Good morning, I’m David Lago-Cachón, from the University of Oviedo and today I’mgoing to talk about our work in applying magnetic nanoparticles to biologicalapplications.

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As this is a very multidisciplinary task, this work has been carried out (performed) in the Physics Department of the University, the Central Hospital of Asturias and a company in the sensoring field, Healthsens.

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In fact, our objective is to develop an early detection method of illneses usingmagnetic nanoparticles as tags, and a magnetic sensor to detect them.

Briefly, the method consists of the following steps:First, a liquid sample from the patient (i.e. blood) has to be obtained.Second the sample is mixed with magnetic nanoparticles (MNP). Those MNP must be functionalized so that they join specifically that we want to detect.Third, the remanent MNP are then removed.

Finally, are detected by a magnetic sensor which allows detection and quantificationof the biological entity.

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So, we have two parallel jobs: On one hand, the development of convenientlyfunctionalized magnetic nanoparticles designed for that particular biological issue, which is mainly what i’m going to talk about right now.And on the other, the development of a suitable magnetic sensor to detect thosenanoparticles.

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Let’s start from the beginning. These are the magnetic nanoparticles that we workwith. They are magnetite nanoparticles made by a co-precipitation method and… /next/

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… covered by PolyAcrilyc Acid (PAA).

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The mean size of the nanoparticles can be obtained from electronic microscopy like this one, and it is 10 nm. That’s a sufficiently small diameter for the MNP to be superparamagnetic.

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The SPM state was confirmed by Field Cooling, Zero Field Cooling experiments, as this one, that shows a Blocking Temperature of about 130 K, far below room temperature.

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Now, as we wanted the nanoparticles to tag specifically to a biological entity, we hadto functionalize them. That means, we incorporate an antibody on the surface of thenanoparticles.

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As you probably know, antibodies are a very usefull kind of proteins that have twodetection regions, at these ends, that bind other proteins or molecules with a veryhigh specifity.

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The strategy we follow to functionalize the MNP was a streptavidin-biotin reaction. That protein and that molecule have a great affinity for each other and are frequentlyused to conjugate things. As many antibodies are comercially available with a biotinadded to them, we only focused on covering the nanoparticles with streptavidin… /next/

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… via an amida bound.

To check if our functionalization has been successful we made several Fourier Transform Infra-Red Spectroscopy of the original nanoparticles, covered with polyacrilic acid, and the final nanoparticles covered with streptavidin and the antibodies. The carbonil (C=O), carboxilate groups and –CH2, disappear in the functionalized nanoparticles, at the same time that nitrogen functional groups appear.

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Also, we measured the hidrodinamic size of the nanoparticles before and afterconjugation. As you know, the hydrodynamic size have into account the surfactantand the interaction with the molecules of the solvent. The primary MNP covered withPAA has a main hydrodynamic size of 60 nm, and the magnetite nanoparticlescovered firstly with streptavidin and secondly with an antibody has a bigger size of about 300 nm.Now that the MNP has been characterized I’m going to present you the work we havedone with them.

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We performed some in vitro experiments with a human tumor cell line, the HeLacells. Why have we chosen that cell line? Two reasons. First one, they are easy growing cells, and the most important is that this kind of cells express in their surface a protein, called MICA, that is mainly related to several illnesses. So, now we do our in vitro experiments, and later it can be done with a patient sample.

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I said that HeLa cells “express that protein”, that means which this protein is locatedacross the cell membrane, with a small portion inside the cell but most of the proteinis outside the cell.We used biotinilized antibody againts that MICA protein, we conjugated it with ourstreptavidin covered MNP and as a result, the MNP stick on that protein, thuslabelling the cells.

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How did we label the cells?HeLa cells grow adhered to surfaces, and that simplifies the work. First we removethe medium inside the cells were growing and we add new medium with the MNP. We wait for a short time, only 15 minutes is time enough to the antibodies to stick onthe cells. After that time we remove that medium, we wash usually 3 times to ensurethat every unbound nanoparticle has been taken away. And finnally we added new medium.

After the labelling of the cells, we detacched them using trypsine-edta and I’m goingto show you how that cells have a magnetic behaviour thanks to the presence of MNP. Everyone of this small circles are HeLa cells that have been tagged with MNP. Ifa MFG is applied, the MNP are going to be attracted, and as they are bound to thecells, we’ll see how the cells move.This is important because it could increase the concentration and improve thesensibility of current diagnosis methods.

We could separate the cells in two fractions, the magnetically tagged or possitivefraction and the negative fraction.

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We done TEM to the possitive fraction and we saw how the nanoparticles are internalized inside the cell. How could that happen? The labelling time were short enough to avoid accidental endocitosisis. In opposite, that was a receptor-mediatedendocytosis.

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All this work I showed you have been repeated with non functionalized MNP, and no cell moved. And also it has been done with a different cell line, that do not have MICA in their surface.So, we conclude that…. /next/

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… our nanoparticles specifically tag MICA expressing cells. As it was expected, theantibody have worked, and there were non specific interaction between the MNPsand the cells.

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I have to acknowledge all the partners that have made this work possible.

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Finally, I would like to thank the organization of the congress for that opportunity, and thank you for you attention.

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