EP4162272A1 - Plasmonverstärkte-fluoreszenz-basierende sensor zum nachweis krankheitsspezifischer biomarker - Google Patents
Plasmonverstärkte-fluoreszenz-basierende sensor zum nachweis krankheitsspezifischer biomarkerInfo
- Publication number
- EP4162272A1 EP4162272A1 EP21731955.7A EP21731955A EP4162272A1 EP 4162272 A1 EP4162272 A1 EP 4162272A1 EP 21731955 A EP21731955 A EP 21731955A EP 4162272 A1 EP4162272 A1 EP 4162272A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- biomarker
- fluorophore
- bound
- detection sensor
- aptamer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56905—Protozoa
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to biomarker detection sensors for the detection of disease-specific biomarkers, kits based on these sensors, methods for determining disease-specific biomarkers in body fluids and uses.
- Fluorescence-based techniques are among the most widespread methods in the fields of biotechnology, life sciences and (bio) medical research. Fluorescence probes and fluorescence-labeled bioreceptors are used extensively in optogenetic studies, in cytogenetic assays, in multicolor fluorescent in situ hybridization (multicolor fluorescent in situ hybridization - mFISFI), in bioimaging analysis, for the observation of both the location and the movement of cells or subcellular elements, when researching molecular dynamics, as well as in fluoroimmunoassays for the detection of molecular biomarkers, as enzyme-linked immunosorbet assay (ELISA).
- ELISA enzyme-linked immunosorbet assay
- fluorescence methods for the sensory method is severely restricted by the low yields of many fluorescent dyes and the occurrence of signal interference.
- the detection of very low analyte concentrations is still one of the main problems. Many attempts have been made to amplify the fluorescence signals to enable their applicability to ultrasensitive fluoroimm
- plasmonic nanostructures To meet this challenge - without the use of expensive equipment, specific or toxic reagents, or significant changes to well-established fluorescence-based assays - plasmonic nanostructures have drawn attention in recent years. This is due to the ability of plasmonic nanostructures to influence the spectral properties of nearby fluorescent dyes. This influence depends strongly on the spectral overlap between the fluorescent dye and the plasmonic absorption, as well as on the distance z between the fluorophore and the nanostructure. In particular, due to the Förster resonance energy transfer mechanism (FRET) (i.e.
- FRET Förster resonance energy transfer mechanism
- Two-dimensional (2D) arrays of metallic nanostructures are particularly suitable as plasmon-enhanced fluorescence (PEF - plasmon-enhanced fluorescence) - based biosensing platforms.
- various fluorescence amplifiers such as gold micro-islands or arrays of metallic nano-objects (e.g. nanoparticles, nanotubes, nanotrangles, nanocrystals, nano-antennas and resonant nanocavities) have been tried in order to move the detection limit further down in fluorescence-based assays.
- the object of the present invention was to provide possibilities for the highly sensitive determination of disease-specific biomarkers which no longer have the problems of the prior art. Further tasks emerge for the person skilled in the art when considering the following description and the claims.
- 2D frameworks of ordered and periodic gold nanoparticles (AuNPs), produced via self-organized diblock copolymer nanolithography, represent an effective way of eliminating most of the problems of the prior art that have been identified.
- AuNPs ordered and periodic gold nanoparticles
- the favorable and scalable position and the easy adjustment of their plasmonic properties by adjusting the AuNP size and the interparticle distance by simply varying the lengths of the hydrophilic or hydrophobic part of the copolymers are the main strengths of such a PEF platform.
- the plasmonic behavior of a 2D framework made of well-ordered AuNPs is closely related to the ratio R between the nanoparticle diameter D and the interparticle distance d. When each nanoparticle is in close proximity to its neighbors (i.e.
- LSP localized surface plasmons
- DSP - delocalized surface plasmon delocalized surface plasmon
- room temperature means a temperature of 293.15 Kelvin, i.e. 20 ° C.
- a plasmon-enhanced fluorescence immunosensor for the determination and detection of Plasmodium falciparum lactate dehydrogenase (P / LDFI) - a malaria marker - is described in whole blood samples.
- the analyte determination is achieved within the scope of the present invention by means of oriented antibodies which are immobilized in a tightly packed configuration by means of photochemical immobilization technology (PIT).
- PIT photochemical immobilization technology
- BCMN Block Copolymer Micelle Nanolithography
- PIT enables maximum control over the nanoparticle size and lattice constant, as well as the distance of the fluorophore from the sensor surface.
- the assemblies of the present invention in some embodiments achieve a detection limit of less than 1 pg / ml ( ⁇ 30 fM) with very high specificity without pretreatment of the samples; this is achieved according to the invention in particular in the embodiments in which the gold nanoparticles form a two-dimensional lattice in the form of honeycombs, ie the gold nanoparticles are preferably arranged densely in a hexagonal or hexagonal manner.
