D Dubuc

K Grenier
email: grenier@laas.fr

M-P Rols
email: rols@ipbs.fr

A Tamra



Abstract-This paper presents a 40 GHz microwave biosensor used to monitor and characterize single cells (THP-1) subjected to electrochemotherapy and obtain an electronic signature of the treatment efficiency. This biosensor proposes a non-destructive and label-free technique that first allows, with the rapid measurement of single untreated cells in their culture medium, the extraction of two frequency-dependent dielectric parameters, the capacitance (C (f)) and the conductance (G (f)). Second, this technique can powerfully reveal the effects of a chemical membrane permeabilizing treatment (Saponin). At last, it permits us to detect, and predict, the potentiation of a molecule classically used in chemotherapy (Bleomycin) when combined with the application of electric pulses (principle of electrochemotherapy). Treatment-affected cells show a decrease in the capacitive and conductive contrasts, indicating damages at the cellular levels. Along with these results, classical biological tests are conducted. Statistical analysis points out a high correlation rate (R 2 >0.97), which clearly reveals the reliability and efficacy of our technique and makes it an attractive technique for biology related researches and personalized medicine.

Index Terms-Biosensor, Electrochemotherapy, Microwave dielectric spectroscopy, Single Cell, THP-1 cells

I. INTRODUCTION

LECTROCHEMOTHERAPY (ECT) is a local antitumor treatment that combines the administration of chemotherapeutic drugs with the local application of electric field pulses in order to transiently depolarize the cell membrane, making it permeable, and thus facilitate the delivery of non-permeant or low permeant drugs into cancer cells [START_REF] Mir | Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses[END_REF]- [START_REF] Testori | Electrochemotherapy for cutaneous and subcutaneous tumor lesions: a novel therapeutic approach: ECT for skin tumors[END_REF]. Among chemotherapeutic agents, bleomycin and cisplatin are the best potential candidates for ECT in cancer patients [START_REF] Mir | Therapeutic perspectives of in vivo cell electropermeabilization[END_REF]. Once passed the cellular membrane and inside the cells, bleomycin induces single and double strand DNA breaks that lead to cell death [START_REF] Tounekti | Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized[END_REF]. The increase in the concentration of the drug in the tumor cells (300-700 fold) [START_REF] Gehl | Enhancement of cytotoxicity by electropermeabilization: An improved method for screening drugs[END_REF] allows the dramatic increase of the cytotoxicity of the molecule locally, at the site of the electroporation and leads to neoplastic cell death. The healthy cells of the tissues surrounding the tumor nodules, which are not subjected to electroporation pulses, are protected from toxic action. This local control of the tumor prolongs the lifespan and improves the quality of life of the patients [START_REF] Spugnini | Electrochemotherapy for localized lymphoma: a preliminary study in companion animals[END_REF]. In addition to drug-induced cell destruction, electroporation is responsible for vascular changes in the tumor region. A "vascular lock" effect is induced where blood stops flowing though the tissue that has been electroporated [START_REF] Gehl | Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research[END_REF]. This vascular blocking effect seems to last longer in tumor tissues than in normal ones [START_REF] Sersa | Electrochemotherapy of Tumours[END_REF]. After first successful in vitro studies [START_REF] Orlowski | Transient electropermeabilization of cells in culture: Increase of the cytotoxicity of anticancer drugs[END_REF], ECT has been successfully applied in the first clinical trial, performed in 1990 [START_REF] Mir | Electrochemotherapy, a new antitumor treatment: first clinical trial[END_REF]. Electrochemotherapy is mainly used in treatment of cutaneous and subcutaneous cancers [START_REF] Gothelf | Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation[END_REF]. ECT induces a local yet robust immune response that is responsible for its high effectiveness [START_REF] Maglietti | Electroporation as the Immunotherapy Strategy for Cancer in Veterinary Medicine: State of the Art in Latin America[END_REF]. It is also of great interest for people diagnosed with cutaneous malignancies at advanced ages as it offers an efficient, safe nonsurgical treatment [START_REF] Sersa | Outcomes of older adults aged 90 and over with cutaneous malignancies after electrochemotherapy with bleomycin: A matched cohort analysis from the InspECT registry[END_REF]. Research is still ongoing for treatment of deep-seated tumors such as colorectal cancer [START_REF] Scala | Electrochemotherapy for rectal cancer after neoadjuvant radiotherapy: A case report[END_REF] and liver metastases [START_REF] Edhemovic | Intraoperative electrochemotherapy of colorectal liver metastases[END_REF]. Other modalities, such as calcium electroporation, where cytotoxic drugs are replaced by high concentration level of calcium, also showed great promise [START_REF] Ágoston | Evaluation of Calcium Electroporation for the Treatment of Cutaneous Metastases: A Double Blinded Randomised Controlled Phase II Trial[END_REF]. In clinical assays, it is crucial to use the optimal electrical parameters along with the convenient drug concentrations, in order to obtain the desired effect while minimizing the side effects and the damage of the healthy cells. This comes from the significant differences between different tumor cell types and different patients phenotypes. From here comes the urge to deepen the research on the effects of ECT with various drugs on different cell types.

Many researches are now focalizing on transposing electroporation studies "on-chip" [START_REF] Khine | A single cell electroporation chip[END_REF]- [START_REF] Geng | Microfluidic electroporation for cellular analysis and delivery[END_REF] in order to avoid costly, time-consuming work and invasiveness of the routinely used methods, such as microscopy, flow cytometry and chemical tests. Although very effective, there is an ongoing trend to develop and employ new analysis methods [START_REF] Ye | Single-Cell Electroporation with Real-Time Impedance Assessment Using a Constriction Microchannel[END_REF], [START_REF] Hai | On-chip electroporation, membrane repair dynamics and transient in-cell recordings by arrays of gold mushroom-shaped microelectrodes[END_REF]-

Single Cell Microwave Biosensor for Monitoring Cellular Response to Electrochemotherapy

A. Tamra, A. Zedek, M-P. Rols, D. Dubuc, and K. Grenier E [START_REF] Khine | Single-cell electroporation arrays with real-time monitoring and feedback control[END_REF], among which the electrical spectroscopy analysis.

Microwave dielectric spectroscopy (MDS) is a label-free analysis approach that involves applying microwave waves (40 MHz -40 GHz) to biological samples and measuring their dielectric properties. It has several advantages such as the use of non-ionizing waves for a non-intrusive, direct and contactless analysis. The technique mainly detects changes of the water molecules network and is related to any modification in the ratio of bound water molecules to free ones [START_REF] Grenier | Recent Advances in Microwave-Based Dielectric Spectroscopy at the Cellular Level for Cancer Investigations[END_REF]- [START_REF] Poiroux | Label-free Detection of Mitochondrial Activity with Microwave Dielectric Spectroscopy[END_REF]. Due to the penetration of electromagnetic waves in the cells in the microwave range, intracellular characterization is accessible either with cell suspensions [START_REF] Grenier | Recent Advances in Microwave-Based Dielectric Spectroscopy at the Cellular Level for Cancer Investigations[END_REF], [START_REF] Artis | Microwaving Biological Cells: Intracellular Analysis with Microwave Dielectric Spectroscopy[END_REF], [START_REF] Grenier | Integrated Broadband Microwave and Microfluidic Sensor Dedicated to Bioengineering[END_REF] or at the single cell level [START_REF] Yang | Distinguishing the viability of a single yeast cell with an ultra-sensitive radio frequency sensor[END_REF]- [START_REF] Ma | Ultra-Wideband Impedance Spectroscopy of a Live Biological Cell[END_REF]. The MDS technique permits therefore to sense and monitor pathological transformations [START_REF] Levy | Dielectric Response of Cytoplasmic Water and Its Connection to the Vitality of Human Red Blood Cells: I. Glucose Concentration Influence[END_REF], [START_REF] Poiroux | Label-free Detection of Mitochondrial Activity with Microwave Dielectric Spectroscopy[END_REF]. In the context of electroporation, it has been used to study the conductivity of the cell suspension [START_REF] Chen | Conductivity in Jurkat cell suspension after ultrashort electric pulsing[END_REF], the cytoplasm and the nucleoplasm [START_REF] Garner | Ultrashort electric pulse induced changes in cellular dielectric properties[END_REF] of Jurkat cells exposed to nanosecond electric pulses nsPEF. The kinetic of cells reactions submitted to different electric stresses has also been monitored [START_REF] Tamra | Microwave Monitoring of Single Cell Monocytes Subjected to Electroporation[END_REF], [START_REF] Li | Correlation Between Optical Fluorescence and Microwave Transmission During Single-Cell Electroporation[END_REF] with microwave signals. However, to our knowledge, there is no work reporting the study of electrochemotherapy effect on biological cells, using MDS analysis. This work results from our previous paper showing a microwave and microfluidic-based biosensor, able to perform dielectric spectroscopy of the single cell and to monitor the intracellular effects of both reversible and irreversible electroporation [START_REF] Tamra | Microwave Monitoring of Single Cell Monocytes Subjected to Electroporation[END_REF]. In this paper, we present the ability of our biosensor to detect the effects induced by bleomycin treatment coupled with electroporation, i.e. electrochemotherapy, on THP-1 cells. The ultimate goal is to propose a method that can be used in hospital laboratories, which can be applied to evaluate and predict the effect of different types of treatment on cells, in a non-invasive way, without the addition of dyes or fluorochromes.

