domain: phys sdv

text:
Marie Mertens

Maede Chavoshi

Olivia Peytral-Rieu

Katia Grenier

Dominique Schreurs

Dielectric Spectroscopy: Revealing the True Colors of Biological Matter

Introduction

Accurate characterization of biological matter, for example, in tissue, cells, and biological fluids, is of high importance. For example, early and correct detection of abnormalities, such as cancer, is essential as it enables early and effective type-specific treatment, which is crucial for mortality reduction [START_REF]Cancer[END_REF]. Moreover, it is imperative to investigate the effectiveness and toxicity of pharmaceutical treatments before administration in clinical practice [START_REF] Pan | 3D cell-based biosensor for cell viability and drug assessment by 3D electric cell/matrigel-substrate impedance sensing[END_REF]. However, biological matter characterization still faces many challenges. State-of-the-art imaging and characterization methods have drawbacks, such as the requirement to attach difficult-to-find and costly labels to the biological target (e.g., COVID-19 rapid test), expensive equipment (e.g., magnetic resonance imaging or MRI), low accuracy (e.g., ultrasound), use of ionizing radiation (e.g., X-rays), and invasiveness [START_REF] Aldhaeebi | Review of microwaves techniques for breast cancer detection[END_REF]. The characterization of biological matter using microwave (µW), millimeter wave (mmW), and Terahertz (THz) spectroscopy is a promising alternative: it is label-free, does not require ionizing radiation, and can be non-invasive.

Moreover, there is a significant difference in how different biological materials absorb, reflect, and transmit electromagnetic (EM) waves [START_REF] Mehrotra | EM-wave biosensors: A review of RF, microwave, mmwave and optical sensing[END_REF] that is due to the difference in their dielectric properties. The dielectric properties are described by the frequency-dependent material parameter called the complex permittivity 𝜺(𝒇), which expresses how the material responds to an external oscillating electric field. The complex permittivity of a material determines how the material absorbs, reflects, and transmits EM waves at different frequencies (Fig 1 .). Since each biological material's permittivity spectrum is different, it acts as an electromagnetic fingerprint. A material's complex permittivity can be calculated from the reflection and transmission of electromagnetic waves through the material, described by the scattering (S)-parameters, which can be measured using a Vector Network Analyzer (VNA) transmitting and receiving EM waves over a range of frequencies. The amplitude and phase of the transmitted and reflected EM waves at different frequencies are influenced by different underlying biological effects at different scales. That causes the entire spectrum to provide information from the supracellular to the molecular and even atomic scale.

Figure 1. Conceptual illustration of how biological tissues (e.g., healthy and cancerous tissue) absorb, reflect, and transmit EM waves differently caused by the difference in their dielectric spectra. Spectroscopy using µW, mmW, and THz EM waves has shown potential to sense the concentration of important biomarkers, such as glucose in diabetes patients, DNA and proteins that inform about genetic disorders, and cancer biomarkers to distinguish healthy matter from cancerous matter [START_REF] Mehrotra | EM-wave biosensors: A review of RF, microwave, mmwave and optical sensing[END_REF]. These applications will be further elaborated on in a later section on Dielectric Spectroscopy Techniques.

Dielectric Spectrum

In this section we explain the meaning and interpretation of the dielectric spectrum, representing complex dielectric permittivity as a function of frequency. The physical phenomena underlying the spectrum's most important features are elaborated on in the subsections on relaxations and resonances.

Dielectric permittivity

As opposed to most non-biological materials, the dielectric permittivity depends heavily on the frequency of the applied electric field for biological matter. Indeed, at different frequencies, the material will absorb, reflect, and transmit EM waves differently. Moreover, permittivity is a complex material parameter:

𝜀(𝑓) = 𝜀 ′ (𝑓) -𝑗𝜀 ′′ (𝑓) (1)
A schematic representation of both the real and imaginary parts of the dielectric permittivity is shown in Figure 2. The real part of permittivity describes how well the electric dipole moments inside the material align to the electric field. The imaginary part of the permittivity describes the EM losses into the surrounding media. These EM losses can be considered to originate in the friction caused by the movement of the charges aligning to the electric field.

More fundamentally, the permittivity describes polarization, and the magnitude and phase describe its extent and delay, respectively. The dielectric spectrum shows relaxation and resonance phenomena. Figures 3 and4 illustrate the relaxation and resonance phenomena.

At the frequency of the relaxation phenomena, also called dispersion, there is a decrease in the real part of permittivity, whereas at the frequency of the resonance phenomena, a sharp resonance is seen in the real part of the permittivity. These phenomena will be further explained in the following section.

Permittivity relaxations

An intuitive explanation for the relaxations (or dispersions) in the real part of the permittivity is given here: an external oscillating electric field causes the alignment of dipole moments (due to the motion of ions, atoms, and molecules) inside the material to the electric field. The larger this alignment, the larger the total electric field and the larger the real part of the permittivity. When the charges' inertia causes their inability to follow the electric field's oscillations, their contribution to the total electric field drops to zero. This can be seen in the spectrum as a drop in the real part of the permittivity and is called a relaxation [START_REF] Aldhaeebi | Review of microwaves techniques for breast cancer detection[END_REF], which is visualized in Figure 3. At each dispersion, there is a peak in the dielectric losses, which can be seen in the imaginary part of the permittivity. This frequency range is indeed where the charges are most out of phase, delayed to the external electric field, causing the presence of an imaginary component in the polarization. The relaxation event does not occur at one single frequency but over a range, as biological matter is highly heterogeneous and the different ions, atoms, and molecules have slightly different relaxation frequencies. The various relaxation mechanisms (𝛼-, 𝛽and 𝛾-dispersion) are determined by distinct polarization mechanisms for the different constituents of matter (cells and cell membranes, ions, molecules, atoms, electrons) [START_REF] Schwan | Electrical Properties of Tissue and Cell Suspensions[END_REF]. As such, the spectrum informs us about the different constituents of matter in different frequency bands. It is essential to understand the fundamentals behind the relaxation phenomena to understand how one could extract information on the biological and physiological state of a material from the frequency spectrum.

𝛼-dispersion

The first relaxation is called 𝛼-dispersion. It is caused by the inability of ions to respond to a fast-changing field, and occurs at Hz to kHz frequencies. Unfortunately, due to high measurement uncertainty through unwanted measurement electrode polarization, 𝛼dispersion can often not be interpreted accurately in terms of permittivity [START_REF] Maenhout | Dielectric detection and ablation techniques for cancer theranostics[END_REF].

𝛽-dispersion

The second relaxation is called 𝛽-dispersion. It is caused by the disappearance of Maxwell-Wagner polarization at the frequency of the dispersion at kHz to MHz frequencies. This

Maxwell-Wagner polarization is caused by the presence of physical interfaces (e.g., cell membranes) that constitute the border between two different media, such as the intracellular and the extracellular fluid. At frequencies lower than the 𝛽-dispersion, and upon the effect of an external electric field, charges accumulate at the border and create a space charge region, which contributes to the permittivity. At frequencies higher than the 𝛽relaxation, these interfaces are shortened, and they become invisible to the electric field, which can now penetrate to the insides of the cell [START_REF] Mehrotra | EM-wave biosensors: A review of RF, microwave, mmwave and optical sensing[END_REF], [START_REF] Maenhout | Dielectric detection and ablation techniques for cancer theranostics[END_REF], see Figure 5. As such, when investigating permittivity at frequencies beyond the 𝛽-relaxation, examination of the cells' contents (e.g., intracellular fluid and particles) becomes possible. Because of the disappearance of the space charge region, there is a decrease in polarization and as such a decrease in permittivity.

𝛾-dispersion

Finally, 𝛾-dispersion occurs at frequencies near 24 GHz. This dispersion is highly influenced by the water content of the material, which is often high for biological matter. At frequencies below the 𝛾-dispersion, the dipole constituted by the asymmetric shape of a free water molecule vastly contributes to the induced polarization when it aligns to the external electric field. However, around 24 GHz, these molecules are unable to align with the external electric field [START_REF] Mehrotra | EM-wave biosensors: A review of RF, microwave, mmwave and optical sensing[END_REF], [START_REF] Maenhout | Dielectric detection and ablation techniques for cancer theranostics[END_REF]. The higher the free water content, the higher the orientation polarization and the higher the permittivity before the 𝛾-relaxation. Given that water content varies between different organs, tissues and cells, measuring permittivity around the 𝛾-relaxation can help in differentiating between different organs for microwave imaging, between live and dead cells, or between cancerous and healthy cells, and so forth. A separate dispersion is attributed to water molecules that are bound to other molecules. Given the complexity of this 𝛿-dispersion, it will not be elaborated on here.