- This detection limit is several orders of magnitude lower than what is achieved in rapid malaria tests or even commercial ELISA kits.
- the confirmation can be made by redundant, second signals or the detection of a second antigen and the detection can be, for example, down to 50 pM for a green fluorescent fluorophore and up to 260 fM for a red fluorescent fluorophore.
- the arrangements of the present invention i.e. the biomarker detection sensors, can be used as a substrate in multititer plates.
- the efficiency is considerably improved compared to conventional fluorescence immunoassays.
- the present invention relates to a biomarker detection sensor for the detection of disease-specific biomarkers comprising or consisting of a substrate with metal nanoparticles bound thereon in branched two-dimensional structures and / or a two-dimensional lattice, in particular gold nanoparticles, and anti-biomarker receptors bound to the metal nanoparticles, in particular gold nanoparticles.
- the detection takes place via fluorescence analyzes, since these can be implemented particularly well in continuous operation.
- the electronic structure of the biomarker detection sensors is changed by the attachment of the disease-specific biomarkers in such a way that they can be measured in their fluorescence emission or absorbance spectrum.
- the substrates of the biomarker detection sensors according to the present invention can be any substrates on which the nanoparticles can be deposited.
- these are known assay wells, surfaces based on polyacrylates (e.g. Plexiglas®), polystyrene or glass surfaces.
- all substrates are suitable that are stable under the experimental conditions (including the Fier position), are transparent for excitation and fluorescence in the respective investigated / applied wavelength range and have no intrinsic fluorescence.
- the metal nanoparticles to be deposited on the substrates can, in principle, be used on all materials that are non-corrosive under the production and investigation conditions to be chosen.
- the materials can be selected from the group consisting of noble metals, in particular gold, silver, platinum, copper, chromium, alloys thereof and Janus particles of these metals. It is particularly advantageous and preferred if the metal nanoparticles are gold nanoparticles.
- the disease to be detected is malaria.
- the disease-specific biomarker is Plasmodium falciparum L-lactate dehydrogenase (PA.DH).
- the anti-biomarker receptors bound to the gold nanoparticles are anti-pLDH antibodies.
- these anti-pLDH antibodies are fixed on the gold nanoparticles, i.e. bound to them.
- the present invention therefore includes biomarker detection sensors of the aforementioned type in which the anti-biomarker receptors are anti-pLDH antibodies which are bound to the gold nanoparticles via thiol groups.
- these anti-pLDH antibodies are anti-pLDH antibodies which have been pretreated with UV radiation.
- UV radiation as a pretreatment, it is possible to modify the anti-pLDH antibodies in such a way that thiol groups become available through the cleavage of disulfide bridges.
- the UV irradiation results in a selective photoreduction of the disulfide bridges in specific cysteine-cysteine / tryptophan triads (in which the presence of the tryptophan activates the disulfide bridge in this triad, so to speak).
- the cleavage of these Cys-Cys bonds in both antibody fragments leads to a total of four free thiol groups, two of which can interact with the surface of the gold nanoparticles to form a covalent bond.
- the UV treatment is carried out for 30 seconds with a radiation output of 1 watt / cm 2 and a wavelength of 254 nm or with a radiation output of 6 watt / cm 2 and a wavelength of 254 nm.
- the present invention also includes biomarker detection sensors in which the ratio R of the diameter D of the nanoparticles to the distance d between the individual gold nanoparticles (edge-to-edge distance) in the two-dimensional grid is> 2.3.
- This ratio is preferably between 2.3 and 3.5, and particularly preferably 2.5 or 3.0.
- the gold nanoparticles form a two-dimensional lattice in the form of honeycombs, i.e. the gold nanoparticles are preferably arranged hexagonally or hexagonally densely.
- the gold nanoparticles form a two-dimensional branched structure, ie the gold nanoparticles are preferably arranged in branched, ribbon-like structures, with only a few particles (2-4) ever have tight packing.
- the gold nanoparticles form a combined two-dimensional structure in which both parts of the structure form lattices in the form of honeycombs, ie the gold nanoparticles are preferably hexagonal or hexagonal in density in these parts arranged, and at the same time parts of the structure form two-dimensional branched structures, ie the gold nanoparticles are preferably arranged in these parts in branched, ribbon-like structures with only a few particles (2-4) always having a dense packing and otherwise being randomly distributed.
- the densely packed nanoparticles in the band-like structures can generate absorptions at 680 nm (delocalized surface plasmons) and the particles, which are only closely spaced with a few neighbors, can generate absorptions at approx. 530 nm (localized surface plasmons LSPR).