II. MATERIALS AND METHODS

A. Description of the microwave biosensor and its fabrication

The MDS biosensor, initially developed by Chen et al. [START_REF] Chen | Microwave biosensor dedicated to the dielectric spectroscopy of a single alive biological cell in its culture medium[END_REF], is composed of two different parts (Fig. 1a). The first part is a coplanar waveguide (CPW) that consists in a central golden ribbon (signal) and two ground planes, with a capacitive gap of 10 µm in the center of the central conductor. It is fabricated using an evaporated titanium and gold layer, which is obtained with a lift-off technique and presents a thickness of 0.3 μm. The waveguide is realized on a borosilicate glass substrate, which exhibits low dielectric losses in the microwave range with a loss tangent of 0.0047 and a permittivity of 4.3. On top of the CPW, a 50-µm thick microfluidic channel is perpendicularly placed to the golden waveguide and includes a mechanical trap located just apart the capacitive gap (Fig. 1b). With this trap, one single cell is captured at a time allowing it to interact with the electromagnetic fields, which are focalized in the capacitive gap. Both the microfluidic channel and the trap are fabricated with SU-8 polymer using photolithography technique, whereas the initial technology developed by Chen et al. [START_REF] Chen | Microwave biosensor dedicated to the dielectric spectroscopy of a single alive biological cell in its culture medium[END_REF] integrated a fluidic part realized in polydimethylsiloxane (PDMS). The change of fluidic material presents several improvements. The PDMS-elastomer imposes a manual placement of the fluidic part on the microwave sensing electrodes, whereas SU-8 polymer is precisely patterned with photolithographic equipment. The misalignment between the capacitive gap and the cell trap is therefore drastically minimized. In addition, PDMS exhibits

Microwave measurements Exclusion test (Trypan blue)

THP-1 cells low stiffness relative to the polymer. While inadvertent sticking of the PDMS blocker to the substrate may occur, the use of a rigid SU-8 structure allows for better microfluidic stability. Primarily, a sacrificial layer of 5 μm thick in LOR photoresist is used to realize the air gap localized at the bottom of the mechanical trap. Its high curing temperature of 180°C enables to stack the subsequent two SU-8 layers, which are deposited and patterned. The first one defines both the cell blocker and the fluidic walls, whereas the second SU-8 layer corresponds to the lid of the fluidic structure. The first SU-8 layer is deposited by spin-coating with conditions suitable to reach a final thickness of 50 μm after hard baking. Once its development is done, the second SU-8 is deposited by rolling. After photolithography and development of the lid, the sacrificial layer is removed by wet etching before drying the structure with ethanol.

The electrical circuit associated with our biosensor is presented in Fig. 1c, where the coplanar waveguide access lines, the SU8 microfluidic walls and the capacitive sensing zone are considered. A de-embedding procedure permits to remove the impact of the access lines and the fluidic walls. Its description is indicated in section 3.1. The individual cell under test is modeled by an electrical circuit, which includes a capacitor C in parallel to a conductance G.

B. Cells and reagents

In this study, THP-1 cells (ATCC # TIB-202) are employed for three reasons. First, they are human cells derived from monocytic leukemia and constitute an important biological model for studies in oncology. Their electroporation parameters are also known [START_REF] Tamra | Microwave Monitoring of Single Cell Monocytes Subjected to Electroporation[END_REF]. Finally, as circulating cells, they exhibit a low tendency to aggregate, which is appropriate for analyzing only individual cells in the microwave sensor. They are grown in Roswell Park Memorial Institute Medium (RPMI) (Gibco-Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal calf serum FCS and a mixture of antibiotics 100 U / ml penicillin and 100 µg / ml streptomycin. The cells are incubated at 37°C under 5% CO 2 for proliferation.

The chemical treatments used are saponin, supplied by Sigma-Aldrich (Lyon, France) and bleomycin, supplied by Merck Millipore (Darmstadt, Germany).

C. Electrochemotherapy

The protocol used is shown in Fig. 2. THP-1 cells are harvested, re-suspended and incubated for 5 minutes in the drug-electroporation buffer mixture. The composition of the buffer is the following, 10 mM K₂HPO₄/KH₂PO₄, 1 mM MgCl₂, 250 mM of sucrose, pH 7.4, conductivity 1.7 S/m. The final concentration of the Bleomycin in the solution is 250 nM (250 nMol/L). Eight electric pulses, applied at 1 kV/cm, are generated by a Betatech generator (ELECTRO cell S20, France), where the duration and frequency are fixed as 100 µs and 1 Hz [START_REF] Marty | Electrochemotherapy -An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study[END_REF]. After electroporation, the cells are incubated with the culture medium for 24 hours, at 37°C. At the end of this incubation time, the cells are either analyzed with the biological exclusion test (Trypan blue test, which is a specific dye that instantly penetrates into the cells with permeabilized/damaged cytoplasmic membranes), or injected in the microwave biosensor in order to extract the electrical responses. The experiments are carried out in triplicate. Five groups of cells are tested:

1. Untreated cells, negative control 2. Cells with 0.02% saponin, positive control 3. Cells with bleomycin alone (250 nM) 4. Cells with electric pulses alone (1 kV/cm) 5. Cells with ECT (250 nM bleomycin + 1 kV/cm).

D. Microwave measurements

Before proceeding to the microwave measurements, a crucial step of de-embedding is performed, in order to remove the effects of the access lines and the walls and to be able to extract the capacitive and conductive contributions of the cell alone. After injecting and measuring the culture medium alone, which represents the reference medium, the cell suspension is injected in the microfluidic channel. With the help of the mechanical trap, one single cell is blocked right above the sensing zone, whereas all other cells are removed by flushing the culture medium in the fluidic channel. Measurement of S parameters is performed once stabilization is electrically observed. When the sensor is loaded with the culture medium and then with an individual cell, C reference medium , G reference medium , and C cell and G cell are extracted respectively. Since C cell and G cell are proportional to the contrasts of the real and imaginary parts of the relative permittivity of the sample, it will therefore be relevant to extract and study these two parameters and their contrast with respect to a reference medium (medium not containing the studied sample: culture medium without cell in our case). This will allow us to observe the changes directly related to the presence of the sample. The differential method is particularly interesting because it takes the drift of the measurement chain over time into account, while improving the repeatability and reproducibility of the tests. As a result, we calculate the capacitive and conductive contrast of the cell relative to the reference medium (culture medium), according to the following equations:

(1) .

(

) 2
The microwave measurements are performed on wafer in a range of frequencies from 40 MHz to 40 GHz. Further details on the method of extraction of these two readouts could be found in [START_REF] Tamra | Microwave Monitoring of Single Cell Monocytes Subjected to Electroporation[END_REF]. The contrast spectra obtained at the end of this calculation are presented as a function of frequency. All of the procedures described allow us to perform microwave dielectric spectroscopy MDS, delivering the ΔC and ΔG spectra over a wide frequency range of the microwave domain.

III. RESULTS AND DISCUSSION

A. Dielectric properties of THP-1 cells

After having recovered all the measurements of the S parameters, a data processing is applied with Matlab software. It allows us to extract the contrasts of capacitance and conductance as a function of frequency. As an illustration of such data, Fig. 3 (a,b) presents the measured S parameters as well as the extracted capacitance and conductance, while the sensor is loaded by the host medium of the cells. Fig. 4 (a,c) shows, respectively, capacitive contrast (ΔC) and conductive contrast (ΔG) spectra obtained for untreated THP-1 cells. Only a representative number of spectra is shown in all the graphs of this paper. Each spectrum represents the electrical response of a single captured cell over the frequency range from 40 MHz to 40 GHz. As far as the differential method presented earlier is concerned, the corresponding culture medium (RPMI) is considered as the reference. The obtained spectra show that, compared to the culture medium alone, the cell has a significantly different spectral response. Indeed, in living cells, the plasma membrane provides regulation between the intra-and extracellular medium while maintaining a heterogeneity between the concentrations of their constituents. This will result in an increase, in absolute value, of the capacitive and conductive contrasts, resulting from the wellexpressed difference between the relatively simple composition of the culture medium, compared to the living cell with all its complexity. On the other hand, Fig. 4 (b,d) shows ΔC and ΔG spectra obtained for saponin-treated THP-1 cells (0.02%). Saponins are qualified as detergent molecules because of their ability to solubilize the plasma membrane, creating a permanent irreversible damage [START_REF] Podolak | Saponins as cytotoxic agents: a review[END_REF], while maintaining the cell's architecture: the nucleus, the mitochondria and other cellular organelles are always present. A significant difference is observable between an untreated cell and a treated cell, on both parameters (ΔC and ΔG). The saponin-treated cells have less contrast than the untreated cells. In the case of cells treated with saponin, which will be considered as our positive control for the rest of the study, the cell membrane becomes permeable. Under the effect of the osmotic pressure linked to the difference in concentrations between the intra-and extracellular species, an equilibrium is created between the two compartments. The cytosolic composition is therefore greatly impacted, which results in a lower contrast between the extracellular and intracellular media compared to untreated cells. This results in a decrease (in absolute values) of the capacitive and conductive contrasts. Thus, using our biosensor, we are able to highlight the presence of a living cell in its liquid culture medium, as well as permeabilized cells, presenting different dielectric responses. Table 1 summarizes the capacitive and conductive contrasts, at 5 GHz and 30 GHz respectively, of THP-1 cell line, with standard deviation values. Around fifty cells have been tested for each condition. These frequencies are particularly chosen to have the maximum value of the two parameters and are consequently considered in the next sections specifically. This table also shows the value of the threshold values for C and G contrast parameters defining the living and dead (i.e., permeabilized) characters of cells. These threshold values were defined in the middle of the measured dynamics of the living cell batches versus the saponin treated ones. This threshold will be used in the rest of the study for cell screening depending on the viability.

