GB2261729A

GB2261729A - Method and optical probe for the determination of reduced nicotinamide adenine dinucleotide in a sample

Info

Publication number: GB2261729A
Application number: GB9121574A
Authority: GB
Inventors: Ashutosh Sharma
Original assignee: Individual
Current assignee: Individual
Priority date: 1991-10-11
Filing date: 1991-10-11
Publication date: 1993-05-26
Also published as: GB9121574D0

Abstract

Use of a fluorophore for determining NADH is described, preferably the quenching of the fluorophore by NADH is measured. Preferably an optical probe is constructed using immobilised indicator on to a support which may be exposed to an excitation radiation. A number of analytes (substrates) can be determined by using corresponding enzymes and co- factor in conjunction with the indicator.

Description

Method and Optical probe for the lletermination of Reduced Nicotinamide Aflenine niniicleotide in a Sample Field of Invention This invention relates to a method and an optical probe for measuring reduced nicotinamide adenine dinucleotide (NADH) content in liquid media, and more particularly relates to a method and probe for measuring NADH content utilizing luminescence quenching.

Background of the Invention The measurement of reduced nicotinamicle adenine dinucleotide (NADH) is of importance in several bio-analytical applications including those in clinical and in bio-reactors. The amount of NADH which is produced or consumed during a cofactor-dependent enzymatic reaction provides means to monitor such a reaction. This forms the basis of several immulloassay schemes and a variety of biosensors. Further, during the last several years there has been an increasing interest in the development of miniaturized fluorescence probes for use in biotechnology and in clinical applications.These probes were based on the measurement of the intrinsic fluorescence of NADH (ci. 46() ntn) or on a chemiluminescent reaction involving NADH as one of the reactants. Several applications of such probes have been explored including studies of enzymatic reactions. suspended-cell metabolism, suspended cell biomass concentrations and detection of cell-toxic compounds. Another important application of particular interest is the measurement of NADH in tie whole blood matrix. Currently, this is not possible due to the strong fluorescence and absorption properties of the endogenous compounds in the blood. It is therefore highly desirable to having a method for the measurement of NADH which can overcome these difficulties.

One such technique which can be adulated to construct an optical sensor with minimal interference from the background is fluorescence quenching (provided the indicator is chosen carefully). The requirement of full reversibility can be met by selecting an indicator which undergoes dynamic quenching by the analyte.

It is, therefore, an object of the present invention to provide an improved, accurate and highly sensitive method for determining NADH concentrations in a sample.

It is another object of the present invention to provide an improved, accurate and highly sensitive method for determining NADH concentrations during the enzymatic reactions.

It is an additional object to provide an improved method for determining NADH concentration continuously.

It is a further object of the present invention to provide an improved apparatus for determining the NADH concentration in a sample having increased sensitivity and which may be used continuously.

A still further object of the present invention is to provide an improved NADH measuring device employing luminescence quenching (fluorescence and/or phosphorescence) as its operational principle and utilizing a fibre optic probe in combination with a relatively simple optical system in association with photo detectors and an electronic computing circuit driven by said photodetectors and arranged to provide a direct analog computation of NADH based on said luminescence quenching as detected by said optical system.

These and other objects and advantages of the present invention will become more readily apparent upon reading the following descriltion.

Therefore, there is a need in the art for a rapid, sensitive and reliable method for monitoring NADH concentration in liquids. Further, there is a need for indicators which can offer above noted advantages.

Brief Summary of The Invention The present invention is based on the discovery that NADH quenches the luminescence of certain indicators (fluorescent or phosphorescent) extremely efficiently.

In the present invention, the quenched luminescence of the luminescent indicator is monitored to quantitatively or qualitatively determine the presence of NADH in a example. The present invention also provides an apparatus capable of carrying out this method.

Brief DescriPtion of the Drawings Figure 1 is a diagrammatic view of an apparatus in accordance with the present invention; Figure 2 is a diagrammatic view of another apparatus in accordance with the present invention; and Figure 3 is the Stem-Volmer Plot for the fluorescence quenching of the indicator methylene blue by NADH in water.

Figure 4 is a diagrammatic view of a NADH measurement based secondary probe in accordance with the present invention.

