WO2024058756A1 - A method of obtaining a biosensor with a biomaterial integrated metasurface - Google Patents

A method of obtaining a biosensor with a biomaterial integrated metasurface Download PDF

Info

Publication number
WO2024058756A1
WO2024058756A1 PCT/TR2023/050943 TR2023050943W WO2024058756A1 WO 2024058756 A1 WO2024058756 A1 WO 2024058756A1 TR 2023050943 W TR2023050943 W TR 2023050943W WO 2024058756 A1 WO2024058756 A1 WO 2024058756A1
Authority
WO
WIPO (PCT)
Prior art keywords
meta
antibody
exosome
nano
protein
Prior art date
Application number
PCT/TR2023/050943
Other languages
French (fr)
Inventor
Fatih INCI
Esma DERIN
Original Assignee
Bilkent Universitesi Ulusal Nanoteknoloji Arastirma Merkezi
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Bilkent Universitesi Ulusal Nanoteknoloji Arastirma Merkezi filed Critical Bilkent Universitesi Ulusal Nanoteknoloji Arastirma Merkezi
Publication of WO2024058756A1 publication Critical patent/WO2024058756A1/en

Links

Classifications

    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates to a method of obtaining a biosensor for detecting biomarkers included in body fluid by means of metasurfaces obtained by integrating biomaterials into inert optical discs.
  • Optical biosensors are one of the most frequently used strategies for the development of bedside diagnostic tests. It is suitable to develop platforms that can take fast and sensitive measurements without the need for any molecular labelling. This application area is among the most important reasons for its widespread use.
  • Another advantage of optical biosensors is that microfluidics can be integrated easily. Thereby, fluid passage can be provided easily and the use of required sample and solution can be significantly reduced.
  • Metamaterials and metasurfaces are nanostructured interfaces comprising thin layers of plasmonic or dielectric materials with sub-wavelength thicknesses that manipulate the light. Therefore, they are widely used in construction of optical sensors. Metamaterials are not found in nature directly. They can be obtained as nanostructures designed by combining different materials based on their shape and material combination. With these nanostructures, new properties (such as light control capability) are obtained. Different strategies are used for generation of meta surfaces, but most of them are highly complex and expensive procedures. Biomaterial integrated meta surfaces are used as biosensors in order to detect potential biomarkers (from serum, blood, saliva, urine, faeces, lavage fluid, interstitial fluid and other body fluids) that can be used to detect any disease. These systems can be called metamaterial plasmonic sensors as well.
  • exosomes The release of exosomes from cells is associated with various diseases. Recent studies have also revealed the potential of exosomes as biomarkers. Proteins, which are also known as three different tetraspanins, are included in the membrane of exosomes. Exosomes can be captured and detected by using antigens specific to tetraspanins. Since tetraspanins are present in exosomes in a large amount, they are one of the most commonly used markers for detection of exosomes.
  • the Chinese patent document no. CN102037358 an application in the state of the art, discloses use of an optical disc with surface activation in biomolecule detection.
  • An objective of the present invention is to realize a method of obtaining a biosensor for detecting biomarkers included in body fluid by means of meta surfaces obtained by integrating biomaterials into inert optical discs.
  • Another object of the present invention is to realize a method of obtaining a low- cost biosensor by means of nanostructures obtained from inert optical discs.
  • Another object of the present invention is to realize a method of sensitive biomarker determination by means of gold nanoparticles (AuNP) and nanoislands chemically integrated into inert optical discs.
  • AuNP gold nanoparticles
  • Another object of the present invention is to realize a method of obtaining sensitive biosensors whereby oxidation of sensors having a silver top surface is prevented by the presence of a Poly L Lysine (PLL) layer, gold nanoparticle and nano-island structures on the surface thereof.
  • PLL Poly L Lysine
  • Another object of the present invention is to realize a method of developing bedside testing devices instead of centralised diagnostic platforms in order to reduce workload and cost in hospitals.
  • Figure l is a flow chart of the inventive method.
  • Figure 2 is a schematic expression of the fabrication stages of metasurfaces, (a) AFM and SEM images and AFM profile of the plasmonic sensor cretated by a plurality of metal coatings (Titanium: 10 nm; Silver: 30 nm; Gold: 15 nm) on a DVD plastic mould, (b) AFM and SEM images and AFM profile of the plasmonic sensor formed by integrating gold nanoparticles (1 : 10 dilution) on the blank metasurface through the PLL layer, (c) AFM and SEM images and AFM profile with nano-island-structure plasmonic sensor created by seeding using chloroauric acid (HAuCh) and hydroxylamine hydrochloride (HONH2.HCI) on the PLL layer directly, (d) AFM and SEM images and AFM profile of the plasmonic sensor created by 65 nm silver metal coating on a DVD plastic mould, (e) AFM and SEM images and AFM profile of the plasmonic sensor created by integrating gold nanoparticles (1
  • Figure 3 is review of the performance of the plasmonic sensor with blank gold and silver top meta surfaces, (a) Changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing final measurement analysis for the sensor with blank gold top meta surface, (b) Changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing final measurement analysis for the sensor with a blank silver top meta surface, (c) Representation of the heat map of the changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing the final measurement analysis for the sensor with empty gold top meta surface, (d) Representation of the heat map of the changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing the final measurement analysis for the sensor with empty silver top meta surface, (e) Representation of the plasmonic resonance value of sensors with gold and silver metasurfaces plotted as a function of time for different glycerol solutions, (f) Linear representation of the effect
