Marie-Pierre Brenier-Pinchart

Denis Filisetti

Sophie Cassaing

Emmanuelle Varlet-Marie

Florence Robert-Gangneux

Laurence Delhaes

Juliette Guitard

Hélène Yéra

Patrick Bastien

Hervé Pelloux

Multicenter Evaluation of the Toxoplasma RealCycler Universal PCR Assay on 168 Characterized Human Samples

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Toxoplasmosis is a worldwide endemic protozoan disease; 30% of the global human population is chronically infected with large disparities across the world. [START_REF] Bigna | Global, Regional, and Country Seroprevalence of Toxoplasma gondii in Pregnant Women: a Systematic Review, Modelling and Meta-Analysis[END_REF] Toxoplasma gondii infection is usually asymptomatic or mildly symptomatic except in immunocompromised patients, and congenital patients. [START_REF] Robert-Gangneux | Epidemiology of and Diagnostic Strategies for Toxoplasmosis[END_REF]

Congenital toxoplasmosis (CT) occurs in infants following infection

Molecular diagnosis of toxoplasmosis : multicenter evaluation of the Toxoplasma

RealCycler  Universal PCR assay on 168 characterized human samples Marie-Pierre Brenier-Pinchart, 1,10, * Denis Filisetti, 2,10 Sophie Cassaing, [START_REF] Martino | Toxoplasmosis after Hematopoietic Stem Cell Transplantation[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Emmanuelle Varlet-Marie, [START_REF] Dard | Management of Toxoplasmosis in Transplant Recipients: an Update[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Florence Robert-Gangneux, [START_REF] Furtado | Ocular Toxoplasmosis I: Parasitology, Epidemiology and Public Health[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Laurence Delhaes, [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Juliette Guitard, [START_REF] Sterkers | Diversity and Evolution of Methods and Practices for the Molecular Diagnosis of Congenital Toxoplasmosis in France: a 4-Year Survey[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Hélène Yéra, [START_REF] Morelle | Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Patrick Bastien, [START_REF] Varlet-Marie | Characterization and Multicentric Validation of a Common Standard for Toxoplasma Gondii Detection Using Nucleic Acid Amplification Assays[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Hervé Pelloux, [START_REF] Bigna | Global, Regional, and Country Seroprevalence of Toxoplasma gondii in Pregnant Women: a Systematic Review, Modelling and Meta-Analysis[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] Yvon Sterkers [START_REF] Varlet-Marie | Characterization and Multicentric Validation of a Common Standard for Toxoplasma Gondii Detection Using Nucleic Acid Amplification Assays[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] during pregnancy and may result in neurologic and neurocognitive deficits, chorioretinitis, fetal abortion and death. Several forms of toxoplasmosis can be observed due to primary infection or to reactivation of chronic toxoplasmosis in the context of immunodepression.

Disseminated and pulmonary toxoplasmosis is a life-threatening opportunistic infection that mainly affects hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients. [START_REF] Martino | Toxoplasmosis after Hematopoietic Stem Cell Transplantation[END_REF][START_REF] Dard | Management of Toxoplasmosis in Transplant Recipients: an Update[END_REF] Cerebral toxoplasmosis is a common cause of central nervous system infection in patients with HIV/AIDS who do not take appropriate prophylaxis and in patients with other immune deficiencies.

Lastly, ocular toxoplasmosis occurs in immunocompromised and immunocompetent patients; toxoplasmic retinochoroiditis is the most common form of posterior uveitis in many countries, particularly in South America. [START_REF] Furtado | Ocular Toxoplasmosis I: Parasitology, Epidemiology and Public Health[END_REF] Molecular detection of T. gondii plays a crucial role in the diagnosis of these different forms of toxoplasmosis and PCR has significantly improved the management of this parasitic disease. Initially, Toxoplasma PCR methods (conventional PCR and real-time PCR (qPCR)) used in medical diagnostic laboratories were laboratory-developed or 'inhouse' PCR assays. [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF][START_REF] Sterkers | Diversity and Evolution of Methods and Practices for the Molecular Diagnosis of Congenital Toxoplasmosis in France: a 4-Year Survey[END_REF] Although they represent sensitive methods [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF][START_REF] Morelle | Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Varlet-Marie | Characterization and Multicentric Validation of a Common Standard for Toxoplasma Gondii Detection Using Nucleic Acid Amplification Assays[END_REF] they suffer from lack of standardization. [START_REF] Sterkers | Diversity and Evolution of Methods and Practices for the Molecular Diagnosis of Congenital Toxoplasmosis in France: a 4-Year Survey[END_REF] Since several years the growing number of commercially available methods has been facilitating the muchneeded standardization, but not all techniques are equal, and it is essential to correctly evaluate them before their implementation in routine diagnosis. [START_REF] Morelle | Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Döşkaya | Comparison of an in House and a Commercial Real-Time Polymerase Chain Reaction Targeting Toxoplasma Gondii RE Gene Using Various Samples Collected from Patients in Turkey[END_REF][START_REF] Ammar | Multicentric Evaluation of the Bio-Evolution Toxoplasma gondii Assay for the Detection of Toxoplasma DNA in Immunocompromised Patients[END_REF][START_REF] Nourrisson | Evaluation of Two Commercial Kits on the Automated ELITe InGenius PCR Platform for Molecular Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF] To this aim, available commercialized PCR assays are evaluated by the "Molecular biology" group of the French National Reference Centre for Toxoplasmosis (http://cnrtoxoplasmose.chu-reims.fr, last access December 28, 2021). [START_REF] Morelle | Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF] Here, analytical performances, clinical sensitivity and specificity of the Toxoplasma RealCycler  Universal assay (Progenie Molecular, Valencia, Spain) were assessed for the diagnosis of toxoplasmosis using 168 characterized human samples in 8 referent centers. In this study, the analytical detection threshold of this commercial qPCR assay was first assessed using calibrated lyophilized T. gondii samples; then, its performances in medical diagnosis were compared with qPCR methods used in routine molecular diagnosis in distinct clinical settings: CT (amniotic fluid, placenta and umbilical cord blood), toxoplasmic chorioretinitis (aqueous and vitreous humor), and toxoplasmic infection in immunocompromised patients (blood sample, cerebrospinal fluid, bronchoalveolar lavage fluid and miscellaneous samples). To our knowledge, this is the first evaluation of this kit in a clinical context.

Materials and methods

Proficiency of the participating laboratories

The eight participating laboratories are proficient in detecting T. gondii in clinical specimens; they are members of the "Molecular biology" group of the French National Reference Centre for Toxoplasmosis (http://cnrtoxoplasmose.chu-reims.fr, last access December 28, 2021) and participate to the external quality assessment (EQA) for Toxoplasma-PCR. [START_REF] Sterkers | Diversity and Evolution of Methods and Practices for the Molecular Diagnosis of Congenital Toxoplasmosis in France: a 4-Year Survey[END_REF][START_REF] Roux | Clinical Relevance of Placenta Examination for the Diagnosis of Congenital Toxoplasmosis[END_REF] Moreover, seven of them hold an agreement from the Ministry of Health (Regional Health Agency) to perform prenatal diagnosis for congenital toxoplasmosis. Samples Toxoplasma Lyophilized standards and External Quality Control samples Four participating centers (centers C, E, G, H) used a calibrated Toxoplasma (type II) suspension produced by the Molecular Biology Study Group of the NRCT (University Hospital of Montpellier). [START_REF] Varlet-Marie | Characterization and Multicentric Validation of a Common Standard for Toxoplasma Gondii Detection Using Nucleic Acid Amplification Assays[END_REF] DNA was extracted from the Toxoplasma suspension in each center using their routine method for molecular diagnosis. The four centers compared the sensitivities of detection of serial dilutions (from 10,000 to 0.1 or 0.01 tachyzoites (Tg)/mL) after amplification using their own in-house qPCR method (here after termed 'reference' PCR method) and the Toxoplasma RealCycler  Universal kit (Progenie Molecular, Valencia, Spain), following the manufacturer's instructions. All dilution points were amplified at least in triplicate. Additionally, three of these centers also evaluated at least three samples of External Quality Control samples (EQCs) from QCMD® (Glasgow, UK). 17 Clinical samples. DNAs extracted from clinical samples, were used to evaluate the commercial kit, as previously described. 18 Overall, 168 clinical samples were included in the study, collected within the frame of routine practice in the eight participating centers for the diagnosis of congenital, ocular, cerebral, disseminated and acute toxoplasmosis. They comprised 58 amniotic fluid (AF), 15 placenta, 14 umbilical cord blood, 1 foetal biopsy, 20 cerebro-spinal fluid (CSF), 6 brain biopsy, 2 cerebral abscess, 21 aqueous humor, 1 vitreous humor, 15 blood, 9 bronchoalveolar lavage fluid (BALF), 1 bone marrow, 1 serum, 1 plasma and 1 lymph node biopsy samples. In total, 141 positive and 27 negative samples were included (Table 1). To be included in the study, all samples had to be characterized and associated to a clinical G), the MagNA Pure Compact with (Center F), and the Nuclisens easyMag system using the NucliSens Magnetic kit (bioMérieux, Craponne, France) which was optimized by a pretreatment step using proteinase K (Center B). 20,21 Center H used manual methods, the Tween-Nonidet-NaOH (TNN; 0.5% Tween 20, 0.5% Nonidet P40, 10 mM NaOH) lysis buffer method for paucicellular fluids 22 and a treatment by proteinase K (Euromedex, Souffelweyersheim, France) in Tris-SDS (2 to 3 µL/100µL of final solution) (56˚C, 12 hours), followed by boiling at 100˚C for 10 min and protein precipitation solution kit (Protein precipitation A7951, Promega, Charbonnieres, France) for tissues or cellular samples (Table 2). [START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF]23 Reference PCR methods The laboratory-developed qPCR used by six participants targeted the non-coding repetitive DNA sequence rep529 (GenBank accession numbers AF146527 and AF146550) and have been previously evaluated and published (Centers B,C,D,E,F,G,H). [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF][START_REF] Varlet-Marie | Characterization and Multicentric Validation of a Common Standard for Toxoplasma Gondii Detection Using Nucleic Acid Amplification Assays[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF]24,25,26 One center used an 'in-house' real-time PCR targeting the B1 region (center A). [START_REF] Lavrard | Pulmonary Toxoplasmosis In HIV-Infected Patients: Usefulness of Polymerase Chain Reaction and Cell Culture[END_REF] Center F used a commercialized Toxoplasma-PCR kit : TIB MolBiol ® assay (LightMix® Kit Toxoplasma gondii (EC) order no. 40-0217-32). [START_REF] Döşkaya | Comparison of an in House and a Commercial Real-Time Polymerase Chain Reaction Targeting Toxoplasma Gondii RE Gene Using Various Samples Collected from Patients in Turkey[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF] 1). the commercialized BioEvolution® test (BioEvolution®, Bussy-Saint-Martin, France) (Centers A, B). [START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF] PCR apparatuses used in each center are described in table 2. Depending on the center and the type of sample, DNA extracts were analyzed in one to four wells. Among the 168 included samples, 16 (9.5%) were analyzed in simplicate at the time of routine clinical diagnosis (centers A and D). After qPCR, DNA samples were then frozen and stored at -20 °C or at -80°C as recommended elsewhere. 18 DNA samples should not have been thawed prior to inclusion in this study.

