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Available online at www.sciencedirect.com

Sensors and Actuators B 128 (2007) 1–4

Letter to the Editor

agnetoresistive biosensors based on active guiding of magnetic particles towards the sensing zone

bstract

Magnetoresistive biosensors, using superparamagnetic particles as a label, have shown to be promising candidates for highly sensitive biosensors.ne challenging task remains a further increase of their sensitivity. In the past, most research groups have realized this by increasing the sensitivityf the sensor. In this letter, we present a novel detection mechanism for these biosensors that increases their sensitivity by releasing specificallyound magnetic particles followed by their active guiding towards the sensing zone. For this, we first bind particles specifically to the sensor

urface using a sandwich assay. In a following step, we release these particles by breaking the biomolecular bonds using a biochemical regenerationolution. Finally, the particles are repositioned to that location that gives rise to a theoretical maximum signal. Experimentally, a sudden increasen the sensor signal is observed during the latter step, which demonstrates the enhancement technique.

2007 Elsevier B.V. All rights reserved.

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eywords: Magnetoresistive sensors; Magnetic biosensors; Superparamagnetic

. Introduction

Recently, magnetoresistive technology in combination withuperparamagnetic particles has shown to be a promising plat-orm for the realization of highly sensitive biosensors. In such aiosensor, superparamagnetic particles are bound to the surfacet places where the analyte was bound to the sensing layer. In aext step, these particles are magnetized using an external mag-etic field which allows the detection of their presence using aagnetic sensor (e.g., magnetoresistive sensor).In 1998, Baselt et al. introduced the BARC-platform, a mag-

etoresistive biosensor that uses a GMR multilayer as a magneticensor [1]. The introduction of the BARC-platform inspired aumber of research groups to improve this already sensitiveiosensor technology in order to further lower its detection limit.n the past, improving biosensor sensitivity has been mainlyealized by modifying the sensor geometry or by increasing theensitivity of the magnetic sensor itself. The latter can easily beealized by replacing the GMR multilayer by a more sensitivepin-valve sensor.

In addition to on-chip detection, magnetic particles offer thenique possibility that they can be manipulated by applyingontrolled magnetic forces. In this regard, Graham et al. haveeported on the combined transport and sensing of particles inrder to increase the assay speed [2].

However, when spin-valve sensors were proposed as a moreensitive alternative to GMR multilayers, it became clear that

hese sensors cannot be used in combination with the preferrederpendicular magnetization field, as a result of their linear trans-er curve in response to an applied magnetic field curve. In a

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925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2007.05.023

cles; Particle manipulation

revious paper, we have described a model that is capable ofredicting the response of a spin-valve sensor for a single parti-le with a perpendicular magnetization crossing the sensor alongts sensitive axis [3]. Fig. 1(a) shows the result of such a simu-ation of the sensor signal, plotted as a function of the in-planeistance between the sensor and the particle. From this figure its clear that, due to the dipolar character of the particle signal,he signal of a particle bound along one side of the sensor cane partially cancelled by a particle placed at the opposite sidef the sensor. Consequently, the signal of perfectly randomizedarticles is expected to become zero from theory.

A second point of concern, which is also observed fromig. 1(a), is the strong dependence of the particle signal on theistance. This dependency causes the average signal of random-zed particles to become lower than the theoretical maximumhose value is equal to the peak value of the signal shown inig. 1(a).

Some research groups have circumvented the first problemy replacing the perpendicular magnetizing field by an in-planeeld [4,5]. However, as magnetoresistive sensors are mainlyensitive to in-plane magnetic fields, its magnitude needs to beimited in order not to saturate the sensor. As this reduction of the

agnetic field also lowers the magnetic moment of the super-aramagnetic particle, the sensitivity increase resulting from these of spin-valves is partially sacrificed. Other than enhancinghe sensitivity of the biosensor by improving the detection ofound particles, we present in this paper a detection mecha-

ism that is based on the repositioning of particles towards anptimal sensing location. The data presented in this paper willemonstrate that this alternative detection mechanism not only

2 Letter to the Editor / Sensors and Actuators B 128 (2007) 1–4

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ig. 1. (a) Simulated sensor signal for a single particle with a perpendicular man-plane and z-component of the magnetic field generated by a bar-shaped cond

olves the problem related to the orientation of the magnetizingagnetic field, it also removes the position dependence of the

article signal and increases it to a theoretical maximal value.