  • Figure 4 is comparison of sensor performances (a) Linear representation of the effect of glycerol solutions in different concentration and refractive index on the blank gold and silver surface, (b) Linear representation of the effect of glycerol solutions of different concentrations and refractive indices on the surface created in the nano-island by providing the same seeding solution on different PLL concentrations, (c) Linear representation of the effect of the amount of gold nanoparticles on the plasmonic sensor resonance with gold top meta surface caused by glycerol solutions of different concentration and refractive index, (d) Linear representation of the effect of glycerol solutions in different concentration and refractive index on the plasmonic sensor resonance with silver meta surface and comparison with blank silver meta surface, (e) Linear representation of the effect of the concentration of the seeding solution on the resonance of the plasmonic sensor with gold top meta surface induced by glycerol solutions of different concentration and refractive index, (f) Linear representation of the effect of glycerol solutions in different concentrations and refractive
  • Figure 6 is comparison of signal enhancement in sensors with silver top meta surface
  • Figure 7 is testing the suitability of different systems by attaching anti-CD63 antibody to surfaces integrated with gold nanoparticles
  • (a) Representation of the performance of 10 8 parti cles/mL on a gold nanoparticle integrated silver top surface sensor with anti-CD81 antibody attached (b) Representation of the performance of 10 8 parti cles/mL on a gold nanoparticle integrated silver top surface sensor with anti-CD63 antibody attached.
  • Clinical applicability test (c) Preparation of exosome samples in artificial urine and representation of its performance on the sensor with gold nanoparticle integrated gold top surface,
  • the inventive method (100) of detecting biomarkers included in body fluid by means of metasurfaces obtained by integrating biomaterials into inert optical discs comprises the steps of
  • the plastic protection layer located on the inert optical disc (DVD: Digital Versatile Disc) is removed by means of a knife and a chemical etching is applied to the exposed nano-periodic structure.
  • the optical disc having a nano-periodic structure subjected to chemical etching is used as a plastic mould and the plastic mould is coated with titanium or chromium (as adhesive layer), silver and gold respectively by means of electron-beam evaporation method in order to obtain a meta surface (biosensor/metamaterial plasmonic sensor) having a gold top surface.
  • the optical disc with nano-periodic structure exposed to chemical etching process is coated with silver by means of sputtering method.
  • the meta surfaces with gold and silver top surface are cut into the desired dimensions and cleaned with ethanol.
  • tse surfaces are functionalized by incubating them in PLL solution with biomaterial at concentrations ranging from 0.05 mg/mL to 1 mg/mL for 8-12 hours at 2-8 °C.
  • the seeding solution chloroauric acid (HAuCU) and hydroxylamine hydrochloride (HONH2.HCI)
  • HAuCU chloroauric acid
  • HONH2.HCI hydroxylamine hydrochloride
  • exosome is isolated from the medium of kidney cells by using microfluidics and the concentration, diameter and size distribution of these exosomes are analysed by means of a laser-based optical technique (NTA Instrument (NS300, Malvern Instruments Ltd., Malvern, Worcestershire, UK) that monitors the Brownian motion of individual particles included in the solution.
  • NTA Instrument N-based optical technique
  • the samples with determined concentration are diluted with PBS so as to be 10 5 -10 9 particles/mL.
  • the wavelength shift is calculated by applying the obtained exosomes to the surface where gold nanoparticles are integrated and nano-island is created. In the light of the wavelength shift information calculated, the presence of exosomes and exosome-related diseases are detected.
  • a second protein (CD-63 antigen) included in the exosome membrane by applying anti-CD63 antibody instead of anti-CD81 antibody to the surface.
  • 50-1000 pg/mL Protein G a direct binding protein, is applied to the sensor with a silver top meta surface wherein gold nanoparticles are integrated by applying PLL solution. Protein G is attached to the meta surface by means of protein-protein interaction.
  • 50 - 500 pg/mL anti-CD63 antibody is applied to the surface and incubated at +2-8 °C for 1-5 hours for the detection of CD63, one of the proteins included in the membrane of exosomes.
  • exosome samples are prepared in artificial urine to demonstrate the clinical applicability of the method (100).
  • the PLL solution is applied and 50 - 1000 pg/mL Protein G, a direct binding protein, is applied to the sensor with a silver meta surface integrating gold nanoparticles. Protein G is attached to the meta surface by means of protein-protein interaction.
  • 50 - 500 pg/mL anti-CD81 antibody is applied to the surface and incubated at 2-8 °C for 1-5 hours for the detection of CD81, one of the proteins included in the membrane of exosomes.
  • Exosomes are isolated from the medium of kidney cells using microfluidics and the concentration, diameter and size distribution of these exosomes are analysed by means of a laser-based optical technique (NTA Instrument (NS300, Malvern Instruments Ltd., Malvern, Worcestershire, UK) that monitors the Brownian motion of individual particles in solution.
  • NTA Instrument N-based optical technique
  • the samples whose concentration is determined are diluted with artificial urine to 10 5 -10 9 particles/mL.
  • the wavelength shift is calculated by providing the obtained exosomes to gold nanoparticle integrated surfaces. In the light of the wavelength shift information calculated, the presence of exosomes and exosome-related diseases are detected.
  • Meta-materials with gold and silver top surface were produced by using nano- periodic structures on the surface of optical discs.
  • the plastic protection layer on the DVD was removed with a knife and the plasmonic effect was obtained by chemical etching and metal coating, respectively.
  • gold top surface titanium or chromium, silver and gold were coated by means of electron-beam evaporation method and metamaterial plasmonic sensors were obtained.
