Nanoparticle activated neutrophils-on-a-chip: A label-free capacitive sensor to monitor cells at work

Highlights

• Sensor monitoring cellular ROS burst in time frames of minutes.

• CMOS is combined with LTCC technology.

• Lable free capacitive response by cells on an insulating surface in a weak electromangnetic field.

• Cell activity in response to external triggers, in this case nanoparticles is measured.

Abstract

Neutrophil granulocytes are the most abundant white blood cells in mammals and vital components of the immune system. They are involved in the early phase of inflammation and in generation of reactive oxygen species. These rapid cell-signaling communicative processes are performed in the time frame of minutes.

In this work, the activity and the response of neutrophil granulocytes are monitored when triggered by cerium-oxide based nanoparticles, using capacitive sensors based on Lab-on-a-chip technology. The chip is designed to monitor activation processes of cells during nanoparticle exposure, which is for the first time recorded on-line as alteration of the capacitance. The complementary metal oxide semiconductor engineering chip design is combined with low temperature co-fired ceramic, LTCC, packaging technology. The method is label free and gently measures cells on top of an insulating surface in a weak electromagnetic field, as compared to commonly used four-point probes and impedance spectroscopy electric measurements where electrodes are in direct contact with the cells.

In summary, this label free method is used to measure oxidative stress of neutrophil granulocytes in real time, minute by minute and visualize the difference in moderate and high cellular workload during exposure of external triggers. It clearly shows the capability of this method to detect cell response during exposure of external triggers. In this way, an informationally dense non-invasive method is obtained, to monitor cells at work.

Introduction

Neutrophil granulocytes, also called neutrophils, are known to be one of the most prominent cell types involved in the early phase of inflammation. Generation of reactive oxygen species (ROS) by these cells plays a crucial role in our defense system fighting intruders such as pathogenic microorganisms [[1], [2], [3], [4]]. Upon contact with a foreign material, the cells may try to phagocytose the intruder, leading to an extensive extracellular release of ROS which may lead to tissue damage [5,6]. This process of frustrated phagocytosis can be harmful for the host and contributes to the ongoing inflammation.

Neutrophils are highly reactive cells and their activation initiated by for example nanoparticles, will release a full set of inflammatory mediators such as myeloperoxidase [4,7] into the extracellular space (Fig. 1). About 10¹¹ neutrophils exit the bone marrow daily under basal conditions [8] and the short half-life in the blood is measured in hours [9].

Monitoring the cell activation during external triggers are of main importance for increased understanding of the mechanistic processes between cells and foreign elements. However, to obtain information on a system, i.e. to view and follow processes without disturbing it by the measurement itself, has been a struggle during scientific development over the years.

The invention of biochips, with focus on the vision of miniaturized laboratories (Lab-on-a-chip (LOC) technology), was first aimed to reduce the cost and to enable bioanalysis to be distributed worldwide [10]. Today the interest is continuously growing, as highlighted in a recent report from the EU commission [11]. Design of immunosensors, such as preclinical immunoassays, have earlier been reported and is an important contribution to the field [12]. Electric impedance spectroscopy (EIS) measurements have been performed by amperometric immunosensors to verify presence of antibodies against the bovine leukemia virus protein gp51 on a pre-immobilized sensor surface with five times higher sensitivity than ELISA [13]. Recently, photoluminescence spectroscopy together with an imaginary capacitor model was used to study the same system now with antigen-antibody complexes adsorbed on TiO₂ nanoparticles deposited on glass [14]. The opportunity to obtain data by non-invasive label free techniques to track and visualize ongoing cellular processes are recently brought to the table. Real-time SPR studies of cancer cells, during drug (daunorubicin) treatment and Au nanoparticle exposure are two such examples [15,16].

Cell-impedance measurements have been used for almost four decades and big leaps were introduced to this technology by micromachining in the 90 s and by microfluidics in the 2000s, which enabled miniaturization of the measurement system [17]. This allowed development of systems for detection and separation of neutrophils from whole blood by using impedance measurements [18,19]. Electrical impedance is the measure of the voltage to current ratio in an AC/DC circuit. When measuring impedance of cells and their activity, the following three techniques are commonly used; 1) electric cell-substrate impedance sensing (ECIS) [20], 2) impedance flow cytometry (IFC) [21] and 3) EIS [[22], [23], [24]]. When the aim is to study cells adhering onto a substrate ECIS is suggested, see earlier studies on apoptosis [25] and cell proliferation [26]. In the literature, the ECIS is predicted to be combined with other liquid handling models and microfluidics to improve the quality and reproducibility of measured data, to enable drug candidate screening, gene function screening etc. [17]. In these impedance measurements the cells are typically in direct contact with the electrodes.

Techniques such as organic electrochemical transistors are used to measure cells [27]. The low voltages used during these measurements are considered safe for the cells but may induce electrochemical side reactions and possible electrode corrosion, masking the pure cell interaction signal.

Cell patch-clamp techniques are used to study exocytosis dynamics [[28], [29], [30]]. This is done by a specially designed pipette tip that measure capacitance over a small area of the cell membrane giving the opportunity to study exocytosis processes over shorter time periods.

A highly interesting and recently developed technique to study cells adhering onto a substrate is based on capacitive measurements [[31], [32], [33]]. Cell morphology changes and release of charged entities are gently monitored through capacitive measurements utilizing a passivation layer on the electrode i.e. the cells, with surrounding media, are separated from the electrical contacts. When cells are activated, they change shape and the area of adhesion on the sensor surface may increase, resulting in a measurable relative capacitive change.

In this work neutrophils and their activity are investigated, when triggered by nanoparticle exposure. This is monitored using a lab-on-a-CMOS chip, packaged by low temperature co-fired ceramic technology (LTCC) [34], see schematic drawing and photo of the setup (Fig. 2, Fig. 3 a, respectively). The method is purely capacitive, without electrodes in contact with the cells and is based on a CMOS chip with an interdigitated electrode array, connected to the second stage of individual three-stage ring oscillators. The capacitance change can be interpreted from the change of oscillation frequency. This lab-on-a-CMOS chip technology [10] enable continuous monitoring of cell adhesion and cell activation as a function of time, in a non-invasive manner, through capacitive measurements.

This low-cost technique is thus label free, non-invasive, reusable and can be used to monitor a wide variety of cell types. The combination of these properties gives it high potential for future applications in cell monitoring. The chip can be used in short measurement in time spans of minutes up to days making it suitable for a whole range of experiments and setups.