ARTICLE IN PRESS

Journal of Magnetism and Magnetic Materials 300 (2006) e315–e319
www.elsevier.com/locate/jmmm

Bio-application of high-Tc SQUID magnetic sensor
Saburo Tanaka, Zarina Aspanut, Hirofumi Kurita, Chika Toriyabe,
Yoshimi Hatuskade, Shinji Katsura
Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
Available online 15 November 2005

Abstract
We propose medical applications using ultra-small magnetic particles and a SQUID magnetic sensor. A high-Tc SQUID system forbiological molecules (DNA) detection is one of that. This system is based on a hybridization process. Two strands in a DNA molecule areheld together by hydrogen bonds between base pairs like a ladder. The two strands are referred to as being complementary to each other.
One strand (sample DNA) was labeled with Fe3O4 ultra-small magnetic particles and the other (probe DNA) was anchored on a glassslide. Then they were hybridized each other on the slide. After washing the excess sample DNA, the hybridized DNA was evaluated in
the presence of excitation AC ﬁeld by high-Tc SQUID. The signal was initially proportional to the concentration of the sample DNA andthen saturated. It means that the hybridization occurred successfully between the sample DNA and the probe DNA.
r 2005 Elsevier B.V. All rights reserved.
PACS: 85.25; 74.76
Keywords: SQUID; Nanoparticles; DNA

1. Introduction
Analysis of biological molecules such as DNA is basedon separation and detection process. The detection method
for the separated molecules determines the total sensitivityof analysis system. To date, several methods have been
introduced for the detection to improve the sensitivity. The
radioisotope labeling method is a one of the most common
methods [1]. However, it requires a special facility, which
conﬁnes the radioisotope. Fluorescence labeling is very
highly sensitive and it can detect single molecules; however,quenching of ﬂuorescence dye inhibits long-time observation [2]. The enzyme labeling method also achieves highsensitivity; however, enzymes are sometime unstable [3]. Alabeling method with small magnetic particles has been
introduced. The SQUID has a large potential to detect
small amount of these particles because of its highsensitivity for magnetic ﬁeld. Detection of small magnetic
particles with a SQUID for immunoassay applications isperformed in several groups [4–7].
Corresponding author. Tel.: +81 532 44 6919; fax: +81 532 44 6929.

E-mail address: tanakas@eco.tut.ac.jp (S. Tanaka).
0304-8853/$ - see front matter r 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jmmm.2005.10.109

Recently, a sensitive DNA chip technique has beendeveloped. Because a DNA chip contains enormous
numbers of different DNA probes which correspond togenetic disease or personal properties, these have been
applied to analysis of polymorphism of human genome. To
apply high-Tc SQUID sensor for DNA chip detection, we
have developed a detection system and a sample preparation method [8]. In this paper, a hybridization of DNA
with relatively short length was investigated. The principleof the detection is shown in Fig. 1. One strand (sampleDNA) was labeled with Fe3O4 ultra-small magnetic
particles and the other (probe DNA) was anchored on aglass slide. They were hybridized with each other on theslide. Then the hybridized DNA was evaluated in thepresence of AC excitation ﬁeld by high-Tc SQUID. Thismethod is classiﬁed into a category of susceptibility
measurement.
2. Detection system
The detection system is schematically drawn in Fig. 2.
The SQUID is made of Y1Ba2Cu3O7y thin ﬁlm. The
junctions utilized in the SQUID are of the bi-crystal type.

ARTICLE IN PRESSS. Tanaka et al. / Journal of Magnetism and Magnetic Materials 300 (2006) e315–e319

e316

Magneticparticles
3`

Probe DNA(knownsequences)

SQUID

G(unknownsequences)

CGCTSample DNA

5` TA5`3`Linker

AGCTTCGAAHYBRIDIZATIONPROCESS
substrateFig. 1. Principle of the detection of DNA sequence.

Function Generator

DNA sample
Excitetion Coil0.01 Hz and band elimination ﬁlter (BEF) with frequencyof 60 Hz were used. The schematic of the sample carrier isshown in Fig. 3. The sample carrier is made of glass and its
dimensions are 7 mm  4 mm  0.9 mm. It has a hole of3 mm in diameter. After ﬁlling the hole with a liquidsample, it was covered with an adhesive tape. When thesample was measured, it was turned over so that the side ofadhesive tape faces the SQUID. The distance between thesample and the SQUID was adjusted at as close as about
0.8 mm. The magnetic ﬂux noise spectrum of the system isshown in Fig. 4. The ﬂux noise in the white noise region
was about 12 mF0/Hz1/2. After applying the excitation
magnetic ﬁeld of 0.43 mT, the white noise was increased bya factor of 20. However neither major ﬂux trapping or ﬂux
jump happened. A blank sample carrier was moved above
the SQUID and the signal was recorded to estimate thebackground noise. Fig. 5 shows the recorded signal trace ofthe blank sample. The peak-to-peak value of the noise isabout 7 mF0.