B. Dielectric properties of THP-1 cells treated with electrochemotherapy

As explained earlier, one of the applications of electroporation is to facilitate the transport of low or nonpermeant molecules through the cell membrane and thus potentiate the cytotoxic effect of anti-tumor drugs (electrochemotherapy). The aim of this study is to evaluate the ability of our microwave biosensor to detect the loss of cell viability induced by the entry of bleomycin molecules after electroporation. Thus, THP-1 cells are treated with either bleomycin alone, electroporation alone, or electrochemotherapy (bleomycin + EP). The different dielectric responses are compared to those of untreated cells (negative control) and saponin-treated cells (positive control). Fig. 5 and Fig. 6 show, respectively, capacitive contrast (ΔC) and conductive contrast (ΔG) spectra obtained for different conditions of treatment. A small but representative number of spectra is presented in these figures, in order to give a general idea of the spectral behavior of the cells as well as the measurement repeatability. By comparing the treated cell spectra to the negative control cell spectra (Fig. 4) in a qualitative manner, we observe a decrease in absolute values of ΔC (Fig. 5.a) and ΔG (Fig. 5.b) for some cells, especially those treated with ECT. It creates a distinct separation between two cell subgroups: living cells and cells affected by the ECT treatment.

In order to consolidate our observations, a quantitative study of the obtained results is made. Therefore, we proceed to a cell sorting and a classification based on the thresholds established earlier. Fig. 6.a and Fig. 6.b show the evolution of the mean values of the capacitive and conductive contrasts, respectively, at the selected frequencies (5 GHz and 30 GHz), as a function of the treatment condition. The percentages of living and affected cells are also shown on the graphs. In comparison to the negative control cells, which feature a viability rate around 80%, we can clearly establish that when cells are treated with bleomycin alone or subjected to the electric field alone, the resulting viability is not significantly affected (~72%, ~80%), whereas treating cells with bleomycin coupled to the electric field causes an increase in the number of cells exceeding the threshold and exhibiting electrical responses characteristic of dead cells (~53%). The same behavior is observed on the conductive contrast ΔG, where ECT induces a clear decrease in viability down to ~36%.

In order to further validate our results and the efficacy of our biosensor, we compare and correlate the evolution of the viability rates obtained with microwave measurements (ΔC 5 GHz and ΔG 30 GHz ) to the standard biological exclusion test with trypan blue. Indeed, there is a concordance in the results obtained with the two techniques, as shown in Fig. 7. Statistical analysis reveals a very good correlation between the two methods of analysis (R 2 ΔC/Exclusion test = 0.97) / (R 2 ΔG/Exclusion test = 0.99), indicating the reliability of microwave dielectric spectroscopy for detecting the effect of electrochemotherapy on THP1 cells.

C. Single cell analysis

Biological studies have been for long performed on groups of cells assuming that all cells of a particular "type" are identical. However, studies on individual cells reveal that this assumption is incorrect. Individual cells within the same population can vary considerably [START_REF] Carlo | Dynamic Single-Cell Analysis for Quantitative Biology[END_REF]- [START_REF] Bakstad | Quantitative measurement of single cell dynamics[END_REF]. This is called "individuality" of cells within the population [START_REF] Zhao | Cell individuality: A basic multicellular phenomenon and its role in the pathogenesis of disease[END_REF]. These differences can have important consequences for the understanding of certain diseases, since certain singular cells play a fundamental role in the pathogenesis of the disease, its evolution over time and in the sensitivity of cells to drugs. New approaches to single-cell analysis are therefore crucial to discover fundamental biological principles and, as a result, improve the detection and treatment of diseases.

The measurements we obtained in our study are represented in a complex plane (ΔC 5 GHz , ΔG 30 GHz ), as shown in Fig. 8.a. We identify a distribution in two clouds of measuring points, based on the threshold values established earlier. The first cloud (1) brings together living cells and the second one (2) contains affected (i.e. dead) cells. By calculating the mean values and the standard deviations of the measurements for each condition (Fig. 8.b), we note that the untreated cells, along with the cells treated with bleomycin or electroporation alone, have a mean value that lies outside the designated affected box, whereas the mean values of the cells treated with saponin or electrochemotherapy lie inside of this box. The mixture of living and affected cells in this population gives a mean value that does not reflect the true state of the two subpopulations of cells: this means that a global study of cell populations masks information when cell suspensions become heterogeneous, hence the interest of a study at the level of the single cell.

IV. CONCLUSIONS

This paper presents an original single cell biosensor based on microwave spectroscopy for the dielectric characterization of human cells subjected to electrochemotherapy. Our biosensor is designed for a label-free detection and an effective measurement of frequency-dependent cell-based parameters (C cell (f) and G cell (f)) of THP-1 cells subjected to different types of treatment. With our biosensor, we are able to reveal the potentiation of the bleomycin molecule with the application of electric pulses (electrochemotherapy). Aside from dielectric measurements, comparison with standard biological tests (based on the exclusion test with trypan blue) points out a strong correlation between the two methods (R 2 >0.97). These findings indicate the robustness and the reliability of microwave dielectric spectroscopy for label-free biological and medical analysis, and that it may be very helpful to provide deeper insights on the effect of electrochemotherapy on cancer and healthy cells and to optimize electric pulses parameters to ensure efficient cancer cell eradication whatever the cell type and population heterogeneity.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Microwave biosensor. (a) The optical microscope image of the microwave biosensor with a microfluidic channel. (b) 2D cross section view of the biosensor. (c) Associated electric scheme of the device.

Fig. 4 .Fig. 3 .

 43 Fig. 4. Capacitive contrast of untreated cells (a) and saponin-treated cells (c). Conductive contrast of untreated cells (b) and saponin-treated cells (d). Cells measurements are performed in culture medium, which serves as reference.

Fig. 5 .

 5 Fig. 5. (a) Capacitive contrast of electroporated cells (yellow color), bleomycin-treated cells (brown) and electrochemotherapy treated cells (green). (b) Conductive contrast of electroporated cells (yellow), bleomycin-treated cells (brown) and electrochemotherapy treated cells (green). Purple dashed lines represent the thresholds.

Fig. 6 .

 6 Fig.6. THP1 response to different treatments estimated by microwave measurements (contrasts of capacitance at 5 GHz and conductance at 30 GHz) and by biological method (exclusion test with trypan blue).

Fig. 8 .

 8 Fig. 8. Position of the different measurements of THP-1 cells undergoing different treatments, in the complex plane (ΔC5GHz, ΔG30 GHz) (a) without and (b) with the standard deviations of measurement.

This work was supported in part by the the Toulouse COMUE and the Region Midi-Pyrénées in France and in part by LAAS-CNRS micro and nano technologies platform, a member of the RENATECH French national network.


Christelle Ghazaly

Krystyna Biletska

Etienne A Thévenot

Philippe Devillier

Emmanuel Naline

Stanislas Grassin-Delyle

Emmanuel Scorsone

Etienne A Thevenot

Development and characterization of electronic noses for the rapid detection of COVID-19 in exhaled breath

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION MATERIALS & METHODS

RESULTS

CONCLUSIONS & PERSPECTIVES

Effect of various experimental conditions on sensor response

Non-invasive and rapid approach is needed for diagnosis of COVID-19. In this work, exhaled breath analysis using e-Nose is presented as an innovative technique to identify the COVID-19 specific VOCs. The analytical performances of Cyranose ® , a commercial e-Nose device, were investigated under controlled conditions towards this goal. Sensitivity, limit of detection and reproducibility of standardized VOCs existing in the breath was assessed. In addition, the effect of various experimental conditions on sensor response was evaluated, including temperature, relative humidity, flow and sampling time, aiming to select the optimal parameters and to validate it in clinical trials to detect the COVID-19 biomarkers. Cyranose ® exhibits high sensitivity and reproducible response towards acetone and nonanal, with a limit of detection of 63 ppb and 20 ppb respectively. Furthermore, results show that the variability of relative humidity, temperature and flow sampling, induced a significant sensors response variation, whereas, varying the sampling time does not affect significantly the sensor response.

1

  CEA, LIST, Laboratoire Capteurs Diamant, 91191 Gif-sur-Yvette, France 2 CEA, LIST, Laboratoire Sciences des Données et de la Décision, 91191 Gif-sur-Yvette, France 3 Département Médicaments et Technologies pour la Santé (DMTS), Université Paris-Saclay, CEA, INRAE, MetaboHUB, Gif-sur-Yvette, France 4 Hôpital Foch, Exhalomics, Département des maladies des voies respiratoires, Suresnes, France *christelle.ghazaly@cea.fr Experimental Setup Evaluation of analytical performances of Cyranose ® sensitivity towards:  Acetone: VOC normally found in the breath of a healthy patient. Limit of Detection: 63 ppb  Nonanal: VOC presents in the exhaled breath of COVID-19 patients*. Limit of detection: 20 ppb Test of an exhaled breath of a control patient  Real-time response of 32 sensors of Cyranose ® : -Sample: exhaled breath of a control patient -Flow rate: 120 mL/min -Baseline: ultra-pure dry nitrogen -Baseline & Sampling time: 60 secondes & 90 secondes  Four sensors exhibit meaningful responses for VOCs in the exhaled breath of a control patient  High repeatability and reproducibility of resistance variations for ten successive measurements Calculation of resistance variation for sensors: S5; S6; S11 and S31

  Breath analysis using Cyranose ® :an array of 32 gas sensors 1-Sampling of an exhaled breath by Cyranose ® 2-A nanocomposite chemiresistive gas sensors 1-Tedlar ® Bag containing:  Exhaled breath of a control patient  Generated classical VOCs (acetone, ethanol…)  Specific vapor identified as a biomarker for COVID-19 (nonanal) processing & Statistical treatment  Pcnose: provided with Cyranose ®  E-NaiR: software tool for e-Nose devices developed by CEA-JOLIOT and used for data processing of clinical trials at Foch Hospital

A-Relative humidity rate B-Sampling flow rate C-Sampling time

		 The highest resistance	Varying sampling
	Increasing relative humidity from 2% to 84% affected positively sensors variations.	variations are obtained by applying a high flow rate of 180 mL/min.  Repeatable variations are obtained under 92% RH for ten successive	time from 20 s to 60 s does not affect significantly sensor's response.
		measurements.


