KR102333797B1 - Novel biomarker microRNA for distinguishing and diagnosing mild cognitive impairment and early Alzheimer's disease - Google Patents

Abstract

The present invention relates to a novel microRNA biomarker for selective diagnosis of mild cognitive impairment and Alzheimer's dementia. The novel miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 obtained by molecular-based analysis has a difference in expression level in the plasma of patients with mild cognitive impairment and Alzheimer's dementia, and there is an effect of reducing the expression level of mild cognitive impairment and Alzheimer's dementia-related proteins. Therefore, the present invention has an effect of enabling selective diagnosis as well as early diagnosis of mild cognitive impairment and Alzheimer's dementia by utilizing novel miRNA present in plasma, which is relatively low in invasiveness, as a molecular diagnostic index.

Description

Novel biomarker microRNA for distinguishing and diagnosing mild cognitive impairment and early Alzheimer's disease

The present invention relates to a novel microRNA biomarker for the screening diagnosis of mild cognitive impairment and Alzheimer's dementia.

Although Alzheimer's dementia is diagnosed through neuropsychological tests and imaging based on cognitive impairment and amyloid hypothesis, recent efforts to develop diagnostic indicators for Alzheimer's dementia from blood, a relatively non-invasive sample, are due to many limitations. This is being actively done.

miRNA is a single-stranded small RNA with about 20 base sequences, a short sequence that can regulate protein through translation inhibition of mRNA by binding to 3'-UTR (3'-untranslated regions) of a specific target mRNA is the RNA of About 1,000 miRNAs have been identified so far in humans, but the functions of most miRNAs remain unknown.

Currently, the analysis of dementia using blood mainly relies on protein, but there is a significant difference between patients compared to structural stability, and there are disadvantages in that the pathology of Alzheimer's dementia cannot be fully reflected in the blood. In order to derive a more sensitive diagnostic index, a smaller size target is required. In particular, in the case of Alzheimer's dementia, since the aggregation of abnormal proteins is induced by various environmental stimuli, rather than by genetic factors, it is more difficult to find a fundamental target by analyzing changes in gene expression regulation prior to protein translation rather than protein results. is presumed to be important. When miRNA is used as a diagnostic index, it is a single-stranded RNA of 21-25 nucleotides present in all living fluids, including blood, urine and saliva, and plays a role in directly controlling gene expression by inhibiting the translation of mRNA. In addition, it is resistant to degradation by RNase, so it is stably present in the blood, and defects in miRNA metabolism or function are closely related to the onset of dementia. have.

For the diagnosis of Alzheimer's disease, it is very important to take an accurate history through the report of a caregiver who knows the patient best. The doctor will check if there is any change in cognitive function, including memory, compared to before, and if so, when and how it appeared, and biochemical tests such as physical examination, neurological examination, mental state examination, daily living function level test, blood test, etc. , brain imaging tests, neuropsychological tests, etc. However, there are still various limitations in diagnosing Alzheimer's disease, making early diagnosis difficult. Mainly, Alzheimer's dementia is diagnosed through neuropsychological examination and imaging based on the cognitive impairment and amyloid hypothesis. is being done

On the other hand, as a diagnosis-related technology for cognitive dysfunction, Korean Patent No. 2033776 discloses a composition for diagnosing Alzheimer's severity including a formulation for measuring the expression level of tau protein, and a method for diagnosing Alzheimer's severity using the same. Although miRNA-based compositions and methods for diagnosing cognitive impairment diseases are disclosed in No. 1992539, novel microRNA biomarkers for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia of the present invention have not yet been disclosed.

The present invention has been derived from the above needs, and the present invention provides a novel micro RNA biomarker for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia, and the novel microRNA biomarker of SEQ ID NO: 1 or SEQ ID NO: 3 according to the present invention The present invention was completed by confirming that there is a difference in the expression level of miRNA in the plasma of the mild cognitive impairment patient group and the Alzheimer's dementia patient group, and can reduce the expression of the Alzheimer's dementia-related protein.

