TWI620934B - Method for identifying parkinson disease dementia from parkinson disease - Google Patents

Abstract

The present invention provides a method for identifying Parkinson's disease or Parkinson's disease dementia with normal cognition in an individual.

Description

Method for identifying Parkinson's disease and Parkinson's disease dementia

The present invention relates to a method of identifying Parkinson's disease and Parkinson's disease dementia.

Parkinson disease (PD) is the second most common neurodegenerative disease outside of Alzheimer's disease. More than 1% of people over the age of 65 have PD. About 10 million people worldwide have PD. The cost of direct and indirect medical care for a PD patient is estimated at $100,000 per year. Many countries, especially the United States, Canada, Europe and Australia, are worried that the cost of medical care will increase and it will be difficult to maintain. A great deal of resources and energy has been invested in the development of diagnostic, therapeutic and vaccines for PD.

The clinical diagnostic criteria for PD are observations of dyskinesias such as bradykinesia, gear-like toughness, resting tremors, and postural instability. Although these clinical features are commonly used, there are several fatal problems in diagnosing PD. For example, other dyskinesias (such as multiple system atrophy, corticobasal degeneration, or progressive supranuclear palsy) may overlap with clinical symptoms of PD. And reduce the accuracy of the diagnosis of PD. In addition, it has been reported that clinical symptoms appear in the basal ganglia (especially the substantia nigra) more than 50% of dopaminergic neurons (dopaminergic After the deterioration of the neurons, the observation of the use of clinical dyskinesia is very difficult for the early diagnosis of PD. Genetic sequence analysis appears to be a better method for early diagnosis of PD. However, only 10% of PD patients are hereditary, and 90% of PD patients are sporadic.

The development of PD as cognitive impairment and dementia is very common and is called Parkinson's Dementia (PDD). Predicting the development of dementia in PD is challenging and has a major impact in this area. Researchers are currently experimenting with biomolecular diagnostics to identify PD and PDD. Alpha-synuclein is the most commonly recognized biomarker in PD or PDD. Since the α-synuclein molecule is phosphorylated, the phospho-α-synuclein molecules are easily aggregated with each other to form Lewy body in dopamine neurons. . Dopamine neurons with Louise body begin to degenerate and lose the ability to exhibit dopamine. The nerve cells in the motor cortex of the brain are damaged by the lack of dopamine, causing movement disorders.

Many findings have shown that the presence of Lewy body in patients with PD or PDD reduces the concentration of alpha-synuclein in cerebrospinal fluid (CSF) compared to healthy subjects. However, the results reported that the changes in the concentration of α-synuclein in the blood were not consistent. The main reason for the inconsistent detection of plasma α-synuclein is the poor detection limit in the detection. According to these reports, α-synuclein currently used for the determination of CSF or plasma is an enzyme-linked immunosorbent assay (ELISA). Alpha-synuclein is expressed and abundantly present in the brain and spinal cord, but is very low in the peripheral blood system. ELISA cannot accurately detect ultra-low concentrations of proteins, such as alpha-synuclein in plasma. Therefore, the detection of α-synuclein by ELISA is used in the biomolecular diagnosis of PD or PDD, and the CSF sample is better than plasma.

CSF is usually collected by lumbar puncture and is high risk and uncomfortable. Early diagnosis by measuring alpha-synuclein in CSF has not been widely accepted by the general public. On the other hand, blood is more easily obtained clinically. Therefore, high sensitivity detection techniques are needed to achieve detection of ultra low amounts of alpha-synuclein in plasma.

