Introduction

Neurodegenerative diseases, referred as to dementia, have become more prevalent in aging societies. Alzheimer’s disease (AD) is the most common form of dementia [1]. Currently, more than 45 million people are diagnosed as probable AD, and an additional 130 million people might have mild cognitive impairment (MCI) due to AD [2,3,4]. The cost of AD-related healthcare is considerable [5, 6]. In the near future, with the availability of disease-modifying drugs, it will be necessary to have conveniently available, accurate diagnostic procedures. Many countries, such as the US, UK, China, Japan, and Germany, have been allocating resources to help researchers develop early-stage diagnostics or therapeutic drugs for AD-related diseases [7,8,9].

According to pathological analysis, AD subjects show amyloid β (Aβ) plaques in the brain cortex [10,11,12,13]. Aβs are peptides of 36–43 amino acids that result from cleavage of the amyloid precursor protein (APP) by the β and γ secretases. Aβ molecules can aggregate to form flexible, soluble oligomers in the brain and can be naturally metabolized. However, certain misfolded oligomers induce the formation of Aβ plaques in the brain [13] and cause damage to neurons, particularly those surrounding the hippocampus, which functions in short-term memory [14, 15]. Thus, subjects show disorders in memory and even cognitive decline. Another possible mechanism for AD is the hyperphosphorylation of Tau proteins, which are abundant in brain neurons and stabilize the microtubules of neurons [17,18,19,20]. The hyperphosphorylation of Tau proteins causes the death of neurons, resulting in the expression of Tau protein from neurons. Meanwhile, due to the death of neurons, neurofibrillary tangles can be observed in the biopsy and the atrophy of the hippocampus occurs [21, 22]. Therefore, three main clinical features occur with AD. The first is Aβ plaques and neurofibrillary tangles in the brains of AD subjects. The second is atrophy of the hippocampus. The third is memory or cognitive disorders. In clinics, amyloid positron emission tomography (amyloid-PET) is used for imaging Aβ plaques [23,24,25]. Tau-PET for imaging neurofibrillary tangles in the brain is under development and can only be used experimentally [26, 27]. Magnetic resonance imaging (MRI) is used for observing atrophy of the hippocampus [28,29,30]. Neuropsychological tests are applied to evaluate memory or cognitive function [31,32,33,34,35,36]. However, there are several disadvantages with the currently used diagnostic procedures. For example, the cost for an amyloid-PET is $4000 (USD). Moreover, the contrast medium of amyloid-PET or Tau-PET is radiological and may have negative effects on subjects. Although MRI can be used in the early-stage diagnosis of AD, it cannot be applied to some subjects, such as those with heart stent or pacemaker. Approximately 3 h are required to complete a detailed subject examination, including neuropsychological tests performed by a neurophysiologist. Hence, neither neuro-imaging nor neuropsychological tests can be applied for large-scale, early-stage screening for AD.

Both Aβ plaques and neurofibrillary tangles are the neuropathological hallmarks of AD [10,11,12,13,14,15,16,17,18,19,20]. Even in the early stage of AD, the concentrations of Aβs and Tau protein in the cerebrospinal fluid (CSF) become abnormal [37,38,39,40]. Most studies indicated that the concentration of Tau protein in the CSF increases, whereas the concentrations of Aβ1–42 in the CSF decreases in AD subjects [41,42,43,44,45]. The diagnostic accuracy of AD using the above-mentioned CSF biomarkers is 80%–95% [46, 47]. Thus, the assay of CSF biomarkers could be used for the early-stage diagnosis of AD. However, it takes a lumbar puncture to obtain CSF. Although the lumbar puncture carriers low risk, it may bring discomfort to subjects. As of yet, there is no safe, comfortable, low-cost, large-scale, early-stage diagnostic method for AD. Such an urgent demand drives a tremendous number of researchers to develop new technologies for clinical uses.

Blood is a very common sample in clinical practice. In contrast to lumbar puncture, venipuncture is rather safe, less uncomfortable, and inexpensive. The difficulty in assaying AD biomarkers in human blood is due to the ultra-low concentrations of biomarkers. The concentrations of AD biomarkers in CSF are sub-ng/ml or several ng/mL [41,42,43,44,45,46,47], while blood biomarkers are at much lower levels of pg/mL [48, 49]. This means that the required limit of detection (LoD) for assaying Aβ or Tau protein in human blood should be several pg/mL. Currently used assay technologies in clinics, such as enzyme-linked immunosorbent assay, turbidimetry, and nephelometry [50,51,52], demonstrate LoD values in the hundreds of pg/mL, which is much higher than the required LoD for assaying blood Aβ and Tau protein. Thus, an ultra-high-sensitivity technology is needed to assay ultra-low-concentration Aβ and Tau protein in human blood for the early-stage diagnosis of AD.

