Françoise Lazarini
email: francoise.lazarini-seran-dour@pasteur.fr

Sarah Levivien
email: sarah.levivien@aphp.fr

Yoann Madec
email: yoann.madec@pasteur.fr

Fabien Taieb
email: fabien.taieb@pasteur.fr

Estelle Mottez
email: estelle.mot-tez@pasteur.fr

Tan-Phuc Buivan
email: tan-phuc.bui-van@pasteur.fr

Audrey Maudoux
email: audrey.maudoux@aphp.fr

Sylvette Wiener-Vacher
email: syl-vette.wiener@gmail.com

Jérôme Nevoux
email: jerome.nevoux@aphp.fr

Thierry Van Den Abbeele
email: thierry.van-den-abbeele@aphp.fr

Pierre Gressens
email: pierre.gressens@in-serm.fr

Pierre-Marie Lledo
email: pierre-marie.lledo@pas-teur.fr

Natacha Teissier
email: nata-cha.teissier@inserm.fr

Thierry Van Den

Natacha Pr

Teissier

Olfactory function in congenital cytomegalovirus infection: a prospective study

Keywords: diagnosis, olfaction, smell, discrimination, odorant mixture, children

Congenital cytomegalovirus (CMV) infection leads to olfactory bulb lesions in the fetus, yet little is known about its impact on olfaction after birth. Here, we have assessed in a prospective study conducted on children in two French hospitals from 2016 to 2019, infection severity and olfactory performance after congenital CMV infection. Children with congenital CMV infection aged 3 to 10 years and healthy controls (CTL) matched for age and sex to CMV children symptomatic at birth (sCMV) were enrolled. Olfactory discrimination was assessed using mono-odorants and binary mixtures. Data were analyzed for 54 children with PCR-confirmed congenital CMV infection, including 34 sCMV (median [IQR] age, 6 [5-8] years; 19 [55.9%] male) and 20 CMV asymptomatic at birth (aCMV, median [IQR] age, 4 [3-6] years;

12 [60.0%] male). sCMV were compared to 34 CTL children. Olfactory scores in CMV-infected children were independent from vestibular deficit and hearing loss. The olfactory score was efficient to discriminate between CTL and sCMV for children >6 years (area under the receiver-operating characteristic curve (AUC, 0.85; P =0.0006), but not for children <7 years. For children >6 years, the proportion of children with total olfactory score <4 differed between sCMV and CTL groups (91.2% and 18.7%, P <0.001), but not between aCMV and age-matched healthy control groups.

Conclusion:

Congenital CMV infection is associated with reduced olfactory performance in children with infection symptoms at birth.

Cytomegalovirus (CMV) is a herpes type 5 virus that can affect the fetal and neonatal brain after in utero infection [START_REF] Coyne | Zika virus -reigniting the TORCH[END_REF]. CMV affects 0.5-2% of newborns and is the leading infectious cause of congenital deafness. Depending on neonatal clinical presentation, children are either categorized as having a symptomatic (sCMV) (presenting with growth retardation, prematurity, jaundice, petechiae, liver and/or hematological anomalies) or asymptomatic (aCMV) (no clinical sign of infection other than possible hearing loss) infection at birth. Prognostic factors for neurosensory sequelae comprise gestational age at infection and sCMV [START_REF] Nicloux | Outcome and management of newborns with congenital cytomegalovirus infection[END_REF][START_REF] Dahle | Longitudinal investigation of hearing disorders in children with congenital cytomegalovirus[END_REF]. 40-60% of sCMV and 10-20% of aCMV children will manifest varying degrees of hearing loss, which can be present at birth or may occur in the first months or years [START_REF] Goderis | Hearing in Children with Congenital Cytomegalovirus Infection: Results of a Longitudinal Study[END_REF]. Although 90% of clinical presentations are silent at birth, no systematic newborn screening has been established to identify aCMV children who are at risk of hearing loss. Human CMV has a specific olfactory receptor expressed on olfactory neurons in the olfactory system that may define viral olfactory cell tropism [5]. Congenital CMV exhibits tropism for neural stem cells of the olfactory system of fetuses, thus lesioning the olfactory bulb [START_REF] Van Den Pol | Cytomegalovirus cell tropism, replication, and gene transfer in brain[END_REF][START_REF] Odeberg | Human cytomegalovirus inhibits neuronal differentiation and induces apoptosis in human neural precursor cells[END_REF][START_REF] Teissier | Cytomegalovirus-induced brain malformations in fetuses[END_REF]. This infection leads to both olfactory and hearing impairments in a mouse model [START_REF] Lazarini | Congenital cytomegalovirus infection alters olfaction prior to hearing deterioration in mice[END_REF]. However, little is known about olfactory dysfunction in CMV-infected children, partly because it is challenging to assess olfaction in toddlers. Many studies have shown the difficulty to reliably test children under 5 years [START_REF] Doty | Olfaction[END_REF][START_REF] Hummel | Olfactory function in children assessed with psychophysical and electrophysiological techniques[END_REF][START_REF] Cameron | Odor identification testing in children and young adults using the smell wheel[END_REF][START_REF] Schriever | Etiologies of olfactory dysfunction in a pediatric population: based on a retrospective analysis of data from an outpatient clinic[END_REF] because of the cognitive and verbal involvement. Discrimination tasks are the most relevant because they are rapid to perform, unlike threshold tasks, and they are requiring minimal cognitive and verbal skills, contrary to identification tasks. New tests based on perception level could constitute useful tools to address olfaction in children. In this regard, mixture based olfactory discrimination tests perform better than standard smell tests in adult humans and in adult and pup animal models [START_REF] Lazarini | Congenital cytomegalovirus infection alters olfaction prior to hearing deterioration in mice[END_REF][START_REF] Hsieh | SMELL-S and SMELL-R: Olfactory tests not influenced by odor-specific insensitivity or prior olfactory experience[END_REF]. Here, we report the olfactory performance of children with a confirmed congenital CMV infection, using a new psychophysical test we have developed. This test aims at measuring the discrimination of monomolecular odorants from the Sniffin' test battery [START_REF] Hummel | Sniffin' sticks': olfactory performance assessed by the combined testing of odor identification, odor discrimination and olfactory threshold[END_REF] and the discrimination of mixture odorants presented in Sniffin' pens. It is non-invasive and rapid to perform, even in very young children, thus requiring little attention and concentration.

Methods

Study Overview and ethical considerations

The main objective of this study was to investigate the association between hearing loss and olfactory performance in children with a congenital CMV infection followed in Robert Debré (Paris) and Bicêtre (Le Kremlin-Bicêtre) hospitals, in France. This prospective study is a nontrial, nondrug study, qualified as exploratory, multicenter, in a paediatric population (Clin-icalTrials.gov number, NCT02782988). It received ethical approval (N° 3372) from Comité de Protection des Personnes (CPP IDF-3). Children were included in the study after explanation of the study and obtaining of written informed consent from both parents.

Enrolment Criteria

Children with confirmed congenital CMV, aged 3 to 10 years, were enrolled in this study during a standard care visit. Proof of congenital infection was ascertained by positive CMV polymerase chain reaction (PCR) in urine and/or blood in the first 3 postnatal weeks, or retrospective diagnosis for the presence of positive PCR on dried blood spots collected at postnatal day 3 to 7.

Exclusion criteria included clinical conditions that may interfere with the study, such as chronic rhinosinusitis, allergic rhinitis, primary ciliary dyskinesia, Kallmans syndrome or other neurologic issues that can impact olfaction.

