Patent Document

PRIORITY CLAIM 
   This application is a Continuation of International Patent Application PCT/CH01/00126, filed on Feb. 28, 2001, which claims priority to German Application No. DE 100 10 539 A1, filed on Mar. 3, 2000, both of which are incorporated herein by reference. 

   BACKGROUND 
   For measuring blood sugar concentration, viscosimetric affinity sensors have, among other things, been developed which can be miniaturized and used in an implanted form (DE 195 01 159) or as transdermal sensors (DE 197 14 087). 
   Viscosimetric affinity sensors for determining sugar levels are based on a sensitive fluid, a concentrated solution consisting of macromolecular branched dextran and the tetravalent bonding protein concanavalin A (ConA), with the specificity of glucose, being situated in a dialysis chamber coupled to a device for measuring viscosity. The viscosity of the sensitive fluid is high when the dextran molecules are cross-linked via their exposed terminal glucose groups by ConA, and is reduced, dependent on concentration, with the free glucose penetrating the dialysis chamber by diffusion from a glycosuric external solution. 
   A particularly favorable method of affinity viscosimetry involves measuring the viscosity, once dialysis has been performed in the segment of a microdialysis fiber, by measuring the flow resistor of a downstream capillary (DE 197 14 087). A known problem in affinity viscosimetry is that the viscosity of the sensitive fluid is dependent not only on the concentration of glucose but to a large extent also on the temperature and on the concentration of the active glycopexic protein (Ballerstädt and Ehwald, Biosensors &amp; Bioelectronics 9: 557-567, 1994; Ehwald et al., Analytical Biochemistry 234: 1-8, 1996). In order to release the signals of a viscosimetric affinity sensor for glucose from this significant temperature-dependency, relative values having low temperature-dependency can be formed (Ballerstädt and Ehwald, Biosensors &amp; Bioelectronics 9: 557-567, 1994). Up until now, only methods for discontinuously determining the relative values by consecutively measuring the viscosity changed by glucose and the reference viscosity have been known. A method for continuously determining such relative values in a sensor on-line has not been known up until now. 
   Developing a viscosimetric affinity sensor which operates on-line requires a method for preparing readings which converts viscosity-dependent measured values provided by the sensor on-line into glucose concentration. In this connection, the aim is that the sensor detects a measured value which is directly dependent, in a linear relationship, on the glucose concentration and is simultaneously independent of the temperature and the concentration of active ConA in the sensitive fluid. The method to this effect has not been known up until now. 
   SUMMARY 
   An object of the invention is to provide a method and a sensor for determining sugar levels by affinity viscosimetry, which allow a parameter which is largely independent of the temperature and the concentration of ConA and directly proportional to the concentration of sugar to be detected on-line. 
   The object is addressed by the method for affinity viscosimetry and by a viscosimetric sensor in accordance with the present invention. 
   In one embodiment, the present invention comprises a method and apparatus for determining solute levels by affinity viscosimetry involving a sensitive fluid, in which the sensitive fluid flows continuously through a first hydraulic resistor in the flow direction of the dialysis chamber, and the sensitive fluid modified by dialysis simultaneously flows through another resistor, wherein the pressure differences between the resistors is determined on-line with the aid of pressure sensors and converted into a relative value which is approximately proportional to the concentration of solute. 
   In one embodiment, the present invention comprises a method and apparatus for determining sugar levels by affinity viscosimetry, in which the sensitive fluid flows continuously through a first hydraulic resistor in the flow direction of the dialysis chamber, and the sensitive fluid modified by dialysis simultaneously flows through another resistor, wherein the pressure differences between the resistors is determined on-line with the aid of pressure sensors and converted into a relative value which is approximately proportional to the concentration of sugar. 
   In accordance with the invention, it is advantageous if the sensitive fluid flows continuously, having a defined sugar content or having no sugar content, through a hydraulic resistor in the flow direction of the dialysis chamber, the reference resistor, and the sensitive fluid modified by dialysis simultaneously flows through another resistor which is approximately isothermal with the reference resistor, the measuring resistor, wherein the pressure difference which drops away across the reference resistor and the measuring resistor is determined on-line with the aid of pressure sensors and converted into a relative value approximately proportional to the concentration of sugar. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  depicts possible variants for the arrangements of the resistors in a sensor in accordance with the invention; 
       FIG. 2  is a schematic representation of a sensor in accordance with the invention for determining sugar levels by affinity viscosimetry; and 
       FIGS. 3   a  and  3   b  are diagrams showing relative viscosity and relative fluidity against glucose concentration. 
   

