WO2013000999A1

WO2013000999A1 - Preparation and use of a microplate functionalised by means of a fluorescent gel for measuring turbidity of suspensions

Info

Publication number: WO2013000999A1
Application number: PCT/EP2012/062558
Authority: WO
Inventors: Erminio MONTELEONE; Caterina DINNELLA; GIovanna FIA
Original assignee: Universita' Degli Studi Di Firenze
Priority date: 2011-06-28
Filing date: 2012-06-28
Publication date: 2013-01-03
Also published as: ITFI20110127A1; EP2726850A1

Abstract

The present invention describes a microplate with transparent bottom wells that were functionalised by means of a fluorescent gel. The above mentioned microplate is used for turbidity assessment in solutions and suspensions by evaluating in which extent haze particles are able to screen gel fluorescent radiation as a function of their concentration. The microplate and the assay performed according to the present invention are extremely advantageous for performing an in vitro assay aimed to the prediction of astringency induced by phenol compounds.

Description

PREPARATION AND USE OF A MICROPLATE FUNCTIONALISED BY MEANS OF A FLUORESCENT GEL FOR MEASURING TURBIDITY OF SUSPENSIONS

FIELD OF THE INVENTION

The present invention relates to the field of methods and devices for determining turbidity of suspensions. In details, the invention relates to a microplate functionalised by means of a fluorescent gel and the assay thereof for determining turbidity of suspensions.

STATE OF THE ART

To determine the concentration of particles suspended in a solution (or suspension) various devices are available such as turbidimeters, nephelometers and turbidity sensors also useful for on-line monitoring of this parameter. Devices with both enhanced light source and sensitivity of signal detector have been developed for answering to specific needs. Devices available on the market or described in patent and non patent literature consist of optic instruments and their accessories all based on the general principle of transmitted/diffuse light detection. Authors of the present invention, recently published (Dinnella, C ; et al Food chemistry 2009, 1 13, 325-330) a method based on the use of two superimposed microplates, one containing a fluorescent standard solution and the other containing the turbid unknown sample. The therein described assay shows technical problems in instrumental response acquisition responsible for the lowering of both test sensitivity and reproducibility. The main issue is related to the need of using the two superimposed microplates. The image magnification, needed for instrumental response acquisition, shows that the microplate superimposition induces the formation of a shadow which is responsible for variations of the analytical response value. Also, the optimization of microplate alignment, in order to limit the shadow presence, considerably lengthens the time needed for performing the test. Overall, this already published fluorimetric device, even if suitable with reference to the proposed chemical-physical principle for determining the parameter of interest, needs long time run and is too complicate to be easily applicable for the routinely monitoring of turbidity values . SUMMARY OF THE INVENTION

The above mentioned drawbacks are overcome in the present invention by means of a , usable for evaluating solution or suspension turbidity, the wells of which, with transparent bottom, were functionalized with a fluorescent gel, the strength of which is not less than 250 g/cm².

Gel strength is such as to avoid the diffusion of gel components towards, and vice versa, the turbid sample which is directly poured on the fluorescent gel.

Subject of the present invention is also a method for functionalising said

microplate.

Furhter subject of the invention is an assay for determining turbidity of solutions/suspensions using the above described functionalised microplate. The principle on which the method is based is related to the capacity of turbid sample to screen the radiation emitted by the fluorescent gel as a function of the suspended particles concentration.

Details and advantages of the present invention are reported herein following. SHORT FIGURE DESCRIPTION

Fig.1 : Scheme of the system used for turbidity evaluation by means of the functionalised microplate according to the invention ;

Fig.2: Fluorescence inhibition percentage values (l%) values as a function of standard formazin turbidity values (NTU) (fluorescence range from 1000 to 7000 lntensity^*mm²) determined according to the assay according to the invention;

Fig.3: Relationship between fluorescence inhibition percentage values and turbidity (NTU) of GSE/mucin mix at different phenol concentration (ranging from

0,39 - 2,91 mg/mL catechin) according to the assay of the invention.

Fig. 4: Relationship between the turbidity of GSE/mucin mix, measured as l%, and the astringency intensity induced by the same GSE solutions.

Fig.5 Effectiveness of wine fining agent assessed as l% values according to a specific application of the assay according to the present invention. ^*p<0.05

DETAILED DESCRIPTION OF THE INVENTION

The fluorescent gel, used for functionalising the microplate wells according to the invention, consists of a gelatinous polymer, with low diffusivity in water, in which was incorporated, by dissolution in an appropriate solvent, a fluorescent compound.

Diffusivity of a gel is inversely related to its solidity; within the purpose of the present invention, a gel with a solidity not lower than 250 g/cm² shows a suitable diffusivity.