  • silver surface silver was coated directly on the nano-periodic structure by sputtering.
  • FIG. 2a shows the SEM, AFM image and profile of the blank surface of the gold top surface.
  • Figure 2d shows the SEM, AFM image and profile of the blank surface of the silver top surface. According to the AFM results, the size of the periodic structure was determined as -740 nm (the Figure 2a-2d). The same characterization procedures were performed for the surfaces with gold nanoparticles integrated and nanoisland formed. In the images obtained, gold particles and nano-islands are clearly visible (the Figure 2b-f).
  • Glycerol solutions prepared at different ratios were used to measure the sensitivity performance of the metasurfaces prepared. Glycerol solution was used to mimic the molecule binding to the surface by changing the refractive index on the surface.
  • a plasmonic sensor with a blank gold and silver top surface was used in the Figure 3 and the resonance shifts are shown in the Figures 3a-d.
  • a user-friendly software based on MATLAB GUI was produced and obtained by time-dependent calculation of resonance shifts (the Figure 3e).
  • a linear graph was obtained by plotting the wavelength shift against the refractive index change (the Figure 3f).
  • the gold and silver surfaces were obtained, they were cut into the desired dimensions and cleaned with ethanol. Then, these surfaces were incubated in different concentrations (0.05-1 mg/mL) of PLL solution at 2-8 °C for 8-12 hours. Then, the same amount of seeding solution was applied on them and nano-island was formed. According to the results obtained, 0.5 mg/mL PLL integrated surfaces gave the best results (the Figure 4b). Surfaces modified with 0.5 mg/mL PPL were used for further processing. The functionalised surfaces were incubated overnight in different concentrations of gold nanoparticle solution. Integration of too many gold nanoparticles disrupted the nano-periodic structure, resulting in signal loss. According to the results obtained, it was observed that the stock gold nanoparticle solution should be diluted 1:2 - 1:50 ratios (the Figure 4c). The same process was repeated with the sensor having a silver top meta surface (the Figure 4d).
  • the nano-island formation was first tested on gold nanoparticle integrated surfaces but no change was observed. Therefore, direct seeding solution (chloroauric acid (HAuCL) and hydroxylamine hydrochloride (HONH2.HCI)) were applied to the PLL functionalized surface.
  • the protocol was optimised using different concentrations of PLL, chloroauric acid (HAuCL) and hydroxylamine hydrochloride (HONH2.HCI) to create nano-islands on the surface. As shown in the Figure 5c, the highest wavelength shift was observed when 20 pM HAuCL: 20 pM HONH2.HCI was mixed 1 : 1 (v:v) on a PLL concentration of 0.5 mg/mL.
  • the sensor surface was functionalized with 1-10 mM 11- mercaptoundodecanoic acid (MUA) overnight at room temperature, thus creating carboxyl groups on the surface. After the incubation period ended, the unbound molecules were removed by washing with ethanol and the surface was dried at room temperature.
  • MUA 11- mercaptoundodecanoic acid
  • the prepared microfluidic chip was combined with the sensor surface and l-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDC) (100 mM) / N-Hydroxy succinimide (NHS) (50 mM) mixture was introduced into the channels and incubated at room temperature for 20-60 min. The channels were then washed with PBS to remove unbound molecules.
  • EDC l-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDC) (100 mM) / N-Hydroxy succinimide (NHS) (50 mM) mixture was introduced into the channels and incubated at room temperature for 20-60 min. The channels were then washed with PBS to remove unbound molecules.
  • Protein G was bound to the succinimide groups formed with EDC/NHS.
  • 100 pg/mL Protein G was prepared in PBS and applied through the channels and in
  • Microfluidic chips placed on the sensor surface were prepared with poly (methyl methacrylate) (PMMA, 2 mm thick) and double-sided adhesive film (DSA, 50 pm thick).
  • PMMA poly (methyl methacrylate)
  • DSA double-sided adhesive film
  • the PMMA and DSA layers were designed separately by using RDWorks software and cut with a laser cutter (LazerFix, Turkey).
  • cavities were cut for fluid inlet and outlet and capillary tubings were fixed to these cavities with epoxy resin. Channels for fluid flow were formed in the DSA layer and combined with PMMA.
  • the microfluidic chip prepared was adhered to the sensor surface with the help of DSA.
  • Exosomes were then isolated from the medium of kidney cells by using microfluidics to serve as a sample.
  • concentration, diameter and size distribution of the isolated exosomes were analysed by using the NTA Instrument (NS300, Malvern Instruments Ltd., Malvern, Worcestershire, UK), a laser-based optical technique that tracks the Brownian motion of individual particles in solution.
  • Concentrated samples were diluted with PBS and artificial urine to 10 5 - 10 9 parti cles/mL. The resulting exosomes were applied to both functionalized surfaces and the wavelength shift was calculated.
  • the wavelength shift was calculated as 1.27 nm (mean 0.94 ⁇ 0.29 nm) as shown in the Figure 5a, while this shift was calculated as 1.82 nm (mean 1.54 ⁇ 0.52 nm) when gold nanoparticles were integrated on the surface (Figure 5b), and 1.7 nm (1.27 ⁇ 0.50 nm) when nano-islands were formed by seeding method (Figure 5c).
  • gold nanoparticle integration increased the signal by 5.5 times compared to the blank surface, while nano-island formation increased the signal by 3.5 times (the Figure 5e).
  • the wavelength shift of the sensor with a blank silver top surface was calculated as 1.11 nm (average 0.77 ⁇ 0.34 nm), while this shift was calculated as 2.32 nm (average 2.47 ⁇ 1.49 nm) when gold nanoparticles were integrated on the surface (the Figure 6b), and 0.43 nm (0.42 ⁇ 0.20 nm) when the nano-island was created by the seeding method (the Figure 6c).
  • gold nanoparticle integration increased the signal by a factor of 2.6 compared to the blank surface, while nano-island formation decreased the signal (the Figure 6e).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

