Biosensors

The employment of magnetic labels brings many advantages for biosensing: for example, it is possible to perform analyte separation from a solution followed by direct measurement and quantification. Considering that other biological cells and compounds are typically non-magnetic, this screening procedure is usually very effective and furthermore the magnetic background is negligible in such a measurement. This kind of particles are often synthetized with a silica coating, which let them be biocompatible for in vivo applications and more easy to functionalize with antibodies or aptamers able to bind a broad range of analytes.

Figure 1.11: Principle of detection for a MR based sensor. (a) DNA probe immobilization, (b) Hybridization of the analyte DNA, (c) binding of the marked magnetic particles ³⁷.

22 A population of MNPs embedded in an external magnetic field tend to have a parallel magnetization with respect to its direction, exerting a stray field on a sensor in their vicinity, whose response will be influenced³⁸. In this respect, for achieving a larger sensitivity, it is crucial to obtain a good compromise between the structure dynamic range and the particles’

magnetization, since it’s necessary to choose an external magnetic field value not so large, in order to avoid saturation of the response, but only not too small because in that case particles’

magnetizations results randomly oriented. Furthermore, it can be useful to add a superimposed AC magnetic field³⁹ and to choose the right working point to have a better signal to noise ratio.

Regarding the latter, considering that the magnetic field generated by a dipole decays as the third power of the distance, it’s necessary to have the particles as close as possible to the sensor’s surface⁴⁰, using, for example, a sandwich-like configuration between a ligand-acceptor pair as sketched in Figure 1.11. Several strategies have been developed for this purpose, starting for example³⁷ with a sensors’ functionalization with a double strand DNA with biotin, able to bind streptavidin present on the MNP, in order to have the particles at a submicrometric distance from the sensing structure.

Figure 1.12: Bead Array Counter (BARC) developed and realized by Baselt et al. in 1998⁴¹. The first idea to employ magneto-resistive structures also for particles’ sensing date back to 1998, when Baselt et al. developed a Bead Array Counter (BARC)⁴¹ based on multiple GMR junctions, in order to perform a fast and easy bio-recognition procedure. Their device is shown in Figure 1.12. Several years later (2004), Schotter et al. performed a comparative study between the performances of GMR-based sensors and standard fluorescent transduction,

23 showing a better sensitivity of the former in the concentration range of interest³⁷. Specifically, the basic sensing element (there are 206 in the device) consists in a double spiral patterned on a GMR stack as shown in Figure 1.13 (a).

This promising result led to the development of a wide number of studies about spintronics devices for biosensing applications, in order to maximize their performances. Since the sensitivity of a GMR based sensor increases while decreasing its volume but, on the contrary, this leads to a smaller available signal, one good compromise could be a layout with a dense array of structures in the sub-micron range, as reported by Wood et al.⁴² in 2005. In this work, a proof of concept of magnetic particles’ detection was performed, where their magnetic moments were simulated with a magnetic SPM tip placed above the sensing surface at the typical distance of a sandwich assay (about 300 𝑛𝑚) and exerting a field comparable with 2 𝜇𝑚 sized commercial particles. However, to evaluate quantitatively bio-recognition events, the markers should be smaller of a couple of orders of magnitude, so that they can bind one or at least few analytes, but in this scenario the magnetic signal becomes smaller and it’s possible to reach the paramagnetic regime, where a stabilization is needed⁴³. Li et al. demonstrated in 2006 the chance to measure quantitatively the presence of magnetic nanoparticles of few nanometers on top of sub-micron optimized GMR structures in the range of 20 − 200 𝑛𝑚, also integrating some permanent magnet in their device as fixed magnetic bias. Further evolution in the nanoscale range with highly dense arrays led to measure quantitatively a solution until the notable detection limit of 20 𝑧𝑚𝑜𝑙 ⁴⁴.

Figure 1.13: Sensing element of the device realized by Schotter et al.³⁷ (a) and the results of comparative studies performed about sensitivity of their GMR based device and fluorescence-based

methods (b).