Herein, the authors describe the development of an ultra-high-sensitivity detection technology for protein: immunomagnetic reduction (IMR) [53, 54]. In IMR, the reagent is a phosphate-buffered saline (PBS) solution having homogeneously dispersed magnetic nanoparticles, which are coated with hydrophilic surfactants (e.g., dextran) and bio-probes (e.g., antibodies) [55]. Under multiple external alternative-current (ac) magnetic fields, magnetic nanoparticles oscillate via magnetic interaction. Thus, the reagent under multiple external ac magnetic fields shows a magnetic property, termed mixed-frequency ac magnetic susceptibility, χ ac, as illustrated in Fig. 1a. Via the bio-probes on the outermost shell, magnetic nanoparticles associate with target molecules to magnetically label the molecules. Because of this association, magnetic nanoparticles become larger, as schematically shown in Fig. 1b. The χ ac response of these larger magnetic nanoparticles to external multiple ac magnetic fields is much less than that of the original individual magnetic nanoparticles. Thus, the χ ac of the reagent is reduced due to the association between magnetic nanoparticles and target molecules. In principle, when greater amounts of target molecules are mixed with a reagent, more magnetic nanoparticles become larger. A larger reduction in χ ac could be expected for reagents. The reduction in χ ac is referred to as the IMR signal, IMR (%), and is quantified via

$${\text{IMR}}(\% ) = \frac{{\chi_{\text{ac,o}} - \chi_{{{\text{ac}},\phi }} }}{{\chi_{\text{ac,o}} }} \times 100\% ,$$
(1)

where χ ac,o and χ ac,ϕ are the ac magnetic susceptibilities of the reagent before and after the association between magnetic nanoparticles and target molecules. In the case of ultra-low concentrations with target molecules, tiny portions of magnetic nanoparticles associate with target molecules. The reduction in the ac magnetic susceptibility of the reagent would be very low. To achieve high-sensitivity detections, a magnetic sensor able to detect the tiny change in the ac magnetic susceptibility of the reagent is required. The high-Tc superconducting-quantum interference-device (SQUID) magnetometer is a promising sensor candidate for IMR measurement. The high-Tc SQUID-based ac magnetosusceptometer for IMR measurement has been developed since 2008 [54]. Currently, the 36-channeled SQUID-based IMR analyzer is available in specific markets. With the aid of antibody-functionalized magnetic nanoparticles and a SQUID-based ac magnetosusceptometer, the LoDs for assaying proteins such as vascular endothelial growth factor (VEGF) and α-synuclein, and viruses like H5N1 are as low as pg/mL [56,57,58]. These results reveal the possibility of assaying Aβ and Tau protein, which might be at the levels of tens of pg/ml, in human blood.

Fig. 1
figure 1

Illustration of the mechanism of immunomagnetic reduction (IMR)

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IMR is not the only one technology reported for the ultra-sensitivity assay. Other technologies such as single molecule assay (SIMOA) or single molecule counting (SMC) have been developed [59,60,61,62]. As compared to SIMOA or SMC, IMR shows the technical advantages. SIMOA and SMC use magnetic beads for the purifications of target molecules (or to concentrate target molecules). This process usually causes loss of target molecules. IMR is a direct measurement of target molecules. Hence, the levels of molecules such as plasma tau protein detected with SIOMA and SMC are lower than that of IMR. Furthermore, SIMOA and SMC are ELISA-based. Sandwich technology is used in SIMOA and SMC. Such technology does not perform well for assaying small molecules. As to IMR, only primary antibody is used. IMR can be widely applied to assay molecules of any sizes.

In this article, the preparation of reagents containing antibody-functionalized magnetic nanoparticles for assaying Aβ and total Tau protein is described. In addition, the design of the high-Tc SQUID-based IMR analyzer is illustrated. Moreover, the characterizations of detecting Aβ and total Tau protein using the reagents and the analyzer are explored. One hundred and twenty-three human plasma samples are analyzed with IMR measurement for Aβ and total Tau protein to investigate the feasibility of discriminating healthy control volunteers from early-stage-AD subjects.

Methods Used in Original Studies

Synthesis of Antibody-Functionalized Magnetic Nanoparticles

The magnetic nanoparticles used are dextran-coated Fe3O4 particles (MF-DEX-0060, MagQu Co., Ltd.), as shown in Fig. 1. These nanoparticles are dispersed in phosphate-buffered saline (PBS) solution. The mean hydrodynamic diameter of the particles is 50–60 nm measured with laser dynamic scattering (Nanotrac 150, Microtrac). The diameter of core Fe3O4 is approximately 25–30 nm according to the θ−2θ X-ray powder diffraction pattern (D-500, Simens) for the (311) peak [55]. The dextran serves as a surfactant to achieve dispersion of the magnetic nanoparticles in the PBS and also acts as a linker between the Fe3O4 particles and antibodies. For antibodies to bind to dextran on the outermost shell of the magnetic nanoparticles, NaIO4 solution is added into the magnetic solution to oxidize dextran to create aldehyde groups (–CHO) [63]. Then, dextran can react with the antibodies via the linking of –CH=N–. Thus, the antibody is bound covalently to dextran. Through magnetic separation, unbound antibodies are separated from the solution. In this work, two types of antibodies, targeting Aβ1–42 and Tau protein, are separately immobilized onto magnetic nanoparticles. For the Aβ1–42 reagent (MF-AB2-0060, MagQu Co., Ltd.), the antibody is monoclonal (A8354, Sigma). The antibody for Tau reagent (MF-TAU-0060, MagQu Co., Ltd.) is also monoclonal (T9450, Sigma) and is against the C terminal region of Tau protein. Thus, total Tau protein is measured. Hereafter, the total Tau protein is referred to as Tau protein in this study. The concentration of reagent is 8 mg Fe/ml, and both reagents demonstrate superparamagnetism. The reagents were stored at 2–8 °C and showed stability at 130 days.

