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The present invention relates to a method of molecular spectroscopy,
particularly for the determination of products of metabolism, in which the
absorption of infrared radiation by a specimen containing a substance to
be determined is measured, as well as to an apparatus for the carrying out
of this method.
The method and the apparatus of the invention can be used in particular for
the determination of glucose in serum or in urine. Such a determination is
of great importance for the recognition of diabetes mellitus and as
check-up on the treatment thereof. The known determination by means of
test strips in urine serves primarily to detect the disease. Although a
well-stabilized diabetic can, in case of regular examination of the urine,
get along with a few spot checks of blood glucose, the determination of
the concentration of glucose in the blood is essentially of importance for
treatment, verification of treatment and adjustment of the daily profile.
The known methods for the determination of glucose can be divided into
three groups, namely biochemical, electrochemical and spectroscopic
methods. Of these methods, the biochemical methods are suitable only as
laboratory procedures; the electrochemical methods, to be sure, at present
afford the most promising prospects for the development of implantable
glucose sensors but are inferior in their precision and specificity to the
spectroscopic methods, as the biochemical methods also are.
The biochemical and electrochemical methods furthermore have the common
disadvantage that the specimen to be measured must be so prepared by
addition of chemicals before the actual measurement process as to form
reaction products which can be detected in the measurement. Thus
continuous measurements are not possible.
In contradistinction to the biochemical and electrochemical methods, the
glucose molecule is not modified in the spectroscopic method.
In addition, there are still a few non-specific methods which are
practically no longer used. Thus, for instance, from German Pat. No. 2 724
543 a method is known for determining glucose based on the long-known
polarimetry, which, however, is today rarely used because of insufficient
specificity, it being suitable at most under favorable conditions for the
determination of sugar in the urine.
A first possibility of determining products of metabolism by spectroscopic
measurement is represented by laser Raman spectroscopy. The frequency of
the exciter radiation is here in the visible spectral range. For the
measurement, the portion in the spectrum of the scattered light which is
shifted towards the red is used.
In measurements in whole blood the difficulty is encountered in this method
of measurement that, due to the hemoglobin and the other chromophoric
substances, blood exhibits strong absorption throughout the entire visible
spectral range, which, to be sure, leads to a reinforcement of the
resonance of the Raman scattering for these molecules but has a high
fluorescence background associated with it and thus makes the detection of
non resonance-reinforced bands difficult if not entirely impossible. This
problem, to be sure, can be solved with the presently available
possibilities of exciting the Raman scattering with short laser pulses in
the subnanosecond range; however, the technical expenditure is so
substantial that it will not be possible in the foreseeable future to find
any practical method with it for samples of whole blood.
Another possibility of avoiding the disturbance by fluorescence of the
chromophores consists in operating in the infrared spectral range, i.e. in
recording directly the infrared spectrum of the specimen. However, since
the preparation of the specimen as well as the recording and evaluation of
the spectrum are time-consuming and complicated, infrared spectroscopy of
the type customary up to now does not constitute competition for the other
methods which exist.
Another spectroscopic method is the so-called attenuated total reflectance
(ATR) spectroscopy, i.e. total reflectance spectroscopy with transversely
attenuated wave.
Thus it is known, for instance, from West German Application for Patent No.
2 606 991 to use a CO.sub.2 laser in combination with the well-known ATR
spectroscopy to determine glucose or else other products of metabolism.
This can be done, however, only in pure solutions; in multi-component
systems or even in whole blood this method necessarily fails, due to basic
difficulties.
Thus, for instance, the Lambert-Beer law for ATR spectroscopy applies only
for an absorption coefficient within the range of 0.1 and less and for an
angle of incidence of approximately 60.degree. or more, referred to the
materials customary in IR, and furthermore only in case of sufficiently
large wave lengths, and even then only approximately for isotropic
specimens. Even upon in-vitro measurements in whole blood therefore
difficulties would already arise caused, for instance, also by the protein
adsorption on the reflection surfaces, which convert the system into
anisotropy.