(a) (b)

24 Exploiting the sub-micron range for sensors dimensions can provide some disadvantages for industrial applications and mass production, because it requires nanofabrication techniques such as EBL which are expensive and slow. For this reason, in order to enhance the sensitivity with structures in the UV Lithography fabrication range, some alternatives were sought, for example by developing sensing circuits more complex than a single magneto-resistor. In this framework, Millen et al. organized a network of GMR structures arranged in a Wheatstone bridge geometry able to quantify the concentration of superparamagnetic particles in the picomolar range⁴⁵. Another approach for optimizing DNA detection with this technology was developed by M. Koets et al. ⁴⁶: by taking advantage of PCR amplification for the LamB gene of E. Coli, it has been possible to measure it quantitatively in the range 4 − 250 𝑝𝑀 with single big GMR sensors (3 𝜇𝑚 x 100 𝜇𝑚). In their devices, the magnetic tags were 300 nm superparamagnetic particles magnetized with on chip current lines, which could provide a bias field due to the Biot-Savart law. In this case, there was a baseline signal due to the current which needed to be subtracted from the particles’ one, obtaining a smooth sensitometric response in the range of interest. Furthermore, magnetic manipulation led to a very fast response compared to previous works, with few minutes needed for obtaining stable results. A further evolution of this kind of system was developed by Zhi et al.⁴⁷, with a modular system consisting by a isothermal amplification module, microfluidic and GMR sensing part with a limit of detection of 10 𝑐𝑜𝑝𝑖𝑒𝑠/𝑚𝐿.

A very useful application of magnetic detection is in the mycotoxin field, considering that this kind of analyte is very small compared to others (< 300 𝑘𝐷), which is usually a problem for other detection methods. In this case, the signal is not provided by mass or volume of the analyte, but only by the magnetic tag: for this reason Mak et al. ⁴⁸ developed an assay able to measure different kinds of mycotoxin in the 𝑝𝑔/𝑚𝐿 range, which is as low as in the case of more complex and expensive methods based, for example, on Surface Plasmon Resonance.

In 2015, Wang et al. ⁴⁹ developed a probe station like system able to multiplex protein detection in a single cartridge in standard clinical concentration range, putting the basis for the application of this technology in mass-produced Point OF Care (POC) devices. A year later, Choi et al.⁵⁰ further optimized and integrated a similar system, obtaining a portable device which can monitor IgG and IgM antibodies levels within one step for self-diagnosis that can be visualized and stored through a simple smartphone interface. The complete device is shown in Figure 1.14

25 and consists in a cartridge reader (similar to the equipment for the home monitoring of glycemia already available on the market) that can be employed with mono-usage cartridge.

Figure 1.14: Complete Point of Care platform for multiple immune-detection in a single cartridge developed by Choi et al.⁵⁰ also equipped with a simple smartphone interface.

The first proof of concept of the possibility to employ Planar Hall Effect based sensors for magnetic particles’ sensing was performed in 2005 by Ejsing et al.⁵¹, whose approach is reported in Figure 1.15. They realized a simple but effective trilayer device able to detect few 2 𝜇𝑚 sized commercial magnetic beads, which were largely employed, after functionalization, in biological laboratory processes such as separation and purification protocols.

Later, it was also demonstrated the chance to detect a single 2.8 𝜇𝑚 particle simulated by the photolithography of a single dot in the center of a PHE based sensor surface⁵², which resulted in a shift in the response during the characterization in a static magnetic field. Also the effective detection of Dynabead M-280 coated with streptavidin was performed in 2007 by Thanh et al.

53 , employing 3 𝜇𝑚 x 3 𝜇𝑚 structures in a spin-valve configuration, while an improved device developed by Quang Hung et al. ⁵⁴ in 2010 was able to get the impressive value of 1450 for the signal to noise ratio for beads detection.

26 Figure 1.15: Planar Hall Effect based device for particles sensing developed by Ejsing et al. in 2005⁵¹. Systematic studies have been performed in order to optimize this kind of devices to pave the way for application in the biosensing field, for example regarding the influence of the thickness of free and pinned layer for the spin valve configuration⁵⁵, obtaining that the larger is the former, the higher sensitivity shows the device, while the latter is correlated to this parameter in the opposite way. Furthermore, it has also been demonstrated that the employment of Wheatstone Bridge geometry can lead to the enhancement of the signal by a geometrical factor⁵⁶. A further improvement in sensitivity has been achieved by Hung et al.⁵⁷ (Figure 1.16), who got the impressive limit of detection value of 4 ⋅ 10 𝑒𝑚𝑢, about 3 orders of magnitude more powerful than expensive and complex systems based on the employment of superconductive quantum interference devices (SQUID).

