DE10254389B3 - Quantifying degree of coating of self-assembled monolayers on gold substrates, useful for quality control of biosensor substrates, by performing gold-catalyzed color-forming reaction - Google Patents

Abstract

Quantifying the degree of coating of self-assembled, thiol group-containing monolayers (SAM) on a gold substrate, in which the coated substrate is subjected to color-forming reaction with a chromophore (I) that reacts chemically with gold to cause a color change. The degree of coating is then determined from the optical density.

Description

The invention relates to a method to quantify the degree of coating self-organizing Monolayer containing thiol groups on a gold substrate, in which controls the gold substrate provided with the monolayer via a color reaction becomes. The gold substrate provided with the monolayer is included brought into contact with a chromophore by a chemical Reaction between the. Gold and the chromophore a color change entry.

Self-organizing monolayers self-assembled monolayer, SAM) of thiol group-containing compounds are widely used in the modification of gold surfaces. such Monolayer can in a simple way by treating the gold surface with the monolayer starting material, he will be produced. The self-organizing Monolayer can followed by the coupling with other functional groups can be modified. The simple manufacture of a monolayer on a gold surface is hence the main reason that these systems in the field of biosensors for the connection of biomolecules common Represent method (X.Kang et al, Electrochemistry Communication (2001) 489-493).

The fields of application of self-organizing monolayers widely used, however, the Assessment of goodness such a monolayer on gold surfaces is a major problem Different methods for the assessment of goodness of monolayers on metallic surfaces are known. So in D. D. Schlereth, Journal of Electroanalytical Chemistry 464 (1999) 189-207 a device for such measurements are described based on the surface plasmon resonance (English surface plasmon resonance, SPR) is based. Likewise from M.Dijksma et al, Langmuir 16 (2000) 3852-3857 an electrochemical measurement method known. However, the disadvantage of these known methods is that they are technical are very expensive, which is a not inconsiderable cost factor leads.

Based on this, it was task the present invention a method for quantifying the degree of coating to provide such a monolayer that is easy to use and is characterized by low costs.

This task is accomplished through the procedure solved with the features of claim 1. Show the further dependent claims advantageous further training.

According to the method of quantification the degree of coating of self-organizing thiol groups Monolayer provided on a gold substrate, with a gold substrate provided with the monolayer is subjected to a color reaction becomes. This color reaction is based on the chemical reaction between the gold-containing components of the substrate and a corresponding one Chromophore, which causes a color change. Based on the optical Density of discoloration is then the Degree of coating determined.

The process is based on the fact that surface of the gold substrate, which is not covered by the monolayer, corresponding Reactions of the gold surface run off with the chromophore and corresponding discoloration can effect.

verwendet. Wird eine Goldoberfläche in eine TMB-Lösung getaucht, kommt es zu einem sofortigen Farbumschlag nach blau, was auf die Oxidation des TMB an der Goldoberfläche zurückzuführen ist. Das TMB wird dabei stufenweise oxidiert und erzeugt unterschiedli che Farbtöne. Das erste Oxidationsprodukt besitzt eine blaue Färbung, während das zweite Oxidationsprodukt eine gelbe Färbung besitzt. Wird dagegen eine Goldoberfläche, die mit einem Monolayer beschichtet ist, mit einer TMB-Lösung behandelt, so ist keine Verfärbungsreaktion festzustellen. Diese Phänomen ist darauf zurückzuführen, daß die reaktive Goldoberfläche durch den Monolayer abgeschirmt wird.A chromophore is preferably chosen as a compound which can be oxidized by gold and which causes a color change. Here, 3,3 ', 5,5'-tetramethylbenzidine (TMB) of the formula I is preferred

used. If a gold surface is immersed in a TMB solution, the color changes to blue immediately, which is due to the oxidation of the TMB on the gold surface. The TMB is gradually oxidized and produces different shades. The first oxidation product has a blue color, while the second oxidation product has a yellow color. If, on the other hand, a gold surface coated with a monolayer is treated with a TMB solution, no discoloration reaction is observed. This phenomenon is due to the fact that the reactive gold surface is shielded by the monolayer.

It is also preferred as a chromophore to use o-phenylenediamines (OPD) of the formula II.

The implementation is preferably carried out the color reaction in such a way that the one provided with the monolayer Gold substrate immersed in the chromophore dissolved in a citrate buffer becomes. The citrate buffer can preferably additionally contain hydrogen peroxide.

The color reaction is preferred by quenching with sulfuric acid canceled.

The optical density is preferred at one wavelength measured in the range between 400 to 500. Is particularly preferred a wavelength of 450 or 492 nm. The evaluation of the degree of coating can preferably by calibration using linear regression of the optical Density.

Using the example below and the following figures are the subject of the invention explained without restricting it to this.

1 shows a representation of the dependence of the optical density (OD) on the calculated degree of coating.

2 shows a representation of the dependence of the degree of coating on the impedance.

example

First, a gold surface was put on a glass substrate by sputtering a target made of gold made with an RF sputtering source. The glass plate treated in this way was washed with 2-propanol and then dried in a vacuum oven. The sputtering process was carried out at 30 mA for 90 seconds. The sputtered gold surface was for the further experiments are used directly.