. Theory

The repositioning mechanism that is presented in this paper,s implemented using the magnetic field (gradient) generated byhe sensor itself. Fig. 1(b) shows a simulation of the y- and z-omponent of the magnetic field generated around a bar-shapedonductor when a current is being sent through. Based on thisimulation, one can calculate that the total field becomes maxi-al on top of the sensor. As a result, in absence of an externaleld particles are attracted towards this location as they are free

o adjust their magnetization. However, when a perpendicularagnetic field is applied, the particles obtain a perpendicu-

ar magnetization and move towards the location where the-component of the magnetic field becomes maximal, which isow located above one of the sensor edges. Fig. 1(a) illustrateshat the particle signal becomes maximal at nearly the sameosition. In other words, a combination of the magnetic fieldradient generated by the sensor and a perpendicular magneticeld positions the particles to that location that gives rise to a

heoretical maximal signal.

. Materials and experimental

In order to demonstrate this physical phenomenon, weesigned a biosensor device that consists out of seven spin-alves connected in parallel. First, the spin-valve structure (Ta.5 nm/Ni80Fe20 4.5 nm/Co90Fe10 0.5 nm/Cu 1.9 nm/Co90Fe10.5 nm/Ir80Mn20 7 nm/Ta 2.0 nm/TiW 5 nm) was sputtered andatterned into rectangular 100 �m × 2 �m structures by ion-illing. The distance between two neighbouring sensor strips

s 12 �m, such that the seven sensors form a rectangular area

f 100 �m × 100 �m. Next, the contact leads (TiW 10 nm/Au50 nm/TiW 10 nm) were evaporated and patterned using a lift-ff process followed by the deposition of the passivation layer250 nm thick PE-CVD Si3N4). We then deposit a thin gold

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ation crossing a spin-valve sensor along its sensitive axis. (b) Simulation of the.

ayer (TiW 10 nm/Au 50 nm) on top of the rectangular sensorrea to act as a seed layer for the alkane thiol-based chem-stry that is used for immobilizing the biomolecules. Finally,he device was packaged and an epoxy ring was deposited inrder to protect the wirebonding. After fabrication, the sensorhows a magnetoresistance ratio of 5.6% and a sensitivity of.5%/(kA m).

In a next step, a sandwich assay was developed for the detec-ion of the stroke marker S100��, which was targeted as a modelystem. To allow coupling of the primary antibody, we firsteposit a mixed self-assembled monolayer (SAM) consisting outf 5% 16-mercapto-1-hexadecanoic acid and 95% 11-mercapto--undecanol [6]. Once the monolayer is formed, the primaryntibody (S53, CanAg Diagnostics AB, Sweden) is covalentlyoupled to the SAM using carbodiimide-coupling chemistry fol-owed by a bovine serum albumin (BSA) and a triethyleneglycollocking step. More specifically, the thiol modified gold samplesere statically incubated for 10 min with a 1:1 (v/v) mixture of.4 M EDC:0.1 M NHS, both dissolved in water, followed by ainse of 10 mM acetate buffer (pH 5.0). The activated samplesere then statically incubated with 100 �g/ml primary antibodyiluted into 10 mM acetate buffer (pH 5.0) for 30 min followedy a blocking step of 10 mg/ml BSA diluted into 10 mM acetateuffer (pH 5.0) for 1 h. Next, the samples were blocked with 1 Mriethyleneglycol monoamine dissolved into water for 30 min.amples were washed with HBS* (HEPES buffered saline with0 mM HEPES, 150 mM NaCl, 1 mM CaCl2 and 0.005% TW-0) in between the different incubations steps. Following thelocking steps, the samples were stored O/N at 4 ◦C. The com-ination of the SAM and the primary antibody plus blocking iseferred to as the sensing layer of the biosensor. In a next step,his sensing layer allows the binding of the analyte (100 ng/ml100��), followed by the binding of a biotinylated secondaryntibody (S36, CanAg Diagnostics AB, Sweden). To this end,he samples were incubated with S100�� in HBS* for 90 min

ollowed by an incubation step of 100 ng/ml biotinylated S36 inBS* for 90 min. The biotin molecules present on the secondary

ntibody ensure a strong coupling between the sandwich assaynd the 300 nm streptavidin coated particles (Bio-Adembeads,

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Letter to the Editor / Sensors

demtech, France). The samples were incubated with 1 mg/mltreptavidin-modified magnetic particles for 30 min. After thearticle incubation step, particles that did not bind specificallyo the assay were washed away using HBS*. Non-specific bind-ng of particles was evaluated by replacing the primary antibodyith an antibody that specifically recognizes human transferrin

HT). In this case, very little particles were found to bind to theurface.

In order to allow specifically bound particles to be repo-itioned, these particles need to be released by breakingregenerating) the antibody–analyte bonds that fix them to theurface. The release of the particles can be achieved by applyingegeneration solutions commonly used in protein purification7]. In addition to the release of particles, this regeneration solu-ion also has to be chosen such that it does not suppress the

obility of particles [8]. For our experiment, we found that a0 mM NaOH solution (combining a high pH value and a lowonic strength) forms the most suitable regeneration solution.lthough such a regeneration step would in principle allow re-se of the sensor devices, we have chosen to use new chips forach measurement reported.

