AU655519B2

AU655519B2 - Measuring system for the determination of the concentration of inorganic and organic compounds in a sample

Info

Publication number: AU655519B2
Application number: AU29918/92A
Authority: AU
Inventors: Joachim Bargon; Werner Groh; Werner Prass; Thomas Stehlin; Andreas Zimmermann
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1991-12-07
Filing date: 1992-12-04
Publication date: 1994-12-22
Anticipated expiration: 2012-12-04
Also published as: DE59208267D1; EP0546472A1; DE4140411A1; CA2084540A1; KR100232781B1; EP0546472B1; KR930013718A; BR9204846A; AU2991892A; JPH05256784A

Description

V i-quwu/ 1 2=191S Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 1

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT 4 44 £9 4 *4 0.* 0 Application Number: Lodged: Invention Title: MEASURING SYSTEM FOR THE DETERMINATION OF THE CONCENTRATION OF INORGANIC AND ORGANIC COMPOUNDS IN A

SAMPLE

.5.4 The following statement Is a full description of this Invenifon, Including the best method of performing It known to :U U 32 HOECHST AKTIENGESELLSCHAFT HOE 91/F 380 Fr.Dr.MI/fe Description Measuring system for the determination of the concentration of inorganic and organic compounds in a sample There are various methods for determining organic or inorganic traces. Among those known for a long time are, for example, the titrimetric determinations silver nitrate determination according to Fajans), photometric determinations and, for example, potentiometric methods using ion-selective electrodes (described e.g. in: Cammann, Das Arbeiten mit ionenselektiven Elektroden [Working with ion-selective electrodes], Springer, 1977, p. 72 et seq.).

The determination of chloride in clinical analysis is 15 generally carried out with the help of photometric and S" potentiometric methods. The titrimetric and photometric determinations are very accurate, but rather laborious and not suitable for continuous measurement. The methods using ion-selec 've membranes are suitable for continuous S 20 measurement, but have drawbacks because of their low specificity and their sometimes poor long-term stability.

S" Furthermore, every potentiometric measurement requires a second electrode as a reference electrode.

An effective determination of the concentration of halides and acyl halides is possible, for example, with the aid of a measuring system comprising a sensor element to be brought into contact with the sample, said sensor element containing an optical fluorescent indicator which, upon irradiation by an excitation light source, fluoresces as a function of the halide concentration present (AT-B-384,891). The indicator which ir present in cationic form is a member, in this case, of the group of the (hetero)aromatic, polycyclic compounds and polyene dyes, and is bound covalently as a thin layer to the surface of a solid support via so-called spacer groups

LI

i i -2 which are arranged between the surface of the support and the indicator molecules. The support material is a member of the group of organic polymers, such as poly(meth)acrylates, poly(meth)acrylamides, polyacrylonitriles, polystyrene or cellulose. The spacer groups used in this case are those molecular groups wiich increase the spacing between the solid support and the indicator molecule by at least the length of a CH 2 unit or a phenyl ring.

The main function of the spacer groups is to avoid interaction between the dye component and the support material, which interection gives rise to restricted mobility and thus to reduced sensitivity of the dye I 0. component.

15 The immobilized dye in this case can only be adequately o; quenched in cationic form, because the hydrophilicity of the support is not sufficient to allow the chloride ions to penetrate into the support material; consequently, *0 fluorescence quenching of the dye only takes place on the 20 support surface. Because the dye is only present in small amounts, the hydrophilicity of the support is only very slightly influenced by electrostatic interactions of the 0 dye cations.

s* :The drawback of the methods described in the prior art is 25 that some of them are very laborious, and that they are not suitable for continuous measurement. Furthermore, their specificity and sensitivity is low, and their longterm stability is sometimes poor.

The object of the invention is therefore, essentially, to overcome said drawbacks of the known procedures or systems for carrying out said procedures, and in particular to provide a measuring system which is notable for high sensitivity and accuracy, which enables a continuous measuring method, and whose geometry can be adapted to -3the various application fields.

