WO2011047662A2

WO2011047662A2 - Sers substrate

Info

Publication number: WO2011047662A2
Application number: PCT/DE2010/001210
Authority: WO
Inventors: Robert MÖLLER; Jürgen Popp; Thomas Schüler; Katharina Strelau
Original assignee: Friedrich-Schiller-Universität Jena
Priority date: 2009-10-20
Filing date: 2010-10-15
Publication date: 2011-04-28
Also published as: WO2011047662A3; EP2491373A2; DE102009050287B4; DE102009050287A1

Abstract

The invention relates to a novel SERS substrate, a method for the production thereof, a method for the examination thereof, and the use thereof. The aim of the invention, which is to provide novel SERS substrates that can be produced in a cost-effective manner compared to clean-room SERS substrates and that have substantially ordered particle structures compared to randomly arranged metal-particle SERS substrates, is solved in that the SERS substrates are composed of metal nanoparticles that are enzymatically deposited onto a substrate surface, and the SERS activity is characterized by means of electrical conductivity measurements or gray-scale analysis.

Description

SERS Substrate Description The invention relates to a novel SERS substrate, a method for its production, a method for its verification and its use.

Enzymes for the production of nanoparticles are known, for example, from US Pat. No. 6,670,113. According to this disclosure, enzymes are used to precipitate or accumulate metal particles (reaction of enzymes with metal ions to achieve metal deposition).

According to US Pat. No. 6,670,113, the following procedure is followed:

- Use of the enzyme horseradish peroxidase

- Apply the enzyme to the surface, allow to dry

Incubation with 1 ml of 0.2% silver acetate in water for 3 min

- Wash 2x with water

- Add 1 ml solution consisting of 2.5 mg / ml hydroquinone, 1 mg / ml silver acetate and 0.06% hydrogen peroxide in 0.1M citrate buffer pH 3.8

- Silver deposit visible within a minute

Use of this principle for biological applications, e.g. Immunostaining,

- detection of bacteria, in situ hybridization, detection of single genes,

- Visualization with the naked eye

- Visualization by microscope

- reflection

- Electron microscopy

- Polarographic, electrochemical, electrical detection

- X-ray spectroscopy

- Chemical tests

- Detection of the mass

- Light scattering - Magnetic detection

- Autometallography

- Scanning Probe Microscopy

From the publication R. Möller, R.D. Powell, J.F. Hainfeld, W. Fritzsche, Nano Lett. 2005, 5, 1475-1482 nanoparticles are known for electrical detection. According to this publication, an enzyme reaction is used as an alternative to the modified spherical nanoparticles for electrical DNA detection.

The procedure is as follows:

Linkage of streptavidin - modified horseradish -

Peroxidase to biotin - modified DNA

Addition of the EnzMet ™ - Kits- enzymatic silver deposition Detection by reading the electrical conductivity of the individual DNA spots

The method of using an enzyme to attach nanoparticles to DNA is an alternative method to streptavidin-modified spherical gold nanoparticles. The advantage of using enzymes is the reduction of the background signal due to the highly specific reaction of the enzyme, resulting in a higher sensitivity.

US 2003/0211488 Al discloses the use of nanoparticles as SERS substrates.

The procedure is as follows:

Modification of Gold Nanoparticles with Thiol - Modified

DNA with internal Raman label

Use of this DNA-nanoparticle complex as a label for sequence-specific DNA detection

Detection by Surface Enhanced Raman Spectroscopy

Increasing the amplification and thus increasing the signal by additional silver deposition on gold nanoparticles

The additional silver reinforcement additionally enables an optical evaluation The disadvantages of this technical solution are that a complicated modification of the gold nanoparticles with thiol - modified DNA must be made, no statements can be made about the duration of the silver deposition and no statements are possible whether the SERS signal drops after a certain silver deposition time.

From the publication Z. Wang, L.J. Rothberg, Appl. Phys. B 84, 289-293 (2006) it is known that a dependence of the SERS gain on the density of the silver films depends on a surface, with low occupancy of the surface with silver (individual silver particle islands) only a very low SERS gain occurs, with As the silver particle islands increase in size and density, there is an increase in SERS enhancement and, in the event that a conductive layer of the silver particle islands forms, the SERS enhancement falls off again.