   DETAILED DESCRIPTION 
     FIG. 1  schematically shows a few of the possible variants for the arrangements of the resistors, the dialysis chamber and the pressure sensors needed for measuring. The arrow represents the pressure pump (Variants  1 ,  2  and  4 ) or suction device (Variant  3 ) with the generated flow direction of the sensitive fluid or solution. 
   The reference resistor Rr, the dialysis chamber D and the measuring resistor Rm can lie in succession on a flow path ( FIG. 1 , Variants  1  to  3 ), or the reference resistor lies on one flow path and the dialysis chamber and the measuring resistor lie together on a parallel flow path ( FIG. 4 , Variant  4 ). 
   If the reference resistor and the measuring resistor lie on one flow path, one pump or suction device is sufficient, and the drop in pressure across the two resistors can be detected by a suitable arrangement of pressure sensors P 1  and P 2  ( FIG. 1 , Variants  1  to  3 ). If the reference resistor and the measuring resistor lie on two parallel flow paths, they are connected in accordance with the invention to one or more pump or suction devices which maintain a constant relation between the flows on the two flow paths ( FIG. 1 , Variant  4 ). 
   To simultaneously measure the drop in pressure across the reference resistor and the drop in pressure across the flow resistor, pressure sensors can be suitably arranged on the flow path, wherein the deformable membranes of these pressure sensors must lie either between the atmosphere and a measuring point on the flow path ( FIG. 1 , Variants  1 ,  3  and  4 ) or between two different measuring points on the flow path (P 1  in  FIG. 1 , Variant  2 ). 
   If the measuring resistor and the reference resistor are measured simultaneously, the ratio of measuring resistor and reference resistor is known to provide a relative, temperature-dependent viscosity (Ballerstädt and Ehwald, Biosensors &amp; Bioelectronics 9: 557-567, 1994), which does not, however, decrease linearly with the glucose concentration and is not suitable for calculating glucose levels from the measured resistor values in a sensor. The relative fluidity (“RF”) calculated in accordance with the invention therefore represents the quotient between the drop in pressure at the reference resistor and the sum of the drop in pressure at the measuring resistor and a correction value leading to a linearization of the correlation with the glucose concentration. The relative fluidity is a relative value which is independent of temperature and ConA concentration and which has a linear relationship to the glucose concentration. 
   It is important for forming the temperature-independent relative value for the flow resistors cited to be kept isothermal. This may be achieved by contacting the two resistors with the body or with an additionally temperature-stabilized device, or by having the two resistors in joint contact with a good heat conductor. For the method in accordance with this embodiment, it is furthermore necessary for the expandable volume content of the flow path between the reference resistor and the measuring resistor to be smaller than the volume of sensitive fluid moved by the pump or suction device within a particular period of time corresponding to the measuring task, since otherwise the change in pressure at the measuring resistor responds too slowly to the change in viscosity. This period of time should not be longer than 15 minutes. 
   Exemplary Application: 
   In the equipment shown in  FIG. 2 , sensitive fluid is moved through a flow channel at a constant speed (5 μl/h) by means of a pump, wherein a pressure sensor for measuring the pressure p 1 , the hydraulic reference resistor  3 , a pressure sensor  4  for measuring the pressure p 2 , a dialysis probe  5 , the hydraulic measuring resistor  6  and a collecting vessel  7  for the used sensitive fluid are situated in succession on said flow channel. In short intervals, the pressures p 1  and p 2  are measured simultaneously and stored, assigned to the time of measurement, by means of a programmable evaluation unit  8 . The difference p 1 −p 2  is then the drop in pressure across the reference resistor, and p 2  the drop in pressure across the measuring resistor. From these pressure values, the evaluation unit calculates the relative fluidity and/or, with the aid of settable constant calibration parameters, the glucose concentration, and displays this on a display. 
   If the quotient Q is the ratio of the drop in pressure across the reference resistor to the drop in pressure across the measuring resistor, RF may be calculated according to the formula:
 
 RF=Q /(1+ kQ )  (Equation 1) 
 
where the constant k is a linearization parameter dependent on the sensitive fluid and the ratio of the resistors, and is determined iteratively for the best correlation between the RF values and the corresponding values of a glucose concentration series ( FIG. 3   b ). As opposed to the relative viscosity ( FIG. 3   a ), the relative fluidity defined by Equation 1 is proportional to the glucose concentration.
 
   In the foregoing description embodiments of the invention have been presented for the purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form or steps disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiments were chosen and described to provide the best illustration of the principals of the invention and its practical application, and to enable one of ordinary skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention as determined by the appended claims when interpreted in accordance with the breadth they are fairly, legally, and equitably entitled.

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