REVIEW

Detection of Plasma Biomarkers UsingImmunomagnetic Reduction: A Promising Methodfor the Early Diagnosis of Alzheimer’s Disease

Shieh-Yueh Yang . Ming-Jang Chiu . Ta-Fu Chen . Herng-Er Horng

Received: February 20, 2017� The Author(s) 2017. This article is an open access publication

ABSTRACT

Alzheimer’s disease (AD) is the most commonform of dementia. The development of assaytechnologies able to diagnose early-stage AD is

important. Blood tests to detect biomarkers,such as amyloid and total Tau protein, areamong the most promising diagnostic methodsdue to their low cost, low risk, and ease ofoperation. However, such biomarkers in bloodoccur at extremely low levels and are difficult todetect precisely. In the early 2000s, a highlysensitive assay technology, immunomagneticreduction (IMR), was developed. IMR involvesthe use of antibody-functionalized magneticnanoparticles dispersed in aqueous solution.The concentrations of detected molecules areconverted to reductions in the ac magneticsusceptibility of this reagent due to the associ-ation between the magnetic nanoparticles andmolecules. To achieve ultra-high sensitivity, ahigh-Tc superconducting-quantum-interfer-ence-device (SQUID) ac magnetosusceptometerwas designed and applied to detect the tinyreduction in the ac magnetic susceptibility ofthe reagent. Currently, a 36-channeled high-TcSQUID-based ac magnetosusceptometer isavailable. Using the reagent and this analyzer,extremely low concentrations of amyloid andtotal Tau protein in human plasma could bedetected. Further, the feasibility of identifyingsubjects in early-stage AD via assaying plasmaamyloid and total Tau protein is demonstrated.The results show a diagnostic accuracy for pro-dromal AD higher than 80% and reveal thepossibility of screening for early-stage AD usingSQUID-based IMR.

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Shieh-YuehYang (&)MagQu Co., Ltd., New Taipei City 231, Taiwane-mail: syyang@magqu.com

Ming-JangChiu � Ta-FuChenDepartment of Neurology, National TaiwanUniversity Hospital, College of Medicine, NationalTaiwan University, Taipei 110, Taiwan

Ming-JangChiuGraduate Institute of Brain and Mind Sciences,College of Medicine, National Taiwan University,Taipei 110, Taiwan

Ming-JangChiuDepartment of Psychology, National TaiwanUniversity, Taipei 110, Taiwan

Ming-JangChiuGraduate Institute of Biomedical Engineering andBioinformatics, National Taiwan University, Taipei116, Taiwan

Herng-ErHorngInstitute of Electro-Optical Science and Technology,National Taiwan Normal University, Taipei 116,Taiwan

Neurol Ther (2017) 6 (Suppl 1):S37–S56

DOI 10.1007/s40120-017-0075-7

Keywords: Alzheimer’s disease;Immunomagnetic reduction; Magneticnanoparticles; Plasma biomarkers; SQUID

INTRODUCTION

Neurodegenerative diseases, referred as todementia, have become more prevalent in agingsocieties. Alzheimer’s disease (AD) is the mostcommon form of dementia [1]. Currently, morethan 45 million people are diagnosed as proba-ble AD, and an additional 130 million peoplemight have mild cognitive impairment (MCI)due to AD [2–4]. The cost of AD-relatedhealthcare is considerable [5, 6]. In the nearfuture, with the availability of disease-modify-ing drugs, it will be necessary to have conve-niently available, accurate diagnosticprocedures. Many countries, such as the US, UK,China, Japan, and Germany, have been allo-cating resources to help researchers developearly-stage diagnostics or therapeutic drugs forAD-related diseases [7–9].

According to pathological analysis, AD sub-jects show amyloid b (Ab) plaques in the braincortex [10–13]. Abs are peptides of 36–43 aminoacids that result from cleavage of the amyloidprecursor protein (APP) by the b and c secre-tases. Ab molecules can aggregate to form flex-ible, soluble oligomers in the brain and can benaturally metabolized. However, certain mis-folded oligomers induce the formation of Abplaques in the brain [13] and cause damage toneurons, particularly those surrounding thehippocampus, which functions in short-termmemory [14, 15]. Thus, subjects show disordersin memory and even cognitive decline. Anotherpossible mechanism for AD is the hyperphos-phorylation of Tau proteins, which are abun-dant in brain neurons and stabilize themicrotubules of neurons [17–20]. The hyper-phosphorylation of Tau proteins causes thedeath of neurons, resulting in the expression ofTau protein from neurons. Meanwhile, due tothe death of neurons, neurofibrillary tanglescan be observed in the biopsy and the atrophyof the hippocampus occurs [21, 22]. Therefore,three main clinical features occur with AD. Thefirst is Ab plaques and neurofibrillary tangles in

the brains of AD subjects. The second is atrophyof the hippocampus. The third is memory orcognitive disorders. In clinics, amyloid positronemission tomography (amyloid-PET) is used forimaging Ab plaques [23–25]. Tau-PET for imag-ing neurofibrillary tangles in the brain is underdevelopment and can only be used experimen-tally [26, 27]. Magnetic resonance imaging(MRI) is used for observing atrophy of the hip-pocampus [28–30]. Neuropsychological tests areapplied to evaluate memory or cognitive func-tion [31–36]. However, there are several disad-vantages with the currently used diagnosticprocedures. For example, the cost for an amy-loid-PET is $4000 (USD). Moreover, the contrastmedium of amyloid-PET or Tau-PET is radio-logical and may have negative effects on sub-jects. Although MRI can be used in theearly-stage diagnosis of AD, it cannot be appliedto some subjects, such as those with heart stentor pacemaker. Approximately 3 h are requiredto complete a detailed subject examination,including neuropsychological tests performedby a neurophysiologist. Hence, neitherneuro-imaging nor neuropsychological testscan be applied for large-scale, early-stagescreening for AD.

