WO2023080340A1 - Novel microrna biomarker for selective diagnosis of mild cognitive impairment and alzheimer dementia - Google Patents

Abstract

The present invention relates to a novel microRNA biomarker for the selective diagnosis of mild cognitive impairment and Alzheimer dementia. The novel miRNA of SEQ ID NO: 1 or SEQ ID NO: 3, secured by molecular basis analysis, exhibits different expression levels in plasmas from a patient group with mild cognitive impairment and Alzheimer dementia and has the effect of reducing the expression of a protein related to mild cognitive impairment and Alzheimer dementia. Therefore, the present invention has the effect of enabling the selective diagnosis as well as early diagnosis of mild cognitive impairment or Alzheimer dementia by utilizing as a molecular diagnosis indicator the novel miRNA, which is present in relatively low-invasive plasma.

Description

Novel microRNA biomarkers for screening diagnosis of mild cognitive impairment and 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 disease is diagnosed through neuropsychological testing and imaging based on the cognitive impairment and amyloid hypothesis, due to many limitations, recent efforts have been made to develop a diagnostic index for Alzheimer's disease from blood, a relatively non-invasive sample. This is actively being done.

miRNA is a single-chain small RNA with a sequence of about 20 bases. It is a short sequence that binds to the 3'-UTR (3'-untranslated regions) of a specific target mRNA and regulates proteins through translational inhibition of mRNA. is the RNA of About 1,000 miRNAs have been identified in humans, but most miRNAs have unknown functions.

Currently, dementia analysis using blood mainly relies on proteins, but there are disadvantages in that the variation between patients is severe compared to structural stability and the pathology of Alzheimer's dementia cannot be fully reflected in blood. In order to derive a more sensitive diagnostic index, a target with a smaller size is required. In particular, in the case of Alzheimer's disease, abnormal protein aggregation is not caused by genetic factors alone, but by various environmental stimuli. It is presumed to be important. When miRNA is used as a diagnostic indicator, it is a single-stranded RNA of 21-25 nucleotides present in all biological fluids including blood, urine, and saliva, and plays a role in directly controlling gene expression by inhibiting mRNA translation. In addition, it is resistant to degradation by RNase and stably exists in the blood. Defects in metabolism or function of miRNA are closely related to the onset of dementia, and it is known to have high specificity and sensitivity depending on the disease, so it can be used as the most optimal diagnostic index. there is.

In the diagnosis of Alzheimer's disease, it is very important to take an accurate medical history through the report of the guardian who knows the patient best. The doctor checks whether there is a change in cognitive function, including memory, compared to the previous one, when and how it appeared, and biochemical tests such as physical examination, neurological examination, mental status examination, daily living function level examination, blood examination, etc. However, there are still various limitations in diagnosing Alzheimer's disease, making early diagnosis difficult. Mainly, Alzheimer's disease is diagnosed through neuropsychological tests and imaging diagnosis based on the cognitive impairment and amyloid hypothesis, and recently, efforts to develop indicators for diagnosing Alzheimer's dementia from blood, a non-invasive sample, have been actively pursued. It is being done.

On the other hand, as technology related to the diagnosis of cognitive dysfunction, Korean Patent No. 2033776 discloses a composition for diagnosing the severity of Alzheimer's disease, including an agent for measuring the expression level of tau protein, and a method for diagnosing the severity of Alzheimer's disease using the same. No. 1992539 discloses a composition and method for diagnosing a miRNA-based cognitive disorder disease, but the novel microRNA biomarker for screening diagnosis of mild cognitive impairment and Alzheimer's dementia has not yet been disclosed.

The present invention was 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 provides a 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 miRNA has a difference in expression level in the plasma of patients with mild cognitive impairment and patients with Alzheimer's dementia and can reduce the expression of proteins related to Alzheimer's disease.

In order to achieve the above object, the present invention provides a biomarker composition for screening and diagnosing mild cognitive impairment and Alzheimer's dementia, comprising novel microRNA consisting of 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 diagnosing 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 screening diagnosis of mild cognitive impairment and Alzheimer's dementia. It has the effect of reducing the expression of sexual dementia-related proteins. Therefore, the present invention utilizes novel miRNAs present in plasma with relatively low invasiveness as a diagnostic index, thereby enabling early diagnosis of mild cognitive impairment and Alzheimer's disease as well as discrimination between mild cognitive impairment and Alzheimer's dementia.

Figure 1 shows the stem loop structure and sequence information of the novel miRNA4 identified in the present invention.

Figure 2 shows the stem loop structure and sequence information of the novel miRNA5 identified in the present invention.

Figure 3 shows mimic and repressor synthesized sequences of miRNA4 (A) and miRNA5 (B) to confirm the function of the novel miRNA of the present invention.

Figure 4 is a Western blot result confirming the change in the expression level of MAP1A (microtubule associated protein 1A), which is encoded by the target gene of the novel miRNA4 of the present invention. **, *** means that there is a statistically significant difference in the expression level of the target gene between the control group that was not treated with anything and the group that was treated with the miRNA mimic or miRNA inhibitor of the present invention, ** is p<0.01, and *** is p<0.001.