Toxoplasma RealCycler  Universal method

The Toxoplasma RealCycler  Universal assay (Progenie Molecular, Valencia, Spain) was performed according to the manufacturer's instructions, using 6 µL of DNA extract and 17 µL of mix. In the manual of this kit, the use of ABI 7500 (Thermo Fisher Scientific, Waltham, MA, USA) and CFX96™ (Bio-Rad, Marne-La-Coquette, France) PCR devices were proposed. In this study, Toxoplasma RealCycler  Universal amplification was also performed using StepOnePlus and StepOne (Thermo Fisher Scientific, Waltham, MA, USA), LighgtCycler 480 (Roche Diagnostic, Meylan, France) and MX3005 (Stratagene, La Jolla, CA, USA) as suggested by the manufacturer. If the initial result obtained with the reference qPCR at the time of diagnosis was confirmed by this new run of reference qPCR, the Toxoplasma RealCycler  Universal PCR was performed again with this sample. This flow chart is reported in figure 1.

Ethical approval and informed consent

Clinical samples were obtained from the participating centers in compliance with the Quality Assurance system and legal policies. This work was carried out in accordance with the French guidelines and regulations; it does not include potentially identifying patient/participant information. The study corresponds to a non-interventional retrospective study and is in agreement with the French Health Public Law (CSP Art L1121-1.1). Written consent was obtained before any prenatal AF sampling.

Data and statistical analysis

The results were expressed as a "PCR performance score", corresponding to the number of positive amplifications over the total number of PCR reactions performed [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | Impact of Pre-Analytic Step Duration on Molecular Diagnosis of Toxoplasmosis for Five Types of Biological Samples[END_REF] and as a mean of the crossing point values ± standard deviation (Cp ± SD). Results were analyzed using Fisher's exact test; a p value of 0.05 or less was considered to be significant. For serial dilutions of Toxoplasma standard, the performances of the PCR were also evaluated by the linear measuring range (regression analysis, R 2 ), and the efficiency (%) [E=(10 (-1/slope) -1) x100]. In each center, the agreement between the commercial PCR assay and the reference PCR method (methods A and B) was analyzed using the Bland and Altman plot, [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] this consists in a graph presenting on the abscissa the average of the values obtained by both methods, i.e. (A + B)/2 and on the ordinate the difference between the values obtained by both methods A and B, i.e. (A -B). The upper and lower limits of agreement are estimated by the bias ± 1.96 SD and represented on the plot. The Bland-Altman plots were produced using GraphPad Prism 8.0.2.

Results

Evaluation of the detection threshold using calibrated lyophilized T. gondii samples

Four centers (C, E, G, H) compared the Toxoplasma RealCycler  Universal method and their reference diagnostic PCR method by testing serial dilutions of the common standard of the NRC-T network ("TG25040, 10 4 parasites per mL, type II strain, Montpellier 2014"). [START_REF] Varlet-Marie | Characterization and Multicentric Validation of a Common Standard for Toxoplasma Gondii Detection Using Nucleic Acid Amplification Assays[END_REF] At least three reactions were used for testing each of the standard's concentrations (10,000, 1000, 100, 10, 1, 0.1 Tg/mL and 0.01 Tg/mL for center H) (Table 3). The threshold of Toxoplasma DNA detectiondefined as the lowest concentration where >50% of the reactions remain positive [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF] varied depending on the center from 0.01 to 1 Tg/mL for the Toxoplasma RealCycler  Universal method and from 0.1 to 10 Tg/mL for the reference PCR assays (Table 3). For two centers (E, G), this threshold was 1 Tg/mL and was identical between the two PCR methods. For centers C and H, the threshold using the Toxoplasma RealCycler  Universal method was 1 Tg/mL and 0.01 Tg/mL, respectively, which was lower by one dilution than the thresholds obtained by the reference PCR methods used by these same centers (10 Tg/mL and 0.1 Tg/mL respectively). The relationship between the Cp and the DNA concentration was linear over 4 log units (r²>0.99) for the commercial PCR kit; PCR efficiencies were satisfactory (Eff=89% to 104%) and were slightly better than those of the reference PCR methods for two centers (centers B and G) (Table 3). Furthermore, according to the center, the performance scores were 73% to 81.5% and 70% to 92.6% for reference PCRs and commercial PCR, respectively (Table 3).

Clinical performances of the Toxoplasma RealCycler  Universal PCR method from characterized human samples analyses

One hundred and sixty-eight characterized human samples were included; 160 of those were analyzed in duplicate by the Toxoplasma RealCycler  Universal PCR method and 8 were so in simplicate. The commercial qPCR assay results were concordant with those of the reference qPCR methods for 160 out of the 168 initial samples, whereas the results obtained for 8 DNA samples from patients with toxoplasmosis were discrepant. Among these, 7 were re-analyzed with the reference qPCR methods in each center involved and one sample was in insufficient amount to be retested and was excluded (Figure 1). Among these 7 initially discrepant DNAs, 4 were excluded from statistical analysis, since the qPCR results were negative after re-testing with the reference qPCR, suggesting DNA degradation. For the 3 remaining samples, the control results using the reference qPCR assay were concordant with the initial diagnosis even though PCR reactions were inconstantly positive with delayed amplification (Cp>35) (Table 4). These 3 samples -amniotic fluid, brain biopsy and plasma-from 3 different centers were still negative after re-testing in duplicate with the commercial qPCR method (Table 4). No false positive results were observed using the commercial method (0/27 samples).

Ultimately, the statistical analysis of this study was performed on 163 clinical samples; 136 from patients with toxoplasmosis and 27 from patients without toxoplasmosis (Table 1). The sensitivity and the specificity of Toxoplasma RealCycler  Universal PCR method were 97.8 % 97.8-100 (133/136) and 100 % 87.2-100 (27/27), respectively. For each center, Bland-Altman analysis was used to study the agreement between commercial PCR and reference qPCR assays, considering that Cp is the quantitative measured parameter. It included from 7 to 30 concordant positive samples according to the center. The agreement between the reference method of each center and the commercial method is good, with no sample (centers A, B, C and E), or only 1 (centers D, F and H) or 2 (center G) samples outside the confidence intervals (Figure 2). Furthermore, 11 ECQ samples were analyzed in 3 centers (centers C, E, G) and in 10/11 cases, the results of the Toxoplasma concordant with the results obtained in the first run (reference qPCR and QCMD® results). Following the protocol, this discrepant sample was re-tested in duplicate with both qPCR methods (Figure 1): the two PCR reactions were positive with the reference PCR method while only 1 out of 2 was positive with the commercial qPCR assay (Table 4).