The present invention achieves this object and relates to a measuring system for the determination of the concentrations of inorganic and organic compounds in a sample, having a sensor to be brought into contact with the sample, which sensor contains an optical fluorescent indicator which, upon excitation via an excitation light source, fluoresces as a function of the present concentration of the inorganic or organic compound to be detected. The measuring system is further configured for measuring and evaluating the incoming radiation and the reemitted fluorescent light.

The measuring system comprises a sensor element to be brought into contact with the sample, said sensor element being compos3d of a polymer matrix which is prepared from a) a matrix component derived from one or more units of the formula I) eC)r ur rs\nc o \ori,

R

2 Rop3

COCI

(I)

in which

R

1 is hydrogen, deuterium or methyl;

R

2 and R 3 independently of one another, are hydrogen, deuterium or methyl, and the degree of polymerization n is an integer in the range from 80 to 2500, preferably from 200 to 2000; b) a dye component which is immobilized covalently on\ the acyl chloride groups of the matrix component, 4 and c) one or more swelling components which are bound covalently at the remaining free acyl chloride groups of the matrix component.

Suitable matrix compounds are compounds of the formula I or copolymers of these compounds, preferably those in which R 1

R

2 and/or R 3 are hydrogen or methyl, particularly compounds in which R 1

R

2 and/or R 3 are hydrogen or deuterium. The matrix component is prepared by radical-initiated polymerization of one or more acyl chlorides of the formula II by means of a radical initiator.

1 R. R1 0

IRI

SC =C-C

(II)

Ci Suitable radical initiators are, for example, azobisiso- 15 butyronitrile (AIBN), tert-butyl peroxide, m-chloroperbenzoic acid and dicumyl peroxide, preferably AIBN. The linear main chain of the matrix component, in a following step, has functional units, in this case the dye component, for example 4- or 5-aminofluorescein, attached to it by covalent immobilization.

t. The dye is used in highly substoichiometric amounts, preferably less than 2 mol particularly in the range from 0.01 to 1 mol based on the matrix component Suitable dye components are, for example, fluorescein or cyanine dyes, but also the known pH indicators and chemoluminescent dyes, particularly -dyes containing hydroxyl or amino groups.

Examples to be mentioned in this instance are fluoresceinamine, rhodamines, coumarins, the phthalo- and carbocyanines and further representatives of the cyanine dyes.

Suitable swelling components are, in general, all the alcohols, in particular straight-chain or branched Ci-Cgalcohols, and glycols, for example glycol Ci-C 4 -monoalkyl ether, preference being given to methanol, ethanol, propanol or butanol, and ethylene glycol monomethyl ether and di- and triethylene glycol monoethyl ether. With the aid of the swelling component, which is used in excess, preferably in 2- to 6-fold excess, based on the matrix component the remaining free acyl chloride groups of the matrix component (approx. 99%) which do not yet have a dye molecule attached to them, are esterified. The °purpose of the swelling component is to exert a dominating influence the swelling capacity of the overall o. 15 system. By means of varying the alkyl residue (methyl, ethyl via propyl to butyl), the swelling capacity of the matrix and thus the response time (time constant) of the sensor is controlled. Thus the permeability of the polymer matrix by means of variation of the swelling 20 component, can be specifically adjusted to the hydrophilicity of the dye and of the substance to be detected.

If glycols are used, it is even possible to produce a hygroscopic polymer matrix.

9 An effective swelling capacity of the matrix component 25 can be achieved, for example, by using methanol. The choice of the swelling component in this case depends on the nature of the dye component, but is independent of its concentration. In the case of hydrophilic dyes, higher alcohols are generally used as the swelling component, whereas increasing hydrophobicity of the dye component means that preference is given to shorter-chain alcohols and glycols. As the swelling capacity of the matrix component rises, the time constants for the response time of the sensor become shorter, the response times being in the range from 0.3 seconds to 25 minutes, particularly in the range from 1 second to 20 minutes. In 6 this way it is possible for the substance to be detected which causes fluorescence quenching of the dye, to penetrate into the polymer matrix, as a result of which said polymer matrix is utilized as a sensitive element over its whole volume range.

In a further embodiment it is possible to bind several swelling components in succession to the matrix polymer in different concentrations.

