Technical Field

The present invention relates to an optical sensor and kits containing the said sensor, that allows early diagnosis of Alzheimer’s, Parkinson’s, and dementia diseases in a short time in a simple, and selective way with high accuracy while the patient is still alive, and before taking medication.

Prior Art

Neurodegenerative diseases develop depending on various cellular, molecular, and genetic factors, or by being triggered by a currently existing neurological disorder. With increasing age, the human brain shrinks (atrophy), however, it becomes more pronounced with the serious neuronal loss associated with neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and other types of dementia, and the effects are more intense.

Of these diseases, in the developing pathophysiological processes of Alzheimer’s disease, initially, the neurons lose their functions, and they fail to communicate with each other, and subsequently, the neurons lose their viability in the long term. In the initial stage of the disease, particularly the hippocampus area, which functions as memory in the brain, is affected, and typical symptoms of Alzheimer’s disease are seen in time together with the neuronal loss in other areas of the cerebral cortex. Distinctive pathological formations in the diagnosis of Alzheimer’s disease are the extracellular accumulation of amyloid-beta peptides in the form of plaques, and the formation of neurofibrillary tangles by the microtubule-binding Tau protein. Amyloid-beta peptide (Ab) is a member of amyloid-beta precursor proteins, that are neuron surface receptors and provide neuronal development together with other cells. Although the physiological role of amyloid-beta peptides is not fully understood, it has been seen that the absence of these peptides does not lead to any physiological impairment. Apart from causing the formation of senile plaques between neurons in Alzheimer’s disease, amyloid-beta peptides also cause cerebral amyloid angiopathy by accumulating in blood vessels in the brain. Its role in Alzheimer’s pathology is forming beta-amyloids through cleaving by the beta-secretase enzyme of the amyloid precursor protein, which is a physiologically functioning cell surface receptor, and transformation of the resulting beta-amyloid peptides into oligomer forms that are toxic to neurons by misfolding. The resulting toxic oligomers accumulate between neurons and prevent synaptic transmission in time. Amyloid-beta peptides derived in the brain can occur in numerous forms at the same time, and not every amyloid-beta peptide changes into a plaque. However, the amyloid-beta plaques play a big part in the development of Alzheimer’s disease, together with the neurofibrillary tangles formed by the Tau proteins.

Another neurodegenerative disease, Parkinson’s disease, is a common neurodegenerative disease pathologically characterized by abnormal accumulation of a-synuclein, called Lewy bodies and Lewy neurites in the cytoplasm of certain neurons in several brain areas. Lewy pathology initially occurs in cholinergic and monoaminergic brain stem neurons, and neurons in the olfactory systems, but also appears in limbic and neocortical brain areas, which is a disease progression. A particular pattern of a-synuclein pathology concentrated essentially in limbic areas of the brain is seen in patients with Alzheimer’s pathology. Protein clusters consisting substantially of a- synuclein exist in all patients with Parkinson’s disease, and this strongly supports the opinion that a-synuclein is a key player in Parkinson’s disease. The normal neuronal function of the 140- amino acid a-synuclein protein is not fully understood. However, it occurs in the cytosol, possibly in the mitochondrion and nucleus as well, and most likely plays a part in synaptic vesicle dynamics, mitochondrial functions, and intracellular trafficking, and is a potential regulator, a-synuclein acquires neurotoxic properties during a pathological process in which soluble a-synuclein monomers initially form oligomers, then gradually aggregate and form small protofibrils and eventually large and insoluble a-synuclein fibrils (i.e. aggregates forming Lewy pathology). The relative over-production of the protein, the presence of mutations that increase the likelihood of misfolding and oligomerization, natural degradation and degradations in the molecular pathways, and increased age-related decline in proteolytic defense mechanisms in the aging brain may play a critical part in a-synuclein accumulation. Thus, the disease development and progression can be characterized by accumulated a-synuclein. Motor defects are the main clinical characteristics of the disease, however, non-motor symptoms are also common. Of these, dementia is the most evident one.

There are not any distinctive diagnostic tests that can give clear information for the diagnosis of neurodeg enerative diseases such as Alzheimer’s, Parkinson’s, and dementia, as mentioned in detail above in the state of the art. Thereby, various medical diagnostic tests are evaluated together in diagnosing these diseases. Various screenings that measure neurological functions, balance, sense, behavior, memory, and reflexes are performed on the patients. Practices such as blood tests, ultrasonography, computed tomography (CT), magnetic resonance imaging (MR), etc. to support the diagnosis and eliminate the possibility of the presence of similar diseases, and personality screening tests to examine depression can also be performed. Performing gene screening may be necessary to study these diseases since Alzheimer’s disease may show similar symptoms to some genetic diseases.