- the diameter D of the nanoparticles of the biomarker detection sensors is between 40 and 60 nm, preferably between 44 and 55 nm, particularly preferably between 46 and 52 nm and particularly preferably about 48 or about 50 nm. In other embodiments, is the diameter D of the nanoparticles of the biomarker detection sensors between 50 and 70 nm, preferably between 55 and 65 nm, particularly preferably 60 nm.
- the distance d between the individual gold nanoparticles (the edges of the individual particles) of the biomarker detection sensors is preferably between 16 and 24 nm, more preferably between 17 and 22 nm, particularly preferably between 18 and 21 nm and particularly preferably 19 or 20 nm.
- the size information was determined by means of scanning electron microscopy.
- An example of a microscope that can be used is a Zeiss® Leo 1550 VP.
- the corresponding information denotes mean diameters; Naturally, the effective diameters in these orders of magnitude scatter a little around the specified specific value. This in turn also means that the distance d between the particles scatters around a central value. This is known to the person skilled in the art.
- the deviation in embodiments is plus / minus 0.14 nm.
- the deviation in embodiments is plus / minus 0.19 nm.
- the specific structure of the AuNP on the substrate can be controlled in the manufacture of the biomarder detection sensors of the present invention.
- a simple way of switching between hexagonal structures and branched structures is to regulate the deposition rate; if the speed is increased above a certain value, the particles no longer have sufficient time to ideally arrange themselves and the resulting structure moves away from the ideal hexagonal arrangement.
- the deposition can be influenced by targeted modification of the substrate, for example by roughening, hydrophobizing / hydrophilizing, functionalizing or the like.
- biomarker detection sensor kits comprising or consisting of
- C) optionally further analysis material preferably cuvettes, pipettes, light source (s), fluorescence detector, in particular fluorescence spectrometer.
- biomarker detection sensor kits comprising or consisting of A) at least one biomarker detection sensor as set out above,
- Bl at least one further preparation comprising at least one identical biomarker-specific aptamer with another fluorophore bound to it, and / or
- C) optionally further analysis material preferably cuvettes, pipettes, light source (s), fluorescence detector, in particular fluorescence spectrometer.
- B2i) at least one, preferably exactly one, different biomarker-specific aptamer for the detection of the same biomarker but binding to a different epitope of the biomarker with a different fluorophore bound to it
- B2M at least one, preferably exactly one, other biomarker-specific aptamer for the detection of a further biomarker with another fluorophore bound to it, and are selected accordingly from B2i) and / or B2M).
- B2i confirms the detection of this biomarker by binding two aptamers to the same biomarker and is therefore even more reliable than B1) and that B2M) can detect a second biomarker.
- kit does not necessarily have to be suitable for the "fin pocket”. Rather, it is intended to express that, prior to the analysis, both the biomarker detection sensor as described above and the preparations comprising biomarker-specific aptamer (s) are present separately from one another, although they are coordinated and associated with one another. In addition, the kit should also be able to include several detection sensors and several aptamer preparations, which are also designed for different biomarkers.
- the biomarker detection sensor kit it is possible for the biomarker detection sensor kit to be configured as a transportable kit.
- a transportable light source is then configured, for example, in the form of a flashlight and the fluorescence detector is correspondingly configured in a comparatively handy form.
- the fluorescence detector only displays a fluorescence signal, but cannot necessarily represent its intensity quantitatively. This would be an example of a rapid test kit.
- the biomarker detection sensor kit on a laboratory scale is designed, ie in particular both the fluorescence light source and the fluorescence detector are suitable and designed for permanent laboratory operation.
- biomarker-specific aptamer is in principle selected appropriately for any disease-specific biomarker.
- biomarker-specific aptamer is directed to the same biomarker in accordance with the anti-biomarker receptor bound to the gold nanoparticles, with aptamer and antibodies directed to different epitopes of the biomarker (in the event that that aptamer and antibody would be directed to the same epitope if they would compete with each other and worsen the result).
- biomarker-specific aptamer is a malaria-specific aptamer.
- biomarker-specific aptamer, the malaria 2008 aptamer is particularly preferred.
- any fluorophore that can be chemically bound to this biomarker is suitable.
- fluorophores that can be used are 5-FAM (5-carboxyfluorescein), cyanine 7, cyanine 5, cyanine 3b or also quantum dots.
- 5-FAM and / or cyanine 5 is bound as a fluorophore to the biomarker-specific aptamer.
- biomarker-specific aptamer in the context of the present invention is therefore the malaria 2008 aptamer with cyanine 5 bound to it, provided that a single fluorophore is used.
- 5-FAM and Cy5 are combined as fluorophores.
