RU2796202C1 - Method for manufacturing a biosensor structure - Google Patents

Abstract

FIELD: sensor devices.

SUBSTANCE: invention is related to a technology for manufacturing sensor structures based on a solid-state semiconductor and a functional organic coating and can be used to create enzyme biosensors based on field-effect transistors or "electrolyte-dielectric-semiconductor" structures. The method for manufacturing a biosensor structure includes modification of a semiconductor electrochemical converter to create an effective negative electrostatic charge, as well as layer-by-layer adsorption of a layer of polycationic polymer molecules and a layer of polycationic enzyme molecules from their aqueous solution. A plate of single-crystal silicon with a hole type of conductivity is used, and its modification is carried out by boiling the semiconductor plate in an ammonium peroxide solution NH₄OH/H₂O₂/H₂O=1/1/4, and in the process of adsorption of enzyme molecules or immediately before the process of adsorption onto the surface of the Si/SiO₂/polyethyleneimine structure, the structure is illuminated from the solution side with an intensity sufficient to change the charge density of the surface of the semiconductor structure during adsorption.

EFFECT: increase of sensitivity to the analyte due to the increase in the number of molecules in the monolayer of the enzyme on the modified surface of the semiconductor converter.

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Description

The invention relates to a technology for manufacturing sensor structures based on a solid-state semiconductor and a functional organic coating and can be used to create enzyme biosensors based on field-effect transistors or electrolyte-dielectric-semiconductor structures, providing high sensitivity and selectivity of the biosensor.

A known method for producing single- or multilayer film elements by layer-by-layer adsorption, which consists in the deposition of polyionic molecules from solution onto charged substrates, that is, polymer molecules with an effective charge in solution (see patent for invention US 5208111, IPC B32B7/04, publ. May 4, 1993). The method includes modifying the substrate in such a way that negatively charged ions or ionizable negatively charged compounds are located over the entire surface area of the substrate, preparing an aqueous solution of polycationic molecules, adsorbing polycationic molecules on the substrate, washing in deionized water, and drying the substrate with the deposited layer in a dry air stream. The charge of the first organic layer is opposite to that of the substrate. To deposit the next layer, the substrate is placed in a solution with molecules having a charge opposite to that of the last deposited layer. If one or several layers in such multilayer polyelectrolyte coatings are enzyme molecules placed between the layers of polyelectrolytes, which have the corresponding electrical charge, then such multilayer polyelectrolyte structures can be used as sensitive coatings for biosensors.

However, this method does not take into account the specifics of the formation of the effective charge of the semiconductor substrate, considering only the charge of OH groups on the surface of silicon dioxide after modification of the silicon substrate, and even more so, the factors influencing the sign of the charge of surface electronic states at the Si/SiO ₂ and SiO ₂ interfaces are not taken into account. /polyelectrolyte. This method also provides for an increase in the number of adsorbed molecules of a certain type per unit area only by changing the number of deposited layers, and a monolayer of molecules with an opposite charge should be adsorbed between the layers of enzyme molecules.

Closest to the present invention is a method for manufacturing sensor structures, including modification of a semiconductor electrochemical converter to create an effective negative electrostatic charge, as well as layer-by-layer adsorption of a layer of polycationic polymer molecules and a layer of polyanionic enzyme molecules from their aqueous solution. In this case, a single-crystal silicon wafer with an electronic type of conductivity is used, and its modification is carried out by boiling the semiconductor wafer in an ammonia-peroxide solution NH ₄ OH/H ₂ O ₂ /H ₂ O = 1/1/4, and in the process of adsorption of enzyme molecules, or beforehand immediately before the adsorption process, on the surface of the “ n -Si/SiO ₂ /polyethyleneimine” structure, the structure is illuminated from the side of the solution with an intensity sufficient to change the charge density of the surface of the semiconductor structure during the adsorption time (see patent for the invention of the Russian Federation No. 2644979, IPC H01L51/40, published 15.02.18).

The disadvantage of this method of manufacturing a biosensor structure is the use of only one type of semiconductor signal converter and enzyme, which is insufficient to expand the range of detectable substrates. Most enzymes are polyelectrolytes with a predominantly negative or positive charge in neutral pH solution. Therefore, only the presence of an electric charge on the enzyme in solution is essential for the experiment.

The technical result of the invention is an increase in the density of immobilized HRP molecules compared to samples without a buffer layer and made without the use of illumination. Expanding the arsenal of tools will improve the technical characteristics of virtually any enzyme biosensors operating on the field effect.