Discussion

The molecular diagnosis of toxoplasmosis still relies on a targeted diagnostic approach performed in proficient laboratories, since no syndromic molecular panels include the detection of Toxoplasma DNA. In the absence of a syndrome-based approach, it remains important to continue the optimization of the preanalytical and analytical steps and to evaluate the performances of the laboratorydeveloped methods and commercialized kits of qPCR assays targeting specifically T. gondii. [START_REF] Morelle | Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Döşkaya | Comparison of an in House and a Commercial Real-Time Polymerase Chain Reaction Targeting Toxoplasma Gondii RE Gene Using Various Samples Collected from Patients in Turkey[END_REF][START_REF] Ammar | Multicentric Evaluation of the Bio-Evolution Toxoplasma gondii Assay for the Detection of Toxoplasma DNA in Immunocompromised Patients[END_REF][START_REF] Nourrisson | Evaluation of Two Commercial Kits on the Automated ELITe InGenius PCR Platform for Molecular Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF]18,20,21,[START_REF] Brenier-Pinchart | Impact of Pre-Analytic Step Duration on Molecular Diagnosis of Toxoplasmosis for Five Types of Biological Samples[END_REF][START_REF] Robert | Molecular Diagnosis Of Toxoplasmosis: Recent Advances and a Look to the Future[END_REF] These are the objectives of the "Molecular biology" group of the French National Reference Centre for Toxoplasmosis (http://cnrtoxoplasmose.chureims.fr, last access December 28, 2021).

Initially, the laboratory-developed methods (reference PCR in this study) of several participants had been compared to detect low amounts of T. gondii from simulated specimens, [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF] and used to validate a common standard for T. gondii. [START_REF] Varlet-Marie | Characterization and Multicentric Validation of a Common Standard for Toxoplasma Gondii Detection Using Nucleic Acid Amplification Assays[END_REF] To date, 4 commercialized PCR kits have been evaluated by our group or others authors: AMS94/F; Clonit, (Clonit, Milan, Italy), Bio-Evolution® kit (Bio-Evolution, Bussy-Saint-Martin, France), ELITe MGB Real-Time PCR assay (Elitech, Puteaux, France) and LightMix® Kit Toxoplasma gondii assay (TIB MolBiol®, Berlin, Germany). [START_REF] Morelle | Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Döşkaya | Comparison of an in House and a Commercial Real-Time Polymerase Chain Reaction Targeting Toxoplasma Gondii RE Gene Using Various Samples Collected from Patients in Turkey[END_REF][START_REF] Ammar | Multicentric Evaluation of the Bio-Evolution Toxoplasma gondii Assay for the Detection of Toxoplasma DNA in Immunocompromised Patients[END_REF][START_REF] Nourrisson | Evaluation of Two Commercial Kits on the Automated ELITe InGenius PCR Platform for Molecular Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF] The Toxoplasma RealCycler  Universal assay was evaluated for the first time in this multicentric study. Four calibrated T. gondii standards, 11 ECQ and 168 clinical samples drawn for diagnosis of congenital, ocular, cerebral, disseminated and acute toxoplasmosis during routine practice of eight laboratories were used. For routine molecular diagnosis of toxoplasmosis, these centers used laboratory-developed PCR methods [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF][START_REF] Sterkers | Diversity and Evolution of Methods and Practices for the Molecular Diagnosis of Congenital Toxoplasmosis in France: a 4-Year Survey[END_REF] or two other commercial kits of qPCR which have been previously evaluated. [START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Döşkaya | Comparison of an in House and a Commercial Real-Time Polymerase Chain Reaction Targeting Toxoplasma Gondii RE Gene Using Various Samples Collected from Patients in Turkey[END_REF][START_REF] Ammar | Multicentric Evaluation of the Bio-Evolution Toxoplasma gondii Assay for the Detection of Toxoplasma DNA in Immunocompromised Patients[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF] All centers involved in the present study participate to the national external quality assessment on a regular basis with consistent results. [START_REF] Sterkers | Diversity and Evolution of Methods and Practices for the Molecular Diagnosis of Congenital Toxoplasmosis in France: a 4-Year Survey[END_REF]17 The analytical detection threshold for the Toxoplasma RealCycler  Universal assay complied with the usual requirements for this molecular diagnosis: it varied from 0.01 to 1 Tg/mL depending on the center and was below to the target threshold proposed previously. [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF] Costa et al reported that the parasitic loads are low during toxoplasmosis infection and sometimes less than 1 Tg/mL of amniotic fluid during CT. [START_REF] Costa | Real-Time PCR as a Prognostic Tool for Human Congenital Toxoplasmosis[END_REF] Therefore, the "Molecular biology" group of the French National Reference Centre for Toxoplasmosis recommended that, for a best sensitivity of the molecular diagnosis of T. gondii, the detection threshold should tend toward 0.5 Tg per reaction tube, corresponding to 0.75 to 2.5 Tg/ml of AF. [START_REF] Sterkers | Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens[END_REF] With the same methodology, this threshold was 1 Tg/mL for the TIB MolBiol assay (TIB MolBiol®, Berlin, Germany) and varied from 1 Tg/mL to 10 Tg/mL depending on the center for the ELITe MGB kit (Elitech, Puteaux, France). [START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF] Initially, 168 DNAs extracted from characterized human samples and 11 ECQs were included and tested with the commercial qPCR method. Nine discrepant results (for eight clinical and one ECQ samples) were observed between the commercial and reference PCR methods; and these samples were re-tested by each center according to the flow chart. Five of these eight samples were excluded from the global analysis. Among them, two samples (AH, BALF) were inconstantly positive using the reference PCR assay at the time of diagnosis, one (serum) sample tested in simplicate had shown a high Cp, and for the remaining sample (CSF), the Cp mean was 32.72. We previously showed that freezing and storage at -20 °C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis. 18 However, a weak DNA degradation starting from a low parasitic load might explain that these samples were not positive after storage. Furthermore, only DNA extracted from AF was studied in Delhaes et al and one cannot rule out that T. gondii DNA extracted from human 'cellular' samples may be less stable. 18 Among the four false negative samples re-tested with the commercial PCR assay, the ECQ sample was weakly (Cp>35) and inconstantly positive (1 positive /2 reactions) after this control; and three samples yielded persistently discrepant results. The parasitic loads in these three clinical samples (AF, brain biopsy, plasma) were low using reference PCR assays (Cp>35) which may in part explain the negativity of the commercial qPCR. In total, the sensitivity and the specificity of Toxoplasma RealCycler  Universal PCR assay were 97.8% (133/136) and 100% (27/27), respectively; and the 11 ECQs were correctly detected only if the PCR was performed in quadriplicate. These clinical performances were better or close to those reported for the BioEvolution® kit (Se: 86% and Sp: 100%), ELITe MGB® kit (Se: 89% and Sp: 100%), TIB MolBiol® (Roche®) assay (Se: 98.1% and Sp: 98.5%) with a comparable methodology [START_REF] Filisetti | Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Brenier-Pinchart | on behalf of the Molecular Biology Pole of the National Reference Center for Toxoplasmosis. Multicenter Comparative Assessment of the TIB Molbiol ® Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis[END_REF] Only in the multicentric retrospective evaluation of the BioEvolution® assay during immunocompromised patients follow up, Ait Ammar et al reported a higher sensitivity and specificity, at 98.8% and 100%, respectively. [START_REF] Ammar | Multicentric Evaluation of the Bio-Evolution Toxoplasma gondii Assay for the Detection of Toxoplasma DNA in Immunocompromised Patients[END_REF] The good performances of this commercial kit are probably partly linked to the use of a number of 'good practices': detection in multiplicate in this study, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. No PCR inhibitors were detected by the kit, however no DNA samples with PCR inhibitors detected with reference PCR methods were specifically included in this study. It should be noticed that the kit could be used with several PCR apparatuses. The manufacturer proposes some technical recommendations depending on the type of validated samples (amniotic fluid, culture, plasma and serum), DNA extraction or PCR devices, but specifies that this kit is potentially compatible with other options. The only drawbacks noted at the time of the study were the absence of uracil-Nglycosylase in the assay and the lack of CE-IVD marketing yet. In conclusion, provided that PCR reactions are performed at least in duplicate to detect low parasitic loads, the Toxoplasma RealCycler  Universal PCR assay showed suitable performances for the molecular diagnosis of the different clinical forms of toxoplasmosis.

Figure 1 :

 1 Figure 1 : Flow chart of the 168 initially included human samples and 11 External Quality Control (EQC) samples. * after DNA freezing and storage at -20 °C. In grey: positioning of the commercial PCR kit in the flow chart.

Figure 2 :

 2 Figure 2 : Bland-Altman plots for each center. The red lines and the dotted black lines represent the means difference (bias) and ±1.96 standard deviations (95%limits of agreement), respectively.