Mechanism of Immunomagnetic Reduction

The detailed mechanism of immunomagnetic reduction (IMR) using the antibody-functionalized magnetic nanoparticles is described here. The frequency-dependent ac magnetic susceptibility χ ac of the reagent under an external magnetic field is expressed as

$$\chi_{\text{ac}} = \chi_{\text{o}} \left[ {\frac{{1 }}{{1 + (2\pi f\tau_{\text{eff}} )^{2} }} + \frac{{(2\pi f\tau_{\text{eff}} )^{2} }}{{1 + (2\pi f\tau_{\text{eff}} )^{2} }}} \right]^{1/2} {\text{e}}^{i\theta } ,$$
(2)

where χ o is the dc magnetic susceptibility, f is the frequency of the external magnetic field, and τ eff is the effective time constant of magnetic relaxation of magnetic nanoparticles in reagent. τ eff can be written as

$$1/\tau_{\text{eff}} = 1/\tau_{B} + 1/\tau_{N} ,$$
(3)

where τ B and τ N are the time constants of Brownian relaxation and Néel relaxation. τ B and τ N are expressed as [64, 65]

$$\tau_{B} = \frac{{3\eta V_{\text{H}} }}{{k_{B} T}},$$
(4a)
$$\tau_{N} = \exp \left( {\frac{{\kappa V_{\text{M}} }}{{k_{B} T}}} \right),$$
(4b)

where η is the viscosity of the water, V H is the hydrodynamic volume of a nanoparticle, k B is the Boltzmann constant, T is the temperature in Kelvin, κ is the anisotropy constant of nanoparticles, and V M denotes the volume of the magnetic core of a nanoparticle. When the viscosity of water (=10−3 Pa-s), the hydrodynamic diameter of the magnetic nanoparticles (~55 nm), the measurement temperature (~293 K), the anisotropy constant of magnetic nanoparticles (~30 kJ m−3), and the core diameter of magnetic nanoparticles (~30 nm) are known, the τ B is estimated to be microseconds and τ N is approximately 1061 s. Since τ N is much larger than τ B , the second term on the right-hand side of Eq. (3) can be neglected. In the current case, τ eff can be regarded as τ B . Thus, the normalized amplitude of ac magnetic susceptibility, |χ ac|/χ o, in Eq. (2) as a function of frequency of an external magnetic field can be numerically analyzed with the known values of η, V H, k B and T in Eq. (4a). Figure 2 plots the relationship between the normalized amplitude of ac magnetic susceptibility and the frequency of the applied ac magnetic field, i.e., the |χ ac|/χ o–f curve. For a given mean hydrodynamic diameter of particles, say 50.8 nm, the |χ ac|/χ o continuously decreases with increasing frequency. On the other hand, as the mean hydrodynamic diameter of the particles increases, the |χ ac|/χ o–f curve moves downward. These results contribute to the reduction in the ac magnetic susceptibility of the reagent as antibody-functionalized magnetic nanoparticles associate with target molecules.

Fig. 2
figure 2

Frequency-dependent normalized amplitude of ac magnetic susceptibility of reagents with various sizes of nanoparticles [52]. The frequency is that of the applied ac magnetic field

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Equation (2) gives the ac magnetic susceptibility of the reagent under only one applied ac magnetic field. In IMR, two magnetic fields, H 1 and H 2, are applied to the reagent. These two applied magnetic fields have different frequencies, f 1 and f 2. The amplitudes of the two applied magnetic fields are weak, e.g., <1 Gauss. Thus, the effective ac magnetic susceptibility of the reagent can be expressed as [53]

$$\chi_{\text{ac,eff}} = 0.32(\chi_{{{\text{ac,H}}_{1} }} +\chi_{{{\text{ac,H}}_{2} }} )/k_{B} T - 0.12(\chi_{{{\text{ac,H}}_{1} }} +\chi_{{{\text{ac,H}}_{2} }})^{3}/( k_{B} T)^{3}+ \ldots ,$$
(5)

where χ ac,H1 is attributed to H 1 with frequency f 1, χ ac,H2 results from H 2 with frequency f 2, k B is the Boltzmann constant, and T is the absolute temperature.

The frequencies shown in the first term on the right-hand side are f 1 and f 2, whereas the second term exhibits frequencies of f 1, f 2, 3f 1, 3f 2, f 2−2f 1, f 2 + 2f 1, f 1−2f 2, f 1 + 2f 2. One can select any linear combination of f 1 and f 2. In IMR, the frequency of f 1 + 2f 2 is of interest. Thus, the amplitude (denoted as χ ac,amp) for χ ac,eff at f 1 + 2f 2 is proportional to the product of χ ac,H1 at f 1 and (χ ac,H2)2 at f 2;

$$\chi_{\text{ac,amp}} = \chi_{{{\text{ac,H}}_{1} }} \cdot (\chi_{{{\text{ac,H}}_{2} }})^2$$
(6)

Once the antibody-functionalized magnetic nanoparticles associate with the target molecules, the hydrodynamic volume of the associated nanoparticles increases. Meanwhile, both χ ac,H1 and χ ac,H2 show a given frequency decrease. Thus, this results in a reduction in the ac magnetic susceptibility of the reagent [66].