Both Abplaques and neurofibrillary tangles arethe neuropathological hallmarks of AD [10–20].Even in the early stage of AD, the concentrationsof Abs and Tau protein in the cerebrospinal fluid(CSF) become abnormal [37–40]. Most studiesindicated that the concentration of Tau protein inthe CSF increases, whereas the concentrations ofAb1–42 in the CSF decreases in AD subjects [41–45].The diagnostic accuracy of AD using theabove-mentioned CSF biomarkers is 80%–95%[46, 47]. Thus, the assay of CSF biomarkers couldbe used for the early-stage diagnosis of AD.However, it takes a lumbar puncture to obtainCSF. Although the lumbar puncture carriers lowrisk, 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 anurgent demand drives a tremendous number ofresearchers to develop new technologies for clin-ical uses.

Blood is a very common sample in clinicalpractice. In contrast to lumbar puncture,venipuncture is rather safe, less uncomfortable,

S38 Neurol Ther (2017) 6 (Suppl 1):S37–S56

and inexpensive. The difficulty in assaying ADbiomarkers in human blood is due to theultra-low concentrations of biomarkers. Theconcentrations of AD biomarkers in CSF aresub-ng/ml or several ng/mL [41–47], whileblood biomarkers are at much lower levels ofpg/mL [48, 49]. This means that the requiredlimit of detection (LoD) for assaying Ab or Tauprotein in human blood should be several pg/mL. Currently used assay technologies in clin-ics, such as enzyme-linked immunosorbentassay, turbidimetry, and nephelometry [50–52],demonstrate LoD values in the hundreds of pg/mL, which is much higher than the requiredLoD for assaying blood Ab and Tau protein.Thus, an ultra-high-sensitivity technology isneeded to assay ultra-low-concentration Ab andTau protein in human blood for the early-stagediagnosis of AD.

Herein, the authors describe the develop-ment of an ultra-high-sensitivity detectiontechnology for protein: immunomagneticreduction (IMR) [53, 54]. In IMR, the reagent isa phosphate-buffered saline (PBS) solutionhaving homogeneously dispersed magneticnanoparticles, which are coated with hydro-philic surfactants (e.g., dextran) and bio-probes(e.g., antibodies) [55]. Under multiple externalalternative-current (ac) magnetic fields, mag-netic nanoparticles oscillate via magneticinteraction. Thus, the reagent under multipleexternal ac magnetic fields shows a magneticproperty, termed mixed-frequency ac magneticsusceptibility, vac, as illustrated in Fig. 1a. Viathe bio-probes on the outermost shell, magneticnanoparticles associate with target molecules tomagnetically label the molecules. Because ofthis association, magnetic nanoparticles

become larger, as schematically shown inFig. 1b. The vac response of these larger mag-netic nanoparticles to external multiple acmagnetic fields is much less than that of theoriginal individual magnetic nanoparticles.Thus, the vac of the reagent is reduced due to theassociation between magnetic nanoparticlesand target molecules. In principle, when greateramounts of target molecules are mixed with areagent, more magnetic nanoparticles becomelarger. A larger reduction in vac could beexpected for reagents. The reduction in vac isreferred to as the IMR signal, IMR (%), and isquantified via

IMRð%Þ ¼vac;o � vac;/

vac;o� 100%; ð1Þ

where vac,o and vac,/ are the ac magnetic sus-ceptibilities of the reagent before and after theassociation between magnetic nanoparticlesand target molecules. In the case of ultra-lowconcentrations with target molecules, tiny por-tions of magnetic nanoparticles associate withtarget molecules. The reduction in the ac mag-netic susceptibility of the reagent would be verylow. To achieve high-sensitivity detections, amagnetic sensor able to detect the tiny changein the ac magnetic susceptibility of the reagentis required. The high-Tc superconduct-ing-quantum interference-device (SQUID)magnetometer is a promising sensor candidatefor IMR measurement. The high-Tc SQUID--based ac magnetosusceptometer for IMR mea-surement has been developed since 2008 [54].Currently, the 36-channeled SQUID-based IMRanalyzer is available in specific markets. Withthe aid of antibody-functionalized magnetic

Fig. 1 Illustration of the mechanism of immunomagnetic reduction (IMR)

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nanoparticles and a SQUID-based ac magneto-susceptometer, the LoDs for assaying proteinssuch as vascular endothelial growth factor(VEGF) and a-synuclein, and viruses like H5N1are as low as pg/mL [56–58]. These results revealthe possibility of assaying Ab and Tau protein,which might be at the levels of tens of pg/ml, inhuman blood.

IMR is not the only one technology reportedfor the ultra-sensitivity assay. Other technolo-gies such as single molecule assay (SIMOA) orsingle molecule counting (SMC) have beendeveloped [59–62]. As compared to SIMOA orSMC, IMR shows the technical advantages.SIMOA and SMC use magnetic beads for thepurifications of target molecules (or to concen-trate target molecules). This process usuallycauses loss of target molecules. IMR is a directmeasurement of target molecules. Hence, thelevels of molecules such as plasma tau proteindetected with SIOMA and SMC are lower thanthat of IMR. Furthermore, SIMOA and SMC areELISA-based. Sandwich technology is used inSIMOA and SMC. Such technology does notperform well for assaying small molecules. As toIMR, only primary antibody is used. IMR can bewidely applied to assay molecules of any sizes.

In this article, the preparation of reagentscontaining antibody-functionalized magneticnanoparticles for assaying Ab and total Tauprotein is described. In addition, the design ofthe high-Tc SQUID-based IMR analyzer is illus-trated. Moreover, the characterizations ofdetecting Ab and total Tau protein using thereagents and the analyzer are explored. Onehundred and twenty-three human plasmasamples are analyzed with IMR measurementfor Ab and total Tau protein to investigate thefeasibility of discriminating healthy controlvolunteers from early-stage-AD subjects.

