Method for grafting a coloured indicator onto a solid substrate and implementation kit

Abstract

The present invention relates to a method for grafting, in a covalent manner, a coloured indicator having at least one aromatic primary amine function onto the surface of a solid substrate, including the steps that consist of: (a) preparing, from the coloured indicator having at least one aromatic primary amine function, the corresponding diazonium salt; (b) placing the solid substrate in contact with a solution containing the diazonium salt prepared in step (a) and subjecting the solution to non-electrochemical conditions for an optimal grafting time whereby organic chains in which the units are derivatives of the diazonium salt are grafted, in a covalent manner, onto the surface of the solid substrate; (c) washing the solid substrate thus grafted; (d) repeating steps (b) and (c) or possibly steps (a) to (c) at least once. The present invention also relates to a kit for implementing such a method.

Claims

1. A method for grafting, in a covalent manner, onto a surface of a solid substrate, a coloured indicator having at least one aromatic primary amine function, comprising the following steps: a) preparing, from said coloured indicator having at least one aromatic primary amine function, a corresponding diazonium salt; b) placing the solid substrate in contact with a solution containing the diazonium salt prepared in step (a) and subjecting said solution to non-electrochemical conditions for an optimal grafting time whereby organic chains comprising units that are derivatives of said diazonium salt are grafted, in a covalent manner, onto the surface of said solid substrate; c) washing the solid substrate thus grafted; d) repeating steps (b) and (c) or possibly steps (a) to (c) at least once; wherein said optimal grafting time is determined for a given coloured indicator by a method involving: ) carrying out a plurality of grafting experiments, each grafting experiment being carried out for a period of time T, regularly subdivided into n sub-periods, n being a whole number greater than or equal to 1 that is different for each grafting experiment, each sub-period corresponding to a step of free-radical chemical grafting of the coloured indicator onto the surface of a solid substrate and each grafting step being followed by at least one washing step, ) after the period of time T, determining a quantity of coloured indicator grafted for each grafting experiment, normalising the quantity determined for each grafting experiment with respect to a quantity determined for the grafting experiment in which n is equal to 1, and determining an experiment E for which the normalised quantity is maximum whereby the optimal grafting time is T/n.sub.e with n.sub.e corresponding to a value of n during the experiment E, and T corresponding to the period of time T during the experiment E.

2. The method according to claim 1, wherein said coloured indicator is sensitive to an element chosen from the group consisting of a hydrogen ion, a halide ion, a calcium ion, a sodium ion, a potassium ion and oxygen.

3. The method according to claim 2, wherein said coloured indicator is sensitive to a chloride ion.

4. The method according to claim 1, wherein said coloured indicator comprises, in its native chemical structure, said at least one aromatic primary amine function.

5. The method according to claim 1, wherein said coloured indicator is a coloured indicator sensitive to pH chosen from the group consisting of Neutral Red, Rhodamine 560, Rhodamine 123, methyl violet 6B, Congo Red and 2-aminophenol.

6. The method according to claim 1, wherein said at least one aromatic primary amine function has been introduced into a native chemical structure of the coloured indicator via conventional organic chemistry techniques.

7. The method according to claim 1, wherein said step (a) involves placing said coloured indicator in a presence of either NaNO.sub.2 in an acidic aqueous medium or NOBF.sub.4 in an organic medium.

8. The method according to claim 1, wherein said non-electrochemical conditions are chosen from the group consisting of thermal conditions, kinetic conditions, chemical conditions, photochemical conditions and combinations thereof.

9. The method according to claim 1, wherein each grafting step in determining the optimal grafting time is followed by a first step of washing in water, then a second step of washing in ethanol, and finally a third, last step of washing in acetone, these three washing steps being carried out in a presence of ultrasounds.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the IR spectrum of Neutral Red grafted onto a thin gold slide.

(2) FIG. 2 shows the IR spectrum of Rhodamine 560 grafted onto a thin gold slide.

(3) FIG. 3 shows the normalised IR transmittances as a function of various sequencings for Neutral Red (FIG. 3A) and Rhodamine 560 (FIG. 3B).

(4) FIG. 4 shows the height of the IR band as a function of the number of cycles for Neutral Red (FIG. 4A) and Rhodamine 560 (FIG. 4B).

(5) FIG. 5 shows the increase in the sensitivity (absorbance) according to the thickness of the sensitive layer deposited.

(6) FIG. 6 shows the absorption spectrum, in the visible range, of Neutral Red grafted onto a thin gold slide at various pH values (FIG. 6A) and the variation in the absorbance peak at 550 nm according to the pH (FIG. 6B).

(7) FIG. 7 shows the absorbance at 560 nm according to the pH measured on a glass slide and Rhodamine 560.

(8) FIG. 8 shows the IR spectrum of the gold slide coated with PMMA (PMMA) and compared to the spectrum of the gold slide (Au).

(9) FIG. 9 shows the IR spectrum of a gold slide coated with PMMA (PMMA), of a neutral-red-grafted normal gold slide (Au+RN) and of a gold slide coated with PMMA and neutral-red-grafted (PMMA+RN).