The invention further relates to a method for the detection of organic and inorganic traces with the aid of the measuring systems according to the invention.

f 4@ 0' Detection is generally possible in three different ways: a) by fluorescence quenching: Sin the presence of organic or inorganic compounds, 15 the fluorescence intensity of the dye molecules is reduced in this case. This so-called fluorescence quenching is reversible, i.e. the fluorescence ""intensity only regains its maximum value if all the *"quenching molecules" have been removed from the solution; b) by a spectral change of the fluorescence light, i.e.

the position of the maximum of the fluorescence light being shifted in the presence of inorganic or or'.nic compounds; or else c) the sensor element contains a precursor of a fluorescent dye morin) which forms a fluorescent complex with the species to be detected Al 3 said complex then being detected.

Figure 1 shows a diagram of a possible simple measuring system according to the invention. Exciting light from a 7 7 light source after passing through a primary filter impinges on the sensor element and then passes through the sample chamber The fluorescent light produced, after passing through the secondary filter (F 2 whose purpose is to filter out the exciting light, impinges onto a photo-detector The electrical signal generated here is amplified in an amplifier fed to a processor unit if required, and then subjected to analog or digital evaluation It is also possible to measure the fluorescent light not after it has passed through a sample chamber, but in reflectance (Figure 2).

The sample chambers may be constructed, for example, as single-shot measuring chambers or as flow-through cells.

Possible light sources include incandescent lamps, lasers q cor LED-type configurations, delivering continuous or 't, r pulsed light. The filters used can be absorption filters S.or interference filters. Possible photometric devices are photoelectric cells, electron multipliers or photodiodes.

The processor unit is used to determine the concentration 0 of the substance to be measured; to this end, it is often 0 tnecessary in practice to reduce the incoming measured signal or the measured fluorescence intensity by a background component or to carry out a normalization, in order to obtain the actual measured signal.

Ii In specific cases, particular requirements may lead to i "'various configurations, and result in other components being used. It may be necessary, for example, to conduct the light from the light source to the sensor element, and away from the sensor element to the detectors, with the aid of optical waveguides (OWG) (see Figure If required, a coupler unit may be interposed in this configuration.

It is also possible to place semipermeable membranes in front of the sensor element in order to make the diffu- 8 -8sion of the quenchers to the indicator layers more selective. In order to separate out the fluorescence signal of the sensor from any interfering fluorescence of the sample material, it may be necessary to incorporate opaque separating layers between sample and sensor, which layers must of course be permeable to the quenchers.

With the aid of the measuring system according to the invention it is possible, for example in the context of environmental technology, to detect inorganic contaminants such as, for example, nitrates, nitrites, halides, in particular chlorides, bromides and iodides, metal ions and heavy metal ions, for example of lead, cobalt, copper, arsenic, antimony, cadmium, chromium, mercury, molybdenum, thallium, nickel and selenium, but also S'1 15 organic contaminants such as, for example, amino and 4' t nitro compounds, organic halides, for example bromoit S methane, chlorodibromomethane, ethylene dibromide, t hexachlorobutadiene, bromoform, and glycols.

In a further embodiment, the measuring system according Oi' 20 to the invention can be used for pH determination.

The system is further suitable, in particular in the field of medical technology, for the determination of Sconcentrations of therapeutically important drugs (drug monitoring). Examples of this are phenytoin, phenobarbital, primidone, carbamazepine, ethosuximide, valproic acid, lidocaine, procainamide, N-acetylprocainamide, quinidine, disopyramide, theophylline, caffeine, amikacin, gentamicin, netilmycin, tobramycin, methotrexate, chloramphenicol and cyclosporin, in particular theophylline, phenytoin, phenobarbital, lidocaine, amikacin, gentamicin, methotrexate and cyclosporin.

Thus, for example, some drugs having a very low therapeutic index, such as some anti-epileptics, xanthines, cardiac glycosides, antiarrhythmic drugs and aminoglyco- _I _I

I

9 sides, must be dosed very accurately. To this end, the blood count, the blood level, the urine level and the liver function of the patient are usually closely monitored. Sensors with the capability of being used in the field of drug monitoring must have high accuracy and a rapid response time.