In spectroscopic measurements in the infrared spectral region, the
absorption contribution of the solvent or embedment material for the
substance to be determined is customarily compensated for by the use of a
reference ray in addition to the actual measurement ray, infrared
radiation of the same wavelength being used in both rays. The required
reproducibility of the measurement in the reference ray, however, requires
pretreatment of the specimen and is not directly applicable to substances
to be examined which are present in the natural biological milieu.
The object of the present invention is now to provide a method for
determining products of metabolism which makes possible quantitative
reproducible measurement in the natural biological milieu without
pretreatment of the specimen and therefore also without consumption of
chemicals, and in addition requires only small amounts of substance.
Proceeding from the basis of the method described in the preamble to claim
1, this object is achieved in the manner that measurement is effected
simultaneously at two different wavelengths of the infrared radiation, the
first wavelength being selected in such a manner that upon changes in
concentration of the substance to be determined in the specimen no change
in the infrared absorption or only a negligible change occurs, while the
second wavelength is so selected that it lies in the region of a
substance-specific absorption band of the substance to be determined and
that the quotient of the absorption values measured at these two
wavelengths is formed.
The method of the invention can therefore be characterized as a
two-wavelength method in which the first wavelength lies in a
"quasi-isosbestic" range while the other wavelength lies in the range of a
substance-specific absorption band, in such a manner that in the case of
this latter wavelength a change in absorption is caused only by a change
in concentration of the substance examined. In general, wavelengths of
these properties can be readily selected on basis of existing infrared
absorption spectra of the specimens in question since in addition to the
substance-specific absorption bands there are quasi-isosbestic ranges also
in multi-component mixtures.
By means of the method of the invention it is possible, for instance, to
determine glucose in human whole blood rapidly and dependably. It is also
possible to measure other products of metabolism such as, for instance,
ethyl alcohol, urea, uric acid, creatinine, peptide decomposition
products, polystyrol and lipids in the blood or in other body liquids. The
measurement can also be effected in dialysates, i.e. in liquids which are
not body fluids but contain metabolism products. One such dialysate is,
for instance, the liquid used for dialysis in kidney patients.
The method of the invention can be carried out by determining the
absorptions at the two different wavelengths by means of transmission
spectroscopy or by means of reflectance spectroscopy, and particularly ATR
spectroscopy. Transmission measurements have the advantage over
measurements by means of ATR spectroscopy, for instance in the case of the
determination of glucose in whole blood, that the Lambert-Beer law applies
here and that they can be calibrated by biochemical absolute-determination
for the obtaining of quantitative measurement values.
The sample to be measured is advisedly present in the form of a smear or
film on a disposable or throw-away specimen support of plastic. The
support material is so selected that it has only a slight absorption of
its own in the region of the first and second wavelengths. The specimen
support can be developed as a flat microscope slide, but also possibly as
a flow-through or trough cell.
As already mentioned, there are also quasi-isosbestic regions in
multi-component mixtures so that a wavelength can be found for which even
upon changes in concentration of several substances in the specimen no
change in the infrared radiation absorption takes place or only a
negligibly small change. In this way, several substances in the specimen
can be determined one after the other in one and the same apparatus by
changing the first wavelength in such a manner that it lies in each case
within the region of a substance-specific absorption band. In each case
the measurement signal is standardized, the specimen itself serving as
reference.
An apparatus for the carrying out of the method of the invention is
characterized by a source of infrared radiation for the production of an
infrared radiation beam which consists of infrared radiation of at least a
first and a second wavelength, the first wavelength being so selected that
upon changes in concentration of the substance to be determined in the
specimen no change in the infrared radiation absorption takes place or
else only a negligibly small change while the other wavelength is so
selected that it lies in the range of a substance-specific absorption band
of the substance to be determined, by a detector device for the separate
measurement of the absorption values of the infrared radiation of the
different wavelengths, and by a division circuit arranged behind the
detector device for forming the quotient of the absorption values
ascertained.