Therefore, the inventors developed an immunodetection technique called immunomagnetic depletion (IMR) for quantitative detection of biomolecules at very low concentrations (eg, 1-10 pg/ml or lower). The main cause of the ultra-high sensitivity of IMR is the use of magnetic nanoparticles functionalized by antibodies. These magnetic nanoparticles are uniformly dispersed in the reagent and can capture the target biomolecule anywhere in the sample to be tested. In addition, since the particles are of a nanometer size, the total bonding area is very large. Therefore, the antibody immobilized on the surface of the magnetic nanoparticles can be very efficiently bound to the target biomolecule, making the immunodetection using the IMR extremely sensitive. IMR has been shown to be useful for the determination of very low concentrations of beta-amyloid and tau in human plasma. By detecting plasma beta-amyloid and tau, it was confirmed that there is a clear distinction between healthy individuals and patients with mild cognitive impairment caused by Alzheimer's disease. These results prompted us to investigate the feasibility of detecting very low concentrations of alpha-synuclein in human plasma to achieve biomolecular diagnosis of PD or PDD, or to distinguish between PD and PDD based on plasma alpha-synuclein concentrations. In the present invention, an agent for detecting α-synuclein by IMR is prepared, and the characteristics of the reagent are examined and α-synuclein is detected. For comparison, the detection characteristics of α-synuclein using ELISA were examined. Finally, preliminary results of identifying PD patients, PDD patients, and healthy individuals by detecting plasma alpha-synuclein were reported. Although the cross-sectional studies completed in this study did not address the progression of PDD in PD, the results may indicate the potential use of this measured plasma alpha-synuclein to identify PD and PDD.

The present invention provides a method for identifying Parkinson's disease or Parkinson's disease dementia having normal cognition in an individual, comprising: (a) detecting alpha-synuclein in a blood sample of the individual An in vitro immunomagnetic depletion (IMR) signal, wherein the IMR signal of the α-synuclein is produced by binding α-synuclein to magnetic nanoparticles containing an anti-α-synuclein antibody; (b) The IMR signal of the α-synuclein detected in the step (a) is aligned with a logic function (I) to calculate the concentration of α-synuclein in the blood sample: Where IMR (%) is the IMR signal of the _α- synuclein, φ _α-syn is the concentration of the α-synuclein, the parameter A is the background value, the B is the maximum, φ _o is The IMR signal is equal to the concentration of _α- synuclein at ((A+B)/2), and the slope of the curve φ _o at the data point φ _o with φ _α-syn as the x-axis and IMR (%) as the y-axis. And (c) comparing the concentration of the α-synuclein obtained in step (b) with a predetermined threshold range, wherein the concentration of the α-synuclein above the predetermined threshold range represents Parkinson's Symptoms of dementia, the concentration of the α-synuclein between the predetermined threshold ranges represents Parkinson's disease with normal cognition, and the concentration of the α-synuclein is lower than the predetermined threshold range health.

In a preferred embodiment, the material of the magnetic nanoparticles is selected from the group consisting of Fe ₃ O ₄ , Fe ₂ O ₃ , MnFe ₂ O ₄ , CoFe ₂ O _{4 ,} and NiFe ₂ O ₄ .

In another preferred embodiment, the material of the magnetic nanoparticles is Fe ₃ O ₄ .

In a further preferred embodiment, the blood sample is plasma.

Figure 1 depicts the bioactivity assay of antibodies immobilized on magnetic Fe ₃ O ₄ nanoparticles using immunomagnetic depletion.

Figure 2 depicts the α-synuclein concentration-dependent IMR signal (solid line) and the light absorption density at 450 nm, O.D. 450 nm (dashed line).

Figure 3 illustrates the detection dynamic range α- synuclein Analysis α- synuclein concentration φ _α-syn are _{converted, IMR} PBS solution of α- synuclein concentration increment φ _α-syn Figure.

Figure 4 depicts the use of IMR Detecting for healthy individuals, PD patients, and PDD patients. Plasma alpha-synuclein concentration was measured.