In order to achieve the above object, the present invention provides a biomarker composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia comprising a novel microRNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.

In addition, the present invention provides a biosensor composition for screening and diagnosing mild cognitive impairment and Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

In addition, the present invention provides a kit for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia comprising the biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia as an active ingredient.

The present invention relates to a novel microRNA biomarker for the selective diagnosis of mild cognitive impairment and Alzheimer's dementia, wherein the novel miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 according to the present invention is expressed in the plasma of a group of patients with mild cognitive impairment, Alzheimer's It has the effect of reducing the expression of senile dementia-related proteins. Therefore, the present invention utilizes the novel miRNA present in plasma with relatively low invasiveness as a diagnostic index, thereby enabling early diagnosis of mild cognitive impairment and Alzheimer's dementia as well as distinguishing between mild cognitive impairment and Alzheimer's dementia.

1 shows the stem loop structure and sequence information of the novel miRNA4 identified in the present invention.
Figure 2 shows the stem loop (stem loop) structure and sequence information of the novel miRNA5 identified in the present invention.
Figure 3 shows mimic and inhibitor synthesis sequences of miRNA4 (A) and miRNA5 (B) for confirming the novel miRNA function of the present invention.
4 is a Western blot result confirming the change in the expression level of MAP1A (microtubule associated protein 1A) encoded by the novel miRNA4 target gene of the present invention. **, *** indicates that there is a statistically significant difference in target gene expression level between the control group untreated and the group treated with miRNA mimic or miRNA inhibitor of the present invention, ** is p<0.01, *** is p<0.001.
5 is a Western blot result confirming the change in the expression level of SHANK1 (SH3 and multiple ankyrin repeat domains protein 1) encoded by the novel miRNA5 target gene of the present invention. **, *** indicates that there is a statistically significant difference in the target gene expression level between the control group untreated and the group treated with miRNA mimic or miRNA inhibitor of the present invention, ** is p<0.01, *** is p<0.001.
6 is a result of confirming the change in the expression levels of MAP1A and SHANK1, respectively encoded by the novel miRNA4 and miRNA5 target genes of the present invention, in the plasma of the mild cognitive impairment group (MCI) and Alzheimer's dementia group (AD). *, *** indicates that there is a statistically significant difference in the target protein expression level of the mild cognitive impairment group (MCI) and Alzheimer's dementia group (AD), * is p<0.05, *** is p<0.001 .

The present invention relates to a biomarker composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia comprising a novel microRNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.

In the present invention, micro RNA (miRNA) refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting translation thereof. MiRNAs represented by specific nucleotide sequences as well as precursors of the miRNAs (pre-miRNA, pri-miRNA), miRNAs having biological functions equivalent to them, such as homologues (i.e., homologs or orthologs), variants such as polymorphisms, and derivatives.

The novel microRNA is preferably derived from human plasma, but is not limited thereto.

The novel micro RNA of SEQ ID NO: 1 reduces the expression level of the MAP1A (microtubule associated protein 1A) gene, and the novel micro RNA of SEQ ID NO: 3 reduces the expression level of the SHANK1 (SH3 and multiple ankyrin repeat domains protein 1) gene. is characterized by

'Diagnosis' of the present invention is to determine the susceptibility of an object, that is, a test subject to a specific disease or disorder, to determine whether an object currently has a specific disease or disorder, Concepts involving determining the prognosis of an affected subject or therametrics (eg, monitoring a subject's condition to provide information on treatment efficacy).

The 'selective diagnosis' of the present invention refers to early diagnosis of mild cognitive impairment and Alzheimer's dementia, as well as distinguishing between mild cognitive impairment and Alzheimer's dementia.

In addition, the present invention relates to a biosensor composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting a microRNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

The agent comprises an antisense oligonucleotide complementary to the microRNA; primer; probe; and a loop-forming oligonucleotide to which a phosphor or quencher is linked; It may include any one selected from, but is not limited thereto.

