Patent Application: US-3797687-A

Abstract:
improved low - potential electrochemical sensors and method for rapid , accurate , in vitro and in vivo measurement of the concentration of carbohydrates in organic or biological fluids by cyclic voltammetric or coulometric scan within a restricted voltage domain and identifying one or more oxidation and / or reduction current peaks , with the concentration of the carbohydrate being a linear function of the current output . two well defined , sharp , distinctly separated , specific , reproducable and interference - free peaks have been discovered in the low - potential voltage domain of - 0 . 9v to - 0 . 2v , cathodic reduction peak 1 and anodic oxidation peak 2 in the range of - 0 . 70v to - 0 . 90v . the scan is pulsed or a steady sweep , and sensitivity increases with increased scan rate in the range of 30 - 50 mv / sec . pulses may range on the order of 20 - 50 millisecond in duration , and sampling during the last 1 . 5 milliseconds of the pulse restricts values to pure oxidation / reduction currents . cyclic and / or pulsed scanning regenerates the electrodes making the sensor drift free . a system is disclosed employing the sensor as part of an implantable insulin pump which is microprocessor controlled , thus functioning as an artificial pancreas . the sensors can also be used in bedside monitoring systems as an indwelling sensor introduced by catheterization , for in - line extracorporeal shunt systems , or for non - invasive measurement of carbohydrate levels .

Description:
the invention is more particularly described below in connection with the preferred embodiment which illustrates , without limitation , the principles of the invention . while we discuss glucose in most detail , the comments apply equally to other carbohydrates , such as ( but not limited to ) fructose and mannose ( see e . g ., fig4 a and 4b ) for their respective peaks 1 and 2 . fig1 shows in schematic the improved electrode assembly 1 of this invention in which a smooth platinum wire 2 is embedded in a glass rod 4 to form the working / sensing electrode . the bottom tip 5 of the platinum wire is exposed and is in contact with the inner surface of the semipermeable membrane 6 . wound around the exterior surface of the glass rod 4 is a first counter electrode 8 of smooth platinum wire , and a second reference electrode 10 . the two electrode windings are spaced from each other , and are medial of the two ends of the glass rod with the reference electrode being between the counter electrode and the lower end of the glass rod . the lead wires of all three electrodes run up through the glass rod , and emerge through cap 12 which keeps them spaced apart so that they do not short out . the three leads are connected to the cyclic voltammetry and telemetry system 20 which is described in more detail with reference to fig2 . the glass rod electrode holder 4 is disposed within a tubular housing 14 , preferably of glass or an inert plastic material such as polytetrafluoroethylene or trichlorofluoroethylene . apertured plug 16 retains the electrode rod 4 rigidly in position . the lower end of the electrode rod 4 may be held in position by spacers if required . the plug may also be made of the same inert material as the housing 14 . the lower end of the housing wall has a groove 18 to receive an o - ring 19 which retains the membrane 6 across to lower opening of the housing 14 . the annular space is filled with a krebs - ringer phosphate buffer ( krpb ), ph 7 . 4 . the semipermeable membrane 6 is typically a dialysis - type or ultrafiltration - type membrane , and is selected to admit glucose or other low molecular weight carbohydrate to the working electrode 2 , while excluding both middle and high molecular weight substances . suitable membrane materials include cellulose dialysis membranes , hydrogel films , or comparable films . fig2 is a schematic diagram of the apparatus system 20 employed in this invention for the low potential pulsed cyclic voltammetry or coulommetry . the electrode system 1 as described above in detail with respect to fig1 is immersed in a solution 21 containing various concentrations of glucose . for in vitro studies , a solution is contained in beaker 22 . for the in vivo system , this may be implanted , or the body fluid being tested can be passed through an extracorporeal shunt , in which case 22 represents a cuvette or other chamber containing the miniature electrode assembly 1 . digital function generator 23 provides a variable voltage driving mechanism for potentiostat 24 pursuant to the predetermined cyclic program . power is supplied to the potentiostat by power supply 25 , and the scan cycle is controlled by the scan and pulse duration control unit 26 . we prefer to use an eco model 567 digital function generator and an eco 549 potentiostat . the reference electrode was ag / agcl from in vivo metric systems , haroldsburg , calif . the test examples herein were performed at 37 ° c . at scan rates between 30 - 50 mv / sec although we expect a broader scan rate to produce equivalent results . the domain for scanning ranged from - 0 . 2 v to - 0 . 