Permittivity resonances

The physical events underlying the resonances in the THz part of the spectrum are rather complicated. However, we will attempt to describe the fundamental idea: when the external electric field is oscillating at THz frequencies (1 000 000 000 000 oscillations per second!), only few very small biological structures are still able to align to the electric field and can as such contribute to the polarization. These small structures are (1) the electrons in the electron cloud surrounding their positive core, and (2), atoms that are attached to each other in a molecule. The electric field not only rotates them but also pulls apart the positive and the negative parts, after which a restoring force acts to bring them back together; the structures then can be considered as if they were springs [START_REF] Pan | 3D cell-based biosensor for cell viability and drug assessment by 3D electric cell/matrigel-substrate impedance sensing[END_REF]. At their resonance frequency, the distance between the positive and the negative parts reaches its maximum, first in the direction of the external electric field (local permittivity maximum), then, because of delay to the electric field, in the opposite direction (lower the total electric field, local permittivity minimum). This is illustrated in Figure 4. There is a peak in losses because of the energy absorption that comes with the separation of the charges. Changes in the concentration of some important biomolecules can be observed as a shift in resonant frequency in the dielectric spectrum as their vibrational frequency lies in the frequency range of 300 GHz to 1 THz [START_REF] Mehrotra | EM-wave biosensors: A review of RF, microwave, mmwave and optical sensing[END_REF].

To conclude, the 𝛼-, 𝛽-, and 𝛾dispersions and the electronic and atomic resonances that appear in biological matter's frequency spectrum are influenced by different biological events. Researchers can measure the whole frequency spectrum and deduct information about all these biological events at the same time. Indeed, researchers can characterize biological matter based on fundamental characteristics of the spectra, such as how broad each dispersion is, and at which frequency each relaxation occurs. Examples of how scientific research used these characteristics for biological matter characterization are given in the following section. However, in practice, not many have succeeded to really link these parameters to specific biological events and so the field still needs to advance in this area.

Moreover, the frequency of characterization can be chosen according to which biological events are of interest to the research (e.g., 𝛽-frequencies to obtain information on number of cells and cell membranes, and 𝛾-frequencies when water content is of interest).

Finally, before diving into the practical aspects of permittivity measurements, we ought to clarify one thing: one might wonder why we choose to consider dielectric constant rather than impedance. Some [START_REF] Hwang | Label-free noninvasive cell characterization: A methodology using broadband impedance spectroscopy[END_REF] prefer to consider impedances which, as opposed to dielectric permittivity, are directly measurable, and no difficult mathematical transformations or calculations are required to obtain them. For the characterization of two or more cells, which always comprise the same geometry and are measured with the exact same method, one is completely right to consider impedances. However, as "dielectric electrical (oxymoron)

engineers" [START_REF] Hwang | Label-free noninvasive cell characterization: A methodology using broadband impedance spectroscopy[END_REF], we consider dielectric permittivity to be the fundamental parameter. It is a material characteristic and should as such, after de-embedding, be independent of the measurement method and size and shape of the MUT, which is not true for impedances. Even though still to be improved in practice, the dielectric permittivity of cells, tissues and liquids of different sizes should thus reflect only the content of the biological matter rather than the size, shape and volume of the cell.

Dielectric characterization methods

Method

Broadband/narrowband Planar/Non-planar Frequency Range

Example applications

Coaxial Probe Broadband, non-planar 10 MHz-110 GHz Mainly tissue characterization: e.g., breast tissue [START_REF] Lazebnik | A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries[END_REF], liver tissue [START_REF] O'rourke | Dielectric properties of human normal, malignant and cirrhotic liver tissue: in vivo and ex vivo measurements from 0.5 to 20 GHz using a precision open-ended coaxial probe[END_REF], renal calculi [START_REF] Yilmaz | Microwave dielectric spectroscopy of renal calculi: A large scale study on dielectric properties from 500 MHz to 18 GHz[END_REF] Coplanar Waveguide Broadband, planar < 1 GHz -sub-THz integration with microfluidics for measurements of small volumes: e.g., single cell detection and analysis [START_REF] Tamra | Impact of a chemical stimulus on two different cell lines through microwave dielectric spectroscopy at the single cell level[END_REF], [START_REF] Tamra | Single cell microwave biosensor for monitoring cellular response to electrochemotherapy[END_REF], detection of mitochondrial activity [START_REF] Poiroux | Label-free detection of mitochondrial activity with microwave dielectric spectroscopy[END_REF] detection of skin melanoma [START_REF] Dubuc | In vitro and vivo investigations toward near-field microwave-based detection of melanoma[END_REF] Microwire Broadband, non-planar

Sub-THz

Detection of protein resonances [START_REF] Nardecchia | Out-of-Equilibrium Collective Oscillation as Phonon Condensation in a Model Protein[END_REF], [START_REF] Lechelon | Experimental evidence for long-distance electrodynamic intermolecular forces[END_REF] Planar Goubeau Lines Broadband, planar Sub-THz-THz frequencies Lysosome dielectric spectroscopy [START_REF] Laurette | Co-integrated microfluidic and THz functions for biochip devices[END_REF] Free-space measurements Broadband, non-planar 50 GHz-THz

Upcoming technology for FDS

Cavity resonator

Narrowband, non-planar Water-based solutions such as NaCl [START_REF] Mao | A noncontact microwave sensor based on cylindrical resonator for detecting concentration of liquid solutions[END_REF] Planar resonators: SRR, CSRR, … Narrowband, planar Bacteria [START_REF] Jain | Passive microwave biosensor for real-time monitoring of subsurface bacterial growth[END_REF], [START_REF] Narang | Sensitive, real-time and non-intrusive detection of concentration and growth of pathogenic bacteria using microfluidic-microwave ring resonator biosensor[END_REF], [START_REF] Mohammadi | Real-time monitoring of Escherichia coli concentration with planar microwave resonator sensor[END_REF], [START_REF] Jain | Differential microwave resonator sensor reveals glucose-dependent growth profile of E. coli on solid agar[END_REF], [START_REF] Jain | Rapid and real-time monitoring of bacterial growth against antibiotics in solid growth medium using a contactless planar microwave resonator sensor[END_REF], glucose [START_REF] Omer | Low-cost portable microwave sensor for non-invasive monitoring of blood glucose level: novel design utilizing a four-cell CSRR hexagonal configuration[END_REF], [START_REF] Omer | Portable radar-driven microwave sensor for intermittent glucose levels monitoring[END_REF], [START_REF] Omer | Compact honeycell CSRR-based microwave newline biosensor for monitoring glucose levels[END_REF], [START_REF] Omer | Non-invasive real-time monitoring of glucose level using novel microwave biosensor based on triple-pole CSRR[END_REF], [START_REF] Omer | Wearable CSRR-based sensor for monitoring glycemic levels for diabetics[END_REF], colon tissue [START_REF] Park | Design of a split ring resonator integrated with on-chip terahertz waveguides for colon cancer detection[END_REF], [START_REF] Maenhout | Flexible, segmented tubular design with embedded complementary split-ring resonators for tissue identification[END_REF], skin tissue [START_REF] Bai | Flexible microwave biosensor for skin abnormality detection based on spoof surface plasmon polaritons[END_REF], cells [START_REF] Watts | Microwave dielectric sensing of free-flowing, single, living cells in aqueous suspension[END_REF], [START_REF] Liu | Microfluidics-based hairpin resonator biosensor for biological cell detection[END_REF] 3. Dielectric spectroscopy techniques Dielectric spectroscopy (DS) is the measurement of the material's fundamental dielectric properties as a function of frequency. These properties can be interpreted as explained above and employed to characterize biological matter. The general methodology consists of (1) the measurement of the reflection and/or transmission of EM waves as S-parameters through the material under test (MUT), followed by ( 2), a mathematical conversion to dielectric permittivity. The former will be discussed below. The latter depends on the geometrical setup of the measurements, as well as the size and the shape of the MUT and the calibration method, and will not be explained in detail further in this article. The basic principle is that the measurement results will be de-embedded i.e., the effects of the setup will be removed, until only the effect of the MUT's permittivity is visible. Alternatively, an analytical description of the effect of the sample's permittivity in the measurement results can be employed, from which the permittivity can directly be extracted.

The most common methodologies for measurement of S-parameters of biological matter for reflection and transmission are explained below and illustrated in Figure 6 and Table 1. Some applications are given as well, and it is shown how the methods have been selected and adapted for measurements on biological matter. For example, biocompatibility of the measurement device must be guaranteed and possible EM-heating effects must be prevented. The preferred method depends on the frequency, the physical and chemical attributes of the biological MUT (liquid/solid, size, etc.), and the requirements in terms of sensitivity and limit of detection among others. Moreover, the operating bandwidth of the measurements plays a key role in selecting the technology. Broadband characterization provides a global measure on a broad range of frequencies, and thus lets us know about the constituents of the matter on different scales, but requires technology that is appropriate for the whole spectrum. The technology can be hard to develop, and the instrumentation can be costly and fragile. Narrowband characterization, on the other hand, focuses on one to a few discrete frequencies so that the device can be designed to be optimized to these frequencies using cheaper and simpler components. These measurements are often more precise and easier to develop. Below we will first elaborate on broadband dielectric spectroscopy (DS), and then the main approaches and applications for narrowband characterization will be explained.

Broadband Dielectric Spectroscopy

In this subsection the most common approaches for broadband dielectric spectroscopy of biological matter are discussed. More specifically, coaxial probes and coplanar waveguides are first explained in depth, after which we summarize the upcoming technologies for sub-THz and THz broadband characterization.

Open-ended coaxial probe

Figure 7. Open-ended coaxial probe method.