CMV infected children were divided into two groups according to neonatal characteristics consistent with recognized clinical definitions: sCMV and aCMV at birth. Healthy controls (CTL) matched for age and sex to the sCMV group were enrolled among children consulting for other ear, nose, throat (ENT) non-rhinological pathologies, anaesthesiology or orthopaedic appointments. CTL children had no history of congenital infection and presented with transient evoked otoacoustic emissions <20 dB for each ear.

Clinical and Radiologic Symptoms

Prenatal and neonatal clinical signs and virological data in favour of congenital CMV infection were recorded. Postural developmental milestones, vestibular canal and otolithic function were assessed as previously described [START_REF] Maudoux | Vestibular impact of Friedreich ataxia in early onset patients[END_REF]. Magnetic resonance imaging of the brain and the inner ear was performed to assess cerebral lesions (see the Supplemental Information for details).

Hearing Evaluation

Children with congenital CMV underwent either objective auditory brainstem response or subjective behavioral audiometry tests to assess auditory thresholds. Hearing deficit was defined by an auditory threshold of the most affected ear ≥25dB. In CTL, normality of hearing was assessed using evoked otoacoustic emissions.

Olfactory Evaluation

Olfaction was assessed in a 15-minute session with 18 pen-like odour-dispensing devices (Sniffin' Sticks, Burghardt, Wedel, Germany) [START_REF] Hummel | Sniffin' sticks': olfactory performance assessed by the combined testing of odor identification, odor discrimination and olfactory threshold[END_REF]. Two series of 3-odorant discrimination tasks were performed: the first with simple odorants (monomolecular odorant test), and the second with binary mixtures of odorants (mixture odorant test). For each task, 3 Sniffin' Sticks were sequentially presented to the subject, two contained the same odorant and one contained a different associated odorant. The child was requested to smell each stick and indicate the stick that smells differently (forced choice between three possibilities). A correct or incorrect answer resulted in a score of 1 or 0, respectively.

Monomolecular Odorant Test

The sticks for the first task contained isoamylacetate (one stick) and anethol odorant (two sticks). The sticks for the second task contained limonene (one stick) and citronellal odorant (two sticks). The sticks for the third task contained anethol (one stick) and eugenol odorant (two sticks). The total score for this test ranged from 0 (no correct response) to 3 (all correct responses). Binary variables were defined using the threshold of 2.

Mixture Odorant Test

The sticks for the first task contained a mixture of L-carvone and D-carvone at a 2:8 proportion (one stick) and mixture of L-carvone and D-carvone at a an 8:2 proportion (two sticks).

The sticks for the second task contain a mixture of isoamylacetate and anethol in an 8:2 proportion (one stick) and mix of isoamylacetate and anethol at a 2:8 proportion (two sticks). The sticks for the third task contain a mixture of anethol and eugenol at an 8:2 proportion (one stick) and mix of anethol and eugenol at a 2:8 proportion (two sticks). The total score for this test ranged from 0 (no correct response for the 3 problems) to 3 (correct responses for the 3 problems). Again, binary variables were defined using the threshold of 2.

Olfactory Score Calculation

The total olfactory score (TOS) was calculated by adding the monomolecular odorant score to the mixture score. It ranged from 0 (no correct response for the 6 problems) to 6 (correct responses for the 6 problems). Binary variables were defined by a total score <4, this threshold was retained as it corresponds to a majority of incorrect responses.

Statistical Analysis

Quantitative variables were summarized as median with interquartile range (IQR) and compared across groups using Mann-Whitney non-parametric test. Categorical data were expressed as percentages and compared between groups using Fisher exact test. The accuracy of olfactory tests was evaluated by applying data to receiver-operating characteristic (ROC) curves. To study the associations between children characteristics and olfaction, the Spearman non-parametric test was used. Statistical analyses were performed using Stata 16 (StataCorp LLC, Texas, USA) and Prism software (GraphPad, version 9, San Diego, USA), significance was considered at the level 5%.

Results

Child Characteristics

From May 2016 to December 2019, we recruited 34 sCMV children (median [IQR] age, 6 [5-8] years; 19 [55.9%] male, Tables 1, S1, S2). We also recruited 34 healthy matched-CTL. As a supplementary control, we included aCMV children. However, due to absence of CMV newborn screening in France, enrolment of aCMV was complex, particularly in the 7-10 year age group, and only 20 aCMV were enrolled (median [IQR] age, 4 [START_REF] Dahle | Longitudinal investigation of hearing disorders in children with congenital cytomegalovirus[END_REF][START_REF] Goderis | Hearing in Children with Congenital Cytomegalovirus Infection: Results of a Longitudinal Study[END_REF][5][START_REF] Van Den Pol | Cytomegalovirus cell tropism, replication, and gene transfer in brain[END_REF] years (only 5 children aged 7-10); 12 [60.0%] male. Thus, we ultimately essentially compared sCMV to CTL children because we did not reach the targeted number of aCMV children. Fig. 1 shows the flow chart of the selection process.

Among the 54 children with congenital CMV infection, 23 presented hearing or vestibular deficit at inclusion. Hearing deficits were reported in 19 children (12 in the sCMV group and 7 in the aCMV group). Three presented with profound congenital hearing loss at birth (1 in the sCMV group and 2 in the aCMV group).

Olfactory Performance

Among CTL, both the monomolecular odorant discrimination score and the TOS were positively correlated with age (r=0.42, P =0.012; and r=0.48, P =0.004, respectively). In CTL, TOS was significantly higher in children 7-10 years than in those 3-6 years (median (IQR): 4.0 [4.0-5.0] and 3.0 [1.0-4.0], P =0.002), and in consequence the proportion with a TOS <4 was significantly lower in CTL 7-10 years than in CTL 3-6 years (18.75% and 66.7%, respectively; P =0.007, Table 2). Considering the monomolecular odorant discrimination score, the proportion with a score <2 was significantly lower in controls aged 7-10 years than in controls aged 3-6 years (6.3% and 55.6%, respectively; P =0.003). Considering the mixture odorant discrimination score, the proportion with a score <2 was not different between CTL aged 7-10 and 3-6 years (37.5% and 61.1%, respectively; P =0.30). There was no association between olfactory scores and sex or with passive smoking.

ROC curve analysis revealed that the TOS was efficient to discriminate between CTL and sCMV for children 7-10 years (area under the ROC curve [AUC]=0.857, P=0.0006, Fig. 2b), but not for children 3-6 years (AUC=0.519, Fig. 2a). Moreover, for children >6 years, the mixture score alone was efficient to discriminate between CTL and sCMV (AUC=0.809, P=0.003, Fig. 2d), but not the monomolecular odorant score (AUC=0.588, Fig. 2c).

Overall, the proportion of children with a TOS <4 was significantly higher in the sCMV group than in the CTL group (73.5% and 44.1%; P=0.025). Considering only the monomolecular odorant discrimination score, there was no difference between the two groups (Fig. 3b). For the only mixture scores, the proportion of children with a score <2 was significantly higher in the sCMV group than in the CTL group (76.5% and 50.0%, respectively, P =0.043).

Stratifying by age, the difference in the proportion of children with a TOS <4 was highly significant between sCMV and CTL in children 7-10 years of age (91.2% and 18.7%, P <0.001), but not in younger children (Fig. 3d).