- the plasmon resonance is designed in such a way that it is coupled with the emission peak of the 5-FAM with both the excitation peak and the emission peak of the Cy5, which means, through emission rate improvement, a 160-fold fluorescence amplification and, through a dual mechanism (excitation and emission rates ), a 5200-fold signal gain is achieved.
- the detection concentration range expands by two orders of magnitude compared to detection of only a single fluorophore in complex matrices such as human blood.
- the confirmation of the analyte binding by two redundant fluorescence signals improves the reliability of the signal and reduces false positive signals due to unspecific binding.
- the proposed approach can also be used for simultaneous monitoring of different biomarkers at low concentrations, paving the way for potential multiplex and floch throughput analysis.
- two different biomarker-specific aptamers can be used, or else the same.
- biomarker-specific aptamers with the same fluorophores or different fluorophores can be used in order to detect different biomarkers at the same time, or the same biomarker-specific aptamer with different fluorophores for the detection of the same biomarker but at different wavelengths.
- the present invention can therefore cover many different variants which allow a high degree of flexibility, in particular when used in the form of kits. It is noteworthy that the variant of the present invention with two different fluorophores, in particular 5-FAM and Cy5, extends the accessible detection range by using both fluorophores, Cy5 being more sensitive in the lower concentration range (up to 0.1 pM), while 5-FAM covers large concentrations (up to 1 pM). In this way, for example, over seven orders of magnitude can be measured instead of four or five for one fluorophore alone.
- BCMN block copolymer micellar nanolithography
- branching patterns of plasmon-coupled, honeycomb-shaped AuNPs which generate a collective mode, the resonance of which is in the far red range
- interspersed plasmon-decoupled AuNPs which localize a narrow one
- LSPR surface plasmon resonance
- Said preparation comprising at least one biomarker-specific aptamer with a fluorophore bonded to it can be this aptamer per se, but in addition to the aptamer with a fluorophore bonded to it can also contain other substances, such as those for the use of such aptamers with fluorophores bonded to it in the Are known in the art.
- the fluorophore-labeled aptamer can be present in aqueous solutions, it being possible for these aqueous solutions to be buffered, for example, or also to contain preservatives or the like.
- 10 millimolar phosphate buffered saline solutions (PBS) are well suited in embodiments of the present invention. This applies accordingly to all variants B0), Bl) and B2).
- biomarker detection sensors are designed as described above. This is because the combinations of the biomarker detection sensors as described above with the preparations comprising at least one biomarker-specific aptamer enable a high level of selectivity.
- the selectivity for the P / LDH is achieved in particular by the combination of the anti-pLDH antibody, which is bound to the gold nanoparticles via thiol groups, and the Malaria 2008 aptamer with cyanine 5 bound to it as a fluorophore.
- the fluorescence intensity depends on the concentration of the biomarker.
- the intensity increases with the increasing concentration of the biomarker and asymptotically approaches a limit value. This limit value is reached when all antibodies are covered by biomarkers bound to them.
- biomarker concentrations between 0.001 femtomoles and 100 nanomoles, in particular between 0.01 femtomoles and 10 nanomoles, can be determined quantitatively.
- Flatter concentrations can be qualitatively determined.
- Qualitative determination means that the presence of the biomarker is indicated by fluorescence, but the exact amount is not determined or, in the case of complete coverage of all antibodies, cannot be determined.
- the qualitative determination is usually sufficient.
- the quantitative determination is usually used for more precise clinical examinations.
- the order symmetry of the two-dimensional lattice of the gold nanoparticles can influence the plasmon resonance.
- a particularly good plasmon resonance was achieved with hexagonal or hexagonal tightly packed gold nanoparticles;
- the present invention also encompasses cubically densely packed, randomly densely packed or other symmetries of order.
- the dual amplification for the variant of the hexagonal arrangements and with a biomarker-specific aptamer and a fluorophore bound to it reaches its maximum due to the plasmon resonance at a distance of about 10 nm from the particle surface.
- this particularly preferred embodiment of the present invention achieves a particularly strong dual amplification and particularly high selectivity and efficiency.
- the present invention is greatly improved with regard to sandwich assays known from the prior art.
- the present invention also relates to a method for determining disease-specific biomarkers in body fluids. This procedure includes or consists of the following steps:
- Step i) consists in providing a biomarker detection sensor as described above.
- a body fluid sample is then applied to the sensor and mixed with at least one biomarker-specific aptamer with a fluorophore coupled to it.
- the sample applied to the sensor is then irradiated in step iii) by means of a light source.
- the light source is selected in such a way that the fluorophore used is particularly well excited.
