organism - a very low content of the absorbing substance is specified, only a highly sensitive spectrometer with a high opto-electronic quality of the measurement signal recording and amplification can enable a sufficient accuracy of the measurement of the content. The selected device should therefore be constructed as simply as possible, ie without moving parts and in particular be miniaturized. The device must also contain the power of a large and low-noise opto-electronic gain in order to achieve the required sensitivity and accuracy. In addition, the measurement should be largely independent of the temperature of the substance to be examined, since these influences can be found directly in the measurement signal. So far, none of the above-mentioned patents has become the basis for a well-known marketable technical development. The methods and devices described in the patents do not seem to be able to meet the requirements mentioned, even for an ex vivo measurement.

The object of the teaching according to the invention is to combine measuring arrangements known in principle or in parts in a manner that is not obviously suitable and to improve them considerably in terms of measuring accuracy, the problems mentioned being solved in a technically effective and simple manner and the requirements mentioned for a detector for glucose for measurement in body fluids should be met. The task is solved according to the requirements. To measure the glucose content in body fluids, a dialysate of the tissue fluid, which is freshly brought into equilibrium, is continuously fed to a measuring chamber with the aid of microdialysis. This can be done in such a way that at least one wall of the measuring cuvette is a diaphragm or the liquid to be measured is fed to the measuring cuvette via a pump system with an exchange path which is separated from the solution to be measured by a diaphragm. A protein-free dialysate of the tissue fluid obtained in this way reduces the cross-sensitivities, since all molecules which are larger than the separation limit of the dialysis membrane and can have a disruptive effect are retained by the membrane. Independence from the temperature is achieved, for example, by a "symmetrical" structure with a measuring and a reference path, because then temperature-dependent changes compensate each other.

The design of the measuring method according to the invention comprises the following characteristics: The modulated, quasi monochromatic electromagnetic beams emanating from radiation sources are divided into two partial beams by a suitable beam splitter and their intensities are detected by detectors (e.g. photodiodes) which are largely independent of the wavelength in a certain wavelength band. Their output signals (photo currents) are transformed into voltages and these are then electronically offset against one another to form a highly stable difference or quotient signal. Such a difference or quotient formation eliminates intensity fluctuations in the source radiation (s). Above all, however, the difference or quotient signals are also modulated, which enables the additional use of the known “lock-in” amplifier technology. This further increases the Sensitivity possible by a factor of up to 10 ³ compared to "conventional" electronic amplifier mechanisms.

The core of the spectrometric measuring method according to the invention and the device used therefor is shown in FIG. The essential elements are a beam splitter (2), which divides the beam generated by a radiation source or sources (1) into two beams (measuring and reference beam), a sample cell (3) and a reference cell (4), each in the beam direction behind the beam splitter , as well as two detectors (5 and 6), each of which detects a partial beam behind a cuvette and converts its intensity into electrical signals. These are then processed as required in signal processing.

Figure 2 shows an embodiment of the measuring arrangement according to the invention. The time-dependent intensity (l (t)) of the (practically) monochromatic source radiation from the radiation source (1, e.g. a laser diode) is modulated sinusoidally:

l (t) = lo • sin (ω-t) + lκ

Inside the reference cuvette (4) there is at all times unchangeable ^ measured material with_ analytes in a concentration that is similar to or equal to the concentrations to be measured in the sample cuvette. According to the invention, the intensity-splitting ratio of the beam splitter (2) is chosen such that the two intensities lιw (t) and lR (t) striking the detectors (5 and 6), while there is no substance (analyte) to be detected in the sample cuvette (3), have the same value, this is the so-called balanced state. The two subsequent current-voltage converters (7 and 8) transform the photo currents generated by the detectors into voltages (Uwι (t) and UR (t)) and at the same time separate the DC voltage component. The two following multipliers (9 and 10) with gains n and - n are to be regarded as one of the essential features of this device. One of the two multipliers inverts the signal, the second is used for analog compensation of signal propagation times that may occur in the first signal inverter - it doesn't matter which of the two has the gain n or - n. The two output voltages of the multipliers are used as supply voltages for a WHEATSTONE measuring bridge or as supply voltages for a voltage divider measuring circuit - this is another important feature of the device according to the invention. While in balanced state, the bridge voltage (UBΓ), which is tapped between the two resistors (11 and 12) and the zero potential of the electronic circuit, is zero, during each analysis (inside the sample cuvette there is analyte) there is a non-zero Bridge voltage (UBΓ) on.

The bridge voltage is during the analysis

= A ^■ n • Uo • sin (ω-t) - n ^■ Uo • sin (ω-t) = (A - 1) • n • Uo - sin (ω-t)

A is the ratio between the values of U (.) During the analysis (at the respective concentration of the analyte) and the balanced state.

