Luce Barbat Du Closel

Nathalie Bonello-Palot

| Yann Péréon

| Andoni Echaniz-Laguna

| Jean

Philippe Camdessanche

Aleksandra Nadaj-Pakleza

Jean-Baptiste Chanson

Simon Frachet

Laurent Magy

Julien Cassereau

Pascal Cintas

Ariane Choumert

Perrine Devic

Sarah Leonard Louis

| Robinson

Gravier Dumonceau

| Emilien Delmont

Emmanuelle Salort-Campana

Françoise Bouhour

Philippe Latour

Tanya Stojkovic

Shahram Attarian
email: shahram.attarian@ap-hm.fr

Luce Barbat

Closel

Jean Philippe Camdessanche

Emilien Delmont

Clinical and electrophysiological characteristics of women with X-linked Charcot-Marie-Tooth disease

Background: X-Linked Charcot-Marie-Tooth disease type 1 (CMTX1) is characterized by gender differences in clinical severity. Women are usually clinically affected later and less severely than men. However, their clinical presentation appears to be heterogenous. Our aim was to extend the phenotypic description in a large series of women with CMTX1.

Methods

: We retrospectively evaluated 263 patients with CMTX1 from 11 French reference centers. Demographic, clinical, and nerve conduction data were collected. The severity was assessed by CMT Examination Score (CMTES) and Overall Neuropathy

Limitations Scale (ONLS) scores. We looked for asymmetrical strength, heterogeneous motor nerve conduction velocity (MNCV), and motor conduction blocks (CB).

INTRODUC TI ON

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of hereditary sensory-motor polyneuropathies characterized by slowly progressive symmetrical distal involvement associated with muscular atrophy. It affects about 1 in 2500 individuals at birth [START_REF] Skre | Genetic and clinical aspects of Charcot-Marie-Tooth's disease[END_REF], and more than 100 genes are involved [START_REF] Rossor | Clinical implications of genetic advances in Charcot-Marie-Tooth disease[END_REF]. Inheritance can be either autosomal dominant, autosomal recessive, or X-linked. CMT is classified according to the mode of inheritance, the causative gene, and the median motor nerve conduction velocity (MNCV) dividing the type of neuropathy into axonal, intermediate, or demyelinating form.

X-Linked inherited neuropathy (CMTX) with a mutation in GJB1 is the second most common subtype of CMT, after CMT1A, accounting for 10%-11% of CMT [START_REF] Fridman | CMT subtypes and disease burden in patients enrolled in the inherited neuropathies consortium natural history study: a cross-sectional analysis[END_REF][START_REF] Murphy | Charcot-Marie-Tooth disease: frequency of genetic subtypes and guidelines for genetic testing[END_REF].

CMTX1 is caused by a mutation in the GJB1 gene [START_REF] Bergoffen | Connexin mutations in X-linked Charcot-Marie-Tooth disease[END_REF], which leads, in most cases, to a loss of normal function of the connexin 32 protein [START_REF] Shy | CMT1X phenotypes represent loss of GJB1 gene function[END_REF]. Cx32, present in the peripheral nervous system, is an intrinsic membrane protein in gap junctions [START_REF] Bruzzone | Connections with connexins: the molecular basis of direct intercellular signaling[END_REF][START_REF] Balice-Gordon | Functional gap junctions in the Schwann cell myelin sheath[END_REF]. Six connexins assemble to form a hemichannel, or a connexon, arranged around a central pore. Two connexons form gap junction channels and facilitate the diffusion of metabolites across the layers of the myelin sheath [START_REF] Bihel | CMTX disorder and CamKinase[END_REF] and play a role in the homeostasis of myelinated axons. Cx32 is also expressed by oligodendrocytes, coupling them to astrocytes [START_REF] Rash | Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons[END_REF].