The following examples are not intended to be limiting, but are merely representative of various aspects and features of the invention.

method

The reagent for the detection of α-synuclein is a functionalized monoclonal antibody (sc-12767, Santa Crusz) made of magnetic Fe ₃ O ₄ nanoparticles (MF-DEX-0060, MagQu) against α-synuclein. Biotech). The detailed steps for immobilizing antibodies on magnetic Fe ₃ O ₄ nanoparticles are described in Hong et al. (Horng et al., IEEE Trans Appl Supercond. 2005; 15: 668-671) and Yang et al. (Yang et al., J Magn Magn Mater. 2008; 320: 2688-2691). The antibody-functionalized magnetic Fe ₃ O ₄ nanoparticles were dispersed in phosphate buffered saline (PBS) at pH-7.2, and the particle distribution was analyzed using a dynamic laser scatterometer (Nanotrac-150, Microtrac) using a vibrating sample magnetometer. (Hyster Mag, MagQu) The magnetic concentration of the reagent was measured, and the biological activity of the antibody on the magnetic nanoparticle was examined using an IMR analyzer (XacPro-S, MagQu). The IMR analyzer is an AC magnetosusceptometer equipped with a high T _c superconducting quantum interference device (SQUID) magnetic quantity meter as a magnetic sensor. Details of the AC magnetic permeability meter are described in Yang et al. (Yang et al., IEEE Trans Appl Supercond. 2013; 23: 1600604-1600607) and Xie et al. (Chieh et al., J Appl Phys 2008, 103: 014703- 1-6) Works. Preparation of α-synuclein (ab51189, Abcam) spiked in PBS solution to establish the correlation between IMR signal and α-synuclein concentration. For each IMR signal measurement, 80 -μl reagent is mixed with 40-μl α-synuclein solution, and then the IMR signal is detected by IMR analyzer (XacPro-S, MagQu), and the IMR signal of each concentration of α-synuclein solution is performed. Repeat the measurement. In addition to IMR signal measurement, a commercially available ELISA kit (KHB0061, Novex) was used to find the α-synuclein concentration-dependent light absorption value.

A list of major systemic diseases, surgeries, and hospitalizations for volunteers participating in the present invention was given to volunteers to report uncontrolled medical conditions, including heart failure, recent myocardial infarction (past 6 months), malignancy ( Volunteers (HbA1C>8.5) have been under control for the past 2 years, and volunteers have also undergone physical examinations. The invention has been approved by the review committee of the University Hospital Ethics Committee.

Participants were asked to provide a 10-ml non-fasting venous blood sample (K3 EDTA, purple tube), each sample was assigned a registration number in the order of sampling, so the laboratory personnel on the individual's clinical status and demographic data Without understanding, the blood sample was centrifuged (2500 g, 15 minutes) within 1 hour of collection, plasma was aliquoted into a cryotube, and stored at -80 ° C (less than three months) until thawed by IMR measurement, 80- The μl reagent was mixed with 40-μl plasma to measure the α-synuclein concentration by IMR, and each plasma sample was repeatedly measured.

Use 9 human plasma samples from healthy individuals aged 38 to 73, 9 human plasma samples from PD patients (38-85 years old), and 14 human plasma samples from PDD patients (60-81 years old), using IMR Alpha-synuclein detection was performed. Clinical symptoms are used to identify PD and PDD patients. It should be noted that all patients with PD are cognitively normal. All registered subject informed consent was provided prior to the acceptance of the procedure, and the invention was approved by the Research Ethics Committee of the Taiwan University Hospital.

The average of the hydrodynamic diameters of the antibody-functionalized magnetic Fe ₃ O ₄ nanoparticles was 55.5 nm, while the standard deviation of the hydrodynamic diameter of the particles was 12.7 nm. The average diameter of the antibody-functionalized magnetic Fe ₃ O ₄ nanoparticles was ~40 nm obtained by scanning electron microscopy. The reagent was superparamagnetic and had a saturation magnetization of 0.3 emu/g. According to a previously published article (J Appl Phys 2013, 144903-1-5), the number of antibody-functionalized nanoparticles in the 1-ml reagent at 0.3 emu/g is about 10 ¹³ . In the 1-ml reagent, the antibody functionalized magnetic nanoparticles have a total surface area of about 1000 cm ² . 80-μl reagent was used in the experiment. For each assay, the total surface area of the antibody-functionalized magnetic nanoparticles in the 80-μl reagent was approximately 80 cm ² . The binding area between the antibody of each well and the target biomolecule was 0.45 cm ² compared to the 96-well ELISA plate. Therefore, the binding area of IMR is almost 180 times larger than that of ELISA.