The bridge voltage can be changed by changing the source intensity of the radiation source; it is (its amount) independent of the position of the sample or reference cuvette, both are interchangeable. The bridge voltage caused by the analyte is detected by a "lock-in" amplifier (13), the output signal (a direct voltage UGI) of which is passed on to a measured value processing unit (14), which in the simplest case consists of a visual display device The output signal of the "Lock-in" amplifier applies:

UGi = (2 / π) - (A - 1) - n - v - Uo

v is the signal amplification of the "Lock-In" amplifier. The sensitivity of the end signal of the entire measuring arrangement is the first derivative of this function according to the quantity A, which is dependent on the concentration of the analyte.

dUci / dA = (2 / π) - n - v - Uo

The sensitivity can be adjusted on the one hand by increasing the source intensity and on the other hand by varying the amplification factors n and v. It is only limited by the signal-to-noise ratio of the signal processing and, in particular, is independent of changes in intensity caused by absorption. The advantage achieved by the measuring arrangement according to the invention lies in the simple and symmetrical structure, which (largely) eliminates both intensity fluctuations in the source radiation and also changes in absorption due to temperature changes in the measured material. In addition, it enables the use of “lock-in” amplifier technology, the well-known advantages of which can be used, ie the detection of very small signal changes with simultaneous elimination of external (electrical or optical) disturbance variables which act on the measuring and / or reference path ,

FIG. 3 shows a preferred extended configuration of the measuring arrangement according to the invention as a two-wavelength spectrometer. The two quasi-monochromatic source radiation of the radiation sources (1a and 1b, e.g. laser diodes) have different wavelengths. The temporal courses of the intensities of the source radiation are modulated sinusoidally and have an invariable temporal phase shift of 180 ° (= π) to each other. All of these are essential features of this measuring arrangement. The two initial intensities then have the following time profile:

H (t) = lo, ι - sin (ω-t) + lκ, ι l2 (t) = lo, 2 • sin (ω-t + π) + lκ, 2 = - lo, 2 ^■ sin (ω- t) + lκ, 2 _

The two beams are combined into one beam with the total intensity I, e.g. by coupling the individual beams into the two inputs of a "Y" light guide cable (1c) with a statistical distribution of the light guide fibers. The combined beams then leave the common exit of the light guide, the intensities add up. If the two amplitudes of the intensity summands ( lo, ι and lo, 2) the same, the light intensity (l (t)) at the output of the light guide is constant.

Inside the closed reference cell (4), the substance to be detected is continuously and unchangeably present in a concentration that is similar to the expected concentration in the sample. The division ratio (IM / IR) of the radiation intensities at the beam splitter (2) is selected so that in the balanced state, ie with the material to be measured without the substance to be detected inside the sample cell (3) (that is, with different analyte contents in the reference and the measuring cuvette) still hits the same intensity on the detectors (5 and 6). The intensities are detected by the detectors, whose photocurrents are transformed into voltages by two current-voltage converters (7 and 8) and at the same time the DC voltage components are separated. The subsequent multipliers (9 and 10) with the gains n and - n amplify these voltages, so that the voltages Uwι (t) and UR (t) are present at the outputs of the multipliers. The two output voltages (Uιvι (t) and UR (Q) are used as bridge supply voltages for a WHEATSTONE measuring bridge or as supply voltages for a measuring voltage divider circuit. While in the balanced state, the bridge voltage (Ußr) between the two resistors (11 and 12) and the zero Potential of the circuit is zero, during the analysis (ie the analyte is in a certain concentration in the measuring cuvette) a non-zero bridge voltage is set in. During the analysis there are changes in intensity in the measuring section caused by the one to be detected On the other hand, the intensity (IR (t)) that hits the detector remains constant in the reference path. The bridge voltage during the analysis results according to:

= n • (A • Uo ^• sin (ω-t) - B • Uo • sin (ω-t)) - n ^• (Uo ^• sin (ω-t) - Uo ^• sin (ω-t)) = ( A - B) • n • Uo ^• sin (ω-t)

A and B are the conversion factors for the two wavelengths between the values of Uwι (t) in the analysis (at the respective concentrations of the analyte) and Uwι (t) in the balanced state. B is proportional to A, substituting B with x ^• A (B = x • A) results in:

UBΓ = A ^• (1 - x) ^• Uo ^• sin (ω-t)

The structure is symmetrical, sample cuvette (3) and reference cuvette (4) and thus the measuring and reference sections of the detector are interchangeable. The bridge voltage caused by the analyte is detected by a "lock-in" amplifier (13) and its output signal UGI is passed on to a measured value processing unit (14). The following applies:

UGI = (2 / π) ^■ A ^• (1 - x) • n • v ^• Uo v is the signal gain of the "Lock-In" amplifier. The sensitivity of the output signal of the entire measuring arrangement is the first derivative of this function according to the quantity A, which depends on the concentration of the analytes:

dUGi / dA = (2 / π) ^■ (1 - x) • n • v • Uo

It is dependent on the one hand on the source intensity of the radiation source and on the other hand on the amplification factors n and v and can therefore be changed. It is also a factor of x, i.e. depends on the difference in absorption between the two wavelengths.