For integrating Planar Hall effect-based sensors in a Lab On Chip platform, it is necessary to implement also a microfluidic module able to handle small volumes of fluid containing the magnetic nanoparticles (MNPs), specifically to drive them very close to the sensing surface, considering the third power trend of reduction of a dipole magnetic field. One example in this kind of applications is the work of Jeong et al. ⁵⁸, who demonstrated the capability of detection of 2 ⋅ 10 𝑒𝑚𝑢 in presence of a thick passivation layer between sensing surface and particles, necessary to avoid fluid leakage which can lead to shortcut between terminals, so obtaining a perfectly reusable device.

27 Furthermore, by increasing the packaging of the structure with a meander-like shape arranged in Wheatstone bridge geometry, Hansen and Rizzi have demonstrated the possibility to measure particles’ concentration down to 4 𝑝𝑀 with this kind of sensing circuit, after an optimization of measurement parameters, especially the frequency⁵⁹.

Figure 1.16: SEM image of the PHE ring realized by Hung et al.⁵⁷, able to measure very low magnetic moment and resulted more sensitive than SQUID technology.

The first theoretical proof of concept of magnetic particles detection by Tunnel Magneto-Resistance junctions was performed by Schepper et al. in 2004⁶⁰. In this work, micromagnetic simulations technique was employed to study the influence of magnetite superparamagnetic beads on the response of tunnel magneto resistance structures consisting in a Permalloy free layer, an 𝐴𝑙𝑂 barrier and a 𝐶𝑜𝐹𝑒 reference layer in two configurations of magnetization: in plane and out of plane. They also studied the spin texture of the system in the first case. They showed, in principle, that the use of TMR sensors could lead to a limit of detection of some tens of particles, an impressive result for that period, especially considering the micrometer in plane dimensions of the structures. One year later (2005), Grancharov et al.⁶¹ performed the experimental detection of magnetic nanoparticles using Magnetic Tunnel Junctions (MTJs) above a functionalized 𝑆𝑖𝑂 passivation layer, with the observation of a change in the shape of the response Vs an applied magnetic field.

As in the previous cases, the optimization in the development of TMR based devices covered both the sensors performances and the realization of complex sensing circuits able to reduce thermal drift and to enhance sensitivity. For example, Shen et al.⁶² developed a Wheatstone bridge-based device, where each component consists in a series of 16 MTJs. In this condition,

28 they were able to get a limit of detection of few 𝑛𝑀, corresponding to about 30 𝑝𝑔/𝜇𝐿 of target DNA strand. MTJs’ employment in liver cancer detection showed promising results in Lei et al. work⁶³, since they showed an excursion dependence of the response as a function of the magnetic field with different numbers of cancer cells immobilized onto the surface with antibodies.

An alternative sensing method, closer to fluorescent imaging and quantification than the simple measurement of magnetoresistance, was proposed by Chan et al.⁶⁴ in 2011. Their readout technique was similar to the standard employment of TMR in hard drive read head, letting such a device scan a functionalized matrix able to immobilize MNPs bounded with an analyte to be revealed. In this case, any “pixel” shows a signal proportional to particles’ number, which can be integrated over a square of 100 𝜇𝑚 x 100 𝜇𝑚 area to obtain the map of the local concentration of the solution spotted. They obtained in this way a limit of detection of 10 𝑝𝑀.

An improvement of this method was developed by Vyas et al.⁶⁵ through the employment of an Anderson Loop for the local magnetic field detection, which lets their system able to obtain a noise reduction in the signal acquisition, achiving a limit of detection for the particles stray field of about 0.006 %/𝑂𝑒. Another possible technique of detection was studied both theoretically and experimentally by Albisetti et al.³⁹ in 2013 who derived the signal obtained by the superposition of an AC magnetic field and a DC: the latter was used to define the working point of the sensor, while the former was employed for magnetizing the particles. In this case, they found that the highest sensitivity (for MNPs detection) is at a working point where the product between the field and the second derivative of the resistance is maximum.

Figure 1.17: Flexible TMR based device realized by Chen et al.⁶⁶ in 2017.

29 During the last years, one of the most important challenge in biosensing field is the development of wearable devices, in order to have the chance to monitor in real time people’s activities. In 2017, Chen et al.⁶⁶ developed an innovative procedure able to obtain powerful TMRs’

structures over a flexible substrate (Figure 1.17), with high reusability (over 1000 bending cycles without losing in performance), proving the possible applications of MTJs also in this growing field.

Chapter 2: Fabrication and characterization of Magneto-Resistive