A biosensor (10) for measuring changes in viscosity, density and/or mass in a fluid to be examined, for example a blood coagulation sensor or immunoassay system. According to the invention, the reagents necessary for the test are contained in a support (13) which is put onto the measurement surface (18) of a piezoelectric element (8). As a blood coagulation sensor, the measurement surface (18) may according to the invention itself have a coagulation activator action or else be coated with coagulation activators.

The invention relates to a biosensor having a piezoelectric element as 
resonant circuit, a measurement surface of which is exposed to components 
of a fluid to be examined by reaction with reagents necessary for the 
test, viscosity and/or density changes in the fluid to be examined and/or 
mass changes due to deposition on the measurement surface of the 
piezoelectric element leading to changes in the resonant circuit 
parameters and being evaluated by a corresponding electronic evaluation 
circuit. 
General comments on quartz crystal resonators are found in the article by 
Zack A. Shana and Fabien Josse in Anal. Chem. 1994, 66, pages 1955-1964, 
entitled "Quartz Crystal Resonators as Sensors in Liquids Using the 
Acoustoelectric Effect". 
EP-A-0,177,858 describes an arrangement with a biosensor, with which it is 
intended to measure the coagulation time of blood. A quartz crystal, which 
is connected as a resonant circuit to an oscillator with fixed frequency, 
is arranged in a measurement chamber that can be sealed with a lid. Before 
introduction into the measurement chamber, the blood to be examined is 
thoroughly mixed with reagents necessary for coagulation, and thereupon 
introduced into the measurement chamber. The coagulation time is 
subsequently measured by evaluating a decrease in amplitude at the quartz 
crystal due to the damping or detuning of the resonant circuit by the 
coagulating blood. The time elapsing before coagulation is measured using 
an electronic stopwatch. This known biosensor has the disadvantage that, 
after a measurement process has been completed, its measurement chamber or 
the measurement surface of the quartz crystal can only be cleaned with 
great difficulty and, in addition, it is necessary to pay particular 
attention to and monitor the time which elapses from mixing the blood with 
reagents necessary for coagulation until application to the measurement 
surface, in order to avoid erroneous measurements. 
The article "A quartz crystal viscosity sensor for monitoring coagulation 
reaction and its application to a multichannel coagulation detector" by 
Muramatsu et al. in Biosensors & Bioelectronics 6 (1991) pages 353-358 
describes a device for determining blood coagulation, in which it is 
necessary to mix the test-specific components, preincubate them at a 
predetermined temperature and apply this mixture to the mass-sensitive 
test face of the quartz crystal. A disadvantage in the case of this method 
is that the influences of manual handling can lead to inaccuracies. 
Biosensors for detecting antigen-antibody reactions, which operate using 
piezoelectric elements, for example a quartz crystal, are also described. 
In the case of such biosensors an antigen-antibody is applied on the 
measurement surface of the piezoelectric element, and the piezoelectric 
element is subsequently immersed the fluid to be examined, in order to 
measure and evaluate the detuning which then takes place of the 
piezoelectric element as resonant circuit. Such biosensors are described, 
for example, in U.S. Pat. Nos. 4,236,893 and 4,735,906. However, these 
described biosensors having an immunocomponent on the measurement surface 
have the disadvantage that the measurement surface of the piezoelectric 
element or quartz is already modified by the coating, so that it is no 
longer possible to measure the frequency of oscillation of the 
piezoelectric element in the unloaded state, which is highly 
disadvantageous in measurements where the piezoelectric element is used as 
mass-sensitive element. 
This group of biosensors, in which application of biological components is 
employed, also includes the following: 
EP-A-0,494,896 describes immunoassays using modified quartz crystal 
microbalances having piezoelectric crystals. These are modified by 
applying biological components on the crystal surface or on a polymer 
intermediate layer. Detection takes place by means of an enzymatic 
amplification mechanism which produces a product that causes a change in 
mass on the crystal surface due to adsorption or due to a reaction with 
the polymer intermediate layer. EP-A-0,408,578 describes immunoassays in 
which an analyte fixing reagent is bound on a substrate surface that is 
fixed by means of a spacer above the mass-sensitive face. Detection then 
takes place as described in EP-A-0,494,896. EP-A-0,295,965 describes 
immunoassay methods in which particle-enhanced tests are carried out. The 
particle-enhanced immunocomplexes are compacted on a modified sensor 
surface, on which binding partners have been immobilized, after an 
immunoreaction by drying or application of a magnetic field, in the case 
of magnetic particles, on the sensor surface. 