Multi-Channeled SQUID-Based ac Magnetosusceptometer

The signal-channeled SQUID-based ac magnetometer was developed in 2008 [54]. The SQUID-based ac magnetosusceptometer consists of three parts: sample magnetization, flux coupling, and superconducting sensing, as shown in Fig. 3. The sample-magnetization component is a set of coils, including two excitation coils (referred to as excitation coil 1 and excitation coil 2, respectively) and one pick-up coil. The inductances and resistance of the three coils are listed in Table 1. The three coils are assembled coaxially, with the pick-up coil being innermost. Excitation coils are driven with an ac signal generator, generating two independent ac voltages at two different frequencies, f 1 and f 2, to excitation coils 1 and 2. The pick-up coil is an axial gradiometer. The ac magnetic signal from the sample is detected by the pick-up coil and is transmitted to the flux coupling component.

Fig. 3
figure 3

Configuration of the single-channel high-Tc SQUID ac magnetosusceptometer for IMR measurement [44]

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Table 1 Inductance and resistance coils of the high-Tc SQUID-based ac magnetosusceptometer
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The flux-coupling component is a pair of twisted wires, as illustrated in Fig. 4. One end of the wires is connected to the pick-up coil. The other end of the wires is terminated with a coil. Once the output ac voltage of the pick-up coil is activated, an ac electric current I is induced along the wires, and an ac magnetic field B is generated at the coil terminal. The ac magnetic field B at the coil terminal is detected with the high-Tc SQUID magnetometer, which is the superconducting–sensing component. The mutual inductance between the coil terminal and the SQUID magnetometer is 1.6 µH. By utilizing the flux-coupling coil, the ac magnetic flux originally generated at the sample component is efficiently transferred to the sensor component of the SQUID-based ac magnetosusceptometer. With this setup, the SQUID magnetometer is not seriously disturbed by the two excitation fields, because the excitation fields are distant from the SQUID magnetometer. Thus, the system is very stable and is suitable for long-term operation. Note that the flux-coupling coil is enveloped with electromagnetically shielded shells.

Fig. 4
figure 4

Illustration of the working principle of the flux-coupling module used with the high-Tc SQUID ac magnetosusceptometer

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The SQUID magnetometer and the coil terminal are immersed in liquid nitrogen. The dewar is placed inside an electromagnetically shielded box, showing 100 dB for the shielding factor at the operating frequency (~20 kHz). The SQUID magnetometer is controlled electronically, and the output signals are sent to a personal computer (PC).

With the need for a high-throughput assay, the channel numbers of the SQUID-based ac magnetosusceptometer were increased to 4 in 2010 [67, 68], to 8 in 2014, and finally to 36 in 2016. An image of the 36-channel SQUID-based ac magnetosusceptometer is shown in Fig. 5. With increases in the channel numbers, more electrical technologies are integrated into the magnetosusceptometer. For example, to prevent electromagnetic crosstalk among coil sets, two neighboring coil sets are separated by 15 cm or each coil set is surrounded with an electromagnetic absorber. The coil sets are activated in sequence. Only one coil set is activated at one instance. To manipulate the activation of each coil set, low-noise electric switches are cascaded between the ac signal generator and excitation coils and between the pick-up coil and the flux-coupling coil component. A 105-channeled SQUID-based ac magnetosusceptometer is under development.

Fig. 5
figure 5

Photo of the 36-channel high-Tc SQUID ac magnetosusceptometer for IMR measurement

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Recruitment of Subjects

This article is based on previously conducted studies [22, 24, 28] and does not involve the performance of any new studies of human or animal subjects. In the previous studies, all subjects were recruited at National Taiwan University Hospital. AD subjects and healthy control volunteers were enrolled from a memory clinic and a mild-cognitive-impairment (MCI) project, respectively. The demographic information of the subjects is listed in Table 2. All subjects had completed primary-school education, and none displayed depressive symptoms, i.e., Geriatric Depression Scale score >8. Subjects with major systemic diseases possibly affecting cognitive function, such as cardiopulmonary failure, hepatic or renal failure, poorly controlled diabetes (HbA1C >8.5), head injury, stroke or other neurodegenerative disease, were excluded. All subjects have been diagnosed by experienced physicians at National Taiwan University Hospital.

Table 2 Demographic information of enrolled subjects
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Several clinical diagnostic tests were performed for the subjects to classify them as healthy controls, MCI due to AD, or AD. The clinical workup included medical history, physical and neurological examinations, laboratory tests, and neuroimaging studies. The neurological examinations included the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating (CDR), the Activities of Daily Living (ADL) scale, the Instrumental ADL scale, and the Wechsler Memory Scale-III (WMS-III). Subjects with dementia meet the diagnostic guidelines for probable AD dementia proposed by the National Institute on Aging-Alzheimer’s Association (NIA-AA) workgroups in 2011. The diagnosis of MCI due to AD also follows the recommendations of the NIA-AA. For the diagnosis of MCI due to AD, a formal cognitive test is used, with a cutoff value at or below the fourth percentile (lower than 1.5 times the standard deviation) of the scale score for the age- and education-matched control. Additionally, all subjects with MCI due to AD demonstrated hippocampal atrophy as observed by magnetic resonance imaging [22].

