WO2021033789A1 - An aptamer-functionalized contact lens and an early diagnosis method of disease using the same - Google Patents

An aptamer-functionalized contact lens and an early diagnosis method of disease using the same Download PDF

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WO2021033789A1
WO2021033789A1 PCT/KR2019/010489 KR2019010489W WO2021033789A1 WO 2021033789 A1 WO2021033789 A1 WO 2021033789A1 KR 2019010489 W KR2019010489 W KR 2019010489W WO 2021033789 A1 WO2021033789 A1 WO 2021033789A1
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contact lens
aptamer
biomarker
amyloid
modified
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PCT/KR2019/010489
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French (fr)
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In Sik SON
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Nexmos Co., Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14507—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue specially adapted for measuring characteristics of body fluids other than blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4076—Diagnosing or monitoring particular conditions of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6801—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be attached to or worn on the body surface
    • A61B5/6813—Specially adapted to be attached to a specific body part
    • A61B5/6814—Head
    • A61B5/6821—Eye
    • 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
    • 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
    • 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
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G02—OPTICS
    • G02C—SPECTACLES; SUNGLASSES OR GOGGLES INSOFAR AS THEY HAVE THE SAME FEATURES AS SPECTACLES; CONTACT LENSES
    • G02C7/00—Optical parts
    • G02C7/02—Lenses; Lens systems ; Methods of designing lenses
    • G02C7/04—Contact lenses for the eyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/103—Detecting, measuring or recording devices for testing the shape, pattern, colour, size or movement of the body or parts thereof, for diagnostic purposes
    • A61B5/1032—Determining colour for diagnostic purposes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4076—Diagnosing or monitoring particular conditions of the nervous system
    • A61B5/4082—Diagnosing or monitoring movement diseases, e.g. Parkinson, Huntington or Tourette
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4076—Diagnosing or monitoring particular conditions of the nervous system
    • A61B5/4088—Diagnosing of monitoring cognitive diseases, e.g. Alzheimer, prion diseases or dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein

Definitions

  • the present invention relates to an aptamer-functionalized contact lens and an early diagnosis method of disease using the same.
  • Aptamers are ssDNA, recognized as alternatives to antibodies as they can bind to target molecules with high affinity and specificity. Advantageous over antibodies, aptamers can detect small molecules such as disease-related metabolites and even targets as small as heavy metal ions (Jo et al., Oligonucleotides. 2011 Apr; 21(2): 85-91).
  • AD Alzheimer's disease
  • omics has also profiled AD-specific proteomic signatures in tear fluid which include lipocalin-1, demcidin, lysozyme-C, and lacritin (Kallo et al., 2016, PLoS ONE 11(6): e0158000.).
  • Amyloid plaques were observed in the retina and lens of AD patients, implying amyloid- ⁇ aggregates are potentially released into tear fluid.
  • the correlation between amyloid- ⁇ accumulation of the brain and that of the retina was reported in an animal model (Nadav et al. Acta Neuropathol (2016) 132:767-787 ).
  • Typical non-invasive ways of collecting tear are using the Schirmer's strips and microcapillaries.
  • these techniques have limitations that may prevent accurate interpretation (Rentka et al. Ann. Clin. Biochem. 54:521-529, 2017).
  • contact lenses are considered as suitable diagnostic devices to measure biomarkers in tears.
  • the present invention has been made in view of needs to resolve the above problems, and an object of the present invention is to provide a contact lens as suitable diagnostic devices to measure biomarkers in tears.
  • Another object of the present invention is to provide a new diagnostic method for neurodegenerative diseases.
  • the present invention provides a contact lens for detecting a molecule in tear fluid, wherein said contact lens surface functionalization with aptamer targeting biomarker.
  • aptamer refers to an oligonucleic acid or peptide molecule that binds to a specific target molecule.
  • the oligonucleic acid may be single or double stranded and may have a fold or other conformation where part of the aptamer associates with another part of the aptamer.
  • Biomarkers that may be targeted by an aptamer associated with a contact lens may include, but are not limited to, those associated with Neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease) such as alpha-synuclein, Amyloid Beta, Tau protein, Insulin Degrading Enzyme and amyloid precursor protein.
  • Neurodegenerative diseases e.g. Parkinson's disease, Alzheimer's disease
  • alpha-synuclein e.g. Parkinson's disease, Alzheimer's disease
  • Amyloid Beta e.g., Alzheimer's disease
  • Tau protein e.g. Alzheimer's disease
  • Insulin Degrading Enzyme e.g., Alzheimer's disease
  • aptamers known in the art for various biomarkers include, but are not limited to, those described in the following: 1. Rahimi, J Vis Exp, 2010 13(39):1955 (amyloid beta) 2. Wang X, Yang Y, Jia M, et al. The novel amyloid-beta peptide aptamer inhibits intracellular amyloid-beta peptide toxicity. Neural Regeneration Research. 2013; 8(1):39-48. doi:10.3969/j.issn.1673-5374.2013.01.005. 3. Takahashi et al., Mol Biosyst. 2009 September; 5(9):986-91 (amyloid beta) 4.
  • the present invention provides a method of detecting biomarker in tear fluid, the method comprising applying to an eye of a subject a functionalized contact lens the present invention for a period of time for biomarker in the tear fluid to bind with aptamer, wherein biomarker binds to the aptamer to produce a sample-loaded device; and subjecting said sample-loaded device to a detection process.
  • said detection process comprises subjecting said sample-loaded device with chemical reagents that produce a colorimetric detectable signal.
  • Said colorimetric detectable signal includes, but is not limited to, fluorescence, luminescence, chemiluminescence, or a color formation, or changes in any of the foregoing, including changes in intensity, duration, anisotropy or polarization, or color change.
  • a method of determining a disease state involves applying to an eye of a subject a functionalized contact lens that includes an aptamer associated therewith for a period of time for an amyloid- ⁇ indicative of the disease state to bind with said aptamer to produce a sample-loaded device; subjecting said sample-loaded device to a detection process; and determining the disease state based on presence or amount of said amyloid- ⁇ .
  • the disease state may pertain to, but is not limited to, a neurological condition.
  • the present invention provides a method for preparing contact lens surface functionalization with aptamer targeting amyloid- ⁇ monomer comprising a) The 5'-end of an aptamer for amyloid-- ⁇ monomer is modified by photocleavable Amino C6 (AmC6); b) the surface of the Poly(methyl methacrylate) (PMMA) of contact lens is modified with a carboxyl group(-COOH); and c) said modified surface of contact lens is reacted with 5'-modified end of said aptamer.
  • AmC6 photocleavable Amino C6
  • PMMA Poly(methyl methacrylate)
  • said b) is comprising that the contact lens is soaked into the NaOH solution in methanol, but is not limited thereto.
  • the aptamer-functionalized contact lens enables the detection of multiple biomarkers simultaneously during the wearing time, which overcomes the limitation of the one-time collection of tears, eventually enhancing the sensitivity of diagnosis.
  • Fig.1 Structure of aptamer tagged with Amino C6. Aptamers for amyloid- ⁇ monomer is attached with Amino C6 at the 5' end.
  • Fig. 4 Surface of PMMA contact lens, after modification.
  • the surface of the untreated control contact lens is (a), and NaOH treated contact lens is (b).
  • the material of this lens is Poly(methyl methacrylate) (PMMA) as a major component.
  • PMMA Poly(methyl methacrylate)
  • the carbonyl group in PMMA is used for aptamer attachment.
  • the 5'-end of the aptamer for amyloid-- ⁇ monomer is modified by photocleavable Amino C6 (AmC6) to make it bind to the carboxyl-modified surface of a contact lens (Fig.1)
  • the surface of the PMMA contact lens is modified with a carboxyl group(-COOH).
  • PMMA contact lens was placed in a methanol solution of sodium hydroxide (5%) for 10 min to hydrolyze the surface ester groups to form -COOH groups (Varma et al. Biomaterials 24 (2003) 297-303). Then they were washed extensively with distilled water to remove the alkali and then dried in a vacuum oven.
  • Second, to convert carboxylic acid groups on the sample surfaces to activated esters samples were incubated in an equimolar mixture of EDC/NHS (15 mM) in 0.1 M MES at room temperature for 90 minutes. After two washing cycles in 0.1 M MES buffer, the samples were then incubated overnight at room temperature in 1.5 ⁇ mol/L of aptamers in PBS. (Fig. 2)
  • 6-Carboxyfluorescein (6-FAM) to the 3'-end of the aptamer.
  • the aptamer functionalized contact lenses were compared with native contact lens via fluorescent spectroscopy.
  • 2.94 ⁇ 1.12 nM of aptamers was attached on the surface of PMMA contact lens using H 2 SO 4 method
  • 3.86 ⁇ 0.86 nM of aptamers was attached on the surface of PMMA contact lens using NaOH method (Fig.3).
  • the surface of the native contact lens and the aptamer functionalized lens were observed with microscopy (Fig. 4), it shows that the surface of the contact lens was not much damaged after the modification.