METHODS USED IN ORIGINALSTUDIES

Synthesis of Antibody-FunctionalizedMagnetic Nanoparticles

The magnetic nanoparticles used are dex-tran-coated Fe3O4 particles (MF-DEX-0060,

MagQu Co., Ltd.), as shown in Fig. 1. Thesenanoparticles are dispersed in phosphate-bufferedsaline (PBS) solution. The mean hydrodynamicdiameter of the particles is 50–60 nm measuredwith laser dynamic scattering (Nanotrac 150,Microtrac). The diameter of core Fe3O4 is approxi-mately 25–30 nm according to the h-2h X-raypowder diffraction pattern (D-500, Simens) for the(311) peak [55]. The dextran serves as a surfactantto achieve dispersion of the magnetic nanoparti-cles in the PBS and also acts as a linker between theFe3O4 particles and antibodies. For antibodies tobind to dextran on the outermost shell of themagnetic nanoparticles, NaIO4 solution is addedinto the magnetic solution to oxidize dextran tocreatealdehydegroups (–CHO) [63].Then,dextrancan react with the antibodies via the linking of–CH=N–. Thus, the antibody is bound covalentlyto dextran. Through magnetic separation,unbound antibodies are separated from the solu-tion. In this work, two types of antibodies, target-ing Ab1–42 and Tau protein, are separatelyimmobilized onto magnetic nanoparticles. For theAb1–42 reagent (MF-AB2-0060, MagQu Co., Ltd.),the antibody is monoclonal (A8354, Sigma). Theantibody for Tau reagent (MF-TAU-0060, MagQuCo., Ltd.) is also monoclonal (T9450, Sigma) and isagainst the C terminal region of Tau protein. Thus,total Tau protein is measured. Hereafter, the totalTau protein is referred to as Tau protein in thisstudy. The concentration of reagent is 8 mg Fe/ml,and both reagents demonstrate superparamag-netism. The reagents were stored at 2–8 �C andshowed stability at 130 days.

Mechanism of ImmunomagneticReduction

The detailed mechanism of immunomagneticreduction (IMR) using the antibody-function-alized magnetic nanoparticles is described here.The frequency-dependent ac magnetic suscep-tibility vac of the reagent under an externalmagnetic field is expressed as

vac ¼ vo1

1 þ ð2pf seffÞ2þ ð2pf seffÞ2

1 þ ð2pf seffÞ2

" #1=2

eih;

ð2Þ

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where vo is the dc magnetic susceptibility, f isthe frequency of the external magnetic field,and seff is the effective time constant ofmagnetic relaxation of magnetic nanoparticlesin reagent. seff can be written as

1=seff ¼ 1=sB þ 1=sN ; ð3Þ

where sB and sN are the time constants ofBrownian relaxation and Neel relaxation. sB andsN are expressed as [64, 65]

sB ¼ 3gVH

kBT; ð4aÞ

sN ¼ expjVM

kBT

� �; ð4bÞ

where g is the viscosity of the water, VH is thehydrodynamic volume of a nanoparticle, kB isthe Boltzmann constant, T is the temperature inKelvin, j is the anisotropy constant ofnanoparticles, and VM denotes the volume ofthe magnetic core of a nanoparticle. When theviscosity of water (=10-3 Pa-s), the hydrody-namic diameter of the magnetic nanoparticles(*55 nm), the measurement temperature(*293 K), the anisotropy constant of magneticnanoparticles (*30 kJ m-3), and the corediameter of magnetic nanoparticles (*30 nm)are known, the sB is estimated to be microsec-onds and sN is approximately 1061 s. Since sN ismuch larger than sB, the second term on theright-hand side of Eq. (3) can be neglected. Inthe current case, seff can be regarded as sB. Thus,the normalized amplitude of ac magnetic sus-ceptibility, |vac|/vo, in Eq. (2) as a function offrequency of an external magnetic field can benumerically analyzed with the known values ofg, VH, kB and T in Eq. (4a). Figure 2 plots therelationship between the normalized amplitudeof ac magnetic susceptibility and the frequencyof the applied ac magnetic field, i.e., the |vac|/vo–f curve. For a given mean hydrodynamicdiameter of particles, say 50.8 nm, the |vac|/vo

continuously decreases with increasing fre-quency. On the other hand, as the meanhydrodynamic diameter of the particles increa-ses, the |vac|/vo–f curve moves downward. Theseresults contribute to the reduction in the ac

magnetic susceptibility of the reagent as anti-body-functionalized magnetic nanoparticlesassociate with target molecules.

Equation (2) gives the ac magnetic suscepti-bility of the reagent under only one applied acmagnetic field. In IMR, two magnetic fields, H1

and H2, are applied to the reagent. These twoapplied magnetic fields have different frequen-cies, f1 and f2. The amplitudes of the twoapplied magnetic fields are weak, e.g.,\1 Gauss.Thus, the effective ac magnetic susceptibility ofthe reagent can be expressed as [53]

vac;eff ¼ 0:32ðvac;H1þ vac;H2

Þ=kBT � 0:12ðvac;H1

þ vac;H2Þ3=ðkBTÞ3 þ . . .; ð5Þ

where vac,H1 is attributed to H1 with frequencyf1, vac,H2 results from H2 with frequency f2, kB isthe Boltzmann constant, and T is the absolutetemperature.

The frequencies shown in the first term onthe right-hand side are f1 and f2, whereas thesecond term exhibits frequencies of f1, f2, 3f1,3f2, f2-2f1, f2 ? 2f1, f1-2f2, f1 ? 2f2. One canselect any linear combination of f1 and f2. InIMR, the frequency of f1 ? 2f2 is of interest.Thus, the amplitude (denoted as vac,amp) forvac,eff at f1 ? 2f2 is proportional to the product ofvac,H1 at f1 and (vac,H2)2 at f2;

vac;amp ¼ vac;H1� ðvac;H2

Þ2 ð6Þ

1.00

0.80

0.60

0.40

0.20

0.000 5000 10000 15000 20000 25000

f (Hz)

Mean diameter 50.8 nm 51.4 nm 52.6 nm 57.2 nm 57.3 nm

| χac

|/χo

Fig. 2 Frequency-dependent normalized amplitude of acmagnetic susceptibility of reagents with various sizes ofnanoparticles [52]. The frequency is that of the applied acmagnetic field

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Once the antibody-functionalized magneticnanoparticles associate with the targetmolecules, the hydrodynamic volume of theassociated nanoparticles increases. Meanwhile,both vac,H1 and vac,H2 show a given frequencydecrease. Thus, this results in a reduction in theac magnetic susceptibility of the reagent [66].