An open-ended coaxial probe is commonly used for measurements of the reflection of EM waves (S11) from a biological MUT in the frequency range from ±0.1 -10 GHz (and sometimes even for measurements up to 110 GHz). As the coaxial transmission line is cut off at the border with the MUT, its field fringes into the liquid or semi-solid biological matter, which affects the reflection of the EM waves [START_REF] Technologies | Basics of Measuring the Dielectric Properties of Materials[END_REF]. The basic concept is illustrated in Figure 7.

The conversion method from reflection to permittivity is well established, and the method requires minimal sample preparation and is easy to use [START_REF] Aydinalp | Characterization of open-ended coaxial probe sensing depth with respect to aperture size for dielectric property measurement of heterogeneous tissues[END_REF]. The coaxial probe has mainly been used to characterize biological tissue, from normal, benign, and malignant breast or liver tissue to renal calculi and muscle [START_REF] Lazebnik | A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries[END_REF], [START_REF] O'rourke | Dielectric properties of human normal, malignant and cirrhotic liver tissue: in vivo and ex vivo measurements from 0.5 to 20 GHz using a precision open-ended coaxial probe[END_REF], [START_REF] Yilmaz | Microwave dielectric spectroscopy of renal calculi: A large scale study on dielectric properties from 500 MHz to 18 GHz[END_REF]. The measurement methodology had to be adapted largely for biological tissue when researchers noticed that the practices were not proper for biological matter. Indeed, it was found necessary to control the precise position and pressure of the probe on the tissue, as compressing the semi-solid would change its dielectric properties [START_REF] Maenhout | Dielectric detection and ablation techniques for cancer theranostics[END_REF]. Moreover, in a large-scale breast tissue investigation performed by Lazebnik et al. [START_REF] Lazebnik | A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries[END_REF], it became clear that an analysis of the content of excised tissue before measurement was of critical importance. A large difference was found between dielectric properties of normal breast tissue and malignant tissue, but it appeared to be largely determined by the adipose (fatty) content of the tissue sample, which was higher in the excised healthy samples. The differences between healthy and malignant tissue were far smaller when this adipose content was accounted for [START_REF] Lazebnik | A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries[END_REF]. Nevertheless, other studies still showed promising results: O'Rourke et al. [START_REF] O'rourke | Dielectric properties of human normal, malignant and cirrhotic liver tissue: in vivo and ex vivo measurements from 0.5 to 20 GHz using a precision open-ended coaxial probe[END_REF] showed that the dielectric properties of ex vivo malignant liver tissues are 19 to 30% higher than normal tissue. Moreover, the renal calculi (kidney stone) category can be statistically differentiated by dielectric properties over the frequency range from 500 MHz to 18 GHz [START_REF] Yilmaz | Microwave dielectric spectroscopy of renal calculi: A large scale study on dielectric properties from 500 MHz to 18 GHz[END_REF].

Planar transmission lines

Transition from non-planar setups to planar ones enables measurements on smaller samples and as such is interesting for the measurement of extracted biological liquid and tissue samples, and cell suspensions. Dielectric matter characterization can be performed using many types of transmission lines, such as coplanar waveguides (CPW), microstrip lines (ML), substrate integrated waveguides (SIW), and planar Goubau lines (PGL) for higher frequencies.

However, most of these methods, as for example ML and SIW, are mainly used for characterization of the dielectric substrate that they are fabricated on, as their modes of propagation show maximum electric field distributions in the substrate (often between a metallic ground plane and the transmission line). They thus rely on the deposition of metallic lines onto the MUT. Characterization using CPW (or PGL, see next section) can be performed by putting the sample -contained in a biocaptor-on top of, and thus directly in contact with the metallic lines that are deposited onto another low-loss dielectric material. Gold is often used for these metallic lines as it allows for maximum biocompatibility, resistance against oxidation when measuring ionic substances, and a good transmission of the EM waves. In CPW the electric field lines in the dominant mode of propagation, quasi-TEM mode, are oriented as shown in Figure 8 (a) [START_REF] Rainee | Coplanar Waveguide Circuits, Components, and Systems[END_REF]. The propagation of the electric field will be affected by any biological matter put on top of the transmission lines. The effective permittivity of the CPW lines is a mathematically complex combination of the permittivity of the dielectric and the permittivity of the material on top of the lines (the unknown editor of Microwaves101 [START_REF] Editor | Coplanar Waveguide[END_REF] provides us with the rule of thumb that it is approximately the average). In most devices, a microfluidic channel, sometimes with hydrodynamic traps, functions as biocaptor and is constructed on top of the CPW lines (Figure 8 (b))-mainly on the signal line and gaps where the field is largest -to allow the electric field to be affected by the biological matter that is pumped through the channel [START_REF] Artis | Microwaving biological cells: Intracellular analysis with microwave dielectric spectroscopy[END_REF], [START_REF] Grenier | Recent advances in microwave-based dielectric spectroscopy at the cellular level for cancer investigations[END_REF]. In fact, as the position of the biological object (e.g., cell, organelle) must be controlled to maximize the sensitivity, "trapping" structures can be added to the microfluidic structure [START_REF] Du | Ultra-wideband impedance spectroscopy of the nucleus in a live cell[END_REF], [START_REF] Peytral-Rieu | Microwave sensor dedicated to the determination of the dielectric properties of 3D biological models from 500 MHz to 20 GHz[END_REF]. As CPW operates in the GHz frequency range, where the electric field can probe the inside of the cells through the shortening of the capacitive effect of the cell membrane, it is used extensively in investigating the intracellular components of different cell lines and their reaction to chemical and physical treatment [START_REF] Du | Ultra-wideband impedance spectroscopy of the nucleus in a live cell[END_REF], [START_REF] Zedek | Microwave permittivity extraction of individual biological cells submitted to different stimuli[END_REF], [START_REF] Tamra | Impact of a chemical stimulus on two different cell lines through microwave dielectric spectroscopy at the single cell level[END_REF], [START_REF] Tamra | Single cell microwave biosensor for monitoring cellular response to electrochemotherapy[END_REF]. Moreover, CPW lines can be adapted to measure the dielectric properties of biological matter of different sizes: from in-vivo tissue characterization down to single cells, making it a promising technique to monitor the effects of drugs on different biological entities in a label free, non-invasive, and non-destructive way [START_REF] Artis | Microwaving biological cells: Intracellular analysis with microwave dielectric spectroscopy[END_REF], [START_REF] Grenier | Recent advances in microwave-based dielectric spectroscopy at the cellular level for cancer investigations[END_REF]. Different studies have shown the possibility to monitor cell electroporation, electrochemotherapy, and the effect of saponin and mitochondrial activity at single cell or cells in suspension level as represented respectively in the first and second images of Figure 9 [23], [START_REF] Tamra | Impact of a chemical stimulus on two different cell lines through microwave dielectric spectroscopy at the single cell level[END_REF], [START_REF] Tamra | Single cell microwave biosensor for monitoring cellular response to electrochemotherapy[END_REF], [START_REF] Poiroux | Label-free detection of mitochondrial activity with microwave dielectric spectroscopy[END_REF]. For instance, in [START_REF] Artis | Microwave dielectric spectroscopy of cell membrane permeabilization with saponin on human B lymphoma cells[END_REF], it was found that saponin, which affects the cell membrane and allows for exchange of contents through the membrane, effectively caused a reduction in dielectric contrast between the extracellular and intracellular medium. Furthermore, mitochondrial activity can be monitored. This is important as it can be an indicator for how cells will react to therapy.

Moreover, melanomas (skin cancers) have been studied directly in vivo and can be detected using microwave spectroscopy with a sensor in contact with the skin of the subject, as represented in the fourth image of Figure 9 [START_REF] Dubuc | In vitro and vivo investigations toward near-field microwave-based detection of melanoma[END_REF]. Finally, the dielectric characterization of microtissue (3D cell aggregates as a model for cancer tissue) is emerging with the device represented in the middle of Figure 9 [19], [START_REF] Peytral-Rieu | Microwave Microfabricated Sensor Dedicated to the Dielectric Characterization of Biological Microtissues[END_REF]. In conclusion, CPW allows studies in multiple bio-sample scales as summarized in Figure 9. [START_REF] Peytral-Rieu | Microwave sensor dedicated to the determination of the dielectric properties of 3D biological models from 500 MHz to 20 GHz[END_REF], [START_REF] Artis | Microwave dielectric spectroscopy of cell membrane permeabilization with saponin on human B lymphoma cells[END_REF], [START_REF] Dubuc | In vitro and vivo investigations toward near-field microwave-based detection of melanoma[END_REF], [START_REF] Peytral-Rieu | Microwave Microfabricated Sensor Dedicated to the Dielectric Characterization of Biological Microtissues[END_REF], [START_REF] Chen | Microwave biosensor dedicated to the dielectric spectroscopy of a single alive biological cell in its culture medium[END_REF]. for dielectric characterization showing the versatility of CPW sensors in biological applications [START_REF] Peytral-Rieu | Microwave sensor dedicated to the determination of the dielectric properties of 3D biological models from 500 MHz to 20 GHz[END_REF], [START_REF] Artis | Microwave dielectric spectroscopy of cell membrane permeabilization with saponin on human B lymphoma cells[END_REF], [START_REF] Dubuc | In vitro and vivo investigations toward near-field microwave-based detection of melanoma[END_REF], [START_REF] Peytral-Rieu | Microwave Microfabricated Sensor Dedicated to the Dielectric Characterization of Biological Microtissues[END_REF], [START_REF] Chen | Microwave biosensor dedicated to the dielectric spectroscopy of a single alive biological cell in its culture medium[END_REF].