Benoît Sabot
email: benoit.sabot@cea.fr

Chavdar Dutsov

Philippe Cassette

Krasimir Mitev

Performance of portable TDCR systems developed at LNE-LNHB

Keywords:

The triple to double coincidence ratio (TDCR) liquid scintillation measurement technique is commonly used in national metrology institutes (NMIs) to perform standardization of pure beta emitters. The LNE-LNHB historical device, RCTD1, is a quite large device, which has been developed and commonly used over the past 30 years with its associated electronics for measurements of various radionuclides. During the last 4 years LNE-LNHB has developed two new portable TDCR devices. Such portable instrumentation gives end-users access to a reference measurement method that can be used for a large number of radionuclides. It addresses a wide range of industrial and medical applications for radionuclide metrology, including calibrations of solutions containing short-lived radionuclides, avoidance of radioactive source transportation, and performing on-site comparison to promote radionuclide metrology harmonization. In this paper, we will present the newly developed portable TDCR liquid scintillation measurement systems. Two kinds of devices have been developed and designed at LNE-LNHB and built using fused deposition modelling (FDM) 3D printing: a mini-TDCR (25 cm diameter, 10 cm height) and a micro-TDCR (16 cm diameter, 10 cm height). After a detailed discussion of the design and the possibilities offered by 3D printing for their conception, this paper will present the performance of the devices obtained for several radionuclides. The results will be compared with the RCTD1 in order to validate the performance of the new devices. They exhibit improved performance, such as higher detection efficiency, and include various useful tools for proper on-site metrology. The first tests, which were performed in Orsay Hospital (CEA/SHFJ) and in another laboratory (IRSN), allowed us to show the very good overall performance of the systems including their outstanding linearity, which was tested in the range from 430 000 s -1 down to 160 s -1 in the double coincidences channel. Applications of the developed systems for high and low activity measurements are also discussed. Finally, the portable device has been used for half-life measurements in order to check for impurities in a radio-pharmaceutical solution.

Introduction

The triple-to-double coincidence ratio (TDCR) technique is a direct measurement method regarded by the international metrology community as a primary measurement technique for many radionuclides (pure beta or alpha emitter, electron capture, etc.) [START_REF] Broda | Radionuclide metrology using liquid scintillation counting[END_REF]. The measurand is the activity of the scintillating source, which can be traced to the mass activity of the solution if the scintillating sources are prepared by precision weighing. The radionuclides that can be calibrated by this method are those for which it is possible to estimate the energy spectrum absorbed by the scintillating source following a decay. In general, for the simplest decay schemes (i.e. a pure beta emitter that decays directly to the ground state) the TDCR measurement is easier than for a complex scheme, and for highest detection efficiency the TDCR measurement is more accurate as it is less dependent on the model. This concerns in particular radionuclides decaying by a pure beta transition or by electron capture to the fundamental level of the daughter nucleus. Application of the TDCR method requires the use of three photomultiplier tubes (PMTs). The design of such a device with high detection efficiency is a key prerequisite for high accuracy measurements, and achieving this was one of the primary goals of this work.

Previous studies performed by different national metrology institutes (NMIs), including the Laboratoire National Henri Becquerel (LNE-LNHB) from the Commissariat à L'énergie Atomique (CEA), have shown the importance of having portable TDCR devices in order to perform on-site calibrations, especially for the measurement of 3 H in nuclear power plants [START_REF] Cassette | Development of Portable Liquid Scintillation Counters for On-Site Primary Measurement of Radionuclides Using the Triple-to-Double Coincidence Ratio Method[END_REF]. In addition, some radionuclides, such as those used for positron emission tomography examinations ( 18 F with a half-life of 1.8 h; 11 C with a half-life of 20 min; 15 O with a half-life of 2 min), are difficult to transfer from the production site (typically a Hospital equipped with a cyclotron) to an NMI for primary calibration because of their short half-life. Hence the requirement for on-site primary Fig. 1. 3D illustration of the mini-TDCR device. Its large size is due to additional components that can be included in the system: an external photon-emitting source with a collimator and a gamma detector (NaI(Tl) or Ce(Br) 0.5 inch size and CdTe). With these components the system can be used as a Compton spectrometer. measurement techniques in order to comply with French regulation [3]. A primary measurement technique available on site will not only allow calibration of the dose calibrator in the numerous geometries required in the radiopharmaceutical production laboratory, but also, provide an on-site technique to respond to specific metrology requests for the development of new radiopharmaceuticals, such as alpha emitters or those decaying by electron capture.

Secondly, for some radionuclides such as 222 Rn in water, where the half-life is longer (3.8232 d), source transportation, preparation and sampling are quite complex, requiring on-site calibration of the liquid scintillation counters. In practice, transportation and regulations related to the use of radioactive sources are becoming more and more difficult [4], so having on-site capability, involving the use of a transportable and reference device, becomes a useful tool for metrology purposes.

Finally, with the recent development of the Extended International Reference System for pure 𝛽-particle emitting radionuclides (ESIR) [START_REF] Coulon | The international reference system for pure beta-particle emitting radionuclides: An evaluation of the measurement uncertainties[END_REF], we believe that such a well-known and well characterized portable device could be used for portable reference systems similar to the International Reference System Transfer Instrument (SIR-TI) [START_REF] Bipm | The SIRTI : A new Tool Developed at the BIPM for Comparing Activity Measurements of Short-Lived Radionuclides World-Wide[END_REF]. ESIR and SIR-TI aim to demonstrate the international equivalence of primary standards from NMIs, so that such portable TDCR devices could be used in the same way as travelling instruments between NMIs. This work presents the progress made in the last four years, starting in 2016, for the conception and validation of two portable TDCR devices. After a detailed description of the devices and their properties, their validation will be discussed with different comparisons performed in the laboratory with the historical device RCTD1 [START_REF] Cassette | Experimental evaluation of TDCR models for the 3 PM liquid scintillation counter[END_REF] and associated electronics [START_REF] Bouchard | MAC3: an electronic module for the processing of pulses delivered by a three photomultiplier liquid scintillation counting system[END_REF], In addition, on-site comparisons with a portable device [START_REF] Mitev | Design and performance of a miniature TDCR counting system[END_REF] of a laboratory from Sofia University ''St. Kliment Ohridski'', Bulgaria (SUBG), will be presented. Finally, on-site measurements with the developed devices are presented and discussed.

Materials and methods

The two measurement devices have been designed and developed at LNE-LNHB. The first one, named mini-TDCR, is based on the feedback of a previous study which had shown that the Hamamatsu R7600U photo-multiplier tubes (PMTs) proposed by the Italian Metrology Institute (ENEA) [START_REF] Cassette | Development of Portable Liquid Scintillation Counters for On-Site Primary Measurement of Radionuclides Using the Triple-to-Double Coincidence Ratio Method[END_REF] seemed to be well suited to the design of such devices. A specific feature of the mini-TDCR is the inclusion of a Compton-TDCR system [START_REF] Cassette | The compton source efficiency tracing method in liquid scintillation counting: A new standardization method using a TDCR counter with a compton spectrometer[END_REF] with gamma spectrometer. The second one, named micro-TDCR, uses new and more efficient Hamamatsu H11234-203 MOD PMTs and is a more compact device.

Design considerations

The design of the two portable instruments was primarily oriented by the geometry of the selected PMTs in order to maximize the detection efficiency. To do so, the following characteristics and assumptions were considered:

• Typical measurement sample: 20 mL liquid scintillation vial.

Among all types of vials available in the laboratory (glass, plastic, etc.), the maximum external diameter is less than 28 mm, and the maximum height with the cap is 68 mm. The position of the meniscus in a vial filled to 10 mL is 20 mm above the bottom of the vial. • The meniscus of the liquid scintillation vial, with a 10 mL filling, was placed in the centre of the PMTs to maximize light collection.

The light output at the meniscus level is strong as the light is totally reflected at the liquid-air interface. • In order to minimize light loss between the emission point and the PMTs, the distance between the sample and the PMT window was optimized to account for their small size. • The addition of different neutral density filters may be necessary to vary the detection efficiency; therefore, sufficient space was provided to accommodate these filters between the vial and the PMTs. • The shape of the optical chamber is optimized to favour reflection of the light and direct it towards the PMTs. • The system is compact and includes a miniature high voltage power supply, equipped with a safety device to avoid damaging the PMTs during a sample change. This safety device turns off the high voltage if the apparatus is opened, allowing light to enter. • The instrument must allow for easy sample changing, while being perfectly light-tight once the measurement has started. • The device must not be sensitive to surrounding electromagnetic fields and is therefore shielded against these disturbances. • The thresholds of the photomultipliers must be easily adjustable in order to allow the detection of single photons, a condition inherent to the measurement method. • It must be possible to easily combine the TDCR devices with other detectors in order to perform coincidence measurements or to use a Compton spectrometer.

The limitations being well established, new ways of production were considered in view of the complexity of the elements required, not necessarily achievable by standard manufacturing. The laboratory has thus been equipped with FDM 3D printers [START_REF]Volumic3D, Volumic3D FDM Printer[END_REF]. The FDM technique, which consists of adding material deposited by fusion, offers the best quality/versatility/price ratio possible with 3D printing. More than a hundred readily available materials can be used with this technique. The printed parts do not require any treatment, and the use of commercial 3D printers is relatively trouble-free. This manufacturing process allows complex shapes to be produced which are not easy to manufacture using computer numerical controlled (CNC) machining. At the very least, with machining, it would be necessary to break down the main parts of the system into several parts to be manufactured and assembled. Hence, the design of the optical chamber and enclosure was developed assuming that everything could be produced by 3D printing.

Prior to the design of the detector, studies were performed on the different materials available for 3D printing, resulting in polylactic acid (PLA) filaments been chosen, mainly because of their availability in both white and black, which facilitates the printing of opaque material.