US 20040180379 discloses SERS substrates and their characterization as a biosensor for the detection of intracellular analytes by means of SERS, wherein the SERS substrate is produced by nanosphere lithography or FON (film over nanospheres).

From the publication N. Felidj, J. Aubard, and G. Levi, J.R. Krenn, A. Hohenau, G. Schider, A. Leitner, and F.R. Aussenegg, Appl. Phys. Lett., Vol. 82, no. 18, it is known that SERS substrates can be produced by electron beam lithography, whereby a highly reproducible SERS substrate can be formed which is tunable to a narrow band wavelength range.

The disadvantages of the previously known SERS substrates are:

highly reproducible SERS substrates (made, for example, with EBL) are very expensive,

favorable SERS substrates are not reproducible over a large area and therefore have to be characterized

- For the characterization, the SERS substrate is usually covered with a Raman reporter and measured the SERS signal of this reporter, then the reporter must again be removed so that the SERS substrate can be used for the analysis of other analytes,

- Often a batch of SERS substrates only characterizes a single one and its results are transferred to the others, since the Raman reporters can usually not be completely removed

- SERS - Substrates only for a certain (usually quite narrow band) wavelength range (100-200nm) can be used.

In addition, u.a. from the publications Pupils, T., Steinbrück, A., Festag, G., Moller, R. and Fritzsche, W. "Enzyme-induced growth of silver nanoparticles studied on single particle level", Journal of Nanoparticle Research (2008) and Festag, G., Schüler, T., Möller, R., Csaki, A. and Fritzsche, W. "Growth and Percolation of Metal Nanostructures in Electrodecapses leading to Conductive Paths for Electrical DNA Analysis, Nanotechnology (2008), 19 the Enzyme-induced silver particle deposition is known per se.

The object of the present invention is to avoid the disadvantages of the prior art, in particular to provide novel SERS substrates which are inexpensive to produce compared to clean-room SERS substrates and which are largely arranged in relation to randomly arranged metal particle SERS substrates Have particle structures, and to provide a method for their preparation and a method for their verification.

According to the invention, this object is achieved by the features of the 1st, the 6th and the 12th claim. Further favorable embodiments of the invention are specified in the subordinate claims.

The essence of the invention is that SERS substrates in the form of enzymatically produced nanoparticles are provided on substrate surfaces. The enzymatically produced nanoparticles according to the invention, in particular enzymatically produced silver manopar particles, can be applied to any desired 2D or 3D substrate surfaces. In addition to conventional 2D surfaces / materials, such as. Glass or silicon, polymers or hydrogels can be used. In principle, any material that has the possibility of immobilizing an enzyme on the carrier surface according to the invention is suitable.

On a substrate, for example glass, silicon, polymers, according to the invention, linker molecules, for example single-stranded DNA, are anchored between two electrodes to which enzyme molecules, such as, for example, a streptavidin-horseradish peroxidase complex, are bound. by means of whose enzyme activity a deposition of characteristically shaped silver nanoparticles on the substrate takes place.

The enzymatic reaction can be catalyzed by various enzymes. In addition to the enzymatic deposition of silver, the production of other metal nanoparticles is possible. For example. In addition to horseradish peroxidase-induced silver deposition, glucose oxidase or alkaline phosphatase may also be used.

The characteristic form of the enzymatically deposited (grown) silver particles is characterized by a desert rose-like growth. The formed particles (with desert rose shape) have pointed, angular structures, which differ significantly from conventional spherical metallic nanoparticles. As can be seen in the SEM image in FIG. 2, the individual particles consist of different platelets which grow into one another and next to one another and thereby form the distinct desert roses of similar formations (FIG. 3).

Due to this particle growth, these enzymatically produced particles can be used as SERS substrate even at high particle densities, even to a closed conductive layer become. Their angular and rough nanostructures prevent the collapse of SERS activity due to local elevations of the electromagnetic field. This makes it possible to characterize the SERS activity of these enzymatically produced metallic nanostructures with the aid of a simple electrical conductivity measurement.