Sub-THz and THz measurements

In this paragraph, we list interesting technologies for broadband sub-THz and THz measurements (0.1 THz -10 THz). These frequency ranges show great potential for dielectric characterization. THz Time Domain Spectroscopy (TDS) has been shown to be able to detect drug effectiveness through comparison of absorption coefficients and refractive indices in [START_REF] Nourinovin | Impact of Drug Treatment on the Electromagnetic Properties of Basal Cell Carcinoma Cancer in the Terahertz Band[END_REF]. It was found that these coefficients and indices showed the trend towards healthy tissue when a drug was administered and the negative effect on the healthy cells, respectively.

Moreover, in [START_REF] Wu | Biomedical applications of terahertz spectra in clinical and molecular pathology of human glioma[END_REF] promising results were obtained showing significant differences in the THz absorption spectrum for different histological types, pathological grades, and glioma-specific biomarkers in measurements of healthy and cancerous brain tissue. In [START_REF] Yu | Myocardial Amyloidosis Detection with Terahertz Spectroscopy[END_REF] myocardial amyloidosis (deposition of specific protein causing heart stiffness) was detected using THz-TDS, combined with convolutional neural networks. Finally, as mentioned in the explanation of dielectric permittivity, the frequency spectrum overlaps with the resonance frequencies of important proteins and biomolecules in this range. To extend these methods for a more accurate frequency domain analysis with higher frequency resolution, there is a need to establish Frequency Domain Spectroscopy (FDS) methods at THz frequencies. Technologies are being developed to cope with the high loss of EM power into lossy biological MUT and the high noise that is inherent to these frequency bands. For instance, [START_REF] Nardecchia | Out-of-Equilibrium Collective Oscillation as Phonon Condensation in a Model Protein[END_REF] showed the use of a microwire to detect resonating proteins at 0.314 THz excited by visible light. Moreover, a protein concentration change effectively caused a change in the measured resonance frequency in [START_REF] Lechelon | Experimental evidence for long-distance electrodynamic intermolecular forces[END_REF]. Planar alternatives are being researched as well, such as the Planar Goubau line (PGL), a fine microstrip line with no ground onto which the Goubau mode can be excited through CPW to PGL transition. This mode shows high confinement around the line and can be used for measurement of extremely small structures close to the line. In [START_REF] Laurette | Co-integrated microfluidic and THz functions for biochip devices[END_REF] transmission through lysozymes was successfully measured using PGL. More research is necessary to investigate the further capabilities of PGL. Finally, research is ongoing for the use of Free Space Measurements (FSM) of biological cells and tissues. Interestingly, the sub-THz to THz measurements are hovering on the border between THz spectroscopy and visible light spectroscopy, slightly further from our field of knowledge, but definitely not less important.

Researchers are exploring the borders of both the microwave and light (towards quantum)

theories and verifying how it can be crossed.

Narrowband Characterization of Biological Matter

The technologies and applications discussed so far are mainly used for broadband dielectric spectroscopy of biological matter, i.e., in a wide frequency range. However, broadband permittivity information is not necessarily needed in many cases; for example, if one is interested in studying the presence or absence of a specific substance, solution concentrations, and viability and growth of cells or organisms like bacteria, among others, at a single frequency or in a small frequency range, narrowband techniques can be more precise and faster to investigate dielectric properties of materials.

Narrowband measurements often employ a resonance-based microwave sensor that concentrates the electric field at frequencies near a specific frequency, depending on the application. The term "resonance" in this section is different than the one used in the prior sections; here, we mean the resonance of a circuit, due to its components. Depending on the structure of a sensor, it can be modelled as a resonant circuit, including an inductance L, capacitance C, and a term relating to conductivity (or inversely, resistance) G or R. Depending on modelling, they can be connected in a series or parallel form. This is how these resonant sensors come into play: the sensor behaves as a series or a parallel RLC circuit with its components tuned by the MUT's properties. For instance, the resonance frequency 𝑓 𝑟𝑒𝑠 and the quality factor 𝑄 define the resonance behavior of the sensor. Although the precise relation depends on sensor configuration and calibration, we can approximately assume that the resonance frequency (determined by the equivalent inductance L and capacitance C) is related to the permittivity as

𝑓 𝑟𝑒𝑠 = 1 2𝜋√𝐿𝐶 ∝ 1 𝜀 (2)
and the quality factor can be inversely related to the losses as

𝑄 ∝ 1 tan 𝛿 (3)
Thus, the interaction of the highly concentrated field and the MUT determines the system's frequency response by changing

𝑓 𝑟𝑒𝑠 0 → 𝑓 𝑟𝑒𝑠 1 , 𝑎𝑛𝑑 𝑄 0 → 𝑄 1 (4) (4)
Nevertheless, challenges in resonance-based biological matter measurements arise from the fact that a considerable portion of them consists of a highly lossy substance: water! The loss decreases the quality factor, flattening the resonance dip and making it difficult to find the resonance frequency.

Various types of planar and non-planar microwave resonant sensors are used for dielectric spectroscopy of different biological matter, ranging from aqueous biological solutions, like glucose or saline solutions, to cells and tissues. Here, we subdivide the narrowband technologies into non-planar cavity resonators and planar resonators.

Non-planar Cavity Resonators

In the case of non-planar cavity resonators, devices such as coaxial resonators have been developed to study the concentration of solutions. As the name implies, a cavity consists of a hollow space filled with the sample, and the resonance behavior changes according to the sample's permittivity.

As discussed previously, in wideband open-ended coaxial probe measurements, the sample is in contact with the end of the probe, changing the impedance. Instead of relying only on the conventional commercially available probes, the idea of designing a closed-ended coaxial resonator by replacing the inner dielectric of the coaxial probe with the MUT is proposed in [START_REF] Mohammed | 3D printed coaxial microwave resonator sensor for dielectric measurements of liquid[END_REF]. In both cases, the sample is in contact with the probe, which makes the probe susceptible to contamination and the measurements prone to error. To avoid any direct contact with the sample, cavities for contactless measurement have been introduced [START_REF] Mao | A noncontact microwave sensor based on cylindrical resonator for detecting concentration of liquid solutions[END_REF], [START_REF] Hassan | Continuous characterization of permittivity over a wide bandwidth using a cavity resonator[END_REF]. In these cavities, a container is placed inside or close to the cavity, and the electric field is perturbed when the container is loaded with the sample. Figure 10 illustrates a cavity resonator with a container for contactless liquid characterization.

For instance, a cylindrical resonator for contactless measurement of NaCl solution (0%-5%) was proposed in [START_REF] Mao | A noncontact microwave sensor based on cylindrical resonator for detecting concentration of liquid solutions[END_REF]. This sensor includes a probe inserted into a cavity and a container to be filled with the saline solution. Among the main propagation modes (TE111, TM010, and TE011), the one with the highest quality factor and the largest separation from the other modes is chosen (TE011). Based on the perturbation theory, the variation in permittivity Δ𝜀 is obtained. Figure 10. A cylindrical resonator with a container for loading the sample for contactless measurement of liquid concentration using dielectric spectroscopy [START_REF] Mao | A noncontact microwave sensor based on cylindrical resonator for detecting concentration of liquid solutions[END_REF].

Planar resonators

Despite having a higher quality factor, non-planar resonant sensors are often bulky, whereas in biomedical applications of dielectric spectroscopy, an appropriate platform for samples with a volume as small as nL or μL is required. Although bulk sensors can be integrated with microfluidics [START_REF] Watts | Microwave dielectric sensing of free-flowing, single, living cells in aqueous suspension[END_REF], miniaturized planar sensors are more easily compatible with various microfluidic systems such as lab-on-a-chip, organ-on-a-chip, cell sorting and trapping, and flexible electronics and wearables.

In fact, basically any structure that forms an RLC circuit on a planar technology, such as CPW or microstrip, can be utilized as a planar resonant sensor; including but not limited to Split Ring Resonators (SRR), their dual counterparts Complementary Split Ring Resonators (CSRR), and interdigitated capacitors combined with other mentioned structures or solely with an inductance.

In this section, we will discuss how various microwave planar resonators have been introduced to the fields of chemistry and biology, focusing on more recent advances in applications of SRRs and CSRRs.

Split Ring Resonators

Planar split ring resonators make it possible to observe the occurrences close to the surface of a sensor, thanks to interaction of a biomatter with the highly concentrated electric field in the slit of a split ring. One newly developed example is a hairpin resonator for melanoma cell detection [START_REF] Liu | Microfluidics-based hairpin resonator biosensor for biological cell detection[END_REF]. As the cell is trapped in a channel, the electromagnetic wave goes through the growth medium, enters the cells, and passes through the growth medium again to reach the other electrode. Although it is usually preferred to evaluate the change in transmission amplitude, the reflection (S11) change is more pronounced than the transmission (S21) change in this case. A higher variation in reflection due to the non-homogeneous interface between the cell and the medium explains this difference.