The drawback with this printing technique is the fabrication method, which consists of fusing filaments in successive layers whose thicknesses range from 25 μ m to 300 μ m. Each layer is made of 400 μ m wide molten plastic fibres fused together side by side. It appears that thin PLA layers are partially transparent to light. This effect is due either to the material thickness or to light passing between two fibres that may not be fused properly. In our case, we have observed that it was necessary to have a minimum thickness of 2 mm of PLA to avoid external light penetration.

Mini-TDCR system

The mini-TDCR (Fig. 1) uses three Hamamatsu R7600U-200 PMTs [START_REF] Hamamatsu | Photomultiplier Tubes R7600U Series[END_REF] with an ultra bialkali photocathode. The supply voltage is positive so that the photocathode is at ground potential, which reduces the background noise [START_REF] Hamamatsu | Photomultiplier Tubes -Basics and Application[END_REF]. The PMTs provide good quantum efficiency with a maximum of 40% for wavelengths between 300 nm and 650 nm, which is compatible with the emission spectrum of liquid scintillators.

The face of the PMTs is 30 mm square, and they are 53 mm long. These dimensions allow them to be brought as close as possible to the light source, i.e. the 20 mL scintillation vial, as shown by the crosssection in Fig. 2. A diffusive layer of adhesive tape is placed on the PMT windows to minimize optical reflections due to the difference between the refractive indices of air and glass. The active surface of these PMTs is an 18 mm square, so a large part of the PMT window is insensitive to light.

As can be seen in Fig. 2, 3D printing allows the formation of recesses, which are used here to minimize the absorption of radiation in the materials of the measuring device, especially for the gamma detectors integrated in this mini-TDCR device.

Micro-TDCR system

The micro-TDCR uses Hamamatsu H11934-203 PMTs [START_REF] Hamamatsu | Photomultiplier Tubes R11265U and H11934 Series[END_REF] with a 30 mm square window and a depth of 32 mm. The sensitive surface is a 23 mm square, larger than the previous PMTs. These PMTs also have an ultra bialkali photocathode like the preceding ones, with the same quantum efficiency, but the window of the PMTs is made of quartz and is diffusive. The smaller size of these PMTs allows a smaller device to be built, as shown in Fig. 4.

Compared to the mini-TDCR, the micro-TDCR cannot accommodate any other detectors and is only intended for standard TDCR measurements. A 3D vertical cross section of this micro-TDCR with the vial position are shown in Fig. 3. The meniscus of the vial is placed in the middle of the PMT in order to collect maximum light. The minimum distance between the vial wall and the PMT is 1 mm, allowing sufficient space to place neutral density filters as described below. Below the vial, the material height is 2 mm in order to prevent light from entering the chamber. As can be seen in Fig. 5, only six different 3D printed parts are needed to build the device and the components are inside the enclosure. The optical chamber is made of a different material and can easily be exchanged.

Optical chamber

Both devices, mini-TDCR and micro-TDCR, use photomultipliers with the same window sizes and accommodate the same types of liquid scintillation vials (20 mL). Consequently their optical chambers have been designed with exactly the same shape and dimensions and the vial-PMT distance are identical for both version of the TDCR. The optical chamber is made as a single piece, possible only with 3D printing. White PLA is used in this case, although this results in a surface that is not very reflective. Ideally, PC-PTFE, which has a good reflective index, could have been used, but tests showed that it was not possible to make an optical chamber with the mechanical and dimensional constraints necessary for its use (i.e. to be able to place the liquid scintillation vial properly inside the chamber). As a result, white PLA was used and improvements to the surface reflectivity were made using additional surface treatment, such as painting or aluminium or reflective foil coating. The chamber was first designed with the assumption that the use of reflected light is never total, so it is better to rely solely on direct light. As it can be seen in Figs. 6 and7, the distance between the PMT window and the vial is therefore as small as possible.

To ensure that the vial is always positioned in the same manner, a small cylindrical cut-out has been made on the bottom of the optical chamber. The radius of the cut-out is only slightly larger than the size of a standard vial, thus minimizing the possible movement of the vial inside the chamber. A drawback is that part of the bottom of the vial is not directly visible to the PMTs and some small light losses are possible. We then decided to optimise the shape of the optical chamber by treating its surface as a perfect mirror. While the upper and lower ends were modified to form a surface inclined at 45 • , various geometries were tried between the PMTs. Triangular, cylindrical and flat areas between PMTs were less efficient than two concave areas to focus the photons on the PMT windows. Accordingly, in the 3D design software used here [START_REF] Systèmes | Solidworks User Manuel[END_REF], we used the sketch function to trace the path of a photon using simple geometric constraints on the sketch line as shown in Fig. 7. Gross optimisation was performed assuming that the surface was a reflecting concave mirror and the changes in index between materials were neglected.

With this design, the chamber can easily be changed, which is convenient for modification and testing of new shapes and especially for testing different surface treatments to improve light reflection. We decided to use a highly reflective foil, enhanced specular reflector (ESR) [START_REF]3M Enhanced Specular Reflector (3M ESR)[END_REF], in order to improve the detection efficiency as will be shown in Section 3.2. This foil is commonly used for Liquid-crystal display (LCD) applications and provides a reflectivity higher than 98% for all the visible spectrum.

Neutral density filters concept

There are three types of efficiency modification methods commonly used in TDCR measurements: chemical quenching, PMT defocusing, and neutral density filters [START_REF] Cassette | Analysis of detection efficiency variation techniques for the implementation of the TDCR method in liquid scintillation counting[END_REF]. However, these methods differ in the way the scintillation light is reduced.

• Efficiency variation by chemical quenching, e.g. with nitromethane: This requires preparing a series of scintillating sources with different amounts of quenching agent. The quenching agent may act differently on the fast and the delayed fluorescence, which is not optimal. This effect was noticed with the mini-TDCR and 3 H sample with different liquid scintillators in a comparison of efficiency variation by chemical quenching and neutral density filters [START_REF] Dutsov | In quest of the optimal coincidence resolving time in TDCR LSC[END_REF]. • Changing the focusing voltage of the PMTs: This requires a fairly simple setup on the voltage divider of the PMTs but necessitates access to a focusing electrode. Moreover, this change of voltage on the PMTs directly influences their operation, which can change the active area of the photocathodes and induce an asymmetry in the detector [START_REF] Arenillas | Implementation of the TDCR liquid scintillation method at CNEA-LMR, Argentina[END_REF].

Fig. 7. Cross-section of the optical chamber, with the reflective surface treated as perfect concave mirror to optimize the shape by simple ray tracing in Solidworks [START_REF] Systèmes | Solidworks User Manuel[END_REF].

• Use of neutral density filters: These filters can be inserted between the vial and the PMTs in order to reduce the number of photons reaching the PMTs, thus reducing the detection efficiency. However, they can be quite thick, which would adversely affect our optimization of the measurement system (PMTs very close to the vial). However, unlike the other two methods for varying the detection efficiency, this one does not require any modification of the photon source or the detectors. We have therefore designed filters with different neutral density adapted to the devices. These filters consist of a plastic film on which different levels of grey are printed by a standard laser printer. The grey areas simply correspond to a level of transparency generated by any image processing software. The sheet is then printed and cut to the desired size. The 3D printed assembly tool shown in Fig. 8 provides a cylindrical filter with a diameter suitable for the measuring device and the 20 mL liquid scintillation vial. The limitation of this assembly is the glue joint of the plastic film, as shown in Fig. 8 with a poorly glued example. In order to overcome this problem, a retaining ring with a coding device that fits into the cap of the measuring device to position this non-uniform glue joint exactly between two PMTs was designed. In this way the number of photons reaching the three PMTs is reduced to the same degree with one single sample. These tools are identical for the mini-TDCR and micro-TDCR, as they both use the same optical chamber and cap.

It is also possible to completely absorb the light coming from the sample by using an opaque filter made with a piece of BK5 tissue with 120 μ m thickness [START_REF]Blackout and Laser Safety Materials[END_REF]. We use this to check if there is any direct detection of high energy radiation by the PMTs which was the case for Sr/Y-90 sample.

These neutral density filters have also been modified to act only on one PMT, as shown in Fig. 9. Such an option was used to increase the asymmetry of the PMTs and see if the correction algorithm was working for pure beta and pure electron capture radionuclides. The results are described in another paper [START_REF] Kossert | On the photomultipliertube asymmetry in TDCR systems[END_REF] where we have demonstrated that the asymmetry correction works as expected, so that non-identical PMTs can be used for the TDCR method.

Complete device and electronics

The complete micro-TDCR device is shown in Fig. 10. The power supplies for each device are locally-made, providing +900 V for the mini-TDCR and +1000 V for the micro-TDCR with a current of 1 mA for both. The three PMTs are powered by the same power supplies, and a voltage divider placed inside the TDCR device is used to deliver the same voltage to each PMT. For both systems, the PMTs are not selected, thus their efficiencies are not identical, but it was decided to supply them with the same voltage and to apply an asymmetry correction during the measurement analysis.

The electronics for signal processing is a nanoTDCR module [START_REF]nanoTDCR Triple-to-Double Coincidence Ratio Liquid Scintillation Counting System (TDCR) and MCA Manual[END_REF]. This module was developed in parallel for the portable devices and was tested with the mini-TDCR. The nanoTDCR has many advantages over the reference MAC3 unit that has been implemented in many NMIs [START_REF] Bouchard | MAC3: an electronic module for the processing of pulses delivered by a three photomultiplier liquid scintillation counting system[END_REF], which with new developments have been made, such as the ability to run four acquisitions in parallel -with two different userselectable coincidence windows (CW) and two different user-selectable base dead-time (DT) durations. A single measurement with this device, therefore provides four results at the same time (2 CW and 2 DT). This module was used for all the results presented previously, as the nanoTDCR has been validated and compared with the MAC3 unit in studies with various radionuclides and detector systems (RCTD1 and mini-TDCR) [START_REF] Mitev | Design and performance of a miniature TDCR counting system[END_REF][START_REF] Jordanov | Development and applications of a miniature TDCR acquisition system for in-situ radionuclide metrology[END_REF]. The nanoTDCR thresholds were set in the valley before the single electron peak and after the thermal noise, in order to allow the detection of single photons. The settings were made using irradiated glass tubes produced in a gamma ray irradiator. The glass of the irradiated tubes has defects which turns the glass into brown colour (Fig. 11). These defect produces single photons in large quantities, up to 4000 s -1 with the one irradiated with the highest doses we have. These irradiated glasses allow easy adjustment of the threshold of the photomultipliers, using the nanoTDCR device.