The enzymatic reaction can be catalyzed by various enzymes according to the invention. In addition to the deposition of silver, the production of other metal nanoparticles is possible. For example, in addition to horseradish peroxidase-induced silver deposition, glucose oxidase or alkaline phosphatase can also be used.

Since the enzymatic deposition of the metal nanoparticles, in particular the silver nanoparticles on the substrate between the two electrodes, depends on the concentration of the linker molecules, the metal nanoparticle density, in particular the silver nanoparticle density (optimally about 50 to 70%) according to the invention by the concentration of Linker molecules (for example, single-stranded DNA of any sequence that is terminally amine- and biotin-modified) are set.

For example. can be adjusted by a linker concentration of 5μΜ a particle density of about 60%.

Immobilization of the enzyme on the substrate surface requires a linker molecule. The linker molecule must possess in its capacity a binding functionality for the enzyme (eg biotin). And additionally, another functionality to bind to the support surface (eg, an amino modification). Thus, for example, DNA can be used as linker molecule, which terminal is biotin as well as amino-modified.

As a result of further growth of the enzymatically produced silver nanoparticles on the substrate, according to the inventive method generates a metallic connection between the two electrodes.

This metallic compound between the two electrodes is used according to the invention to determine the SERS activity of the novel SERS substrates by a simple conductivity measurement, since it was surprisingly found that a relationship between the SERS activity and the conductivity of the SERS substrates according to the invention ( enzymatically deposited metal nanoparticles, especially silver manoparticles, on a substrate between two electrodes).

The SERS substrates according to the invention show their highest SERS activity between about 1 to 10 μs, whereas in the case of SERS substrates according to the prior art, the SERS activity already breaks down at about 1 μ8.

In the context of the invention, however, apart from this electrical SERS substrate analysis, too, the characterization of the SERS activity lies above gray value analysis (optical analysis).

The particular advantage of the SERS substrates according to the invention in the form of enzymatically produced metal nanoparticles, in particular enzymatically produced silver nanoparticles, is that they are used to characterize the SERS activity via electrical conductivity or gray value analysis, whereby a characterization of the SERS substrate surface in a very short time is possible and does not have to take place directly before its intended use (contrary to the state of the art, no cumbersome characterizations using standard molecules necessary), or that no expensive, produced in clean room SERS substrates are necessary to perform good SERS measurements can ,

This is possible because of the desert rose-like structure of the enzymatically deposited silver mane particles (Figure 3). This structure prevents by the many needle-like projections a drop in the SERS activity in the formation of a conductive layer of enzymatically deposited silver nanoparticles. This makes it possible to characterize the SERS activity of the enzymatically deposited silver nanoparticles even with larger particle densities. This is in contrast to the behavior of spherical nanoparticles. This property allows a simple and rapid characterization of the SERS substrates via the electrical conductivity. In addition, an optical characterization via the analysis of the gray values is possible.

Furthermore, the enzyme-deposited metal nanoparticles according to the invention, in particular silver nanoparticles, show a broad absorption range. While the absorption range of spherical nanoparticles is very narrow (50-200 nm on average), the absorption range of the enzymatically deposited silver nanoparticles ranges from the near UV to the IR range.

This allows the use of the enzymatically deposited silver nanoparticles with the most diverse excitation frequencies, since they must lie in the absorption region of the metal substrate.

It is also possible to use these SERS substrates for quantitative analysis. For this purpose, several SERS substrates are produced. By measuring the electrical conductivity, the SERS substrates are characterized in terms of their SERS activity in order to make the measurements on different SERS substrates comparable. Subsequently, the SERS substrates are incubated with different concentrations of the analyte solution and measured. Using the calibration series thus determined, unknown concentrations can subsequently be determined.

The SERS substrates according to the invention can be used, for example, for the detection of low-molecular-weight substances (for example dyes, antibiotics) of biomolecules (for example DNA, PNA, proteins), of natural substances and of organic polymers.

The invention will be explained in more detail below with reference to the drawings and the embodiments. Show it: 1 shows a schematic representation for carrying out the method according to the invention for producing the SERS substrates according to the invention (immobilization of horseradish peroxidase molecules in the electrode gap and enzymatic deposition of metal nanoparticles, in particular silver nanoparticles, on a substrate surface),

2: a scanning electron micrograph of a

Embodiment of the SERS substrate according to the invention

(Metal particles in the form of silver particles with desert rose-like shape on substrate surface, prepared according to the method schematically shown in Figure 1)

3 shows a schematic representation of an embodiment of the SERS substrate according to the invention (metal particles on substrate surface).

