DE102009050287A1 - SERS substrate - Google Patents

Abstract

The invention relates to a novel SERS substrate, a method for its production, a method for its testing and its use. The object of the invention to provide novel SERS substrates which can be produced inexpensively in comparison to clean room SERS substrates and which have largely ordered particle structures compared to randomly arranged metal particle SERS substrates is achieved in that the SERS substrates are made from enzymatically A substrate surface deposited nanoparticles made of metal existing electrical conductivity measurements or gray value analysis takes place.

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, for example, from US 6,670,113 known. According to this disclosure, enzymes are used to precipitate or enrich metal particles (reaction of enzymes with metal ions to achieve metal deposition).

• – Verwendung des Enzyms Meerrettich-Peroxidase – Aufbringen des Enzyms auf Oberfläche, eintrocknen lassen – Inkubation mit 1 ml 0,2% Silberacetat in Wasser für 3 min – 2x mit Wasser waschen – 1 ml Lösung bestehend aus 2,5 mg/ml Hydrochinon, 1 mg/ml Silberacetat und 0,06% Wasserstoffperoxid in 0,1 M Citratpuffer pH 3,8 zugeben – Silberabscheidung innerhalb einer Minute sichtbar
• – Verwendung dieses Prinzips für biologische Anwendungen wie z. B. Immunfärbung,
• – Detektion von Bakterien, in Situ Hybridisierung, Detektion von einzelnen Genen, – Visualisierung mit dem bloßen Auge – Visualisierung mittels Mikroskop – Reflektion – Elektronmikroskopie – Polarographische, elektrochemische, elektrische Detektion – X-ray Spektroskopie – Chemische Tests – Detektion über die Masse – Lichtstreuung – Magnetische Detektion – Autometallographie – Scanning Probe Microscopy

According to US 6,670,113 The procedure is as follows:

• - use of the enzyme horseradish peroxidase - application of the enzyme to surface, allowed to dry - incubation with 1 ml of 0.2% silver acetate in water for 3 min - 2x with water washing - 1 ml solution consisting of 2.5 mg / ml hydroquinone, Add 1 mg / ml silver acetate and 0.06% hydrogen peroxide in 0.1 M citrate buffer pH 3.8 - silver deposit visible within one minute
• - Use of this principle for biological applications such. B. 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 - mass detection - light scattering - Magnetic Detection - Autometallography - Scanning Probe Microscopy

From the publication R. Moller, RD Powell, JF 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.

• – Anbindung von Streptavidin – modifizierter Meerrettich – Peroxidase an Biotin – modifizierte DNA
• – Zugabe des EnzMet^TM – Kits → enzymatische Silberabscheidung
• – Detektion über Auslesen der elektrischen Leitfähigkeit der einzelnen DNA-Spots

The procedure is as follows:

• - Attachment of streptavidin - modified horseradish peroxidase to biotin - modified DNA
• - Addition of the EnzMet ^TM kit → 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 A1 discloses the use of nanoparticles as SERS substrates.

• – Modifizierung von Goldnanopartikeln mit Thiol – modifizierter DNA mit internem Raman-Label
• – Nutzung dieses DNA-Nanopartikel-Komplexes als Label für einen sequenzspezifischen DNA-Nachweis
• – Detektion über Oberflächenverstärkte Raman Spektroskopie
• – Erhöhung der Verstärkung und damit Erhöhung des Signals durch zusätzliche Silberabscheidung an Goldnanopartikeln
• – Die zusätzliche Silberverstärkung ermöglicht zusätzlich eine optische Auswertung

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 via surface enhanced Raman spectroscopy
• - Increasing the gain and thus increasing the signal by additional silver deposition on gold nanoparticles
• - The additional silver reinforcement additionally allows 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 as to whether the SERS signal falls off after a certain silver deposition time.

From the publication Z. Wang, LJ 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 little occupancy of the surface with silver (individual silver particle islands) only a very low SERS gain, with increasing size and density of the silver particle islands an increase in the SERS reinforcement, and in the event that a conductive layer of the silver particle islands forms, the SERS gain drops again.

US 2004 018 03 79 For example, SERS substrates and their characterization are known as biosensors for the detection of intracellular analytes by SERS, the SERS substrate being prepared by Nanosphere Lithography or FON (film over nanospheres).