Monte-Carlo Model

To meet all the needs of the measurement method (corrective factors or efficiency calculation), geometrical models for Monte Carlo simulations corresponding to both devices were created using PENE-LOPE 2018 code [START_REF] Salvat | PENELOPE-2018: A code system for Monte Carlo simulation of electron and photon transport[END_REF]. PENELOPE is known to have an accurate model for low energy electrons, positrons and photons transport [START_REF] Asai | The PENELOPE physics models and transport mechanics. Implementation into Geant4[END_REF], which allows simulation of the energy absorbed by the scintillator to perform the proper TDCR calculations. Fig. 12 shows the model with two cross-sections.

This model corresponds to a 3D representation of the mini-TDCR or micro-TDCR device. It includes all the material of the vial, cap, liquid scintillator, PMTs and optical chamber. It is important to model all the interactions between the ionizing radiation and the detector, especially to take into account scattering of high-energy gamma photons. This Monte-Carlo model calculates the energy spectrum absorbed in the scintillator for discrete energy emissions, or from a spectrum input (probability density as a function of energy), or for the full decay scheme of a given radionuclide using the PenNuc function of PENE-LOPE 2018. The latter uses data from the Decay Data Evaluation Project (DDEP) [START_REF] Kellett | The decay data evaluation project (DDEP) and the JEFF-3.3 radioactive decay data library: Combining international collaborative efforts on evaluated decay data[END_REF]. As an example, Fig. 13 shows the simulated absorption spectrum of 18 F in an Ultima Gold liquid scintillator.

This simulation allows evaluation of the components of each emission produced by the decay of the radionuclide. The energy deposition in the scintillator is calculated from:

• The beta particles with a spectrum identical to 18 F (green in Fig. 13); as expected, these electrons produce a large energy deposition. The PENELOPE input spectrum data were obtained with BetaShape 2.0 code [START_REF] Mougeot | Betashape: A new code for improved analytical calculations of beta spectra[END_REF]; • The positron annihilation (purple in Fig. 13); the Compton scattering of the 511 keV photons produces a lower energy deposition than the electrons; • The positron particle of 18 F (red in Fig. 13); the spectrum is broader than the one from 𝛽 -particles due to the stacking of the scattered photons and the positron emission spectrum. The PENE-LOPE input spectrum data were also obtained with BetaShape 2.0 code [START_REF] Mougeot | Betashape: A new code for improved analytical calculations of beta spectra[END_REF]; • 18 F (blue in Fig. 13); it has a shape almost identical to the red spectrum but this time the simulation takes into account the decay path of the radionuclide, i.e. the emission probabilities from possible electron captures (always present for 𝛽 + decays). The PENELOPE input data were obtained with the PenNuc module.

The first three spectra help to understand phenomena taking place in the scintillator. They give the contributions of the different types of radiation in the scintillator and help to refine the calculations for the TDCR method. The last spectrum results from the absorption of energy in the scintillator following the 18 F decay (all emissions combined, taking into account the emission intensities). Nevertheless, this is not the spectrum to use for a TDCR measurement, as the coincident energy depositions cannot be simply added, due to the scintillator non-linearity.

Such models must be detailed in order to have the complex path of high energy electrons escaping from the vial, which may interact outside on the wall of the optical chamber and deposit only a fraction of the energy in the scintillator.

Results

Neutral density filter characterisation

These filters, produced using the previously described technique, were tested in the micro-TDCR device with a 3 H source in 10 mL of Ultima Gold scintillating liquid. The reduction of the T/D ratio was measured as a function of the transparency level. The results, shown in Fig. 14, prove that the filter is reducing the efficiency properly. In this test the filter level was limited to 68% transparency, as the T/D ratio becomes too small for measurements below this level.

Therefore, it is easy to produce any efficiency variation with various levels of neutral density filters. In case we need higher efficiency reduction, we can decrease the transparency of the filter quite easily.

These different levels of neutral density filters were then used to perform the efficiency variation and activity calculation on the 3 H sample in order to derive the proper kB value (ionisation quenching parameter from Birks model [START_REF] Birks | The Theory and Practice of Scintillation Counting[END_REF]) as shown in Fig. 15.

Table 1

Comparison of efficiency between the RCTD1 from LNE-LNHB and the new portable devices. TiO 2 is for the optical chamber with titanium oxide paint on its surface, DPMTs is for diffusive PMTs, ESR is for the optical chamber with highly reflective foil on its surface. The kB value was here evaluated with a coincidence window of 40 ns and a dead time of 50 μ s. The activity was calculated with an updated version of the TDCR07c code [START_REF] Cassette | Detection Efficiency Calculation for Pure-Beta Radionuclides, Program with Short Tutorial[END_REF] (TDCR18), yielding the results T/AB, T/BC, T/AC to take into account the asymmetry of the PMTs. The optimal kB value found with both the mini-TDCR and micro-TDCRs is 0.01 cm MeV -1 , which is the same as the value obtained with the RCTD1 counter.

Nuclide

Response of the TDCR devices and efficiency

Table 1 shows the different TDCR values obtained for the same batch of samples during the development of the devices. The 3 H and 14 C sources were in a toluene PPO scintillator and a glass vial, the 63 Ni source was in UG and a glass vial, and the 55 Fe source was in UGAB and a PE-PTFE vial. The results were compared between RCTD1 from LNE-LNHB and the newly developed mini-TDCR and micro-TDCR with different surface reflectivity of the optical chamber. The RCTD1 is here considered as the reference device of LNE-LNHB, it uses old Burle PMTs while today some Hamamatsu PMTs are better.

From these results we can note that the mini-TDCR has a lower efficiency than RCTD1. Even if the window of the PMTs is made diffusive in order to increase the efficiency, it still remains lower. However, this is not the case with the micro-TDCR, which has the same optical chamber as the mini-TDCR, but different PMTs. The efficiency with this device is actually higher, which shows a real improvement in the results. The reflective foil used here [START_REF]3M Enhanced Specular Reflector (3M ESR)[END_REF] has more than 98% reflectivity for visible light and is the best reflector found so far. We can clearly see a great improvement in the detection efficiency depending on the energy emission. While there is a little improvement at high energy, we can see that detection efficiency becomes very close to 1 for 14 C.

There is, however, a trade-off with these new compact devices: as shown in Table 1 they have a higher blank counting rate than the RCTD1 device. This difference is due to two phenomena: their higher efficiency and their inherent shielding. The RCTD1 device consists of an aluminium optical chamber a few centimetres thick, while the portable devices are made only of plastic with lower external radiation shielding capacity. This is nevertheless not a hindrance to the method, as standard practice is to perform a blank measurement before each sample measurement, also if necessary these portable instruments can be placed in a shielded cell, unlike other bulky instruments.

In Table 2 we present the efficiency results of both portable devices obtained for various samples measured with the devices. Here we show the T/D corresponding to each sample and the calculated activity with both devices. T/D is used as one of the indicators for the ESIR system [START_REF] Coulon | The international reference system for pure beta-particle emitting radionuclides: An evaluation of the measurement uncertainties[END_REF] for pure beta emitters. In these results the 241 Pu sample contains 2.7% of 241 Am and the 32 P sample contains 1.3% of 33 P, and the calculated activities are corrected for these impurities. The other samples do not have any detected impurities.

All these results are presented for the mini-TDCR and micro-TDCR systems, both with reflective foil on the optical chamber surface. From these results we can clearly see the performance of the systems as well as the differences between both.

Comparison and validation of the systems

Samples of 3 H and 14 C in toluene PPO scintillator were measured in a glass vial using all three devices: RCTD1, mini-TDCR and micro-TDCR. The measurements were performed at LNE-LNHB. These radionuclides, commonly measured with the TDCR method, were chosen because they are low energy emitters, so their measurement is very sensitive to the detection efficiency. For each of these measurements, the activity was calculated with the TDCR18 [START_REF] Cassette | Detection Efficiency Calculation for Pure-Beta Radionuclides[END_REF] code with the measured T/AB, T/BC and T/AC ratio measurements in order to correct for any asymmetry. For each measurement, the value kB = 0.010 (1) cm MeV -1 was used. The results calculated with 10 consecutive measurements are presented in Table 3.

Table 2

Summary of TDCR values for various radionuclides measured using both devices mini-TDCR and micro-TDCR. The activity is calculated with the same code, the nuclear data for EC radionuclides and beta spectra are calculated with BetaShape 2.0 [START_REF] Mougeot | Betashape: A new code for improved analytical calculations of beta spectra[END_REF][START_REF] Mougeot | Towards high-precision calculation of electron capture decays[END_REF]. The difference 𝛥 between the mini-TDCR and micro-TDCR is calculated as (micro-TDCR -mini-TDCR)/mini-TDCR. The vial type P stands for PTFE coated PE, G stands for glass vial, and G f stands for frosted glass vial. A slight difference can be observed in the results. It seems to be correlated to the efficiency of the devices, the higher the efficiency, the higher the activity of the final result. However, this variation is not significant in view of the measurement uncertainties, as all the results are coherent. This comparison allows us to validate the proper operation of our new devices in comparison with the RCTD1 counter.