. Results and discussion

Following sandwich assay formation and binding of mag-etic particles, the biosensor chip is placed under a microscope,quipped with a CCD camera to optically monitor the movementf the particles, and connected to a battery-powered home-madeead-out system. This system sends a stable current through theensor, removes the DC offset and amplifies the sensor signalith a factor 100 before being read by a DAQ card installed in the

C. During the measurement, a uniform perpendicular magneticeld of 6.4 kA/m was applied to magnetize the particles.

Fig. 2 shows the result of the experiment. First, the group ofensors is linearized using a current of 20 mA. As this current

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ig. 2. Sensor signal at different steps of the experiment. In step 1, the sensor outpuolution is carefully applied releasing the particles from the surface. The release of phe sensors. As the particle signal becomes maximal at this location, the signal increaurface causing the sensor signal to return to zero.

Actuators B 128 (2007) 1–4 3

s also responsible for repositioning the particles, the currentirection is chosen such that the field generated is oriented alonghe same direction as the magnetic stray-field originating fromhe pinned layer of the spin-valve. During the initial step, theeservoir of the biosensor, formed by the epoxy ring that protectshe wire bonds, remains filled with HBS buffer, as drying theurface was found to create problems for releasing the particles.ig. 2 illustrates that the sensor signal of a random distributionf particles is close to zero as is expected from theory (step 1).n a following step, we carefully replace the HBS buffer with a0 mM NaOH solution using a pipette. The release of particles,riggered by adding this NaOH solution, is immediately followedy a movement of the particles towards one edge of the sensors.his movement of particles is accompanied by a sudden changef the sensor signal, which increases to about 6 mV (step 2).he repositioning of particles is illustrated on the microscopic

mages in Fig. 2. In a final step, we use a pipette to generate atrong flow that flushes all particles away from the sensor area.s no particles remain present on top of the sensor (step 3), weefine this sensor signal to be the zero signal.

The first advantage of this repositioning technique is thathe sensitivity of the biosensor is enhanced as the particle signalncreases to a maximal value. Another advantage results from theact that regenerating the biomolecular bonds should only affectpecifically bound particles. Non-specifically bound particlesre therefore not released and contribute less to the total signal.s such, it may also improve the specificity of the biosensor.ne possible drawback is that repositioning the particles willost likely lead to a faster saturation of the sensor area, whichost likely limits the dynamic range.We conclude that the ability to combine both manipulation

nd detection, which is a unique property of magnetic parti-les that has not been fully explored, is a promising techniqueo improve on an already sensitive magnetoresistive biosensorechnology.

t is small due to a random distribution of particles. In step 2, the regenerationarticles is immediately followed by a repositioning above one of the edges ofses to a theoretical maximal value. In step 3, all particles are flushed from the

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Letter to the Editor / Sensors

cknowledgements

R. Wirix-Speetjens and R. De Palma thank the Institute for theromotion and Innovation through Science and Technology inlanders (IWT-Vlaanderen) for financial support. The authors

hank Stijn de Jonge and Erwin Vandenplas for assistance inrocessing, INESC-MN (Portugal) for the deposition of spin-alve structures and Dr. Olle Nilsson (CanAg Diagnostics AB,weden) for valuable discussions on the S100 assay and forindly supplying the necessary reagents.

eferences

1] D.R. Baselt, G.U. Lee, M. Natesan, S.W. Metzger, P.E. Sheehan, R.J. Colton,A Biosensor based on magnetoresistance technology, Biosens. Bioelectron.13 (1998) 731–739.

2] D.L. Graham, H.A. Ferreira, P.P. Freitas, Magnetoresistive based biosensorsand biochips, Trends. Biotechnol. 22 (2004) 455–462.

3] R. Wirix-Speetjens, W. Fyen, J. De Boeck, G. Borghs, Single magneticparticle detection: experimental verification of simulated behaviour, J. Appl.Phys. 99 (2006) 103903.

4] H.A. Ferreira, N. Feliciano, D.L. Graham, L.A. Clarke, M.D. Amaral, P.P.Freitas, Rapid DNA hybridization based on ac field focusing of magneticallylabeled target DNA, Appl. Phys. Lett. 87 (2005) 013901.

5] G. Li, V. Joshi, R.L. White, S.X. Wang, J.T. Kemp, C. Webb, R.W. Davis, S.Sun, Detection of single micron-sized magnetic bead and magnetic nanopar-ticles using spin valve sensors for biological applications, J. Appl. Phys. 93(10) (2003) 7557–7559.