Motor
Compensation Coil

Phase
Shifter

7 mmmm

SQUID

4 mm

Φ3Ref. in

SQUID Electronics

Lock−in Amplifier
PCHPF
PC Recorder

Adhesive tape
BEF 60 HzFig. 2. Schematic diagram of the DNA detection system. SQUID output
signal is lock-in ampliﬁed and recorded by a PC recorder.

Aldehyde coated
SubstrateFig. 3. Schematic drawing of the sample carrier. The size of the glass slideis 7 mm  4 mm  0.9 mm. It has a hole of 3 mm in diameter. After ﬁllingthe hole with a liquid sample, it was covered with an adhesive tape.

1
10Flux noise [φ0 / Hz1/2]

The washer size of the SQUID is about 5  5 mm2 and theeffective area is 0.05 mm2. The SQUID was operated in aﬂux-locked loop with a ﬂux modulation frequency of256 kHz. The cryostat (not shown in the ﬁgure) wasspecially designed for a SQUID microscope. The SQUIDwas located inside a vacuum and separated by a quartz
window. A more detailed description can be foundelsewhere [9,10]. A set of excitation coil is mounted above
the SQUID microscope. This excitation coil generates
magnetic ﬁeld at 100 Hz and modulates the SQUID signals.
The maximum applied magnetic ﬁeld was 0.43 mT at peakto-peak value. The relative position of the SQUID and theexcitation coil was carefully adjusted so that the SQUID
output signal without particles became zero. A compensation coil wound around the SQUID was employed to beable to cancel the residual vertical component of theexcitation ﬁeld, which could not be canceled by theadjustment. A phase shifter was used for adjusting thephase of the ﬁeld current. In the measurement, the sample
was driven with a speed of 10 mm/s by a stepping motor,
which was installed outside a magnetically shielded room.
When the sample passed above the SQUID, the SQUID
output signal was lock-in ampliﬁed and recorded by PC
recorder. A high pass ﬁlter (HPF) with frequency ofLiquid Sample-1

Applied Magnetic Fieldof 0.43mT10-2-310-410-510Without magnetic field-610110100
Frequency [Hz]1000

Fig. 4. Flux noise spectrum of the detection system. White noise is about
12 mF0/Hz1/2. Microphonic noise at 40 Hz, power frequency noise at 60 Hz
and their harmonics can be seen.

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Magnetic flux [mΦo ]Magnetic flux [mΦo ]3020100-10-20e317

50250-25-50-75-300102030 40Distance [mm]5060Fig. 5. Signal trace of the blank sample. The peak-to-peak value of thebackground signal is about 7 mF0.

01020 30 40 50
Distance [mm]60Fig. 6. Typical signal trace of a liquid sample with particles. This sample
contains 0.73 pmol of nanoparticles.

3.1. Magnetic nanoparticles
We used nanoparticles from Meito Sangyo Co., Ltd. The
core of the particle is iron oxide Fe3O4 (magnetite) which iscoated with a dextran [11]. The averaged core diameter andthe outer dimension of the particles were 11 and 65 nm,respectively. The particles had superparamagnetic properties. The COOH group is attached around the surface ofthe dextran. The DNA will be connected to the COOH
group via a proper linker. The particles were supplied in
the form of an aqueous magnetic ﬂuid. The concentrationof the particles in the original ﬂuid is 36 mM. The original
ﬂuid was diluted with distilled water to have the desiredconcentrations. We took 4 ml of each liquid sample withdesired concentration and put it on the glass sample holder
shown in Fig. 3. The holder was covered with an adhesive
tape and then moved above the SQUID. The recorded
typical signal trace is shown in Fig. 6. This sample contains
0.73 pmol of nanoparticles. The peak-to-peak value showsmagnetic ﬁeld of 100 mF0. Fig. 7 shows the magnetic signal
measured by the SQUID when the number of the particles
is changed. The signal is almost proportional to thenumber of the particles.
3.2. DNA sample
We employed single-strand DNA (ssDNA) with relatively short length, 30 base pairs (Human Papillomavirus
Probes 33). The sequence of the strand is CATAG TCGTA
CAGTA CCTTA CTGCA AAATG and its complement.
One ssDNA named sample DNA was modiﬁed with aminogroup (NH2) at the 50 terminal. This amino group can beconnected to the carboxyl group COOH of the nanoparticle via a crosslinker [12]. The crosslinker which consists ofEDAC (1-ethyl-3-3-dimethylaminoproply carbodiimidehydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide) acts as a connector between the ssDNA andmagnetic nanoparticle. The other ssDNA named probe
DNA was labeled with NH2 at 50 terminal and ﬂuorescent
Cy3 at 30 terminal. This NH2 acts as a linker between

Peak to peak value [mΦo ]103

3. Measurement

1021011000.01

0.1
Mol of particles [pmol]

1Fig. 7. Magnetic signal vs. number of particles. Volume of the sample is
4 ml. The signal is almost proportional to the number of the particles.