Nuclide

The mini-TDCR device was then moved to Sofia University, in order to perform a comparison with the portable device, called TDCR-SU [START_REF] Mitev | Design and performance of a miniature TDCR counting system[END_REF]. This portable TDCR uses the same PMTs as the mini-TDCR, but the optical chamber is made of PTFE and has a different shape. The main difference is that the PMTs are not placed as close to the vial as in the mini-TDCR. The sources measured in this experiment were two standard Wallac sources ( 3 H and 14 C) and two sources calibrated by LNE-LNHB and RCTD1 in 2008 (LKB 3 H №2 and Amersham 14 C). All sources were in a toluene based cocktail in glass vials covered by diffusive tape. One of the 14 C sources was measured with and without diffusive tape, but the difference in the calculated activity was less than 0.08%.

The LKB 3 H source №2 was measured in the past with mesh filters on the TDCR-SU, and it was noticed that the optimal kB value was abnormally high (above 0.018 cm MeV -1 ), indicating some problems with the sample or the measurement. In order to identify the cause of this issue, it was decided to measure this sample with various neutral density filters in the mini-TDCR. The efficiency variation method with the same neutral density filters also was performed also on the LKB 3 H source №1 with the TDCR-SU detector in order to see if the problem came either from the TDCR-SU or from the filters. The results are presented in Fig. 16.

From this analysis we can see that the kB obtained with the LKB 3 H №1 source is 0.015 cm MeV -1 with the TDCR-SU (similar to the value with that device system and the mesh filters). There is no problem in the kB evaluation in this case. However, we again found the same unexpectedly high value of kB for the LKB 3 H №2 source, confirming that the problem is with this sample. Table 4 summarizes the comparison between each device for the different sources.

One can see a difference in the detection efficiencies between both devices. Whilst it is less than 1% for the 14 C samples, it increases to almost 12% for the 3 H sample. This shows that the design, material and reflectivity of the chamber, have a major impact on the efficiency. It should also be noted that at the time of this comparison, the mini-TDCR chamber was painted with TiO 2 paint, rather than lined with an ESR foil, as is now the case, so the detection efficiency is now higher as shown in Table 1. The Table 5 shows the D count rate of each blank sample, the LKB №1 is the blank for LKB 3 H №1 and LKB 14C. We can observe an increase in the blank D count rate on the sample in the mini-TDCR compared to the TDCR-SU. This increase seems to be due to the difference in detection efficiency as both devices are unshielded and both are made of some centimetres of plastic only. The difference of the background compared to the results obtained in the LNE-LNHB laboratory can be explained by the verified higher indoor radon concentration in Sofia University compared to that in LNE-LNHB, which is due to the soil composition. The activities obtained in this experiment were compared with the certified activity and the results are presented in Table 6. Good agreement can be seen between the current measurements and the source certificates.

We must note that, as confirmed by the filter measurements, the LKB 3 H №2 sample shows a high kB and may be compromised.

On-site test for high activity and half-life measurement

Measurements were performed in the Orsay Hospital in CEA/SHFJ (Service Hospitalier Frédéric Joliot), where 11 C and 18 F are produced for PET imaging. The aim was both to check the linearity of our device during the measurements and to show that it is possible to use the device to measure the presence or absence of impurities in the solution. These measurements were carried out on samples of 11 C and 18 F prepared from spare ready to inject solutions mixed with Ultima Gold scintillating cocktail. Thanks to the quality control performed by SHFJ, we knew that these colloidal solutions were free from impurities. Successive series of measurements were carried out on these two samples. Follow-up counting rates of the logical sum of double coincidences are presented in Fig. 17. These results were obtained with a coincidence window of 40 ns and a dead-time of 50 μ s.

All counting rates are corrected for accidental coincidences according to the methodology proposed in [START_REF] Dutsov | Evaluation of the accidental coincidence counting rates in TDCR counting[END_REF], which represents a correction of the order of 0.03% to 0.04% for 11 C and 0.03% to 0.15% for 18 F. For these radionuclides, the detection efficiency is high, leading to a low proportion of accidental coincidences despite the high count rate. The highest accidental coincidence corrections occur when the counting rate is the highest and again when the counting rate is close to the background counting rate. The smallest corrections are obtained for activity between 10 kBq and 20 kBq, which could be regarded as the optimum activity value in order to minimize the accidental coincidence correction for an activity measurement with this device and these two radionuclides. The series of measurements performed for both radionuclides were fitted with Eq. ( 1):

𝑓 (𝑥) = 𝐷 0 𝑒 ( -𝑙𝑛(2)𝑡 𝑇 1∕2
)

+ 𝐷 𝐵𝑙𝑘 (1)
Where, 𝐷 𝐵𝑙𝑘 is the background counting rate, 𝐷 0 is the counting rate at the beginning of the experiment, 𝑡 is the time and 𝑇 1∕2 is the measured half-life. The fitting is performed with 𝜒 2 minimization using the nonlinear least squares Levenberg-Marquardt algorithm implemented in Gnuplot [START_REF]Gnuplot Fitting Algorithms Manual[END_REF].

The result obtained for 11 C, 20.333 [START_REF] Cassette | Experimental evaluation of TDCR models for the 3 PM liquid scintillation counter[END_REF] min, is consistent with the DDEP value of 20.361 [START_REF] Jordanov | Development and applications of a miniature TDCR acquisition system for in-situ radionuclide metrology[END_REF] min [START_REF] Chisté | Table des radionucleides C-11, Table Radionucleides[END_REF]. In a similar way, the counting analysis was carried out for the 18 F sample; the half-life thus measured was 1.82870 [START_REF]Blackout and Laser Safety Materials[END_REF] h, in excellent agreement with the DDEP value 1.827890 [START_REF] Jordanov | Development and applications of a miniature TDCR acquisition system for in-situ radionuclide metrology[END_REF] h [START_REF] Chisté | Table des radionucleides F-18, Table Radionucleides[END_REF]. The uncertainty presented here correspond only to the fit uncertainty, a complete study of the half-life measurements and corresponding uncertainty budgets will be presented in another publication.

It is especially important to note that the first measurements were carried out with an extremely high count rate of 430 000 s -1 . These measurements were performed down to 160 s -1 , thereby demonstrating that the micro-TDCR combined with nanoTDCR device can operate over a very wide range of counting rates with very good linearity of the detection device. An impurity measurement was also performed after the total decay of the 11 C (24 h) and 18 F (7 days) respectively with liquid scintillation. The counting rate obtained 𝐷 = 4.85 (8) s -1 corresponds to the rate from the blank vial, 𝐷 = 4.87 (8) s -1 , so no long-lived radionuclides were present in the samples, as we could suspect the presence of 3 H. From these results, we deduced that the solution is free of impurities, the Table 7 presents the activity and activity concentration measurement results. The uncertainty of the measured volume is quite large compared to typical high precision mass measurements such as performed in the metrology laboratory, which are not feasible in the hospital. This explains the higher-than-normal uncertainty of the activity concentration.

The uncertainty budget also shows the importance of the exact measurement time, which is critical for the measurement of 11 C. Its short half-life has a huge impact on the results considering the chosen reference time. It should be noted that no access to an internet time server was possible during this measurement, and the corresponding uncertainty component could be eliminated if a time server were available. On the other hand, while 18 F has a longer half-life, there are some non-negligible electron capture decays that increase the uncertainty arising from the decay data. For activity calculation with the TDCR model, we used the results of PENELOPE 2018 simulations in order to get the interaction probability of 511 keV (Fig. 13). The calculated detection efficiency with the fully detailed model was 0.07% lower compared to a simple vial model.

On-site test for low activity and radon calibration

A first test, dedicated to in-situ measurement of radon in water, was performed at the Institut de Radioprotection et Sureté Nucléiare (IRSN) in the framework of their proficiency test preparation [START_REF] Tsoupko-Sitnikov | Results of the Proficiency Test 163 RN 300[END_REF]. The final goal was to calibrate their commercial scintillation detectors. The problem of 222 Rn in water is mainly related, apart from the measurement itself, to the preparation, transport and storage of liquid scintillation samples. The gas has its own behaviour in the water/scintillator mixture and can escape from the sample. By performing primary measurements in-situ on samples prepared directly within the laboratory, we eliminated all these problems and were able to directly calibrate the secondary scintillation devices. To do this, the micro-TDCR device was transported to the Environmental Analysis and Metrology Service of the IRSN/Vésinet and set up inside a shield as show in Fig. 18. This shielding was developed for an HPGe detector, but the small size of the micro-TDCR device, allows it to be placed inside the shielding in order to reduce the background. The shielding was composed of 10 cm of low-level lead and 2 mm of copper. This type of shielding is widely used for the measurement of environmental samples in gamma-ray spectrometry and allowed us to reduce the Dcount rate for the samples from 23.8 (1) s -1 to 2.1 (1) s -1 . The samples were prepared in a 20 mL PE-PTFE coated liquid scintillation vials with 10 mL of radon-loaded water (deionized water for the blank) and the rest of the volume filled with UG LLT scintillator.

The measurements were performed the day after the preparation of the samples and radon spiking, so equilibrium was reached between radon and decay products. Due to the long half-life of 210 Pb (22.23 (12) years), the decay scheme process is considered to be stopped at this radionuclide; in the present case it represents only 0.008% of the radon activity. Using the decay data, we can calculate the equilibrium ratio between 222 Rn and its progenies up to 210 Pb (considered stable in our case). This ratio is 5.025, i.e. for one radon decay there are 5.025 detectable events in liquid scintillation with an almost unitary detection efficiency as they are high energy alpha or beta particles.