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

(10) I. General Observations

(11) The inventors used the radical chemical grafting method as described in the international application WO 2008/078052 [5].

(12) The reference substrate in this method is a gold slide generally in the form of a glass slide onto which a layer of chromium of approximately 15 nm is deposited and itself coated with a layer of gold of approximately 200 nm.

(13) However, the work presented below has been carried out on a thin gold slide in the form of a glass slide onto which a layer of chromium of approximately 3 nm is deposited and itself coated with a layer of gold of approximately 27 nm.

(14) II. Sequencing

(15) It should be noted that all the solutions used during the sequencing are mechanically stirred and that the concentrations of coloured indicators used are 2 g.Math.l.sup.1, or 7.4 mmol.Math.l.sup.1, and 0.2 g.Math.l.sup.1, or 0.5 mmol.Math.l.sup.1, respectively, for Neutral Red and for Rhodamine 560.

(16) II.1. Determining the Optimal Duration of a Bath

(17) A. Neutral Red

(18) In a beaker: 25 ml of Neutral Red solution (0.1 g of neutral red in 25 ml of HCl 0.1 mol.Math.l.sup.1)+25 ml of NaNO.sub.2 solution (1 molar equivalent with respect to the neutral red, or 0.024 g in 25 ml HCl 0.1 mol.Math.l.sup.1) (M(neutral red)=269 g.Math.mol.sup.1, M(NaNO.sub.2)=69 g.Math.mol.sup.1). In this solution, the neutral red is transformed into the corresponding diazonium salt.

(19) An excess of iron powder corresponding to the non-electrochemical condition as defined in the method according to the invention is added (approximately 1 g) in order to reduce the diazonium salt.

(20) 7 gold slides are placed in contact with the reactive solution for a total reaction time of 30 min and, for each of the slides, with a precise sequencing namely:

(21) 1) 1 bath of 30 min

(22) 2) 2 baths of 15 min

(23) 3) 3 baths of 10 min

(24) 4) 6 baths of 5 min

(25) 5) 10 baths of 3 min

(26) 6) 15 baths of 2 min

(27) 7) 30 baths of 1 min

(28) Between two baths, each of the slides (2) to (8) is rinsed with water, ethanol and acetone, in the presence of ultrasounds.

(29) Then, each slide is passed through an infrared spectrometer, and the transmittance of the aromatic band at 1600-1650 cm.sup.1 is measured (FIG. 1).

(30) B. Rhodamine 560

(31) A protocol comparable to that implemented with the neutral red (7 slides+same sequencing) was used with rhodamine 560 under the following conditions: 25 ml of solution of rhodamine 560 (0.01 g of rhodamine 560 in 25 ml of (HCl 0.5 mol.Math.l.sup.1+ethanol 1:1 volume))+25 ml NaNO.sub.2 solution (2 molar equivalents with respect to the rhodamine 560, or 0.0038 g in 25 ml (HCl 0.5 mol.Math.l.sup.1+ethanol 1:1 volume)) (M(rhodamine 560)=367 g.Math.mol.sup.1, M(NaNO.sub.2)=69 g.Math.mol.sup.1).

(32) Then an excess of iron powder is also added (approximately 0.2 g).

(33) Each slide is passed through an infrared spectrometer, and the transmittance of the band caused by the CO carbonyl of the carboxylic acid function at 1770 cm.sup.1 is measured (FIG. 2).

(34) C. Results

(35) These experiments were repeated four times for each coloured indicator. In order to compare the results, all the transmittances were normalised with respect to the transmittance obtained for the slide (1) i.e. a single bath of 30 min.

(36) The results presented in FIG. 3 clearly show that the optimal length of the bath is 5 min for neutral red (FIG. 3A) and 1 min for rhodamine 560 (FIG. 3B).

(37) II.2. Multiplication of the Number of Baths

(38) A. Neutral Red

(39) A solution containing the aryl diazonium salt corresponding to neutral red is prepared as described in point II.1.A, hereinafter designated diazonium solution.

(40) To 10 ml of diazonium solution, an excess of iron powder and a gold slide are added for 5 min. Then, the slide is cleaned with water, ethanol and acetone, in the presence of ultrasounds before the aromatic IR band at 1600-1650 cm.sup.1 is measured.

(41) The same cycle is repeated 17 times i.e. 17 baths of 5 min during which the gold slide is put in contact with the aryl diazonium salt derived from the neutral red in the presence of a non-electrochemical condition (iron powder) with, between each bath, rinsing of the slide and IR measurement.

(42) B. Rhodamine 560

(43) A solution containing the aryl diazonium salt corresponding to rhodamine 560 is prepared according to an alternative of the method described in point II.1.B namely by placing 0.02 g of rhodamine and 0.0076 g of NaNo.sub.2 in 100 ml of (HCl 0.5 M+EtOH 1:1 volume).

(44) This solution is put in contact with a gold slide in the presence of an excess of iron powder for 1 min before being cleaned with water, ethanol and acetone, in the presence of ultrasounds. The IR measurement is carried out every 3 baths of 1 min and 17 IR measurements are carried out.