As shown in Table 2, MMSE scores varied among groups. MMSE was 28.6 ± 1.1 for healthy controls, 24.6 ± 2.2 for MCI due to AD subjects, and 22.2 ± 2.9 for early-stage AD subjects. Significant group differences were observed between healthy controls and MCI due to AD or early-stage AD subjects (p < 0.01). Furthermore, significant group differences between MCI due to AD and early-stage AD subjects at a significance level of p < 0.01 were found.

Preparation of Human Plasma

Subjects were asked to provide a 10-mL non-fasting venous blood sample (K3 EDTA, lavender-top tube). Each sample was blinded to colleagues in the laboratory. The blood samples were centrifuged (2500 g for 15 min) within 1 h of draw, and plasma was aliquoted into cryotubes and stored at −20 °C.

Measurement of IMR Signals

Plasma (40–60 µL) was mixed with 80–60 µL reagent at room temperature for the detection of an immunomagnetic reduction signal, IMR (%). For each sample, IMR signal measurements were performed in duplicate. The IMR signals were converted into biomarker concentrations via standard curves. All plasma samples were blinded for IMR measurements.

Discussion

Consistency of IMR Signals Among Channels

Tau-PBS samples (100-pg/mL) were used for IMR detection by the 36-channel SQUID-based ac magnetosusceptometer. The IMR signal of each channel is plotted in Fig. 6. The maximum IMR signal is 5.14%, while the minimum IMR signal is 5.02%. The mean value and the standard deviation of the 36 IMR signals are 5.07% and 0.03%, respectively. The coefficient of variation in the 36 IMR signals is obtained as 0.62%, revealing the high consistency in IMR signals among channels.

Fig. 6
figure 6

Measured IMR signals of the 36-channel high-Tc SQUID ac magnetosusceptometer. The detected sample is 100-pg/mL Tau protein

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Concentration-Dependent Biomarker IMR Signals

Amyloid β 1–42 (Aβ1–42) (10 µL of 0.1-mg/mL) (Cat. no. A9810; SIGMA-ALDRICH,) was spiked into 9990 µL of PBS (pH 7.4) to produce a 100 ng/mL (=100,000 pg/mL) Aβ1–42-PBS stock-A sample. The stock-A sample (100 µL) was spiked into a 9900-µL PBS buffer pool to obtain a 1 ng/mL (=1000 pg/mL) Aβ1–42-PBS stock-B sample. Stock-A and stock-B samples were mixed with PBS in various volume ratios to obtain nine Aβ1–42-PBS samples of 1, 10, 50, 100, 1000, 2500, 5000, 20,000, and 50,000 pg/mL, which were used for IMR measurements. The IMR signals of these nine Aβ1–42-PBS samples are plotted with dots in Fig. 7. Clearly, Fig. 7 shows the IMR signal increases as Aβ1–42 concentration increases.

Fig. 7
figure 7

Aβ1–42-concentration dependency of IMR signal (solid line) and optical density (dashed line)

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The IMR signals in Fig. 7 for Aβ1–42 concentrations from 1 to 50,000 pg/mL are used for exploring the analytic relationship, which follows the logistic function [69, 70]

$${\text{IMR}}(\% ) = \left[ {\frac{A - B}{{1 + \left( {\frac{{\phi_{{{\text{A}}\beta_{1 - 42} }} }}{{\phi_{\text{o}} }}} \right)^{\gamma } }} + B} \right] \times 100\% ,$$
(7)

where A, B, γ and ϕ o are fitting parameters. By fitting the Aβ1–42 concentration-dependent IMR signals in Fig. 7 to Eq. (18.1), the parameters are found to be A = 1.91, B = 8.09, γ = 0.49 and ϕ o = 14,157. The parameter ϕ Aβ1–42 is the Aβ1–42 concentration. The fitted logistic function is plotted with a solid line in Fig. 7, with a coefficient of determination (R 2) of 0.999. Remarkably, significant IMR signals can could be detected for the 1-pg/mL Aβ1–42-PBS sample. This implies that the minimal detection limit of assaying Aβ1–42 using IMR is on the pg/mL level.

The IMR signal as a function of Tau-protein concentration from 1 to 5000 pg/mL, i.e., IMR (%)-ϕ Tau curve, was measured. Samples of various concentrations of Tau protein were prepared by the following processes: 50 µL of purified human Tau protein ladder with six isoforms (30 µg/mL; Cat. no. T7951; SIGMA-ALDRICH) was mixed with 250 µL of PBS (pH 7.4) to reach a final concentration of 5 µg/mL Tau protein; 20 µL of Tau protein solution (5 µg/mL) is spiked into a 9980-µL PBS (pH 7.4) pool to reach a 10 ng/mL (=10,000 pg/mL) Tau-PBS stock-A sample; 1000 µL of the stock-A sample was spiked into a 9000-µL PBS pool to obtain a 1 ng/mL (=1000 pg/mL) Tau-PBS stock-B sample. Then, the stock-A and stock-B samples are mixed with PBS with various volume ratios to obtain Tau-PBS samples containing 0.1–2500 pg/mL of total Tau protein.