In sCMV children, there was no difference in the TOS between children presenting with and those without neurological involvement (Fig. S1). There was no difference for the TOS between sCMV children presenting with hearing loss and those with normal hearing (Fig. 3e).

There was no difference in the proportion of children with a TOS<4 between aCMV, subset of age-matched sCMV and subset of age-matched CTL children in the 7-10 year age group as well as in younger children (Fig. S2).

There was no difference in the olfactory scores between children who received antiviral treatment after CMV detection (n=7) and those without treatment (n=38) (Table 2).

Discussion

This is the first study to assess olfactory function in children with congenital CMV infection and to report the severity of their altered olfaction ability. The strengths of this study are: i) PCR-confirmed congenital CMV infection, ii) the documentation of clinical, radiologic and vestibular symptoms as well as concomitant evaluation of hearing and iii) enrolment of age and sex-matched CTL.

Reduced olfactory score was frequent in congenital CMV infection, occurring in 91.2% of our sCMV patients aged 7-10 years, thus becoming the most frequent sensorineural deficit in our series. 44.1 % of these patients experience other sensorineural deficits (hearing loss in 35.3%, vestibular deficit in 38.2%). Conversely, 5 aCMV children aged 7-10 years demonstrated normal olfaction. The most likely explanation of this observation is the probable link between olfactory performance and the severity of congenital CMV infection. A recent retrospective study demonstrated that 67% of children with olfactory dysfunction were of congenital origin, whereas 12% were due to head trauma [START_REF] Schriever | Etiologies of olfactory dysfunction in a pediatric population: based on a retrospective analysis of data from an outpatient clinic[END_REF]; the role of congenital infection being to date unknown, the responsibility of CMV has certainly not yet been evaluated. In previous studies, loss of smell in infants has been linked to neurodevelopmental disorders, including attention deficit/hyperactivity disorders and autism spectrum [START_REF] Doty | Olfaction[END_REF][START_REF] Rozenkrantz | A Mechanistic Link between Olfaction and Autism Spectrum Disorder[END_REF]. Olfaction is essential for food information, safety, emotion regulation, scaffolds environment perception and memory, mother-child attachment, and social cognition [START_REF] Schaal | Olfaction scaffolds the developing human from neonate to adolescent and beyond[END_REF]. However, there is no absolute correlation between neurodevelopmental disorders and olfactory scores, as we do not find a link between these two in our present series.

Olfactory loss can also be observed after other post-viral infections such as rhinovirus, parainfluenza virus, coronavirus (CoV) 229E and Epstein-Barr virus [START_REF] Suzuki | Identification of viruses in patients with postviral olfactory dysfunction[END_REF]. Olfactory discrimination and thresholds were preserved in these latter infections, compared to identification [START_REF] Whitcroft | Patterns of olfactory impairment reflect underlying disease etiology[END_REF]. Olfactory loss can be an early sign of coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome CoV-2; this dysfunction can persist several months and be associated to an olfactory bulb hypometabolism [START_REF] Niklassen | COVID-19: Recovery from Chemosensory Dysfunction. A Multicentre study on Smell and Taste[END_REF][START_REF] De Melo | COVID-19-related anosmia is associated with viral persistence and inflammation in human olfactory epithelium and brain infection in hamsters[END_REF][START_REF] Guedj | Long COVID and the brain network of Proust's madeleine: targeting the olfactory pathway[END_REF]. Fetopathological studies have demonstrated the presence of CMV in neural stem cells of the olfactory bulb underlining the specific targeting of the pluripotent cells, rather than olfactory neurons [START_REF] Teissier | Cytomegalovirus-induced brain malformations in fetuses[END_REF].

Olfactory scores in our CMV-infected children were independent from age, contrasting with CTL children. Improved olfactory performance in healthy children is correlated with the maturation of the olfactory system with better ability to discriminate with age. This is not observed in sCMV-infected children, possibly due to the viral targeting of pluripotent cells [START_REF] Teissier | Cytomegalovirus-induced brain malformations in fetuses[END_REF]. Olfactory scores in our CMV-infected children were independent from hearing loss or vestibular deficit. These findings contrast with an epidemiological study where a correlation was found between hearing loss and olfactory dysfunction, but infection, in particular congenital, was not considered as an influential factor [START_REF] Park | Epidemiological association of olfactory dysfunction with hearing loss and dysphonia in the Korean population: A cross-sectional study[END_REF]. The incidence of cranial neuropathies is higher in patients with post-viral olfactory loss compared to a control population [START_REF] Jitaroon | Evaluation of the Incidence of Other Cranial Neuropathies in Patients With Postviral Olfactory Loss[END_REF]; however, we found no difference for the olfactory score between children presenting neurological manifestations and those without neurological involvement. These findings suggest that peripheral (audiovestibular) and central (cerebral) lesions are independent and that neurological damage did not induce vulnerability to olfactory dysfunction in our sCMV infants.

CMV host entry is probably systemic, associated with macrophage infection [START_REF] Farrell | Cytomegalovirus host entry and spread[END_REF]. To date, there is no evidence of CMV spread to the brain through the cribriform plate.

Another insight of our study is the greater efficiency of the mixture discrimination tests in assessing olfactory function in children compared to the mono-odorant testing. While the monomolecular test evaluates the ability to discriminate between two single odorants of similar concentration, the mixture test is a more difficult perception test with discrimination of mixtures presenting the same two odorants but in different concentration. Of note, the odorant mixture discrimination score only discriminates between CTL and CMV from the age of 7, which strongly limits its use in clinics. The lower discrimination efficacy in younger children may be due to the subtler olfactory difference between scent pens that children 3-6 may be less attentive to.

Limitations of our study include the use of olfactory tests that have not been validated

for children in this version before and a predefined cut-off value that was not based on previous observations in a control group. The cut-off value first appears in the initial statistical plan of the study's protocol, that was subject to no change. This cut-off of 4 points to distinguish between normosmia and olfactory dysfunction was retained in the initial statistical plan of this study as it corresponds to a majority of incorrect responses. This cut-off leads to a high percentage of children in the control group with reduced olfactory function. Another limit of our study is the small sample size of the human cohort, especially for aCMV patients. Extending these investigations to a larger group of children, including controls, would allow specifying these first findings. Moreover, this study would benefit from additional approaches to characterize the olfactory function, by using tests of perception and identification of odorants.

In conclusion, this study highlighted the high incidence of olfactory impairment in children with congenital sCMV infection. As olfactory loss can impact nutrition, social interaction, safety and quality of life, early detection of olfactory disorders may lead to olfactory rehabilitation programs in order to limit neurodevelopmental consequences: recent studies have demonstrated the importance of olfactory training to improve the olfactory function in adults [START_REF] Addison | Clinical Olfactory Working Group Consensus Statement on the Treatment of Post Infectious Olfactory Dysfunction[END_REF][START_REF] Zhang | Smell disorders in COVID-19 patients: role of olfactory training: A protocol for systematic review and meta-analysis[END_REF] and children [START_REF] Mahmut | Olfactory training in 8-year-olds increases odour identification ability: a preliminary study[END_REF]. Olfactory score is the sum of monomolecular and mixture odorant discriminations. Passive smoking is defined by exposition to more than a tobacco pack per day; Hearing deficit is defined by auditory threshold of the most affected ear ≥25dB; Controls had normal hearing (inclusion criterion).

Legends of the figures

Fig. 1 .