FIG. 4 shows a modification of the measuring arrangement shown in FIG. 3. Instead of the multipliers (9 and 10) and the subsequent measuring bridge or voltage division circuit (1 1 and 12) in the measuring arrangement according to FIG. 3, an electronic ratio generator (15) can also be used. The output voltages of the two current-voltage converters (7 and 8) serve as input signals of the ratio generator. The DC voltage components are not separated. The following results for the output voltage of the ratio generator (U (t)):

, = [Uo • sin (ω-t) ^■ A • (1 - x) + UK • A • (1 + x)] / [2 • UK]

Following the ratio generator is a "lock-in" amplifier (13) whose output signal UGI is passed on to a measured value processing unit (14) for further processing. With Uκ> Uo> O the following applies:

UGI = (1 / π) • A • (1 - x) • v • Uo / UK

v is the signal amplification of the "Lock-In" amplifier. The sensitivity of the output signal of the entire measuring arrangement is the first derivative of this function according to the quantity A, which is dependent on the concentration of the analyte:

dUci / dA = (1 / π) • (1 - x) • v • Uo / UK As in the previous arrangement, it depends on the one hand on the source intensity and the amplification factor v and on the other hand on the factor x (difference in absorption of the two wavelengths) and can therefore also be changed.

FIG. 5 and FIG. 6 show an expansion of the measuring arrangements described with reference to FIGS. 3 and 4. Instead of the two quasi-monochromatic radiation sources, a larger number of radiation sources can be used. The intensities of the individual radiation sources can be superimposed, for example, by multi-arm light guides (FIG. 5) or by dielectric beam splitters (FIG. 6). The temporal phase shift of the individual source intensities for three sources is 2-π / 3 (120 °), for four sources π / 2 (90 °) and generally for k radiation sources accordingly (2-π / k, k e N). The transfer of the solution form shown in FIGS. 3 and 4 to design using several sources is within the knowledge of the person skilled in the art.

The advantage of using multiple sources (simultaneous measurement at multiple wavelengths) is on the one hand a significant increase in specificity, and on the other hand, with a suitable choice of the individual wavelengths (increase and decrease in transmission), the signal change (through the difference formation) increases. _

Further advantages are the compact as well as the symmetrical construction: on the one hand only two photosensitive elements are required for the detection of k sources and for the elimination of the fluctuations in the source intensities, on the other hand absorption changes due to temperature fluctuations are eliminated.

An example of a device for carrying out the method according to the invention is shown in FIG. This shows the diagram of an arrangement of components according to the device shown in FIG. 3. The radiation sources (1 a) were a laser diode ("FNLD 1450", LASER GRAPHICS, Kleinostheim) with a wavelength of 1450 nm and an optical output power of max. 4 mW and (1b) an LED ("LED 16", LASER GRAPHICS, Kleinostheim) with a wavelength of 1580 (+ 150) nm and an optical output power of max. 1, 2 mW. Both radiation sources were supplied by current sources (19 and 20: "Model LDC 220", PROFILE, Karlsfeld), the output currents of which were provided by two function generators (21 and 22: "HM 8131-2", HAMEG, Frankfurt am Main) sinusoidal shape. An interference filter (17: "Model 42-6197", COHERENT, Dieburg) with a central wavelength of 1620 nm was arranged behind the LED (1b). The radiations from the LED and the laser diode were each in one leg of a "Y" -shaped Optical fiber (1 c; a special design with a diameter of 1 mm, LOT-ORIEL, Darmstadt) made of quartz fibers that are permeable to infrared radiation, and the combined beam, which leaves the common end of the optical fiber, from a subsequent collimator lens (18: “DIV-THR -Optik-LWL ", LASER 2000, Wessling) bundled and focused. The subsequent beam splitter prism (2:" 44-3861 ", COHERENT, Dieburg) divided the beam into two equally powerful partial beams that separated the following cuvettes (3 and 4; special designs with a layer thickness of 1 mm and a volume of 50 μL, HELLMA, Müllheim). Two InGaAs PIN photodiodes ("G 5832-01", HAMAMATSU, Herrsching) served as detectors (5 and 6), downstream power amplifiers (7 and 8: "DLPCA-100", FEMTO, Berlin) amplified their photo currents. The output signals of the current amplifiers were amplified by subsequent voltage amplifiers (9 and 10 "4-channel INH amplifiers", SCIENCE PRODUCTS, Hofheim) and their output voltages at two resistors (11 and 12: "metal layer 1, 2 MΩ", RS, Mörfelden -Walldorf). A "lock-in" amplifier (13: "LIA-MV-150", FEMTO, Berlin) and the output signal showed the voltage between the two resistors against circuit zero and its output signal was shown by a digital storage oscilloscope (14: "9304" , LECROY, Heidelberg).