Multi-Channeled SQUID-Based acMagnetosusceptometer

The signal-channeled SQUID-based ac magne-tometer was developed in 2008 [54]. TheSQUID-based ac magnetosusceptometer con-sists of three parts: sample magnetization, fluxcoupling, and superconducting sensing, asshown in Fig. 3. The sample-magnetizationcomponent is a set of coils, including twoexcitation coils (referred to as excitation coil 1and excitation coil 2, respectively) and onepick-up coil. The inductances and resistance ofthe three coils are listed in Table 1. The threecoils are assembled coaxially, with the pick-upcoil being innermost. Excitation coils are drivenwith an ac signal generator, generating twoindependent ac voltages at two different fre-quencies, f1 and f2, to excitation coils 1 and 2.The pick-up coil is an axial gradiometer. The acmagnetic signal from the sample is detected bythe pick-up coil and is transmitted to the fluxcoupling component.

The flux-coupling component is a pair oftwisted wires, as illustrated in Fig. 4. One end ofthe wires is connected to the pick-up coil. Theother 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 inducedalong the wires, and an ac magnetic field B isgenerated at the coil terminal. The ac magneticfield B at the coil terminal is detected with thehigh-Tc SQUID magnetometer, which is thesuperconducting–sensing component. Themutual inductance between the coil terminaland the SQUID magnetometer is 1.6 lH. Byutilizing the flux-coupling coil, the ac magneticflux originally generated at the sample compo-nent is efficiently transferred to the sensorcomponent of the SQUID-based ac magneto-susceptometer. With this setup, the SQUIDmagnetometer is not seriously disturbed by thetwo excitation fields, because the excitationfields are distant from the SQUID magnetome-ter. Thus, the system is very stable and is suit-able for long-term operation. Note that theflux-coupling coil is enveloped with electro-magnetically shielded shells.

The SQUID magnetometer and the coil ter-minal are immersed in liquid nitrogen. Thedewar is placed inside an electromagneticallyshielded box, showing 100 dB for the shieldingfactor at the operating frequency (*20 kHz).The SQUID magnetometer is controlled elec-tronically, and the output signals are sent to apersonal computer (PC).

Pick-up coil

Flux-transfer line

Sample

Function generator (f1)

Function generator (f2)

Excitation coil 2 Excitation coil 1 Sponge

Magnetically shielded box

Sponge rf shielding room

Dewar Liquid N2

HTS SQUID Flux coupled to SQUID

SQUID electronics

Readout electronics

Fig. 3 Configuration of the single-channel high-TcSQUID ac magnetosusceptometer for IMR measurement[44]

Table 1 Inductance and resistance coils of the high-TcSQUID-based ac magnetosusceptometer

Inductance (mH) Resistance (X)

Excitation coil 1 4.21–4.26 18.71–18.80

Excitation coil 2 2.72–2.77 14.94–15.02

Pick-up coil 0.190–0.198 2.56–2.62

B

Time I

B

M

Time MI

TTimmee

I Flux-coupling coil

SQUID magnetometer

Pick-up coil

Fig. 4 Illustration of the working principle of theflux-coupling module used with the high-Tc SQUID acmagnetosusceptometer

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With the need for a high-throughput assay,the channel numbers of the SQUID-based acmagnetosusceptometer were increased to 4 in2010 [67, 68], to 8 in 2014, and finally to 36 in2016. An image of the 36-channel SQUID-basedac magnetosusceptometer is shown in Fig. 5.With increases in the channel numbers, moreelectrical technologies are integrated into themagnetosusceptometer. For example, to pre-vent electromagnetic crosstalk among coil sets,two neighboring coil sets are separated by 15 cmor each coil set is surrounded with an electro-magnetic absorber. The coil sets are activated insequence. Only one coil set is activated at oneinstance. To manipulate the activation of eachcoil set, low-noise electric switches are cascadedbetween the ac signal generator and excitationcoils and between the pick-up coil and theflux-coupling coil component. A 105-channeledSQUID-based ac magnetosusceptometer isunder development.

Recruitment of Subjects

This article is based on previously conductedstudies [22, 24, 28] and does not involve theperformance of any new studies of human oranimal subjects. In the previous studies, allsubjects were recruited at National TaiwanUniversity Hospital. AD subjects and healthycontrol volunteers were enrolled from a mem-ory clinic and a mild-cognitive-impairment(MCI) project, respectively. The demographicinformation of the subjects is listed in Table 2.All subjects had completed primary-schooleducation, and none displayed depressivesymptoms, i.e., Geriatric Depression Scale score[8. Subjects with major systemic diseases pos-sibly affecting cognitive function, such as car-diopulmonary failure, hepatic or renal failure,poorly controlled diabetes (HbA1C [8.5), headinjury, stroke or other neurodegenerative dis-ease, were excluded. All subjects have beendiagnosed by experienced physicians atNational Taiwan University Hospital.

Several clinical diagnostic tests were per-formed for the subjects to classify them ashealthy controls, MCI due to AD, or AD. Theclinical workup included medical history,physical and neurological examinations, labo-ratory tests, and neuroimaging studies. Theneurological examinations included theMini-Mental State Examination (MMSE) andClinical Dementia Rating (CDR), the Activitiesof Daily Living (ADL) scale, the InstrumentalADL scale, and the Wechsler Memory Scale-III(WMS-III). Subjects with dementia meet thediagnostic guidelines for probable AD dementiaproposed by the National Institute onAging-Alzheimer’s Association (NIA-AA) work-groups in 2011. The diagnosis of MCI due to ADalso follows the recommendations of theNIA-AA. For the diagnosis of MCI due to AD, aformal cognitive test is used, with a cutoff valueat or below the fourth percentile (lower than 1.5times the standard deviation) of the scale scorefor the age- and education-matched control.Additionally, all subjects with MCI due to ADdemonstrated hippocampal atrophy as observedby magnetic resonance imaging [22].