The biological activity of the antibody immobilized on the magnetic nanoparticles was investigated by measuring the IMR signal generated by the binding between the α-synuclein and the antibody on the magnetic nanoparticles. The time-dependent AC susceptibility χ _ac of the reagent after mixing the recording reagent and the solution to be tested is shown in FIG. 1 . Two samples to be tested were prepared: one was a pure PBS solution and the other was a 3.1-fg/ml alpha-synuclein solution. The dotted line 1 in the figure indicates the time-dependent AC susceptibility χ _ac of the mixture of the reagent and the PBS solution. Obviously, the temporality of the dotted line χ _ac remains almost unchanged. However, for the solid line corresponding to the mixture of the reagent and the 3.1-fg/ml α-synuclein solution, the temporal χ _ac decreased at 45 minutes and then reached a lower value. It was observed that the AC susceptibility χ _{ac of the} reagent was significantly lowered due to the binding between α-synuclein and the antibody on the magnetic nanoparticle.

In order to quantify the decrease in the AC susceptibility χ _ac of the reagent, the start/final 之前 before/after the binding between the α-synuclein and the antibody on the magnetic nanoparticle is calculated according to the temporal χ _ac shown in FIG. 1 . _Ac . As described in the previously published paper (ACS Chem Neurosci. 2011; 2: 500-505; J Appl Phys 2010, 107: 074903-1-5), the confidence interval of the AC susceptibility χ _ac of the assay reagent is in Figure 1. The time-dependent AC susceptibility χ _ac is shown in the first and last 40-50 minutes. In the present invention, the data of the AC susceptibility χ _ac of the reagents in the first and last 45 minutes is used to determine the decrease in χ _ac .

As shown in Fig. 1, for the PBS solution, the p- value of the alternating magnetic susceptibility χ _ac between the first and last 45 minute intervals was 0.046. It was observed that the AC magnetic susceptibility χ _ac of the reagent mixed with PBS was slightly reduced. For the 3.1-fg/ml alpha-synuclein solution, the p- value of the alternating magnetic susceptibility χ _ac between the first and last 45 minute intervals was 0.007. A significant reduction in the time-dependent AC susceptibility χ _ac of the reagent after mixing with the α-synuclein solution was demonstrated.

The starting χ _{ac is} expressed as χ _ac,o , which is the average value of χ _ac in the first 45 minutes. The final χ _ac is called χ _{ac, φ} , which is the average of the χ _ac in the last 45 minutes. The reduction of the AC susceptibility χ _ac of the reagent, such as the IMR signal, is obtained by the following formula: IMR(%)=(χ _ac,o -χ _ac,φ )/χ _ac,o ×100% (1)

Through the equation (1), the IMR signals of the dotted line and the solid line in Fig. 1 are calculated to be 1.56 and 2.13%, respectively, and the results in Fig. 1 show the background value of the IMR detection. This background value is mainly caused by the electronic noise of the detection system. The IMR signals for the PBS solution were 1.56 and 1.65% based on repeated measurements. Therefore, the background value of the IMR signal is 1.61% and the standard deviation is 0.06%.