 1 Fig. 1. Enrolment in the INFECSMELL-CLIN study. This study was performed between May

Fig. 2 .

 2 Fig. 2. ROC curves for the discrimination of children with congenital cytomegalovirus infection

Fig. 3 .

 3 Fig. 3. Olfactory scores in children with congenital cytomegalovirus infection and controls.

Table 1 .

 1 Characteristics of the children with congenital, PCR-confirmed, CMV infection

	Severity of congenital CMV infection

Table 1 .

 1 Characteristics of the children with congenital, PCR-confirmed, CMV infection (continued).

	Severity of congenital CMV infection

Table 1 .

 1 Characteristics of the children with congenital, PCR-confirmed, CMV infection (continued). Viral symptoms at birth: one at least of the following neonatal symptoms: intrauterine growth retardation, prematurity, petechiae, organomegaly, icteriae, thrombocytopenia; b Maternal primary infection: Cases with high IgG avidity in the first trimester were considered as non-primary infections. Cases with seroconversion and/or positive IgG positive IgM, and low or intermediate IgG avidity in first trimester were considered as primary infections in the first trimester. Cases with negative IgG and IgM levels in the first trimester (at 12 to 14 weeks) were classified in either the second or third trimester groups, depending on the date of seroconversion.

	Severity of congenital CMV infection

a c

Hearing deficit: auditory threshold of the most affected ear ≥25dB.

d

In those with hearing deficit and no implant. e Cochlear implants were usually performed in the early infancy, before 6.

Table 2 .

 2 Olfactory scores by characteristics in controls and CMV-infected childrenNo.

(%) Variable Monomolecular odor- ant discrimination Score <2 Mixture odorant discrimination Score <2 Total olfactory Score <4

	Controls (n=34)
	Age group, y
	≤6 years (n=18)	10 (55.6)	11 (61.1)	12(66.7)
	>6 years (n=16)	1 (6.3)	6 (37.5)	3 (18.75)
	P Value	0.003	0.30	0.007
	Sex
	Girls (n=15)	5 (33.3)	6 (40.0)	6 (40.0)
	Boys (n=19)	6 (31.6)	11 (57.9)	11 (57.9)
	P Value	>0.999	0.49	0.49
	Passive smoking
	Yes (n=8)	2 (25.0)	2 (25.0%)	1 (12.5)
	No (n=26)	9 (34.6)	15 (57.7%)	14 (53.9)
	P Value	>0.999	0.22	0.053
	CMV-infected children (n=54)
	Age group, y
	≤6 years (n=34)	20 (60.6)	19 (57.6)	22 (66.7)
	>6 years (n=21)	5 (23.8)	18 (85.7)	15 (71.4)
	P Value	0.012	0.038	0.772
	Sex
	Girls (n=23)	11 (47.8)	19 (82.6)	20 (86.7)
	Boys (n=31)	14 (45.2)	18 (58.1)	17 (54.8)
	P Value	>0.999	0.077	0.017
	Passive smoking
	Yes (n=10)	4 (40.0)	7 (70.0)	6 (60.0)
	No (n=44)	24 (54.6)	30 (68.2)	31 (70.5)
	P Value	0.49	>0.999	0.71
	Antiviral treatment after CMV detection
	Yes (n=7)	3 (42.9)	6 (85.7)	7 (100.0)
	No (n=38)	18 (47.4)	25 (65.8)	23 (60.5)
	P Value	>0.999	0.407	0.077
	Hearing deficit
	Yes (n=19)	11 (57.9)	14 (73.7)	14 (73.7)
	No (n=35)	16 (45.7)	26 (74.3)	25 (71.4)
	P Value	0.57	>0.999	>0.999
	Vestibular deficit
	Yes (n=20)	9 (45.0)	1 (75.0)	13 (65.0)
	No (n=31)	13 (41.9)	19 (61.3)	20 (66.7)
	P Value	>0.999	0.37	>0.999

Supplementary information

Supplementary methods. Table S1. Congenital CMV diagnostic confirmation of the 54 cytomegalovirus-infected children.

Table S2. Neonatal viral symptom characteristics of the 34 cytomegalovirus-infected children symptomatic at birth.

Competing interests

The odorant mixtures are the subject of a patent (WO2017198816A1 published on November


Fabrice R A Onofri
email: fabrice.onofri@univ-amu.fr

Isaac Rodríguez-Ruiz

Fabrice Lamadie
email: fabrice.lamadie@cea.fr

Isaac Rodriguez-Ruiz
email: isaac.rodriguezruiz@toulouse-inp.fr

Microfluidic lab-on-a-chip characterization of nano-to microparticles suspensions by light extinction spectrometry