Detailed Description of The Rreferred Embodiment Referring to the drawings, and more particularly, Fig. 1, a probe in accordance with the present invention is illustrated. The probe contains a luminescence molecule 4, whose luminescence is quenched by NADH.The luminescent molecule 4 may be an organic, inorganic or an organometallic complex. In accordance with a preferred embodiment of the invention, the luminescence molecule is selected from the group of molecules consisting of methylene blue, thiazine dyes, conjugated organic compounds with -N- bridge structures, coumarines, flavaones, quinones, acridines, azo dyes, fluorescence redox-indicators, tri-phenyl nceth.ule dyes. The indicator can also be chosen from the various derivatives of these indicators.

TT'he luminescent molecule 4 is immobilized on support means 5. Although immobilizing the luminescent molecule on a support is not required to practice the invention, it is preferred in a transducer type application. An enclosure 6, with its material having high permeability for NADH is desirable to increase exposure of individual luminescent molecules to NADH.

As the permeability of the material enclosing the indicator increases, the response time of the probe becomes faster.

It is preferred that the probe is provided with an optical isolation 7, which made out of a NADH permeable material. This optical layer will ensure that no extemal light is entering in to the probe, thus reducing the noise.

Solid support means 5. for carrying the luminescent molecules 4, may be determined by routine testing. Preferred polymer materials include silicone rubber, polyisoprene, cellulose, silica gel, polyvinyichloride, and Amberlite XAD4, anonionic, hydrophobic polymer available from Rollm and Haas. Examples of others are Gas Chrom Q, Amberlite XAD2, XAD8; Dow XFS4()22; Johns-Manville Chromsorb Nos. N, P ,PS, Q, R, S, QS, T; Hamilton Co. PRP-1.

Acrilamide polymer, glass, nylon mesh, dialysis membrane or the like.

The luminescent molecule can be chemically immobilized on the support means by using standard methods of covalent bonding or by using ion exchange. Immobilization of the luminescent molecules can also be accomplished physically by various methods, including entrapping indicator molecules in a polymer support, adsorbing the molecules on a polymer/solid support surface, vaporizing the molecules and depositing them on a support surface, absorbing the indicator into a support material such as paper, and forming a molecular layer or layers using the Langimur-Blodgett technique.

blue method chosen to immobilize the luminescent molecule on the support means depends oji e sT specific molecule used and oil the nature of the support means. The use of chemical immobilization is dependant upon the functional groups available on the luminescent molecule used and on the support means. Physical immobilization techniques are dictated by properties of the support means, such as solubility. temperatule and surface tension, and by properties of the luminescent molecule. such as melting point and adsorption properties. It is not necessary that the luminescent molecule 4 be immobilized on a support means to practice the present invention. The luminescent molecule can be used in solid form or in solution.A polymer which dissolves the molecule may also be used in such a case fluorophore is dissolved in the polymer and smeared onto ;t solid support. However, it may be necessary that the chosen polymer allows diffusion of NADH through it. If use of optical fibre is made simple dip an < l dry technique may he used Referring again to Fig. 1, a bifurcated optical fibre bundle 8 is attached to the support means 5. The optical fibre bundle 8 carries excitation radiation to luminescent molecule 4 via fibre 9 and collects the luminescence emitted from the indicator molecules upon excitation via optical fibre l() which carries the light to a measuring instrument (see Fig. 3).

Another embodiment of the apparatus of the present invention is shown in Fig. 2. As discussed previously. luminescent molecule 1] is immobilized on support means 12. Molecule 11 and support means 12 form the base of chamber 12.

The liquid test sample containing NAD4 flows through chamber 13 via tube 14, which has an inlet 15 and an outlet 16. Chamber 13 is sealed by an O-ring 17, and is further enclosed by housing 18. A bifurcated oplical fibre bundle 19 is attached to an optically transparent wall of chamber 13.

The apparatus of the present invention can also be used with a single optical fibre. In that case. both the optical excitation and emission radiation are carried by the single fibre. When a single fibre is used. it may be necessary to use :s means such as a splitter plate or the like for splitting the beam.