The technical result is achieved by the fact that in the method of manufacturing a biosensor structure, including modification of the substrate to create an effective negative electrostatic charge, preparation and adsorption of a layer of polyelectrolyte molecules on the substrate from an aqueous solution, preparation and adsorption of a layer of organic enzyme molecules from an aqueous solution, washing in deionized water and drying the substrate in a stream of dry air after each layer is applied, single-crystal silicon with a layer of silicon dioxide tunneling transparent to electrons is used as the substrate, polyethyleneimine is used to prepare an aqueous solution of polyelectrolyte molecules, during the adsorption of enzyme molecules on the substrate, the substrate is illuminated from the side of the solution with white light according to the invention , polycationic molecules are chosen as polyelectrolyte molecules, horseradish peroxidase is used as _organic enzyme molecules, and single-crystal silicon is used as a substrate with a hole type of conductivity, the silicon substrate is additionally illuminated during the adsorption of polyelectrolyte molecules with a light intensity sufficient to change the charge density of the surface of the semiconductor structure.

In the process of manufacturing a biosensor structure, a change occurs in the electrostatic interactions between its layers, which is retained during its operation. A change in the morphology and surface potential of a semiconductor signal converter due to the use of illumination leads to a change in the physicochemical parameters of organic layers, a redistribution of charges in polyionic enzyme molecules, aimed at increasing their biocatalytic activity.

The invention is illustrated by drawings.

In FIG. 1 shows a diagram illustrating the essence of the claimed invention: 1 - the stage of preliminary preparation of the silicon surface, 2 - the stage of applying a layer of polycationic molecules, 3 - the stage of applying enzyme molecules by the proposed method. During the stages, the following processes occur: activation of negatively charged OH groups on the surface of silicon 4 after stage 1; the formation of a positive charge on the surface due to the adsorption of molecules of the cationic polyelectrolyte polyethyleneimine (PEI) 5 after step 2; partial compensation of the negative charge of the surface by the charge of polycationic molecules of the horseradish peroxidase enzyme 6. 7 - space charge region (SCR), the width of which varies at stages 1-3 depending on processes 4-6, 8 - tunnel thin layer of SiO ₂ .

In FIG. 2 - shows two images obtained using atomic force microscopy (AFM) and surface profiles of samples: p -Si/SiO ₂ /HRP, after dark (a) and light (b) deposition of the enzyme on a silicon substrate from an aqueous solution.

In FIG. 3 - average roughness observed after dark and light deposition of HRP on p -Si/SiO ₂ /PEI from an aqueous solution, where 9 - p -Si/SiO ₂ , 10 - p -Si/SiO ₂ /PEI (under illumination), 11 - p -Si/SiO ₂ /PEI/HRP (in the dark), 12 - p -Si/SiO ₂ /PEI/HRP (under illumination).

In FIG. 4 - AFM images and surface profiles of samples: p -Si/SiO ₂ /PEI/HRP, after dark (a) and light (b) deposition of the enzyme on a silicon substrate from an aqueous solution.

The method is implemented as follows.

First, a stage of preliminary preparation of the surface of single-crystal silicon with a layer of natural oxide is carried out to remove organic and inorganic contaminants and create a uniform negative charge on the surface due to the activation of negatively charged OH groups on the oxide surface. Preliminary preparation of silicon wafers includes peroxide-ammonia treatment (boiling at 75°C for 10-15 minutes in a solution of NH ₄ OH/H ₂ O ₂ /H ₂ O in a volume ratio of 1/1/4, respectively). After boiling in an ammonia-peroxide solution, the substrates are thoroughly washed in deionized water with a resistivity of 18 MΩ·cm for 20 minutes and dried in a stream of dry air.

The next step is the application of a layer of polycationic molecules from an aqueous solution onto a cleaned silicon wafer, due to which the enzyme molecules are fixed by layer-by-layer adsorption. To do this, prepare an aqueous solution of a cationic polyelectrolyte, for example, polyethyleneimine concentration of 1-3 mg/ml. The single-crystal silicon plate cleaned at the previous stage is immersed in the prepared solution for 10 minutes. This time is sufficient for the adsorption of one continuous monolayer of polyelectrolyte molecules. Next, the silicon wafer is washed in deionized water for 10 minutes to remove those molecules that are not adsorbed on the surface of the silicon wafer due to electrostatic interaction, and dried in a stream of dry air.