As shown in Table 2, MMSE scores variedamong groups. MMSE was 28.6 ± 1.1 for

Fig. 5 Photo of the 36-channel high-Tc SQUID acmagnetosusceptometer for IMR measurement

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healthy controls, 24.6 ± 2.2 for MCI due to ADsubjects, and 22.2 ± 2.9 for early-stage AD sub-jects. Significant group differences wereobserved between healthy controls and MCIdue to AD or early-stage AD subjects (p\0.01).Furthermore, significant group differencesbetween MCI due to AD and early-stage ADsubjects at a significance level of p\0.01 werefound.

Preparation of Human Plasma

Subjects were asked to provide a 10-mLnon-fasting venous blood sample (K3 EDTA,lavender-top tube). Each sample was blinded tocolleagues in the laboratory. The blood sampleswere centrifuged (2500 g for 15 min) within 1 hof draw, and plasma was aliquoted into cry-otubes and stored at -20 �C.

Measurement of IMR Signals

Plasma (40–60 lL) was mixed with 80–60 lLreagent at room temperature for the detectionof an immunomagnetic reduction signal, IMR(%). For each sample, IMR signal measurementswere performed in duplicate. The IMR signalswere converted into biomarker concentrationsvia standard curves. All plasma samples wereblinded for IMR measurements.

DISCUSSION

Consistency of IMR Signals AmongChannels

Tau-PBS samples (100-pg/mL) were used for IMRdetection by the 36-channel SQUID-based acmagnetosusceptometer. The IMR signal of eachchannel is plotted in Fig. 6. The maximum IMRsignal is 5.14%, while the minimum IMR signalis 5.02%. The mean value and the standarddeviation of the 36 IMR signals are 5.07% and0.03%, respectively. The coefficient of variationin the 36 IMR signals is obtained as 0.62%,revealing the high consistency in IMR signalsamong channels.

Concentration-Dependent Biomarker IMRSignals

Amyloid b 1–42 (Ab1–42) (10 lL of 0.1-mg/mL)(Cat. no. A9810; SIGMA-ALDRICH,) was spikedinto 9990 lL of PBS (pH 7.4) to produce a100 ng/mL (=100,000 pg/mL) Ab1–42-PBSstock-A sample. The stock-A sample (100 lL)was spiked into a 9900-lL PBS buffer pool toobtain a 1 ng/mL (=1000 pg/mL) Ab1–42-PBSstock-B sample. Stock-A and stock-B sampleswere mixed with PBS in various volume ratios toobtain nine Ab1–42-PBS samples of 1, 10, 50,

Table 2 Demographic information of enrolled subjects

Group Healthy control MCI due to AD Early-stage AD

Numbers 68 24 31

Age (years) 62.7 ± 9.7 71.0 ± 10.3 72.1 ± 10.7

CDR 0 0.5 0.5–1

MMSE 28.6 ± 1.1 24.6 ± 2.2* 22.2 ± 2.9*,�

/Ab1–42 (pg/mL) 15.82 ± 0.73 17.52 ± 1.22 18.61 ± 1.55

/Tau (pg/mL) 13.38 ± 6.85 33.33 ± 7.77 53.57 ± 22.87

CDR clinical dementia ranking, MMSE Mini-Mental State Examination� Significant group difference between MCI due to AD and early-stage AD groups at a significance level of p\0.01* Significant difference between the healthy control and MCI due to AD or early-stage AD groups by ANOVA at asignificance level of p\0.01

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100, 1000, 2500, 5000, 20,000, and 50,000 pg/mL, which were used for IMR measurements.The IMR signals of these nine Ab1–42-PBS sam-ples are plotted with dots in Fig. 7. Clearly,Fig. 7 shows the IMR signal increases as Ab1–42

concentration increases.The IMR signals in Fig. 7 for Ab1–42 concen-

trations from 1 to 50,000 pg/mL are used forexploring the analytic relationship, which fol-lows the logistic function [69, 70]

IMRð%Þ ¼ A� B

1 þ /Ab1�42

/o

� �c þ B

264

375� 100%; ð7Þ

where A, B, c and /o are fitting parameters. Byfitting the Ab1–42 concentration-dependent IMRsignals in Fig. 7 to Eq. (18.1), the parameters arefound to be A = 1.91, B = 8.09, c = 0.49 and/o = 14,157. The parameter /Ab1–42 is the Ab1–42

concentration. The fitted logistic function isplotted with a solid line in Fig. 7, with a coef-ficient of determination (R2) of 0.999. Remark-ably, significant IMR signals can could bedetected for the 1-pg/mL Ab1–42-PBS sample.This implies that the minimal detection limit ofassaying Ab1–42 using IMR is on the pg/mL level.

The IMR signal as a function of Tau-proteinconcentration from 1 to 5000 pg/mL, i.e., IMR(%)-/Tau curve, was measured. Samples of vari-ous concentrations of Tau protein were pre-pared by the following processes: 50 lL ofpurified human Tau protein ladder with six

isoforms (30 lg/mL; Cat. no. T7951; SIG-MA-ALDRICH) was mixed with 250 lL of PBS(pH 7.4) to reach a final concentration of 5 lg/mL Tau protein; 20 lL of Tau protein solution(5 lg/mL) is spiked into a 9980-lL PBS (pH 7.4)pool to reach a 10 ng/mL (=10,000 pg/mL)Tau-PBS stock-A sample; 1000 lL of the stock-Asample was spiked into a 9000-lL PBS pool toobtain a 1 ng/mL (=1000 pg/mL) Tau-PBSstock-B sample. Then, the stock-A and stock-Bsamples are mixed with PBS with various vol-ume ratios to obtain Tau-PBS samples contain-ing 0.1–2500 pg/mL of total Tau protein.