The IMR signal is a function of the concentration of alpha-synuclein, i.e., IMR (%) - φ _α-syn curve, which is shown in Figure 2. As the concentration of _α- synuclein φ _{α-syn increased} from 3 × 10 ^-4 pg/ml (= 0.3 fg / ml), the IMR signal also increased. φ _α-syn dependent IMR (%) follows the logic function represented below Where A, B, φ _o and γ are the alignment parameters. By comparing the data points in Fig. 2 with equation (2), it is found that the alignment parameters are A = 1.94, B = 3.95, φ _o = 49.7, γ = 0.26. The alignment curve is shown in the solid line in Fig. 2, and the corresponding coefficient of determination R ² is 0.998. R ² very close to 1 means that φ _α-syn dependent IMR (%) is indeed controlled by the logic function.

The parameter A in the formula (2) is a value of IMR (%) when φ _α-syn approaches zero. Usually, the value A is slightly higher than the background value. For example, A is 1.94% and the background value here is 1.61%. The difference between A and the background value is mainly due to the noise caused by the dynamic balance of the binding between the protein molecule and the antibody functional magnetic nanoparticles. However, A is not a low-detection limit. Generally, the low detection limit is defined as three times the standard deviation of the IMR signal when the IMR signal is higher than the A and the low concentration test, that is, the 3-σ criterion. In this experiment, the standard deviation of the low concentration Celsius was 0.028%. Therefore, the low detection limit is the concentration with a 2.02% IMR signal. The low detection limit for detecting α-synuclein was 0.3 fg/ml via the formula (2).

The α-synuclein concentration-dependent light absorption density at 450 nm by ELISA, OD 450 nm, is shown in the cross symbol in Figure 2. Experimental data conforms to logic functions

The alignment parameters A ' , B ' , φ _o ' and γ ' in the formula (3) are 0.189, 5.070, 13566.08 and 1.44, respectively. The logic function of equation (3) is shown in the dashed line in FIG. The coefficient of determination R ² between the cross symbol and the broken line is 0.999. Using the 3-σ criterion, the low detection limit for detecting α-synuclein using ELISA was 79.04 pg/ml. For the detection of α-synuclein, IMR was significantly 250,000 times more sensitive than ELISA. As mentioned above, the sensitivity of IMR detection was 200 times higher than that of ELISA after considering the reaction surface. Another 1250 times may be due to the extremely low noise magnetic sensor, the high T _c superconducting quantum interference device (SQUID) magnetic meter. The high T _c SQUID magnetic meter shows a noise value of 50fT/Hz ^1/2 , which is three orders of magnitude lower than the magnetic signal generated by a single magnetic nanoparticle. This means that the reduction of the AC magnetic signal caused by the combination of the single magnetic nanoparticle with the target biomolecule can be detected by the high T _c SQUID magnetic meter. Therefore, the very low noise high T _c SQUID magnetic meter is very sensitive to the reduction of the AC magnetic signal of the reagent and exhibits an extremely high sensitivity in the determination of biomolecules.

In addition to the low detection limit, the use of IMR to determine the dynamic range of alpha-synuclein is also an important feature. To test this dynamic range, the experimental IMR signal in Figure 2 was converted to the concentration of alpha-synuclein via equation (2). The conversion concentration of _α- synuclein is represented by φ _{α-syn, IMR} . Verify the correlation between φ _{α-syn, IMR} and φ _α-syn as shown in Figure 3. In Fig. 3, a linear relationship between φ _{α-syn, IMR} and φ _α-syn can be obtained. According to regulations issued by the US Food and Drug Administration (FDA), the slope of the linearity in Figure 3 must be between 0.90 and 1.10. In Fig. 3, if φ _{α-syn, IMR} is used for the α-synuclein concentration φ _α-syn of 0.31 fg/ml to 31 ng/ml, then φ _{α-syn, IMR} -φ _α-syn curve The slope was 0.77 and the coefficient of determination R ² was 0.991, as shown by the dotted line in FIG. The slope of the dotted line in Figure 3 does not meet the requirements of the US FDA, and the range of α-synuclein concentrations used to investigate the dynamic range of detection should be reduced. Therefore, the highest φ _{α-syn, IMR in} Fig. 3 is ignored, i.e., φ _α-syn is a value of 31 ng/ml. The linear curve between φ _{α-syn, IMR} and φ _α-syn is plotted in Figure 3 as a dashed line in the range of 0.31 to 3.1 ng/ml. The slope of the dotted line is 1.48, and the coefficient of determination R ² is 0.999. The slope of the dotted line is much higher than the requirements of the US FDA. It seems that the second highest φ _{α-syn in} Figure 3 _{, IMR} should also be ignored. The linear curve between φ _{α-syn, IMR} and φ _α-syn is in the range of 0.31 fg/ml to 310 pg/ml to achieve the effect shown in Figure 3. The slope of the solid line is 0.93, and the coefficient of determination R ² is 0.999. It is worth noting that the slope of the solid line meets the requirements of the US FDA. Therefore, the dynamic range of α-synuclein concentration detected by IMR is from 0.3 fg/ml to 310 pg/ml.

