Development of a Stable, Multi-Use Creatinine Biosensor with Extended Use Life

Whole blood creatinine level is one of the major markers for renal function.  For risk assessment of patients with chronic kidney disease (CKD) in radiology, or frequent monitoring of critically ill patients for acute kidney injury, a rapid whole blood creatinine testing at point of care test (POCT) environment is gaining more and more attention recently [1, 2, 3].  It is believed the readily available creatinine results could benefit the radiology operation and in general improve the quality and timeliness of patient care [4].

                GEM Premier 4000 (Instrumentation Laboratory) is a whole blood analyzer for critical care and point of care testing (POCT).  The multi-use cartridge contains a sensor array and calibration reagents for measured parameters of pH, blood gases (pO2, and pCO2), electrolytes (Na⁺, K⁺, Ca⁺⁺, and Cl^-), metabolites (glucose, and lactate), hematocrit, and Co-oximetry.  We have developed a creatinine biosensor that accurately and rapidly measures creatinine concentrations directly in whole blood.  This creatinine sensor can be integrated into the current cartridge and provide POCT creatinine results.

                Through cascading reaction mechanism, the creatinine sensor uses three enzymes (creatininase, creatinase and sarcosine oxidase) to convert creatinine in whole blood sample to hydrogen peroxide.  The resulting hydrogen peroxide is detected by amperometric oxidation at a platinum electrode.  A creatine sensor comprised of only creatinase and sarcosine oxidase is used to correct for interference from endogenous creatine in blood sample using a differential measurement.  Other interfering compounds such as ascorbic acid and acetaminophen are rejected by an electro-polymerized barrier layer generated in situ over the platinum electrode.  To extend the sensor’s linearity and use life, a diffusion-limiting polymeric outer membrane is applied over the enzyme.

                Creatinine biosensors typically suffer from short use life due to limited enzyme stability in the challenging test environment.  This was no exception during the early stage in our sensor development.  When cartridges were installed in the analyzer, the creatinine sensor showed high initial sensitivity but it decreased rapidly over time.  When multiple creatinine sensors were fabricated in the sensor array, the one closer to the counter electrode lost sensitivity much faster than the one further away from the counter electrode.  In our multi-sensor array, a common silver counter electrode was shared among all amperometric sensors of glucose, lactate, pO2, creatinine, and creatine.  The half reaction on the counter electrode was determined by the net current passing the electrode.  Our hypothesis is that silver ions produced at the counter electrode can reach the enzyme layer by permeating through the outer sensor membrane.  Because silver ion is a potent inhibitor to many enzymes including creatininase, it is likely to be responsible for the sensor’s sensitivity loss over its use life.  To confirm this hypothesis, surface elemental analysis (LA-ICPMS) were performed on multiple creatinine sensor arrays before, and after the end of their use life.  Various levels of silver were detected on all creatinine sensors.  Strong correlations were found between the level of silver and the rate of sensitivity loss of each sensor, and much higher silver levels were found on the sensors closer to the counter electrode.

                As a result of the investigation, a solution was implemented by replacing the silver counter electrode with gold.  Multiple batches of sensors were produced with gold counter electrode and the stability of creatinine sensor was significantly improved.  No adverse effects on the other sensors were observed.

                Stable analytical performance of the new creatinine sensor was demonstrated in GEM Premier 4000 analyzers after appropriate modifications to calibration solutions and software were made to accommodate the creatinine and creatine sensors.  Heparinized whole blood samples from healthy individuals were collected and spiked with a concentrated creatinine solution to cover a concentration range from 1.0 to 15 mg/dL.  Using a sample volume of 150 µL, the creatinine concentration readings were available from the analyzer 80 seconds after the sample introduction.  The samples were tested in parallel on an ABL 800 Flex analyzer (Radiometer) as reference method.  A total of 303 whole blood samples were tested on 11 cartridges during the third week of sensor’s use life.  Good agreement was found between the GEM and the ABL.  The linear regression results yielded a slope of 1.027 with an intercept of 0.015 (r²=0.990) over the tested range. 

                The creatinine sensor, when integrated into the GEM Premier 4000 system, showed excellent analytical performance in whole blood with results comparable to a commercially available analyzer.  The creatinine sensor developed in the improved sensor array as described above showed a use life of at least three weeks in continuous use, and was compatible with other sensors in the cartridge.

REFERENCES

[1] S. Haneder, A. Gutfleisch, C. Meier, J. Brade, D. Hannak, S. O Schoenberg, C. R Becker, H. J Michaely, World J Radiol, 4(7), 328-334 (2012)

[2] J. A. Straseski, M. E. Lyon, W. Clarke, J. A. DuBois, L. A. Phelan, and A. W. Lyon,  Clin. Chem. 57(11), 1566–1573 (2011)

[3] K. L Schnabl, S Bagherpoor, J Dubois, and P. Yip, Clin Biochem. 43(12), 1026-1029 (2010)

[4] E. Lee-Lewandrowski, C. Chang, K. Gregory, K. Lewandrowski, Clin Chim Acta. 413 (1-2), 88-92 (2012)