Embodiment 1

Production of an embodiment of the Invention according to

substrate

Wash the support materials for 5 min in an ultrasound bath in acetone, ethanol and distilled H ₂ 0

Drying under nitrogen

Wet chemical activation with a 1: 1: 1 (v / v / v) mixture of conc. HCl: H ₂ 0 ₂ : H ₂ O for 10 min at 20 ° C

Thorough cleaning with distilled H ₂ 0

Drying with nitrogen and finally in a drying oven at 80 - 100 ° C for 5 min

alternatively physical activation with the help of a

Plasma transporters at 1 mbar at 400 W for 30 min - Alternatively activation by means of plasma etching in one

two-step process: 30 sec in argon plasma at 50 W and 5 Pa and 5 min in oxygen plasma at 50 W and 5 Pa

- Silanization with 3-glycidyloxypropyltrimethoxysilane (GOPS) in a 10 mM solution of GOPS in anhydrous toluene for 6 h at 70 ° C with permanent stirring

- Wash twice in toluene, ethanol and water for 5 min

- Spotting the biotin-modified capture DNA in 3 x SSC or 150 mM phosphate buffer (PB) each with pH 8.5

Incubate the spotted support materials for 3 h in a humid chamber at 37 ° C

- 5 min washing in a 0.1% aqueous Triton X-100 solution

- Purification for 2 x 2 min in HCl pH 4.0 and 10 min in a 100 mM KCl solution

- wash briefly with distilled H ₂ 0

- Incubate for 15 min in a 50 mM ethanolamine solution with 0.1% SDS in Tris pH 9.0

Wash briefly with distilled H ₂ O and under nitrogen

dry

- Attachment of the enzyme complex in a 1: 1000 dilution in PBS pH 7.4 / 0.05% Tween 20 for 1 h at 20 ° C.

- Wash with PBS pH 7.4 / 0.05% Tween 20 for 6 x 5 min

- Rinse with distilled H ₂ 0

- Dry with nitrogen

Incubate the support surface with EnzMet ™ Kit for

Silver deposition for 5 min

Stopping the reaction by washing with distilled H ₂ 0

- dry under nitrogen

Verification / characterization

- Characterization of the SERS activity by measuring the electrical conductivity

- If the electrical conductivity of the substrates is between 1 μ8 and 10μ8, the substrate can be used Embodiment 2

SERS measurements with the SERS substrate prepared according to Example 1 for the detection of low molecular weight substances in the form of dyes

Incubation of the SERS substrate with dye solution (e.g., crystal violet or carboxyfluorescein) of defined concentration for 1 h at room temperature

- Blowing the dye solution with compressed air and drying the

SERS substrates

Measuring the SERS Spectra of the Dye with Raman Microscope and Frequency-Doubled Nd: YAG Laser By measuring different concentrations, it is possible to set up a calibration line that can be used in the

Connection allows the determination of unknown analyte concentrations

Embodiment 3

SERS measurements with the SERS substrate prepared according to Example 1 for the detection of low molecular weight substances in the form of antibiotics

Incubation of the SERS substrate with solution of the

Antibiotic (e.g., vancomycin or ciprofloxacin) of defined concentration for 1 h at room temperature. Blowing the antibiotic solution with compressed air and drying the SERS substrates

Measuring the SERS Spectra of the Antibiotic Using Raman Microscope and Frequency-Doubled Nd: YAG Laser By measuring various concentrations, it is possible to set up a calibration line, which then allows the determination of unknown analyte concentrations Embodiment 4

Preparation of SERS substrates and their use for SERS measurements for DNA detection

Option A)

- Washing the support materials for 5 min in an ultrasonic bath in acetone, ethanol and distilled H ₂ 0

- Drying under nitrogen

Wet-chemical activation with a 1: 1: 1 (v / v / v) mixture of conc. HCl: H ₂ 0 ₂ : H ₂ O for 10 min at 20 ° C