The present invention relates to a method (100) of obtaining a biosensor for detecting biomarkers included in body fluid by means of metasurfaces obtained by integrating biomaterials into inert optical discs.

Description

A METHOD OF OBTAINING A BIOSENSOR WITH A BIOMATERIAL INTEGRATED METASURFACE
Technical Field
The present invention relates to a method of obtaining a biosensor for detecting biomarkers included in body fluid by means of metasurfaces obtained by integrating biomaterials into inert optical discs.
Background of the Invention
With the aim of reducing the workload and costs in hospitals, the healthcare system switches from centralised diagnostic platforms to point-of-care testing platforms and thus, it is aimed to reach more individuals. In this context, biosensor platforms have a crucial impact on disease diagnosis as an alternative or complement to these existing tests.
Today, convetional disease diagnosis methods such as ELISA, Simoa, Luminex and Multiplex Searchlight are used for biomarker determination. However, the fact that these methods are complex, long-term and expensive is the major handicaps. Therefore, there is an urgent need to develop semi-/partially invasive tests with low-cost, easy-to-use, high accuracy and sensitivity for early diagnosis. The World Health Organisation (WHO) states that bedside diagnostic systems should be (i) low-cost, (ii) sensitive, (iii) specific, (iv) user-friendly, (v) rapid, (vi) durable, (vii) do not require complex equipment and (viii) easy to use. For this reason, these characteristics should be considered when developing bedside tests.
Optical biosensors are one of the most frequently used strategies for the development of bedside diagnostic tests. It is suitable to develop platforms that can take fast and sensitive measurements without the need for any molecular labelling. This application area is among the most important reasons for its widespread use. Another advantage of optical biosensors is that microfluidics can be integrated easily. Thereby, fluid passage can be provided easily and the use of required sample and solution can be significantly reduced.
Metamaterials and metasurfaces are nanostructured interfaces comprising thin layers of plasmonic or dielectric materials with sub-wavelength thicknesses that manipulate the light. Therefore, they are widely used in construction of optical sensors. Metamaterials are not found in nature directly. They can be obtained as nanostructures designed by combining different materials based on their shape and material combination. With these nanostructures, new properties (such as light control capability) are obtained. Different strategies are used for generation of meta surfaces, but most of them are highly complex and expensive procedures. Biomaterial integrated meta surfaces are used as biosensors in order to detect potential biomarkers (from serum, blood, saliva, urine, faeces, lavage fluid, interstitial fluid and other body fluids) that can be used to detect any disease. These systems can be called metamaterial plasmonic sensors as well.
The release of exosomes from cells is associated with various diseases. Recent studies have also revealed the potential of exosomes as biomarkers. Proteins, which are also known as three different tetraspanins, are included in the membrane of exosomes. Exosomes can be captured and detected by using antigens specific to tetraspanins. Since tetraspanins are present in exosomes in a large amount, they are one of the most commonly used markers for detection of exosomes.
In light of this information, in order to overcome the above-mentioned drawbacks, there is a need for a method of obtaining a biomarker which enables to detect biomarkers included in body fluid by means of biosensors (meta surface/metamaterial/metamaterial plasmonic sensor) with increased sensitivity by integrating biomaterials; solving manufacturing difficulties related to cost, biocompatibility and complexity; and providing low-cost easy detection.
The Chinese patent document no. CN102037358, an application in the state of the art, discloses use of an optical disc with surface activation in biomolecule detection.
Summary of the Invention
An objective of the present invention is to realize a method of obtaining a biosensor for detecting biomarkers included in body fluid by means of meta surfaces obtained by integrating biomaterials into inert optical discs.
Another object of the present invention is to realize a method of obtaining a low- cost biosensor by means of nanostructures obtained from inert optical discs.
Another object of the present invention is to realize a method of sensitive biomarker determination by means of gold nanoparticles (AuNP) and nanoislands chemically integrated into inert optical discs.
Another object of the present invention is to realize a method of obtaining sensitive biosensors whereby oxidation of sensors having a silver top surface is prevented by the presence of a Poly L Lysine (PLL) layer, gold nanoparticle and nano-island structures on the surface thereof.
Another object of the present invention is to realize a method of developing bedside testing devices instead of centralised diagnostic platforms in order to reduce workload and cost in hospitals.
Detailed Description of the Invention “A Method of Obtaining a Biosensor with Biomaterial Integrated Metasurface” realized to fulfil the objectives of the present invention is shown in the figures attached, in which:
Figure l is a flow chart of the inventive method.
Figure 2 is a schematic expression of the fabrication stages of metasurfaces, (a) AFM and SEM images and AFM profile of the plasmonic sensor cretated by a plurality of metal coatings (Titanium: 10 nm; Silver: 30 nm; Gold: 15 nm) on a DVD plastic mould, (b) AFM and SEM images and AFM profile of the plasmonic sensor formed by integrating gold nanoparticles (1 : 10 dilution) on the blank metasurface through the PLL layer, (c) AFM and SEM images and AFM profile with nano-island-structure plasmonic sensor created by seeding using chloroauric acid (HAuCh) and hydroxylamine hydrochloride (HONH2.HCI) on the PLL layer directly, (d) AFM and SEM images and AFM profile of the plasmonic sensor created by 65 nm silver metal coating on a DVD plastic mould, (e) AFM and SEM images and AFM profile of the plasmonic sensor created by integrating gold nanoparticles (1 : 10 diluted) on the blank silver metal surface through the PLL layer, (f) AFM and SEM images and AFM profile of the nano-island- structured plasmonic sensor created by seeding using chloroauric acid (HAuCh) and hydroxylamine hydrochloride (HONH2.HCI) on the PLL layer directly.
Figure 3 is review of the performance of the plasmonic sensor with blank gold and silver top meta surfaces, (a) Changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing final measurement analysis for the sensor with blank gold top meta surface, (b) Changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing final measurement analysis for the sensor with a blank silver top meta surface, (c) Representation of the heat map of the changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing the final measurement analysis for the sensor with empty gold top meta surface, (d) Representation of the heat map of the changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by performing the final measurement analysis for the sensor with empty silver top meta surface, (e) Representation of the plasmonic resonance value of sensors with gold and silver metasurfaces plotted as a function of time for different glycerol solutions, (f) Linear representation of the effect of glycerol solutions in different concentrations and refractive indices on sensors with gold and silver meta surfaces.
Figure 4 is comparison of sensor performances (a) Linear representation of the effect of glycerol solutions in different concentration and refractive index on the blank gold and silver surface, (b) Linear representation of the effect of glycerol solutions of different concentrations and refractive indices on the surface created in the nano-island by providing the same seeding solution on different PLL concentrations, (c) Linear representation of the effect of the amount of gold nanoparticles on the plasmonic sensor resonance with gold top meta surface caused by glycerol solutions of different concentration and refractive index, (d) Linear representation of the effect of glycerol solutions in different concentration and refractive index on the plasmonic sensor resonance with silver meta surface and comparison with blank silver meta surface, (e) Linear representation of the effect of the concentration of the seeding solution on the resonance of the plasmonic sensor with gold top meta surface induced by glycerol solutions of different concentration and refractive index, (f) Linear representation of the effect of glycerol solutions in different concentrations and refractive indices on the resonance of the plasmonic sensor with silver meta surface of the determined seeding solution and comparison with blank silver meta surface.