The apparatus of the present invention can also be used without fibre optics. If such a system is used, the luminescent molecule can be immobilized on an optically transparent support means in place of the distal end o1 the fibre optic bundle. The transparent support means may be glass, quartz, a polymer such as, plexi glass, acrylic, or any other optically transparent material.

In this case, the luminescent molecules ale excited by focusing a collimated beam of light on the transparent plate which is incorporated in a flow through apparatus window. A laser or LED source can also be used for this purpose. The resulting luminescence is then collected at the same angle as that of excitation or at an angle other than that of excitation on either side of the plate using, for example. a collimating device.

The detection or measurement of NAUH can also be made by injecting the chosen luminescent molecule into a stream containing NADH at constant rate so as to have a generally uniform and constant concentration of the chosen luminescent molecule in the stream (such as in combination with flo injection system). An optical window can be provided to allow luminescence measul-clllents Jt might be necessary to find a common solvent both for the sample and the indicalor.

Method of the present invention can also be used in combination with an enzymatic reaction where NADH is consumed or produced to determine the concentrations of another analyte. To construct such secondary probes it may be necessary to first immobilise the fluorescent indicator alongwith a suitable enzyme onto a solid support. A protective analyte permeable coating or membrane may be required. These secondary probes according to the method of the present invention can also be constructed by modifying the enzyme using known methods and labelling it with tllt illdicat(l- without affecting its activity.Method of the present invention can also be implemented, simply by adding the suitable enzyme along with appropriate substrate and the indicators in the test sample. The aqueous enviromnent for the enzymic activity or for solubility reasolls may be necessary. These secondary probes can also be constructed by holding the enzyme solution in a semipermeable membrane such as dialysis membrane which will allow the analyte to diffuse through to the enzyme but will retain the enzyme and indicator. If no suitable membrane which can retain both the enzyme as well as the indicator is available, it may be necessary to first immobilise the indicator on to the solid support and then expose it to the enzyme contained in a semipermeable membrane.If an enzyme labelled with the indicator is used, it can simply be held in the membrane in front of a solid support for example the end of an optical fibre.

The method of the present invention call also be used in conjunction with a bioreactor, in such a case the fluid containing the NADH will be brought in contact with the indicator. One another way to implement the method of the present invention in the measurement of an analyte is to immobilize the indicator and a suitable enzyme along with NADA and the indicator on the inner walls of an optically transparent capillary tube. Sample, when brought in contact with the capillary tube will rise ill the tube and then the tube can be exposed to the light as to observe the luminescence quenching. When a sample containing NADH is to be analyzed use of the enzyme is not necessary. Use of the capillary will provide a cost effective method and will require small amount of samples.

One such analyte for which a secondly probe can be developed in accordance to the present invention is glucose. Glucose concentrations in a sample can be measured by using the method of the present invention. Enzyme glucose dehydrogenase (GDH) along with the co-enzyme NAD+ and the NADH sensitive tluorophore are brought in contact with the sample containing glucose. Following enzymatic reaction takes place: Glucose + NAD+ --GDH--- > Gluconolactone + NADH amount of the NADH produced then provides a mean to measure the glucose concentrations.

Methods and probes for a variety of analytes can be developed by using corresponding enzymes and the indicator chosen according to the present invention. A variety of enzymatic reactions and immuno assay systems involving production or consumption of NADH are known in literature. Some important analytes which can be measured using the method of the present invention include : alcohol, penicillin, lactate, urea, bile acids, glutamate.

The apparatus and method of the present invention may be used to determine the presence of NADH in a sample qualitatively, or it may be used to determine the NADH content in a sample quantitatively by luminescence quenching. Fluorescence quenching is given by the Stern-Volmer equation: = /1 = 1 -e Ksv [NADH] which relates to fluorescence intensity T of the fluorescence to concentration [NADH] of NADH (the quencher), where lo is the fluorescence intensity in the absence of the quencher and Ksv is the quenching constant.

Therefore, by measuring the fluorescence intensity I of the luminescent molecule 1 for known concentrations of NADH, the quenching constant Rv, for the luminescent molecule can be determined. If the relationship between the NADH concentration and the fluorescence intensity of the luminescent molecule is liDe:lr. NAVh concentration can be easily determinetl using the above equation. However. if the relationship is non-linear, Rv will change with varying NADH concentrations.In this instance, it will be necessary to plot intensity versus NADH concentrations to obtain a curve which can be used to determine the amount of NADH in a sample based on the luminescence intensity I of luminescent molecule 1.