From the publication N. Felidj, J. Aubard, and G. Levi, JR Krenn, A. Hohenau, G. Schider, A. Leitner, and FR Aussenegg, Appl. Phys. Lett., Vol. 82, no. 18 For example, 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.

• – hochreproduzierbare SERS-Substrate (hergestellt bspw. mit EBL) sind sehr teuer,
• – günstige SERS-Substrate sind nicht großflächig reproduzierbar und müssen somit charakterisiert werden,
• – für die Charakterisierung wird das SERS-Substrat üblicherweise mit einem Ramanreporter belegt und das SERS-Signal dieses Reporters gemessen, anschließend muss der Reporter wieder entfernt werden, damit das SERS-Substrat für die Analyse von anderen Analyten genutzt werden kann,
• – häufig wird aus einer Charge SERS-Substrate nur ein einzelnes charakterisiert und dessen Ergebnisse auf die anderen übertragen, da sich die Ramanreporter meist nicht vollständig entfernen lassen
• – SERS-Substrate immer nur für einen bestimmten (meist recht schmalbandigen) Wellenlängenbereich (100–200nm) einsetzbar.

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 must therefore be characterized
• For the characterization, the SERS substrate is usually coated with a Raman reporter and the SERS signal of this reporter is measured, then the reporter has to be removed again 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, the publications include students, T., Steinbrück, A., Festag, G., Moller, R. and Fritzsche, W. "Enzyme-induced growth of silver nanoparticles studied at 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 per se known.

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 ordered 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 nanoparticles, can be applied to any 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 sharp, angular structures, which are significantly different from conventional spherical metallic nanoparticles. As in the REM recording in 2 can be seen, the individual particles consist of different platelets, which grow into each other and next to each other and thereby form the distinct desert roses similar formations ( 3 ).

Due to this particle growth, these enzymatically produced particles can be used as SERS substrate even at high particle densities, down to a closed conductive layer. 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, is dependent on the concentration of the linker molecules, the metal nanoparticle density, in particular the silver nanoparticle density (optimally about 50 to 70%), is determined by the concentration of the linker Möleküle (for example, single-stranded DNA of any sequence that is terminally amine- and botin-modified) set. For example. can be adjusted by a linker concentration of 5 uM, 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 a further growth of the enzymatically produced silver nanoparticles on the substrate, a metallic compound is generated between the two electrodes according to the method according to the invention.

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 nanoparticles, 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 SERS substrates according to the prior art the SERS activity already breaks down at about 1 μS.

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 grayscale analysis, whereby a characterization of the SERS substrate surface in a very short time is possible and not directly before their intended use must be made (contrary to the prior art no cumbersome characterizations using standard molecules necessary), or that no expensive, manufactured in clean room SERS substrates are needed to perform good SERS measurements can , This is due to the desert rose-like structure of the enzymatically deposited silver nanoparticles ( 3 ) possible. This structure prevents, due to the many needle-like protrusions, a decrease in SERS activity in the formation of a conductive layer of enzymatically deposited silver nanoparticles. Characterized a characterization of the SERS activity of the enzymatically deposited silver nanoparticles is possible even at 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 for their SERS activity 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, for example, for the detection of low molecular weight substances (eg, dyes, antibiotics) of biomolecules (eg, DNA, PNA, proteins), natural products, and organic polymers.

The invention will be explained in more detail below with reference to the drawings and the embodiments. Show it:

1 : a schematic representation for carrying out the method according to the invention for the production of 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 FIG. 2: a scanning electron micrograph of an embodiment of the SERS substrate according to the invention (metal particles in the form of silver particles with desert rose-like form on substrate surface, produced according to the method described in US Pat 1 schematically illustrated method) and

3 : 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 SERS substrate according to the invention