- this light source is a light source that emits in the red range of the visible spectrum; particularly advantageous results are achieved when the light source in a wavelength range between 610 and 670 nm, preferably between 625 and 655 nm, in particular, in the case of cyanine 5 as the fluorophore, emitted at 625 nm.
- 5-FAM as the fluorophore
- particularly preferred results are achieved when the light source emits wavelengths of 450 nm to 510 nm, in particular 470 nm.
- step iv the fluorescence emission of the sample is detected.
- this detection can be carried out using any of the methods known to those skilled in the art. If the method according to the invention is carried out with one of the biomarker detection sensor kits according to the invention, this can also be a purely visual detection by the person performing the method; respectively.
- the detection preferably takes place with dedicated fluorescence spectrometers, for example those based on CMOS or sCMOS photodetectors.
- the at least one biomarker-specific aptamer with a fluorophore coupled to it to be mixed with the body fluid sample is as described above and can also be in the form of the preparation described for the biomarker detection sensor kit which contains this biomarker-specific aptamer with a fluorophore bound to it.
- the analysis of the fluorescence emission can take place as an optional step v) in the method according to the invention.
- This analysis can be quantitative, qualitative, or both.
- This analysis is expediently carried out by means of commercially available computers and fluorescence data processing programs known to those skilled in the art.
- the determined result of the fluorescence emission analysis can be stored or displayed, or both.
- the body fluid is not limited to a specific body fluid in the method for determining disease-specific biomarkers according to the present invention, it is a matter of course for the person skilled in the art that he selects the body fluid according to whether the disease-specific biomarkers he is looking for occur in the respective body fluid.
- the body fluid is blood samples.
- the body fluids Before being used in the method of the present invention, the body fluids can be worked up or prepared in accordance with the methods customary in the art.
- the method of the present invention is particularly suitable and intended for examining body fluid samples that are already present as such, i.e. the removal of body fluids is not part of the method of the present invention.
- the anti-biomarker receptors are anti-pLDFI antibodies
- the biomarker-specific aptamers are malaria 2008 aptamers, which are preferred 5-FAM and / or cyanine 5 is / are bound as a fluorophore; in accordance with the above statements on the biomarker detection sensor or the biomarker detection sensor kit of the present invention.
- the pre-treatment of the anti-pLDFI antibodies with UV radiation activates them.
- the antibodies are converted into a conformation so that they bind to the gold nanoparticles in a way that makes the binding epitopes of the antibody easily accessible for the biomarker to be examined.
- the present invention also relates to uses of the biomarker detection sensors, the biomarker detection sensor kits or the method according to the invention for determining disease-specific ones Biomarkers in body fluids for the qualitative or quantitative determination of disease-specific biomarkers.
- this use is encompassed for the determination of malaria biomarkers and in particular in body fluid samples, most preferably in blood samples.
- Another preferred use according to the present invention is the use of the biomarker detection sensors according to the present invention as a substrate in automated multititer plates.
- biomarker detection sensors according to the present invention can be analogous to Lohmüller, T., Aydin, D., Schwieder, M. et al., "Nanopatterning by block copolymer micelle nanolithography and bioinspired applications", Biointerphases 6, MR1-MR12 (2011), https://doi.Org/10.1116/l.3536839.
- FIG. 1 is a schematic representation of the procedure for producing a biomarker detection sensor according to the present invention.
- These micelles can then be used on a Substrate 1, for example by means of a dip coater, are deposited. Due to their structure, the micelles then arrange themselves in regular structures on the surface of the substrate. The micelles around the gold precursors are then removed, for example by means of plasma furnace treatment. In the process, the gold precursors 9 are also reduced to form the gold nanoparticles 2. A two-dimensional grid of gold nanoparticles 2 (unfilled circles) then remains on the surface.
- Section B illustrates how a solution of anti-pLDH antibodies 10 is pretreated by means of UV radiation (shown as a flash) and then subsequently this treated aqueous solution 10a is poured onto the substrate produced in section A) with gold nanoparticles located thereon where the antibodies bind to the gold nanoparticles.
- the substrate 1 illustrated in section C) is then obtained with gold nanoparticles 2 located thereon, to which the anti-biomarker receptors are bound, which is represented here by filled circles.
- This production method is also described, for example, in Lohmüller, T., Aydin, D., Schwieder, M. et al., "Nanopatterning by block copolymer micelle nanolithography and bioinspired applications", Biointerphases 6, MR1-MR12 (2011), https: // doi.Org/10.1116/l.3536839.
- FIG. 1 shows the arrangement of the particles on the substrate only schematically and by way of illustration and is not restricted to a specific arrangement.
- the production method illustrated in this figure can be used both for the production of substrates with hexagonally arranged nanoparticles (as illustrated) and for the production of branched nanoparticles or also for mixed forms.