The data shown in Figure 2 demonstrates that the IMR assay is very sensitive and may be able to detect alpha-synuclein in human plasma. Previous studies collected plasma samples from 9 healthy individuals, 9 PD patients, and 14 PDD patients, using IMR to distinguish healthy individuals, PD patients, and PDD patients. The demographic information of the 33 subjects collected is listed in Table 1. The detection concentration of _α- synuclein in human plasma is φ _{α-syn, IMR is} shown in Figure 4. The plasma φ _{α-syn, IMR} range of PDD patients is 0.1 to 100 pg / ml, while the plasma φ _{α-syn, IMR of} healthy individuals is much lower than 0.1 pg / ml. The plasma φ _{α-syn and IMR of} PD patients were distributed between healthy individuals and patients with PDD. The p- value of plasma φ _{α-syn, IMR} between healthy individuals and PD patients was 0.005, indicating the fact that the plasma α-synuclein concentration was higher in PD patients than in healthy individuals. In Figure 4, a significant difference in plasma φ _{α-syn, IMR} between PD patients and PDD patients was observed ( p < 0.001). According to the results of Figure 4, plasma alpha-synuclein concentrations continue to rise in healthy individuals with PD and progress to PDD. It is worth noting that the age between healthy individuals and PD patients ( p > 0.05) and between PD patients and PDD patients ( p > 0.05) is consistent.

PD: Parkinson's disease; PDD: Parkinson's disease dementia; MMSE: mini-mental state examination; SD: standard deviation

Previous studies have shown that alpha-synuclein is released from neurons into body fluids through exocytosis, including CSF and plasma, which contributes to intercellular transmission of alpha-synuclein pathology in the brain. Many studies have focused on examining the value of all or oligomeric alpha-synuclein in plasma samples from PD patients and comparing them to healthy controls, but the results are contradictory. Since phosphorylation and fibrillar alpha-synuclein are the major pathological forms of proteins, a recent study found that phospho-α-synuclein in early PD-deficient samples was compared to the control group. Higher plasma values, these observations suggest that alpha-synuclein values in plasma (either in either oligomeric or phosphorylated form) may partially reflect the feasibility of alpha-synuclein pathology in the brains of PD patients. Sex and potential. Furthermore, cortical Louise/neuritis pathology is more extensive in PDD than in PD without dementia, which means that alpha-synuclein loading in plasma is more severe in PDD than in PD. Our results support the hypothesis that plasma alpha-synuclein values are significantly higher in PDD than normal The cognitive PD has a slightly higher value than the healthy control group. Pathological features of Alzheimer's dementia: amyloid beta plaques and tau neurofibrillary tangles can also be observed and associated with cognitive status in patients with PDD, and future studies need to be combined to assess PDD The patient's phospho-α-synuclein, amyloid-like beta, total plasma and phospho-tau values are better understood for the pathophysiology of PDD.