The disease classically begins in childhood, with difficulty running or repeated ankle sprains. Clinically there is usually a distal motor deficit of the lower and upper limbs, associated with muscle atrophy and bone deformities. In addition to peripheral neuropathy, CMTX1 is also characterized by acute episodic dysfunctions in the central nervous system [START_REF] Yuan | Genetic and phenotypic profile of 112 patients with X-linked Charcot-Marie-Tooth disease type 1[END_REF][START_REF] Al-Mateen | The central nervous system phenotype of X-linked Charcot-Marie-Tooth disease: a transient disorder of children and young adults[END_REF][START_REF] Hardy | X-linked Charcot-Marie-Tooth disease presenting with stuttering stroke-like symptoms[END_REF][START_REF] Liang | Recurrent episodes of reversible posterior leukoencephalopathy in three Chinese families with GJB1 mutations in X-linked Charcot-Marie-Tooth type 1 disease: cases report[END_REF][START_REF] Tziakouri | Transient, recurrent central nervous system clinical manifestations of X-linked Charcot-Marie-Tooth disease presenting with very long latency periods between episodes: is prolonged sun exposure a provoking factor?[END_REF][START_REF] Stancanelli | Unusual features of central nervous system involvement in CMTX associated with a novel mutation of GJB1 gene[END_REF][START_REF] Vivekanandam | CNS phenotype in X linked Charcot-Marie-Tooth disease[END_REF][START_REF] Yang | Expansion of the phenotypic spectrum of X-linked Charcot-Marie-Tooth (CMT) disease[END_REF]. Nerve conduction studies show in male patients intermediate slowing of conduction and distal axonal loss [START_REF] Nicholson | Intermediate nerve conduction velocities define X-linked Charcot-Marie-Tooth neuropathy families[END_REF].

Previous studies have shown that men and women with CMTX1 exhibit phenotypic differences, with women usually being clinically affected later and less severely than men, with less slowed MNCV and axonal loss [START_REF] Yuan | Genetic and phenotypic profile of 112 patients with X-linked Charcot-Marie-Tooth disease type 1[END_REF][START_REF] Panosyan | Cross-sectional analysis of a large cohort with X-linked Charcot-Marie-Tooth disease (CMTX1)[END_REF][START_REF] Birouk | X-linked Charcot-Marie-Tooth disease with connexin 32 mutations: clinical and electrophysiologic study[END_REF][START_REF] Kleopa | How do mutations in GJB1 cause X-linked Charcot-Marie-Tooth disease?[END_REF][START_REF] Dubourg | Clinical, electrophysiological and molecular genetic characteristics of 93 patients with X-linked Charcot-Marie-Tooth disease[END_REF]. However, the phenotype of women with CMTX1 appears to be very heterogeneous. Siskind et al. [START_REF] Siskind | Phenotype expression in women with CMT1X[END_REF] suggested that there were various phenotypic subgroups in women, but this hypothesis has not been verified.

We aimed to extend the phenotypic description of women with CMTX1, delineate different subgroups of patients, and report the atypical clinical presentation and motor conduction results compared to men and other types of CMT patients. Patients were included if they had a mutation in the GJB1 gene or neuropathy with a first-degree family history of a GJB1 mutation.

PATIENTS AND ME THODS

Subjects and clinical assessment

Variants not observed in public databases, including ClinVar and the Hereditary Peripheral Neuropathy Mutation Database, were considered new variants. Data concerning sex, age, age at disease onset, mode of onset, clinical examination, presence of walking aids (orthosis, canes, crutches, wheelchair), history of stroke-like syndrome or orthopedic surgery of the lower limbs, and treatment for neuropathic pain (gabapentin, pregabaline, duloxetine, or transcutaneous electrical neurostimulation) were collected. We considered that patients were clinically asymmetrical if at least two points of difference were in the Medical Research Council (MRC) scale testing between each side for the same muscle. We also looked for associated signs, such as scoliosis, tremor, deafness, optic nerve atrophy, or cognitive disorders. We noted deafness if an audiogram was performed and

Results:

The study included 137 women and 126 men from 151 families. Women had significantly more asymmetric motor deficits and MNCV than men. Women with an age of onset after 19 years were milder. Two groups of women were identified after 48 years of age. The first group represented 55%, with women progressing as severely as men, however, with a later onset age. The second group had mild or no symptoms. Some 39% of women had motor CB. Four women received intravenous immunoglobulin before being diagnosed with CMTX1.