Figure 5 is comparison of signal enhancement in sensors with gold top metasurface (a) Exosome capture of 108 parti cles/mL and signal representation of the sensor with blank gold top surface, (b) Exosome capture of 108 particles/mL and signal representation of the sensor with gold nanoparticle integrated gold top surface, (c) Capture of 108 particles/mL of exosomes and signal representation of the sensor with nano-island-formed gold top surface by seeding, (d) Nonparametric Kruskal-Wallis statistical analysis of the data (no statistical difference was observed (n=3, p>0.05)). (e) Comparison of all modifications with normalisation of the data (between 0-1). Figure 6 is comparison of signal enhancement in sensors with silver top meta surface (a) Exosome capture of 108 parti cles/mL and signal representation of the sensor with blank silver top surface, (b) Exosome capture of 108 parti cles/mL and signal representation of the sensor with silver top surface integrated with gold nanoparticles, ((c) Exosome capture and signal representation of 108 particles/mL exosomes of the sensor with nano-island-formed silver top surface by seeding, (d) Non-parametric Kruskal-Wallis statistical analysis of the data (statistical difference indicated by an asterisk (n=3, p<0.05)). (e) Comparison of all modifications with normalisation of the data (between 0-1).
Figure 7 is testing the suitability of different systems by attaching anti-CD63 antibody to surfaces integrated with gold nanoparticles (a) Representation of the performance of 108 parti cles/mL on a gold nanoparticle integrated silver top surface sensor with anti-CD81 antibody attached, (b) Representation of the performance of 108 parti cles/mL on a gold nanoparticle integrated silver top surface sensor with anti-CD63 antibody attached. Clinical applicability test, (c) Preparation of exosome samples in artificial urine and representation of its performance on the sensor with gold nanoparticle integrated gold top surface, (d) Preparation of exosome samples in artificial urine and representation of its performance on the sensor with gold nanoparticle integrated silver surface.
The components illustrated in the figures are individually numbered, where the numbers refer to the following:
100. Method
The inventive method (100) of detecting biomarkers included in body fluid by means of metasurfaces obtained by integrating biomaterials into inert optical discs comprises the steps of
- obtaining a meta surfacing by coating inert optical discs with metal (101),
- functionalizing the surface by integrating biomaterial into the prepared meta surface (102), - creating nano-island on the surface by providing the seeding solution into the functionalized meta surface (103),
- administrating antibody of the exosome membrane protein to the meta surface provided with seeding solution (104), and
- detecting disease by capturing the exosome of the antibody with a meta surface containing the antibody placed into a body fluid (105).
In the step of obtaining a meta surfacing by coating inert optical discs with metal (101) of the inventive method (100), the plastic protection layer located on the inert optical disc (DVD: Digital Versatile Disc) is removed by means of a knife and a chemical etching is applied to the exposed nano-periodic structure. In one embodiment of the invention, the optical disc having a nano-periodic structure subjected to chemical etching is used as a plastic mould and the plastic mould is coated with titanium or chromium (as adhesive layer), silver and gold respectively by means of electron-beam evaporation method in order to obtain a meta surface (biosensor/metamaterial plasmonic sensor) having a gold top surface. In another embodiment of the invention, the optical disc with nano-periodic structure exposed to chemical etching process is coated with silver by means of sputtering method.
In the step of functionalizing the surface by integrating biomaterial into the prepared meta surface (102) of the inventive method (100), the meta surfaces with gold and silver top surface are cut into the desired dimensions and cleaned with ethanol. Then, tse surfaces are functionalized by incubating them in PLL solution with biomaterial at concentrations ranging from 0.05 mg/mL to 1 mg/mL for 8-12 hours at 2-8 °C.
In the step of creating nano-island on the surface by providing the seeding solution into the functionalized meta surface (103) of the inventive method (100), the seeding solution (chloroauric acid (HAuCU) and hydroxylamine hydrochloride (HONH2.HCI)) is applied to the functionalised meta surface directly directly with the PLL solution. In case where 1 : 1 (v:v) 10-100 pM HAuCh: 10-100 pM HONH2.HCI is applied on 0.05-1 mg/mL PLL concentration on the surface, nanoisland formation occurs on the surface of the meta surface.
In the step of administrating antibody of the exosome membrane protein to the meta surface provided with seeding solution (104) of the inventive method (100), 50-1000 pg/mL Protein G, which is a direct binding protein, is applied to the meta surface created in the nano by applying the PLL solution. Protein G is attached to the meta surface by protein-protein interaction. Then, 50-500 pg/mL anti-CD81 antibody is applied to the surface for the detection of CD81, one of the proteins included in the membrane of exosomes, and incubated at 2-8 °C for 1-5 hours.
In the step of detecting disease by capturing the exosome of the antibody with a meta surface containing the antibody placed into a body fluid (105) of the inventive method (100), exosome is isolated from the medium of kidney cells by using microfluidics and the concentration, diameter and size distribution of these exosomes are analysed by means of a laser-based optical technique (NTA Instrument (NS300, Malvern Instruments Ltd., Malvern, Worcestershire, UK) that monitors the Brownian motion of individual particles included in the solution. The samples with determined concentration are diluted with PBS so as to be 105-109 particles/mL. The wavelength shift is calculated by applying the obtained exosomes to the surface where gold nanoparticles are integrated and nano-island is created. In the light of the wavelength shift information calculated, the presence of exosomes and exosome-related diseases are detected.
In one embodiment of the invention, in order to demonstrate the applicability of the method (100) to different diseases, it is aimed to use a second protein (CD-63 antigen) included in the exosome membrane by applying anti-CD63 antibody instead of anti-CD81 antibody to the surface. 50-1000 pg/mL Protein G, a direct binding protein, is applied to the sensor with a silver top meta surface wherein gold nanoparticles are integrated by applying PLL solution. Protein G is attached to the meta surface by means of protein-protein interaction. Then, 50 - 500 pg/mL anti-CD63 antibody is applied to the surface and incubated at +2-8 °C for 1-5 hours for the detection of CD63, one of the proteins included in the membrane of exosomes.
In one embodiment of the invention, exosome samples are prepared in artificial urine to demonstrate the clinical applicability of the method (100). The PLL solution is applied and 50 - 1000 pg/mL Protein G, a direct binding protein, is applied to the sensor with a silver meta surface integrating gold nanoparticles. Protein G is attached to the meta surface by means of protein-protein interaction. Then, 50 - 500 pg/mL anti-CD81 antibody is applied to the surface and incubated at 2-8 °C for 1-5 hours for the detection of CD81, one of the proteins included in the membrane of exosomes. Exosomes are isolated from the medium of kidney cells using microfluidics and the concentration, diameter and size distribution of these exosomes are analysed by means of a laser-based optical technique (NTA Instrument (NS300, Malvern Instruments Ltd., Malvern, Worcestershire, UK) that monitors the Brownian motion of individual particles in solution. The samples whose concentration is determined are diluted with artificial urine to 105-109 particles/mL. The wavelength shift is calculated by providing the obtained exosomes to gold nanoparticle integrated surfaces. In the light of the wavelength shift information calculated, the presence of exosomes and exosome-related diseases are detected.
The entire experimental process followed to reach the said method (100) is carried out as follows.