The source of infrared radiation can contain a strong continuum radiator or
one or more lasers, in which connection gas or solid lasers can be used.
For the separating out of the two wavelengths used for the measurement
interference filters can be provided or suitably developed beam splitters.
It is advantageous to use only one detector on the reception end and to
make the radiation beams of different wavelength distinguishable by
different modulation.
By the formation of the quotient of the absorption values which are
measured for the two wavelengths one obtains a standardized measurement
signal, the specimen itself serving as reference.
Further features of the invention are set forth in the subordinate claims.
The invention will be described in further detail below with reference to
FIGS. 1 to 12 of the accompanying drawings, in which:
FIG. 1 is an infrared absorption spectrum of water;
FIG. 2 is an infrared absorption spectrum of heparin;
FIG. 3 is an infrared absorption spectrum of D-glucose;
FIG. 4 is an infrared absorption spectrum of dried human whole blood the
glucose content of which is within the normal physiological range;
FIG. 5 is an infrared absorption spectrum of dried human whole blood which
has been enriched with D-glucose to such an extent that the glucose
content lies within the pathological range;
FIG. 6 shows a first embodiment of an apparatus in accordance with the
invention;
FIG. 7 shows a second embodiment of an apparatus;
FIG. 8 shows a third embodiment of the invention with a modified source of
infrared radiation;
FIG. 9 shows a fourth embodiment of the invention;
FIG. 10 shows a fifth embodiment of the invention;
FIG. 11 shows transmission curves of two single-band interference filters
such as used in the embodiments of FIGS. 6, 7 and 9; and a transmission
curve of a double-band interference filter such as used in FIG. 8; and
FIG. 12 shows the transmission curves of a wavelength-selective beam
splitter such as provided in the embodiments of FIGS. 7 and 9.
The selection of the two wavelengths used for the measurement will be
described in connection with FIGS. 1 to 5, using the determination of
glucose as example. One essential difficulty in this determination in
whole blood or urine is that body fluids consist of water to a high
percentage. Water, however, has an absorption coefficient of 700 cm.sup.-1
in the region of the glucose absorption in the infrared. Glucose, on the
other hand, has an absorption coefficient of merely about 0.1 cm.sup.-1 in
this range. This difficulty, which is present in general in IR
measurements in aqueous solutions, is customarily avoided by a so-called
double-beam method. In it the absorption of the solvent or the embedment
means is compensated for by a reference ray path in which a specimen which
consists only of water is present. By the use of lasers instead of the
conventional sources of light this problem can be reduced further.
The double-beam method, however, has the fundamental disadvantage that a
complicated preparation of the specimen is necessary in order to make
meaningful use of the reference ray path possible.
In the method of the invention, a double-wavelength method is used instead
of the double-beam method. In it, the absorption of the specimen is
determined simultaneously at two different wavelengths. The two
wavelengths are so close to each other that dispersive effects can be kept
small. This permits of measurement in the biological medium without
complicated and tedious prior treatment of the specimen.
The first wavelength .lambda..sub.1 is selected in such a manner that upon
changes in concentration of the substance to be examined in the specimen
only a negligibly small change of the absorption, if any, takes place,
i.e. .lambda..sub.1 should lie at the "isosbestic point" or in a
"quasi-isosbestic" region.
Such a quasi-isosbestic region is indicated in FIGS. 4 and 5 by the lines
41 and 42. It can be seen that in this region upon a change of the glucose
concentration there is only a negligibly small change in the absorption.
This absorption, therefore, corresponds to the fundamental absorption of
the specimen and is suitable for the standardization of the measurement
signal.
The line 41 corresponds to the value 940 cm.sup.-1 and the line 42 to the
value 950 cm.sup.-1.