Dissolution under heating means that the temperature ranges from 50 and 100°C, depending on the polymer melting point and on the heat stability of the fluorescent compound.

The fluorescent gel preferably fills 30-50% of the total available volume in each single well

Gelatinous polymer is preferably chosen among agarose, acrylamide and gelatin. Fluorescent compound is preferably chosen among 5(6)-carboxyifluorescein and 4-metilumbelliferon.

Fluorescent gel could incorporate preservative compounds (such as sodium azyde; salicylic acid, sorbic acid) in order to improve its stability during cold storage (4°C) in the dark.

The present invention for an aspect relates to a method for preparing a microplate as above described; this method comprising the following steps: a. preparing a solution comprising a fluorescent compound and a polymer with a gelatinous texture in a solvent, which is suitable for dissolving both components; for preparing the solution, the mixture may be optionally heated at a temperature which is compatible with the stability of the fluorescent compound;

b. dispensing the solution from step (a) in the wells of the microplate;

c. resting at appropriate temperature and over a period of time for solidifying the gel.

The solution for preparing the fluorescent gel, as described in the step (a), must have the following characteristics: - solvent used to dissolve the polymer must present physical-chemical characteristics (pH, ionic strength) compatible with both the fluorescent compound solubility and the right gel solidification;

- the amount of fluorescence emitted by the gel must be optimised as a function of the resolving capacity of the method used to detect the signal;

- when heating is used to solubilize the polymer, polymer melting point must be compatible with heat stability of the fluorescent compound and polymer solidification temperature should be low in order to facilitate the pouring of the gel in the microplate wells;

- polymer concentration must be low enough to permit the pouring of small gel volumes in the microplate wells by using a micropipette;

Fluorescent compound solution is preferably prepared using a aqueous solution buffered at a pH value suitable for the fluorescent compound/polymer mix. As an example, the combination 5(6)-carboxyfluorescein/agarose-low melting point (65- 75 °C) should be prepared in a buffered solution at pH 7.0, using, as an example, citrate-phosphate buffer. In the case of a solution 5(6)-carboxyfluorescein/agarose at low melting point should preferably be heathen at 65-75 °C in order to obtain an homogeneous solution.

The solution containing a fluorescent compound and a polymer is preferably prepared at a polymer concentration ranging from 0.5mM e 2.0mM (depending on the amount of fluorescence emitted by the compound) and at a gelatinous polymer concentration ranging from 1 .0 %w/v e 1 .5 %w/v (especially in the case of agarose).

Fluorescent compound/polymer compound is poured in the microplate wells, optionally hot, by using a micropipette. The volume poured in the wells should preferably be 30-40% of the total well volume.

The micro-plate in which the fluorescent compound-polymer gelling agent solution was dispensed is then allowed to stand, in a horizontal position, for a time and at a temperature sufficient to solidify the gel. The microplate is preferably left to stand at room temperature (20-25 °C) for 8-10 min or at 0-4 °C fro 3-7 min.

The present invention also refers to an assay for determining a solution or suspension turbidity, said method comprising the use of a microplate as above described, wherein a predetermined volume of a solution (or suspension) with an unknown turbidity is introduced into one of the wells of the microplate directly above the fluorescent gel.

This assay involves the preparation of a reference sample consisting of a well (functionalised with the fluorescent compound) containing the same volume of the unknown solution/suspension of the same solvent used for preparing the unknown solution/suspension.

The response of the assay for turbidity determination by using the functionalised microplate according to the present invention can be obtained using instruments widely available on the market an currently used, even with different purpose, in analysis laboratories. Moreover, in respect to current turbidity determinations based on the use of turbidimeter and nephelometer, the present method allows the acquisition of the response of several samples at one time and very small amounts (microliters) of samples and reagents are needed in this assay.

The amount of fluorescence screened by haze particles can preferably be determined by using a microplate fluorescence counter or by using a system for image acquisition and analysis (Gel Doc™ 2000, Bio-Rad, Hercules, CA, USA) fitted with a transilluminator as schematised in fig 1 .

The turbidity value of unknown samples is expressed as transmitted fluorescence inhibition percentage (l%) computed as reported in the following: l% = 100 - (Fl_s x 100 / Fl_r)

Fl_s= intensity of fluorescence measured in the wells containing the samples with unknown turbidity

Flr= intensity of fluorescence measured in the wells containing deionised water

The turbidity range able to induce a linear response of l% was determined by using 12 solutions of standard formazin with increasing turbidity (ranging from 10 to 650 NTU). Turbidity values were determined either by using a nephelometer HACH 2001 N Laboratory Turbidimeter (Hach Co, Loveland, USA), and by using the assay FLUO-HAZE by means of the functionalised microplate. The obtained data showed an increasing of 1% values as a function of the turbidity values of standard formazine samples. A significant linear relationship (y=0,0518x+5,5021 ; r² = 0,9601 ) was found relating the turbidity values of standard formazine samples, expressed as NTU, and l% values (Fig.2) The fluorescence range within the linear relationship between the turbidity values of samples, expressed as NTU, and l% values was verified is comprised between 1000 and 7000 (lnt^*cm²).