Conclusions:

We identified two subgroups of women with CMTX1 who were over 48 years of age. Additionally, we have demonstrated that women with CMTX can exhibit an atypical clinical presentation, which may result in misdiagnosis. Therefore, in women presenting with chronic neuropathy, the presence of clinical asymmetry, heterogeneous MNCV, and/or motor CB should raise suspicion for X-linked CMT, particularly CMTX1, and be included in the differential diagnosis.

K E Y W O R D S

Charcot-Marie-Tooth, clinical trial, CMTX, connexine 32, hereditary peripheral neuropathy optic atrophy if the patient had undergone visual evoked potential testing. Similarly, cognitive disorders were only recorded if they had been objectively assessed through neuropsychological testing.

The CMT Examination Score version 2 (CMTESv2), a standardized and validated composite score for CMT patients assessing clinical and functional severity, was established for each patient [START_REF] Shy | Reliability and validity of the CMT neuropathy score as a measure of disability[END_REF]. This score was calculated during the consultation or was retrospectively calculated for patients for whom the relevant data were available. This scale comprises seven items, each scored from 0 to 4, with a maximum score of 28. The Overall Neuropathy Limitations Scale (ONLS) score [START_REF] Graham | A modified peripheral neuropathy scale: the overall neuropathy limitations scale[END_REF] was also calculated for each patient. On this scale, impairment in upper limbs is scored from 0 to 5, and lower limbs from 0 to 7. The total maximum ONLS score is 12. For both CMTES and ONLS, a higher score indicates a more severe phenotype. Phenotypes were classified according to their CMTES score as mild (CMTES 0-7), moderate (CMTES 8-16), and severe (CMTES >16) [START_REF] Murphy | Reliability of the CMT neuropathy score (second version) in Charcot-Marie-Tooth disease[END_REF].

Motor nerve conduction study data were collected, including MNCV, compound muscle action potential (CMAP), distal motor latency, and F wave latency on the ulnar and median nerves. We also looked for the presence of conduction blocks (CB) and temporal dispersion in the forearm. Motor CB was considered if there was a reduction in amplitude of >30% between the proximal and the distal negative CMAP peak (excluding the tibial nerve, the fibular nerve at the fibula, and the ulnar nerve at the elbow) if the distal CMAP was >1 mV [START_REF] Van Den Bergh | European Academy of Neurology/Peripheral Nerve Society guideline on diagnosis and treatment of chronic inflammatory demyelinating polyradiculoneuropathy: report of a joint Task Force-Second revision[END_REF]. Motor CB was classified as severe if this reduction in amplitude was >50%. Temporal dispersion was defined as an increase in the duration of >30% between the proximal and distal negative CMAP peak [START_REF] Van Den Bergh | European Academy of Neurology/Peripheral Nerve Society guideline on diagnosis and treatment of chronic inflammatory demyelinating polyradiculoneuropathy: report of a joint Task Force-Second revision[END_REF]. Patients with median MNCV below 25 m/s were categorized as having 'demyelinating' neuropathy, those with more than 38 m/s had 'axonal' neuropathy, and those between 25 and 38 m/s were classified as having 'intermediate' neuropathy [START_REF] Harding | The clinical features of hereditary motor and sensory neuropathy types I and II[END_REF].

We compared the right and left MNCV in both ulnar and median nerves and considered MNCV to be asymmetric if there was more than a 10 m/s difference between the right and left nerves. This threshold was chosen because 10 m/s is more than two standard deviations of the mean MNCV [START_REF] Nandedkar | Revising nerve conduction reference limits[END_REF].

Statistical analysis

For our analyses, we artificially subdivided patients according to their median current age (Figure 3) or median age of onset (Figure 5). We used the medians because the distributions were not normal.

RE SULTS

General population

Two hundred and sixty-three patients (137 women and 126 men), from 151 families, followed for CMTX1 were included in the study.