Abstract

Disclosed herein is to develop a contact lens-based wearable device for early diagnosis of diseases by surface functionalization with a disease specific single strand DNA aptamer. The aptamer-functionalized contact lens enables the detection of multiple biomarkers simultaneously during the wearing time, which overcomes the limitation of the one-time collection of tears, eventually enhancing the sensitivity of diagnosis.

Description

AN APTAMER-FUNCTIONALIZED CONTACT LENS AND AN EARLY DIAGNOSIS METHOD OF DISEASE USING THE SAME
The present invention relates to an aptamer-functionalized contact lens and an early diagnosis method of disease using the same.
Aptamers are ssDNA, recognized as alternatives to antibodies as they can bind to target molecules with high affinity and specificity. Advantageous over antibodies, aptamers can detect small molecules such as disease-related metabolites and even targets as small as heavy metal ions (Jo et al., Oligonucleotides. 2011 Apr; 21(2): 85-91).
Recent metabolomic analyses on tear fluid have identified various sets of Alzheimer's disease (AD) biomarkers such as lysophosphocholine (Li et al., Clinical Biochemistry 43 (2010) 992-997), desmosterol (Sato et al., Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring 1 (2015) 67-74), and ornithine (Liang et al., RSC Adv., 2015, 5, 96074). The development of omics has also profiled AD-specific proteomic signatures in tear fluid which include lipocalin-1, demcidin, lysozyme-C, and lacritin (Kallo et al., 2016, PLoS ONE 11(6): e0158000.). Amyloid plaques were observed in the retina and lens of AD patients, implying amyloid-β aggregates are potentially released into tear fluid. Interestingly, the correlation between amyloid-β accumulation of the brain and that of the retina was reported in an animal model (Nadav et al. Acta Neuropathol (2016) 132:767-787 ).
Recently, it has been reported that the secretion of proteins into basal tears of Parkinson's disease (PD) patients can be altered by changes in nerve function. The total α-synuclein (α-Syn Total), oligomeric α-synuclein (α-Syn Oligo), CCL-2 and DJ-1/PARK-7 level of PD patient was compared with healthy control. It was found that a significant difference in PD patient α-Syn Oligo and α-Syn Total levels relative to healthy controls, and these values may be able to distinguish PD patient tears from those of healthy controls (Sarah F Hamm-Alvarez et al. 2019 Biomarkers in Medicine, doi.org/10.2217/bmm-2019-0167). Aforementioned findings strongly suggest that tear fluid can serve as biomarker pools for disease diagnosis.
However, the lack of standardized sampling methods and limited sampling volume set roadblocks on the further utilization of this invaluable biofluids for diagnostic purpose.
Typical non-invasive ways of collecting tear are using the Schirmer's strips and microcapillaries. However, these techniques have limitations that may prevent accurate interpretation (Rentka et al. Ann. Clin. Biochem. 54:521-529, 2017). To overcome this limitation, contact lenses are considered as suitable diagnostic devices to measure biomarkers in tears.
The present invention has been made in view of needs to resolve the above problems, and an object of the present invention is to provide a contact lens as suitable diagnostic devices to measure biomarkers in tears.
Another object of the present invention is to provide a new diagnostic method for neurodegenerative diseases.
In order to accomplish the above object, the present invention provides a contact lens for detecting a molecule in tear fluid, wherein said contact lens surface functionalization with aptamer targeting biomarker.
The term "aptamer" as used herein pertains to an oligonucleic acid or peptide molecule that binds to a specific target molecule. When the aptamer includes an oligonucleic acid the oligonucleic acid may be single or double stranded and may have a fold or other conformation where part of the aptamer associates with another part of the aptamer.
Biomarkers that may be targeted by an aptamer associated with a contact lens may include, but are not limited to, those associated with Neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease) such as alpha-synuclein, Amyloid Beta, Tau protein, Insulin Degrading Enzyme and amyloid precursor protein.
Examples of specific aptamers known in the art for various biomarkers include, but are not limited to, those described in the following: 1. Rahimi, J Vis Exp, 2010 13(39):1955 (amyloid beta) 2. Wang X, Yang Y, Jia M, et al. The novel amyloid-beta peptide aptamer inhibits intracellular amyloid-beta peptide toxicity. Neural Regeneration Research. 2013; 8(1):39-48. doi:10.3969/j.issn.1673-5374.2013.01.005. 3. Takahashi et al., Mol Biosyst. 2009 September; 5(9):986-91 (amyloid beta) 4. Tsukakoshi et al, Biotechnol Lett. 2010 May; 32(5):643-8 (alpha synuclein) 5. Krylova et al., Febs Letters, 2005, Volume 579, Issue 6, Pages 1371-1375 (tau) 6. DE 102010038842 A1 (tau) 7. Farrar C T, William C M, Hudry E, Hashimoto T, Hyman B T (2014) RNA Aptamer Probes as Optical Imaging Agents for the Detection of Amyloid Plaques. PLoS ONE 9(2): e89901 8. Lee et al., Scientific Reports (Impact Factor: 5.58). 06/2015; 5:10757 (tau) 9. Sarell et al., J Biol Chem. 2014 Sep. 26; 289(39):26859-71 (Amyloid precursor protein) 10. US Pat Pub 20130022538 alpha-1-antitrypsin 11. EP 2316935 A1 (IL-7)