• - Washing the support materials for 5 min in an ultrasonic bath in acetone, ethanol and distilled H ₂ O.
• - Drying under nitrogen
• Wet-chemical activation with a 1: 1: 1 (v / v / v) mixture of conc. HCl: H ₂ O ₂ : H ₂ O for 10 min at 20 ° C
• - Thorough cleaning with distilled H ₂ O
• - Drying with nitrogen and finally in a drying oven at 80-100 ° C for 5 min
• Alternatively, physical activation using a plasma stripper at 1 mbar at 400 W for 30 min
• Alternatively, activation by means of plasma etching in a two-stage process: 30 seconds in argon plasma at 50 W and 5 Pa and 5 minutes 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 × 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
• - 5 min washing in a 0.1% aqueous Triton X-100 solution
• Purification for 2 × 2 min in HCl pH 4.0 and 10 min in a 100 mM KCl solution
• - Wash briefly with distilled H ₂ O.
• - 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 6 × 5 min with PBS pH 7.4 / 0.05% Tween 20
• - Rinse with distilled H ₂ O
• - Dry with nitrogen
• Incubation of the support surface with EnzMet ^™ silver deposition kit for 5 min
• - stopping the reaction by washing with distilled H ₂ O.
• - 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 μS and 10 μS, 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 (eg.
• 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, the establishment of a calibration line is possible, which then 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 (eg 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 with Raman microscope and frequency doubled Nd: YAG laser
• - By measuring different concentrations, the establishment of a calibration line is possible, 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 O.
• - Drying under nitrogen
• Wet-chemical activation with a 1: 1: 1 (v / v / v) mixture of conc. HCl: H ₂ O ₂ : H ₂ O for 10 min at 20 ° C
• - Thorough cleaning with distilled H ₂ O
• - Drying with nitrogen and finally in a drying oven at 80-100 ° C for 5 min
• Alternatively, physical activation using a plasma stripper at 1 mbar at 400 W for 30 min
• Alternatively, activation by means of plasma etching in a two-stage process: 30 seconds in argon plasma at 50 W and 5 Pa and 5 minutes 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 × 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
• - 5 min washing in a 0.1% aqueous Triton X-100 solution
• Purification for 2 × 2 min in HCl pH 4.0 and 10 min in a 100 mM KCl solution
• - Wash briefly with distilled H ₂ O.
• - 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 support surface for hybridization
• Incubation for two hours at the hybridization temperature in the hybridization oven
• 5 min each in 2x SSC / 0.1% SDS; Wash 2 x SSC and 0.2 x SSC, blow dry with nitrogen and refrigerate at 4 ° C
• - Record 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 support surface for hybridization
• Incubation for two hours at the hybridization temperature in the hybridization oven
• 5 min each in 2x SSC / 0.1% SDS; Wash 2 x SSC and 0.2 x SSC, blow dry with nitrogen and refrigerate at 4 ° C
• - 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 6 × 5 min with PBS pH 7.4 / 0.05% Tween 20
• - Rinse with distilled H ₂ O
• - Dry with nitrogen
• Incubation of the support surface with EnzMet ^™ silver deposition kit for 5 min
• Stopping the reaction by washing with distilled H ₂ O
• - under nitrogen drying
• - Characterization of the SERS activity by measuring the electrical conductivity
• - If the electrical conductivity of the substrates is between 1 μS and 10 μS, the substrate can be used
• Detection of the Raman-active molecule by means of a conventional micro-Raman spectrometer

Variant b)

• - Washing the support materials for 5 min in an ultrasonic bath in acetone, ethanol and distilled H ₂ O.
• - Drying under nitrogen
• Wet-chemical activation with a 1: 1: 1 (v / v / v) mixture of conc. HCl: H ₂ O ₂ : H ₂ O for 10 min at 20 ° C
• - Thorough cleaning with distilled H ₂ O
• - Drying with nitrogen and finally in a drying oven at 80-100 ° C for 5 min
• Alternatively, physical activation using a plasma stripper at 1 mbar at 400 W for 30 min
• Alternatively, activation by means of plasma etching in a two-stage process: 30 seconds in argon plasma at 50 W and 5 Pa and 5 minutes 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 × 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
• - 5 min washing in a 0.1% aqueous Triton X-100 solution
• Purification for 2 × 2 min in HCl pH 4.0 and 10 min in a 100 mM KCl solution
• - Wash briefly with distilled H ₂ O.
• - 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
• Single-stranded sample DNA in 5x 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 2x SSC / 0.1% SDS; Wash 2 × SSC and 0.2 × SSC, blow with nitrogen dry and store at 4 ° C in the refrigerator
• - 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 6 × 5 min with PBS pH 7.4 / 0.05% Tween 20
• - Rinse with distilled H ₂ O
• - Dry with nitrogen
• Incubation of the support surface with EnzMet ^™ silver deposition kit for 5 min
• - stopping the reaction by washing with distilled H ₂ O.
• - dry under nitrogen
• - Record 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 support surface for hybridization
• Incubation for 2 h at the optimized hybridization temperature in the hybridization oven
• 5 min each in 2x SSC / 0.1% SDS; Wash 2 x SSC and 0.2 x SSC, blow dry with nitrogen and refrigerate at 4 ° C
• - Characterization of the SERS activity by measuring the electrical conductivity
• - If the electrical conductivity of the substrates is between 1 μS and 1 μS, the substrate can be used
• Detection of the Raman-active molecule by means of a conventional micro-Raman spectrometer