We identified 84 different GJB1 mutations; among these, 27 were new mutations (Figure 1). Clinical characteristics are shown in Table 1. The median age at clinical assessment was 45 years (IQR 32-58; range: 18-84 years), 48 years for women (IQR 33-60; range: 18-83 years), and 40 years for men (IQR 30-56; range: 18-84 years) and did not differ significantly between the genders (p = 0.060). Age at disease onset was 19 (IQR 10-32) years for women versus 12 (IQR 7-15) years for men (p < 0.001).

CMTES score was significantly lower in women (mean score 7.5 [±5.3]) than in men (mean score 11.0 [±4.5], p < 0.001). This significance was reinforced after adjustment for patient age (p < 0.001).

The gender difference was statistically significant for each item of the CMTES score, as shown in Figure 2, except for the first item relating to subjective sensory symptoms. Some 19.2% (23/120) women followed a treatment for neuropathic pain, compared with 12.6% (14/111) of men; and the difference was not significant. The phenotype (mild, moderate, or severe) differed significantly between the genders. Seventy-one of 137 (51.8%) women had a mild phenotype, whereas 87/126 (69%) men had a moderate phenotype. The ONLS score, correlated to the CMTES score, showed a similar trend in which men were more severely affected than women (mean ONLS score was 2.3 for women and 3.5 for men, p < 0.001). The difference was also significant when the upper and lower limbs ONLS scores were compared separately between genders. Ten percent (13/127) of women and 16% (19/118) of men used canes or walkers. A wheelchair was used by 3% (3/118) of men but not by women. Twenty-five percent (32/127) of women and 36% (42/118) of men had moderate or severe difficulty in buttoning. Four percent (5/127) of women and 16% (19/118) of men were unable to cut most foods.

Fifteen percent (18/120) of women had asymmetrical strength, compared with only 3.6% (4/111) of men (p = 0.003). Associated signs (scoliosis, tremors, deafness, optic nerve atrophy, stroke-like syndrome, cognitive disorders) did not differ between genders.

Five women and three men received an inaccurate diagnosis before a GJB1 mutation was detected. Of these patients, four women and two men were initially diagnosed with CIDP, leading to the prescription of immunoglobulin or steroids. One woman was diagnosed with traumatic lower plexus injury associated with bilateral entrapment syndrome, and one man was initially diagnosed with spinal muscular atrophy type IV.

Women's phenotype

Women were more often asymptomatic at diagnosis than men; 17.2% (21/130) and 0.8% (1/124), respectively (p < 0.001). When women were symptomatic, symptoms at disease onset were not different from those of men ( We identified two groups among patients over the age of 48 years (Figure 3). Of the women aged over 48 years, 45% had a mild phenotype (CMTES 0-7), while 55% had a moderate (CMTES 8-16) or severe (CMTES >16) phenotype. We chose 48 years as the cutoff since it represents the median age of the women included in this study. The group of 30 minimally symptomatic women represents 45% of women aged over 48 years (30/67). For this subgroup, the CMTES score was 3.2, and the age of onset was 40 years. For the remaining 37 women aged over 48 years (37/67 patients, 55%), with moderate or severe phenotype the CMTES score was 12.7, and the age of onset was 19 years. For men over 48 years, CMTES was 12.9, and the age of onset was 13 years. For the more severely affected women, the CMTES score did not differ significantly from that of men over 48 years. However, their age of onset was later and differed significantly (p < 0.001).

Women who developed the disease before the age of 19 years had a CMTES score of 9.5, while those who developed the disease after the age of 19 years had a score of 5.6 (Figure 4a, p < 0.0001). We chose 19 years as the cutoff since it represents the median age of the women in this study's disease onset. The ONLS score was 2.8 for women who developed the disease before the age of 19years and 1.6 for women who developed the disease after the age of 19 years (Figure 4b, p < 0.001). CMTES score correlated significantly with age for women (p < 0.001), as shown in Figure 3. The CMTES score correlated significantly with the age of symptom onset (Figure 5, p < 0.001).