The detected IMR signals for Tau-PBS samples are plotted with crosses in Fig. 8. These experimental data are fitted to Eq. (7) by replacing ϕ Aβ1–42 with ϕ Tau. The values of the fitting parameters were found to be A = 2.28, B = 7.34, γ = 0.33 and ϕ o = 39.03. The coefficient of determination (R 2) was 0.999. The fitted logistic function is plotted as the solid line in Fig. 8. Similar to the case in Fig. 7, the Tau-protein solution at pg/mL provides a significant IMR signal. The low-detection limit of assaying Tau protein using IMR at the level of pg/ml.

Fig. 8
figure 8

Tau protein concentration dependency of IMR signal (solid line) and optical density (dashed line)

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To reveal the ultra-high sensitivity of IMR, the Aβ1–42 and Tau-protein concentration-dependent signals (OD450) were explored using the most frequently used laboratory assay, enzyme-linked immunosorbent assay (ELISA), which is plotted as dashed lines in Figs. 7 and 8 for Aβ1–42 and Tau protein, respectively [59]. Clearly, for both Aβ1–42 or Tau protein, the OD450 is trivial for concentrations lower than 100 pg/mL. The results in Figs. 7 and 8 provide evidence that the IMR assays are much more sensitive than ELISA. Samples with Aβ1–42 or Tau-protein concentrations at several tens of pg/mL can be assayed with IMR, but not with ELISA.

Interference Tests for IMR Assay on Aβ1–42 and Tau Protein

For human plasma, many kinds of chemical and biological molecules co-exist with Aβ1–42 and Tau protein. It is necessary to investigate the interference of these molecules in the detection of Aβ1–42 and Tau protein using IMR. Some typical molecules were selected as interfering materials and mixed with Aβ1–42 or Tau protein. For the interference tests on Aβ1–42, a sample of pure 100-pg/mL Aβ1–42 and samples of 100-pg/mL Aβ1–42 with either 1000-µg/mL hemoglobin, 600-µg/mL conjugated bilirubin, 30,000-µg/mL intra lipid, 200-µg/mL uric acid, 500-IU/mL rheumatoid factor, 60,000-µg/mL albumin, 500-µg/mL acetylsalicylic acid, 300-µg/mL ascorbic acid, 1000-µg/mL ampicillin sodium, 100-pg/mL Aβ1–40, or 1000-ng/mL heterophilic antibody were assayed using IMR. The measured concentration of the pure Aβ1–42was 115.74 pg/mL. The detected Aβ1–42 concentrations of other samples with interfering materials ranged between 104.31 and 123.80 pg/mL. The resulting recovery rates were 90.1%–108.7%. According to the FDA guideline, the accepted recovery rate is 90%–110%. Hence, there is no significant interference of these typical molecules with the assay of Aβ1–42 using IMR.

For the interference tests on Tau protein, a sample of pure 100-pg/mL Tau protein and samples of 100-pg/mL Tau protein with either 1000-µg/mL hemoglobin, 600-µg/mL conjugated bilirubin, 30,000-µg/mL intra lipid, 200-µg/mL uric acid, 500-IU/mL rheumatoid factor, 60,000-µg/mL albumin, 500-µg/mL acetylsalicylic acid, 300-µg/mL ascorbic acid, 1000-µg/mL ampicillin sodium, or 1000-ng/mL heterophilic antibody were assayed using IMR. The measured concentration of the pure Tau protein was 99.85 pg/mL. The detected Tau-protein concentrations of the other samples with interfering materials ranged between 91.67 and 108.51 pg/mL. The resulting recovery rates were 91.8%–108.7%. Hence, there is no significant interference of these typical molecules with the assay of Tau protein using IMR.

The reason for the high specificity of the IMR assay is discussed in Ref. [70]. Interference with the assay is partly caused by non-specific binding between antibodies and non-target molecules. Once non-target molecules associate with antibodies immobilized on magnetic nanoparticles, the IMR signal is detectable and a false positive occurs. According to the mechanism of IMR, the magnetic nanoparticle is rotating regardless of whether non-target or target molecules associate with the magnetic nanoparticles via antibodies. The bound molecule is experiencing the centrifugal force because of particle rotation. The strength of the centrifugal force can be enhanced by increasing the rotation frequency of the nanoparticles. This can be done by adjusting the frequency of the applied ac magnetic fields. The binding force between the antibodies and non-target molecules is weaker than that between antibodies and target molecules. Hence, by suitably adjusting the rotating frequency of the applied ac magnetic fields, the strength of the centrifugal force is manipulated to be higher than the binding force for non-target molecules, but lower than that of target molecules. Thus, the binding between antibodies and non-target molecules is broken, whereas the binding between antibodies and target molecules is maintained. Therefore, the interference of non-target molecules is inhibited in IMR.