come

Introduction

Microfluidics has become an essential tool in many areas from life sciences [START_REF] Whitesides | The origins and the future of microfluidics[END_REF][START_REF] Sipos | Microfluidic platform for monitoring Saccharomyces cerevisiae mutation accumulation[END_REF] to chemistry [START_REF] Demello | Control and detection of chemical reactions in microfluidic systems[END_REF] and environmental analysis [START_REF] Jokerst | Advances in microfluidics for environmental analysis[END_REF]. In chemical engineering [START_REF] Mitchell | Microchip-based synthesis and total analysis systems (µSYNTAS): chemical microprocessing for generation and analysis of compound libraries[END_REF], microfluidics improves the space-time yields, selectivity, reaction residence times and conversion performances of fundamental synthetic transformations in solution. This makes possible, for instance, to synthetize cadmium sulphide [START_REF] Edel | Microfluidic routes to the controlled production of nanoparticles[END_REF][START_REF] Hung | Alternating droplet generation and controlled dynamic droplet fusion in microfluidic device for CdS nanoparticle synthesis[END_REF], gold [START_REF] Ishizaka | Dynamic control of gold nanoparticle morphology in a microchannel flow reactor by glucose reduction in aqueous sodium hydroxide solution[END_REF], titania [START_REF] Wang | Preparation of titania particles utilizing the insoluble phase interface in a microchannel reactor[END_REF], or CdSe-ZnS [START_REF] Wang | Continuous synthesis of CdSe-ZnS composite nanoparticles in a microfluidic reactor[END_REF] nanoparticles of controlled sizes and morphologies. Nevertheless, several challenges remain to be overcome before microfluidics becomes a truly multipurpose and practical tool. One of the main current difficulties is obtaining real-time, high-performance, quantitative information on processes at the same micrometre scale [START_REF] Huo | Recent Advances on Optical Detection Methods and Techniques for Cell-based Microfluidic Systems[END_REF]. Most lab-on-a-chip (LoC) setups still rely on bulky and expensive benchtop instruments for analyte/product characterization. This limits their attractiveness compared with standard protocols and techniques, notably in real-world applications outside controlled laboratory environments [START_REF] Rodríguez-Ruiz | Photonic Lab-on-a-Chip: Integration of Optical Spectroscopy in Microfluidic Systems[END_REF]. In the quest for compact analytical platforms, different detection and characterization systems have already been integrated in LoC devices, based either on electrochemical [START_REF] Kimmel | Electrochemical sensors and biosensors[END_REF], magnetic [START_REF] Hoshino | Microchip-based immunomagnetic detection of circulating tumor cells[END_REF], or optical [START_REF] Balslev | Lab-on-a-chip with integrated optical transducers[END_REF] transducers. While these devices have already been successful in the characterization of continuous (fluid) phases or effective media [START_REF] Rodríguez-Ruiz | Photonic Lab-on-a-Chip: Integration of Optical Spectroscopy in Microfluidic Systems[END_REF][START_REF] Mark | Microfluidic lab-ona-chip platforms: requirements, characteristics and applications[END_REF][START_REF] Rodríguez-Ruiz | Uranium(VI) On-Chip Microliter Concentration Measurements in a Highly Extended UV-Visible Absorbance Linearity Range[END_REF][START_REF] Rodríguez-Ruiz | A multiple path photonic lab on a chip for parallel protein concentration measurements[END_REF], their development for the characterization of disperse phases (droplets, bubbles, crystals or aggregates) is still in its infancy. Classical approaches based on the temporal coherence of light (Dynamic Light Scattering, DLS) are limited in terms of flow dynamics and temperature control, while those based on angular scattering (Multi-Angle Static Light Scattering, MASLS) are difficult to implement at the microscale and limited to microparticles. Additionally, it is worth noting that both approaches do not provide absolute particle concentrations [START_REF] Chastek | Miniaturized dynamic light scattering instrumentation for use in microfluidic applications[END_REF][START_REF] Chastek | A microfluidic platform for integrated synthesis and dynamic light scattering measurement of block copolymer micelles[END_REF][START_REF] Destremaut | Microfluidics with on-line dynamic light scattering for size measurements[END_REF][START_REF] Sentis | Organic photo sensors for multi-angle light scattering characterization of particle systems[END_REF][START_REF] Gao | Analysis of noisy multi-angle dynamic light scattering data[END_REF]. To address these issues, we report a novel approach to characterize particle size distributions (PSDs) and concentrations of nano-to microparticles in static as well as flowing suspensions. This approach is based on the principle of light extinction spectrometry (LES) [START_REF] Deepak | Forwardscattering corrections for optical extinction measurements in aerosol media. 1: Monodispersions[END_REF][START_REF] Deepak | Forwardscattering corrections for optical extinction measurements in aerosol media. 2: Polydispersions[END_REF][START_REF] Ku | Determination of refractive indices of Mie scatterers from Kramers-Kronig analysis of spectral extinction data[END_REF][START_REF] Ferri | Modified version of the Chahine algorithm to invert spectral extinction data for particle sizing[END_REF][START_REF] Su | Optimization of regularization parameter of inversion in particle sizing using light extinction method[END_REF][START_REF] Horváth | Measuring contact angles of small spherical particles at planar fluid interfaces by Light Extinction[END_REF][START_REF] Ceolato | Aerosol light extinction and backscattering: A review with a lidar perspective[END_REF][START_REF] Onofri | Development of an in situ ITER dust diagnostic based on extinction spectrometry: Dedicated light scattering models[END_REF][START_REF] Barbosa | An introduction to light extinction spectrometry as a diagnostic for dust particle characterisation in dusty plasmas[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF][START_REF] Onofri | On the Optical Characterisation of Nanoparticle and their Aggregates in Plasma Systems[END_REF][START_REF] Thomas | Retrieval of aerosol refractive index from extinction spectra with a damped harmonic-oscillator band model[END_REF][START_REF] Gienger | Extinction spectra of suspensions of microspheres: determination of the spectral refractive index and particle size distribution with nanometer accuracy[END_REF] and a photonic lab-on-a-chip with three scale-probing channels, see Figure 1. After this brief introduction, section 2 summarizes the background and basic requirement of LES. Section 3 presents the testing procedure and experiment set up to evaluate this first prototype on nano-and microparticle calibration standards, while the results are presented and discussed in section 4. Section 5 is a general conclusion with perspectives. I  and P I  , respectively, after its passage through the analysed particulate medium. These spectra are compared via a minimization process to those predicted by light absorption and scattering models of particle extinction (based for example on Lorenz-Mie theory (LMT) for spherical and multilayered particles [START_REF] Onofri | Electromagnetic scattering from a multilayered sphere located in an arbitrary beam[END_REF] or the T-Matrix method for spheroids and aggregates [START_REF] Onofri | Development of an in situ ITER dust diagnostic based on extinction spectrometry: Dedicated light scattering models[END_REF][START_REF] Mackowski | Calculation of the T matrix and the scattering matrix for ensembles of spheres[END_REF]). The basic LES equation for a homogeneous and optically dilute particulate medium with particle concentration, v C , a dimensionless quantity, and volume-normalized PSD, D v is [START_REF] Barbosa | An introduction to light extinction spectrometry as a diagnostic for dust particle characterisation in dusty plasmas[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF][START_REF] Onofri | On the Optical Characterisation of Nanoparticle and their Aggregates in Plasma Systems[END_REF]:

( ) ( ) max min ,, 3 ln / , 2 D th v m D D D LC T Q D v dD   =-  (1)
where ,, mD Q

 
is the extinction efficiency factor of a particle with diameter D and relative refractive index m=m0/ms; m0/ms is the ratio of the refractive indexes of the particle and the solvent at the wavelength in air, ; = ms is the local wavelength; and L is the optical path length. The discretized and algebraic forms of this equation, its variables and the reconstructed transmission are detailed in [START_REF] Barbosa | An introduction to light extinction spectrometry as a diagnostic for dust particle characterisation in dusty plasmas[END_REF]. Eq. 1 is indeed the linearized and generalized form of the Beer-Lambert equation which, for a monodisperse sample of spherical particles, is simply:

( ) ,, exp 3 / 2 . th v m D T LC Q D    =- (2)
Eq. 1 is also a Fredholm equation of the first kind, such that LES is an inverse method [START_REF] Su | Optimization of regularization parameter of inversion in particle sizing using light extinction method[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF]. Controlling the optical path length is crucial because as shown by Eq. ( 2), the dynamic range and sensitivity of LES are exponentially dependent on this parameter. The harmful effects of multiple scattering can also be detected, to some extend, by comparing the transmissions obtained with different path lengths. The difference between the measured and theoretical transmittances ( exp T  and th T  ) can be minimized by least squares, 2 exp th TT to determine the PSD and, afterwards, the particle concentration by volume (or number). If the minimization is performed by fixing certain parameters, this typically only yields the moments of the PSD (e.g., the standard deviation and mean value for a log-normal distribution) and possibly its boundaries. In contrast, full regularization methods (e.g., Phillips-Twomey or Tikhonov) allow the real shape of the distribution to be retrieved provided the data are not too noisy [START_REF] Su | Optimization of regularization parameter of inversion in particle sizing using light extinction method[END_REF][START_REF] Onofri | Development of an in situ ITER dust diagnostic based on extinction spectrometry: Dedicated light scattering models[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF][START_REF] Onofri | In-situ characterization of dust mobilized by laser cleaning methods and loss of vacuum accident[END_REF]. Another key assumption of LES is that the amount of scattered light detected (scattered by the sample or any component of the optical setup) is negligible [START_REF] Deepak | Forwardscattering corrections for optical extinction measurements in aerosol media. 1: Monodispersions[END_REF][START_REF] Barbosa | An introduction to light extinction spectrometry as a diagnostic for dust particle characterisation in dusty plasmas[END_REF]. In practice, this sets upper limits on the size of the particles and the optical density of the medium. Inelastic scattering processes [START_REF] Mitri | A compact optical sensor for explosive detection based on NIR luminescent quantum dots[END_REF] and polarization effects [START_REF] Mitri | Optical radiation force circular dichroism spectroscopy[END_REF][START_REF] Onofri | On the size and morphological characterization of needle-shaped TiO2 nanoparticles in suspension[END_REF] are also neglected in the standard working principles of LES. Knowledge of the composition and morphology of the particles are also required, even if both can be inferred in some situations [START_REF] Ku | Determination of refractive indices of Mie scatterers from Kramers-Kronig analysis of spectral extinction data[END_REF][START_REF] Onofri | Development of an in situ ITER dust diagnostic based on extinction spectrometry: Dedicated light scattering models[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF][START_REF] Thomas | Retrieval of aerosol refractive index from extinction spectra with a damped harmonic-oscillator band model[END_REF][START_REF] Gienger | Extinction spectra of suspensions of microspheres: determination of the spectral refractive index and particle size distribution with nanometer accuracy[END_REF]. Although LES is mainly used to characterize particulate media in large facilities (e.g. nanocrystallite nucleation and growth in plasma [START_REF] Barbosa | An introduction to light extinction spectrometry as a diagnostic for dust particle characterisation in dusty plasmas[END_REF][START_REF] Onofri | On the Optical Characterisation of Nanoparticle and their Aggregates in Plasma Systems[END_REF], fractal aggregation in aerosols or deposited on an interface [START_REF] Ceolato | Aerosol light extinction and backscattering: A review with a lidar perspective[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF]), it has two considerable advantages for the microscale characterization of nano-to microsuspensions. First, its detection scheme is rather simple and fast, and its sensitivity can be controlled via the optical path length. Second, and crucially for many processes and studies, LES provides absolute measurements of particle concentrations, in stationary as well as flowing or transient suspensions.