- Thorough cleaning with distilled H ₂ 0

- Drying with nitrogen and finally in a drying oven at 80 - 100 ° C for 5 min

- alternatively physical activation with the help of a

Plasma transporters at 1 mbar at 400 W for 30 min

- Alternatively activation by means of plasma etching in one

- Wash twice in toluene, ethanol and water for 5 min

Incubate the spotted support materials for 3 h in a humid chamber at 37 ° C

Wash in a 0.1% aqueous Triton X-100 solution for 5 min

- Purification for 2 x 2 min in HCl pH 4.0 and 10 min in a 100 mM KCl solution

- wash briefly with distilled H ₂ 0

- Incubate for 15 min in a 50 mM ethanolamine solution with 0.1% SDS in Tris pH 9.0 Wash briefly with distilled H ₂ O and dry under nitrogen

take up single-stranded sample DNA in 5 × SSC pH 7.0 / 0.1% SDS and then add to the carrier surface for hybridization

Incubation for two hours at the

Hybridization temperature in the hybridization oven

5 min each in 2 x SSC / 0.1% SDS; Wash 2 x SSC and 0.2 x SSC, blow dry with nitrogen and heat at 4 ° C

Store the refrigerator

single-stranded label DNA modified at the 3 'or / and 5' end with a Raman-active molecule in 5 × SSC pH 7.0 / 0.1% SDS and then added to the carrier surface for hybridization

Incubation for two hours at the

Hybridization temperature in the hybridization oven

Store the refrigerator

Attachment of the enzyme complex in a 1: 1000 dilution in PBS pH 7.4 / 0.05% Tween 20 for one hour at 20 ° C either in the hybridization chamber

Wash 6x5 min with PBS pH 7.4 / 0.05% Tween 20

Rinse with distilled H ₂ 0

dry with nitrogen

Incubate the support surface with EnzMet ™ Kit for

Silver deposition for 5 min

Stop the reaction by washing with distilled H ₂ O under nitrogen

Characterization of the SERS activity by measuring the electrical conductivity

If the electrical conductivity of the substrates is between 1 μ§ and 10 μ8, the substrate can be used

Detection of the Raman-active molecule by means of a

conventional micro-Raman spectrometer Variant b)

- Drying under nitrogen

- Thorough cleaning with distilled H ₂ 0

- Drying with nitrogen and finally in a drying oven at 80 - 100 ° C for 5 min

- alternatively physical activation with the help of a

Plasma transporters at 1 mbar at 400 W for 30 min

- Alternatively activation by means of plasma etching in one

- Wash twice in toluene, ethanol and water for 5 min

Incubate the spotted support materials for 3 h in a humid chamber at 37 ° C

Wash in a 0.1% aqueous Triton X-100 solution for 5 min

- Purification for 2 x 2 min in HCl pH 4.0 and 10 min in a 100 mM KCl solution

- wash briefly with distilled H ₂ 0

Wash briefly with distilled H ₂ O and under nitrogen

dry

- take single-stranded sample DNA in 5 x SSC pH 7.0 / 0.1% SDS and then add to the support surface for hybridization Incubation for two hours at the

Hybridization temperature in the hybridization oven

5 min each in 2 x SSC / 0.1% SDS; Wash 2 x SSC and 0.2 x SSC, blow dry with nitrogen and refrigerate at 4 ° C

- Wash with PBS pH 7.4 / 0.05% Tween 20 for 6 x 5 min

- Rinse with distilled H ₂ 0

- Dry with nitrogen

Incubate the support surface with EnzMet ™ Kit for

Silver deposition for 5 min

Stopping the reaction by washing with distilled H ₂ 0

- dry under nitrogen

- Take up single-stranded label DNA modified at the 3 'or / and 5' end with a Raman-active molecule in 5 × SSC pH 7.0 / 0.1% SDS and then add to the carrier surface for hybridization

- Incubation for 2 h in the optimized

Hybridization temperature in the hybridization oven

- If the electrical conductivity of the substrates is between 1 μ8 and 10μ8, the substrate can be used

Detection of the Raman-active molecule by means of a

conventional micro-Raman spectrometer

Variant c)