The detected IMR signals for Tau-PBS sam-ples are plotted with crosses in Fig. 8. Theseexperimental data are fitted to Eq. (7) byreplacing /Ab1–42 with /Tau. The values of thefitting parameters were found to be A = 2.28,B = 7.34, c = 0.33 and /o = 39.03. The coeffi-cient of determination (R2) was 0.999. The fittedlogistic function is plotted as the solid line inFig. 8. Similar to the case in Fig. 7, theTau-protein solution at pg/mL provides a sig-nificant IMR signal. The low-detection limit ofassaying Tau protein using IMR at the level ofpg/ml.

6.00

5.00

4.00

3.00

2.00

1.00

0.000 5 10 15 20 25 30 35

Channel No.

IMR

(%)

Fig. 6 Measured IMR signals of the 36-channel high-TcSQUID ac magnetosusceptometer. The detected sample is100-pg/mL Tau protein

0.800.6

5.00 6.0

4.00 4.0

3.00 2.0

2.00 0.0

1 10 100 100010000 φAβ1-42 (pg/ml)

IMR

(%)

OD

450

Fig. 7 Ab1–42-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 Ab1–42 and Tau-protein concentration-de-pendent signals (OD450) were explored usingthe most frequently used laboratory assay,enzyme-linked immunosorbent assay (ELISA),which is plotted as dashed lines in Figs. 7 and 8for Ab1–42 and Tau protein, respectively [59].Clearly, for both Ab1–42 or Tau protein, theOD450 is trivial for concentrations lower than100 pg/mL. The results in Figs. 7 and 8 provideevidence that the IMR assays are much moresensitive than ELISA. Samples with Ab1–42 orTau-protein concentrations at several tens ofpg/mL can be assayed with IMR, but not withELISA.

Interference Tests for IMR Assay on Ab1–42and Tau Protein

For human plasma, many kinds of chemical andbiological molecules co-exist with Ab1–42 andTau protein. It is necessary to investigate theinterference of these molecules in the detectionof Ab1–42 and Tau protein using IMR. Sometypical molecules were selected as interfering

materials and mixed with Ab1–42 or Tau protein.For the interference tests on Ab1–42, a sample ofpure 100-pg/mL Ab1–42 and samples of 100-pg/mL Ab1–42 with either 1000-lg/mL hemoglobin,600-lg/mL conjugated bilirubin, 30,000-lg/mLintra lipid, 200-lg/mL uric acid, 500-IU/mLrheumatoid factor, 60,000-lg/mL albumin,500-lg/mL acetylsalicylic acid, 300-lg/mLascorbic acid, 1000-lg/mL ampicillin sodium,100-pg/mL Ab1–40, or 1000-ng/mL heterophilicantibody were assayed using IMR. The measuredconcentration of the pure Ab1–42was 115.74 pg/mL. The detected Ab1–42 concentrations of othersamples with interfering materials rangedbetween 104.31 and 123.80 pg/mL. The result-ing recovery rates were 90.1%–108.7%. Accord-ing to the FDA guideline, the accepted recoveryrate is 90%–110%. Hence, there is no significantinterference of these typical molecules with theassay of Ab1–42 using IMR.

For the interference tests on Tau protein, asample of pure 100-pg/mL Tau protein andsamples of 100-pg/mL Tau protein with either1000-lg/mL hemoglobin, 600-lg/mL conju-gated bilirubin, 30,000-lg/mL intra lipid,200-lg/mL uric acid, 500-IU/mL rheumatoidfactor, 60,000-lg/mL albumin, 500-lg/mLacetylsalicylic acid, 300-lg/mL ascorbic acid,1000-lg/mL ampicillin sodium, or 1000-ng/mLheterophilic antibody were assayed using IMR.The measured concentration of the pure Tauprotein was 99.85 pg/mL. The detectedTau-protein concentrations of the other sam-ples with interfering materials ranged between91.67 and 108.51 pg/mL. The resulting recoveryrates were 91.8%–108.7%. Hence, there is nosignificant interference of these typical mole-cules with the assay of Tau protein using IMR.

The reason for the high specificity of the IMRassay is discussed in Ref. [70]. Interference withthe assay is partly caused by non-specific bind-ing between antibodies and non-target mole-cules. Once non-target molecules associate withantibodies immobilized on magnetic nanopar-ticles, the IMR signal is detectable and a falsepositive occurs. According to the mechanism ofIMR, the magnetic nanoparticle is rotatingregardless of whether non-target or targetmolecules associate with the magneticnanoparticles via antibodies. The bound

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S46 Neurol Ther (2017) 6 (Suppl 1):S37–S56

molecule is experiencing the centrifugal forcebecause of particle rotation. The strength of thecentrifugal force can be enhanced by increasingthe rotation frequency of the nanoparticles.This can be done by adjusting the frequency ofthe applied ac magnetic fields. The bindingforce between the antibodies and non-targetmolecules is weaker than that between anti-bodies and target molecules. Hence, by suitablyadjusting the rotating frequency of the appliedac magnetic fields, the strength of the cen-trifugal force is manipulated to be higher thanthe binding force for non-target molecules, butlower than that of target molecules. Thus, thebinding between antibodies and non-targetmolecules is broken, whereas the bindingbetween antibodies and target molecules ismaintained. Therefore, the interference ofnon-target molecules is inhibited in IMR.