Experimental setup and procedure

We designed a glass LoC with three sensing microchannels of different optical path lengths (L = 1.5, 3.5 and 15 mm respectively), with width 650 µm and depth 250 µm, see Figure 1 and Figure 2). Glass was chosen, over PDMS for instance [START_REF] Rodríguez-Ruiz | Photonic Lab-on-a-Chip: Integration of Optical Spectroscopy in Microfluidic Systems[END_REF], because of its optical and mechanical properties regarding, notably, chemical exposure and irradiation [START_REF] Mattio | Photonic Labon-a-Chip analytical systems for nuclear applications: optical performance and UV-Vis-IR material characterization after chemical exposure and gamma irradiation[END_REF]. This monolithically integrated device was manufactured by FEMTOPRINT® following our specifications, with an innovative femtosecond laser pulses micromachining technology for ultra-fast micromolding of fused silica. With a high repeatability and alignment precision (< 1µm) and a low arithmetical mean deviation of the assessed profile (< 100nm), a surface accuracy of better than  is obtained after additional chemical etching. The glass LoC includes six self-aligned channels for optical fibres and six self-aligned pairs of 2D microlenses (with an effective focal length of 925 µm, optimized with a ray tracing procedure introduced in a previous work [START_REF] Rodríguez-Ruiz | A multiple path photonic lab on a chip for parallel protein concentration measurements[END_REF]) for beam collimation and detection angle control. The channels are all 650 µm wide and 250 µm deep to limit the amount of suspension required for the analyses. The optical channels end with optical grade parallel windows. Light is transmitted to the LoC and the spectrometers along solarization-resistant fibres (200 µm core) with a low numerical aperture (NA=0.22).

The three channels are lighted simultaneously by splitting the output of a fibre-optic balanced deuterium-halogen light source (A AvaLight-DH-S-BAL from Avantes, not shown for the sake of clarity). The spectra are recorded simultaneously with three integrated Czerny-Turner spectrometers equipped with high resolution and high sensitivity linear CCDs (AvaSpec-ULS4096CL-EVO from Avantes). As is commonly done, the signal-to-noise ratio (SNR) of the measured transmittance was improved by reducing the CCD exposure time as much as possible (to 11-17 ms depending on the optical path) and averaging 10 successive spectra, giving a measurement rate of nearly 6 per second. For the sake of simplicity, the potential of LES for LoC microfluidics was evaluated using six monodisperse particle standards provided by Thermo Scientific with NIST certificates based on SEM analyses of dry samples. The six standards are aqueous suspensions of polystyrene nano-and microparticles with mean diameters of 30 nm, 50 nm, 80 nm, 0.20 µm and 0.40 µm, and silica (SiO2) microparticles with mean diameter 0.50 µm, with nominal concentrations in mass ranging from 0.1 to 1.0 wt%. Polystyrene suspensions with a maximum concentration range of 1, 5, 10, 50, 100, 150, 250, 400, 500, 750 and 1000 ppm were then prepared by successive dilutions with Type I ultrapure water obtained by distillation, deionization and reverse osmosis (Fisher Scientific Accu100). Concentrations of silica suspensions were limited to 50, 100, 250 and 500 ppm. Because of the high concentration of the initial suspensions and the successive dilutions, the estimated relative error on the concentration of the suspensions ranged from 3.5% for the more concentrated suspensions (above 50 ppm) up to 23 % for the 1 ppm suspensions. According to the manufacturer of the particle standards, the standard deviation at 2- of the PSDs is less than 3 nm for the polystyrene and 11 nm for the SiO2 beads. The monodispersity of the samples was confirmed by benchtop DLS (Litesizer from Anton Paar) analyses (data not shown). Typically, each spectrophotometric acquisition consisted in recording spectra of the background ( B I  , light off), of the reference obtained with the solvent ( S I  , here water, light on) and of the particle sample ( P I  , light on), such that the experimental transmittance was calculated as

( ) ( ) exp /. P B S B T I I I I      = - - (3)
To this end, the LoC was filled alternately with two computer-controlled syringes, one for the solvent and on for the analysed particle sample, after sonication. This procedure was repeated to obtain several transmittance signals for each suspension and channel. Spectral transmittances were analysed in a reduced spectral range (300-600 nm, out of a full spectrometer range of 195-1110 nm). Below 300nm, it was to avoid the effects of the unspecified mixture of surfactants and/or bases used as stabilizing additives and, above 600 nm, to prevent the detection of residual contributions from higher orders of the diffraction gratings. To account for the limited spectral resolution of the spectrometers, the theoretical transmittances th T  were convoluted (symbol ) as ,

th th T T R    → (4
) with the spectral-impulse-response R  of the spectrometers, before performing the minimization process. This spectral-impulse-response R  was estimated by analysing several laser outputs, take the form of Gaussian peaks whose standard deviations increase almost linearly from 3.6 to 4.1nm between 405 and 633 nm (extrapolated down to 300nm).

The data were analysed using LMT to calculate the kernel of Eq. 1, followed by least squares minimization assuming a normal PSD defined by its mean and standard deviation (i.e., parameters of the two-parameter model, as detailed in previous articles [START_REF] Barbosa | An introduction to light extinction spectrometry as a diagnostic for dust particle characterisation in dusty plasmas[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF]). The boundaries of the PSD, Dmin and Dmax, were imposed at 1% of the mode [START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF]. For the refractive index dispersion of the materials (polystyrene, silica and water), we used relevant databases and softwares [START_REF] Palik | Handbook of Optical Constants of Solids[END_REF][START_REF]Software for the Design and Manufacture of Optical Thin Film Coatings[END_REF][START_REF] Holmgren | X Steam, Thermodynamic properties of water and steam -Water properties A complete IAPWS IF-97 standard[END_REF]. The minimization process was performed iteratively for mean sizes and standard deviations varied from 1 to 1000 nm and 1 to 50 nm (i.e., ranges of the two-parameter model), respectively, in 1 nm steps (for more details, see Ref. [START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF]). Several tests were used to reject transmittance signals with features that were (i) too weak ( exp T  < 0.05, with a high risk of collecting scattered light) or (ii) too strong ( exp T  > 0.995, potentially too noisy), or (iii) with an excessive high-frequency noise level (for previous reasons plus some effects of contaminants), or (iv) giving a reconstructed transmittance with a too high residual deviation (>3%) [START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF]. The validation rate of the testing procedure ranged from 0 to 80%, depending on the channel, the size and concentration considered, with a mean validation rate for all samples of 20%.