90 v . while we can use normal and differential pulse voltammetry , we prefer to use square wave voltammetry as we can sample the current at peaks 1 and 2 more quickly , and accordingly more nearly continuously . normal and differential pulse techniques typically run at scan rates of 1 - 10 mv / sec . the square wave voltammetry permits scan rates up to 1 v / sec or more , thus permitting a determination of glucose concentration in a matter of seconds . accordingly , we employ a programmable pulse waveform generator 23 for the input pulses ( of duration on the order of 20 - 50 milliseconds ) to the glucose sensing electrode via the potientiostat 24 . we prefer to use an eg & amp ; g princeton applied research model 384b polarographic analyzer . in the technique we use , a symmetrical square wave is superimposed on a staircase wave form where the forward pulse of the square wave ( pulse direction same as the scan direction ) is coincident with the staircase step . the reverse pulse of the square wave occurs halfway through the staircase step . the current is sampled twice during each square wave cycle , once at the end of the forward pulse and once at the end of the reverse pulse . this technique discriminates against charging or capacitance current by delaying the current measurement to the last 1 . 5 milliseconds or so of the pulse . the difference current between the two measurements is plotted versus the potential staircase . as a result , the pulse technique is more sensitive to oxidation or reduction currents , called faradaic currents , than conventional dc voltammetry . the differential pulse voltammetry yields peaks for faradaic currents rather than a sigmoidal wave form of conventional dc voltammery . similarly , square wave voltammetry yields peaks for faradaic processes , and the peak height is directly proportional to the concentration of the species in solution . the current and voltage values as sensed by the electrode may be displayed on combined or separate ammeter and coulometer unit 27 of fig2 . pulsed coulometry is similar , except we measure the charge accumulated during the pulse . we integrate the current during the last 1 . 5 milliseconds or so of the pulse to focus on the purely oxidation and / or reduction currents . the counter and working electrodes provide a varying current which is then plotted on the y axis of a cyclic voltammogram in response to the current cycle impressed by the digital function ( programmable pulse wave form ) generator 23 and potentiostat 24 . similarly , the voltages of the reference and working electrodes are sampled throughout the domain in response to the voltage scan program of the digital function generator 23 , and the output values are plotted on the x axis of the cyclic voltammogram . fig3 and 4a show a series of superimposed cyclic voltammograms of increasing concentrations of carbohydrate in the above - identified in vitro situation . vary sharp oxidation current peaks are seen at - 0 . 72 v for glucose ( fig3 ) and - 0 . 74 v for fructose ( fig4 a ); this is peak 2 . a reduction peak , peak 1 , is seen at - 0 . 80 v for glucose ( fig3 ) and - 0 . 84 v for fructose fig4 a ). both peaks are sharp , distinct , and reproduceable . as shown in fig3 and 4a , the peak height increases with increasing concentration , and the separation of the peaks is extremely clear . unlike all other signals , the peak voltages of these two signals do not shift position ( i . e . and drift free ) with variation in carbohydrate concentrations . the absence of multiple or overlapping peaks in this region indicates that the chemistry is a simple redox . the two clear inflection points indicate the voltages at which the oxidation and reduction currents switch . for comparison to the carbohydrate cyclic voltammogram traces , fig3 and 4a show in dashed lines the trace for the electrolyte - alone buffer . note the complete absence of peaks at - 0 . 80 v and - 0 . 84 v . when the peak current height is plotted against carbohydrate concentration , the relationship is shown to be linear . as shown in fig4 b , 5 , 6 , 7 , 9 and 10 , this linearity is clearly demonstrated for the medically useful range of 0 - 400 mg / dl and beyond . fig4 b shows the relationship for fructose in vitro for both peaks through the range of 600 mg / dl . by the linearity of the plot , precise determinations of carbohydrate concentration can be determined by interpolation on the graph . this can be built into the programming of the output from the sensor ( see fig1 and 2 ) so that there can be direct digital readout of carbohydrate concentration , which permits nearly instantaneous measurement of the concentration of carbohydrate sensed ( glucose , fructose , mannose , etc .). similar results were obtained with both fructose and mannose . the concentrations of the carbohydrates were varied between 0 and 600 mg / dl in krpb . a linear response of current versus carbohydrate concentration was obtained for both substances at both peaks 1 and 2 (- 0 . 84 v and - 0 . 74 v ) with good sensitivity . in vivo glucose sensor use was simulated by placing the electrode in human serum dialysate . human serum ( sera - chem , a normal clinical chemistry control serum from fisher scientific company , pittsburgh , pa ) was dialysed against an equal volume of krebs - ringer phosphate buffer solution ( krpb ) at ph 7 . 