Among women with CMTX1, the most commonly associated symptoms were tremors (20/ We identified three women with the stroke-like syndrome. The symptoms included a right hemiparesis associated with dysarthria for 20 min, left upper limb hemiparesis for 1 h, and flaccid tetraparesis for 10 min, recurrent. Two patients had brain MRIs showing T2-weighted images with white matter hyperintensities without gadolinium enhancement. Unfortunately, we do not have access to the MRI images. The third patient had a normal MRI. There was no mention of triggering factors for any of the three patients.

We found that women who presented with a missense variant in the lipid bilayer domain had a more severe clinical presentation and earlier onset of disease compared to those with variants located in the intra-and extracellular domains (respectively, CMTES 9.1 and 6.6, p = 0.009; ONLS 2.9 and 1.8, p = 0.001; the age of onset 17.3 and 26.3 years, p = 0.001). However, there was no significant association between gene domain localization and asymmetry.

Nerve conduction study

We collected 128 motor nerve conduction study data: 66 for women and 62 for men (

DISCUSS ION

This study describes the phenotype of 137 women with CMTX1.

Among them, we identified two subgroups of women over the age of 48 years. The first subgroup, representing 55% of the cohort,

F I G U R E 2

TA B L E 2

Symptoms present at the onset of the disease by gender.

had a disease progression as severe as that observed in men but with a later onset age. The second subgroup, comprising the remaining 45% of women, had either mild or no symptoms. Notably, women who developed the disease after the age of 19 years had a significantly milder phenotype than those with an earlier onset age. Moreover, we observed that women had significantly more asymmetric motor deficits and heterogeneous MNCV than men.

Additionally, 39% of women in our cohort had motor conduction blocks.

General population: women's phenotype compared to men

Our results regarding the general phenotypic description of women compared to men are in agreement with previous CMTX1 cohorts [START_REF] Yuan | Genetic and phenotypic profile of 112 patients with X-linked Charcot-Marie-Tooth disease type 1[END_REF][START_REF] Panosyan | Cross-sectional analysis of a large cohort with X-linked Charcot-Marie-Tooth disease (CMTX1)[END_REF][START_REF] Dubourg | Clinical, electrophysiological and molecular genetic characteristics of 93 patients with X-linked Charcot-Marie-Tooth disease[END_REF][START_REF] Hong | Clinical characterization and genetic analysis of Korean patients with X-linked Charcot-Marie--Tooth disease type 1[END_REF][START_REF] Lu | Clinical and genetic features of Chinese X-linked Charcot-Marie-Tooth type 1 disease[END_REF][START_REF] Gouvea | New novel mutations in Brazilian families with X-linked Charcot-Marie-Tooth disease[END_REF]. It is noteworthy that due to the large sample size of this study, we were able to identify significant differences in each item of the CMTES score, except for the first item, which assesses neuropathic pain. The results for this item were consistent with the

|

comparable percentage of patients, both men and women, who used analgesic treatment. Consistent with our findings, Panosyan et al. [START_REF] Panosyan | Cross-sectional analysis of a large cohort with X-linked Charcot-Marie-Tooth disease (CMTX1)[END_REF] previously reported similar frequencies of paresthesia and burning feet between men and women. Although this study has a limitation of retrospective calculation of CMTES scores, the results

were consistent with a previous study [START_REF] Yuan | Genetic and phenotypic profile of 112 patients with X-linked Charcot-Marie-Tooth disease type 1[END_REF][START_REF] Panosyan | Cross-sectional analysis of a large cohort with X-linked Charcot-Marie-Tooth disease (CMTX1)[END_REF].

We did not observe significant differences in the signs associated with the disease between men and women. However, women tended to exhibit tremors less frequently (16.3% vs. 23.7%). This difference could be attributed to the less severe neuropathy and lower degree of disability observed in women.

We identified three women with a stroke-like syndrome.