Figure 5 is a Western blot result confirming the change in the expression level of SHANK1 (SH3 and multiple ankyrin repeat domains protein 1), which is encoded by the target gene of the novel miRNA5 of the present invention. **, *** indicates that there is a statistically significant difference in the target gene expression level between the control group treated with nothing and the group treated with the miRNA mimic or miRNA inhibitor of the present invention, ** indicates p<0.01, and *** is p<0.001.

Figure 6 is the result of confirming the changes in the expression levels of MAP1A and SHANK1, respectively, encoded by the target genes of the novel miRNA4 and miRNA5 of the present invention in the plasma of the mild cognitive impairment group (MCI) and the Alzheimer's dementia group (AD). *, *** indicates that there is a statistically significant difference in the target protein expression level between the mild cognitive impairment group (MCI) and the Alzheimer's dementia group (AD), * is p <0.05, and *** is p <0.001 .

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

In the present invention, microRNA (miRNA) refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting their translation. Not only miRNAs represented by specific nucleotide sequences, but also precursors (pre-miRNA, pri-miRNA) of the miRNAs, miRNAs with biological functions equivalent to these, for example, 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 microRNA of SEQ ID NO: 1 reduces the expression level of the MAP1A (microtubule associated protein 1A) gene, and the novel microRNA of SEQ ID NO: 3 reduces the expression level of the SHANK1 (SH3 and multiple ankyrin repeat domains protein 1) gene characterized by that

'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, to a specific disease or disorder Concepts include determining the prognosis of an affected subject or therametrics (eg, monitoring the condition of a subject to provide information about treatment efficacy).

The 'selective diagnosis' of the present invention refers to not only early diagnosis of mild cognitive impairment and Alzheimer's disease, but also discrimination between mild cognitive impairment and Alzheimer's dementia.

In addition, the present invention relates to 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.

The agent may include an antisense oligonucleotide complementary to the microRNA; primer; probe; and a loop-forming oligonucleotide linked to a fluorophore or quencher; 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 caused by normal aging. In other words, it refers to a state in which cognitive ability is lowered compared to those of the same age group, and it is different from dementia 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 disease is often accompanied by mental behavior symptoms such as personality change, agitation, depression, delusions, hallucinations, increased aggression, and sleep disturbance, as well as cognitive decline. It is a disease that causes physical complications such as infection, pressure sore, etc., and progresses very slowly in the beginning, so it may be difficult to recognize the onset.

The biosensor composition used in the present invention is a term used in the sense that it can sense (sens) the selection of mild cognitive impairment and Alzheimer's dementia, and is used in the same or similar meaning as 'biomarker composition'.

In addition, the present invention includes, as an active ingredient, a biomarker composition for screening and diagnosing mild cognitive impairment and Alzheimer's dementia, which includes, as an active ingredient, an agent capable of detecting novel microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 It relates to a kit for screening and diagnosing 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 all of total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA. In addition to the above nucleic acids, the kit of the present invention may include known nucleic acids or nucleic acids that can be found in the future that enable screening and diagnosis of mild cognitive impairment and Alzheimer's dementia, Antibodies for measuring select diagnostic markers may also be included. The nucleic acids included in the kit may be individually or arbitrarily combined and packaged in different containers.

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, for example, a solid phase. Examples of the material of the solid phase include plastic, paper, glass, silicon, and the like, and a material of the solid phase that is preferable from ease of processing may be plastic. The shape of the solid phase is arbitrary, and may be, for example, square, circular, rectangular, or film-like.

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

The cartridge in the multi-point diagnosis device may be composed of a thermostat, a stirrer, a disposable tip, a sample injection container, etc., and an all-in-one device with a built-in micro-sized whole blood separator, fluorescence detector, and liquid handler. An algorithm from which the exponent' is derived and a fully automatic detection system may be included.

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

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

(3) checking whether the miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed in the plasma isolated in step (2);

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

1. Obtaining samples from patients with Alzheimer's disease

After collecting blood from patients classified as MCI and AD into EDTA tubes, plasma was separated and stored at -70 °C until use at the Neurobiology Laboratory of Gyeongsang National University.

2. Total miRNA extraction

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

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

3. Next Generation Sequencing (NGS)

A small RNA-seq library was created 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. Samples prepared with the NovaSeq 6000 sequencing platform were sequenced according to the SMARTer smRNA-Seq Kit manual.

4. Screening miRNA data through raw data analysis

Contaminated or low-quantity data in plasma was removed in the order of adapter, quality, and length trimming from the raw data obtained by NGS analysis. miRNA Deep2* sequencing was performed with clean data from which unnecessary information was removed through this trimming process.

5. miRNA Deep2* sequencing

miRNA Deep2* is a software that can identify new miRNAs from refined 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 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 score for the conserved seed sequence.

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

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

To verify the function of novel miRNAs and how they are associated with Alzheimer's disease, screening was performed using miRDB software that scores target genes (miRNAs) based on miRNA sequences. Among the target genes with high gene scores, it was verified through expression analysis of genes related to Alzheimer's disease.