In case findings that raise suspicions about the said neurodegenerative diseases are reached as a result of all these tests, a disease can be diagnosed after carrying out the tests for the evaluation of cognitive functions. Despite all these pre-diagnostic tests and extreme problems, except the post-mortem detection of senile plaques (beta-amyloid) and neurofibrillary tangles, there is no definitive diagnosis of Alzheimer’s disease. The disease cannot be completely suppressed and/or treated since the definitive diagnosis of Alzheimer’s disease can only be made post-mortem, and the diagnosis process takes a very long time. Similarly, there is not any particular test to identify Parkinson’s disease; the diagnosis of Parkinson’s is made when other disease possibilities with similar symptoms are eliminated or when the patient responds to Parkinson’s medication.

In light of the known state of the art, as described in detail above, it is seen that there is a need for a non-invasive method and/or a biosensor and/or a kit that allows early diagnosis of Alzheimer’s, Parkinson’s, and dementia diseases in a short time in a simple, and selective way with high accuracy while the patient is still alive, and before taking medication. Figures Describing the Invention

Figure 1: Kit according to the invention

1: Eppendorf tube

2: CdTe quantum dot

3: Polymer layer having specific cavities to the histidine-tagged MDVFMI<GLSI<AI<EG (SEQ ID No: 1) peptide sequence (a-synuclein protein). The histidine-tagged MDVFMKGLSKAKEG (SEQ ID No: 1) peptide sequence also means the 1-14 peptide sequence of the a-synuclein protein.

4: Graphene quantum dot

5: Silica layer

6: Polymer layer having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites

7: Buffer solution

Figure 2: Scheme showing the preparation and working principle of graphene quantum dots having specific cavities to beta-amyloid plaques with thiol aptamer recognition sites

201 : Citric acid

202: Graphene quantum dots (GQDs)

203: Vinyl-modified graphene quantum dots 204: Beta-amyloid oligomer

204: Thiol aptamer

206: Beta-amyloid plaques with thiol aptamer recognition sites

207: Fluorescence quenching

208: High fluorescence emission

2A: 185 °C, Pyrolysis

2B: sol-gel method

2C: Self-assembly

2D: Polymerization

2E: Separation of template molecule

2F: Rebinding

Figure 3: Scheme showing the preparation and working principle of CdTe quantum dots having specific cavities to the a-synuclein protein

Detailed Description of the Invention

The present invention relates to an optical sensor suitable for use in the early diagnosis of neurodeg enerative diseases, such as diseases such as Alzheimer’s, Parkinson’s, dementia, etc. that allows specifically detecting the beta-amyloid plaques with thiol aptamer recognition sites and/or a-synuclein protein. Optical sensors according to the invention are preferably graphene quantum dot- and/or CdTe quantum dot-based optical sensors.

Since the formation of beta-amyloid plaques, and fibrillary tangles caused by the aggregation of beta-sheets in the protein structure increases with age, and similarly a-synuclein protein accumulates in certain areas of the brain, the development of neurodegenerative diseases such as Alzheimer’, Parkinson’s, etc. is linearly dependent on the aggregation of these proteins. Consequently, recognizing beta-amyloid plaques and fibrillary tangles as biomarkers of Alzheimer’s disease, and a-synuclein protein as biomarkers of Parkinson’s disease has led to promising actions for the early diagnosis and treatment of the most severe neurodegenerative diseases of the time.

Accordingly, the invention relates to a biosensor and a diagnostic kit containing the said biosensor, that allows co-detection of Alzheimer’s and Parkinson’s disease.

The biosensor according to the invention is characterized in that it comprises a) Graphene quantum dots having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites, and b) CdTe quantum dots having specific cavities to the a-synuclein protein.

Graphene quantum dots having specific cavities to beta-amyloid plaques in the biosensor according to the invention consist of a graphene quantum dot core in the center, and around this, a polymer layer having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites.

CdTe quantum dots having specific cavities to the a-synuclein protein in the biosensor according to the invention consist of a CdTe quantum dot core in the center, and around this, a polymer layer having specific cavities to the a-synuclein protein.