The sensitivity of the sensor element of the measuring system according to the invention can be as low as ng/ml, in particular 5 ng/ml, coupled with response times from 1 second to 20 minutes, depending on the type of the dye and the swelling component used, and on the substance to be detected.

Examples 1) Preparation of poly(acryloyl chloride) The preparation of the poly(acryloyl chloride), in contrast to other methods described in the literature, employed chlorobenzene instead of dioxane, and instead of precipitating the polymer in n-heptane, unreacted monomer was removed on a rotary evaporator. By means of these S 20 measures, it was possible to avoid a contact with air humidity, and to increase the yield from 10% to 77% and the degree of polymerization from an initial 133 to 1733.

Batch: 200 g of acryloyl chloride 2.2 mol (previously distilled at 92-960C (Fluka)) 200 g of chlorobenzene 50% solution 723.4 mg of AIBN (recrystallized) 0.2 mol In a 1 liter 3-necked flask equipped with an oil bath, thermometer, N 2 inlet, reflux condenser and drying tube, the acyl chloride is mixed with chlorobenzene and AIBN, and the flask is purged with nitrogen for one hour at room temperature. Both the starting compounds and the i nitrogen were dried before use. The mixture is then polymerized at 70 0 C for 7 hours, the solution is then diluted with 200 ml of dioxane, and stored overnight in a refrigerator at 4 0

C.

Work-up: Unreacted acyl chloride is removed on a rotary evaporator at 70 0 C under reduced pressure (20-400 mbar) until the distillate no longer smells of acyl chloride. As soon as the polymer precipitates, a further 250 ml of dioxala are added. The polymer solution, well sealed, is then stored in a refrigerator.

Assay: 8.808 g of the polymer solution are separated and esterified with 40 ml of methanol under reflux. The methanol is separated from the poly(methyl acrylate) (PMA) generated, and the PMA is dried in vacuo and weighed.

Yield: 1.6316 g of polymer (PMA) S Free acyl chloride: 125.5 g 17.5% Yield of the polymerization: 62.8% Determination of molecular weight: Description of the Walter GPC apparatus: Columns: Ultrastyrogel columns 3 units: 500 A, 103 A, 104 A RI detector Walter 410 Pump: Walter model 510 HPLC pump Solvent: Tetrahydrofuran (Riedel analytical grade) Software: Walters Maxima 820 Standard: PMMA from PSS Polymer Standards Service, ~I :ii Rb~--s 11 (molecular weights: 1080000, 710000, 530000, 280000, 152000, 96000, 52200, 24400, 13100, 6400, 2030, 1210) 158278 g/mol 1.64 Weight average: Polydispersity: 2) Immobilization of fluoresceinamine: t ce t C9 t C4 41 t ft's If t *It ft ftl f t i Each time, 90 g of the polymer solution are diluted to this end with 400 ml of dioxane (Riedel, dried), a fluoresceinamine solution (Fluka) in dioxane is added, and the mixture is stored while stirring. In the following batches A-E, different fluoresceinamine concentrations are reacted under otherwise identical preparation conditions.

15 Batch Acyl chloride Fluoresceinamine solution [mol in dioxane [mol] [mg] [mmol] [ml] A 90.26 0.24 9.33 0.027 0.010 mmol B 90.05 0.24 41.3 0.119 0.041 mmol C 90.10 0.24 82.5 0.238 0.082 mmol D 89.95 0.24 175.0 0.504 0.1.71 mmol E 90.33 0..24 838.7 2.415 100 0.818 mmol 12 Fluoresceinamine, used in amounts which are highly substoichiometric, is completely acylated (as checked by thin layer chromatography (TLC)) by stirring at room temperature for a week.

3) Esterification of the excess acyl chloride groups: As the low concentration of the fluoresceinamine results in the amination of only relatively few acyl chloride groups (batch A: 1.0x10 2 mol batch E: 0.8 mol the remaining acyl chloride groups which are still free are esterified in separate tests using 3 different alcohols.

To this end, each solution (batch A-E) is divided into 3 equal parts, and each part-solution is esterified with methanol, ethanol or butanol. The alcohol is used in fold excess and is heated for 7 hours, together with the 15 polymer solution, under reflux to 60 80 0

C.