4 , below - 4 ° c . for over 24 hrs . the composition of the original serum , the dialysed serum , and the dialysate was determined by independent ( conventional ) analysis . among others , interfering and / or inhibiting substances present in the dialysate included : urea , amino acids , creatinine , and uric acid . both 50 and 100 ml volumes of the serum dialysate were placed in a testing cell of the type shown in fig2 . cyclic voltammetric studies were carried out within a scan range of - 0 . 80 v to + 0 . 80 v versus ag / agcl at 30 mv / sec scan rate . we found that only the low potential redox signal peaks reported herein as peaks 1 and 2 were uninhibited by substances present in the dialysate . while there is a minor alteration of the peaks , fig5 shows that plots of the redox currents versus glucose concentrations of peaks 1 and 2 are linear in the 100 - 200 mg / dl range . all other peaks that we had observed , in particular the peak at + 0 . 70 v , were completely inhibited by substances present in the serum dialysate . we have tested in serum dialysate beyond 500 mg / dl , and although precisely linear for such high concentrations , we have not found evidence of electrode poisoning or decrease in output . this is extremely important as it permits operation of in vivo , bedside , or extracorporeal infusion delivery system for patients in hyperglycemic shock , e . g . with glucose levels as high as 1000 - 1200 mg / dl . the sensor can sense &# 34 ; over limit &# 34 ; ( out of normal range ) conditions , and infuse relatively large amounts of insulin to quickly bring the values within range . thereafter the system can sense glucose levels &# 34 ; on scale &# 34 ; within the normal 0 - 400 mg / dl range and can administrate innfusion at a preprogrammed rate to control the glucose level within a proper metabolic range . fig6 is a plot of the current peak heights for various concentrations of glucose in krpb for the reduction peak , peak 1 , of - 0 . 80 v , for both long scan times and short scan rates . the long scan is 30 mv / sec , whereas the short scan rate is 50 mv / sec . the linear relationship between glucose concentration and current peak height is clearly demonstrated by both scan rates . fig7 shows plots of the peak currents for peaks 1 and 2 (- 0 . 80 v and - 0 . 72 v , respectively ) with respect to the square roots of the scan rates . the linearity clearly demonstrates that the redox is under diffusion control . further , since the curves ( lines ) follow the randles - sevcik relation very well , and the peak - to - peak separation at a scan rate of 30 mv / sec is 0 . 08 v at 37 ° c ., the reaction can be considered a simple , direct , reversible , one electron transfer process which does not involve non - electrochemical or secondary reactions . fig8 shows , in schematic form , an entire prosthetic system employing the glucose sensor of the present invention . the blood glucose sensor assembly ( as described in more detail above in connection with fig1 and 2 ) may be implanted in vivo , for example , in contact with the blood stream , in contact with extracellular fluid , or directly in contact with tissue . the output signal 31 , typically in digital form , is fed to a microprocessor controller which through preprogrammed logic compares the output signal to normal metabolic demand baseline . where the glucose concentration is determined to be too low or too high , the appropriate amount of insulin , and possibly glucagon or glucose , may be metered into the blood stream by one or more infusion pump ( s ) 34 operating at the appropriate command signal 33 in response to the algorithm in the controller 32 . the insulin may be input directly into the portal vein 35 to affect carbohydrate metabolism and glycogen storage in the liver 36 which restores the proper level of blood and tissue glucose 39 via normal circulation 38 . when the blood glucose is low , glucagon and glucose can be directly metered through the systemic vein 37 to restore the blood and tissue glucose level 39 . in turn , the blood glucose sensor assembly 30 continuously samples the flowing blood or tissue fluid 40 to maintain the proper physiologic control of metabolism in patients . alternatively , insulin may be input into a body or tissue space such as the peritoneal cavity , a systemic vein , or the like . fig9 and 10 demonstrate the sensor can precisely determine glucose concentration in the ultrafiltrate of human serum . in these figures , the concentration in mg / dl is plotted against the current in milliamperes for both peaks 1 ( fig9 ) and 2 ( fig1 ) for a scan rate of 30 mv / sec . the temperature of the krpb solution in which the glucose was dissolved was 37 °± 3 ° c . it should be understood that various modifications within the scope of this invention can be made by one of ordinary skill in the art without departing from the spirit thereof . we therefore wish our invention to be defined by the scope of the appended claims as broadly as the prior art will permit , and in view of this specification if need be . 1 . blackshear , p . j ., rohde , t . d ., grotting , j . c ., dorman , f . d ., perkins , p . r ., varco , r . l ., buchwald , h . diabetes . 1979 ; 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