Assay of Plasma Biomarkersfor Diagnosing Alzheimer’s Dementia

The IMR assay is applied to detect the concen-trations of Ab1–42 and Tau protein in humanplasma. According to the clinical diagnosis,there were 68 healthy controls, 24 subjects withMCI due to AD, and 31 early-stage-AD subjects.MCI due to AD is the transition stage fromhealth to early-stage AD [22, 24, 48]. Figure 9agives the age-dependent Ab1–42 concentrationfor healthy controls and subjects suffering fromeither MCI due to AD or early-stage AD. Eachpoint in Fig. 9a denotes the Ab1–42 concentra-tion of one subject. For healthy controls, theAb1–42 concentration in human plasma is15.82 ± 0.73 pg/mL. Note that the plasmaAb1–42 concentration (plotted with dots) inhealthy controls truly exists below the low-de-tection limit of ELISA but above that of IMR.This explains why IMR is suitable for applica-tion to the assay of ultra-low-concentrationbiomarkers of AD in human plasma. Once sub-jects present with MCI due to AD or early-stageAD, the concentration of plasma Ab1–42 (plottedwith crosses) increases (18.14 ± 1.51 pg/mL). Asignificant difference (p\0.001) in plasmaAb1–42 concentration between healthy controlsand subjects is observed in Fig. 9a. Through

analysis of the receiver operating characteristic(ROC) curve shown in Fig. 9b, the cut-off valueof the plasma Ab1–42 concentration for dis-criminating subjects and healthy controls wasfound to be 16.42 pg/mL. The corresponding

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clinical sensitivity and specificity were 0.945and 0.926, respectively. The area under thecurve (AUC) was 0.976, as listed in Table 3.

As shown in Fig. 10a, using IMR, the detectedconcentration for the Tau protein in humanplasma was (13.38 ± 6.85) pg/mL for healthycontrols and (40.74 ± 13.72) pg/mL for subjects.An increase in plasma Tau protein in subjects witheither MCI due to AD or early-stage AD was clearlyobserved. Through t test analysis, the p value interms of concentration of plasma Tau proteindifferentiating healthy controls from subjects wassmaller than 0.001, revealing a significant differ-ence in the concentration of Tau protein inhuman plasma between subjects and healthycontrols. According to analysis of the ROC shownin Fig. 10b, the cut-off value of plasma Tau-pro-tein concentration for differentiating subjectswith MCI due to AD or early-stage AD from heal-thy controls was found to be 25.20 pg/mL. Thecorresponding clinical sensitivity and specificitywere 0.964 and 0.956, respectively. The AUC was0.993, as listed in Table 3.

The results of t test and ROC-curve analysesshow the high consistency between the levels of

plasma Ab1–42 and Tau protein and clinicaldiagnosis for discriminating healthy controlsand subjects with early-stage AD or MCI due toAD. This implies a blood test via IMR ispromising as an initial step before performingcomplicated and high-cost clinical diagnosis.

In the past, several research groups haveapplied ELISA or modified ELISA to assay plasmaAb1–42 and Tau protein [71–76]. The resultsdemonstrated no significant difference betweenhealthy controls and AD subjects, possibly dueto the ELISA technique’s lack of ultra-sensitiv-ity. The results shown in Figs. 9a and 10a evi-dence that the levels of plasma Ab1–42 and Tauprotein are lower than 100 pg/mL, which isbelow the low-detection limit of ELISA.

Combined Plasma Biomarkersfor Differentiating MCI due to ADfrom Early-Stage AD

In addition to demonstrating the feasibility ofdifferentiating healthy controls from AD sub-jects, the discrimination between MCI due to

Table 3 Cut-off value, clinical sensitivity, specificity, and area under the curve for discriminating healthy controls and ADsubjects and for differentiating disease severity via ROC curve analysis using biomarker concentrations in human plasma

Plasma biomarker Feature Healthy controlsvs. AD subjects

MCI due to ADvs. early-stage AD

Ab1–42 Cut-off value 16.41 pg/mL 17.68 pg/mL

Sensitivity 0.945 0.742

Specificity 0.926 0.750

AUC 0.976 0.762

Tau protein Cut-off value 25.20 pg/mL 37.93 pg/mL

Sensitivity 0.964 0.742

Specificity 0.956 0.792

AUC 0.993 0.822

Ab1–42XTau protein Cut-off value 455.49 (pg/mL)2 643.96 (pg/mL)2

Sensitivity 0.945 0.774

Specificity 0.971 0.837

AUC 0.995 0.841

AD subjects include those with MCI due to AD and early-stage ADAUC area under the curve

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AD from early-stage AD using plasma biomark-ers was investigated. Remarkably, the subjectsin this study with a high degree of AD severityshowed lower scores on the MMSE. Hence, therelationship between plasma biomarker levelsand MMSE score was investigated.

For plasma Ab1–42, the subjects with MCI dueto AD showed a concentration of (17.52 ± 1.22)pg/mL, while (18.61 ± 1.55) pg/mL was foundfor subjects with early-stage AD. The p value ofthe plasma Ab1–42 concentration between MCIdue to AD and early-stage AD was calculated tobe 0.003, indicating a significant differencebetween MCI due to AD and early-stage ADwhen using plasma Ab1–42 as a diagnosticparameter. Through ROC analysis shown inFig. 11a, the cut-off value to differentiate ear-ly-stage AD from MCI due to AD was 17.68 pg/mL. The clinical sensitivity and specificity were0.742 and 0.750, respectively. The area underthe curve was 0.762, as listed in Table 3. Theseresults reveal an accuracy of identifying diseaseseverity of approximately 75% based on theAb1–42 plasma concentration.