The above described assay is very useful for determining turbidity development in beverages from fruit and vegetables, with particular reference to wine. Turbidity represents a critical parameter for the physical-microbiological stability of these products. Production processes involve fining steps with the aim of reducing/preventing turbidity development.

The effectiveness of such as steps is currently checked by nephelometric responses and by the use of sensors for on-line monitoring.

The assay proposed in the present invention is very useful for determining optimal conditions for fining when several samples have to be analysed.

Moreover, fining steps are performed in red wine production with the aim of removing phenol compounds responsible for product astringency. Fining steps represent a critical point in red wine production since a too high extent of phenol removal can induce a lowering of wine sensory properties as well as colour losses. Removal of colloidal aggregates responsible for haze development and wine colour losses represent the main goal of fining steps. This step needs to be optimized in order allow the selective removal of colloidal aggregates with low stability while leaving phenol compounds needed for a proper wine maturation and development of its sensory profile in terms of right intensity of taste and mouth-feel sensations.

In a particularly preferred embodiment the assay of the present invention is combined with an in vitro test for predicting the astringent potential of phenol compounds in wine, this in vitro test is based on the development of turbidity in a reaction mixture of phenol/mucin which has proved to be proportional to the astringency intensity perceived when tasting wine. This correlation between turbidity development in a phenol/mucin mixture and the intensity of perceived astringency was demonstrated by proponents of the present invention (Monteleone, et al., Food Quality and Preference, 2004, 15, 761 -769). The use of the phenol/mucin reactivity test in combination with the functionalised microplate test, according to the present invention, is here proposed a useful tool to evaluate the effectiveness of different oenological fining agent for the removal of phenols potentially more unstable and more astringent.

A further aspect of the present invention is related to the realization of a "ready-to- use" kit for turbidity/astringency assessment in food products. This kit comprising the functionalised microplate according to the present invention and at least one of following components: turbidity standards (one or more), mucin and/or buffered solutions. The buffered solution should be used for preparing (immediately before use) the mucin solution and should have a pH of 3.5. A C18 cartridge for phenol extraction should be included in the kit when it is devoted to wine analysis.

The following applicative examples are reported with the aim to better illustrate the possible uses of the present invention..

EXPERIMENTAL PART

Microplate Functionalization

A black colour commercial microplate (Greiner, Labortechnik, USA), with 96 wells with a flat and transparent bottom (volume 250 μΙ per well) was modified by inclusion of an agarose gel containing 5(6) carboxyfluorescein as fluorescent compound. Low melting point agarose (Low Melt Agarose, Bio-Rad) (1 % p/v) was dissolved in a citrate-phosphate buffered solution pH 7.0 containing 5(6) carboxyfluorescein (Sigma) (1 mM). This step was performed at 70 °C. The mix was stirred for 3 min, then poured in the microplate wells (100 μΙ) and left to solidify at room temperature for 10 min. This step can be fastened by storing the microplate at 4°C for 5 min. The microplate is ready to be used immediately after gel solidification. These microplates were used in the in vitro test for phenol induced astringency prediction.

example 1 - performing the assay

A grape-seeds phenol commercial extract (GSE) produced by Intec (Verona) was used to prepare phenol solutions at different concentration for astringent potential evaluation. The commercial extract was dissolved in ethanol giving a 1 %(w/v) solution. The phenol content of this solution was determined by Folin-Ciocalteau reagent according to the official method (Off. J. Eur. Communitie, 1992). Eight GSE solutions at pH 5.0 with a concentration range from 0.48 to 3.6 g/L (expressed as catechin) were prepared.

Mucin from submascellar bovine glands (Sigma-Aldrich) was used as model protein dissolved in citrate-phosphate buffer pH 3.5 at a concentration of 0.2% (w/v).

The reaction mix was prepared by pouring in the microplate well, directly over the gel, 120 μΙ_ of phenol and 30 μΙ_ of mucin solutions. A reference sample was prepared by pouring 150 μΙ of water in a well. The microplate was then left at 37^0 for 1 min.

Microplate was then irradiated with UV-light in a transilluminator fitted with a system for image acquisition and analysis (Gel Doc™ 2000, Bio-Rad, Hercules, CA, USA). The resulting image was then acquired by a camera placed above the transilluminator and connected to a computer. Data acquisition and analysis were performed with Quantity One v. 4.3.0 (Bio-Rad) software. The fluorescence intensity of selected image area, corresponding to the microplate wells, was computed and expressed as lntensity^*mm².