Jn the practice of the method of the present invention. luminescent molecules l are exposed to the sample to be analyzed. While luminescent molecule l is exposed to the sample, the luminescent molecule is also exposed to radiation having a wavelength where luminescent molecule 1 shows analytically determinable absorption. Preferably, the wavelength used is the wavelength where the absorption of luminescent molecule l is maximum. However, this condition of exposing the fluorophore to sample and radiation, simultaneously is not necessary if the mechanism for quenching is static, i.e., if the indicator reacts with NADH in its ground state resulting in a non-luminescent product formation.The luminescence given off by luminescent molecule 1 is then measured at a wavelength where the luminescent molecule shows analytically determinable emission. Either fluorescence or phosphorescence should be measured. Again it is preferred to use the wavelength where the emission is at a maximum. The luminescence should also be measured in the complete absence of NADH to obtain Io value. Finally. in quantitative determinations, the luminescence intensity obtained from the luminescent molecule exposed to the sample is used with the previously calculated K,v and 1o or a previously generated curve based on known NADH concentrations to obtain the NADH content of the sample.The NADH concentration can also be determined by measuring changes in the luminescence decay time of luminescent molecule 1 due to the presence of NADH. However. this technique is restricted to indicators undergoing dynamic quenching of the fluorescence.

Example This example illustrates the generation of a Stem-Volmer plot for the quenching of a luminescent molecule by NADH. The luminescent molecule used was methylene blue and the luminescence intensity measurements were made for various NADH concentrations in water.

A I 0 M solution of the indicator methylene blue in distilled water was made. this solution was placed into a 1x1 cm rectangular quartz cell and was kept at a temperature of 22 'C.

A white light excitation source was used in combination with a photomultiplier as detector.

NAOH in varying concentrations (by diluting a stock solution of NADH in water) up to 400 was xmas added to the solution of water and indicator in the cell. Excitation light having a wavelength of 654 nm was focused on the solution, and the resulting fluorescence was measured at a wavelength of 698 nm.

The results are shown in Fig. 3. The relationship between I and Ksv is linear in the initial part of the plot, with Ksv of 1029 M-'.

Simultaneous measurement of fluorescence and phosphorescence can be employed.

Claims

1. Use of a fluorophore for the dCtLeti()n and/on assay of NADH.

2. A method of detection and/or assay (if NADH comprising measuring luminescence quenching of a fluorophore by NADH.

3. An optical probe comprising a support means including a fluorophore immobilised onto it.

4. A fibre optic probe comprising fibre optic, said fibre optic includes a fluorophore immobilised on the distal end of the libie optic or immobilised on to the core of the fibre or a fluorophore held close to tlic disttl cnd of the fibre optic or close to the fibre optic with the core of the fibre optic exposed to lhL indicator niolecules

5.A method in accordance with claims 1-2 wherein the said method is modified to detect and/or assay an analyte (substrate) by using a suitable enzyme and a co-factor to convert the substrate into a product and NADH.

6. A method according to claim 5 wherein the NADH is consumed during the enzymatic reaction.

7. A probe in accordance with claims 3-4 wherein the said probe is modified to detect and/or measure an analyte (substrate) by using t stuitable enzyme and a co-factor to convert the analyte into a product and NADH.

8. A probe in accordance with claim 7 wherein NADH is consumed during the enzymatic reaction.

9. A method and/or a probe in accordance with claims 1-4 for the detection/monitoring of NADH and as substantially described herein wilh reference to figs 1-3.

10. A method and /or probe in accordance with claims 5-8 for the detection of an analyte substantially as described herein with reference to fig. 4.

11. A method and /or a probe in accordance with claims 1-10 wherein the fluorophore used is chosen from the fluorescent indicators belonging to the group of thiazine dyes and in their acids and salts, in particular methylene blue its analogs and derivatives.

12. An indicator in accordance will claims l wherein the indicator is chosen from the group of fluorescent redox indicators, thiazine dyes, az(l dyes, tri-phenyl methane dyes.