The next step is the application of a layer of polycationic enzyme molecules, such as horseradish peroxidase (HRP). The claimed technical result is achieved due to the fact that at this stage the semiconductor substrate, on which a layer of polycationic molecules has been previously applied, is illuminated with monochrome or polychrome ("white") light with wavelengths from the intrinsic absorption region of the semiconductor.

An example of a specific implementation.

As a semiconductor signal converter, plates of single-crystal Si p -type were used, which were processed in an ammonia-peroxide solution of NH ₄ OH:H ₂ O ₂ :H ₂ O=1:1:4 for 10 minutes at a temperature of 75°. Then the samples were washed in deionized water and dried in a stream of dry air.

Horseradish peroxidase is the enzyme used in the experiment. HRP molecules were precipitated from aqueous solutions with a protein concentration of 0.5 mg/ml. Interest in this enzyme is due to its wide application, difference from the enzyme glucose oxidase, increased amphoteric properties and relatively small size.

To deposit organic layers, prepared substrates were either immediately immersed in an aqueous solution of horseradish peroxidase, or first in an aqueous solution of PEI (concentration 1 mg/mL) and then in an HRP solution. The adsorption time of organic molecules was constant and amounted to 10 minutes. During adsorption, some of the samples were illuminated. The samples were then washed in deionized water for 10 minutes and dried in a stream of dry air to remove loose molecules.

To further evaluate the influence of the field effect and illumination on the adsorption of polyelectrolytes, the level of photosensitivity of the Si wafers used was measured. In all experiments, a Schott fiber illuminator with a halogen lamp was used, the emission spectrum of which contained wavelengths from the absorption spectrum of Si. The illumination at the center of the light spot was approximately 20,000 lux.

Following the technological algorithm, coatings with reproducible parameters were obtained, which was confirmed by measurements of the obtained samples using the INTEGRA Spectra installation.

Results of adsorption of HRP from an aqueous solution under illumination and in the dark on a pure Si p -type substrate and on a substrate with a PEI buffer layer

The buffer polyelectrolyte layer of PEI was deposited under illumination on a silicon substrate from an aqueous solution (pH≈6.5).

By the magnitude of the change in the average height of irregularities (H _cf ) and roughness (Sa), one can judge the success of the immobilization of HRP molecules (Fig. 2). The deposition of organic molecules leads to an increase in the average height of irregularities, while the change in _Hav correlates with the size of the deposited molecules. The change in roughness, which is essentially the root-mean-square deviation of the probabilistic distribution of heights on the scan, depends both on the number of adsorbed particles and on the homogeneity (uniformity) of the surface layer.

In FIG. 3 shows the absolute changes in average roughness after applying HRP over a pre-applied PEI layer. The average roughness changed more with photostimulated HRP application.

The PEI layer was applied under illumination, so it had a very insignificant effect on the average height of the irregularities and the average roughness. Obviously, the PEI layer increased the HRP adsorption - the number of immobilized HRP molecules increased, but their distribution over the substrate surface became more inhomogeneous. Photostimulated adsorption (PSA) HRP increased the roughness. Lighting in this case (in the presence of a PEI sublayer) increases both the average height of irregularities and the average roughness.

Also from FIG. It can be seen from Fig. 4 that when a PEI layer is preliminarily applied, under the same adsorption conditions, a much higher density of immobilized HRP molecules is obtained compared to samples without a buffer layer.

Thus, the sensitivity to the analyte increases due to the increase in the number of molecules in the monolayer (density) of the enzyme on the modified surface of the semiconductor converter.

Claims (1)

A method for manufacturing a biosensor structure, which includes modifying the substrate to create an effective negative electrostatic charge, preparing and adsorbing a layer of polyelectrolyte molecules on the substrate from an aqueous solution, preparing and adsorbing a layer of organic enzyme molecules from an aqueous solution, washing in deionized water, and drying the substrate in a stream of dry air after each layer is applied, single-crystal silicon with a layer of silicon dioxide tunneling transparent to electrons is used as a substrate, polyethyleneimine is used to prepare an aqueous solution of polyelectrolyte molecules, during the adsorption of enzyme molecules on the substrate, the substrate is illuminated from the side of the solution with white light with an intensity sufficient the time of change in the charge density of the surface of the Si/SiO ₂ structure during the adsorption of enzyme molecules, characterized in that polycationic molecules are chosen as polyelectrolyte molecules, horseradish peroxidase is used as organic molecules of the enzyme, and single-crystal silicon with a hole type of conductivity is used as a substrate, additionally the silicon substrate is illuminated during the adsorption of polyelectrolyte molecules with a light intensity sufficient to change the charge density of the surface of the semiconductor structure.

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