- FIG. 2a is a scanning electron microscope image of a substrate provided with ordered gold nanoparticles according to a variant of the present invention. It can be seen from this recording that imperfections can certainly occur during the manufacture of the sensors. However, these flaws are not detrimental to the overall performance of the biomarker detection sensor, since they are, so to speak, averaged out in the context of plasmon resonance or dual amplification.
- FIG. 2b are two scanning electron microscope images of another substrate provided with ordered gold nanoparticles according to another variant of the present invention. From these recordings it can be seen that here in the short-range order there are still hexagonal structures, but the long-range order essentially has branched structures as well as isolated nanoparticles. The upper picture shows a structure before and the lower picture one after the AuNP growth.
- Figure 3 is a diagram in which the two narrow curves represent the excitation spectrum (left, dashed curve) and the emission spectrum (right, dotted curve) of cyanine 5 (Cy5) and the broad curve the experimentally determined extinction spectrum of a malaria biomarker detection sensor ( prepared according to Example A) described below). It can be seen that the broad extinction spectrum allows the plasmonic resonance to be superimposed on both the extinction and emission spectrum of cyanine 5. The wavelength in nm is plotted on the x-axis and the extinction (left) and the normalized excitation / emission (right) on the y-axis.
- FIG. 3b for a substrate produced in accordance with Example B) described below shows the experimental (solid line) extinction spectrum.
- the plasmon fluorophore overlap with 5-FAM (emission spectrum, small dashed line // excitation spectrum, large dashed line) and Cy5 (emission spectrum, dash-dot line // excitation spectrum, dotted line) dyes is shown.
- the experimental extinction spectrum shows two plasmonic resonances at 524 nm and 675 nm. Isolated AuNPs contribute to the resonance at smaller wavelengths, as was to be expected based on 30 nm gold nanospheres in air, whereas AuNPs arranged along the branches lead to a collective resonance at higher wavelengths.
- the extinction is plotted on the left-hand axis, and excitation / emission on the right-hand axis.
- the wavelength in nm is plotted on the horizontal axis.
- FIG. 4 is a diagrammatic representation of the particle size distribution of gold nanoparticles produced according to Example A) described below became.
- the mean diameter D of the gold nanoparticles in this example is about 48 nm (47.67 + 0.14 nm).
- FIG. 5 is a diagrammatic representation of the inter-particle distances in nanometers in relation to the respective centers of the nanometers.
- the mean inter-particle distance, measured center-to-center, is about 68 nm (68.47 + 0.19 nm), resulting in an inter-particle distance, measured particle edge to particle edge, of about 20 nm (produced according to Example A described below) ).
- FIG. 6 shows schematically the mode of operation of the present invention on a single gold nanoparticle (AuNP).
- the basis is a gold nanoparticle 2 arranged on a substrate 1 to which antibodies 3 (shown here as Y) are bound.
- antibodies 3 shown here as Y
- the way in which the antibodies 3 are represented makes it clear that they are bound to the gold nanoparticles 2 in a conformation which makes the binding epitopes of the antibodies easily accessible to the respective biomarker. This is illustrated by the fact that in each case one “arm” of the antibody 3 is oriented away from the gold nanoparticle 2.
- the biomarker 4 (Pfl_DH) to be examined is shown (only one, for the sake of clarity, in the form of a cloud), which is here is shown as binding to the binding epitope of the middle antibody 3.
- An aptamer 5 is then shown on the other side of the biomarker 4, which thus, as it were, sandwiches the biomarker 4 together with the antibody 3
- the fluorophore 6 is shown as a star, which is intended to represent its fluorescence emission, on the opposite side of the aptamer 5.
- the distance between the fluorophore 6 and the surface 1 is approximately 10 nm, which means that with this arrangement both high sensitivity and high selectivity is achieved.
- FIG. 7 shows an image of the fluorescence points of a sensor structure with a detected biomarker according to example B), below.
- the picture shows a reproduction in which the red and green channels, which result from the detection with two color channels, ie two different fluorophores (5-FAM and Cy5), are shown combined (here in black and white for reproduction purposes).
- two color channels ie two different fluorophores (5-FAM and Cy5)
- Au-S gold salt especially HAuCU
- Diblock copolymers P18226-S2VP and P3807-S2VP, respectively
- Toluene (99.8%), gold (III) chloride trihydrate (HAUCU * 3H 2 0), silver nitrate (AgNCb) and ascorbic acid were purchased from Sigma-Aldrich; Acetone (> 99.0%), 2-propanol (> 99.5%) and ethanol (> 99.5%) were used by Merck Millipore bought; Hexadecyltrimethylammonium bromide (CTAB) (> 99.0%) was purchased from Fluka; Bovine serum albumin (BSA) (Fraction V IgG-free, low in fatty acids) came from Gibco. Pure deionized water, which was used for all aqueous solutions, was dosed from a Milli-Q® system (18.2 megohms specific resistance).