Table 1 )

 1 . Commercial manual extraction kits, used according to the manufacturer's specifications, were QIAamp DNA Micro, Mini, FPPE DNA tissue Kits (Qiagen, Courtaboeuf, France) (centers A, B, D, E). Four automated DNA extraction methods were used : the MagNA Pure 96 DNA with Viral NA Small Volume Kit (Center C) or with High Pure PCR template (Roche Molecular Biochemicals, Meylan, France) (Center

	diagnosis. Congenital toxoplasmosis (CT) cases
	were clinically classified as defined by the
	European Research Network on Congenital
	Toxoplasmosis	group. 19	Cases	of
	toxoplasmosis	in	immunocompromised
	patients were assessed according to the
	European Group for Blood and Marrow
	Transplantation Infectious Diseases Working
	Party classification. 3 The diagnosis of
	toxoplasmosis disease was excluded for the 27
	negative PCR results.
	Molecular techniques.
	DNA extraction

Table 1 : Description of human clinical samples and type of toxoplasmosis diseases

 1 Two centers included samples analyzed with different qPCR methods because they switched from in-house PCR assays targeting rep529 and the B1 gene to . *Number in brackets : number of samples included in statistical analysis according the flow chart (Figure

	Samples	Number	of	Toxoplasmosis	Absence	of
		samples			toxoplasmosis
	Congenital toxoplasmosis
	Amniotic fluid (AF)	58		48	10
	Placenta	15		14	1
	Umbilical cord blood	14		11	3
	Foetal biopsy	1		1	0
	Cerebral toxoplasmosis
	Cerebro-spinal fluid (CSF)	20 (19)*		19 (18)	1
	Brain biopsy	6		6	0
	Cerebral abcess	2		2	0
	Ocular toxoplasmosis
	Aqueous humor (AH)	21(19)		18 (16)	3
	Vitreous humor	1		1	0
	Disseminated/pulmonary
	toxoplasmosis
	Blood/buffy-coat	15		12	3
	Broncho alveolar lavage fluid	9 (8)		5 (4)	4
	(BALF)
	Bone marrow aspiration	2		1	1
	Serum	2(1)		1 (0)	1
	Plasma	1		1	0
	Acute toxoplasmosis
	Lymph node biopsy	1		1	0
	Total of human samples	168 (163)		141 (136)	27
				[84% (83.5%)]	[16% (16.5%)]

Table 2 : Details of DNA extraction, PCR methods used in each center in routine diagnosis of toxoplasmosis, and PCR apparatuses used with the Toxoplasma RealCycler

 2 TNN : Tween-Nonidet-NaOH (0.5% Tween 20, 0.5% Nonidet P40, 10 mM NaOH) lysis buffer method 22 † DNA extraction technique described inSterkers Y et al, 2012 23 Each clinical samples as well as the QCMD® EQC samples were analyzed in two wells. If the result of Toxoplasma RealCycler  Universal PCR was discordant with that of the previous routine diagnosis, the DNA sample was re-tested with the reference PCR. The sample was excluded from the statistical analysis if this PCR reference result did not confirm the initial result or if the sample DNA was not in sufficient quantity to be re-tested.

	Toxoplasma PCR in routine diagnosis

 Universal PCR assay.

*

ACKNOWLEDGMENTS

This study was in part funded by the Ministry of Health and "Santé Publique France" through financial support to the French National Reference Centre for Toxoplasmosis (Centre National de Référence de la Toxoplasmose).The authors would like to thank Valérie Martin (Grenoble, France), Sylvie Douzou, France Joullié and Martine Brun (Montpellier, France), Chloé Gommenginger (Strasbourg, France), Cédric Rondot and Yacine Garaoun (Cochin Hospital, Paris, France) for their technical support. National Reference Center for Toxoplasmosis Network. Evolution of Toxoplasma-PCR Methods and Practices: a French National Survey and Proposal for Technical Guidelines. Int J Parasitol 2018, 48:701-707. 17. Bastien P, Jumas-Bilak E, Varlet-Marie E, Marty P. Three Years of Multi-Laboratory External Quality Control for the Molecular Detection of Toxoplasma gondii in Amniotic Fluid in France. Clin Microbiol Infect 2007, 13:430-433. 18. Delhaes L, Filisetti D, Brenier-Pinchart MP, Pelloux H, Yera H, Dalle F, Sterkers Y, Varlet-Marie E, Touafek F, Cassaing S, Bastien P. Freezing and Storage at -20 Degrees C Provides Adequate Preservation of Toxoplasma gondii DNA for Retrospective Molecular Analysis. Diagn Microbiol Infect Dis 2014, 80:197-9. 19. Lebech M, Joynson DH, Seitz HM, Thulliez P, Gilbert RE, Dutton GN, Ovlisen B, Petersen E. Classification System and Case Definitions of Toxoplasma gondii Infection in Immunocompetent Pregnant Women and Their Congenitally Infected Offspring. European Research Network On Congenital Toxoplasmosis. Eur J Clin Microbiol Infect Dis 1996, 20. Faucher B, Miermont F, Ranque S, Franck J, Piarroux R. Optimization of Toxoplasma gondii DNA Extraction from Amniotic Fluid Using Nuclisens Easymag and Comparison with QIA Amp DNA Mini Kit. Eur J Clin Microbiol Infect Dis 2012, 31:1035e9. 21. Yera H, Ménégaut L, Brenier-Pinchart MP, Touafek F, Bastien P, Dalle F, Molecular Biology study group of the French National Reference Centre for Toxoplasmosis. Evaluation of Five Automated and One Manual Method for Toxoplasma and Human DNA Extraction from Artificially Spiked Amniotic Fluid. Clin Microbiol Infect 2018, 24:1100.e7-1100.e11. 22. Hohlfeld P, Daffos F, Costa JM, Thulliez P, Forestier F, Vidaud M. Prenatal Diagnosis of Congenital Toxoplasmosis with a Polymerase Chain Reaction Test on Amniotic Fluid. N Engl J Med 1994;331:695-699. 23. Sterkers Y, Pratlong F, Albaba S, Loubersac J, Picot MC, Pretet V, Issert E, Boulot P, Bastien P. Novel Interpretation of Molecular Diagnosis of Congenital Toxoplasmosis According to Gestational Age at the Time of Maternal Infection. J Clin Microbiol 2012, 50:3944-3951. 24. Reischl U, Bretagne S, Kruger D, Ernault P, Costa JM. Comparison of Two DNA Targets for the Diagnosis of Toxoplasmosis by Real-Time PCR Using Fluorescence Resonance Energy Transfer Hybridization Probes. BMC Infect Dis 2003, 3:7. 25. Talabani H, Asseraf M, Yera H, Delair E, Ancelle T, Thulliez P, Brézin AP, Dupouy-Camet J. Contributions of Immunoblotting, Real-Time PCR, and the Goldmann-Witmer Coefficient to Diagnosis of Atypical Toxoplasmic Retinochoroiditis. J Clin Microbiol 2009, 47:2131-2135.


Mei Mei

Claude Le Men

Karine Loubière

Gilles Hébrard

Dr Nicolas Dietrich
email: nicolas.dietrich@insa-toulouse.fr

Taylor bubble formation and flowing in a straight millimetric channel with a cross-junction inlet geometry Part II: Gas-liquid mass transfer

Keywords: Bubble formation, cross-junction, Taylor bubbles, mass transfer, concentration fields

Using the resazurin-based colorimetric technique and advanced image acquisition, the equivalent oxygen concentration fields inside the liquid slugs were measured during and after the bubble formation stage at a cross-junction in a straight millimetric channel. Firstly, two different mechanisms were identified for the development of oxygen concentration fields depending on the two-phase Reynolds numbers (ReTP). Under low ReTP, a jet-like central oxygen concentration 'finger' occurred between the newly formed bubble and the gas finger at the bubble pinch-off point. Right after the bubble pinch-off, the dissolved oxygen was transported first by the entering liquid from two side inlets, and later by the developing recirculation loop inside the liquid slug. Under higher ReTP, two highly concentrated oxygen concentration spots were formed near the bubble rear and the channel wall region, and a much more complex flow structure in the liquid slug appeared. Then, whatever the operating conditions, it was observed that the averaged oxygen concentrations inside the liquid slug followed a nearly linear relation as a function of the axial position in the channel, and that the related mass flux density decreased as far as the bubbles flowed along the channel's length. The bubble formation process could be decomposed into three stages when considering the temporal evolution of the cumulated oxygen mass and depending on the development of the liquid slug. At last, the overall volumetric liquid side mass transfer coefficients (kL a) were deduced from the concentration fields, and found to linearly increase with the recirculation frequencies, leading to a scaling law.

Introduction

Microstructured continuous-flow technology is now considered as one of the effective approaches for process intensification and gained many interests for a wide range of applications [START_REF] Geng | Multiphase Microfluidics: Fundamentals, Fabrication, and Functions[END_REF][START_REF] Liu | Manipulation of gas-liquid-liquid systems in continuous flow microreactors for efficient reaction processes[END_REF][START_REF] Yan | Microreaction processes for synthesis and utilization of epoxides: A review[END_REF], especially for multiphase reactions (e.g., hydrogenation and (photo)oxidation). They could indeed offer several advantages compared to conventional batch processes, such as the enhanced heat and mass transfer rates, narrow residence time distribution, high exchange surface areas, and higher yields and selectivity [START_REF] Willms | Microreactor studies for efficient organic oxidation processes[END_REF][START_REF] Mariotti | The role of flow features and chemical kinetics on the reaction yield in a T-shaped micro-reactor[END_REF][START_REF] Xu | Numerical investigation on the hydrodynamics of Taylor flow in ultrasonically oscillating microreactors[END_REF][START_REF] Yang | Enhancement of gas-liquid mass transfer and mixing in zigzag microreactor under ultrasonic oscillation[END_REF].