Results and discussion

Typical raw signals (reference, sample signal and background) are shown in Figure 3 (a), while Figure 3 (b) and Figure 3 (c) compare the experimental transmissions with those reconstructed for increasing channel path lengths and particle concentrations respectively. Figure 4 allows estimating the global resolution and dynamic range of all size measurements obtained with the device. The standard deviations of individual PSDs are not shown because they were below 1-4 nm at 1-. Each statistic is obtained from 10 to 55 measurements depending on the validation rate. Suspensions with particle sizes of 30, 50 and 400nm were measured on static samples, and the others, on flowing samples (50 µL/ min). Looking at Figure 4, we found that the diameters measured with the photonic LoC are generally in good agreement with the reference values. The spread of the data, reflected by the length of the error bars (rmsd on all measured diameters for a given nominal diameter), is larger for the smallest particles. The fact is that smaller particles have lower extinction coefficients and thus higher and noisier transmittance. This is highlighted in Figure 3 which shows the transmittance signals for the 80 nm polystyrene particles at 5 and 250 ppm measured in the three channels. A part of the dispersion of the data may also be due to adventitious contamination of the samples, which were prepared and analysed in a conventional laboratory rather than in a clean room facility. Similarly, the water used for the dilutions was probably not completely free of nanocontaminants. The increase in the rmsd of the data for the largest diameters is harder to explain, except as being due to the detection of scattered light. Figure 5 compares the volume concentrations measured for all samples (validated signals only) with the expected values (i.e. deduced from manufacturer information and the dilution process). The measured concentrations are consistent with the expected values, despite a certain amount of dispersion that can be attributed to the same processes pointed out for the results in Figure 4. Each channel covers about two orders of magnitude in terms of suspension concentration. Such that in combination, the three channels can be used to analyse suspensions with concentration changes of up to three orders of magnitude, which is already promising for numerous applications. However, this dynamic range is not obtained for all particle sizes, depending on the SNR of the signals which highly depends on the optical path length but also on the quality of the suspension considered.

Conclusion and perspectives

We introduce and evaluate a new LoC approach to measure particle sizes (from 30 nm to 0.5 µm) and volume concentrations (from 1 to 1000 ppm) of optically dilute colloidal suspensions. This simple and effective spectrophotometric approach is applicable to both stationary suspensions and dynamic particulate flows, with potential applications in various fields of research and engineering. The design of the LoC, and more especially the detection channels, could be further optimized to fit the optical properties, size and concentration of the particles (i.e. the optical thickness of the samples) and to better control the SNR of the transmittance signals. Increasing substantially the optical path length [START_REF] Singh | Towards on-chip mid infrared photonic aerosol spectroscopy[END_REF] and integrating a microfluidiccontrolled optical router [START_REF] Dietvorst | Microfluidic-controlled optical router for lab on a chip[END_REF] for multiplexed analyses with a single spectrometer would also be a benefit for practical applications. The consideration of particles with more complex shapes and compositions [START_REF] Ceolato | Aerosol light extinction and backscattering: A review with a lidar perspective[END_REF][START_REF] Onofri | Sizing highlyordered buckyball-shaped aggregates of colloidal nanoparticles by light extinction spectroscopy[END_REF][START_REF] Thomas | Retrieval of aerosol refractive index from extinction spectra with a damped harmonic-oscillator band model[END_REF][START_REF] Gienger | Extinction spectra of suspensions of microspheres: determination of the spectral refractive index and particle size distribution with nanometer accuracy[END_REF], as well as multiple scattering or polarization effects [START_REF] Mitri | Optical radiation force circular dichroism spectroscopy[END_REF][START_REF] Onofri | On the size and morphological characterization of needle-shaped TiO2 nanoparticles in suspension[END_REF], is also part of the perspectives of the present work.

Figure 1

 1 Figure 1 Schematic diagram of the photonic LoC, with three channel paths and light extinction capabilities over a broadband spectral range, for characterizing the particle size distribution and absolute concentration of colloidal suspensions.

  collimated light beam with incident and final intensities 0

Figure 2 (

 2 Figure 2 (a-f) Lab-on-a-chip: (1) Glass LoC in its (2) housing (hatch open); (3) computer controlled syringe pumps (x2) feeding the LoC via its (4) inlet and (5) outlet; suspension (4) inlet and (5) outlet; (6) incoming and (7) outgoing optical fibres; (8) the CCD spectrometers (x3); (c-e) Expanded views of (9) the self-alignment channels (x6) for optical fibres; (10) the 2D microlense assemblies (x6) for collimating and controlling optical fibre outputs and inputs; (11) the optical channels (x3) with path lengths L=1.5, 3.5 and 15 mm, width 650 µm and depth 250 µm; (12) the ends of the channels with parallel optical-grade windows; and (13) air channels to avoid probing channels cross-talking during simultaneous acquisition. The scratches visible in part (b) are those on the breadboard underneath the LoC. False colours were added in parts (b, d, e) to highlight the paths of the fluids (light blue) and probing beams (multicoloured). (f) Setup with (2) LoC housing cover removed.

Figure 3

 3 Figure 3 LES (a) raw signals; (b, c) experimental and reconstructed transmissions for suspensions of polystyrene nanoparticles of 80nm in diameters. (a) Typical raw signals for a concentration of 250 ppm and the 3.5 mm probing length. (b) Evolution of the transmissions for increasing channel path lengths when the particle concentration is fixed to 250 ppm. (c) Evolution of the transmissions for increasing particle concentrations when the optical path length is fixed to 15mm. The parameters estimation is performed on the spectral 300-600 nm (highlighted in grey in (a)).

Figure 4

 4 Figure 4 Mean diameter measured with all channels and for all prepared suspensions versus the mean diameter estimated by SEM analyses on the source suspensions. The error bars show the spread (rmsd) of the measured mean diameters. Static samples 30, 50 and 400nm, flowing samples: 80 and 500nm.

Figure 5

 5 Figure 5 Volume concentration of aqueous suspensions of polystyrene (PS) and Silica particles versus the concentration calculated from the dilution process and the manufacturer-specified concentration of the starting suspension. Static samples: 30, 50 and 400nm, flowing samples: 80 and 500nm.

Fundings.

This research was supported by the Energy Division of the CEA (DES-DPE-CYN/PRATA) and was partially funded by the French National Research Agency (ANR) under grant numbers ANR-13-BS09-0008-02, Labex MEC (ANR-11-LABX-0092), and A*MIDEX (ANR-11-IDEX-0001-0).

Data availability.

Disclosures.

The authors declare no conflicts of interest.

The LES analysis software and the data that support the findings of this study are available from the authors upon reasonable request.