Assay of Plasma Biomarkers for Diagnosing Alzheimer’s Dementia

The IMR assay is applied to detect the concentrations of Aβ1–42 and Tau protein in human plasma. According to the clinical diagnosis, there were 68 healthy controls, 24 subjects with MCI due to AD, and 31 early-stage-AD subjects. MCI due to AD is the transition stage from health to early-stage AD [22, 24, 48]. Figure 9a gives the age-dependent Aβ1–42 concentration for healthy controls and subjects suffering from either MCI due to AD or early-stage AD. Each point in Fig. 9a denotes the Aβ1–42 concentration of one subject. For healthy controls, the Aβ1–42 concentration in human plasma is 15.82 ± 0.73 pg/mL. Note that the plasma Aβ1–42 concentration (plotted with dots) in healthy controls truly exists below the low-detection limit of ELISA but above that of IMR. This explains why IMR is suitable for application to the assay of ultra-low-concentration biomarkers of AD in human plasma. Once subjects present with MCI due to AD or early-stage AD, the concentration of plasma Aβ1–42 (plotted with crosses) increases (18.14 ± 1.51 pg/mL). A significant difference (p < 0.001) in plasma Aβ1–42 concentration between healthy controls and subjects is observed in Fig. 9a. Through analysis of the receiver operating characteristic (ROC) curve shown in Fig. 9b, the cut-off value of the plasma Aβ1–42 concentration for discriminating subjects and healthy controls was found to be 16.42 pg/mL. The corresponding clinical sensitivity and specificity were 0.945 and 0.926, respectively. The area under the curve (AUC) was 0.976, as listed in Table 3.

Fig. 9
figure 9

a Age-dependent Aβ1–42 concentration in human plasma of healthy controls and subjects with MCI due to AD or early-stage AD. Each data point denotes one subject. b ROC curve for discriminating healthy control and subjects with MCI due to AD or early-stage AD using plasma Aβ1–42 concentration as a diagnostic parameter

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Table 3 Cut-off value, clinical sensitivity, specificity, and area under the curve for discriminating healthy controls and AD subjects and for differentiating disease severity via ROC curve analysis using biomarker concentrations in human plasma
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As shown in Fig. 10a, using IMR, the detected concentration for the Tau protein in human plasma was (13.38 ± 6.85) pg/mL for healthy controls and (40.74 ± 13.72) pg/mL for subjects. An increase in plasma Tau protein in subjects with either MCI due to AD or early-stage AD was clearly observed. Through t test analysis, the p value in terms of concentration of plasma Tau protein differentiating healthy controls from subjects was smaller than 0.001, revealing a significant difference in the concentration of Tau protein in human plasma between subjects and healthy controls. According to analysis of the ROC shown in Fig. 10b, the cut-off value of plasma Tau-protein concentration for differentiating subjects with MCI due to AD or early-stage AD from healthy controls was found to be 25.20 pg/mL. The corresponding clinical sensitivity and specificity were 0.964 and 0.956, respectively. The AUC was 0.993, as listed in Table 3.

Fig. 10
figure 10

a Age-dependent Tau protein concentration in human plasma of healthy controls and subjects with MCI due to AD or early-stage AD. Each data point denotes one subject. b ROC curve for discriminating healthy controls and subjects with MCI due to AD or early-stage AD using plasma total Tau-protein concentration as a diagnostic parameter

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The results of t test and ROC-curve analyses show the high consistency between the levels of plasma Aβ1–42 and Tau protein and clinical diagnosis for discriminating healthy controls and subjects with early-stage AD or MCI due to AD. This implies a blood test via IMR is promising as an initial step before performing complicated and high-cost clinical diagnosis.

In the past, several research groups have applied ELISA or modified ELISA to assay plasma Aβ1–42 and Tau protein [71,72,73,74,75,76]. The results demonstrated no significant difference between healthy controls and AD subjects, possibly due to the ELISA technique’s lack of ultra-sensitivity. The results shown in Figs. 9a and 10a evidence that the levels of plasma Aβ1–42 and Tau protein are lower than 100 pg/mL, which is below the low-detection limit of ELISA.

Combined Plasma Biomarkers for Differentiating MCI due to AD from Early-Stage AD

In addition to demonstrating the feasibility of differentiating healthy controls from AD subjects, the discrimination between MCI due to AD from early-stage AD using plasma biomarkers was investigated. Remarkably, the subjects in this study with a high degree of AD severity showed lower scores on the MMSE. Hence, the relationship between plasma biomarker levels and MMSE score was investigated.

For plasma Aβ1–42, the subjects with MCI due to AD showed a concentration of (17.52 ± 1.22) pg/mL, while (18.61 ± 1.55) pg/mL was found for subjects with early-stage AD. The p value of the plasma Aβ1–42 concentration between MCI due to AD and early-stage AD was calculated to be 0.003, indicating a significant difference between MCI due to AD and early-stage AD when using plasma Aβ1–42 as a diagnostic parameter. Through ROC analysis shown in Fig. 11a, the cut-off value to differentiate early-stage AD from MCI due to AD was 17.68 pg/mL. The clinical sensitivity and specificity were 0.742 and 0.750, respectively. The area under the curve was 0.762, as listed in Table 3. These results reveal an accuracy of identifying disease severity of approximately 75% based on the Aβ1–42 plasma concentration.