Meta-materials with gold and silver top surface were produced by using nano- periodic structures on the surface of optical discs. First, the plastic protection layer on the DVD was removed with a knife and the plasmonic effect was obtained by chemical etching and metal coating, respectively. For the gold top surface, titanium or chromium, silver and gold were coated by means of electron-beam evaporation method and metamaterial plasmonic sensors were obtained. For the silver surface, silver was coated directly on the nano-periodic structure by sputtering.
Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to examine the structures of the obtained surfaces. Figure 2a shows the SEM, AFM image and profile of the blank surface of the gold top surface. Figure 2d shows the SEM, AFM image and profile of the blank surface of the silver top surface. According to the AFM results, the size of the periodic structure was determined as -740 nm (the Figure 2a-2d). The same characterization procedures were performed for the surfaces with gold nanoparticles integrated and nanoisland formed. In the images obtained, gold particles and nano-islands are clearly visible (the Figure 2b-f).
Glycerol solutions prepared at different ratios (l%-70%, volume: volume) were used to measure the sensitivity performance of the metasurfaces prepared. Glycerol solution was used to mimic the molecule binding to the surface by changing the refractive index on the surface. A plasmonic sensor with a blank gold and silver top surface was used in the Figure 3 and the resonance shifts are shown in the Figures 3a-d. For time-dependent measurements, a user-friendly software based on MATLAB GUI was produced and obtained by time-dependent calculation of resonance shifts (the Figure 3e). A linear graph was obtained by plotting the wavelength shift against the refractive index change (the Figure 3f). When the results of the plasmonic sensor with a blank gold top surface and the plasmonic sensor with a blank silver top surface were compared, it was observed that the silver surface was more sensitive (the Figure 4a).
After the gold and silver surfaces were obtained, they were cut into the desired dimensions and cleaned with ethanol. Then, these surfaces were incubated in different concentrations (0.05-1 mg/mL) of PLL solution at 2-8 °C for 8-12 hours. Then, the same amount of seeding solution was applied on them and nano-island was formed. According to the results obtained, 0.5 mg/mL PLL integrated surfaces gave the best results (the Figure 4b). Surfaces modified with 0.5 mg/mL PPL were used for further processing. The functionalised surfaces were incubated overnight in different concentrations of gold nanoparticle solution. Integration of too many gold nanoparticles disrupted the nano-periodic structure, resulting in signal loss. According to the results obtained, it was observed that the stock gold nanoparticle solution should be diluted 1:2 - 1:50 ratios (the Figure 4c). The same process was repeated with the sensor having a silver top meta surface (the Figure 4d).
The nano-island formation was first tested on gold nanoparticle integrated surfaces but no change was observed. Therefore, direct seeding solution (chloroauric acid (HAuCL) and hydroxylamine hydrochloride (HONH2.HCI)) were applied to the PLL functionalized surface. The protocol was optimised using different concentrations of PLL, chloroauric acid (HAuCL) and hydroxylamine hydrochloride (HONH2.HCI) to create nano-islands on the surface. As shown in the Figure 5c, the highest wavelength shift was observed when 20 pM HAuCL: 20 pM HONH2.HCI was mixed 1 : 1 (v:v) on a PLL concentration of 0.5 mg/mL. When the refractive index sensitivity was calculated by using equation (1), the highest value was obtained when 20 pM HAuCL: 20 pM HONH2.HCI was seeded 1 : 1 (v:v) onto a PLL concentration of 0.5 mg/mL (the Table 1). Subsequent steps were continued at these optimised concentrations.
Figure imgf000013_0001
Refractive index sensitivity = . (1)
Table 1: Calculation of refractive index sensitivity of the sensor by seeding at different concentrations
Figure imgf000014_0001
Subsequent to the optimised PLL concentration, different concentrations of seeding solutions (10-100 pM) were applied to the surfaces and the difference in signal enhancement was observed. According to the results obtained, the best signal was observed when 10 pM seeding solution was applied (the Figure 4e-f).
The experiment focused on CD81, one of the proteins included in the exosome membrane, and the sensor surface was functionalized with anti-CD81 antibody. For this, it is necessary to perform layer-by-layer surface chemistry to attach the anti-CD81 antigen on the blank gold surface. To create the layer-by-layer chemistry, the sensor surface was functionalized with 1-10 mM 11- mercaptoundodecanoic acid (MUA) overnight at room temperature, thus creating carboxyl groups on the surface. After the incubation period ended, the unbound molecules were removed by washing with ethanol and the surface was dried at room temperature. Then, the prepared microfluidic chip was combined with the sensor surface and l-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDC) (100 mM) / N-Hydroxy succinimide (NHS) (50 mM) mixture was introduced into the channels and incubated at room temperature for 20-60 min. The channels were then washed with PBS to remove unbound molecules. In the third step, Protein G was bound to the succinimide groups formed with EDC/NHS. For this step, 100 pg/mL Protein G was prepared in PBS and applied through the channels and incubated overnight at 2-8 °C and the channels were washed with PBS at the end of incubation. As a final step, 50-500 pg/mL anti-CD81 antigen was applied to the surface and incubated at 2-8°C for 1-5 hours.
Microfluidic chips placed on the sensor surface were prepared with poly (methyl methacrylate) (PMMA, 2 mm thick) and double-sided adhesive film (DSA, 50 pm thick). The PMMA and DSA layers were designed separately by using RDWorks software and cut with a laser cutter (LazerFix, Turkey). In the PMMA layer, cavities were cut for fluid inlet and outlet and capillary tubings were fixed to these cavities with epoxy resin. Channels for fluid flow were formed in the DSA layer and combined with PMMA. The microfluidic chip prepared was adhered to the sensor surface with the help of DSA.
As PLL was applied on the seeded surface, the MUA and EDC/NHS chemistry steps were eliminated and 50-1000 pg/mL Protein G was applied directly. Protein G was attached to the surface by means of protein-protein interaction. Then, SO- SOO pg/mL anti-CD81 antibody was applied to the surface and incubated at 2-8°C for 1-5 hours. The same procedure was performed for anti-CD63 antibody.
Exosomes were then isolated from the medium of kidney cells by using microfluidics to serve as a sample. The concentration, diameter and size distribution of the isolated exosomes were analysed by using the NTA Instrument (NS300, Malvern Instruments Ltd., Malvern, Worcestershire, UK), a laser-based optical technique that tracks the Brownian motion of individual particles in solution. Concentrated samples were diluted with PBS and artificial urine to 105- 109 parti cles/mL. The resulting exosomes were applied to both functionalized surfaces and the wavelength shift was calculated. In the plasmonic sensor with a blank gold top surface, the wavelength shift was calculated as 1.27 nm (mean 0.94 ± 0.29 nm) as shown in the Figure 5a, while this shift was calculated as 1.82 nm (mean 1.54 ± 0.52 nm) when gold nanoparticles were integrated on the surface (Figure 5b), and 1.7 nm (1.27 ± 0.50 nm) when nano-islands were formed by seeding method (Figure 5c). When these data were normalised (0-1), gold nanoparticle integration increased the signal by 5.5 times compared to the blank surface, while nano-island formation increased the signal by 3.5 times (the Figure 5e). As shown in the Figure 6a, the wavelength shift of the sensor with a blank silver top surface was calculated as 1.11 nm (average 0.77±0.34 nm), while this shift was calculated as 2.32 nm (average 2.47±1.49 nm) when gold nanoparticles were integrated on the surface (the Figure 6b), and 0.43 nm (0.42±0.20 nm) when the nano-island was created by the seeding method (the Figure 6c). When these data were normalized (0-1), gold nanoparticle integration increased the signal by a factor of 2.6 compared to the blank surface, while nano-island formation decreased the signal (the Figure 6e).
Within these basic concepts; it is possible to develop various embodiments of the inventive “Method of Obtaining a Biosensor with Biomaterial Integrated Metasurface (100)”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.