While trapping techniques often accommodate single-cell dielectric spectroscopy, Watts et al. [START_REF] Watts | Microwave dielectric sensing of free-flowing, single, living cells in aqueous suspension[END_REF] introduce a biosensor that consists of a double SRR that detects the free-flowing live cells moving in the microfluidic channel along the gap. This sensor cannot be considered entirely planar as it has a copper cavity for stimulation, and the gold ring resonator is deposited on a glass coverslip. Nevertheless, this hybrid configuration produces a large quality factor, even with lossy water.

Following the studies on dielectric spectroscopy for cells and bacteria [START_REF] Piekarz | Planar single and dual-resonant microwave biosensors for label-free bacteria detection[END_REF], [START_REF] Russel | High-frequency, dielectric spectroscopy for the detection of electrophysiological/biophysical differences in different bacteria types and concentrations[END_REF], [START_REF] Bridges | Microwave near-field detection of single biological cells and nanoparticles[END_REF], SRRbased sensors have been developed for real-time monitoring of bacteria concentration and growth [START_REF] Jain | Passive microwave biosensor for real-time monitoring of subsurface bacterial growth[END_REF], [START_REF] Narang | Sensitive, real-time and non-intrusive detection of concentration and growth of pathogenic bacteria using microfluidic-microwave ring resonator biosensor[END_REF], [START_REF] Mohammadi | Real-time monitoring of Escherichia coli concentration with planar microwave resonator sensor[END_REF] and the effect of glucose or antibiotics on this growth [START_REF] Jain | Differential microwave resonator sensor reveals glucose-dependent growth profile of E. coli on solid agar[END_REF], [START_REF] Jain | Rapid and real-time monitoring of bacterial growth against antibiotics in solid growth medium using a contactless planar microwave resonator sensor[END_REF]. As illustrated in Figure 11( SRRs have also been implemented in tissue studies, focusing on the tissues' dielectric characteristics as a key feature of their physiological state. A microstrip ring resonator with CPW access (MRRC) has been devised to measure the dielectric parameters of animal tissues [START_REF] Deshours | Improved microwave biosensor for non-invasive dielectric characterization of biological tissues[END_REF]. The sensor is made up of a microstrip ring resonator (MRR) on the top surface with CPW feed lines on the other side, and the sample is loaded as a superstrate on the MRR. The advantage of the proposed configuration compared to a simple MRR with a microstrip feedline is that the sample-induced losses do not affect the signal propagation in feedlines.

Same as the applications of Goubau line for broadband measurement of bio matter, at higher frequencies (~600 GHz), a THz-imaging technique based on SRR integrated with PGL is proposed in [START_REF] Park | Design of a split ring resonator integrated with on-chip terahertz waveguides for colon cancer detection[END_REF]. With a bare sensor resonance frequency of 596 GHz, healthy and cancerous colonic tissue models cause a 130 and 155 GHz frequency shift to lower frequencies, respectively. Nevertheless, the effective sensing depth of the sensor is limited to a few microns, limiting its performance in deeper analysis of a tissue.

Complementary Split Ring Resonators

CSRRs are one of the most popular planar resonators because of the strong electric field perpendicular to the CSRRs' surface, improving the penetration depth. This is important especially when a sample is not sensed in direct contact to the sensor as, for example, sensing the blood beneath multiple layers of skin or in a container to investigate blood glucose concentration or in-vivo tissue characterization. Omer et al. have developed several glucose sensors in a hexagonal CSRR form (honey cell) [START_REF] Omer | Low-cost portable microwave sensor for non-invasive monitoring of blood glucose level: novel design utilizing a four-cell CSRR hexagonal configuration[END_REF], [START_REF] Omer | Portable radar-driven microwave sensor for intermittent glucose levels monitoring[END_REF], [START_REF] Omer | Compact honeycell CSRR-based microwave newline biosensor for monitoring glucose levels[END_REF] and three circular CSRRs [START_REF] Omer | Non-invasive real-time monitoring of glucose level using novel microwave biosensor based on triple-pole CSRR[END_REF], [START_REF] Omer | Wearable CSRR-based sensor for monitoring glycemic levels for diabetics[END_REF] for wearable applications. An illustration is given in Figure 12. These studies not only present a novel structure compared to a single CSRR but also develop (1) a dipole antenna tag reader for distant sensing and (2) a radar-driven portable sensor to overcome the need for high-cost and bulky VNAs. The sensor's selectivity and specificity remain the main constraints in using dielectric spectroscopy for glycemia. The presence of other substances such as ions, proteins, and other molecules can negatively affect the repeatability and reproducibility of the measurements. However, the glucose concentration in the blood is dominant compared to these other components [START_REF] Omer | Low-cost portable microwave sensor for non-invasive monitoring of blood glucose level: novel design utilizing a four-cell CSRR hexagonal configuration[END_REF], enabling the design of microwave sensors for glycemia monitoring as a potential non-invasive replacement for currently available invasive and minimally-invasive solutions. Similar to the case of blood glucose biosensors, non-invasive in-vivo tissue spectroscopy requires a good penetration depth such that the signal passes through different layers.

Furthermore, flexibility and spatial resolution are essential. For instance, Maenhout et al. [START_REF] Maenhout | Flexible, segmented tubular design with embedded complementary split-ring resonators for tissue identification[END_REF] developed a flexible tubular device using CSRRs with high penetration depth to characterize colorectal tissue. The prototype is designed to be deployed in patients' colon and evaluate the tissue's health or malignancy in six directions (Figure 13). Figure 13. Flexible tubular device for colon tissue dielectric spectroscopy using three CSRRs and their equivalent electrical models [START_REF] Maenhout | Flexible, segmented tubular design with embedded complementary split-ring resonators for tissue identification[END_REF].

Skin tissue and its abnormalities have also been considered a possible application of resonant microwave sensors [START_REF] Bai | Flexible microwave biosensor for skin abnormality detection based on spoof surface plasmon polaritons[END_REF] and CSRRs; as in [START_REF] Sharma | Design and Modeling of the Ring Resonator-Based Microwave Sensor for Skin Cancer Detection[END_REF], performance of a CSRR-based senor on a detection of cancerous cells in a skin tissue phantom is validated using simulations. Variation in the amplitude and phase of reflection and transmission coefficients in the frequency range of 2-18 GHz is correlated to the presence of cancer cells with higher water content in the epidermis of their developed skin model.

The resonant sensors and the characterized biological matter are not limited to what we discussed here; the research in this area is ongoing, aiming to improve the specifications such as quality factor and sensitivity, for a more precise detection.

Conclusions and Future Perspectives

In conclusion, the field of DS is large and growing. This non-ionizing, non-invasive and labelfree characterization technique is already employed for biological matter in the detection of cancer biomarkers, glucose sensing in diabetes patients and many more areas, and can be adapted for the characterization of any type of biological matter on a continuously lowering scale as the operating frequencies rise through the development of new measurement technologies and equipment. The biomedical applications of dielectric spectroscopy are not limited to the ones mentioned here, neither are the technologies only valuable on their own. Combining different DS techniques as well as combining DS techniques with other characterization methods e.g., electrochemical analysis, can allow us to achieve measurements in a broader frequency range [START_REF] Bao | Broadband dielectric spectroscopy of cell cultures[END_REF], [START_REF] Dittami | A multilayer MEMS platform for singlecell electric impedance spectroscopy and electrochemical analysis[END_REF]. Moreover, artificial intelligence has yet to improve this field as drastically as it did a lot of scientific domains in the last decades: Machine Learning algorithms can be applied in the design process [START_REF] Wang | Optimal design of planar microwave microfluidic sensors based on deep reinforcement learning[END_REF], or in data analysis, such as for classification [START_REF] Osterberg | Microwave sensing of yeast cell species and viability[END_REF] or personalized sensor calibration, to improve the performance of dielectric spectroscopy. Despite the advances in sensor miniaturization as well as measurement techniques and instruments, bringing dielectric spectroscopy to point-of-care applications remains challenging. Large, complicated, and costly VNA setups which require calibration before measurements restrain the application of the sensors for untrained users.

Moreover, it causes the devices to be a chip-in-a-lab rather than a lab-on-a-chip. Recent progress in the development of VNA-on-a-chip is considered a game changer in that regard and is expected to bring the devices into the mobile testing domain. Finally, dielectric spectroscopy combined with microwave heating brings up new possibilities in cancer theranostics; the tumor can be detected through dielectric spectroscopy (diagnostics) and specifically targeted as its dielectric properties alter its sensitivity to ablation techniques (therapeutics).

Figure 2 .

 2 Figure 2. Complex permittivity of biomatter. Similarly represented in [4].

Figure 3 .

 3 Figure 3. Conceptual illustration of dispersions in permittivity spectrum: as the frequency

Figure 4 .

 4 Figure 4. Conceptual illustration of resonances in permittivity spectrum: as the frequency

Figure 5 .