The article "Detection of Antistreptolysin O Antibody: Application of an 
Initial Rate Method of Latex Piezoelectric Immunoassay" by Muratsugu et 
al. in Anal. Chem. 1992, 46, pages 2483-2487 describes a method termed 
"Latex piezoelectric immunoassay" wherein, instead of the otherwise 
customary visual detection of the latex agglutination by means of 
turbidimetric and nephelometric methods, use is made of a piezoelectric 
crystal which detects the viscosity or density change in the reaction 
solution. 
The arrangements referred to have the disadvantage that the required 
sensitivities and detection limits can be achieved only by modifications 
of the sensor surface and amplification mechanisms. In addition, operation 
is complex and elaborate. Application for immunoassays requires many 
manual operations which may lead to inaccuracies. Particularly for the 
piezoelectric structures used in coagulation diagnostics, the very complex 
manual operations (mixing the reagents and pre-incubation, application of 
the sample) lead to inaccuracies in the determination of the coagulation 
time of the sample. 
The object of the present invention is to obtain a biosensor having a 
piezoelectric element as resonant circuit, which permits viscosity and/or 
density changes in a fluid to be examined and/or mass changes due to 
deposition on the piezoelectric element, which is of simple design, which 
does not require complex procedural steps during operation and does not 
make prior mixing of the reaction components with the sample to be 
measured necessary, and which leads to an increased measurement accuracy. 
This object is achieved according to the present invention with a biosensor 
according to the preamble of claim 1, in that the biosensor is equipped 
with a coagulation-initiating surface, 
a) it being possible for this surface to be a support which contains the 
reagents necessary for the test, 
b) a coagulation-initiating coating, or 
c) for the support per se to be the surface as such; and 
d) the fluid to be examined can be applied onto the support and comes into 
contact with the reagents and the measurement surface. 
In an advantageous embodiment of the biosensor according to the invention, 
the support is provided with a filter layer, for the separation of 
substances that interfere with the test, on the side remote from the 
measurement surface, or the support is provided with a sample-conditioning 
layer on the side remote from the measuring surface. As a result, any 
interfering suspended matter in the fluids to be examined can be retained 
or the fluid can be conditioned for the test, for example the pH value can 
be adjusted. 
A variant provides that either the sensor surface is used as coagulation 
activator without additional coating or the support itself, without 
containing coagulation activator reagents, already has a coagulation 
activation action, and that the fluid to be examined can be applied onto 
the support and comes into contact with the agents and the measurement 
surface. 
Further advantageous embodiments are described in the claims. 
A biosensor is thereby obtained with which so-called "dry tests" can be 
carried out, i.e. the fluid to be examined can be introduced into the 
biosensor for the test without additional reagents being required, since 
all reagent components or agents are already contained in the biosensor. 
These reagents necessary for the test, already present in the biosensor, 
also do not load the measurement surface of the piezoelectric element, so 
that accurate measurements or tests are possible. 
The biosensor according to the invention is preferably designed as a 
single-use article, which is possible and economical because of the simple 
structure and the concomitant low costs. Preferably, the biosensor can be 
used for determining parameters of the blood coagulation system; it can, 
however, also be used to great advantage for immunoassay. 
When coagulation activator supports are used, the material advantageously 
consists of glass balls, glass particles, glass dust, glass fibers, 
nonwoven glass materials or similar particulate systems which are applied 
directly on the sensor surface or in the measurement space in the form of 
a thin film or in loose form. Use may also be made of other coagulation 
activator substances known per se to the person skilled in the art such 
as, for example, kaolins, feldspars, silicates or other activator 
substances which have a negative surface charge. In the case of such a 
solution it is not detrimental for the substances to rest on the 
measurement surface of the piezoelectric element; surprisingly, this 
contact does not lead to a nominal preloading of the piezoelectric 
element, so that the oscillation parameters can still be measured with 
full accuracy before filling with the fluid to be examined. 