However, one of the decay product of radon has a short half-life (T 1∕2 ( 214 Po) = 162.3 μ s), comparable to the dead time of the electronics. As demonstrated in [START_REF] Cassette | Standardization of 222 rn by LSC and comparison with 𝛼-and 𝛾-spectrometry[END_REF], several measurements with different The results with different dead times are presented in Fig. 19. The D and T counting rates were calculated for a dead time equal to zero using the values obtained from the fit with Eq. ( 2).

These measurements allowed us to calculate the activity of the samples prepared by the IRSN with the method from [START_REF] Cassette | Standardization of 222 rn by LSC and comparison with 𝛼-and 𝛾-spectrometry[END_REF]. The detection efficiency was determined with the T/D value obtained at zero dead time with Betashape 2.0 [START_REF] Mougeot | Betashape: A new code for improved analytical calculations of beta spectra[END_REF] spectra and considering the detection efficiency for alpha equal to 1. The calculation took into account the relative quantum efficiencies of the PMTs. The detection efficiency obtained for D is 5.0108 for radon at equilibrium with its daughter and with kB = 0.01 cm MeV -1 . The main compound of the uncertainty budget arises from the fitting results of the zero dead time extrapolation. The calculated activity of this sample is 243.4 (24) Bq of 222 Rn, this value was used by IRSN to calibrate their Tricarb LS counter.

Conclusions

The two devices developed and manufactured at LNE-LNHB are now operational and have been validated by various comparisons and in-situ measurement tests. These devices are not only portable, but also more efficient than the historical device (RCTD1) of LNE-LNHB, especially in the case of the micro-TDCR which is the smallest of the devices. The optimization of the chamber's shape was done with our only available tool at the beginning of the device development but we believe there is still potential to increase the detection efficiency when using advanced ray-tracing tools based on a comprehensive physical model. The devices provide more flexibility for testing, as we can easily 3D-print different optical chambers or specific tools. As a result, the mini-TDCR and micro-TDCR were also used in various experiments and collaborations in order to improve the TDCR method, such as the compact electronic module nanoTDCR [START_REF] Jordanov | Development and applications of a miniature TDCR acquisition system for in-situ radionuclide metrology[END_REF][START_REF] Dutsov | Study of two different coincidence counting algorithms in TDCR measurements[END_REF], optimal coincidence windows [START_REF] Dutsov | In quest of the optimal coincidence resolving time in TDCR LSC[END_REF], accidental coincidence correction [START_REF] Dutsov | Evaluation of the accidental coincidence counting rates in TDCR counting[END_REF], PMT asymmetry [START_REF] Kossert | On the photomultipliertube asymmetry in TDCR systems[END_REF], measurement of the half-life of excited nuclear states [START_REF] Dutsov | Measurement of the half-life of excited nuclear states using liquid scintillation counting[END_REF] and new studies on the time domain in liquid scintillation [START_REF] Mitev | Time-domain based evaluation of detection efficiency in liquid scintillation counting[END_REF]. The multiple experiments presented here have demonstrated the very good capability and detection efficiency of such complete devices, in particular with excellent linearity for counting rates from 430 000 s -1 to 160 s -1 . We also demonstrated the significance of such portable devices, for example for the measurements in a secondary environmental laboratory, in particular for radon, which allows source preparation and/or transportation difficulties to be overcome for this radionuclide.

We believe that such devices, which are now commonly used in our laboratory for liquid scintillation measurements, are of great interest in order to perform on-site comparison measurements or for a possible future portable Extended International Reference System for pure 𝛽particle emitting radionuclide (ESIR) devices. Finally, following these developments the work will now focus on two areas: firstly to develop the Compton-TDCR device, secondly to combine the portable TDCR with on-site source preparation using direct volume measurement.

CRediT authorship contribution statement

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 2 .

 2 Fig. 2. 3D picture of the mini-TDCR: this cut, normal to the z axis shows the position of the vial and the other detectors.

Fig. 3 .

 3 Fig. 3. Exploded view of micro-TDCR, the components of the device are all inside producing a very compact device.

Fig. 4 .

 4 Fig. 4. 3D picture of the micro-TDCR device, the most efficient and compact system.

Fig. 5 .

 5 Fig. 5. Exploded view of micro-TDCR, the components of the device are all inside producing a very compact device.

Fig. 6 .

 6 Fig.6. 3D image of the optical chamber covered by reflective foil. The complex shape allows the light to be redirected towards the PMT active areas.

Fig. 8 .

 8 Fig. 8. On the left: picture of the filter tool, in the middle: mounted filter, and filter mounted in the cap on the right.

Fig. 9 .

 9 Fig. 9. Filters with different neutral density. The filters shown here were designed to cover only one PMT.

Fig. 10 .

 10 Fig. 10. The whole set of components, including locally-made high voltage supply, can be easily handled and connected to a portable computer.

Fig. 11 .

 11 Fig. 11. Picture of the irradiated glass; the brown colour is due to a defect of the glass after exposure to Co-60 gamma rays.

Fig. 12 .Fig. 13 .

 1213 Fig. 12. Picture of the 3D model from Penelope 2018; on the left: vertical section of the system with colours corresponding to the different materials; on the right: horizontal section with the three PMTs.

Fig. 14 .

 14 Fig. 14. Results of T/D reduction depending on the neutral density filter transparency for a 3 H sample in Ultima Gold and PE-PTFE vial.

Fig. 15 .

 15 Fig. 15. Evaluation of kB for a 3 H sample in 10 mL of Ultima Gold liquid scintillator.

Fig. 16 .

 16 Fig. 16. Efficiency variation of the TDCR-SU detector with the LKB 3 H №1 source (left) and of the mini-TDCR detector with LKB 3 H №2 (right).

Fig. 17 .

 17 Fig.17. Measurement of18 F and 11 C half life on-site, the uncertainties correspond only to fitting adjustment and boundaries. The residuals are defined as the difference between experimental double counting rate and the fit function that was then divided by the standard deviation.

Fig. 18 .

 18 Fig. 18. Picture of the micro-TDCR device inside the shielding from an HPGe detector.

Fig. 19 .

 19 Fig. 19. Analysis of the results obtained with different dead time and extrapolation.

Benoît

  Sabot: Investigation, Methodology, Design, Monte-Carlo, Funding acquisition, Experiments, Writing -original draft. Chavdar Dutsov: Investigation, Software, Formal analysis, Experiments, Manuscript review. Philippe Cassette: Supervision, Conceptualization, Experiments, Methodology, Manuscript review. Krasimir Mitev: Resources, Formal analysis, Experiments, Manuscript review.

Table 3

 3 Comparison between RCTD1, mini-TDCR and micro-TDCR for 3 H and14 C samples. The difference 𝛥 is calculated as a relative difference from RCTD1.

		Activity	𝛥	Activity	𝛥	Blank D	𝛥
		14 C (Bq)		3 H (Bq)		count rate (s -1 )
	Micro-RCTD	6400 (6)	0.22%	1060 (5)	0.43%	10.21 (5)	45%
	Mini-RCTD	6392 (13)	0.10%	1056 (7)	0.00%	10.6 (2)	51%
	RCTD1	6386 (13)	-	1056 (7)	-	7.0 (1)	-

Table 4

 4 Comparison between the mini-TDCR and the portable TDCR-SU for 3 H and14 C samples.

	Samples		kB	TDCR-SU		Mini-TDCR		Relative difference
			(cm MeV -1 )	Efficiency	Activity	Efficiency	Activity	Efficiency	Activity
					(Bq)		(Bq)		(Bq)
	LKB, 3 H, №1	0.013 0.015	0.5594 0.5544	489.4 (24) 493.8 (25)	0.6251 0.6201	490.5 (25) 494.5 (25)	11.7% 11.9%	0.23% 0.13%
	LKB, 3 H, №2	0.013 0.018	0.5557 0.5440	839 (7) 857 (7)	0.6182 0.6070	841 (6) 8576 (6)	11.3% 11.6%	0.29% 0.00%
	LKB, 14 C		0.010	0.9547	1779.4 (36)	0.9630	1780.0 (36)	0.87%	0.03%
	Amersham, 14 C	0.010	0.9531	544.1 (20)	0.9600	543.9 (20)	0.72%	-0.03%
	Table 5
	D count rate for each blank sample and both TDCR devices.
	Blank	TDCR-SU	mini-TDCR	Relative
	sample			deviation
	LKB №1	13.25 (6)	14.21 (6)	7.2%
	LKB №2	13.13 (5)	14.10 (9)	7.4%
	Amersham	17.10 (7)	19.18 (8)	12.2%

Table 6

 6 Comparison between mini-TDCR and the portable TDCR-SU for 3 H and 14 C samples.

	Samples	Device	Activity (Bq)		Relative deviation
			Measurement	Certificate
	LKB, 3 H, №2	TDCR-SU Mini-TDCR	839 (7) 841 (6)	835 (8)	0.40% 0.69%
	Amersham, 14 C	TDCR-SU Mini-TDCR	544.1 (20) 543.9 (20)	543.9 (11)	0.03% 0.00%

Table 7

 7 Results of activity concentration measurements for18 F and 11 C with their corresponding uncertainty budget.

	Relative standard	Relative standard
	uncertainty 18 F	uncertainty 11 C

RESULT, activity concentration (MBq ml

-1 ) 82.3 (10) 9.47 (9)

Acknowledgements

This work was funded by the French national metrology institute: Laboratoire National de Métrologie et d'Essais (LNE). The authors thank them for providing all the funds which were required to build the setup and perform the various studies linked to this project. The authors thank Philippe Gervais and Anne-Laure Teppe for their assistance, for the supply of 11 C and 18 F, and for having allowed us to carry out these experiments at CEA/SHFJ (Orsay Hospital) under very good conditions. The authors thank Vadim Tsoupko-Sitnikov for the radon experiments at IRSN. Finally, the authors thank Margot Corbel and Sophie Morelli for some of the source preparation and Mark A. Kellett, Carole Fréchou, Christophe Dulieu and Marie-Christine Lépy for their useful comments on the document.