The second wavelength .lambda..sub.2 is selected in such a manner that it
lies on a substance-specific absorption band. This condition is satisfied
by wavelengths in the region between the two wavelengths designated 51 and
52 in FIGS. 1 to 5. Line 51 corresponds to a value .lambda..sub.2 of 1090
cm.sup.-1 and line 52 to 1095 cm.sup.-1. From FIGS. 3 and 2 it can be seen
that the region indicated lies on an absorption band of glucose and at the
same time, however, in the region of minimal absorption for heparin. The
mucopolysaccharide heparin is present in whole blood in a relatively high
percentage in the basophilic leucocytes. In the normal case, the influence
is not too high, about 0.5%, but in borderline pathological situations
with increased number of leucocytes or else after administration of
heparin as an anticoagulant a substantial disturbance of the glucose
measurement can take place. By the selection of the wavelength
.lambda..sub.2 shown, disturbance of the measurement by heparin in the
specimen is avoided.
If a CO.sub.2 laser is used as source of radiation the .lambda..sub.2 lines
correspond to the laser lines R (40) to R(52) and the .lambda..sub.1
wavelength range lies between the CO.sub.2 laser lines P(14) to P(26). The
wavelength selection is advisedly effected by suitable interference
filters which can be produced in these regions in accordance with the
prior art with a half width of about 5 cm.sup.-1. Instead of a CO.sub.2
laser there may also be used a semiconductor laser, for instance a
Pb.sub.1-x --Sn.sub.x --Te-- or a Raman laser, or else a continuum
irradiator with frequency selection. The detection of the measurement
signal is effected with the methods and apparatus customary in the prior
art.
The embodiments of devices for the determination of products of metabolism
shown in FIGS. 6 to 10 can be used both for transmission spectroscopy and
for ATR total reflect and spectroscopy with transversely attenuated
infrared light waves. In this connection, the specimen carrier 1, which
has been only schematically indicated, in or on which the specimen 2 is
provided is made preferably either as microscope slide or as cell from,
for instance, a copolymer of polyethylene and polypropylene, depending on
whether the specimen 1 is irradiated vertically or horizontally.
Each of the embodiments shown has a source of infrared radiation 3 and a
detector device 4. Within the infrared radiation beam 5 which forms the
ray path between the source 3 and the detector 4 there is located the
specimen carrier 1 bearing the specimen 2 to be measured.
The source of infrared radiation 3 is developed in such a manner that it
produces the infrared radiation beam 5 which consists of infrared
radiation of a first and a second wavelength .lambda..sub.1 and
.lambda..sub.2 respectively. The detector device 4 is developed in such a
manner that it measures separately the absorption or intensity values of
the infrared radiation of the different wavelengths .lambda..sub.1 and
.lambda..sub.2. Behind the detector device 4 there is provided a divider
circuit 6 to which the two signals I.sub.1 and I.sub.2 determined by the
detector device 4 are fed and which forms the quotient Q=I.sub.2 /I.sub.1,
in which I.sub.2 is the absorption or intensity value corresponding to the
wavelength .lambda..sub.2 while I.sub.1 is the absorption or intensity
value corresponding to the wavelength .lambda..sub.1. Behind this divider
circuit 6 there is an indicating and/or recording device 7 which can be
developed, for instance, as recorder, printer or digital or analog display
instrument or the like.
In the embodiment shown in FIG. 6, the infrared radiation source 3 has a
strong continuum radiator 8 such as, for instance, a Globor or Nernst rod.
By means of a diaphragm 9 a first and a second infrared radiation beam 10
and 11 respectively are stopped out. For the selecting of the two
wavelengths .lambda..sub.1 and .lambda..sub.2 from the continuum spectrum
of the source of radiation 8 a first interference filter 12 is arranged in
the ray path of the individual ray 10, it passing essentially only
radiation of the wavelength .lambda..sub.1, while in the ray path of the
second individual ray 11 there is arranged a second interference filter 13
which passes substantially only radiation of the wavelength
.lambda..sub.2.