The exemplary device according to FIG. 7 gave very precise measurement results even for material to be measured with a low concentration of the substance to be analyzed. FIG. 8 shows a calibration curve for D (+) glucose created with this device. For a concentration of 100 mg / dL in the area of the calibration curve, the measurement results in an absolute error of approximately 5 mg / dL.

Claims

claims

1. Method for long-term stable and easily reproducible spectrometric measurement of the concentrations of the components of aqueous solutions, in particular also

Dialysates of interstitial antifreeze fluids, in which a measuring beam is split into two partial beams by a beam splitter, one partial beam is passed through a measuring cuvette and the other partial beam is passed through a cuvette filled with a reference solution, the light intensities of both partial beams are measured and the measuring signals, if necessary After suitable amplification, a symmetrical signal processing is carried out, characterized in that the intensity of the measuring beam fluctuates periodically uniformly periodically, in the signal processing the signal of each partial beam is first fed to a multiplier, one of which causes a signal inversion, and then a difference or ratio formation he follows.

2. The method according to claim 1, characterized in that the measuring beam is monochromatic radiation.

3. The method according to claim 1, characterized in that the measuring beam consists of several superimposed monochromatic beams.

4. The method according to any one of claims 1 to 3, characterized in that the difference or ratio signal is demodulated.

5. The method according to any one of claims 1 to 4, characterized in that the demodulated signal is detected by a measured value processing unit and the concentration is determined by means of calibration curves stored there.

6. Device for long-term stable and easily reproducible spectrometric measurement of the concentrations of the components of aqueous solutions, in particular also

Dialysates of interstitial tissue fluids, consisting of a radiation source (1) with a periodically uniformly fluctuating intensity, a beam splitter (2), a measuring cuvette (3) arranged in the measuring beam, and a reference cuvette (4) arranged in the reference beam, which contains an unchangeable analyte of similar concentration, as expected for the solution to be measured, behind which Sample and reference cuvette in the beam paths arranged detectors (5 and 6) for measuring the light intensity of the partial beams, current-voltage converters (7 and 8) for converting the signals into electrical signals, one multiplier (9 and 10) for each of the two partial signals, one of which effects a signal inversion, and a signal processing and evaluation device.

7. The device according to claim 6, characterized in that the radiation source (1) provides a monochromatic beam.

8. The device according to claim 6 or 7, characterized in that the

Radiation source (1) consists of several radiation sources, the beams of which are combined into a beam by means of suitable optical components.

9. The device according to claim 8, characterized in that the optical components that combine the individual beams in one beam, dielectric beam splitter or optical

Are light guides.

10. Device according to one of claims 6 to 9, characterized in that the signal evaluation device is a Wheatston measuring bridge or a voltage divider circuit (11 and 12).

1 1. Device according to one of claims 6 to 9, characterized in that the signal evaluation device is a ratio generator (15)

12. Device according to one of claims 6 to 1 1, characterized in that a lock-in amplifier (13) and a measured value processing unit (14) are arranged behind the signal evaluation device.

13. The apparatus according to claim 12, characterized in that the measured value processing unit (14) from a microcomputer and a visual

Display device exists.

14. The apparatus according to claim 13, characterized in that the measured value processing unit (14) consists of a microcomputer with a bidirectional telemetric transmission unit.

15. Device according to one of claims 6 to 14, characterized in that the measuring cuvette (3) separated by a diaphragm from the solution to be measured, or to a pump system with an exchange path which is separated by a diaphragm from the solution to be measured , connected.

16. Use of the device according to claim 6 to 15 for measuring process parameters or for monitoring and regulating process sequences, in particular in chemical production processes.

17. Use of the device according to claim 6 to 15 in microreactors.

18. Use of the device according to claim 6 to 15 it in miniaturized design as an implantable glucose sensor.