However, in the previous cohort, stroke-like attacks were more frequently reported in men [START_REF] Yuan | Genetic and phenotypic profile of 112 patients with X-linked Charcot-Marie-Tooth disease type 1[END_REF][START_REF] Al-Mateen | The central nervous system phenotype of X-linked Charcot-Marie-Tooth disease: a transient disorder of children and young adults[END_REF][START_REF] Hardy | X-linked Charcot-Marie-Tooth disease presenting with stuttering stroke-like symptoms[END_REF][START_REF] Liang | Recurrent episodes of reversible posterior leukoencephalopathy in three Chinese families with GJB1 mutations in X-linked Charcot-Marie-Tooth type 1 disease: cases report[END_REF][START_REF] Tziakouri | Transient, recurrent central nervous system clinical manifestations of X-linked Charcot-Marie-Tooth disease presenting with very long latency periods between episodes: is prolonged sun exposure a provoking factor?[END_REF][START_REF] Stancanelli | Unusual features of central nervous system involvement in CMTX associated with a novel mutation of GJB1 gene[END_REF] than in women. These central symptoms should prompt a discussion of differential diagnoses [START_REF] Koutsis | X linked Charcot-Marie-Tooth disease and multiple sclerosis: emerging evidence for an association[END_REF][START_REF] Ayrignac | Adult-onset genetic leukoencephalopathies: a MRI pattern-based approach in a comprehensive study of 154 patients[END_REF].

Clinical heterogeneity in women

Women with CMTX1 exhibit a more heterogeneous clinical presentation than men. Specifically, 10% of women in our cohort used canes or crutches, whereas 17.2% were asymptomatic.

In our study, 45% of women over the age of 48 years exhibited a mild phenotype (CMTES 0-7). The remaining female CMTX1 patients over 48 years of age (55%) experienced disease progression as severe as that observed in men, albeit with a later onset of symptoms.

These findings corroborate those reported by Siskind et al. [START_REF] Siskind | Phenotype expression in women with CMT1X[END_REF], who studied 31 women with CMTX1. The largest subgroup in their cohort consisted of approximately 60% of female CMTX1 patients with mild symptoms and stable neuropathy. The second subgroup included women with moderate impairment, which worsened with age, similar to male CMTX1 patients. The third group included three asymptomatic women.

In our cohort, the age of symptom onset was more variable in women than in men. We found a significant linear correlation between the age of symptom onset and the CMTES score in women but not in men. Notably, we demonstrated that patients with a late onset of the disease (after 19 years) exhibited a significantly milder phenotype. We can hypothesize that these women are more likely to belong to the subgroup of women with mild symptoms after the age of 48 years. The heterogeneous phenotype observed among women with CMTX1 could be explained by the X inactivation mechanism.

The inactivation of pathogenic X could potentially account for the subgroup of asymptomatic women, a hypothesis that has yet to be tested [START_REF] Siskind | Phenotype expression in women with CMT1X[END_REF].

Asymmetry in women

In our cohort, we observed that 15% of women exhibited an asymmetric motor deficit compared with only 3.6% of men (p = 0.003).

We excluded patients with other causes of motor deficit from these results. Asymmetry in motor or sensory deficits is atypical in CMT patients, as reported in previous clinical descriptions of CMT1X cohorts [START_REF] Panosyan | Cross-sectional analysis of a large cohort with X-linked Charcot-Marie-Tooth disease (CMTX1)[END_REF][START_REF] Birouk | X-linked Charcot-Marie-Tooth disease with connexin 32 mutations: clinical and electrophysiologic study[END_REF][START_REF] Kleopa | How do mutations in GJB1 cause X-linked Charcot-Marie-Tooth disease?[END_REF][START_REF] Siskind | Phenotype expression in women with CMT1X[END_REF]. However, one study of pediatric CMT patients reported asymmetry in foot alignment and ankle flexibility in 3%-5% of patients, with the asymmetry associated with greater severity of neuropathy according to the CMTNS scale, but without analysis according to the type of CMT [START_REF] Burns | Symmetry of foot alignment and ankle flexibility in paediatric Charcot-Marie-Tooth disease[END_REF]. Another study of patients with CMT1A found pinprick or vibration sensory asymmetry in 23/180 patients (12.8%), but no asymmetry was observed for other items in the CMTES score [START_REF] Pelayo-Negro | An observational study of asymmetry in CMT1A[END_REF].