Numerous experimental works [START_REF] Butler | Modelling of mass transfer in Taylor flow: Investigation with the PLIF-I technique[END_REF][START_REF] Yao | Two-phase flow and mass transfer in microchannels: A review from local mechanism to global models[END_REF] were devoted to investigating hydrodynamics [START_REF] Roudet | Hydrodynamic and mass transfer in inertial gas-liquid flow regimes through straight and meandering millimetric square channels[END_REF]Mei et al., 2020a;[START_REF] Xu | Numerical investigation on the hydrodynamics of Taylor flow in ultrasonically oscillating microreactors[END_REF] and mass transfer [START_REF] Yang | Local investigations on the gas-liquid mass transfer around Taylor bubbles flowing in a meandering millimetric square channel[END_REF][START_REF] Abiev | Gas-Liquid and Gas-Liquid-Solid mass transfer model for Taylor flow in micro (milli) channels: a theoretical approach and experimental proof[END_REF][START_REF] Kexel | Taylor Bubble Study of the Influence of Fluid Dynamics on Yield and Selectivity in Fast Gas-Liquid Reactions Chemie Ingenieur Technik[END_REF][START_REF] Schlüter | Visualization and Quantitative Analysis of Consecutive Reactions in Taylor Bubble Flows[END_REF] under reactive conditions [START_REF] Kastens | Test System for the Investigation of Reactive Taylor Bubbles[END_REF][START_REF] Paul | Reaction systems for bubbly flows[END_REF][START_REF] Krieger | Kinetic study of leuco-indigo carmine oxidation and investigation of Taylor and Dean flow superposition in a coiled flow inverter[END_REF][START_REF] Haase | Simultaneous Detection of Hydrodynamics, Mass Transfer and Reaction Rates in a Three-Phase Microreactor[END_REF] for gas-liquid flows in confined geometries, especially for the Taylor flow regime. But most of them were limited to an overall evaluation of the mass transfer characteristics, making it difficult to separate contributions between the bubble formation and the bubble flowing stages. For example, recent works [START_REF] Zhang | CFD simulation of mass transfer intensified by chemical reactions in slug flow microchannels[END_REF][START_REF] Zhang | Dynamic changes in gas-liquid mass transfer during Taylor flow in long serpentine square microchannels[END_REF]Mei et al., 2020b;[START_REF] Liu | Effect of mixing on mass transfer characterization in continuous slugs and dispersed droplets in biphasic slug flow microreactors[END_REF][START_REF] Zhao | Hydrodynamics and mass transfer of Taylor bubbles flowing in non-Newtonian fluids in a microchannel[END_REF] have shown that the liquid side mass transfer coefficient decreased along the microchannel, namely that the mass transfer rates during bubble formation stage were relatively greater than the ones during bubble flowing stage. Therefore, there is a need to develop local measurements enabling simultaneous visualization of both hydrodynamics and mass transfer during the first moments of the Taylor flow, namely when the bubbles are forming and detaching at the inlet.

The bubble formation process is a key point when designing microreactors because the question of "when and how bubble pinch-off happens" directly determines the bubble size, frequency and flow regimes [START_REF] Dang | Formation characteristics of Taylor bubbles in a microchannel with a converging shape mixing junction[END_REF], and thus the mass transfer characteristics. This has been then extensively investigated through experiments, simulations and theoretical analysis methods [START_REF] Fu | Bubble formation and breakup dynamics in microfluidic devices: A review[END_REF][START_REF] Svetlov | Mathematical modeling of the droplet formation process in a microfluidic device[END_REF]. Although the physical properties (density ratio, viscosity ratio and surface tension, etc.) of gas-liquid and liquid-liquid systems are different, the bubble and droplet formation stage in microchannels could be both described by the filling and squeezing stages [START_REF] Garstecki | Formation of droplets and bubbles in a microfluidic T-junction -scaling and mechanism of break-up[END_REF][START_REF] Yao | Two-phase flow and mass transfer in microchannels: A review from local mechanism to global models[END_REF]. However, it is surprising to remark that few works aims at understanding the role of the bubble formation stage into the overall gas-liquid mass transfer process occurring in Taylor flows (Yang et al., 2016a). [START_REF] Sun | Dissolution of carbon dioxide bubbles and microfluidic multiphase flows[END_REF] investigated the initial shrinkage of dissolved CO2 bubble right after the bubble formation within several liquid pairs (e.g., water, methanol, and ethanol) and found that it was governed by the ratio of the diffusion coefficient and Henry's law constant. In the case of the absorption of CO2 in NaOH, [START_REF] Tan | Mass transfer characteristic in the formation stage of gas-liquid segmented flow in microchannel[END_REF] showed that the mass transfer coefficient kL during the bubble formation stage could vary from 1.4´10 -4 to 5.5´10 -4 m s -1 depending on the gas and liquid flow rates and on the intersection angles at the inlet. The intensification factor of experimental kL compared to that calculated according to the Higbie penetration diffusion model [START_REF] Higbie | The rate of absorption of a pure gas into a still liquid during short periods of exposure[END_REF] were identified between 5 and 15, resulting from efficient surface renewal and acceleration of gas phase. Based on the variation of bubble length (due to the fast consumption of CO2 by the chemical reaction), the absorption fraction of the bubble formation stage to the overall mass transfer could reach up to 40%. Later, [START_REF] Yao | An online method to measure mass transfer of slug flow in a microchannel[END_REF] and [START_REF] Yao | Gas-liquid flow and mass transfer in a microchannel under elevated pressures[END_REF] found a lower absorption fraction (from 1.5% to 10%) for CO2 and water (or ethanol solution), which could be due to elevated pressure, lower bubble formation time and lack of chemical reaction in their system. On the contrary, Yang et al. (2016a) quantified a large contribution (45-90%) of the bubble formation stage on mass transfer when compared to the overall bubble flowing stage. Therefore, there is no consensus on the role of the mass transfer occurring during the bubble formation stage as strongly dependent on the fluid pairs, pressure, or flow rates.

In parallel, other researchers [START_REF] Fries | Impact of inlet design on mass transfer in gas-liquid rectangular microchannels[END_REF] specifically investigated the effects of inlet geometry on mass transfer, and showed that a carefully designed inlet geometry could enhance mass transfer by varying unit cell lengths (2 to 8 times of the channel width). [START_REF] Sattari-Najafabadi | Hydrodynamics and mass transfer in liquid-liquid non-circular microchannels: comparison of two aspect ratios and three junction structures[END_REF] designed three kinds of inlet geometries (e.g., cross-shaped junction, cross-shaped T-junction and T-junction), and observed significant differences in terms of mass transfer during the droplet formation stage, depending on the junction configuration. In particular, the cross-shaped junction presented the best performances (also observed by [START_REF] Xu | Enhancement of mass transfer performance of liquid-liquid system by droplet flow in microchannels[END_REF]) and could achieve up to 30% on the enhancement of overall kL a, [START_REF] Soh | A CFD model for the coupling of multiphase, multicomponent and mass transfer physics for micro-scale simulations[END_REF] simulated the evolution of dissolved CO2 in the liquid slug (silicone oil) by the VOF method and obtained the concentration fields inside the liquid slug during the bubble formation process inside a cross-junction. They divided the bubble formation process into the filling and squeezing stages and found that a majority of dissolved CO2 were firstly carried by the liquid phase in the filling stage and then accumulated significantly during the squeezing stage due to the block of the liquid. However, the experimental measurements of the concentration fields during bubble formation process remains challenging and only few studies were reported (Yang et al., 2016a). For example, by applying the micro PLIF technique, [START_REF] Bai | Experimental study of mass transfer in water/ionic liquid microdroplet systems using micro-LIF technique[END_REF] succeeded to measure the mass transfer process during the droplet formation and flowing stages, and calculated the associated volumetric mass transfer coefficients. They obtained relative higher values at the droplet formation stage than those at the droplet flowing stage. But so far, to the best of author's knowledge, there remains a research gap for quantitatively studying the mass transfer process during the filling and squeezing stages of the bubble formation.

In keeping with this context, the Part II of the present work aims at analyzing the mass transfer during the bubble formation process at a cross-junction and after the bubble detachment (flowing stage). This analysis will be drawn on the knowledge of the related bubble dynamics and the hydrodynamics features (flow regimes, pinch-off pattern, bubble and liquid slug lengths and velocities, etc.), that were detailed in the Part I of this work. The resazurin-based colorimetric method will be implemented to measure the transfer of oxygen from the bubble to the liquid phase. Section 2 will describe the experimental setup, the image processing, and the calibration method for mass transfer.

In sub-section 3.1, the equivalent oxygen concentration fields during the bubble formation and flowing stages will be analyzed. The variations of average oxygen concentrations and mass flux density as a function of the axial position in the channel will be presented in sub-section 3.2, while the enhancement factor induced by the colorimetric chemical reaction will be estimated in sub-section 3.3. Then, the temporal evolution of the oxygen mass during the development of liquid slug at the bubble formation will be followed, revealing the occurrence of three stages (sub-section 3.4).

At last, the relationship between the overall volumetric liquid side mass transfer coefficients kL ⸱a and the recirculation frequencies will be pointed out to propose a scaling law (sub-section 3.5) that allowed gathering the database obtained in this work and other works from the literature.