Fig. 11
figure 11

ROC curve for discriminating MCI due to AD from early-stage AD using a plasma Aβ1–42 and b plasma Tau protein as a diagnostic parameter

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Regarding the plasma Tau protein, subjects with MCI due to AD and early-stage AD showed concentrations of (33.33 ± 7.77) pg/mL and (53.57 ± 22.87) pg/mL, respectively. A clear increase in the concentration of plasma Tau protein for early-stage AD compared to MCI due to AD resulted. The p value in terms of plasma Tau-protein concentration for these two groups was calculated to be smaller than 0.001, showing a definite difference in plasma Tau-protein concentration between MCI due to AD and early-stage AD. By performing ROC analysis shown in Fig. 11b, the cut-off value of Tau-protein concentration to discriminate MCI due to AD and early-stage AD was 37.93 pg/mL. The clinical sensitivity and specificity were 0.742 and 0.792, respectively. The area under the curve was 0.822. Compared to plasma Aβ1–42, plasma Tau protein showed higher accuracy in determining disease severity for prodromal AD. This suggests that plasma Tau protein is a promising biomarker not only to discriminate healthy controls and AD subjects but also to identify disease severity for prodromal AD. However, according to published papers [70, 77,78,79,80], elevations of Tau protein in CSF were found for other types of neurodegenerative diseases, such as Parkinson’s disease, frontotemporal dementia, vascular dementia, and brain injuries. The degree of specificity of Tau protein to AD is not high enough. If Tau protein were to be used as the sole parameter for diagnosing prodromal AD, the false-positive occurrence due to other types of dementia or brain injuries would be more than negligible. Fortunately, Aβ1–42 is highly correlated to the pathology of AD. Aβ1–42 should be considered a parameter for diagnosing prodromal AD. To achieve high specificity and accuracy, the combination of Aβ1–42 and Tau protein is suggested as a possible biomarker. In Figs. 9a and 10a, the levels of Aβ1–42 and Tau protein become higher for AD subjects. To enhance the increased biomarker level, the product of Aβ1–42 concentration and Tau-protein concentration, denoted as ϕ Aβ1–42xϕ Tau, is proposed as a diagnostic parameter [49]. The distributions of ϕ Aβ1–42xϕ Tau for healthy controls, MCI due to AD, and early-stage AD are plotted in Fig. 12. The solid lines in Fig. 12 denote the Gaussian distribution.

Fig. 12
figure 12

Distributions of the concentration measurements of Aβ1–42 and Tau protein in human plasma of healthy controls and subjects with MCI due to AD or early-stage AD. Each data point denotes one subject

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Figure 12 clearly shows that the values of ϕ Aβ1–42xϕ Tau for healthy controls range between zero and 400 (pg/mL)2. The ϕ Aβ1–42xϕ Tau values increase for MCI due to AD and are much higher for early-stage AD. Obviously, ϕ Aβ1–42xϕ Tau is promising as a diagnostic parameter not only for discriminating AD subjects but also for identifying disease severity. In this study, MMSE scores decreased with disease severity. Hence, ϕ Aβ1–42xϕ Tau increased with decreasing MMSE score for subjects in this study. Through analysis of the ROC shown in Fig. 13a, the cut-off value for discriminating healthy controls and AD subjects was found to be 455.49 (pg/mL)2, with the corresponding clinical sensitivity and specificity being 0.945 and 0.971, respectively. The area under the curve was 0.995. The cut-off value is plotted with a thinly dashed line in Fig. 12. On the other hand, the cut-off value for differentiating early-stage AD from MCI due to AD was obtained as 643.96 (pg/mL)2 via the ROC analysis shown in Fig. 13b. The clinical sensitivity and specificity and area under the curve were 0.774, 0.837, and 0.841, respectively.

Fig. 13
figure 13

ROC curve using the concentration measurements of Aβ1–42 and Tau protein in human plasma to discriminate a healthy controls from AD subjects, and b MCI due to AD from early-stage AD

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This implies an accuracy of determining the severity of prodromal AD of nearly 85%, which is absolutely applicable in clinics.

Conclusions and Outlook

Immunomagnetic reduction (IMR) is an assay technology involving the use of antibody-functionalized magnetic nanoparticles and a high-Tc SQUID-based ac magnetosusceptometer. When applying SQUID-based IMR to detect the biomarkers (Aβ1–42 and Tau protein) related to Alzheimer’s disease (AD), the low-detection limits are at the pg/mL level. Such a high-sensitivity assay makes it possible to quantitatively detect ultra-low concentrations of Aβ1–42 and Tau protein in human plasma. One hundred and twenty-three subjects, including healthy controls, those with MCI due to AD, and early-stage AD subjects, were enrolled for plasma tests of Aβ1–42 and Tau protein using IMR. The results showed an accuracy of determining the severity of prodromal AD of nearly 85% by determining the concentration levels of Aβ1–42 and total Tau protein in human plasma. Therefore, technically, SQUID-based IMR is suitable for screening early-stage Alzheimer’s disease. SQUID-based IMR is a blood test, which is low-cost, low-risk, simple, and highly acceptable. These advantages are critical for the large-scale diagnosis of early-stage AD. Concerning future large-scale diagnosis, the throughput of the 36-channel analyzer would be a bottleneck. An updated version of the analyzer with tripled throughput is under development. Additionally, the detection of plasma Aβ1–42 and total Tau protein using SQUID-based IMR for global cohorts is necessary, and the authors have been collaborating with groups in the US and Sweden. The detected concentrations of plasma Aβ1–42 or total Tau protein show a high consistency among cohorts from Taiwan, the US and Sweden, and the collaboration has now extended to Japan and Germany.