The present invention provides a method of detecting biomarker in tear fluid, the method comprising applying to an eye of a subject a functionalized contact lens the present invention for a period of time for biomarker in the tear fluid to bind with aptamer, wherein biomarker binds to the aptamer to produce a sample-loaded device; and subjecting said sample-loaded device to a detection process.
In one embodiment of the present invention, said detection process comprises subjecting said sample-loaded device with chemical reagents that produce a colorimetric detectable signal.
According to another embodiment, Said colorimetric detectable signal includes, but is not limited to, fluorescence, luminescence, chemiluminescence, or a color formation, or changes in any of the foregoing, including changes in intensity, duration, anisotropy or polarization, or color change.
In a further embodiment, disclosed is a method of determining a disease state that involves applying to an eye of a subject a functionalized contact lens that includes an aptamer associated therewith for a period of time for an amyloid-β indicative of the disease state to bind with said aptamer to produce a sample-loaded device; subjecting said sample-loaded device to a detection process; and determining the disease state based on presence or amount of said amyloid-β. The disease state may pertain to, but is not limited to, a neurological condition.
The present invention provides a method for preparing contact lens surface functionalization with aptamer targeting amyloid-β monomer comprising a) The 5'-end of an aptamer for amyloid--β monomer is modified by photocleavable Amino C6 (AmC6); b) the surface of the Poly(methyl methacrylate) (PMMA) of contact lens is modified with a carboxyl group(-COOH); and c) said modified surface of contact lens is reacted with 5'-modified end of said aptamer.
In one embodiment of the present invention, said b) is comprising that the contact lens is soaked into the NaOH solution in methanol, but is not limited thereto.
As can be seen through the present invention, the aptamer-functionalized contact lens enables the detection of multiple biomarkers simultaneously during the wearing time, which overcomes the limitation of the one-time collection of tears, eventually enhancing the sensitivity of diagnosis.
Fig.1. Structure of aptamer tagged with Amino C6. Aptamers for amyloid-β monomer is attached with Amino C6 at the 5' end.
Fig.2. The reaction of PMMA with 5-amino-terminated single strand aptamer.
Fig.3. The reaction of PMMA with 5-amino-terminated single strand aptamer.
Fig. 4 Surface of PMMA contact lens, after modification. The surface of the untreated control contact lens is (a), and NaOH treated contact lens is (b).
Hereinafter, in order to assist your understanding of the present invention, the preferred example is presented. But in order to easily understand the following example is the invention, it is provided. The content of the invention is not restricted by the example.
Examples
Example 1.
1) Contact Lens Surface functionalization with aptamer targeting amyloid-β monomer
A. Contact lens: 　Morning-Q RS (Interojo Inc.)
The material of this lens is Poly(methyl methacrylate) (PMMA) as a major component. The carbonyl group in PMMA is used for aptamer attachment.
B. Aptamer:
The 5'-end of the aptamer for amyloid--βmonomer is modified by photocleavable Amino C6 (AmC6) to make it bind to the carboxyl-modified surface of a contact lens (Fig.1)
C. Covalent attachment of AmC6-aptamer to contact lens surface.
First, the surface of the PMMA contact lens is modified with a carboxyl group(-COOH). PMMA contact lens was placed in a methanol solution of sodium hydroxide (5%) for 10 min to hydrolyze the surface ester groups to form -COOH groups (Varma et al. Biomaterials 24 (2003) 297-303). Then they were washed extensively with distilled water to remove the alkali and then dried in a vacuum oven. Second, to convert carboxylic acid groups on the sample surfaces to activated esters, samples were incubated in an equimolar mixture of EDC/NHS (15 mM) in 0.1 M MES at room temperature for 90 minutes. After two washing cycles in 0.1 M MES buffer, the samples were then incubated overnight at room temperature in 1.5 μmol/L of aptamers in PBS. (Fig. 2)
2) Results
To optimize the aptamer crosslinking technique to the surface of the contact lens, two different methods were tested. One is that the contact lens is soaked into concentrated sulfuric acid(H 2SO 4) solution for 20 min. And the other method is that the contact lens is soaked into the 5% NaOH solution in methanol at room temperature for 10 min. Covalent attachment of single-stranded aptamer to contact lens surface provides great control over the orientation of aptamer, enabling its biomarker binding domain (toward 3' end) free to engage with target molecules. To achieve covalent crosslinking of aptamer with contact lens surface, EDC/NHS chemistry was used (procedure and result are discussed in detail in the preliminary result section). In order to confirm modified PMMA surfaces in a relevant in vitro system, we attached 6-Carboxyfluorescein (6-FAM) to the 3'-end of the aptamer. The aptamer functionalized contact lenses were compared with native contact lens via fluorescent spectroscopy. As a result, 2.94±1.12 nM of aptamers was attached on the surface of PMMA contact lens using H 2SO 4 method, while 3.86±0.86 nM of aptamers was attached on the surface of PMMA contact lens using NaOH method (Fig.3). The surface of the native contact lens and the aptamer functionalized lens were observed with microscopy (Fig. 4), it shows that the surface of the contact lens was not much damaged after the modification.