Mild cognitive impairment refers to an intermediate stage between cognitive decline and dementia due to normal aging. In other words, it refers to a state in which cognitive ability is lowered compared to the same age group, and it is different from dementia in that daily life is possible. This mild cognitive impairment is an intermediate stage between normal and dementia, and is known as a high-risk group for dementia.

Alzheimer's dementia is often accompanied by psycho-behavioral symptoms such as personality changes, agitation, depression, delusions, hallucinations, increased aggression, and sleep disturbance as well as cognitive decline. , infection, and even physical complications such as bedsores, it progresses very slowly in the beginning and it may be difficult to recognize the onset.

The biosensor composition used in the present invention is a term used in the sense of being able to sense (sensing) the selection of mild cognitive impairment and Alzheimer's dementia, and is used in an equivalent or similar meaning to a 'biomarker composition'.

In addition, the present invention includes, as an active ingredient, a biomarker composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia comprising an agent capable of detecting a novel microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient It relates to a kit for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia.

The kit is preferably for RT-PCR, real-time PCR, isothermal PCR, northern blotting, RNA protection assay or microarray chip, but is not limited thereto.

The kit of the present invention may additionally include a nucleic acid capable of specifically binding to the microRNA of SEQ ID NO: 1 or SEQ ID NO: 3, which means that the nucleic acid includes both RNA, DNA, or RNA/DNA (chimera), The DNA may include all of cDNA, genomic DNA, and synthetic DNA.

In addition, the RNA may include total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA. In addition to the nucleic acid, the kit of the present invention may contain a known nucleic acid or a nucleic acid that can be discovered in the future that enables the selective diagnosis of mild cognitive impairment and Alzheimer's dementia, and may contain known mild cognitive impairment and Alzheimer's dementia. Antibodies for measuring selectable diagnostic markers may also be included. The nucleic acids included in the kit may be packaged in different containers individually or in any combination.

The kit of the present invention may include a fluorescent material for labeling, an enzyme and medium for nucleic acid amplification, instructions for use, and the like. The kit of the present invention is a device for screening and diagnosing mild cognitive impairment and Alzheimer's dementia in which the nucleic acid is bound or attached to a solid phase, for example. Examples of the solid material include plastic, paper, glass, silicone, and the like, and a preferred solid material from the viewpoint of ease of processing may be plastic. The shape of the solid phase is arbitrary, and may be, for example, a square, a circle, a rectangle, a film shape, and the like.

The kit of the present invention may be included in a device for high-throughput-screening (HTS)-based multiplexed POCT that simultaneously processes a large amount of miRNA samples.

The cartridge in the multi-point diagnosis device can be composed of a thermostat, a stirrer, a disposable tip, a sample injection container, etc., and when human blood is injected with an all-in-one device with an ultra-small whole blood separator, a fluorescence detector, and a liquid handler, it automatically 'risk of dementia' An algorithm from which the index' is derived and a fully automatic detection system may be included.

In addition, the present invention comprises the steps of (1) collecting blood from a subject;

(2) separating plasma from the blood of step (1); and

(3) confirming the expression of miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 in the plasma isolated in step (2); relates to a method for screening and diagnosing mild cognitive impairment and Alzheimer's dementia, including the.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

1. Obtaining samples from Alzheimer's dementia patients

After the blood of patients classified as MCI and AD was collected in an EDTA tube, plasma was separated and stored at -70°C until used in the Neurobiology Laboratory of Gyeongsang National University.

2. Total miRNA extraction

Total RNA in each plasma of the obtained patient group and normal group was extracted using the QIAGEN miRNeasy Serum/Plasma Separation Kit, which can mainly purify miRNA and other small RNAs from a small amount of serum and plasma.

Thereafter, it was confirmed whether a sufficient amount of RNA was secured for library production for NGS analysis using the Bioanalyzer RNA Small RNA chip.