When the support is used for the reagents necessary for the test, the 
support preferably consists of nonwoven material or paper; another 
advantageous solution also consists in the support consisting of a 
synthetic woven or knitted fabric. 
In a further advantageous embodiment of the biosensor according to the 
invention, the coagulation activators are provided with a filter layer, 
for the separation of substances that interfere with the test, on the side 
remote from the measurement surface or the support is provided with a 
sample-conditioning layer on the side remote from the measuring surface. 
As a result, any interfering suspended materials in the fluid to be 
examined can be retained or the fluid can be conditioned for the test, for 
example the pH value can be adjusted. 
If the biosensor according to the invention is used as a blood coagulation 
sensor, then the support contains coagulation-initiating reagents or the 
piezoelectric element is itself used as a coagulation initiator. A further 
variant of the biosensor according to the invention as a blood coagulation 
sensor provides that, instead of a support that carries reagents, a 
coagulation-initiating substance is put onto or applied onto the 
piezoelectric element. 
In the case of immunoassay use, the support contains immunocomponents as 
reagents. The immunocomponents are advantageously bound to particles which 
are known per se to the person skilled in the art from nephelometric or 
turbidimetric methods. These particles can, in similar fashion to the 
coagulation activators, also be put directly onto the measurement surface 
or applied in a readily water-soluble film in the measurement chamber. 
In a preferred measurement arrangement with a biosensor according to the 
invention, an oscillator circuit and a microprocessor circuit as 
electronic evaluation circuit are provided, the piezoelectric element is 
inserted as frequency-determining element into the oscillator circuit and 
the frequency changes are digitally measured by the microprocessor circuit 
and evaluated. In such a case, the microprocessor circuit can take the 
time component into account in the measurement and, by means of 
corresponding programming, carry out a qualitative and/or quantitative 
assessment. In order to obtain particularly stable measurements, the 
biosensor is preferably provided with a temperature-control device for 
keeping its temperature at a constant value. With a measurement 
arrangement with a microprocessor circuit it is possible to employ the 
temperature dependence of the piezoelectric element to measure the 
temperature in the biosensor in order, as a function thereof, to control 
the evaluation or to carry out thermal regulation of the biosensor to a 
constant temperature. 
Further advantageous developments of the invention can be found in the 
subclaims.

The biosensor with sandwich construction as shown in FIG. 1 contains five 
layers. A first layer 1 forms the bottom of the biosensor 2, a second 
layer 2 forms a free space 6 below a piezoelectric element 8, by means of 
a corresponding recess, a third layer 3 serves as support for the 
piezoelectric element 8, a fourth layer forms a measurement chamber 7 
above the piezoelectric element 8, by means of a corresponding recess 9, 
and, finally, a fifth layer 8 forms a seal for the measurement chamber 7 
above the piezoelectric element 8, an access hole 11 in the form of a bore 
permitting the measurement chamber 7 to be filled with the fluid to be 
examined. It is also possible to provide a membrane that is permeable to 
the fluid to be examined instead of the covering layer 5 with a bore 11. 
As can be seen from FIG. 2, the piezoelectric element is arranged on a 
support plate forming the third layer 3, specifically the piezoelectric 
element 8 is bonded into a corresponding recess in the third layer 3 by 
means of an adhesive 12. Both faces of the piezoelectric element 8 are 
provided with electrodes 14 which are connected to terminal faces 16 via 
conductor tracks 15 applied onto the third layer 3. The upper face 
(visible in FIG. 1), given the reference number 18 in FIG. 2, forms the 
measurement surface of the piezoelectric element 8, the face of the 
electrodes 14 being intended to be taken as the density-sensitive or 
viscosity-sensitive or mass-sensitive face. 
A support 13 which rests loosely on the measurement surface 18 of the 
piezoelectric element 8 is fitted into the measurement chamber 7 (see FIG. 