Claims

CLAIMS A method (100) of detecting biomarkers included in body fluid by means of metasurfaces obtained by integrating biomaterials into inert optical discs; characterized in that it comprises the steps of
- obtaining a meta surfacing by coating inert optical discs with metal (101),
- functionalizing the surface by integrating biomaterial into the prepared meta surface (102),
- creating nano-island on the surface by providing the seeding solution into the functionalized meta surface (103),
- administrating antibody of the exosome membrane protein to the meta surface provided with seeding solution (104), and
- detecting disease by capturing the exosome of the antibody with a meta surface containing the antibody placed into a body fluid (105). A method (100) according to Claim 1; characterized in that in the step of obtaining a meta surfacing by coating inert optical discs with metal (101); the plastic protection layer located on the inert optical disc (DVD: Digital Versatile Disc) is removed by means of a knife and a chemical etching is applied to the exposed nano-periodic structure. A method (100) according to Claim 2; characterized in that in the step of obtaining a meta surfacing by coating inert optical discs with metal (101); the optical disc having a nano-periodic structure subjected to chemical etching is used as a plastic mould and the plastic mould is coated with titanium or chromium (as adhesive layer), silver and gold respectively by means of electron-beam evaporation method in order to obtain a meta surface (biosensor/metamaterial plasmonic sensor) having a gold top surface. A method (100) according to Claim 2; characterized in that in the step of obtaining a meta surfacing by coating inert optical discs with metal (101); the optical disc with nano-periodic structure exposed to chemical etching process is coated with silver by means of sputtering method. A method (100) according to any of the preceding claims; characterized in that in the step of functionalizing the surface by integrating biomaterial into the prepared meta surface (102); the meta surfaces with gold and silver top surface are cut into the desired dimensions and cleaned with ethanol and then, these surfaces are functionalized by incubating them in PLL solution with biomaterial at concentrations ranging from 0.05 mg/mL to 1 mg/mL for 8-12 hours at 2-8 °C. A method (100) according to any of the preceding claims; characterized in that in the step of creating nano-island on the surface by providing the seeding solution into the functionalized meta surface (103); the seeding solution (chloroauric acid (HAuCU) and hydroxylamine hydrochloride (HONH2.HCI)) is applied to the functionalised meta surface directly with the PLL solution. A method (100) according to Claim 6; characterized in that in the step of creating nano-island on the surface by providing the seeding solution into the functionalized meta surface (103); in case where 1 : 1 (v:v) 10-100 pM HAuCl4: 10-100 pM HONH2.HCI is applied on 0.05-1 mg/mL PLL concentration on the surface, nano-island formation occurs on the surface of the meta surface. A method (100) according to any of the preceding claims; characterized in that in the step of administrating antibody of the exosome membrane protein to the meta surface provided with seeding solution (104); 50-1000 pg/mL Protein G, which is a direct binding protein, is applied to the meta surface created in the nano by applying the PLL solution. A method (100) according to any of the preceding claims; characterized in that in the step of administrating antibody of the exosome membrane protein to the meta surface provided with seeding solution (104); Protein G is attached to the meta surface by protein-protein interaction and then, 50- 500 pg/mL anti-CD81 antibody is applied to the surface for the detection of CD81, one of the proteins included in the membrane of exosomes, and incubated at 2-8 °C for 1-5 hours. A method (100) according to Claim 8; characterized in that in the step of administrating antibody of the exosome membrane protein to the meta surface provided with seeding solution (104); anti-CD63 antibody, which is a protein included in exosome membrane, is used instead of anti-CD81 antibody. A method (100) according to any of the preceding claims; characterized in that in the step of detecting disease by capturing the exosome of the antibody with a meta surface containing the antibody placed into a body fluid (105); exosome is isolated from the medium of kidney cells by using microfluidics and the concentration, diameter and size distribution of these exosomes are analysed by means of a laser-based optical technique that monitors the Brownian motion of individual particles included in the solution. A method (100) according to Claim 11; characterized in that in the step of detecting disease by capturing the exosome of the antibody with a meta surface containing the antibody placed into a body fluid (105); the samples with determined concentration are diluted with PBS so as to be 105-109 particles/mL. A method (100) according to Claim 12; characterized by in that the wavelength shift is calculated by providing the obtained exosomes to gold nanoparticle integrated surfaces and in the light of the wavelength shift information calculated, the presence of exosomes and exosome-related diseases are detected.
PCT/TR2023/050943 2022-09-12 2023-09-12 A method of obtaining a biosensor with a biomaterial integrated metasurface WO2024058756A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TR2022/014113 2022-09-12
TR2022/014113A TR2022014113A2 (en) 2022-09-12 2022-09-12 METHOD OF OBTAINING A BIOSENSOR WITH A BIOMATERIAL INTEGRATED METASURFACE

