Method of sample analysis

• US 5,158,868 A
• Filed: 10/15/1990
• Issued: 10/27/1992
• Est. Priority Date: 07/17/1987
• Status: Expired due to Fees

• Current Assignee: Il Holding SpA (Werfen SA)
• Original Assignee: Iniziative Marittime 1991 SRL
• Inventors: Geiselman, Theodore S., Bergkuist, Carolyn, Fraticelli, Yvonne
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US07/597,070
• Time in Patent Office: 743 Days
• Field of Search: 435/4, 435/12, 435/25, 435/180, 436/52, 436/68, 436/108, 436/111, 436/518
• US Class Current: 435/4
• CPC Class Codes: C12M 21/18 Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: Y10T 436/117497 with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/171538 Urea or blood urea nitrogen
• CPC Class Codes: Y10T 436/173845 Amine and quaternary ammonium

Abstract

A system for analyzing a biological fluid or the like for a constituent of interest comprises structure defining a sample inlet port, structure defining an analysis region, a measuring system connected in sensing relation to the analysis region, and structure defining a reaction chamber that has an immobilized enzyme capable of modifying the constituent of interest. Control means operates liquid flow means in a unidirectional sample flow mode to initially flow a sample of material to be analyzed from the sample inlet port to the reaction chamber and to the analysis region for a measurement of unmodified sample material, and then operates the liquid flow means in a bidirectional sample flow mode to oscillate the sample material in the reaction chamber and facilitate modification by the immobilized enzyme of the constituent of material in the sample. Modified sample material is then introduced into the analysis region and the measuring system is operated to obtain a measurement of modified sample material, the measurements of modified and unmodified sample material then being used to provide an indication of the amount of the constituent of interest in the sample material.

Claims

1. A method for measuring a constituent of interest of a biological fluid or the like comprising the steps of

providing a reaction chamber that contains an immobilized enzyme capable of modifying a constituent of interest,

providing a measuring system,

placing a first portion of a biological fluid to be analyzed in said reaction chamber and concurrently exposing a second unmodified portion of said biological fluid to said measuring system to provide a first data output,

oscillating said first biological fluid portion with bidirectional flow in said reaction chamber to facilitate modification by said immobilized enzyme of said constituent of interest in said biological fluid,

then exposing said first portion of said biological fluid to be analyzed to said measuring system to provide a second data output, and

modifying said second data output as a function of said first data output to provide an indication of the actual amount of said constituent of interest in said biological fluid.

2. The method of claim 1 wherein said measuring system includes an analysis region and said analysis region and said reaction chamber are connected together in a series flow path, and

said first and second portions of said biological fluid are placed in said reaction chamber and exposed to said measuring system by flowing said biological fluid to be analyzed through said reaction chamber at a flow rate of at least about one hundred microliters per second.

3. The method of claim 1 wherein said constituents of interest are urea and glucose, and two different enzymes are coimmobilized in said reaction chamber.

4. The method of claim 3 wherein said two different coimmobilized enzymes are glucose oxidase and urease and said measuring system includes an ammonium ion selective electrode and an oxygen sensing electrode.

5. A method of measuring a substance capable of being enzymatically converted to a detectable ion with compensation for potassium ion interference comprising the steps of

placing a sample containing a substance capable of being enzymatically converted to a detectable ion in a chamber comprising an immobilized enzyme capable of converting said substance to said detectable ion and detecting means for detecting said detectable ion;

contacting said sample with said detecting means before said immobilized enzyme has converted said substance to said detectable ion and measuring the output of said detecting means to provide a potassium ion interference (pre-enzymatic reaction) output;

oscillating said sample in said chamber in a bidirectional flow mode interval to facilitate conversion by said immobilized enzyme of said substance in said sample to said detectable ion;

contacting said sample with said detecting means after said immobilized enzyme has converted said substance to said detectable ion and measuring the output of said detecting means to provide an apparent substance amount output; and

modifying said post-enzymatic reaction output as a function of said pre-enzymatic reaction output to provide an indication of the actual amount of a substance in said sample using a form of the Nicolsky equation.

6. The method of claim 5 wherein said substance comprises urea and said detectable ion comprises ammonium ion.

7. The method of claim 5 wherein said detecting means comprises an ion selective electrode and a reference electrode, said ion selective and reference

8. A method of measuring a substance capable of being enzymatically converted to a detectable ion with compensation for potassium ion interference comprising the steps of

placing a sample containing a substance capable of being enzymatically converted to a detectable ion in a chamber comprising an immobilized enzyme capable of converting said substance to said detectable ion and detecting means for detecting said detectable ion;

contacting said sample with said detecting means before said immobilized enzyme has converted said substance to said detectable ion and measuring the output of said detecting means to provide a potassium ion interference (pre-enzymatic reaction) output;

oscillating said sample in said chamber with bidirectional flow to facilitate conversion by said immobilized enzyme of said constituent of interest in said sample to said detectable ion;

contacting said sample with said detecting means after said enzyme has converted said substance to said detectable ion and measuring the output of said detecting means to provide an apparent substance amount (post-enzymatic reaction) output; and

modifying said post-enzymatic reaction output as a function of said pre-enzymatic reaction output to provide an indication of the actual amount of a substance in said sample. electrodes are spaced from one another by a flow path portion located so that said reference electrode is downstream of and spaced from said ion selective electrode by a flow path volume greater than the displacement volume that sample material is moved during said bidirectional sample flow mode interval.

9. The method of claim 8 further comprising the step of calibrating said detecting means using first and second calibrators having known concentrations of potassium ions and said substance by contacting said first and second calibrators individually with said detecting means and measuring the output of said detecting means before and after said enzyme has converted said substance in said calibrators to said detectable ion.

10. The method of claim 9 wherein said substance is urea and the actual concentration of urea in said sample is determined by effectively subtracting said pre-enzymatic reaction data from said post enzymatic reaction data using the following form of equation:

space="preserve" listing-type="equation">conc..sub.UN =antilog [(E.sub.S -E.sub.cal

     1)(1/S)] *

(Cal 1_UN +kCal_1k)-(k conc. K)

where

conc._UN =actual concentration of urea in the sample;

E_s =post-enzymatic reaction measurement for the sample in millivolts;

E_cal1 =post-enzymatic reaction measurement for calibrator 1 in millivolts;

Cal 1_UN =actual concentration of urea in calibrator 1 (known);

Cal _1k =actual concentration of potassium in calibrator 1 (known);

k=potassium selectivity factor for the electrode; and

conc. K=concentration of potassium in the sample.

11. The method of claim 10 wherein said ion selective electrode includes nonactin ionophore, and said immobilized enzyme includes urease enzyme.

12. The method of claim 8 that further includes the concurrent measurement of a second substance in said sample, and a second enzyme is coimmobilized in said reaction chamber.

13. The method of claim 12 wherein said detecting means and said chamber are connected together in a series flow path, and

said sample to be analyzed is flowed through said chamber to said detecting means at a flow rate of at least about one hundred microliters per second.

14. The method of claim 13 wherein said second substance in said sample is glucose and said coimmobilized second enzyme is glucose oxidase.

15. The method of claim 14 wherein said ion selective electrode includes nonactin ionophore, and the other coimmobilized enzyme is urease.