SH-SY5Y cells used as a neural function and differentiation model were used, and a miRNA mimic showing the overexpression effect of the selected new miRNA, a miRNA inhibitor suppressing the expression of the new miRNA, 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 plasma collected from patients and normal groups was extracted using the QIAGEN miRNeasy Serum/Plasma Kit, which can purify miRNA and other small RNAs from a small amount of plasma. Subsequently, 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 determine whether a sufficient amount of RNA was secured for library production for NGS.

Example 2. Next Generation Sequencing (NGS)

(1) Building a library and deriving raw data

In generating a small RNA-seq library for sequencing on the Illumina Platform for NGS, the SMARTer smRNA-Seq Kit, which can produce a high-quality miRNA library even with a small amount of RNA, was used. Libraries for total RNA sequencing of patients and normal groups obtained from plasma were prepared, and raw data were derived by sequencing using the prepared libraries and the NovaSeq 6000 sequencing platform.

(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 amounts of data.

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

(3) miRNA Deep2* sequencing

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

The RNA fold generation graphic contains 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 previously known miRNAs and new miRNAs by mapping using an RNA database.

The reads finally processed 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 was then used for miRDeep2 analysis using the genome sequence. Known/novel miRNAs predicted by miRDeep2 and other small RNAs matching RNAcentral were aligned using Bowtie (target small RNA, <50nt) and Bowtie2 (target smRNA, ≥50nt).

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

- Bowtie: software commonly used for sequence alignment and sequence analysis

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

To predict novel miRNAs, unique clustered reads were separately aligned to 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 minimum free energy secondary structure of an RNA sequence using a nearest-neighbor thermodynamic model. The graphics generated by RNAfold include real in silico-folded hairpins, read counts for each part of the hairpin, the minimum free energy score, and the randfold score, as well as the score of the conserved seed sequence.

miRDeep2* derived novel miRNAs through a score of -10 to 10. The novel miRNAs derived above were expressed in both the mild cognitive impairment group and the Alzheimer's disease group. Among them, miRNA 4 species having a miRDeep2* score of 1 or more, an RNA fold of "yes", and a mature read number of 10 or more were selected.

The stem loop structures of the selected novel miRNA4 and miRNA5 are shown in Figs. 1 and 2, and sequence information is shown in Fig. 3.

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

In order to verify the association of the new miRNA with Alzheimer's disease, miRDB software was used to screen the target gene by score based on the sequence of the miRNA. Among the target genes having 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, and after transfection of miRNA mimics that show the overexpression effect of selected new miRNAs, miRNA inhibitors that inhibit the expression of new miRNAs, and negative control miRNAs into SH-SY5Y , The protein expression of the target gene was confirmed by Western blot.

The miRNA mimic is actually a miRNA that can function like a novel miRNA in vivo, and is designed with a double bond. The miRNA inhibitor is a complementary sequence of the derived miRNA, and is a miRNA that can inhibit the miRNA produced by single binding. On the other hand, the negative control miRNA is a miRNA composed of miRNAs that do not regulate genes and serves as a standard for verification. mimics of the novel miRNA4 and miRNA5; and miRNA inhibitors are disclosed in FIG. 3 .

[Gene screening using miRDB software]

Among the genes controlled by novel miRNAs, in order to select targets highly related to Alzheimer's disease, miRNA4 mimic among novel miRNAs is based on sequence 5'-GGGGGUGUGGGGUAAAAAAU-3' (SEQ ID NO: 1), miRNA5 mimic is sequence 5' Based on -GGGGGAGAGAAGGGAAAAG-3' (SEQ ID NO: 3), genes were screened by score using 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 neurology and Alzheimer's dementia were selected.

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

Human neuroblastoma SH-SY5Y was transfected with miRNA4 mimic for overexpression of novel miRNA4 and miRNA4 inhibitor for suppression of function for 24 hours at each concentration. Thereafter, proteins were separated and subjected to Western blotting.

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 increased through the removal of miRNA4 binding to 3'-UTR with an inhibitor miRNA (FIG. 4).

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

Mimic miRNA5 for overexpression of novel miRNA5 and inhibitor miRNA5 for suppression of function were transfected into human neuroblastoma SH-SY5Y in a concentration dependent manner (1nM, 10nM, 100nM) for 48 hours, and proteins were separated and Western blotting was performed. did As miRNA5 binding to 3'-UTR increased due to overexpression of novel miRNA5, it was confirmed that the expression of SHANK1, a candidate target gene protein, was significantly reduced. By removing miRNA5 binding to 3'-UTR with inhibitor miRNA5, It was confirmed that the expression of the candidate target gene protein SHANK1 increased (FIG. 5).

Meanwhile, target sequencing was performed to analyze the expression levels of new miRNA4 and miRNA5 in the plasma of patients classified as MCI and AD to confirm whether there was a difference in the expression level of each group in the blood of the new miRNA. Significant differences in the expression levels of miRNA4 and miRNA5 were confirmed between the mild cognitive impairment (MCI) group and the Alzheimer's dementia (AD) group.

Claims (11)

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

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