- BSA Bovine serum albumin
- PBS phosphate-buffered saline
- NaF PO 10 mM NazFIPO 10 mM, MgCh 1 mM, pH 7.1
- Pan malaria antibody anti-pLDH monoclonal antibody clone 19g7 was produced by Vista Laboratory Services (Langley, USA).
- Plasmodium falciparum lactate dehydrogenase (P / LDH) and PvLDH were obtained by bacterial expression.
- the Malaria 2008s aptamers labeled with 5-FAM or cyanine-5-tag (5'-5-FAM (Cy5) -CTG GGC GGT AGA ACC ATA GTG ACC CAG CCG TCT AC-3 ') were provided by Friz Biochem GmbH ( Neuried, Germany).
- Millex® syringe filters (pore size 0.20 ⁇ m) with hydrophobic polytetrafluoroethylene membrane were purchased from Merck Millipore; Superslip® coverslips (borosilicate glass, 0.13-0.17 mm thick) were purchased from Thermo Fisher Scientific and cut by a diamond-tipped glass cutter.
- Nanolithography based on the self-assembly of block copolymers has been used to produce arrays of ordered AuNPs with adjustable density, size and interparticle spacing.
- 29.2 mg of the diblock copolymers P18226-S2VP were added to 15 ml of toluene with vigorous stirring and under controlled conditions (argon inert gas, O2 ⁇ 1 ppm, H 2 q ⁇ 0.1 ppm) and held for 72 hours in order to obtain a homogeneously dispersed, to obtain inverted micelles with a hydrophilic core and an outer hydrophobic shell (spherical).
- the substrates were cleaned by ultrasound for five minutes in succession in acetone, 2-propanol and pure ethanol and then immersed in toluene To “make” the surface non-polar so that the hydrophobic shells could adhere to it. Subsequently, the substrates were dipped into the solution containing PS-AuNPs by means of a dip coater with careful adjustment of the dipping speed. It was found that an immersion speed of 0.6 mm / s enabled a particularly good coating of the glass surface both with regard to the density of the arrangement of the AuNPs and with regard to the large-area uniformity of the deposition.
- the PS-AuNPs were transferred to the non-polar glass surface by hydrophobic interaction, whereby a hexagonal arrangement took place by means of self-organization is also illustrated in FIG.
- the copolymers were then etched by means of oxygen plasma treatment (0.8 mbar pressure, 200 W, 30 minutes), as a result of which the AuNPs were immobilized at prefixed positions on the glass surface.
- the plasmonic resonance depends strongly on the ratio R between the AuNP diameter D and the interparticle distance d, higher values of R guarantee greater coupling between the AuNPs.
- R> 2/3 the plasmonic resonance is dominated by the collective behavior of the AuNPs, and several advantages for metal-enhanced fluorescence (MEF) result.
- CEF metal-enhanced fluorescence
- the functionalization of the AuNPs with pan-malaria antibodies was carried out using photochemical immobilization technology (PIT).
- PIT photochemical immobilization technology
- 1 ml of aqueous solution of anti-pLDH (25 pg / ml) was irradiated by means of a UV lamp for 30 seconds and then poured onto the substrate.
- the UV source consisted of two U-shaped low-pressure mercury lamps (2 W at 254 nm) into which a standard quartz cuvette could be inserted. Under consideration of Geometry of the lamps and the proximity to the cuvette, the radiation intensity that was used to generate the thiol groups was approximately 1 W / cm 2 .
- the samples were rinsed with ultrapure water (dosed using a Milli-Q® system) in order to remove unbound antibodies.
- bovine serum albumin (BSA) solution 50 pg / ml was applied to cover the free gold surface and to protect it from non-specific adsorption.
- Plasmodium falciparum lactate dehydrogenase (Pfl_DH) was added to 1 ml of a dilute solution of uninfected human blood (dilution 1: 100 in 25 mM Tris buffer).
- the functionalized substrates were incubated with 1 ml of contaminated blood solution of the same dilution for 2 hours at room temperature.
- a rocker shaker was used to accelerate the binding kinetics and improve analyte diffusion. Concentrations between 0.01 femtomol and 10 nanomol were measured.
- the samples were rinsed copiously with Tris buffer (25 mM) and with ultrapure water to remove unbound proteins.
- the samples were rinsed copiously with PBS and ultrapure water to remove unbound aptamers.