 5 Figure 5. An illustration of the concept of 𝛽-relaxation, where the Maxwell-Wagner

Figure 6 .

 6 Figure 6. Illustration of different types of microwave sensors for dielectric characterization

Figure 8 .

 8 Figure 8. (a) Propagation of electric field in quasi-TEM mode. (b) microfluidic channel on

Figure 9 .

 9 Figure 9. Examples of microwave transmission-based sensors depending on bio-sample scale

  a), the resonance frequency changes the most when the sample is placed over the ring gap, and so the bacteria growth medium is placed over the sensitive gap in SRR, and the corresponding capacitance and conductance gradually change over time, which is measured through the change in amplitude of the reflection or the transmission as well as the resonance frequency shift. The time-dependent variation of the resonance amplitude for two different volumes of E-coli (Figure 11 (b)) corresponds to the bacterial growth. As the simulation results in Figure 11 (c) and (d) confirm, the changes in the loss tangent contribute to changes in the amplitude, which can be translated into bacterial growth.

Figure 11 .

 11 Figure 11. Planar Split Ring Resonator (SRR) sensor for bacterial growth monitoring in a real-

Figure 12 .

 12 Figure 12. Triple CSRR sensor for non-invasive glucose measurement and equivalent electrical

Table 1 :

 1 Overview of dielectric characterization methods for biological matter and their applications.


text:
Olivia Peytral-Rieu

David Dubuc

Katia Grenier
email: grenier@laas.fr

Microwave-Based Sensor for the Noninvasive and Real-Time Analysis of 3-D Biological Microtissues: Microfluidic Improvement and Sensitivity Study

published or not. The documents may come

Abstract-Microwave dielectric spectroscopy of biological materials is gaining of interest as it presents attractive features such as being non-invasive, rapid, label-free and suitable for intracellular investigations, notably for biological and medical researches. Specific microwave biosensors have consequently been developed to investigate biomolecules, cells in suspension and also tissues. A type of biosensor is however missing for the analysis of 3D aggregates of cells, also called microtissues or spheroids, which constitute an intermediate biological model between 2D cell cultures and tissues or organs. Therefore, we present a microwavebased microsensor suitable for the study of such 3D biological models. It is composed of a coplanar waveguide (CPW) with a central capacitive gap, which allows to measure the capacitive and conductive contrasts between the studied bio-object and a reference liquid, typically the cell culture medium, in the frequency range from 500 MHz to 20 GHz. This sensor also integrates a microfluidic channel to maintain the microtissues in liquid. Two fluidic configurations are investigated, one open and one closed, the latter including a mechanical trap. The advantages of the closed configuration are then highlighted experimentally, enabling accurate and real-time microwave measurements of microtissues maintained in their culture medium and in the vicinity of the sensing area for sensitivity enhancement. In addition, an analytical study on the capacitive gap size performed with both simulations and experiments leads to an optimal gap size for sensitivity improvement. This new sensor consequently offers new non-invasive, non-destructive and dielectric sensing possibilities for biomedical investigations such as drug screening, personalized medicine and also fundamental research.

Index Terms-Biology, coplanar waveguide, dielectric spectroscopy, microwave biosensor microtechnology, microtissues, spheroids.

I. INTRODUCTION

LECTRICAL approaches, especially dielectric spectroscopy techniques, which exploit the interaction between Manuscript received XXX XX, 2023; revised XXX X, 2023; accepted XXX X, XXXX. This work was supported in part by LAAS-CNRS micro and nanotechnologies platform member of the French RENATECH network. (Corresponding author: Olivia Peytral-Rieu) O. Peytral-Rieu, D. Dubuc are with the LAAS-CNRS, Université de Toulouse, CNRS, Université Paul Sabatier, Toulouse, France (e-mails : opeytral@laas.fr, dubuc@laas.fr).

electromagnetic fields and materials, present attractive interests for a broad range of researches including biological ones [START_REF] Stoy | Dielectric properties of mammalian tissues from 0.1 to 100 MHz; a summary of recent data[END_REF]- [START_REF] Mehrotra | EM-Wave Biosensors: A Review of RF, Microwave, mm-Wave and Optical Sensing[END_REF]. RadioFrequency (RF) impedancemetry and microwave dielectric spectroscopy are gaining increasing attention for noninvasive, nondestructive biological investigations, while being label-free and cost-effective [START_REF] Thielecke | Evaluation of impedance spectroscopy for the characterization of small biological samples in tissuebased test systems[END_REF]- [START_REF] Groeber | Impedance Spectroscopy for the Non-Destructive Evaluation of In Vitro Epidermal Models[END_REF]. Electromagnetic waves in the microwave frequency range gather in particular significant interests for biological detection investigations. First, the wavelengths are larger or similar to the dimensions of the living and biological materials under test, resulting in a large variety of possible biological materials to study [START_REF] Schwan | Electrical properties of blood and its constitutents: Alternating current spectroscopy[END_REF], ranging from nanoscale elements to tissues and body investigations. Appropriate electromagnetic instrumentations are therefore available to analyze biological phenomena according to the dielectric properties of the matter. In addition, water, which is the largest constituent of any biological materials or living cells [START_REF] Grenier | Recent Advances in Microwave-Based Dielectric Spectroscopy at the Cellular Level for Cancer Investigations[END_REF], presents its dielectric relaxation in the microwave frequency range (around 20 GHz at ambient temperature), due to its molecular reorientation dynamic. Water molecules are then used as a natural marker of living organisms with microwave dielectric spectroscopy. Finally, this frequency range allows electromagnetic waves to bypass the bilipid cell membrane, giving access to intracellular investigations.

Microwave dielectric spectroscopy has therefore been demonstrated for detecting amino acids, carbohydrates, proteins, and nucleic acids in aqueous solutions, as well as cell suspensions without any requirement of pretreatment, labeling, fixation and/or permeabilization [START_REF] Grenier | Microwave dielectric spectroscopy: An emerging analyzing technique for biological investigations at the cellular level[END_REF]- [START_REF] Mohammadi | A Label-Free, Non-Intrusive, and Rapid Monitoring of Bacterial Growth on Solid Medium Using Microwave Biosensor[END_REF]. Moreover, it may also detect differences between treated and untreated cells (and yeasts) [START_REF] Osterberg | Microwave Sensing of Yeast Cell Species and Viability[END_REF], [START_REF] Artis | Microwaving Biological Cells: Intracellular Analysis with Microwave Dielectric Spectroscopy[END_REF], making it a promising method for testing drug effects. Drug screening in oncology is particularly important to identify potential molecules, that could effectively eliminate tumors. These tumors also constitute specific threedimensional (3D) biological objects with their own defense bio- When a molecule is tested in drug screening, the results must be as accurate as possible, while using biological models as close as possible to the real conditions and environment. Traditionally, 2D cultured cells are used for such experiments, due to their ease of preparation and affordability, although such models are now considered to be quite far from the heterogeneity and the complexity of the in vivo configuration. Mastered 3D cell aggregates, also called spheroids or microtissues, have consequently been introduced, constituting a new intermediate biological model between 2D cultured cells and in vivo investigations [START_REF] Nath | Three-dimensional culture systems in cancer research: Focus on tumor spheroid model[END_REF]. They present both the advantages of 2D cell cultures, i.e a rapid and affordable growth, and those of an in vivo configuration, with its specific complexity of environment and structure. Moreover, the size of the microtissues gives access to different models of tumor. Indeed, when the microtissues are larger than 200 µm in diameter, a hypoxic core (dead cells) is formed, inducing the formation of different layers in the spheroid, a necrotic one in the center, while external and living cells may spread [START_REF] Nath | Three-dimensional culture systems in cancer research: Focus on tumor spheroid model[END_REF]- [START_REF] Veelken | Single cell-based automated quantification of therapy responses of invasive cancer spheroids in organotypic 3D culture[END_REF].

From the study of individual cells to that of organs, including cells in suspension, numerous microwave based-devices exist both in fluidic or other configurations [START_REF] Lazebnik | A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries[END_REF]- [START_REF] Zarifi | Noncontact and Nonintrusive Microwave-Microfluidic Flow Sensor for Energy and Biomedical Engineering[END_REF]. However, for the intermediate size of microtissues, none have been introduced at this particular frequency range. Only a first version of a microwave-based sensor dedicated to the determination of the dielectric properties of spheroids of 300 µm in diameter in the frequency range from 500 MHz to 20 GHz was introduced in [START_REF] Peytral-Rieu | Microwave Sensor Dedicated to the Determination of the Dielectric Properties of 3D Biological Models from 500MHz to 20GHz[END_REF]. This structure exhibits an open fluidic configuration, which leads to significant measurement dispersions due on the one hand to a lack of precision in the position of the microtissue within the sensor and on the other hand to intrinsic evaporation. To overcome these drawbacks, a complete revised version of the sensor is proposed and quantitatively evaluated in this article. It now integrates a hydrodynamic trapping solution to precisely locate the spheroid under test in the close vicinity of the microwave sensing zone and a close microfluidic channel with inlet and outlet, enabling a continuous liquid flow and avoiding any possible evaporation phenomenon. To better highlight the improvements provided by this entirely revised sensor configuration, the first sensor is briefly reminded to understand the disadvantages that motivated the development of the new sensor integrating a fluidic advanced structure. A comparison study is then carried out using both sensor configurations loaded with a liquid medium and with polystyrene beads. To do so, the electrical modeling employed to extract the capacitive and conductive contrasts of the microwave measurements is previously given. Finally, an analytical study of the capacitive gap used to sense the biological element is provided to define the best dimensions of the device to maximize sensitivity. To characterize the fabricated microdevice, this latter is connected to a Vector Network Analyzer using two coplanar probes and two coaxial cables, using a preliminary SOLT calibration step. Measurements are performed from 500 MHz to 20 GHz.

