Method for determination of product and substrate concentrations in a medium

• US 20030119201A1
• Filed: 11/22/2002
• Published: 06/26/2003
• Est. Priority Date: 05/22/2000
• Status: Abandoned Application

• Current Assignee: Forschungszentrum Jã¼Lich GmbH (Bundesrepublik Deutschland), Dasgip Drescher Arnold & Schneider AG
• Original Assignee: Dasgip Drescher Arnold & Schneider AG
• Inventors: Franz, Andreas, Weuster-Botz, Dirk, Arnold, Matthias, Wandrey, Christian, Wolfram, Evelyn
• Application Number:
  US10/301,851
• Publication Number:
  US 20030119201A1
• Time in Patent Office: Days
• US Class Current: 436/178
• CPC Class Codes: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 2001/4016 being a selective membrane, e.g. dialysis or osmosis
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

A method for determining substrate and product concentration in liquid and/or gaseous media is disclosed. Several samples of at least one substance to be analyzed are removed in at least one sampling region by time-controlled diffusion of the analyte between the medium and a diffusion medium, which is fed to the sampling regions through fluid conduits using at least one pump and semipermeable membranes. The diffusion medium is transported to at least one detector while simultaneously new diffusion medium is fed from the sampling region, and analyzed to determine the analyte concentration. The detector provides a temporal concentration distribution or a temporal distribution of a signal proportional to the concentration. A change in the ratio of the signal maximum to the base line is ascertained in the output of the detector signal and, on the basis of this, a change in the diffusion properties of the semipermeable membrane is inferred and a correction factor ascertained.

Claims

1. A method for determining substrate and product concentration in liquid and/or gaseous media in which several samples of at least one substance to be analyzed-the analyte-are removed in at least one sampling region (3) by time-controlled diffusion of the at least one analyte between the respective medium and a diffusion medium which is fed to the sampling regions (3) through fluid conduits (5a, 5b) using at least one pump (6) by semipermeable membranes (2) and subsequently the diffusion medium is transported to at least one detector (7) while simultaneously new diffusion medium is being fed from the sampling region (3) and is analyzed by this to determine the analyte concentration, characterized in that the detector provides a temporal concentration distribution or a temporal distribution of a signal proportional to the concentration, in that a change in the ratio of the signal maximum to the base line is ascertained in the output of the detector signal, and on the basis of this, a change in the diffusion properties of the semipermeable membrane is inferred and a correction factor is ascertained.

2. Method according to claim 1, characterized in that an inference is made as to the analyte concentration in the sampled medium through a calibration of the detector (7) and a corresponding evaluation of detector signals, whereby the maximal rise of the front face of the detector signal, the signal maximum, the surface under the signal curve or the increased baseline following through flow of the peak maximum which results from the diffusion of the analyte into the diffusion medium are adduced for the evaluation.

3. Method according to claim 2, characterized in that several properties of detector signal distribution are used for evaluation at the same time, or ratios of these values toward one another are used.

4. Method according to claim 1, characterized in that two signals at different flow rates of the diffusion medium and/or different diffusion times with resting medium are ascertained in close temporal sequence and compared with one another with respect to their characteristic properties in order to recognize and to correct a possible drift due to change in the diffusion properties.

5. Method according to claim 4, characterized in that in addition a change in analyte concentration over time known on the basis of several measurements and/or a dynamic model is taken into consideration.

6. Method according to claim 1, characterized in that several sampling regions (3) are provided in the manner of a parallel connection and in that the at least one pump (6) operates continuously and a multivalve or multipath arrangement (12) connected in series upstream from the sampling regions (3) in the fluid conduit (5b) provided for the diffusion medium is controlled such that in any given case diffusion medium flows through one of the parallel fluid conduit regions (5b) to the detector (7) and a transport is prevented in the remaining sampling regions in alternation.

7. Method according to claim 6, characterized in that the valve arrangement (12) is controlled such that one of the fluid conduit regions (5b) is basically continuously subjected to through flow in alternation in any given case.

8. Method according to claim 6, characterized in that in the parallel sampling regions (3), the diffusion or sampling time of one region is at all times at least the measuring time of all other parallel sampling regions necessary for signal recording in the detector together.

9. Method according to claim 1, characterized in that a pressure measuring unit is connected in series upstream from the sampling regions (3) in the fluid conduit (5a) for recognizing a disturbance in a conduit segment.

10. Method according to claim 1, characterized in that air or gas bubbles are removed from fluid diffusion medium before reaching the sampling regions (3) using a bubble trap (9).

11. Method according to claim 1, characterized in that the multipath or multivalve arrangement (12) is controlled by a computer (18).

12. Method according to claim 1, characterized in that several parallel connected detectors are provided and diffusion medium coming from the sampling regions is fed to one of the detectors through a multipath or multivalve arrangement.

13. Method according to claim 1, characterized in that in a sample preparation module (16) connected upstream in series from the detector (7), at least one substance which can disturb the detector used is absorbed or reactively transformed into a non-disturbing chemical form.

14. Method according to claim 1, characterized in that in a sample preparation module (16), the analyte is reactively transformed into a form measurable by the detector (7).

15. Method according to claim 1, characterized in that a diffusion medium is used which is basically free from analytes to be detected.

16. Method according to claim 1, characterized in that a diffusion medium is used which contains a known concentration of at least one analyte which lies above the concentration in the medium to be sampled so that a diffusion of the analyte from the diffusion medium into the medium to be sampled takes place in the region of the sampling region.

17. Method according to claim 1, characterized in that the samples obtained from parallel sampling regions through diffusion are gathered with an automatic fraction collector in the output of the sampling region or of the detector for a subsequent off-line analysis.

18. Method according to claim 1, characterized in that, for calibration, the semipermeable membranes are dipped in media of known analyte concentration and measurement data sets are compiled on the basis of which the measured results supplied by the detector are evaluated for determining analyte concentration.

19. Method according to claim 1, characterized in that the semipermeable membranes (2) are dipped for calibration in at least one reaction container with the medium to be used in the experiment, and in that known concentrations of at least one analyte is set through addition of correspondingly calculated volumes of a concentrated standard mixture of at least one analyte and measured data sets are compiled for the various concentrations on the basis of which the measurement results supplied by the detector are evaluated for determining the analyte concentration.

20. Method according to claim 18, characterized in that the end concentration of the at least one analyte at the same time represents the desired start concentration in the experiment mixture.

21. Method according to claim 18, characterized in that the detector (7) issues a value for the concentration of the analyte in the diffusion medium, and an inference is made by calculation about the concentration in the medium at past diffusion times by comparing this measured value with the measured values which were ascertained by the calibration method according to claims 18 to 20 with a known analyte concentration.

22. Method according to claim 1, characterized in that diffusion medium is guided past the sampling regions (3) to the detector (7) through a bypass conduit (20).

23. Method according to claim 22, characterized in that in the region of the bypass conduit (20), a standard medium is injected into the diffusion medium and this segment is transported by connecting the bypass conduit (20) to the detector (7) in order to correct drift phenomena of the detector (intermediate calibration).

24. Method according to claim 22, characterized in that rinsing fluid is fed to the detector through the bypass conduit (20).