The basic course of the transmission function of the interference filters
12, 13 is shown in the upper and middle parts of FIG. 11 in which the
transparency T in percent is plotted over the wavelength .lambda. in
.mu.m. The half-width value .DELTA..lambda. should be smaller than or
equal to 1% of the corresponding wavelength .lambda..sub.1 or
.lambda..sub.2 which is to be transmitted.
The two individual rays 10, 11 are combined by mirrors 14 and a wave-length
selective beam divider 15 to form a single infrared radiation beam 5 after
they have previously been modulated by a chopper 16 with different
frequencies f.sub.1 (modulation frequency of the infrared radiation beam
10) and f.sub.2 (modulation frequency of the infrared radiation beam 11)
respectively. The fact that different frequencies result is indicated in
the manner that the chopper disk 17 of the chopper 16 cuts the infrared
radiation beam 11 at a place located closer to the axis of rotation 18 of
the chopper 16 than the infrared radiation beam 10, and the chopper blade
for the two beams (10, 11) has a different number of segments.
The transmission function of the wavelength-selective beam splitter 15 is
shown in FIG. 12 in which the transparency T and the reflectivity R are
plotted, in each case in percent, over the wavelength in .mu.m, and the
wavelengths .lambda..sub.1 and .lambda..sub.2 entered.
The radiation beam 5 passes through the specimen 2, which may for instance
be a smear of a drop of blood, on a specimen holder 1 developed as a
microscope slide. This slide may consist, for instance, of a copolymer of
polyethylene and polypropylene. After passage through the specimen 2, the
infrared radiation beam 5 passes into the detector device 4 and comes here
against a single detector 19 which has practically the same sensitivity in
the range in which the wavelengths .lambda..sub.1 and .lambda..sub.2 are.
For example, the detector 19 can be a pyroelectric receiver, such as for
instance a triglycin-sulfate crystal, referred to in abbreviated fashion
also as TGS crystal. This practically identical sensitivity is necessary
in order that the following electronic system can be operated with the
same time constants. The output signal of the detector 19 is fed to two
parallel lock-in amplifiers 20, 21 (phase-sensitive rectifiers with
possibly amplifier behind same), one of which is tuned to the modulation
frequency f.sub.1 of the infrared radiation portion of the wavelength
.lambda..sub.1 and the other to the modulation frequency f.sub.2 of the
infrared radiation portion of the wavelength .lambda..sub.2. At the output
of the lock-in amplifier 20 the abovementioned intensity value I.sub.1 or
a signal proportional to it is obtained, while at the output of the
lock-in amplifier 21 there is available the above-indicated intensity
value I.sub.2 or a signal proportional to it. These two signals are fed
into the following division circuit 6 which produces therefrom the
standardized concentration-proportional signal Q=I.sub.2 /I.sub.1.
In the embodiment shown in FIG. 7, the chopper 16 is so arranged and
developed that it modulates two individual rays 10 and 11 with the same
chopping frequency f. Accordingly a second wavelength-selective beam
splitter 22 is provided in the corresponding detector device 4 and it
divides the sole infrared beam 5 impinging upon it into a first individual
beam 23 of the wavelength .lambda..sub.1 and a second individual beam 24
of the wavelength .lambda..sub.2. The first individual beam 23 comes onto
a detector 25 and the second individual beam 24 strikes a second detector
26. These detectors 25, 26 may be of the same type as the detector 19 of
FIG. 6.
The lock-in amplifiers 27 and 28 arranged behind the two detectors 25, 26
respectively, both of which amplifiers are tuned to the modulation
frequency f, in their turn produce at their outputs intensity values
I.sub.1 and I.sub.2 respectively from which the above-indicated quotient Q
is formed in the division circuit 6.
FIG. 8 shows an embodiment in which only a single radiation beam, namely
the infrared radiation beam 5, is stopped-out by the diaphragm 9 from the
radiation of the source of radiation 8, which is also developed as
continuum radiator. This beam passes through a double-band interference
filter 29 and the chopper disk 17 of a chopper 16 and then passes through
the specimen 2. The transmission curve of this double-band interference
filter 29 is shown in the lower part of FIG. 11 and, as can be noted from
a comparison with the middle and upper parts of FIG. 11, represents a
combination of the transmission curves of the two interference filters 12
and 13 which are used in the embodiments of FIGS. 6 and 7.