Claims (8)

  1. A contact lens for detecting a biomarker molecule in tear fluid, wherein said contact lens surface functionalization with aptamer targeting biomarker.
  2. The contact lens of claim 1, wherein said biomarker associated with Neurodegenerative diseases.
  3. The contact lens of claim 1, wherein said biomarker is selected from consisting of alpha-synuclein, Amyloid Beta, Tau protein, Insulin Degrading Enzyme and amyloid precursor protein.
  4. A method of detecting biomarker in tear fluid, the method comprising applying to an eye of a subject a functionalized contact lens according claim 1 for a period of time for biomarker in the tear fluid to bind with aptamer, wherein said biomarker binds to the aptamer to produce a sample-loaded device; and subjecting said sample-loaded device to a detection process.
  5. The method of claim 4, wherein said biomarker is selected from group consisting of alpha-synuclein, Amyloid Beta, Tau protein, Insulin Degrading Enzyme and amyloid precursor protein.
  6. The method of claim 4, wherein said detection process comprises subjecting said sample-loaded device with chemical reagents that produce a colorimetric detectable signal.
  7. A method for preparing contact lens surface functionalization with aptamer targeting amyloid-β monomer comprising a) The 5'-end of an aptamer for amyloid--β monomer is modified by photocleavable Amino C6 (AmC6);
    b) the surface of the Poly(methyl methacrylate) (PMMA) of contact lens is modified with a carboxyl group(-COOH);
    c) said modified surface of contact lens is reacted with 5'-modified end of said aptamer.
  8. The method of claim 7, wherein said b) is comprising that the contact lens is soaked into the NaOH solution in methanol.
PCT/KR2019/010489 2019-08-19 2019-08-19 An aptamer-functionalized contact lens and an early diagnosis method of disease using the same WO2021033789A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113576541A (en) * 2021-08-04 2021-11-02 张希武 Capture method of vesicles in tears and contact lens capture chip

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