The expression “the specific cavity to beta-amyloid plaques containing thiol aptamer recognition sites” used in the context of the present invention means the cavities obtained by applying a “molecularly imprinting” method to a polymer layer.

The expression “a-synuclein protein recognition sites” used in the context of the present invention means the cavities obtained by applying a “molecularly imprinting” method to a polymer layer.

In the graphene quantum dots having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites according to the invention, the polymer layer that is around the graphene quantum dot core and has specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites consists of methacrylic acid (MAAc) monomer and N’,N’- methylenebisacrylamide (MBAAm) crosslinker. In other words, in the graphene quantum dots having specific cavities to beta-amyloid plaques with thiol aptamer recognition sites, the polymer layer that is around the graphene quantum dot core and has specific cavities to betaamyloid plaques containing thiol aptamer recognition sites is the polymethacrylic acid p(MAAc) polymer crosslinked with the N’,N’ -methylenebisacrylamide (MBAAm).

In CdTe quantum dots having specific cavities to the a-synuclein protein according to the invention, the polymer layer around the CdTe quantum dot core, that has specific cavities to the a-synuclein protein consists of oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) and copper(II) acrylate (Cu(II)Ac) monomers, and ethylene glycol dimethacrylate (EGDMA) crosslinker. In other words, in CdTe quantum dots having specific cavities to the a- synuclein protein, the polymer layer around the CdTe quantum dot core, that has specific cavities to the a-synuclein protein is poly oligoethylene methacrylate p(OEGMA) polymer crosslinked with the ethylene glycol dimethacrylate (EGDMA).

One embodiment of the invention relates to a method for preparing the polymer layer around the graphene quantum dot core, that has specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites in the graphene quantum dots having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites. Accordingly, the method used for preparing the polymer layer located on the said graphene quantum dots, that has specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites (Method I) comprises the following steps: a. Dissolving graphene quantum dot cores with a vinyl functional group, and aptamer and betaamyloid oligomers with a thiol group in a buffer solution, such as phosphate-buffered saline (PBS), b. Initiating the polymerization by adding to the resulting mixture methacrylic acid (MAAc) monomer, and N’,N’ -methylenebisacrylamide (MBAAm) crosslinker, and at least one polymerization initiator, c. Separating the graphene quantum dots coated with polymer following the polymerizing reaction (polymerization) from the reaction medium, d. Obtaining graphene quantum dots having specific cavities to these plaques by washing the polymer-coated graphene quantum dots obtained in the previous step with a solution containing alcohol, and organic acid, and removing the beta-amyloid plaques with thiol aptamer recognition sites in the polymer structure.

In other words, in step a) of method 1, while graphene quantum dot cores with vinyl functional groups are dissolved in buffer solution, aptamer and beta-amyloid oligomers with thiol groups are dissolved in another phosphate buffer solution at the same time, and beta-amyloid plaques with thiol aptamer recognition sites are prepared, and two solutions are mixed.

In another aspect, the invention relates to graphene quantum dots having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites that can be obtained by a process according to Method I.

One embodiment of the invention relates to a method for preparing the polymer layer around the CdTe quantum dot core, that has specific cavities to the a-synuclein protein in the CdTe quantum dots having specific cavities to the a-synuclein protein. Accordingly, the method used for preparing the polymer layer located on the said CdTe quantum dots, that has specific cavities to the a-synuclein protein (Method II) comprises the following steps: a. Mixing CdTe quantum dots in acetonitrile, and adding to the resulting mixture histidine- tagged MDVFMKGLSKAKEG (SEQ ID No: 1), b. Adding to the resulting mixture oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), and copper(II) acrylate (Cu(II)Ac) monomers, and ethylene glycol dimethacrylate (EGDMA) crosslinker, and at least one polymerization initiator, c. Initiating the polymerization by heating the resulting mixture to a temperature between 55- 95°C, preferably to a temperature between 65-90°C, d. Separating the CdTe quantum dots coated with polymer following the polymerization from the polymerization medium, e. Obtaining CdTe quantum dots having specific cavities to the a-synuclein protein by washing the polymer-coated CdTe quantum dots obtained in the previous step with a solution containing alcohol, and organic acid, and removing the histidine-tagged MDVFMKGLSKAKEG (SEQ ID No: 1) epitope in the polymer structure.

In another aspect, the invention relates to CdTe quantum dots having specific cavities to the a- synuclein protein that can be obtained by a process according to Method II.