II. MICROWAVE SENSOR DESCRIPTION AND FLUIDIC OPTIMIZATION

A. Description of the Microwave Sensor and Test Setup

B. Fluidic Configuration of the Sensor

In order to maintain the spheroid under test in its culture medium during the experiments, the first microwave device presented in [START_REF] Peytral-Rieu | Microwave Sensor Dedicated to the Determination of the Dielectric Properties of 3D Biological Models from 500MHz to 20GHz[END_REF] has an open channel with two reservoirs on each side. The spheroid placement has to be manual in this configuration and the medium can evaporate. Consequently, dispersions of the dielectric measurements exist. To overcome these two issues, a closed fluidic system is fabricated with the object blocked by a permanent flow and a mechanical trap placed just above the sensing gap. This trap includes three openings. The central one is larger than the two others placed on each side and serves as a guide to the object to be analyzed. The side openings allow the fluid to flow while the central one is closed by the object under test, as indicated by the crosssection of the structure in Fig. 1(f).

For experiments, the fluid channel is loaded with a Phosphate Buffered Saline (PBS) solution at a flow rate of 5 µL/min.

C. Microfabrication of the Device

The fabrication of the microwave biosensor involves standard microtechnology processes. First, the sensor is made on a quartz substrate, which is previously cleaned with a piranha process and an oxygen plasma treatment. The metallization used to define the microwave coplanar waveguide is then performed with a lift-off technique. The obtained waveguide is composed of a titanium flash to enable the metal adhesion to the substrate, followed by an evaporated gold layer of 0.3 μm thick.

A patterning of a photosensitive SU-8 polymer layer is then carried out to obtain a passivation on the device, except at the location of the coplanar accesses and of the sensing gap. A second metallization is also done to create air bridges with the same process as that described for the coplanar waveguide.

D. Electrical Modeling and Dielectric Contrasts Calculation

As the microwave biosensor is a two-port system, an electrical model is defined, as indicated in Fig. 2(a). It includes (1) two-line sections with a characteristic impedance Z0 associated with a phase θ, (2) two normalized admittances which correspond to the microfluidic channels y1 (= Y1Z0), also considered to be symmetrical with respect to the sensing zone, and (3) y2 (= Y2Z0), the normalized admittance of our object under test, also modeled by a capacitor, C, and a conductance, G, placed in a shunt configuration. S11 and S21 corresponds to the measured S parameters when the structure is loaded with a bio-entity, whereas S11empty and S21empty are the S parameters while the device is empty. As explained in [START_REF] Peytral-Rieu | Microwave Sensor Dedicated to the Determination of the Dielectric Properties of 3D Biological Models from 500MHz to 20GHz[END_REF] and considering θ = 0, y2 may be expressed as:

𝑦 𝟐 = 1 2 ( 1- 𝑆 𝟏𝟏 -𝑆 𝟐𝟏 𝑆 𝟏𝟏𝑒𝑚𝑝𝑡𝑦 1+ 𝑆 𝟏𝟏 -𝑆 𝟐𝟏 𝑆 𝟏𝟏𝑒𝑚𝑝𝑡𝑦 -𝑦 𝟏 ) , (1)
Where

𝑦 𝟏 = 1- 𝑆 𝟏𝟏 +𝑆 𝟐𝟏 𝑆 𝟏𝟏𝑒𝑚𝑝𝑡𝑦 1+ 𝑆 𝟏𝟏 +𝑆 𝟐𝟏 𝑆 𝟏𝟏𝑒𝑚𝑝𝑡𝑦 . ( 2
)
Using the calculated y1 and y2, one may extract the C capacitor and the G conductance, which model the object under test with Eq. 3 and Eq. 4 respectively:

𝐶 = 1 2𝜋𝑓 𝐼𝑚𝑔(𝑦 𝟐 ) 50 , (3)
𝐺 = 𝑅𝑒(𝑦 𝟐 ) 50 , ( 4
) where f is the frequency.

To better enhance electrical variations between the host medium and the entity under test, electrical contrasts are considered. Those capacitive and conductive contrasts are obtained using Eq. 5 and Eq. 6 respectively, where CDUT and GDUT are the capacitance and the conductance of the device under test when loaded by an object to be analyzed and CREF and GREF are the capacitance and the conductance obtained with a reference fluid in the sensor, respectively. ∆𝐶 = 𝐶 𝐷𝑈𝑇 -𝐶 𝑅𝐸𝐹 (5) ∆𝐺 = 𝐺 𝐷𝑈𝑇 -𝐺 𝑅𝐸𝐹 (6) The measurements are consequently realized in two different steps, with first the measurement of the device with the reference fluid (here PBS) and then the measurement of the device with the biological object each minute during fifteen minutes.

This procedure is applied to both measured and simulated data. By way of example, Fig. 2(b) and (c) present the photograph of the open-fluidic structure loaded with a microtissue and the extracted mean values of the capacitive and conductive contrasts obtained for three different spheroids, respectively.

E. Experimental Evaluation of the Microfluidic Channel Modification

As explained in the section II.B, the initial microwave sensor presented in [START_REF] Peytral-Rieu | Microwave Sensor Dedicated to the Determination of the Dielectric Properties of 3D Biological Models from 500MHz to 20GHz[END_REF] includes an open fluidic channel. This configuration leads to several drawbacks. Here is evaluated the improvement expected by a closed microfluidic configuration. As the major dispersions with the first sensor are detected with polystyrene beads due to their low weight, this model is also kept in this investigation to better highlight the improvement of the closed fluidic structure.

To perform this evaluation, two beads in PBS are measured in the two fluidic configurations. Due to the dispersions in the available polystyrene beads, these may present slightly different diameters. Microwave measurements are recorded every minute for at least 20 minutes.

A double-regime stabilization is observed on both types of devices, open and closed. The first regime is related to the fluidic stabilization inside the system and lasts approximately 5 min. This first period is not considered in the remainder of this article. The extracted data referring to 0 minute consequently start after this stabilization.

Only in open fluidic configuration, a second period of measurements is linked to the fluctuations induced by the bead movements in the structure. Such variations are not observed in the closed structure. Therefore, to be able to compare the two fluidic configurations, this second period is divided in two stages (0-5 min and 7-11 min), in order to consider the time needed for the final placement of the beads in the open fluidic structure.

The mean values and the standard deviation of the dielectric contrasts are calculated for each half period. The trajectories between the two periods are given in Fig. 3 information on the dispersions and the stability of the measurements. This figure clearly shows that the ΔC and ΔG variations of the open system are larger than those obtained with the closed system. The standard deviation of ΔG indeed presents a value ranging up of 0.78 mS with polystyrene beads in the open configuration, instead of 0.02 mS with the closed structure. Likewise, the standard deviation of ΔC is up to 5.61 fF and less than 0.21 fF for the respective settings. Moreover, the placement of the beads on the sensing gap is also faster and more efficient with the closed system.

A summary of these results is given in Table I with both mean values and standard deviations of ΔC and ΔG over an 11 minutes period. The low standard deviations in the case of the closed configuration confirms its superiority in term of stability of the microwave measurements in comparison with the open structure. The flow in the closed system indeed allows stable localization of the polystyrene bead at the bottom of the mechanical trap, in the close vicinity of the microwave electrodes. In addition, evaporation no longer has an influence. This new closed system is consequently more suitable for accurate measurements.

In addition to the fluidic improvement, an analytical study of the biosensor is performed to enhance its sensitivity.

III. SENSITIVITY OPTIMIZATION OF THE CAPACITIVE SENSING GAP

A previous study performed on a microwave biosensor dedicated to the analysis of individual cells [START_REF] Chen | Parametric study of a microwave sensor dedicated to the dielectric spectroscopy of single particles and biological cells[END_REF] has already demonstrated the influence of the width of the capacitive gap on the sensitivity of the sensor. Therefore, to investigate a 10 µm-diameter cell, a structure with a 2 µm-width gap presents Fig. 4. a) 3D view of the global system at scale with the electrodes in yellow on a quartz wafer in gray, the SU8 passivation in green, the fluidic system in red and the liquid medium in blue. A polystyrene bead placed in front of the fluidic blocker on the electrodes is symbolized in purple. The pink and brown planes crossing the spheroid represent the two sectional views of c), e) and d), f) respectively. b) Zoom on the sensing zone and the gap width g with the four different gaps tested. Representations of the electric field modulus for a gap width of 10 and 60 µm at 5 GHz obtained with HFSS along the (y,z) plane for c) and e) respectively, and along the (x,z) plane for d) and f). The white dotted line represents the border of the polystyrene bead. the highest sensitivity. However, the device presented in this paper aims at studying 3D biological object with a diameter close to 300 µm. An analytical study related to the impact on the sensitivity of the capacitive gap width is consequently conducted in order to determine the optimal value to achieve the maximum dielectric contrasts. A specific object model is initially defined.