3. Next Generation Sequencing (NGS)

A small RNA-seq library was generated on the Illumina Platform using the SMARTer smRNA-Seq Kit, which can produce a high-quality miRNA library even with a small amount of RNA. Prepared samples were sequenced with the NovaSeq 6000 sequencing platform according to the SMARTer smRNA-Seq Kit manual.

4. Selection of miRNA data through raw data analysis

In the order of trimming of adapter, quality, and length of the raw data obtained by NGS analysis, contaminant or small amount of data present in plasma was removed. Through this trimming process, miRNA Deep2* sequencing was performed with clean data from which unnecessary information was removed.

5. miRNA Deep2* Sequencing

miRNA Deep2* is a software that can identify new miRNAs from purified raw data. According to the RNAfold algorithm, miRBase (Pre-miRNA) libraries cut short in mature, star and loop sequences are arranged side by side to predict new miRNAs. The RNAfold algorithm can be derived in a way that predicts the secondary structure of the RNA sequence with the least free energy using the most similar thermodynamic model. The RNA fold generation graphic includes the number of reads for each part of the hairpin, the score for the minimum free energy, the score for the randfold, and the conserved seed sequence score.

The miRDeep2* system can detect previously known miRNAs and detect new miRNAs by mapping using an RNA database.

6. Verification of novel miRNA related to Alzheimer's disease reflected in blood

For functional verification of how novel miRNAs are related to Alzheimer's dementia, they were screened using miRDB software that scores target genes (miRNAs) based on the sequence of miRNAs. It was verified through expression level analysis of genes related to Alzheimer's dementia among target genes with high gene scores.

SH-SY5Y cells used as models for neural function and differentiation were used, and a miRNA mimic showing the overexpression effect of selected new miRNAs, a miRNA inhibitor that suppresses the expression of new miRNAs, and a negative control miRNA were transfected into SH-SY5Y. After injection, the protein expression of the target gene was confirmed by Western blot.

Example 1. Sample acquisition and total RNA extraction

Total RNA in each plasma collected from the patient group and the normal group was extracted using the QIAGEN miRNeasy Serum/Plasma Kit, which can purify miRNA and other small RNAs from a small amount of plasma. Afterwards, it was confirmed that a sufficient amount of RNA was secured for NGS analysis by quantifying RNA using a Bioanalyzer RNA Small RNA chip to see if sufficient amount of RNA was secured for library production for NGS.

Example 2. Next Generation Sequencing (NGS)

(1) Library construction and raw data derivation

In generating a small RNA-seq library for sequencing on the Illumina Platform for NGS, the SMARTer smRNA-Seq Kit, which can produce high-quality miRNA libraries even with a small amount of RNA, was used. A library was prepared for sequencing the total RNA of the patient group and the normal group obtained from plasma, and sequencing was performed using the prepared library and the NovaSeq 6000 sequencing platform to derive raw data.

(2) Selection of miRNA data from raw data

The raw data obtained from NGS analysis was trimmed in the order of Adapter, Quality, and Length to remove contaminated or small amount of data.

In Total Read Count, which is the raw data obtained by sequencing, the presence of adapter (nonAdapter Read Count), appropriate length (Short Read Count), and low quality (Low Quality Read Count) were checked. miRNA Deep2* sequencing was performed using the primary processing data from which information has been removed.

(3) miRNA Deep2* sequencing

miRNA Deep2* predicts new miRNAs by arranging miRBase (Pre-miRNA) libraries cut short in mature, star, and loop sequences according to the software's RNAfold algorithm that can identify newly known miRNAs from the primary processed data. The RNAfold algorithm was derived by predicting the secondary structure of the RNA sequence with the minimum free energy using the most similar thermodynamic model.

The RNA fold generation graphic includes the number of reads for each part of the hairpin, the score for the minimum free energy, the score for the randfold, and the conserved seed sequence score. The miRDeep2* system detected known miRNAs and new miRNAs by mapping using an RNA database.

The final processed reads for each sample were classified into piRNA, tRNA, snRNA, snoRNA, known miRNA and novel miRNA through miRBase v22.1 and non-coding RNA database (RNAcentral release 14.0).