In plasma samples, heterophilic antibodies are a major confounding factor and can interfere with the detection of sandwich ELISA. According to the guidelines of the Clinical and Laboratory Standards Association (CLSI-EP-A2: Clinical Chemical Interference Test), heterophilic antibodies (HA) are defined as one of the common interfering substances in immunoassays. The IMR method has a low interference and high specificity compared to the previously studied ELISA, and the mechanism of selection is based on the centrifugal force generated by the magnetic nanoparticles oscillated in the reagent, which has been discussed in the prior studies. In fact, not only HA, the naturally occurring biomolecules of drugs that are often used in plasma, but also the binding of magnetic nanoparticles can be prevented by this selection mechanism. This makes IMR a highly specific method for clinical analysis of Parkinson's disease biomarkers.

Clinically, the patient is first diagnosed with PD, and may develop into dementia at a later stage of the disease and is therefore diagnosed with PDD. Therefore, biomarkers that predict or diagnose the early course of PDD in PD individuals do have clinical implications. According to the results in Figure 7, the plasma alpha-synuclein value was significantly higher in patients with PDD than in PD patients ( p < 0.001), which means that plasma alpha-synuclein is expected to be used as a clinical monitor for PDD progression in PD patients. parameter.

An agent for measuring α-synuclein was developed by immobilizing an antibody against α-synuclein on magnetic nanoparticles. The dynamic range of α-synuclein was determined to be from 0.3 fg/ml to 310 pg/ml by immunomagnetic depletion (IMR) using a high T _c SQUID magnetic meter. The ultra-sensitivity SQUID-based IMR was applied to the determination of human plasma alpha-synuclein. Preliminary results showed significant differences in plasma alpha-synuclein concentrations between healthy individuals, PD patients, and PDD patients. It seems promising to apply IMR to the diagnosis of PD and PDD by measuring plasma alpha-synuclein.

Claims (4)

A method for identifying Parkinson's disease or Parkinson's disease dementia having normal cognition in an individual, comprising: (a) detecting an immunomagnetic of alpha-synuclein in vitro in a blood sample of the individual An IVR signal, wherein the IMR signal of the α-synuclein is produced by combining α-synuclein with magnetic nanoparticles containing an anti-α-synuclein antibody, and is obtained by the following formula IMR signal of α-synuclein: IMR(%)=(χac,o-χac,φ)/χac, o×100% where χac represents time-dependent AC susceptibility; χac,o denotes start Χac, the average of the system χac in the first 45 minutes; χac, φ is the final χac, the average value of the system χac in the last 45 minutes; (b) the α- detected in step (a) The IMR signal of synuclein is aligned with a logic function (I) to calculate the concentration of α-synuclein in the blood sample: Where IMR (%) is the IMR signal of the _α- synuclein, φ _α-syn is the concentration of the α-synuclein, the parameter A is the background value, the B is the maximum, φ _o is The IMR signal is equal to the concentration of _α- synuclein at ((A+B)/2), and the slope of the curve φ _o at the data point φ _o with φ _α-syn as the x-axis and IMR (%) as the y-axis. And (c) comparing the concentration of the α-synuclein obtained in step (b) with a predetermined threshold range, wherein the concentration of the α-synuclein above the predetermined threshold range represents Parkinson's Symptoms of dementia, the concentration of the α-synuclein between the predetermined threshold ranges represents Parkinson's disease with normal cognition, and the concentration of the α-synuclein is lower than the predetermined threshold range health. The method of claim 1, wherein the magnetic nanoparticle material is selected from the group consisting of Fe ₃ O ₄ , Fe ₂ O ₃ , MnFe ₂ O ₄ , CoFe ₂ O ₄ and NiFe ₂ O ₄ . Group. The method of claim 1, wherein the magnetic nanoparticle material is Fe ₃ O ₄ . The method of claim 1, wherein the blood sample is plasma.