Publications (1)

Publication Number Publication Date
WO2024058756A1 true WO2024058756A1 (en) 2024-03-21

Family

ID=85162120

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/TR2023/050943 WO2024058756A1 (en) 2022-09-12 2023-09-12 A method of obtaining a biosensor with a biomaterial integrated metasurface

Country Status (2)

Country Link
TR (1) TR2022014113A2 (en)
WO (1) WO2024058756A1 (en)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ESMA DERIN: "Integrated Metasurfaces for Biomarker Detection ", MASTER OF SCIENCE THESIS BIOMATERIAL, 1 August 2022 (2022-08-01), XP093151117, [retrieved on 20240412] *
WANG ZHENBIAO, CHEN JUNJIE, KHAN SAYED ALI, LI FAJUN, SHEN JIAQING, DUAN QILIN, LIU XUEYING, ZHU JINFENG: "Plasmonic Metasurfaces for Medical Diagnosis Applications: A Review", SENSORS, MDPI, CH, vol. 22, no. 1, CH , pages 133, XP093151119, ISSN: 1424-8220, DOI: 10.3390/s22010133 *
YANG QINSI; CHENG LIMING; HU LIANG; LOU DOUDOU; ZHANG TING; LI JIAOYUAN; ZHU QINGFU; LIU FEI: "An integrative microfluidic device for isolation and ultrasensitive detection of lung cancer-specific exosomes from patient urine", BIOSENSORS AND BIOELECTRONICS, ELSEVIER SCIENCE LTD, UK, AMSTERDAM , NL, vol. 163, 18 May 2020 (2020-05-18), Amsterdam , NL , XP086196010, ISSN: 0956-5663, DOI: 10.1016/j.bios.2020.112290 *

Also Published As

Publication number Publication date
TR2022014113A2 (en) 2022-10-21

Similar Documents

Publication Publication Date Title
US11519907B2 (en) Label-free detection of renal cancer
Inan et al. Photonic crystals: emerging biosensors and their promise for point-of-care applications
Hong et al. Nanobiosensors based on localized surface plasmon resonance for biomarker detection
Yang et al. Halloysite nanotube-modified plasmonic interface for highly sensitive refractive index sensing
TW201538959A (en) Digital LSPR for enhanced assay sensitivity
JP6248053B2 (en) Electronic nose or tongue sensor
KR20100002960A (en) Surface plasmon resonance sensor chip, method for manufacturing the same, surface plasmon resonance sensor system, and method for detecting analyzed material with surface plasmon resonance sensor system
US11280784B2 (en) Patterned plasmonic nanoparticle arrays for multiplexed, microfluidic biosensing assays
US11911999B2 (en) Plasmonic patch as a universal fluorescence enhancer
CN110286104A (en) Local surface plasma resonance nano-sensor and its protein quantitation methods
Lee et al. Rapid and sensitive detection of lung cancer biomarker using nanoporous biosensor based on localized surface plasmon resonance coupled with interferometry
JP5178049B2 (en) Target substance detection element, target substance detection apparatus, and target substance detection method
US20200173922A1 (en) Detection of biomarkers using plasmonic gratings
WO2024058756A1 (en) A method of obtaining a biosensor with a biomaterial integrated metasurface
JP5835335B2 (en) Method for quantitative measurement of specific analytes using surface plasmon resonance and surface plasmon excitation enhanced fluorescence spectroscopy
Predabon et al. Detection of tumor necrosis factor-alpha cytokine from the blood serum of a rat infected with Pb18 by a gold nanohole array-based plasmonic biosensor
CN115746827A (en) Membranous multilayer quantum dot fluorescent material, preparation method and immunochromatography application thereof
JP5205293B2 (en) Antibody-immobilized substrate, and method and use of the antibody-immobilized substrate
KR102428467B1 (en) Diagnosis method using plasmon phenomenon, diagnostic kit and manufacturing method of diagnostic kit
JP2011169609A (en) Assay method using plasmon excitation sensor
Kemper Non-specific protein-surface interactions in the context of particle based biosensors
Garifullina et al. High-throughput fabrication of high aspect ratio Ag/Al nanopillars for optical detection of biomarkers
Wood et al. Extending lipoarabinomannan detection limitations with plasmonic gratings
TW200427976A (en) Scattering surface plasma and its detecting slide with nano-particls
Lesuffleur et al. Plasmonic nanohole arrays for label-free kinetic biosensing in a lipid membrane environment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23865987

Country of ref document: EP

Kind code of ref document: A1