Graphene quantum dots and CdTe quantum dots within the scope of the invention can be obtained by using methods known in the art, or are available from commercial sources.

Graphene quantum dots according to the invention can be synthesized, for example, by pyrolysis of citric acid.

Graphene quantum dots with the vinyl functional group used in Method 1 according to the invention can be obtained by coating the graphene quantum dots obtained by pyrolysis of citric acid with a silica layer according to the Stober method, and by modification with vinylmethoxysilane.

CdTe quantum dots according to the invention are obtained by the following steps:

• Mixing the CdCb solution with thioglycolic acid (TGA),

• Fixing the pH of the solution to 11 with NaOH,

• Adding to the resulting solution trisodiumcitrate, Na2TeOs, and then NaBFF, and

• Synthesizing TGA-modified CdTe quantum dots by subjecting the reaction solution to the hydrothermal treatment in a hydrothermal reactor.

Any method known in the art, preferably centrifugation, is used to separate the quantum dots from the reaction liquid in step c) of Method I, and step d) of Method II.

Alcohol used in step d) of Method I, and step e) of Method II is selected from the group consisting of ethanol, methanol, isopropanol, with methanol being preferably used.

Organic acid used in step d) of Method I, and step e) of Method II is selected from the group consisting of acetic acid, citric acid, oxalic acid, malic acid, with acetic acid being preferably used.

In a preferred embodiment of the invention, methanol is used as alcohol, and acetic acid as organic acid in step d) of Method I, and step e) of Method II. The methanol and acetic acid used herein (methanol: acetic acid) are used in a ratio between 10:1 and 1: 10, preferably in a ratio between 9: 1 to 1 :9 by volume (v/v).

In one aspect, the present invention relates to a biosensor that allows co-detection of Alzheimer’s and Parkinson’s diseases, and said biosensor comprises: a) Graphene quantum dots having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites that can be obtained by Method I, and b) CdTe quantum dots having specific cavities to the epitope of a- synuclein protein that can be obtained by Method II.

The biosensors according to the invention recognize using their cavities the molecule within a sample. Said recognition process occurs by the attachment of the target molecule within the sample to the cavities on the biosensor according to the invention, and the fluorescence change following this attachment.

Thus, with the biosensor according to the invention, it is possible to detect quickly the presence of both aptamer and beta-amyloid plaques, and a-synuclein protein within a sample. With the use of graphene quantum dots and CdTe quantum dots in the biosensor, it is possible to measure the intensity of fluorescence emitted by said two fluorosensors at different wavelengths independently of each other, which allows achieving results for both biomarkers independently of each other, even though optical measurements are made with both sensors.

The mechanism of co-diagnosis of Alzheimer’s and Parkinson’s diseases by the optical sensor according to the invention is such that the intensities of fluorescence emitted by the sensors at different wavelengths are attenuated as a result of the molecular recognition of the beta-amyloid and a-synuclein proteins accumulated in the blood, by the specific cavities to the biomarkers of these diseases.

In another aspect, the present invention relates to a biosensor for use in the diagnosis of neurodeg enerative diseases such as Alzheimer’s, Parkinson’s, dementia, etc., comprising a) graphene quantum dots having specific cavities to beta-amyloid plaques, and b) CdTe quantum dots having specific cavities to the epitope of a-synuclein protein.

The present invention also relates to a biosensor for use in the diagnosis of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, dementia, etc., comprising graphene quantum dots having specific cavities to the aptamer and beta-amyloid plaques that can be obtained by Method I, and b) CdTe quantum dots having specific cavities to the epitope of a-synuclein protein that can be obtained by Method II.

In another aspect, the present invention relates to a kit suitable for use in the diagnosis of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, dementia, etc., said kit comprising:

- Eppendorf tube

The biosensor according to the invention, comprising graphene quantum dots having specific cavities to beta-amyloid plaques, and b) CdTe quantum dots having specific cavities to the epitope of a-synuclein protein, and

Optionally a buffer solution, such as PBS, or bicarbonate-carbonic acid buffer.

The result obtained from the kit according to the invention is determined by reading with a spectrophotometer, or the fluorescence microscope after contacting it with the patient sample. In a preferred embodiment of the invention, fluorescence spectroscopy is used.

The expression “comprising” within the scope of the invention is also used in the sense of “containing”.

When technically applicable, embodiments of the invention may be combined.