Materials and methods

Experimental setup

The schematic experimental setup is described in detailed in Part Ⅰ. Only the major information will be thus reported in this part. The gas phase (i.e., air) was fed from house compressed air lines and the flow rate was controlled by a DPC17S-V0L6-BB5 airflow controller. Liquid solutions were supplied by the syringe pump (Harvard Apparatus, PHD 22/2000, USA). The way to introduce gas and liquid phases into the main channel through a cross-junction is indicated in Fig. 1. The channel was lighted by a M530L4 LED and gas-liquid flow images were recorded by a Basler acA1920-155µm camera equipped with a 50 mm 1:1.4 Nikkor lens. The image acquisition frequency was set at 400 fps. The width, wh, and depth, wv, of the main channel were 2.19 mm and 2.13 mm, respectively. The hydraulic diameter of the channel, dh (see Part Ⅰ), was 2.16 mm.

Using a specific sliding support design, the camera could be horizontally changed to different region-of-interest (ROI) positions (see Fig. 1). This allowed investigating the mass transfer during the bubble formation stage, but also after the bubble detachment (flowing stage). Note that (i) some of the observing positions (from X6 to X11) were particularly selected to maintain the same flowing time while varying the total superficial velocity for two-phase flow and (ii) Xi represented the starting position of the ROI window.

Resazurin-based colorimetric method

The colorimetric technique proposed by [START_REF] Dietrich | A new direct technique for visualizing and measuring gas-liquid mass transfer around bubbles moving in a straight millimetric square channel[END_REF] was implemented.

The overall scheme of the redox reactions was as follows: in the presence of glucose and sodium hydroxide, resazurin (blue and not fluorescent) was first irreversibly reduced to resorufin (noted RF, pink and highly fluorescent, molecular mass of 213.19 g • mol !" ), which was reversibly reduced to dihydroresorufin (noted DH, colorless, not fluorescent, and highly reactive with oxygen to form RF). The liquid phase was composed of D-glucose anhydrous (Sigma Aldrich, CAS 50-99-7, noted GL) at a concentration of 20 g • L !" , sodium hydroxide (Sigma Aldrich, CAS 1310-73-2) at a concentration of 20 g • L !" , and resazurin (Sigma Aldrich, CAS 199303, purity 93%, noted RZ) at a concentration of 74 mg • L !" (considering purity). Therefore, the maximal equivalent oxygen concentration, C #$% , that could be measured by the resazurin-based method, was equal to 5.2 mg • L !" , calculated as follows.

C #$% = ['(]•+ ! " ,•-#$ (1)
where MO2 (32 g • mol !" ) and MRZ (229.19 g • mol !" ) were the molar masses of O2 and resazurin, respectively. [RZ] was the mass concentration of resazurin, in mg • L !" .

Establishment of the calibration relationship

The calibration relationship linking the equivalent dissolved oxygen concentration and the gray level intensity (or absorbance) should be first established.

For that, five aqueous solutions at different RZ concentrations (purity being considered) were prepared: 0, 9, 19, 37, and 74 mg • L !" (the concentration of glucose and NaOH were kept at 20 g • L !" ). Once prepared, air was injected inside these solutions to fully convert DH (colorless) into RF (pink). A portable dissolved oxygen probe (HQ40d) was used to verify that a fully conversion was reached with excess dissolved oxygen. Then, the "fully converted" solutions (pink) were immediately injected into the millimetric channel. For each RF concentration, 200 frames containing solution without RF or "fully converted" solution were taken, as shown in Fig. 2 (a). This process was repeated three times to reduce experimental uncertainties. At last, for each RF concentration, a time-averaged gray level value image was obtained, allowing to eliminate the slight deviation between instantaneous gray values. Note that this calibration procedure was made again each time when the ROI position (i.e., axial position, Xi) was changed. The absorbance, A, could be deduced from the time-averaged gray level values as follows:

𝐴 = -log + . ̅ . % 0 , (2)
where 𝐼 1 / and 𝐼 ̅ were the averaged gray intensities when no dye was present in the solution ([RF]=0) and at a given RF concentration respectively. They were obtained considering all the pixels inside of channels and for a given ROI position. For reminder, the term "equivalent" was used because in reality the oxygen concentration in the liquid phase was null: indeed, oxygen was fully consumed by DH to form RF, according to an instantaneous oxidation reaction (see Yang et al. (2016b)).

In Fig. 2(b), some slight differences could be observed between the different ROI positions: they were probably caused by the uncertainty of each calibration and the variations of light at different positions. (ii) Converting gray level values in the liquid slugs into the absorbances using Eq.

(2).

(iii) Obtaining equivalent O2 concentrations using the established calibration relationship in Fig. 2 (c).

(iv) Plotting the equivalent O2 concentration fields shown in Fig. 3 (d). As a reminder, the gray level intensity, I, recorded for each pixel was an overall value resulting from the contributions of different x-y planes along the depth direction (i.e., z direction). It is important to recall that, from a theoretical point of view, these gray level intensity values could not be linearly added together (because of the exponential attenuation of light), whereas the absorbance values could be. Therefore, for each pixel, the obtained absorbance corresponded to the summation of the absorbances at different x-y planes along the depth direction. In addition, it should be kept in mind that the Beer-Lambert law should be applied at given wavelength only if the concentration of the absorbing specie (here RZ) is homogeneous in the depth direction. In the present experiments, due to the 3D flow structure in the liquid slug, some gradients of oxygen concentration inevitably existed in the depth direction.

Unfortunately, this level of heterogeneities could not be captured by the method, and all the more that it depended on the operating conditions. The heterogeneity was here defined as a percentage of the standard deviation errors (SD, see Supplementary Material S1) with respect to the averaged equivalent oxygen concentrations inside of the liquid slug. Taking five representative operating conditions under jTP = 28.6 cm⸱ s -1 , and twelve ROI observing positions, the heterogeneities were estimated between 1% and 20% and decreased with the ROI observing positions.

Results and discussion

Equivalent O2 concentration fields

By implementing the colorimetric technique and applying the image processing, the equivalent O2 concentration fields in the liquid slugs could be obtained at the crossjunction and after, at different axial positions in the channel.

The critical Capillary number for the transition between a circular crosssectional shape of the bubble and a square one with curved corners occurred at 10 -2 according to [START_REF] Boden | Synchrotron radiation microtomography of Taylor bubbles in capillary two-phase flow[END_REF] and [START_REF] Fries | Segmented gas-liquid flow characterization in rectangular microchannels[END_REF]. As the two-phase Ca number were below 8.5×10 -3 in this study (see Part I), the liquid film thickness between bubble and channel side walls, 𝛿 2$33 , could be considered as constant according to [START_REF] Fries | Segmented gas-liquid flow characterization in rectangular microchannels[END_REF], and close to 2% of the hydraulic diameter of the channel (i.e., 𝛿 2$33 ≈ 43 𝜇𝑚).

The spatial resolution of the images was at 8.25 μm per pixel, meaning that the liquid film could be visualized by around 5 pixels theoretically. However, it was not possible to experimentally measure the gray level variation between the bubble and channel wall.

Therefore, in this study, the concentration fields in the liquid film close to the wall could not be measured.

During the bubble formation process

The time-variation of equivalent O2 concentration fields during the bubble pinch-off process is described in Fig. 4 for various operating conditions.

Fig. 4 (a) presents a case where the pinch-off process covered an extremely long period of 3975 ms (around half of the whole pinch-off process). Under this condition, the associated CaTP and ReTP were equal to 5.9×10 -5 and 8, respectively. The capillary force was thus dominant compared to the viscous and inertial forces. Before the pinchoff point (-112.5 ms < t < -2.5 ms), some layers of dissolved O2 formed on both sides of the gas finger neck and became gradually thicker. At the bubble pinch-off point, these layers merged, generating a jet-like central oxygen concentration 'finger' between the just-formed bubble and the gas finger. Then the gas finger grew, protruded into the main channel, and finally blocked the main channel (0 < t < 2112.5 ms). During this stage, the dissolved O2 was transported by the entering liquid from inlets. As the Reynolds number was very low, the equivalent O2 field remained highly symmetric with respect to the centerline of the main channel. Once the main channel was fully blocked by the coming gas finger (2612.5 < t < 3862.5 ms), the liquid from the inlets hardly entered into the main channel and the recirculation loop started to develop inside the liquid slug.

Therefore, as the liquid flow inside the liquid slug presented higher velocities along the centerline and a negative velocity near the channel wall, the dissolved O2 concentration was transported from the tip of the gas finger to the rear of the formed bubble. In the meantime, the thickness of the dissolved O2 region near gas finger got thicker, enabling to better disperse oxygen in the radial direction of channel. . In these cases, the whole bubble formation period was shortened due to higher gas and liquid superficial velocities (55 ms, 32.5 ms and 20 ms respectively, against 8247.5 ms in the case (a)). One can observe that the jet-like central oxygen concentration 'finger' at the centerline became no more visible from Fig. 4 (b) to (d), as the times to break up the gas finger were shortened (up to 412 times lower compared to case (a)). Furthermore, when increasing two-phase Reynolds number ReTP, the recirculation loop developed faster in the liquid slug.