The detector device 4 in FIG. 8 can be developed in the manner shown in
FIG. 7.
FIG. 9 shows an embodiment in which the infrared radiation source 3 has as
source of light a laser 8, for instance a CO.sub.2 laser or a Raman laser
with multi-line emission. This laser is provided with a beam-widening
device 30. By means of the diaphragm 9 a single radiation beam, namely the
infrared radiation beam 5 is stopped-out and is modulated before it passes
through the specimen 2, by means of a chopper 16 with a chopping frequency
f.
The detector device 4 is essentially of the same construction as shown in
FIG. 7; however, for purposes of wavelength selection a first interference
filter 12 is arranged in the ray path of the first individual beam 23
between the wavelength-selective beam splitter 22 and the first detector
25 so that the latter receives only infrared radiation of the wavelength
.lambda..sub.1 while a second interference filter 13 which passes only
radiation of the wavelength .lambda..sub.2 is provided in the ray path of
the second individual beam 24 between the wavelength selective beam
splitter 23 and the second infrared radiation detector 26.
The embodiment of FIG. 9 can also be modified in such a manner that by the
splitting up and recombination of the radiation beam emerging from the
beam widening device 30, with selection of the wavelengths .lambda..sub.1
and .lambda..sub.2 and of a double-frequency modulation by means of a
chopper 16 in accordance with the embodiment of FIG. 6, it is possible to
use a detector device 4 of the type shown in FIG. 6 which requires only a
single detector 19.
In FIG. 10 there is shown an embodiment in which two monochromatic lasers 8
are provided, one of which has the emission wavelength .lambda..sub.1 and
the other the emission wavelength .lambda..sub.2. These lasers 8 can, for
instance, be Pb.sub.1-x Sn.sub.x Te lasers so that, after passage of the
laser radiations through the beam-widening device 30, a first individual
beam 10 is produced by the diaphragm 9, which beam contains only infrared
radiation of the wavelength .lambda..sub.1, as well as a second individual
beam 11 which contains only infrared radiation of the wavelength
.lambda..sub.2. These two individual beams are modulated in a manner
similar to that described in FIG. 6 with two different chopping
frequencies f.sub.1 and f.sub.2 respectively and are combined to form a
common infrared radiation beam 5 by a mirror 14 and a wavelength-selective
beam splitter 15. The detector device 4 can in this embodiment be of the
type shown in FIG. 6 but it can also be developed in the manner shown in
FIG. 7 provided that the two individual beams 10, 11 in FIG. 10 are
modulated with the same chopping frequency f.
The specimen space can also be developed as a flow-through cell of small
layer thickness. This is particularly of interest when the method is to be
used for continuous measurement, for instance upon dialysis with an
artificial kidney. In this case of use what is of interest is less the
glucose determination than rather the determination of peptide fission
products, defect hormones, urea, uric acid and creatinine in the
dialysate. The isosbestic or quasi-isosbestic region for .lambda..sub.1 is
in this connection substantially the same as in the glucose measurement
but the second wavelength .lambda..sub.2 must be selected in each case in
accordance with the substances. The optical and electrical construction of
the evaluation apparatus itself is selected in accordance with one of the
embodiments shown.
It is also possible to determine several substances simultaneously with the
method of the present invention. In that case more than two wavelengths
must then be radiated simultaneously through the specimen, one of these
wavelengths lying in an at least quasi-isosbestic region and the others
being selected in each case adapted to the substances to be investigated.
Thus it is readily possible, for instance, to determine glucose, ethyl
alcohol, uric acid and creatinine simultaneously with the use of five
different wavelengths. The standardization is effected in each case by the
fifth wavelength, which lies in a quasi-isosbestic range, for instance in
the range shown in FIGS. 1 to 5.
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