6] F. Frederix, K. Bonroy, W. Laureyn, G. Reekmans, A. Campitelli, W. Dehaen,G. Maes, Enhanced performance of an affinity biosensor interface based onmixed self-assembled monolayers of thiols on gold, Langmuir 19 (2003)4351–4357.

7] K. Anderson, M. Hamalaien, M. Malmqvist, Identification and optimizationof regeneration conditions for affinity-based biosensor assays. A multivariatecocktail approach, Anal. Chem. 71 (13) (1999) 2475–2481.

8] R. Wirix-Speetjens, W. Fyen, K. Xu, J. De Boeck, G. Borghs, A force studyof on-chip magnetic particle transport based on tapered conductors, IEEETrans. Mag. 41 (10) (2005) 4128.

iographies

oel Wirix-Speetjens obtained his masters in electrical engineering at theatholic University of Leuven in 2001 and his PhD in engineering in 2006

rom the same university. His PhD work was focusing on the manipulation andetection of magnetic particles and the design and development of a novel mag-etoresistive biosensor. Currently, he works as an technical consultant in theanking sector.

unter Reekmans obtained his masters in biochemical engineering at theniversity College of Limburg (KHLim) in 1991. His graduate work was on

he purification and partial characterisation of an enzyme called calf pregas-

ric esterase. After graduation, he worked as a research assistant at the Centeror Human Genetics (Catholic University of Leuven) in the laboratory of Prof.uido David. Since 2002, he joined the Biosensor group (currently MCP-ART)

t IMEC, where he is working as a senior technician. Throughout, he has built upxperience in protein/protein interaction chemistry, biosensor research, surface

Actuators B 128 (2007) 1–4

hemistry, molecular biology and tissue culture. Currently he is working on theevelopment of magnetic and optical biosensors for biomedical applications.

andy De Palma obtained his masters in physical and analytical chemistry at theatholic University of Leuven in 2002. His graduate work was on the character-

sation and optimisation of oxide-based biosensor interfaces for immunosensorpplications. Since 2002, he joined the Biosensor group (currently MCP-ART)t IMEC where he is working towards a PhD. Currently he is working on theevelopment of magnetic biosensors and on the synthesis and functionalizationf magnetic nanoparticles for biomedical applications.

hengxun Liu obtained his masters in biophysics from Tsinghua University,hina, where he worked on microfluidic actuation in lab-on-a-chip systems.ince 2004 he is a PhD student at the Catholic University of Leuven. His PhDork is mainly the on-chip manipulation and detection of cells with magnetoflu-

dics and is being conducted at IMEC.

im Laureyn obtained his masters in physical and analytical chemistry at theatholic University of Leuven in 1997 and his PhD in chemistry in 2002 from

he same university. His PhD work was focusing on the characterisation andptimisation of oxide-based biosensor interfaces for immunosensor applicationsnd on the use of interdigitated electrodes for impedimetric biosensing. Forhis work, carried out in collaboration with IMEC, he was awarded with thelaverbel Chem’Award 2002. Currently, he is responsible for several EU andational projects within the MCP-ART Group of IMEC. His current researchnterests are the use of functional nano-bio assemblies and self-assembly forhe development of ultra-sensitive biosensors based on micro-electronics andanotechnology.

ustaaf Borghs obtained his PhD in science (physics group) in 1980 at theniversity of Leuven (Belgium) for research in nuclear physics. From 1981 tille joined IMEC in 1984, he worked in the field of atomic physics and hyperfinenteractions at the same university. After a sabbatical at the CNRS in Sophiantipolis, France in 1985 he was a group head at IMEC responsible for research

n compound semiconductor materials and devices for high frequency applica-ions and solar cells. As a department director he started new research activitiesn magneto-electronics, plastic electronics and bioelectronics. He was associateice-president of the MCP Division till 2003. Currently he heads the MCP-ARTroup, with research activities on biosensors, functional nanoparticles, neuro-

lectronics, magneto-electronics and III–V semiconductors. As IMEC researchellow he is responsible for the long-term scientific strategy of the MCP Divi-ion working on micro- and nano-systems, materials research and packaging. Heublished over 300 papers in refereed journals. Gustaaf Borghs is also part-timerofessor at the University of Leuven.

Roel Wirix-SpeetjensGunter ReekmansRandy De Palma

Chengxun LiuWim Laureyn ∗Gustaaf Borghs

Interuniversity Microelectronics Center (IMEC), Kapeldreef75, Heverlee 3001, Belgium

∗ Corresponding author.E-mail address: Wim.Laureyn@imec.be (W. Laureyn)

13 March 2007

Available online 25 May 2007