Contrary to Fig. 4 (a), at the stage where the main channel was fully blocked by the coming gas finger, two spots of high equivalent O2 concentration could be distinguished near the formed bubble rear and channel wall region, marked by two red circles in Fig.

(b)

. This phenomenon could be attributed to the developed recirculating loop inside of liquid slug. Indeed, in the region between the bubble rear and the channel wall, the liquid flow came from the centerline and turned backwards along the channel wall, thus carrying the dissolved oxygen due to the diffusive transport between the liquid and the bubble rear [START_REF] Abiev | Gas-Liquid and Gas-Liquid-Solid mass transfer model for Taylor flow in micro (milli) channels: a theoretical approach and experimental proof[END_REF]. It was also possible that the liquid coming from the channel corner (surrounding the front bubble) could contribute to these two spots of high equivalent O2 concentration, because of the diffusive transport between the liquid film in the corner and the bubble lateral body, and the convective transport within the liquid film [START_REF] Abiev | Gas-Liquid and Gas-Liquid-Solid mass transfer model for Taylor flow in micro (milli) channels: a theoretical approach and experimental proof[END_REF].

During the bubble flowing process

In Fig. 5, the equivalent O2 concentration fields are plotted for different flowing times at a total two-phase superficial velocity jTP of 28.6 cm s -1 and for different gasliquid superficial velocity ratio ƞ0. The flowing time was calculated by dividing the observing position Xi by the bubble velocity UB, and thus corresponded to the time required for the liquid slug to travel to the axial position Xi. The related CaTP and WeTP numbers were equal to 4.3×10 -3 and 2.4, respectively. Therefore, the effects of ƞ0 on the equivalent O2 concentration fields could be quantified by comparing Fig. 5 (a), (b) and (c). One can observe that the oxygen dissolved inside the liquid slug became higher as far as the bubbles flowed inside the channel (i.e., with increasing axial positions or flowing times). At X2 = 17 mm, where the liquid slug was not far from the bubble formation point, the recirculating flow inside of liquid slug was not yet fully formed.

Along the centerline region and near wall region of the liquid slug, the equivalent O2 concentration presented higher values than the ones of other regions, due to the convective transport of oxygen by the recirculation loop and within the liquid film [START_REF] Abiev | Gas-Liquid and Gas-Liquid-Solid mass transfer model for Taylor flow in micro (milli) channels: a theoretical approach and experimental proof[END_REF]. However, in some cases, the region near the bubble rear along the centerline also presented a lower equivalent O2 concentration, which may be induced by the complex 3D flow structure (combined effects of the horizontal and vertical flow structures). As the bubble flowed along the channel, the tip region near the bubble nose always held higher equivalent O2 concentrations until reaching an almost uniform field, as seen in the last position X12 in Fig. 5 (a). However, in Fig. 5 (b) and (c), even for the last position X12, some gradients of equivalent O2 concentration still existed inside the liquid slug. Therefore, at an identical two-phase superficial velocity jTP (i.e., at almost same bubble velocity), an increase of gas-liquid superficial velocity ratios ƞ0 (i.e., shorter liquid slugs) led to improve the mixing inside the liquid slug, to increase the equivalent O2 concentration and further to enhance the mass transfer from gas phase to liquid phase.

Axial variation of the concentration and mass flux density

The averaged O2 concentrations could be calculated at different observing positions Xi, as follows:

𝐶 4 " ????? (𝑋) =

∭ 6(%,9)•;%•;9•;< = & •> ' " , (4)
where 𝐶 4 " ????? (𝑋) was the equivalent oxygen concentration at an axial location Xi as depicted in Fig. 5 and measured only in the liquid slug, Ls the slug length and dh the hydraulic diameter of the channel (see Part I).

Fig. 6 plots this averaged concentration, 𝐶 4 " ????? , normalized by the maximum oxygen concentration Cmax (i.e., 5.2 mg⸱ L -1 , see section 2.2) as a function of the axial positions, for each two-phase superficial velocity jTP and for different gas-liquid superficial velocity ratio, ƞ0 (other conditions are plotted in the Supplementary Material S2). At a given jTP, an increase of gas-liquid superficial velocity ratio ƞ0 logically increased the amount of oxygen transferred by unit of liquid slug volume, as observed in Fig. 5. When 𝐶 4 " ????? /𝐶 #$% kept below 0.8, the normalized O2 concentrations increased linearly with axial positions. In addition, the mass flux density cumulated in the liquid slugs along the axial positions, noted j ? " , was calculated according to the equation defined in Mei et al. (2020b):

𝜑 ? " (𝑋) = @ ( A ( ;# ! " (B) >B (5)
where the bubble velocity UB and bubble surface SB were obtained in the Part Ⅰ of the article. These parameters were considered constant along the channel length (the bubble nose and rear were assumed as hemispherical shape, see Supplementary Material S3).

As shown in Fig. 7, whatever jTP and h0, the mass flux density, j ? " , sharply decreased over the first millimeters after the cross-junction and finally tended towards a plateau. At given axial position X and jTP, an increase of h0 induced higher mass flux densities, and at given axial position X and h0, an increase of jTP also induced higher mass flux densities. One can note that, for all operating conditions, the mass flux densities became constant after 0.2 m. After this location from the cross-junction (almost 10 times the hydraulic diameter of the channel), the entrance effects (bubble formation stage) had no more influence. Note that the same trends were observed for other values of jTP (see Supplementary Material S4).

Estimation of enhancement factor

Due to the chemical reaction between oxygen and dihydroresorufin, the chemical enhancement factor E (the ratio between the average mass fluxes with the resazurin-based reaction) should be estimated. The Hatta number Ha (the ratio between the reaction rate and the diffusion rate in the liquid film) and E could be calculated by solving two implicit equations, as presented in Mei et al. (2020b) (the related equations are given in the Supplementary Material S5). In the present study, the oxygen concentration at saturation ( 𝐶 ! ! * ) was considered equal to 8.15 mg L -1 (no bubble expansion, pressure drop below 7 kPa).

The related reaction rate constants k2 was initially considered equal to 1.28´10 6

L⸱ mol -1 s -1 (Yang et al. ( 2016b)), and later to 0.67´10 6 L⸱ mol -1 s -1 ( Hoppe ( 2019)). For all the conditions displayed in Fig. 7, whatever the value of k2 considered, when the Hatta numbers ranged between 3 and 8.6, the enhancement factors were between 1.02 and 1.04.

when Ha ranged from 0.65 to 3, E tended towards to unity.

Thus, these calculations would suggest that the enhancement caused by the colorimetric chemical reaction on the mass transfer could be assumed negligible. Thus, E was considered as unity in the following section.

Cumulated oxygen mass during bubble formation process

This sub-section aims at analyzing the oxygen mass that was cumulated inside one liquid slug during the formation of the Taylor bubble. For that, the considered liquid slug was the one in front of the forming bubble. Fig. 8 presents some image sequences relating to the development of the liquid slug. They show that the liquid slug could be divided into three stages:

a. the liquid surrounding the gas finger neck, squeezed the gas finger until the pinch-off of the front bubble.

b. after the pinch-off of the front bubble, the liquid entered the main channel until the back gas finger fully blocked the channel.

c. the liquid slug formed and was separated by the back gas finger and front formed bubble.

Note that, in Fig. 8, the images on the right column correspond to the zones occupied by the liquid phase that were extracted using Matlab® software. The unit cell was here marked by the green dotted box. were estimated according to the displacement of the gas finger nose or bubble nose (see solid red dots in Fig. 8). They showed that, during the stage (a), the gas finger was accelerating until the bubble pinch-off with velocities ranged from 24.8 to 28.1 cm⸱ s -1 , while during the stage (b), the formed bubble velocity was almost constant around 28.6 cm⸱ s -1 . The fast rate of oxygen mass in stage (c) could be thus due to the consequences of the developing recirculation loop in the formed liquid slug and also of the accelerating movement of subsequent gas finger. During the stage (b), the oxygen mass slightly varied, probably due to a 'dilution effect' induced by the fresh liquid coming from side channels.

In the future, deeper studies relating the local variations of mass transfer rates and the gas finger velocities need to be further implemented to have a better understanding.

Overall mass transfer coefficients

The overall volumetric liquid side mass transfer coefficient, kL⸱a, could be deduced from the oxygen mass balance in the liquid phase according to Eq. ( 6).

;6 ) " (B) CCCCCCCCCC

;D

= 𝐸 𝑘 E (𝑋) 𝑎(𝑋) (𝐶 4 " * (X) -𝐶 ? " (𝑋) G3 ??????????? )

where t represented the flowing time of the bubble until reaching the axial position (t = X/UB) and 𝐶 ? " (𝑋) G3 ??????????? was equal to zero because of a full consumption of O2 by DH in the liquid slug. The enhancement factor E was considered equal to 1 (see section 3.3).