4s 0 The excess alcohol is then removed on a rotary evaporator and the residual polymer is dissolved in dioxane. The polymer is precipitated in dried n-heptane and dried in vacuo.

00 From the 5 batches a total of 15 different polymers are thus obtained, which cover 2 orders of magnitude of the fluoresceinamine proportion and which, for each batch, contain 3 different esterified alcohols.

1 4e f

Claims

1. A measuring system for the determination of the concentration of inorganic and organic compounds in a sample, having a sensor to be brought into contact with the sample, which sensor contains an optical fluorescent indicator which, upon excitation via an excitation light source, fluoresces as a function of the present concentration of the inorganic or organic compound to be detected, said measuring system being configured for measuring and evaluating the incoming radiation and the reemitted fluorescent light, wherein the sensor element to be brought into contact with the sample is composed of a polymer matrix which is prepared from a) a matrix component derived from one or more units of the formula I. e-c- r corvi R 2 tR COCI n .I (I) in which R 1 is hydrogen, deuterium or methyl and R 2 and R 3 independently of one another, are hydrogen, deuterium or methyl, and the degree of polymerization n is an integer in the range from 80 to 2500, and b) a dye component which is immobilized covalently Scompnen- t, i on the acyl chloride groups of the matrix component, and c) one or more swelling components which are bound covalently at the remaining free acyl chloride groups of the matrix component. v~C0 n ~e re:O Iose

2. A sensor element contained in a measuring system as claimed in claim 1, wherein the dye component of the polymer matrix is a member of the group of fluores- cein dyes.

3. The sensor element as claimed in claim 2, wherein the dye component is used in the range from 0.01 to 1 mol based on the matrix component

4. The sensor element as claimed in claim 2, wherein the swelling component used is methanol, ethanol, pro::nol and/or butanol.

Th sensor element as claimed in claim 2, wherein theswelling component is used in 2- to 6-fold tees based on the matrix component

6. The sensor element as claimed in claim 2, wherein tetime constants for the response time of the 20 sensor are in the range from 1 second to 20 minutes.

7. The measuring system as claimed in claim 1, wherein the light is conducted from the light source to the 1 14 19sensor element, and away from the sensor element to the detectors, with the help of optical waveguides.

8. The measuring systemi as claimed in cjlaim 1, wherein semipermeable membranes are placed in front of the sensor element.

9. The measuring system as claimed in cla~im 1, wherein the sensitivity of the sensor element can be as low as 0.5 ng/ml. k 15 The measuring system as claimed in claim 1, wherein the system is used for pH determination. DATED this 3rd day of December 1992. HOECHST AKTIENGESELLSCHAFT2 WATERMARK PATENT TRADEMARK ATTORNEYS "THE ATRIUM" 290 BURWOOD ROAD HAWTHORN. VIC. 3122. 0 a9 0 ob 0 0 0 00 **be HOE 91/F 380 Abstract Measuring system for the determination of the concentra- tion of inorganic and organic compounds in a sample Measuring system for the determination of the concentra- tions of inorganic and organic compounds in a sample, having a sensor to be brought into contact with the sample, which sensor contains an optical fluorescent indicator which, upon excitation via an excitation light source, fluoresces as a function of the present concen- tration of the inorganic or organic compound to be detected. The measuring system is configured for measur- ing and evaluating the incoming radiation and the re- emitted fluorescent light. r It The measuring system comprises a sensor element to be S":'0o brought into contact with the sample, said sensor element being composed of a polymer matrix which is prepared from a) a matrix component derived from one or more units of the formula I R2 S 6R 1 'L J n lOOi in which (I) R 1 is hydrogen, deuterium or methyl; R 2 and R 3 independently of one another, are hydrogen, deuterium or methyl, and the degree of polymerization n is an integer in the range from 80 to 2500, preferably from 200 to 2000; i iia 2 HOE 91/F 380 b) a dye component which is immobilized covalently on the acyl chloride groups of the matrix component, and c) one or more swelling components which are bound covalently at the remaining free acyl chloride I groups of the matrix component. 1 6 9 44 4,4 t* t i