(45) C. Results

(46) The IR measurements obtained with neutral red and rhodamine 560 are presented in FIG. 4A and FIG. 4B, respectively.

(47) In both cases, the transmittance increases with the number of cycles which proves that by multiplying the number of cycles, the quantity of dyes on the slides increases.

(48) The sequencing method is therefore effective in increasing the thickness of the layer of coloured indicators and thus the sensitivity and the accuracy of a solid substrate thus grafted for the determination of chemical parameters. For this purpose, the UV-visible spectrum, at a pH of 1, of a gold slide grafted with neutral red after 6 then 12 baths of 5 min should be considered (FIG. 5).

(49) III. Optical Variations in the Grafted Dyes

(50) Since once grafted, the dyes lose the aromatic primary amine naturally present in the dye, it should be verified that the Neutral Red and the Rhodamine 560 have preserved their properties of variations in the absorption of light according to the pH of the medium.

(51) Thus, a gold slide grafted with either neutral red or rhodamine 560 is placed in an optical cell of a UV-Vis spectrometer. The light passes through the cell and the slide which allows the optical signal caused by the grafted dye to be measured. Moreover, by varying the pH in the cell, it is possible to verify the variations in the absorption spectrum.

(52) A. Neutral Red

(53) The absorption spectrum presented in FIG. 6 shows that there are variations in the optical properties of the neutral red, once grafted, according to the pH. Indeed, by comparing this result to those obtained for neutral red dissolved in solutions, two changes can be observed: the maximum absorption is at 550 nm for the grafted neutral red, whereas it is at 520 nm for the neutral red in solution; and the range of pH at which the grafted neutral red can be used is between 1 and 4, whereas this range is between 5 and 8 for neutral red in solution.

(54) These changes are very certainly the consequence of the loss of the aromatic primary amine lost during the grafting, which leads to a change in the conjugated system of the aromatic cycles responsible for the absorption of the neutral red in the visible range.

(55) B. Rhodamine 560

(56) FIG. 7 shows the variations in the absorbance peak, according to the pH and at 560 nm, of the rhodamine 560 grafted onto a glass slide.

(57) IV. Grafting of the Neutral Red onto PMMA

(58) IV.1. Preparation of a Slide of PMMA

(59) In order to obtain a slide of PMMA, approximately 1.5 g of PMMA are dissolved in 100 ml of N,N-dimethylformamide (DMF), then a gold slide is soaked in the PMMA solution.

(60) Then, the slide is taken out of the bath and left to air dry, while using a little acetone, before complete drying, in order to remove the DMF that can remain trapped in the layer of PMMA.

(61) The slide is passed through an infrared spectrometer (FIG. 8). The characteristic band of the CO carbonyl of the PMMA at 1730 cm.sup.1 and the bands caused by the CO bond at approximately 1200-1300 cm.sup.1 are observed, the slide has indeed been coated with PMMA.

(62) IV.2. Grafting of the Neutral Red onto the PMMA Slide

(63) A solution containing 0.2 g of neutral red and 1 equivalent of NaNO.sub.2 (0.048 g) in 50 ml of HCl at 0.1 mol.Math.l.sup.1 is prepared. An excess of iron powder (approximately 1 g) is added to the solution before placing the PMMA slide therein for 30 min.

(64) The slide is passed through an infrared spectrometer and compared to a slide of PMMA with the band CO at 1730 cm.sup.1 and a neutral-red-grafted normal gold slide with the aromatic bands at approximately 1600-1650 cm.sup.1-. Since, for the gold slide previously prepared, both the CO band and the aromatic bands are observed: the neutral red has indeed been grafted onto the layer of PMMA covering the gold slide.

(65) This is also confirmed by the XPS spectrum obtained via the difference between the spectrum of a neutral-red-grafted gold slide coated with PMMA and the spectrum of a gold slide coated with PMMA without grafting. In such a spectrum, only the signal caused by the neutral red remains, and the conventional contributions for neutral red, already observed during the grafting of the neutral red onto a normal gold slide, are observed.

REFERENCES

(66) [1] Noire et al, 2000, A New Sol-Gel Derived Optical Sensor for High Acidity Measurements: Applications in Nuclear Fuel Reprocessing, Journal of Sol-Gel Science and Technology, vol. 17, pages 131-136. [2] Raoufi et al, 2013, Fibre optic pH sensor using optimized layer-by-layer coating approach, IEEE Sensors Journal, vol. 14, pages 47-54. [3] Nguyen et al, 2009, Development of intrinsic optical fiber pH sensors for industrial applications, IEEE Sensors Conference. [4] Baldini et al, 1994, Controlled-pore glasses embedded in plastic optical fibers for gastric pH sensing purposes, Appl. Spectr., vol. 48, pages 549-552. [5] International application WO 2008/078052 on behalf of CEA, published on Jul. 3, 2008. [6] Lyskawa and Blanger, 2006, Direct Modification of a Gold Electrode with Aminophenyl Groups by Electrochemical Reduction of in Situ Generated Aminophenyl Monodiazonium Cations, Chemistry of Materials, vol. 18, pages 4755-4763.