Variant c)

• - Washing the support materials for 5 min in an ultrasonic bath in acetone, ethanol and distilled H ₂ O.
• - Drying under nitrogen
• Wet-chemical activation with a 1: 1: 1 (v / v / v) mixture of conc. HCl: H ₂ O ₂ : H ₂ O for 10 min at 20 ° C
• - Thorough cleaning with distilled H ₂ O
• - Drying with nitrogen and finally in a drying oven at 80-100 ° C for 5 min
• Alternatively, physical activation using a plasma stripper at 1 mbar at 400 W for 30 min
• Alternatively, activation by means of plasma etching in a two-stage process: 30 seconds in argon plasma at 50 W and 5 Pa and 5 minutes 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 × 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
• - 5 min washing in a 0.1% aqueous Triton X-100 solution
• Purification for 2 × 2 min in HCl pH 4.0 and 10 min in a 100 mM KCl solution
• - Wash briefly with distilled H ₂ O.
• - 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 6 × 5 min with PBS pH 7.4 / 0.05% Tween 20
• - Rinse with distilled H ₂ O.
• - Dry with nitrogen
• Incubation of the support surface with EnzMet ^™ silver deposition kit for 5 min
• - stopping the reaction by washing with distilled H ₂ O.
• - dry under nitrogen
• - take up single-stranded sample DNA in 5 × SSC pH 7.0 / 0.1% SDS and then add to the support surface for hybridization
• Incubation for 2 h at the hybridization temperature in the hybridization oven
• 5 min each in 2x SSC / 0.1% SDS; Wash 2 x SSC and 0.2 x SSC, blow dry with nitrogen and refrigerate at 4 ° C
• - Record 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 support surface for hybridization
• Incubation for 2 h at the hybridization temperature in the hybridization oven
• 5 min each in 2x SSC / 0.1% SDS; Wash 2 x SSC and 0.2 x SSC, blow dry with nitrogen and refrigerate at 4 ° C
• - Characterization of the SERS activity by measuring the electrical conductivity
• - If the electrical conductivity of the substrates is between 1 μS 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.

LIST OF REFERENCE NUMBERS

• a

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

  b

  - deposited silver nanoparticles lead to an electrically conductive layer

  c

  Use of the conductive layer as SERS substrate

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 6670113 [0002, 0003]
• US 2003/0211488 A1 [0007]
• US 20040180379 [0011]

Cited non-patent literature

• R. Moller, RD Powell, JF Hainfeld, W. Fritzsche, Nano Lett. 2005, 5, 1475-1482 [0004]
• Z. Wang, LJ Rothberg, Appl. Phys. B 84, 289-293 (2006) [0010]
• N. Felidj, J. Aubard, and G. Levi, JR Krenn, A. Hohenau, G. Schider, A. Leitner, and FR Aussenegg, Appl. Phys. Lett., Vol. 82, no. 18 [0012]
• T., Steinbrück, A., Festag, G., Moller, R. and Fritzsche, W. "Enzyme-induced growth of silver nanoparticles studied at single particle level", Journal of Nanoparticle Research (2008) [0014]
• 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) [0014]

Claims (13)

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 a 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 subsequently metal nanoparticles are deposited on the substrate surface. A method for producing a SERS substrate according to claim 6, characterized in that single-stranded DNA is used as the linker molecules. A process for the preparation of a SERS substrate according to claim 6, characterized in that streptavidin-horseradish peroxidase complex, glucose oxidase or alkaline phosphatase is used as the enzyme molecule. A process for producing an SERS substrate according to claim 6, characterized in that are used as metal nanoparticles silver nanoparticles. A method of testing a SERS substrate according to 1, 2, 3, 4 or 5, wherein the SERS activity is determined by conductivity measurement. Method for checking a SERS substrate according to 1, 2, 3, 4 or 5, wherein the SERS activity is determined by gray value analysis (c). Use of a SERS substrate according to 1, 2, 3, 4 or 5 for the detection of chemical or biological substances. Use of a SERS substrate according to 12, characterized in that the substances are antibiotics or DNA.

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