1) of the biosensor 10, i.e. into the recess 9. The support 13 is used for 
holding reagents necessary for the test, with which the fluid to be 
examined is intended to react. During introduction of the fluid to be 
examined via the access hole or the bore 11, the fluid reacts with the 
reagents situated in the support 13 and, together with the reagents, 
reaches the measurement surface 18 of the piezoelectric element 8. This 
thus means that the start of the measurement is exactly established by 
introduction of the fluid to be examined, and the speed with which the 
fluid to be examined is mixed with the reagents and then introduced to the 
biosensor does not, as previously, depend on the skill of the operator. 
The biosensor 10 in sandwich form, represented in FIGS. 1 and 2, can be 
produced in suitable fashion, for example by bonding together the 
individual layers 1-5. The individual layers 1-5 preferably consist of 
synthetic films; but the use of paper for some of the layers is also 
possible. It is further possible to combine some of the layers 1-5, for 
example layers 1 and 2 as well as 4 and 5, the respective recesses for the 
free space 6 and the measurement chamber 7 being produced as an 
impression. 
The biosensor 10 is very simple and inexpensive to produce, so that it can 
be designed and used as a single-use biosensor. The single-use form has, 
primarily, the advantage that the biosensor 10 can be delivered ready for 
use and provided with the reagents necessary for the test; reuse would not 
be expedient with such a design. 
The measurement arrangement shown in FIG. 3 contains an evaluation circuit 
20 which has an oscillator circuit 21, a microprocessor circuit 22 as well 
as the biosensor 10, the piezoelectric element 8 of which is connected to 
the oscillator circuit 21 via the terminal faces 16. The oscillator 
circuit 21 uses the piezoelectric element 8, which is expediently a quartz 
crystal, as frequency-determining element, while the microprocessor 
circuit 22 has a corresponding frequency-measurement arrangement for 
measuring the frequency of oscillation of the oscillator 21. The 
microprocessor can further be programmed and designed in such a way that 
it evaluates the frequency changes or other parameters of the 
piezoelectric element 8 or of the oscillator circuit 21 while taking into 
account the time component of the corresponding changes. By virtue of 
corresponding programming, a suitable evaluation can be carried out on the 
basis of these measured values and can be output on a display or another 
output unit (not represented). 
Since the oscillation parameters, in particular the frequency of 
oscillation, of the piezoelectric element 8 are temperature dependent, it 
is expedient to keep the temperature of the piezoelectric element at a 
constant value. For this purpose, a suitable temperature control circuit 
for the piezoelectric element is provided, the temperature dependence of 
the piezoelectric element 8 being advantageously used for measuring the 
actual temperature. By virtue of a suitable control circuit, which is 
expediently constructed with the microprocessor that is present, suitable 
temperature control can then be produced, which keeps the operating 
parameters of the piezoelectric element constant in the intervals between 
tests. Such a control circuit is not shown in detail. 
Some examples in conjunction with experiments will now be explained below. 
Exemplary embodiment with reference to the determination of the coagulation 
times (prothrombin time) of lyophilized normal human plasma pool and 
lyophilized controls 
Preimpregnation: a paper from the company Macherey and Nagel (MN 215) which 
is immersed in a 0.5% gelatin solution is used as support material for the 
support 13. Excess liquid is removed by rolling off through two metal 
rollers, and the paper is subsequently dried for 60 minutes at 50.degree. 
C. 0.1M calcium chloride solution is applied according to the same 
procedure. Drying is then carried out for 30 minutes, again at 50.degree. 
C. 
Application of the coagulation reagents: 100 .mu.l phospholipon 25 P (100 
g/l, Natterman) and 100 .mu.l tissue factor (1 g/l, Behringwerke AG) are 
mixed with 25 .mu.l 20% Triton X 100 and incubated for 2 hours at 
-20.degree. C. This solution is diluted at 1:5 with an impregnation buffer 
(0.1% HSA, 0.1% Haemaccel, 0.1% Thiocid in 20 mM Hepes pH 7.3) and applied 
onto the paper, preferably by soaking. Drying is then carried out within 
20 minutes at 37.degree. C. 
Determination of the coagulation time of a plasma sample (prothrombin time) 
(Quick's test): 
The paper prepared in this way is cut to a suitable surface area and put 
directly onto the measurement surface 18 of the piezoelectric element 8. 