Fluorescence inhibition percentage (l%) was computer as in the following:

l% = 100 - (Fl_s x 100 / Fl_r)

Fl_s= Fluorescence intensity detected in phenol/mucin mix containing well

Flr= Fluorescence intensity detected in water containing well An increasing of both NTU and l% values was observed with the increasing of GSE concentration from 0.48 a 3,6 g/L (Fig. 3). NTU and l% were related by a significant linear relationship (y = 0,1662x + 16,103; r² = 0,9413).

The significant variation of GSE/mucin solutions l% values were found in physiologically active phenol concentration range able to induce astringency intensity variation form extremely weak to extremely strong.

Turbidity of GSE/mucin solutions expressed as l% values was related to the astringency intensity values induced by the same GSE solutions. Sensory evaluations were performed by a panel of 23 trained subjects according to Monteleone et al. Food Quality and Preference, 2004, 15, 761 -769. A linear model describes the relationship between astringency intensity and l% values induced by GSE at different concentration (y=0.1704x + 0.0425; r²=0,9202) thus demonstrating that the functionalised microplate instrumental response can be used for predicting astringency potential of wine and grape phenol compounds (Fig.4).

Example 2. Evaluation of effectiveness of different penological fining agent

The effectiveness of four experimental fining agents (Gly A, Gly B, Na₂C0₃ A, Na₂C0₃ B) was compared with gelatin (G) considered as a referent fining agent. Fining was performed on a model phenol solution (grape seed extract 1 .8 mg/ml in ethanol). Experimental fining agents were added to phenol solution giving a final concentration of 0.4 mg/ml, gelatine was added to the model phenol solution at a final concentration 0.2 mg/ml. Samples were filtered after 18 hours and used in the functionalised microplate assay..

Solutions obtained after filtration were tested for reaction with mucin 0.2% (w/v) in citrate-phosphate buffer pH 3.5 in order to evaluate the effectiveness of tested fining agents in removal of unstable and potentially most astringent phenol compounds.

Reaction mix was prepared pouring 120 μΙ of phenol and 30 μΙ of mucin solutions directly over the gel. A reference sample was prepared with 150 μΙ of water. The microplate was then left at 37°C for 1 min.

Data acquisition and analysis were performed as previously described.

Data showed that fining agents exert different effects on model phenol solution properties (Fig.5). In fact l% values of solution added with fining agents were significantly lower in respect to those detected in untreated phenol model solution.

Not significant differences were found between the four experimental fining agents while gelatin resulted, as expected, the most effective fining agent.

The reaction test phenol/mucin combined with the functionalized microplate resulted a useful tool to check, monitor and optimize fining steps in wine production process.

Claims

1 . A microplate, usable for evaluating solution or suspension turbidity, the wells of which, with transparent bottom, were functionalized with a fluorescent gel, the strength of which is not less than 250 g/cm².

2. Microplate according to claim 1 , wherein the fluorescent gel comprises a polymer, having a gelatinous texture and a low water diffusivity, where a fluorescent compound was incorporated by dissolution in an appropriate solvent and/or with heating.

3. Microplate according to claim 2, wherein the polymer is chosen from agarose, acrylamide and gelatin.

4. Microplate according to any one of the claims from 2 to 3, wherein the fluorescent compound is chosen from 5(6)-carboxyfluorescein or 4- methylumbelliferone.

5. Microplate according to any one of the claims from 1 to 4, wherein the gel further comprises preservatives.

6. A method for preparing a microplate according to any one of the claims from 1 to 5, said method comprising the following steps:

a. preparing a solution comprising a fluorescent compound and a polymer with a gelatinous texture in a solvent, which is suitable for dissolving both components; for preparing the solution, the mixture may be optionally heated at a temperature which is compatible with the stability of the fluorescent compound;

b. dispensing the solution from step (a) in the wells of the microplate;

7. An assay for determining the solution or suspension turbidity, said method comprising the use of a microplate according to any one of the claims from 1 to 5, wherein a predetermined volume of a solution (or suspension) with an unknown turbidity is introduced into one of the wells of the microplate according to the invention, directly above the fluorescent gel.

8. A method for determining the development of turbidity in plant-origin beverages, said method wherein the assay according to claim 7 is employed.

9. An in vitro test for predicting the astringent potential of phenolic compounds in wine, said test comprising the use of the assay according to claim 7, said test depending on the development of turbidity in a reaction phenol/mucin mixture proportional to the astringency felt when the wine is consumed.

10. A kit for evaluating the turbidity or astringency features of foodstuff, said kit comprising a microplate according to any one of the claims from 1 to 5 and further comprising at least one of the following components: one or more turbidity standards, mucin and a buffer solution.