Genome mapping was processed by Bowtie and STAR using RSEM. Bowtie then used the genome sequence for miRDeep2 analysis. Known/new miRNAs predicted by miRDeep2 and other small RNAs consistent with RNAcentral were aligned using Bowtie (target small RNA, <50 nt) and Bowtie2 (target smRNA, ≥ 50 nt).

- RSEM (RNA-Seq by Expectation-Maximization): A tool to quantify RNA-Seq transcripts

- Bowtie: software commonly used for sequence alignment and sequencing

- STAR (Spliced Transcripts Alignment to a Reference): fast RNA-seq read mapper

To predict novel miRNAs, unique clustered reads were separately aligned against the reference genome and precursor miRNAs. miRNAs can predict mature star and loop sequences according to the RNAfold algorithm using miRNA Deep2*. The RNAfold function predicted the least free energy secondary structure of the RNA sequence using the nearest neighbor thermodynamic model. Graphics generated from RNAfold contain actual in silico-folded hairpins, as well as the number of reads for each part of the hairpin, minimum free energy score, randfold score and score of the conserved seed sequence.

miRDeep2* derived a novel miRNA through a score of -10 to 10. The derived novel miRNAs were expressed in both the mild cognitive impairment group and the Alzheimer's disease group, among which miRNA4 types with a miRDeep2* score of 1 or more, an RNA fold of "yes", and the number of mature reads of 10 or more were selected.

The stem loop structures of the selected novel miRNA4 and miRNA5 are disclosed in FIGS. 1 and 2 , and sequence information is disclosed in FIG. 3 .

Example 3. Confirmation of the regulation of gene expression level related to Alzheimer's dementia by novel miRNA

In order to verify how the novel miRNA is related to Alzheimer's dementia, miRDB software was used that screens target genes by score based on the sequence of the miRNA. Among the target genes with high gene scores, genes related to Alzheimer's dementia were selected.

SH-SY5Y cells used as a model for neural function and differentiation were used. After transfection of a miRNA mimic that shows the overexpression effect of selected new miRNAs, a miRNA inhibitor that suppresses the expression of new miRNAs, and a negative control miRNA into SH-SY5Y , The protein expression of the target gene was confirmed by Western blot.

miRNA mimic is actually a miRNA that can function like a novel miRNA in a living body, and was made with a double bond. A miRNA inhibitor is a complementary sequence of the derived miRNA, and is a miRNA that is made into a single bond and can inhibit the corresponding miRNA. On the other hand, the negative control miRNA is a miRNA composed of miRNAs that do not regulate genes, and serves as a verification standard. mimic of the novel miRNA4 and miRNA5 produced; and miRNA inhibitors were disclosed in FIG. 3 .

[Genetic screening using miRDB software]

Among the genes controlled by the new miRNA, in order to select a target highly related to Alzheimer's dementia, the miRNA4 mimic among the novel miRNAs is based on the sequence 5'-GGGGGUGUGGGGUAAAAAAU-3' (SEQ ID NO: 1), and the miRNA5 mimic is the sequence 5' Based on -GGGGGAGAGAAGGGAAAAG-3' (SEQ ID NO: 3), genes were screened by score using the target prediction miRDB software. The miRNA inhibitor for the miRNA mimic of SEQ ID NO: 1 is 5'-AUUUUUUACCCCACACCCCC-3' (SEQ ID NO: 2), and the miRNA inhibitor for the miRNA mimic of SEQ ID NO: 3 is 5'-CUUUUCCCUUCUUCUCUCCCCC-3' (SEQ ID NO: 4).

After setting the cut-off value of the gene score of the corresponding miRNA to 80, target genes related to neurological and Alzheimer's disease were selected.

As a target gene for miRNA4, a MAP1A (microtubule associated protein 1A) gene was selected. The MAP1A protein is As a protein capable of binding to tau belonging to the microtubule-related protein family in the brain, it is involved in microtubule assembly, an essential step in neurogenesis, and plays an important role in regulating neuronal homeostasis and synaptic plasticity.