A. Choice of the Object Model Under Study

The object, which is chosen to evaluate the influence of the gap on the sensitivity, corresponds to polystyrene beads with a diameter close to 300 µm. They are indeed easier to manipulate than microtissues during the microwave measurements performed to validate the analytic study. They present a round 3D shape, which is simple to implement in an electromagnetic software. Finally, due to the low permittivity of polystyrene compared to water-based biological entities, high dielectric contrasts are expected. The which correspond to aggregates of cells and thus with constituents similar to those present in the host medium, indeed exhibit low dielectric contrasts compare to the reference fluid.

B. Simulations of the Microwave Sensor

To evaluate the influence of the gap width on the sensitivity of the device, 3D simulations using HFSS are performed at different frequencies (1 GHz, 5 GHz, 10 GHz, 15 GHz and 20 GHz). The schematic of the simulated structure is given in Fig. 4(a). Fig. 4(b) shows the different gaps tested. The permittivities of the different elements used for the simulations comes from [START_REF] Chen | Microwave biosensor dedicated to the dielectric spectroscopy of a single alive biological cell in its culture medium[END_REF]- [START_REF] Grenier | Integrated Broadband Microwave and Microfluidic Sensor Dedicated to Bioengineering[END_REF]. Initially, the HFSS simulations are carried out to get the theorical S parameters of the empty device (𝑆 𝑒𝑚𝑝𝑡𝑦 ) and then of the device loaded by the reference fluid (𝑆 11 and 𝑆 21 ). This enables to obtain the capacitance and conductance of the reference fluid, 𝐶 𝑅𝐸𝐹 and 𝐺 𝑅𝐸𝐹 , with the same calculation as described in section II.D. In a second phase, the S parameters of the device loaded with a polystyrene bead placed in the reference fluid are simulated to obtain the capacitance and the conductance of the device under test, i.e. 𝐶 𝐷𝑈𝑇 and 𝐺 𝐷𝑈𝑇 respectively, for the different gaps and frequencies of interest. Finally, the extracted capacitances and conductances are used to calculate the two capacitive and conductive contrasts, ΔC and ΔG, according to the gap sizes and frequencies.

The results of the simulations are presented in dotted lines in Fig. 5 and Fig. 6 for a 300 µm diameter bead. Fig. 5 presents the capacitive and conductive contrasts versus frequency, while Fig. 6 provides the capacitive and conductive contrasts versus the gap width at 5 GHz and 15 GHz respectively. Maximum contrasts in absolute value are observed for a width of 10 µm.

To confirm these results, experiments are conducted and described in the next section.

C. Experimental Results

The influence of the capacitive gap on the sensitivity of the device is tested with the closed fluidic configuration.

Due to microfabrication constraints, structures with a capacitive gap width equal to or greater than 10 µm are fabricated and tested, ranging from 10 to 60 µm. All devices are microfabricated on the same quartz wafer to limit microfabrication variations from batch to batch.

Three different beads are measured for each size of the capacitive gap. Each measurement is taken every minute for 15 min. As previously mentioned in section II.E, the first four minutes are not considered to allow the stabilization of the microwave measurements. The mean value and standard deviation for each gap width are then extracted based on the 10 minutes long measurements. These results are all gathered in Fig. 5 and Fig. 6 in solid lines. Fig. 5 presents the mean value of the capacitive and conductive contrasts, 𝛥𝐶 and 𝛥𝐺 respectively, as a function of frequency. Each color corresponds to a gap width. The PBS measurement is used as the reference for the two contrasts. Similar to simulation results, the magnitudes of the dielectric contrasts decrease as the gap width rises, except for 𝛥𝐺 above 18 GHz. This result is confirmed by the curves indicated in Fig. 6, which presents the capacitive and conductive contrasts as a function of the gap width at 5 GHz and 15 GHz respectively. The greatest magnitude contrasts are obtained for a gap width of 10 µm. As visible with the 3D views of the electromagnetic simulations of Fig. 4, the electric field is more intense in the 10 µm wide gap and its vicinity in comparison to that of 60 µm (cf. Fig. 4(c) compare to Fig. 4(e). Even if the first temptation would have been to choose large capacitive gaps to better cope with samples size, smaller gaps should however be preferred to maximize the strength of the electromagnetic field, leading to better sensitivity of the elements under test.

IV. CONCLUSION

In this article, an optimized microwave sensor dedicated to the analysis of 3D biological models is described. Due to its closed fluidic configuration with an integrated mechanical trap to block microtissues in the immediate vicinity of the sensing area, the sensor operates both in flow and in real time. A comparison of this structure with the one having an open fluidic configuration clearly highlights the benefits of the new fluidic channel and micro-tissue trapping. A better stability of the microwave measurements is achieved as well as an improved reliability. Moreover, the placement of the microtissue to be tested is obtained more quickly and more efficiently with the closed fluidic system compare to the open one.

In addition, an analytical study of the capacitive sensing area is performed to define the best sensor dimension adapted to improve its sensitivity. Both simulations and experimental results carried out with polystyrene beads lead to the selection of a capacitive gap of 10 µm wide to reach the highest dielectric contrasts in amplitude.

Such a microwave sensor enables real time analysis of microtissues exhibiting a size close to 300 µm in diameter. Due to the configuration of the mechanical trap, the host medium of the spheroid may be modified during the microwave experiments. This capability is particularly interesting for drug screening investigations and opens up new perspectives in biology and medicine with the non-invasive and dielectric analysis of 3D biological models.

Fig. 1 (

 1 a) to (c) and Fig. 1(d) to (f) present the schematics and the photographs of the RF biosensors with open and closed fluidic configurations respectively. They are based on a coplanar waveguide with a capacitive gap of 10-µm width located in the center of the structure. The positioning of the coplanar probes is achieved with 50 Ohm accesses, which present a central conductor of 135 µm wide and slots of 14 µm large. As far as the closed fluidic configuration is concerned, the grounds are also balanced with air bridges. For both structures, the sensing gap is placed just below the object under test in the middle of the coplanar line. While considering the fluidic configurations, the open structure presented in Fig. 1(b) and c) implies to maintain the object under test with the circular shape of the open fluidic wall only, as shown in the cross section of Fig. 1(c). Two reservoirs are placed on each side of

Fig. 1 .

 1 Fig. 1. Presentation of the two versions of the microwave sensor. Schematics of the CPW structures with a) an open fluidic configuration and d) a closed fluidic one, with the gold layer in yellow, the SU8 passivation in green, the fluidic system in brown. Photographs of b) the open fluidic and e) the closed fluidic structures. c) and f) Cross sections of c) the open fluidic and f) the closed fluidic structures, at the location marked by the dotted line in a) and d). The purple sphere or circle represent a 300 µm diameter 3D biological entity. The grey color corresponds to the wafer substrate, while the yellow layer to gold electrodes.

  The next step is dedicated to the elaboration of the fluidic part with the creation of both the spheroid blocker and fluidic channel. These are realized with the superposition and patterning of several dry photoresist films. The total height of the fluidic walls corresponds to 400 µm thick and the top lid to 100 µm thick respectively.

Fig. 2 .

 2 Fig. 2. a) Equivalent electrical circuit used for data analysis. b) Photograph of the open-fluidic structure loaded with a microtissue. c) Extracted mean value of the capacitive and conductive contrasts obtained for three different spheroids and the PBS reference fluid. Spheroids 1, 2 and 3 are indicated by the red, green and purple curves and present a diameter of 262 µm, 266 µm and 364 µm respectively. The solid lines correspond to the capacitive contrasts ΔC, while the dotted lines are for the conductive contrasts ΔG.

  Fig. 3. Comparison of variations between dielectric contrasts with open (a, b, c) and closed (d, e, f) systems. a) and d) correspond to the measurements of PBS whereas b), c), e) and f) of polystyrene beads. The plain symbol corresponds to the data obtained during the 0-5 min period and the hollow one during the 7-11 min period. The arrows indicate the trajectories between the two time periods.

Fig. 5 .

 5 Fig. 5. Experimental mean values of the capacitive and conductive contrasts obtained for single polystyrene beads as a function of frequency for different gap widths (10, 20, 40 and 60 µm) for solid lines and simulation data in dotted line.

TABLE I

 I

	COMPARISON BETWEEN THE OPEN AND CLOSED CONFIGURATIONS
		∆C at 5GHz (fF)	∆G at 20GHz (mS)
		Mean	Standard deviation	Mean	Standard deviation
	PBS opened	-1.09	0.57	-0.02	0.06
	206 µm opened	-65.81	1.60	-10.38	0.22
	260 µm opened	-63.81	5.61	-10.01	0.78
	PBS closed	-1.14	0.15	-0.04	0.01
	292 µm closed	-60.46	0.13	-5.81	0.02
	212 µm closed	-64.12	0.07	-6.95	0.01
	Mean values are calculated by considering the entire eleven-minutes period
	of the different measurements.