ssDNAs and the substrate and Cy3 was used for conﬁrming
whether the ssDNAs were anchored on the substrate.
First, we prepared particles connected with ssDNA. The
concentration of the particles and that of ssDNA are 0.856
and 2.35 mM, respectively. This ratio determines thenumber of ssDNA on the surface of the particle. According
to this ratio 2.35/0.856, it is estimated that one particle has
about three ssDNA on its surface. Then the solution
containing the particles with ssDNA was diluted to prepare
solutions with different concentrations. These solutions
were used as sample DNA through the experiments.
Second, we prepared probe DNA with a concentrationof 10 mM, which is labeled with NH2 at 50 terminal andﬂuorescent Cy3 at 30 terminal. A volume of 2 ml of theprobe DNA was taken and put on an aldehyde-coatedglass substrate, whose dimension is the same as the sample
carrier shown in Fig. 3. A volume of 2 ml corresponds to20 pmol of probe DNA. Here we investigated how muchprobe DNA can be anchored on the substrate before
hybridization. Fig. 8 shows the ﬂuorescence microscope
image from Cy3 of probe DNA on the aldehyde-coatedsubstrate. (a) and (b) show the image of before wash andafter wash respectively and (c) indicates schematic of theDNA on the substrate. All probe DNA including someﬂoating DNA can be seen before wash. After the wash theonly probe DNA anchored to the substrate can be seen, therest having been washed away. Hence, we estimate thatARTICLE IN PRESSe318

S. Tanaka et al. / Journal of Magnetism and Magnetic Materials 300 (2006) e315–e319

Peak to peak value [mΦo ]100101110
Mol of ssDNA [pmol]

100

Fig. 9. Magnetic signal vs. mol of sample DNA. Horizontal axis shows
the amount of sample DNA applied to the probe DNA. Signal showssaturation at about 5 pmol of DNA.

Fig. 8. Fluorescence image of probe DNA on the aldehyde-coatedsubstrate. (a) Before wash. (b) After wash. (c) Schematic drawing of theDNA on the substrate. After washing ﬂoating DNA, the amount of theDNA decreased.

1–5% of probe DNA remained corresponding to0.2–1 pmol of DNA. Although this efﬁciency should beimproved in the future by optimizing the condition of thereaction between NH2 and aldehyde, this anchored DNA
can be used for following hybridization experiments.

of residual sample DNA involved in the hybridization on
the substrate must be smaller than this amount because theexcess DNA was washed before the measurement. Therefore, we estimated the real amount of DNA involved in thehybridization using a relation between the signal and thenumber of particles as shown in Fig. 7. By this estimation,
it is known that the 5 pmol of DNA in Fig. 9 corresponds
to 0.07–0.2 pmol of the nanoparticles. Since one particle
has about three ssDNA, the amount of DNA involved in
the hybridization can be calculated and becomes
0.2–0.6 pmol. This amount is almost consistent with theamount of probe DNA on the substrate after wash. Thus
the properties on Fig. 9 implies that the signal increases
along with the amount of the sample DNA and then shows
the tendency of saturation at the amount of the probeDNA on the substrate. Therefore it is found that the signal
does not increase even if the amount of the applied sample
DNA was increased at more than 0.2–0.6 pmol, because thereaction is determined by the amount of probe DNA on thesubstrate. This result indicates that the hybridization wassuccessfully done.

3.3. Hybridization
Here, we tried to make hybridization on the substrate.
After applying 20 pmol of the probe DNA, unbound DNA
was washed. Then the particles with sample DNA wereapplied to the substrate. After washing away the excess
DNA, which was not hybridized with probe DNA, thesample was measured. Several sample DNA with different
concentration were applied to the probe DNA. The
concentration of the probe DNA was constant and20 pmol. Fig. 9 shows the relation between the magnetic
signal and the mol of sample DNA. Signal shows atendency of slow increment more than at about 5 pmol ofthe DNA.
4. Discussion
The horizontal axis in Fig. 9 shows the amount ofsample DNA applied to the probe DNA. The real amount

5. Conclusions
DNA detection system using a high-Tc SQUID magnetometer was constructed and demonstrated. Single strandDNA (ssDNA) with relatively short length, 30 base pairs(Human Papillomavirus Probes 33) was used. A probe
DNA was anchored on aldehyde-coated substrate via theamino group NH2. Then a sample DNA labeled withnanoparticles Fe3O4 was applied to the substrate so thatthe hybridization occurred. After wash of excess DNA, themagnetic signals from the particles are measured under AC
magnetic ﬁeld by a SQUID. As a result, it is found that thesignal does not increase even if the amount of the applied
sample DNA was increased at more than 0.2–0.6 pmol,
because the reaction is determined by the amount of probe
DNA on the substrate. This result indicates that thehybridization was successfully done. At next stage,

ARTICLE IN PRESSS. Tanaka et al. / Journal of Magnetism and Magnetic Materials 300 (2006) e315–e319

negative results in case of the sample DNA that is notcomplementary each other should be performed.
Acknowledgements
This work was supported in part by a Grant-in-Aid for
Science Research (B) from the Japan Society for thePromotion of Science, and The 21st Century COE Program‘‘Ecological Engineering for Homeostatic Human Activities’’ from the Ministry of Education, Culture, Sports,Science and Technology.
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