From the axial variations of equivalent O2 concentration (Fig. 6), 𝐶 ? " (𝑋) ????????? could be considered as linearly increasing with X (i.e., time t). Using the least square method, the slope coefficient (noted 𝜁) between 𝐶 ? " (𝑋) ????????? and X could be obtained considering a linear relation when 𝐶 4 " ????? /𝐶 #$% was below 0.8. Therefore, the overall volumetric liquid side mass transfer coefficient kL⸱a could be directly deduced from

𝑘 E 𝑎 = H ×@ ( 6 ) " * (7)
As the mass transfer mechanism was mainly affected by the recirculation loops inside the liquid slug, it was interesting to compare these coefficients with the frequencies of loop recirculation, fc, defined by [START_REF] Abiev | Mass transfer characteristics and concentration field evolution for gas-liquid Taylor flow in milli channels[END_REF] as

𝑓 K ≈ @ ( L= + (8)
The numerical values of the overall volumetric liquid side mass transfer coefficients kL⸱a and the recirculation frequencies fc are reported in the Supplementary Material S6. (2020b) in different in-plane spiral-shaped tubes. Whatever the cases, kL⸱a followed a linear increase relation with fc, that could be described by the following correlation proposed by (Mei et al., 2020b):

kL⸱a = ω fc (9)
The coefficient w was found equal to 1.99 × 10 !, in the present straight channel. This coefficient was lower than the ones obtained in the in-plane spiral-shaped milli-reactor As explained by Mei et al. (2020b), the intensification factor for kL⸱a was almost proportional to the increase of O1/á𝜆ñ (λ=2r/dit). For straight channels, the curvature ratio was infinity and O1/á𝜆ñ was thus zero. A scaling law for kL⸱a considering fc and 1/á𝜆ñ was thus proposed to gather all the points for fc < 50, which consisted of two terms as below:

M , $ N - = 2 × 10 !, + ,O1."1 N - %./0 + " áQñ , ".R" , MAPER: 15.0% (10)
The first term was a constant own to straight channels, and the second term accounted for the effects of centrifugal force induced by the channel curvature. 10) led to an acceptable description of all the configurations when recirculation frequencies were under 50.

Conclusions

The present work aimed at investigating the gas-liquid mass transfer around

Taylor bubbles during and after the bubble formation (twelve observing positions) in a straight milli-channel with a cross-junction inlet geometry. Using the resazurin-based colorimetric technique and by improving image acquisition system (monochromatic light and higher magnification), the equivalent oxygen concentration fields were accurately visualized and measured. The main findings were the following:

1) The resazurin-based colorimetric method was implemented using a monochromatic light in the image acquisition system for the first time, thus allowing to apply the Beer-Lambert law to calculate the equivalent oxygen concentrations. However, one should keep in mind that the application of the Beer-Lamber law assumed that the concentration of dissolved species was homogeneous in the vertical direction, which was not systematically the case in the experiments. The induced error was estimated between 1% and 20%.

2) The equivalent oxygen concentration fields could be measured with high spatial and temporal resolution. During the bubble formation stage, it was possible to distinguish different mechanisms for the developing oxygen concentration fields which depended on ReTP. During the bubble flowing stage, the progressive accumulation of the dissolved oxygen inside the liquid slug was quantified as a function of the axial positions.

3) The average oxygen concentrations in the liquid slug increased linearly with the axial position X, while the mass flux density decreased with X until a location of almost 0.2 m. Furthermore, at the same flowing time for liquid slugs, it was found that the averaged oxygen concentrations increased with the gas-liquid superficial velocity ratio at given two-phase superficial velocities, and vice versa.

4) During the bubble formation, the evolution of the cumulated oxygen mass was described by three stages corresponding to (a) the oxygen present in the liquid surrounding the gas finger neck, (b) the oxygen accumulated in the liquid after the pinch-off of the front bubble, (c) the oxygen cumulated in the liquid slug (separated by the front bubble and back gas finger). The rate of cumulated oxygen mass was found the highest for the third stage, which was probably due to the combined effect of the developing recirculation loop and of the accelerating gas finger.

5) The overall volumetric liquid side mass transfer coefficients (kL a) obtained in the present work were compared with the ones measured in the literature. They followed a linear increase with the recirculation frequencies for fc < 50. A scaling law based on recirculation frequency and curvature ratio was at last proposed, allowing to gather the data from different configurations.

Nomenclature

Latin letters

Symbol

Fig. 1

 1 Fig. 1 Geometry of the straight milli-channel with a cross-junction. The 12 axial positions, Xi

Fig. 2

 2 Fig. 2 Calibration process for mass transfer measurements (a) raw images of the millimetric channel

  Note that, contrary to the study presented inMei et al. (2020b), a "global" calibration (instead of a pixel-by-pixel calibration) was implemented here as there was no radial change of the light penetration depth in the present case (the setup involved a square channel and not a tube). Furthermore, a Beer-Lambert law type relationship linking the absorbance to the resorufin concentration [RF] could be applied (instead of a linear one, as in Mei et al. (2020b)) because the light was monochromatic (green LED and NE2R10A absorptive ND filter was used, see Part I). Knowing [RF], the equivalent O2 concentration into the global window of observation was deduced considering the stoichiometry of the reaction (i.e., one mole of DH reacted with 0.5 moles of oxygen to form one mole of RF). Finally, as shown in Fig. 2 (b), whatever the observing position Xi, the following calibration relationship between the equivalent O2 concentration and the absorbance of RF was obtained (corresponding to the dotted line in Fig. 2 (b)): [𝑂 , ] = 3.70 × 𝐴 (3) with a mean absolute percentage error of regression (MAPER, see the Supplementary Material S1) equal to 11%.

Fig. 3

 3 Fig.3presents the main steps of the image processing, starting from a raw image

Fig. 3

 3 Fig. 3 Main steps of the image processing: (a) Raw image of gas-liquid flow. (b) Image with

Fig. 4

 4 Fig. 4 Time-variation of the equivalent O2 concentration fields during the bubble pinch-off process

Fig. 5

 5 Fig. 5 Equivalent O2 concentration fields during the bubble flowing process at jTP = 28.6 cm⸱ s -1

Fig. 6

 6 Fig. 6 Axial variation of normalized averaged equivalent O2 concentrations in the liquid slug for

Fig. 7

 7 Fig. 7 Variation of the mass flux density with the axial positions under different operating

Fig. 8

 8 Fig. 8 Illustration of the development of unit cell, liquid slug and bubble during the bubble formation

Fig. 9

 9 Fig. 9 Evolution of cumulated oxygen mass inside the liquid phase during bubble formation pinch-

Fig. 9

 9 Fig. 9 displays a typical variation of the oxygen mass cumulated in the liquid

Fig. 10

 10 Fig. 10 Relationships between the overall volumetric liquid-side mass transfer coefficients kL a and

Fig. 10

 10 Fig. 10 plots the overall volumetric liquid side mass transfer coefficient, kL⸱a, as

( 2 .

 2 75 × 10 !, and 3.45 × 10 !, ,Mei et al. (2020b)), which was possibly due to the absence of centrifugal force, different cross-sectional shapes (cylindrical tube versus squared channel), and channel dimensions. In addition, the minimal ReTP number in the present study was about 2.4 times of the maximal ReTP number in the spiral-shaped reactor. This emphasized the mass transfer intensification caused by the centrifugal force even under lower ranges of ReTP number.In addition, the data of[START_REF] Roudet | Hydrodynamic and mass transfer in inertial gas-liquid flow regimes through straight and meandering millimetric square channels[END_REF] in straight and meandering channels (2mm, square),[START_REF] Yang | Local investigations on the gas-liquid mass transfer around Taylor bubbles flowing in a meandering millimetric square channel[END_REF] in a meandering milli-channel (2mm, square),[START_REF] Dietrich | A new direct technique for visualizing and measuring gas-liquid mass transfer around bubbles moving in a straight millimetric square channel[END_REF] in a straight milli-channel (2mm, square), and[START_REF] Abiev | Mass transfer characteristics and concentration field evolution for gas-liquid Taylor flow in milli channels[END_REF] in a vertical glass tube (dit, 3mm), were plotted in Fig.10. They also followed a good linearity when the recirculation frequencies were lower than 50. Above this value, the overall volumetric liquid side mass transfer coefficient kL⸱a remained almost constant, which was probably due to other factors (e.g., possibilities of bypass flow, shorter flowing time inside of the milli-channels).

Fig. 11

 11 Fig. 11 Comparison between the predicted (kL a)pre and experimental (kL a)exp for the configuration

Fig. 11

 11 Fig. 11 compares the predicted (kL a)pre with the experimental (kL a)exp for the

fc

  Frequency of recirculation in the liquid slug (fc ≈ UB / 4LS) [s -1 ] I Intensity, i.e., gray value [the gas phase (𝑗 &' = 𝑄 &' /Ω) [m•s -1 ] jL Superficial velocity of the liquid phase (𝑗 ( = 𝑄 ( Mass flux density of oxygen transferred per unit of area [kg•m -2 •s -1 ] w Linear dimensionless coefficient associating kL a and fc [-]

Acknowledgments

The author would like to acknowledge the financial assistance provided by the China Scholarship Council. We also acknowledge the support for this work from the CNRS research federation FERMaT.

Subscripts and Superscripts

Declaration of Competing Interest

The authors report no declarations of interest.