The invention will be described in more detail with reference to examples, which are given below for illustrative purposes only, and should not be construed as limiting the scope of the invention.

EXAMPLES:

Example 1: Contents of the diagnostic kit according to the invention

The diagnostic kit according to the invention, in an Eppendorf tube, comprises:

Graphene quantum dots coated with the polymer layer having specific cavities to beta amyloid plaques containing thiol aptamer recognition sites,

CdTe quantum dots coated with the polymer layer having specific cavities to the epitope of a-synuclein protein.

In addition to this tube, optionally a sealed container with a buffer solution, such as PBS may be contained in the kit.

The diagnostic and detection kit according to the invention also comprises an informative document describing the working principle of the kit and/or a reference solution showing that the test works properly, etc.

Example 2: Preparation of the diagnostic kit

Synthesis of Graphene Quantum Dots and functionalization thereof with vinyl groups:

Graphene quantum dots (GQD) are synthesized by the pyrolysis of citric acid. Graphene quantum dots are coated with the silica layer according to the Stober method and modified with vinyltrimethoxysilane.

Preparation of graphene quantum dots having specific cavities to the aptamer and fa- amyloid oligomers obtained by the molecularly imprinting technique:

Vinyl-functionalized graphene quantum dots (GQD) are dissolved in PBS, thiol-terminated aptamer and beta-amyloid oligomers are dissolved in PBS, and beta-amyloid plaques with aptamer recognition sites are obtained, and after mixing with the solution of graphene quantum dot, to this solution methacrylic acid (MAAc) and N’,N’ -methylenebisacrylamide (MBAAm) are added, and N2 gas at room temperature is passed through the solution. Polymerization is initiated by adding to the solution ammoniumpersulfate (APS), the polymerization initiator, and tetramethylenediamine (TEMED), the catalyst. After centrifuging at a rate of 14000 rpm the polymer-coated GQDs obtained as a result of the polymerization reaction, they are washed with methanol: acetic acid (9:1, v/v) until no beta-amyloid protein containing thiol aptamer recognition sites is observed in the structure (monitored by HPLC). As a result of this process, molecularly imprinted quantum dots having specific cavities to beta-amyloid plaques containing thiol aptamer recognition sites are obtained.

Molecularly imprinted graphene quantum dots with both fluorescence characteristics and dual molecular recognition capability are an optical sensor that can molecularly recognize and separate the beta-amyloid protein plaques from a total of 1 mL of human blood sample in less than 20 minutes with a recovery rate of 94% between 107%. The graphene quantum dots prepared are a sensor and also a method providing a fast, reliable, and easy detection in the early diagnosis of Alzheimer’s disease.

Synthesis of CdTe quantum dots:

The pH of the solution is fixed to 11 with NaOH by mixing the CdCh solution with the thioglycolic acid (TGA) solution. Next, to this solution trisodiumcitrate and Na2TeO, are added, and then by adding NaBH-i the new solution obtained is transferred to the hydrothermal reactor and subjected to hydrothermal treatment for 3 hours, and thus, water-soluble TGA-modified CdTe quantum dots are synthesized.

Synthesis of epitope-imprinted CdTe quantum dots:

CdTe quantum dots are dissolved in acetonitrile. To this, histidine-tagged MDVFMKGLSKAKEG (Seq ID No: 1) is added, and N2 gas at room temperature is passed through the solution, after adding oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) and copper (II) acrylate (Cu(II)Ac) monomers, ethylene glycol dimethacrylate (EGDMA) crosslinker, AIBN initiator. The temperature of the solution is fixed to 90°C, and after centrifuging at a rate of 14000 rpm the polymer-coated CdTe quantum dots obtained after a total of 30 minutes of polymerization, they are washed with methanol: acetic acid (9: 1, v/v) until no epitope is observed in the structure (monitored by HPLC). As a result of this process, molecularly imprinted CdTe quantum dots having specific cavities to a-synuclein are obtained.

Particularly CdTe quantum dots with the capability of molecularly recognizing the protein by the specific cavities to epitopes of a-synuclein protein is an optical sensor that can molecularly recognize and separate a-synuclein protein from a total of 1 mL of human blood sample in less than 20 minutes with a recovery rate of 93% between 103%. The molecularly imprinted CdTe quantum dots prepared are a fluorosensor and also a diagnostic method providing a fast, reliable, specific, and easy detection in the early diagnosis of Parkinson’s disease.