Each miRNA was transfected into human neuroblastoma SH-SY5Y with miRNA4 mimic for overexpression of novel miRNA4 and miRNA4 inhibitor for function inhibition for 24 hours at each concentration. Thereafter, the protein was isolated and Western blot was performed.

It was confirmed that the expression of MAP1A, a candidate target gene protein, was significantly decreased according to the increase of miRNA4 binding to 3'-UTR due to overexpression of miRNA4. In addition, it was confirmed that the expression of the target protein MAP1 A was increased through the removal of miRNA4 binding to 3'-UTR with inhibitor miRNA (FIG. 4).

In addition, as a target gene for miRNA5, SHANK1 (SH3 and multiple ankyrin repeat domains protein 1) was selected. The SHANK1 protein forms a complex with GKAP/PSD-95 and Homer, and is an excitatory synaptic adapter protein in the postsynaptic density (PSD) that connects the actin-based cytoskeleton with the receptors of the post-synaptic membrane including NMDA-type and metabolic glutamate receptors. and synaptic junctions to perform structural and functional roles.

Human neuroblastoma SH-SY5Y was transfected with mimic miRNA5 for overexpression of novel miRNA5 and inhibitor miRNA5 for function inhibition in a concentration-dependent manner (1 nM, 10 nM, 100 nM) for 48 hours, and the protein was isolated and Western blot was performed. did. It was confirmed that the expression of the candidate target gene protein, SHANK1, was significantly reduced as miRNA5 binding to 3'-UTR increased due to overexpression of new miRNA5. By removing miRNA5 binding to 3'-UTR with inhibitor miRNA5, It was confirmed that the expression of the candidate target gene protein SHANK1 was increased (FIG. 5).

Meanwhile, target sequencing was performed to analyze the expression levels of new miRNA4 and miRNA5 in plasma of patients classified as MCI and AD to check whether the expression level of the new miRNA differs by group in the actual patient's blood. A significant difference in the expression levels of miRNA4 and miRNA5 was confirmed in the mild cognitive impairment (MCI) group and the Alzheimer's dementia (AD) group.

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Claims (11)

A biomarker composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia comprising a novel microRNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient. The biomarker composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia according to claim 1, wherein the novel microRNA is derived from human plasma. According to claim 1, wherein the novel micro-RNA of SEQ ID NO: 1 reduces the expression level of MAP1A (microtubule associated protein 1A) gene, and the novel micro-RNA of SEQ ID NO: 3 is SHANK1 (SH3 and multiple ankyrin repeat domains protein 1) gene A biomarker composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia, characterized in that it reduces the expression level of A biosensor composition for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 5. The method of claim 4, wherein the agent comprises an antisense oligonucleotide complementary to the microRNA; primer; probe; and a loop-forming oligonucleotide to which a phosphor or quencher is linked; A biosensor composition for screening diagnosis of mild cognitive impairment and Alzheimer's dementia, comprising any one selected from among. A kit for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia comprising the composition of claim 4 as an active ingredient. The method of claim 6, wherein the kit is RT-PCR, real-time PCR, isothermal PCR, Northern blotting, RNA protection assay or microarray chip for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia kit. The kit for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia according to claim 6, wherein the kit is for a high-throughput-screening (HTS)-based multiplexed POCT device. The kit for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia according to claim 8, wherein the multiplexed POCT device comprises a thermostat, a stirrer, a disposable tip, and a sample injection container. [Claim 9] The kit for screening and diagnosing mild cognitive impairment and Alzheimer's dementia according to claim 8, wherein the multiplexed POCT device is an all-in-one device with a built-in ultra-small whole blood separator, a fluorescence detector and a liquid handler. The kit for screening and diagnosis of mild cognitive impairment and Alzheimer's dementia according to claim 8, wherein the multiplexed POCT device comprises a detection system that calculates a dementia risk index from human blood.

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