- Drying under nitrogen

Wet-chemical activation with a 1: 1: 1 (v / v / v) mixture of conc. HCl: H ₂ 0 ₂ : H ₂ O for 10 min at 20 ° C Thorough cleaning with distilled H ₂ 0

Drying with nitrogen and finally in a drying oven at 80 - 100 ° C for 5 min

alternatively physical activation with the help of a

Plasma transporters at 1 mbar at 400 W for 30 min

alternatively activation by means of plasma etching in one

Silanization with 3-glycidyloxypropyltrimethoxysilane (GOPS) into a 10 mM GOPS solution in anhydrous toluene for 6 h at 70 ° C with permanent stirring

then wash each twice for 5 min in toluene, ethanol and water

Spotting the biotin-modified capture DNA in 3 x SSC or 150 mM phosphate buffer (PB) each at pH 8.5

Incubate the spotted support materials for 3 h in a humid chamber at 37 ° C

Wash for 5 min in a 0.1% aqueous Triton X-100 solution

Purification for 2 x 2 min in HCl pH 4.0 and 10 min in one

100 mM KCl solution

Wash briefly with distilled H ₂ 0

Incubate for 15 min in a 50 mM ethanolamine solution with 0.1% SDS in Tris pH 9.0

Wash briefly with distilled H ₂ O and dry under nitrogen

Attachment of the enzyme complex in a 1: 1000 dilution in PBS pH 7.4 / 0.05% Tween 20 for 1 h at 20 ° C.

Wash 6x5 min with PBS pH 7.4 / 0.05% Tween 20

Rinse with distilled H ₂ 0

dry with nitrogen

Incubate the support surface with EnzMet ™ Kit for

Silver deposition for 5 min

Stopping the reaction by washing with distilled H ₂ 0 dry under nitrogen take up single-stranded sample DNA in 5 × SSC pH 7.0 / 0.1% SDS and then add to the carrier surface for hybridization

Incubation for 2 h at the hybridization temperature in the hybridization oven

Characterization of the SERS activity by measuring the electrical conductivity

If the electrical conductivity of the substrates is between 1 μ8 and 10 μS, the substrate can be used

Detection of the Raman-active molecule by means of a conventional micro-Raman spectrometer

All in the description, the following claims and the drawings illustrated features may be essential to the invention both individually and in any combination.

By the immobilization of horseradish peroxidase molecules in the electrode gap silver particles are deposited

deposited silver particles lead to an electrically conductive layer

Use of the conductive layer as SERS substrate

Claims

SERS substrate consisting of metal nanoparticles deposited enzymatically on a substrate surface.

SERS substrate according to claim 1, characterized in that the metal is silver.

SERS substrate according to claim 1 or 2, characterized in that the nanoparticles have a desert rose-like shape in which they have pointed, angular structures.

SERS substrate according to claim 1, 2 or 3, characterized in that the substrate surface consists of glass, silicon, polymers or hydrogel.

SERS substrate according to claim 1, 2, 3 or 4, characterized in that the substrate surface carries linker molecules to which metal nanoparticles are bound.

A method for producing an SERS substrate according to one or more of the preceding claims, wherein linker molecules are anchored on a substrate surface between two electrodes, to which enzyme molecules are subsequently bound, by means of which metal nanoparticles are subsequently deposited on the substrate surface.

Process for producing an SERS substrate according to claim 6, characterized in that single-stranded DNA is used as linker molecules.

8. A method for producing an SERS substrate according to claim 6, characterized in that as the enzyme molecule Streptavidin-horseradish peroxidase complex, glucose oxidase or alkaline phosphatase is used.

9. A method for producing an SERS substrate according to claim 6, characterized in that as metal nanoparticles

Silver nanoparticles are used.

10. A method of testing a SERS substrate according to 1, 2, 3, 4 or 5, wherein the SERS activity is determined by conductivity measurement.

11. Procedure for

an SERS substrate according to 1, 2, 3, 4 or 5 in which the SERS activity is determined by gray value analysis (c).

12. Use of an SERS substrate according to 1, 2, 3, 4 or 5 for the detection of chemical or biological substances.

13. Use of an SERS substrate according to 12, characterized in that the substances are antibiotics or DNA.