The piezoelectric element 8 is connected to a suitable electronic circuit 
for excitation and measurement of the natural frequency of oscillation. 
The frequency of oscillation is acquired, for example with a frequency 
counter of the company Keithley (Model 775) with a data link to a data 
acquisition PC. 
In order to determine the coagulation time using Quick's test, Standard 
Human Plasma (Ch.-B. 502 546, Behringwerke AG), Pathoplasma I (Ch.-B. 502 
876, Behringwerke AG) and II (Ch.-B. 502 969, Behringwerke AG) were used. 
The sample (20 .mu.l) is applied to the support 13 and the decrease in the 
frequency of oscillation over time is monitored. The coagulation system of 
the plasma sample is activated by thromboplastin and calcium ions. After a 
delay phase, initiation of the coagulation becomes observable through a 
decrease in the frequency of oscillation of the piezoelectric element 8 
towards low values. With the aid of the typical curve profile recognized 
evaluation methods can be used to determine the coagulation time of the 
samples. 
During the series of measurements, different coagulation times were 
established for the plasma samples used. The measurement results are 
represented in the following table: 
Determination of the prothrombin time of the Pathoplasma I sample with the 
biosensor 10 in comparison with the specified set-point values for 
Standard Human Plasma and Pathoplasma II. (The average values of two 
determinations are specified.) 
TABLE 1 
______________________________________ 
Standard Patho- Sample: 
Human Plasma 
plasma II 
Pathoplasma I 
______________________________________ 
Set-point 
% of standard 
98 13 24.3 
values 
Interval 10-16 21.3-27.3 
Sensor % of standard 
98 13 
calibration 
Value % of standard 23.6 
found 
______________________________________ 
Exemplary embodiment with reference to the determination of whole blood 
A nonwoven glass fiber mat from the company Whatman (GFF) was used as 
coagulation activator for the support. 
Determination of the coagulation time of whole blood (recalcified citrated 
whole blood): 
The nonwoven mat is cut to a suitable surface area and applied directly 
onto the measurement surface 18 of the piezoelectric element 8. The 
piezoelectric element 8 is connected to a suitable electronic circuit for 
excitation and measurement of the natural frequency of oscillation. The 
frequency of oscillation is acquired, for example with a frequency counter 
of the company Keithley (Model 775) with a data link to a data acquisition 
PC. 
80 .mu.l of whole blood are pipetted into the measurement chamber and the 
decrease in the frequency of oscillation over time is monitored. The 
coagulation system of the sample is activated by the coagulation activator 
surface of the nonwoven mat. After a delay phase, the incipient 
coagulation cascade becomes observable through a decrease in the frequency 
of oscillation of the piezoelectric element 8 toward low values. With the 
aid of the typical curve profile recognized evaluation methods can be used 
to determine the coagulation time of the samples. 
Exemplary embodiment relating to the determination of the coagulation time 
of whole blood. 
The piezoelectric element 8 is connected to a suitable electronic circuit 
for excitation and measurement of the natural frequency of oscillation. 
The frequency of oscillation is acquired, for example with a frequency 
counter of the company Keithley (Model 775) with a data link to a data 
acquisition PC. 
80 .mu.l of whole blood (recalcified citrated whole blood) are pipetted 
into the measurement chamber which does not contain any coagulation 
activator substances or a support material, and the decrease in the 
frequency of oscillation over time is monitored. The coagulation system of 
the sample is activated by the coagulation activator surface of the 
sensor. After a delay phase, the incipient coagulation cascade becomes 
observable through a decrease in the frequency of oscillation of the 
piezoelectric element 8 toward low values. With the aid of the typical 
curve profile recognized evaluation methods can be used to determine the 
coagulation time of the samples. 
Exemplary embodiment of a latex-enhanced immunoassay for determining 
rheumatoid factors (Rf test) 
The preimpregnated support materials are impregnated with Rf latex 
reagents, as described in the first example, and used in the same 
structure. When a Rf-positive sample is added, the agglutination reaction 
takes place, which is monitored by the decrease in the frequency of 
oscillation of the piezoelectric element 8. The Rf content is then 
determined either by determining the end point or by means of the reaction 
rate, which is connected with the change in frequency as a function of 
time.