Regarding the plasma Tau protein, subjectswith MCI due to AD and early-stage AD showedconcentrations of (33.33 ± 7.77) pg/mL and(53.57 ± 22.87) pg/mL, respectively. A clearincrease in the concentration of plasma Tauprotein for early-stage AD compared to MCI dueto AD resulted. The p value in terms of plasmaTau-protein concentration for these two groupswas calculated to be smaller than 0.001, show-ing a definite difference in plasma Tau-proteinconcentration between MCI due to AD andearly-stage AD. By performing ROC analysisshown in Fig. 11b, the cut-off value ofTau-protein concentration to discriminate MCIdue to AD and early-stage AD was 37.93 pg/mL.The clinical sensitivity and specificity were0.742 and 0.792, respectively. The area underthe curve was 0.822. Compared to plasmaAb1–42, plasma Tau protein showed higheraccuracy in determining disease severity forprodromal AD. This suggests that plasma Tauprotein is a promising biomarker not only todiscriminate healthy controls and AD subjectsbut also to identify disease severity for prodro-mal AD. However, according to publishedpapers [70, 77–80], elevations of Tau protein inCSF were found for other types of neurodegen-erative diseases, such as Parkinson’s disease,frontotemporal dementia, vascular dementia,and brain injuries. The degree of specificity ofTau protein to AD is not high enough. If Tauprotein were to be used as the sole parameter for

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diagnosing prodromal AD, the false-positiveoccurrence due to other types of dementia orbrain injuries would be more than negligible.Fortunately, Ab1–42 is highly correlated to thepathology of AD. Ab1–42 should be considered aparameter for diagnosing prodromal AD. Toachieve high specificity and accuracy, thecombination of Ab1–42 and Tau protein is sug-gested as a possible biomarker. In Figs. 9a and10a, the levels of Ab1–42 and Tau proteinbecome higher for AD subjects. To enhance the

increased biomarker level, the product of Ab1–42

concentration and Tau-protein concentration,denoted as /Ab1–42x/Tau, is proposed as a diag-nostic parameter [49]. The distributions of/Ab1–42x/Tau for healthy controls, MCI due toAD, and early-stage AD are plotted in Fig. 12.The solid lines in Fig. 12 denote the Gaussiandistribution.

Figure 12 clearly shows that the values of/Ab1–42x/Tau for healthy controls range betweenzero and 400 (pg/mL)2. The /Ab1–42x/Tau valuesincrease for MCI due to AD and are muchhigher for early-stage AD. Obviously, /Ab1–42

x/Tau is promising as a diagnostic parameter notonly for discriminating AD subjects but also foridentifying disease severity. In this study, MMSEscores decreased with disease severity. Hence,/Ab1–42x/Tau increased with decreasing MMSEscore for subjects in this study. Through analy-sis of the ROC shown in Fig. 13a, the cut-offvalue for discriminating healthy controls andAD subjects was found to be 455.49 (pg/mL)2,with the corresponding clinical sensitivity and

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specificity being 0.945 and 0.971, respectively.The area under the curve was 0.995. The cut-offvalue is plotted with a thinly dashed line inFig. 12. On the other hand, the cut-off value fordifferentiating early-stage AD from MCI due toAD was obtained as 643.96 (pg/mL)2 via theROC analysis shown in Fig. 13b. The clinicalsensitivity and specificity and area under thecurve were 0.774, 0.837, and 0.841,respectively.

This implies an accuracy of determining theseverity of prodromal AD of nearly 85%, whichis absolutely applicable in clinics.

CONCLUSIONS AND OUTLOOK

Immunomagnetic reduction (IMR) is an assaytechnology involving the use of antibody-func-tionalized magnetic nanoparticles and a high-TcSQUID-based ac magnetosusceptometer. Whenapplying SQUID-based IMR to detect thebiomarkers (Ab1–42 and Tau protein) related toAlzheimer’s disease (AD), the low-detection limitsare at the pg/mL level. Such a high-sensitivityassay makes it possible to quantitatively detectultra-low concentrations of Ab1–42 and Tau pro-tein in human plasma. One hundred and twen-ty-three subjects, including healthy controls,those with MCI due to AD, and early-stage ADsubjects, were enrolled for plasma tests of Ab1–42

and Tau protein using IMR. The results showed anaccuracy ofdetermining the severity ofprodromalAD of nearly 85% by determining the concentra-tion levels of Ab1–42 and total Tau protein inhuman plasma. Therefore, technically, SQUID--based IMR is suitable for screening early-stageAlzheimer’s disease. SQUID-based IMR is a bloodtest, which is low-cost, low-risk, simple, andhighly acceptable. These advantages are criticalfor the large-scale diagnosis of early-stage AD.Concerning future large-scale diagnosis, thethroughput of the 36-channel analyzer wouldbe abottleneck. An updated version of the analyzerwith tripled throughput is under development.Additionally, the detection of plasma Ab1–42 andtotal Tau protein using SQUID-based IMR forglobal cohorts is necessary, and the authors havebeen collaborating with groups in the US andSweden. The detected concentrations of plasmaAb1–42 or total Tau protein show a high consis-tency among cohorts from Taiwan, the US andSweden, and the collaboration has now extendedto Japan and Germany.

ACKNOWLEDGEMENTS

No funding or sponsorship was received for thisstudy or publication of this article. All named

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Fig. 13 ROC curve using the concentration measure-ments of Ab1–42 and Tau protein in human plasma todiscriminate a healthy controls from AD subjects, andb MCI due to AD from early-stage AD

Neurol Ther (2017) 6 (Suppl 1):S37–S56 S51

authors meet the International Committee ofMedical Journal Editors (ICMJE) criteria forauthorship of this manuscript, take responsi-bility for the integrity of the work as a whole,and have given final approval for the version tobe published.

Disclosures. Shieh-Yueh Yang is anemployee of MagQu Co., Ltd., receives a salaryfrom MagQu, and holds MagQu stock. Herng-ErHorng also holds MagQu stock. Ming-Jang Chiuand Ta-Fu Chen have nothing to disclose.

Compliance with Ethics Guidelines. Thisarticle is based on previously conducted studiesand does not involve any new studies of humanor animal subjects performed by any of theauthors.

Data Availability. Data sharing is notapplicable to this article as no datasets weregenerated or analyzed during the current study.

Open Access. This article is distributedunder the terms of the Creative CommonsAttribution-NonCommercial 4.0 InternationalLicense (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommer-cial use, distribution, and reproduction in anymedium, provided you give appropriate creditto the original author(s) and the source, providea link to the Creative Commons license, andindicate if changes were made.

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