B Masenelli

E Tutis

M N Bussac

L Zuppiroli

Numerical model for injection and transport in multilayers OLEDs

Keywords: organic light-emitting diode, numerical model, transport, injection, recombination. -0.7 -0.6 -0

We present a recently developed numerical code for OLED simulation. This code contains a detailed description of contacts, charge transport and recombination. Its efficiency is briefly shown through examples of single-layer and bilayer devices and discussed in more detail through the investigation of the action of a LiF thin film on injection. In particular, we show how the code can help to discriminate between several possible explanations for this phenomenon.

Introduction

Tremendous progress has been achieved concerning injection, transport and recombination of charge densities in OLEDs. Novel devices, using spacers, dipolar layers, new material multilayer stacks, have proved efficient in decreasing the voltage onset as well as increasing the external quantum efficiency. However, the reasons for this efficiency are not fully understood yet, and the behavior of typical single-layer and multilayer systems is not utterly clear. In order to clarify the fundamental mechanisms at stakes as well as to predict typical device behavior, a model is required. Several models, dealing with single layer or multilayer devices, have been proposed [1,2]. Despite their efficiency, they often describe injection in a restricted manner, which may reveal problematic to understand phenomena such as the role of insulating monolayers on injection.

Model presentation

In order to fully account for injection, transport and recombination in OLEDs, we have developed a numerical model. The model first takes advantage of the homogeneity of molecular organic layers. Hence, all significant quantities are calculated at each node of a one-dimensional (1D) array. The distance between two nodes is of physical relevance since it represents the actual distance between two molecules. It is therefore linked to the material density. At each node, energy levels and charge densities for both electrons and holes are to be calculated. The energy of the lowest unoccupied molecular orbital (LUMO) writes:

        m x m x x x , ~E E E E IF 0 (1)
where E 0 is the bare LUMO energy, E IF is the image force contribution due to the presence of metal electrodes, and E(x m , x) is the contribution of charge at node x m . A similar equation can be written for highest occupied molecular orbital (HOMO) levels. It is noteworthy that discreteness effects due to charge localization on molecules are taken into account, especially where they are most important, namely at the nodes near the electrodes (the description of charge localization effects is beyond the scope of this paper and will be published elsewhere [3]). Concerning the charge density evolution, three contributions are to be distinguished (cf. fig. 1): the variation due to injection from electrodes, that due to transport within the organic layers, and the last one due to recombination. Focusing on electrons, one can write:

ion recombinat transport injection                      dt d dt d dt d dt d n n n n (2)
In our framework, injection is treated as a tunnelling current between electrode and several nodes. This accounts for long range tunneling. Transport is modelled as hopping between nearest-neighbor sites in local thermodynamic equilibrium. The hopping frequency is assumed to be constant (at typical electric field) and deduced from the macroscopic mobility value. Recombination is treated in the framework of Langevin theory, as being the result of a competition between the separating drift force and the attractive Coulomb force. As one can see, charge densities and energy levels are coupled in a non-explicit way. Thus, equations ( 1) and ( 2) must be solved self-consistently. Our code fulfills this criterion using a time-dependent procedure.

Results

Simulation of single-layer and bilayer devices

First of all, the model is found to simulate single-layer devices accurately. Among others, we simulated some Ca/Alq 3 /Ca devices fabricated in our laboratory [3] as well as devices made by the Los Alamos group [4]. For these simulations, only a small number of parameters (tunneling integral, position of the first node) had to be determined. This was made possible because available experimental data were numerous enough to provide a strong constraint on these parameters. Our

Nearest-neighbor hopping

Tunneling current recombination Actual LUMO level electrode model also provided an explanation of the pinning of the energy barrier at the metal/organic interface observed by Campbell et al. [5].

In a second step, we used the code to investigate bilayer devices. We thus fabricated several bilayer devices made of an aluminum cathode and a O 2 plasma-treated ITO anode, surrounding an electron transport layer (ETL) of Zn(q-2-COOEt) 2 and an emitting and hole transport layer (HTL) of a CN derivative of bicarbazyl [6]. The thickness of the ETL was varied from 15 nm to 60 nm while keeping the HTL thickness constant. This provided a set of experimental I-V and luminance-V curves that enabled the determination of the few unknown parameters. The code could simulate the experimental data successfully and revealed space charge effects due to hole accumulation in the ETL because of their low mobility in this material [3].

Simulation of the action of a LiF thin film on electron injection.

The simulation of both single-layer and bilayer devices revealed the dramatic influence of the injection interface on the device behaviour. We thus focused on the description on such interfaces, and particularly, on optimized injection interfaces. It is known that adding a thin film of insulating material such as LiF, MgO or self-assembled monolayer can enhance electron injection. Among these materials, the most used is LiF. We thus concentrated on determining its action. Two mechanisms have been proposed to explain its favorable effect on injection. The first one relies on UPS measurements. These measurements showed that adding a small LiF layer on a metal (most of the time Al) decreases its work function [7,8,[START_REF] Mori | SPIE conf. Proceedings[END_REF]. Reported values for this reduction span from 0.5eV to 1.5eV depending on the LiF thickness and on the group. The proposed explanation is the creation of a dipole within the LiF layer. This dipole would lower the metal work function. In the second hypothesis, the insulating film reduces the undesirable effect of the image force. Indeed, the image force induces the existence of a zone where the local electric field is opposed to the external one. This leads to a local current which may flow back to the electrode. By moving the first organic molecule away from the metal surface thanks to the LiF film, the width of this zone as well as the amplitude of the negative local field is reduced. Consequently, the total current coming from the electrode is enhanced. by 1 nm. One can see that it does enhance the injection. But the enhancement is not large enough to reproduce the experimental one obtained with the addition of 0.2 nm (circle symbol) and 0.5 nm (square symbol) of LiF. Hence, the reduction of the backflow current alone cannot explain the action of LiF. We thus investigated the possibility of a dipole effect. We deposited 55nm thick Al layers capped with 0, 0.5, 0.6, 1.5 and 3 nm thick LiF layers. After deposition, the samples were moved from the deposition chamber to a nearby glove box for Kelvin probe measurements. Consequently, we reduced the energy barrier between (Al/LiF) and Alq 3 in the simulation, which was initially of 0.65eV. The simulation of experimental devices with 2 Å and 5 Å thick LiF layers set the barrier height to 0.25 eV (which is in fair accordance with both Kelvin probe measurements and UPS values). The only difference between these two simulations was the tunneling integral value (i.e. the value of the overlapping wave functions of the electron in the metal and on the first molecular site). The tunneling integral value was decreased by a factor 100 for the device having a 5 Å thick LiF layer. This accounts for the insulating action of LiF. The good agreement between the simulations and the experimental data shows that the dipole effect is necessary to explain the enhancement of injection induced by the presence of LiF. However, this dipole effect cannot always be invoked. In particular, for self-assembled organic monolayers which exhibit no dipole or even a dipole opposed to injection [11]. This type of device still needs to be investigated and modelled to fully account for the injection enhancement.

Figure 1 :

 1 Figure 1: energy scheme representing the three mechanisms contributing to charge density variations: long range tunnel injection, nearest-neighbor hopping and recombination

Figure 2 :

 2 Figure 2: simulation of the effect of LiF on injection

  The work function of Al capped layers versus LiF thickness is reproduced in fig.3. It is clear that by adding a LiF layer, the work function of Al is lowered typically by 0.35eV for a 6 Å thick Lif layer, 0.5eV for a 30 Å thick LiF layer. Figure 3: decrease of the Al work function versus LiF thickness (Kelvin probe measurements)