- Fluorescence images were taken with a Zeiss Axio Observer ZI phase-inverted contrast fluorescence microscope, equipped with a Zeiss Colibri.2 LED light source (module 625 nm), Zeiss Plan apochromat objective 10x / 0.45 Ph 1 M27
- Such a value is then subtracted from the original image , smoothing out spatial variations in the background.
- the rolling ball radius was set to 10 pixels, a size sufficiently larger than the size of the largest objects that were not part of the background.
- a threshold value which was slightly higher than the smoothed background, was set in order to segment the image, the total intensity of which was measured by adding up the signal components from all spots. In order to obtain a good and reliable analysis of the fluorescence signals, ten images were taken at random of each sample and their intensity average was determined.
- Bloch BCs (periodic boundary conditions ) were only used for polarization studies to compensate for the phase shift that occurs when reintroduced an electromagnetic interference with a non-zero angle on the opposite side of the workspace.
- Perfectly adjusted layer BCs in the z-direction ensure perfect absorption of the electromagnetic waves that are backscattered by the plane containing the light source and incident through the opposite side of the working environment.
- the working environment was resolved via a grid with a spatial resolution of 0.5 nm, which ensured high accuracy and at the same time kept the simulation time within a few hours.
- the AuNPs were modeled as homogeneous gold hemispheres, whereas the substrate was represented as a thick dielectric layer of silicon dioxide (S1O2).
- the substrates were cleaned by ultrasound for five minutes in succession in acetone, 2-propanol and ethanol and then immersed in a non-polar solvent to prevent the adhesion of hydrophobic polystyrene - Allow envelopes.
- the substrates were then dipped into the solution containing PS-AuNPs by means of a dip coater at a dipping speed of 0.8 mm / s. This immersion speed enables a good coating of the substrate surface with simultaneous prevention of maximum packing density (cf. FIG. 2b, from which it can be seen that in the short-range order there are still hexagonal structures, but the long-range order shows branched structures).
- the copolymers were then etched by means of oxygen plasma treatment (0.8 mbar pressure, 200 W, 30 minutes), as a result of which the AuNPs were immobilized at prefixed positions on the glass surface.
- the substrates were then incubated for 2 hours with exclusion of light with 2 ml of an Au deposition solution (CTAB 190 mM, FIAuCU * 3H 2 0 42 mM, AgNO3 8 mM, ascorbic acid 100 mM).
- the functionalization of the AuNPs with pan-malaria antibodies was carried out using photochemical immobilization technology (PIT).
- PIT photochemical immobilization technology
- 1 ml of aqueous solution of anti-pLDFI (50 pg / ml) was irradiated using a UV lamp for 30 seconds and then poured onto the substrate.
- the UV source (Trylight, Promete Srl) consisted of two U-shaped low-pressure mercury lamps (6 W at 254 nm) into which a 10 mm standard quartz cuvette could be inserted. Taking into account the geometry of the lamps and the proximity to the cuvette, the radiation intensity that was used to generate the thiol groups was approximately 0.3 W / cm 2 .
- Uninfected human blood was diluted 1: 100 in 25 mM Tris buffer to reduce the turbidity of the sample.
- the desired amount of Plasmodium falciparum lactate dehydrogenase (P / LDFI) was added to 1 ml of a dilute solution of the sample in order to achieve analyte concentrations in the range from 1 fM to 1 pm received (based on the undiluted blood).
- the functionalized substrates were incubated with 1 ml of contaminated blood solution for 2 hours at room temperature.
- the recorded fluorescence images were processed with ImageJ software to process the full intensity emanating from the bright points.
- the RGB images were divided into two channels containing the red and green components in order to analyze the proportions of the two fluorophores separately.
- the “rolling balT” algorithm was also used here.
- FIG. 7 shows the resulting image in which the red and green channels are shown combined again (here in black and white for reproduction purposes).
- the specificity of the apta immunosensor was tested against lactate dehydrogenase from Plasmodium vivax (PvLDH), which is 90% identical to P / LDH.
- PvLDH lactate dehydrogenase from Plasmodium vivax
- the Pv DH was added to uninfected human blood (diluted 1: 100 in 1 mL of 25 mM Tris buffer) in order to obtain the highest analyte concentration investigated in the calibration curve (1 mM based on undiluted whole blood).
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DE102020115332.5A DE102020115332A1 (de) | 2020-06-09 | 2020-06-09 | Biomarkersensorbasierende detektion krankheitsspezifischer biomarker |
PCT/EP2021/065114 WO2021249910A1 (de) | 2020-06-09 | 2021-06-07 | Plasmonverstärkte-fluoreszenz-basierende sensor zum nachweis krankheitsspezifischer biomarker |
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