The monolayer was made by immersion the gold-coated glass plate in a solution of undecanthiol in ethanol manufactured. To realize different degrees of coating the coating was at different temperatures at room temperature (25 ° C, 50% humidity) periods carried out. The monolayers produced in this way were used for the color reaction and uses the impedance measurements used as a reference.

The color reaction was carried out by immersing the gold substrates coated with the SAM in a 3,3 ', 5,5'-tetramethylbenzidine (TMB) over a period of 20 minutes. The area of the gold surface was 40 mm ² . The TMB solution was prepared by dissolving TMB in 0.2 M citrate buffer, which also contained hydrogen peroxide. The concentration of hydrogen peroxide (35% solution) was adjusted to be 20% of the final volume. It was found that the reaction of TMB with the gold surface is very strongly influenced by the concentration of the hydrogen peroxide. The prior art typically uses a hydrogen peroxide (35% solution) concentration of approximately 0.02% of the final volume for the reaction of TMB in the immunoassay. It has now been found that, however, the hydrogen peroxide concentration must be more than 100 times as large for the reaction with gold. Table 1 shows the relationships between the concentration of hydrogen peroxide and the result of the TMB color reaction. Table 1

These results show that if the concentration> 20 % and less than 2%, the color reaction drops significantly. The Hydrogen concentration should therefore be between 2 and 20% of the effective amount can be set.

After an incubation over a period of 20 minutes, an equivalent volume of 2 M sulfuric acid was added and the optical density of the solution was then determined at a measuring wavelength of 450 nm and a reference wavelength of 650 nm. The degree of coating was then determined from the measured optical densities as follows: θ = 1 - (OD - Od sat ) / (OD bare - Od sat ).

Here, OD ^{bare means} the optical density of an untreated gold surface and OD ^sat the optical density of a gold surface completely coated with a monolayer. The gold surface fully saturated with a monolayer was prepared by treatment with 1 mM 11-undecanethiol with a reaction time of 24 hours. Under these conditions, both the impedance measurements ( 2 ) as well as the measurements of the optical density the saturation of the surface.

As in 1 It can be seen that a linear relationship between the optical density in the color reaction and the degree of coating calculated according to the above formula could be established. A correlation factor of 73% × cm ² / AU was found in the range of the optical density between 0 and 1.6 AU / cm ² . In the same way, a linear relationship between the optical density and the degree of coating was found when using o-phenylenediamine (OPD) instead of TMB. For the impedance measurements used as a reference ( 2 ) a glass plate provided with a monolayer was connected to an electrochemical cell with a capacity of 100 ml. The area of the gold surface for the impedance measurements was 41.8 mm ² . As a test solution, 2 mM potassium hexacyanopherate, dissolved in 100 mM sodium phosphate buffer, was dissolved at a pH of 7.4. The impedance measurement was carried out at a potential of 0.1 V against Ag / AgCl at a pulse voltage with an amplitude of 10 mV and a frequency range between 10 mHz to 10 kHz. The impedance Z was estimated using an adaptation program of the frequency analyzer. The degree of coating was then calculated from the measured impedance using the following equation: θ = 1 - Z bare / Z SAM

Z ^{bare means} the impedance of an untreated gold surface and Z ^{SAM means} the impedance of a gold surface coated with a monolayer.

The results show that that both the impedance measurements as well as color reactions with TMB to the same Lead results. Allows at the same time the implementation the color reaction, however, that a larger number of samples can be determined much easier than with the Impedance method.

Claims (10)

Method of quantifying the degree of coating self-organizing monolayer containing thiol groups on a gold substrate, in which a gold substrate provided with the monolayer undergoes a color reaction a change in color due to a chemical reaction with gold effecting chromophore and based on the optical density the degree of coating is determined. A method according to claim 1, characterized in that the chromophore can be oxidized by gold and a color change causes. Method according to at least one of the preceding claims, characterized in that as a chromophore 3 , 3 ', 5,5'-tetramethylbenzidine (TMB) is used. Method according to at least one of the preceding Expectations, characterized in that as the chromophore o-phenylenediamine (OPD) is used. Method according to at least one of the preceding Expectations, characterized in that the color reaction by immersing a with the monolayer provided gold substrate in the chromophore dissolved in a citrate buffer is immersed. Method according to at least one of the preceding Expectations, characterized in that the citrate buffer additionally hydrogen peroxide contains in a concentration between 2 and 20%. Method according to at least one of the preceding Expectations, characterized in that the color reaction is terminated by adding sulfuric acid becomes. Method according to at least one of the preceding Expectations, characterized in that the optical density at a wavelength in the range from 400 to 500 nm is measured. Method according to at least one of the preceding Expectations, characterized in that the degree of coating by linear Optical density regression is calibrated. Method according to at least one of the preceding claims, characterized in that the Monolayer was made by coating the gold substrate from a solution of undecanthiol in ethanol.