The heterogeneous phenotype observed among women with CMTX1 may be due to the X inactivation mechanism. When most Schwann cells in a nerve have a pathological X chromosome inactivated, demyelination is expected to be less severe. Therefore, the inactivation of the pathogenic X could explain the subgroup of asymptomatic women, although this hypothesis requires further testing. Siskind et al. in 2011 [24] investigated whether the presence of a GJB1 mutation affected the pattern of X inactivation in white blood cells. They did not observe any difference in the X-inactivation pattern between 14 affected women (CMTNS score ranged from 0 to 19) and the control group. However, the pattern of X inactivation is not homogeneous between different tissues [START_REF] Zito | Heritability of skewed Xinactivation in female twins is tissue-specific and associated with age[END_REF][START_REF] Gale | Tissue specificity of X-chromosome inactivation patterns[END_REF]. Thus, to test this hypothesis, the pattern of X inactivation should be directly evaluated in nerve cells in future investigations. Dubourg et al. in 2001 [START_REF] Dubourg | Clinical, electrophysiological and molecular genetic characteristics of 93 patients with X-linked Charcot-Marie-Tooth disease[END_REF] suggested that the asymmetry in women could also be explained by this mechanism if X inactivation is not homogeneous and is too variable from one nerve to another.

Motor conduction in women

In our study, MNCV and CMAP were less impaired in women than in men, consistent with previous findings [START_REF] Panosyan | Cross-sectional analysis of a large cohort with X-linked Charcot-Marie-Tooth disease (CMTX1)[END_REF][START_REF] Birouk | X-linked Charcot-Marie-Tooth disease with connexin 32 mutations: clinical and electrophysiologic study[END_REF][START_REF] Kleopa | How do mutations in GJB1 cause X-linked Charcot-Marie-Tooth disease?[END_REF][START_REF] Dubourg | Clinical, electrophysiological and molecular genetic characteristics of 93 patients with X-linked Charcot-Marie-Tooth disease[END_REF]. We also found that women exhibited less prolonged distal latency and F wave latency.

MNCV was significantly more asymmetric in women than men, consistent with clinical presentation. Gutierrez et al. in 2016 [START_REF] Gutierrez | Unusual electrophysiological findings in X-linked dominant Charcot-Marie-Tooth disease[END_REF] described five patients from a family with the R15W mutation (two women and three men) who exhibited significant heterogeneity in MNCV in women. Dubourg et al. in 2001 [23] reported greater ulnar and median CMAP amplitude heterogeneity in 37 CMTX1 women compared to male CMTX1 patients and CMT1A patients.

Interestingly, 39% of women in our cohort exhibited motor conduction blocks in the upper limbs. Although these were frequently mild, 31% of patients had blocks greater than 50%. Unfortunately, data on CMAP duration was often missing, preventing an investigation of the rate of temporal dispersion. Some previous studies [START_REF] Nicholson | Intermediate nerve conduction velocities define X-linked Charcot-Marie-Tooth neuropathy families[END_REF][START_REF] Gutierrez | Unusual electrophysiological findings in X-linked dominant Charcot-Marie-Tooth disease[END_REF][START_REF] Tabaraud | Demyelinating X-linked Charcot-Marie-Tooth disease: unusual electrophysiological findings[END_REF] reported a few cases of CMTX1 patients with temporal dispersion, suggesting it as a characteristic of CMTX1 neuropathy.

These results are consistent with previous studies. Some authors have suggested that the high rates of conduction blocks and temporal dispersion are indicative of a primary demyelination process, although this remains a matter of debate [START_REF] Birouk | X-linked Charcot-Marie-Tooth disease with connexin 32 mutations: clinical and electrophysiologic study[END_REF][START_REF] Hahn | Genotype/phenotype correlations in X-linked dominant Charcot-Marie-Tooth disease[END_REF][START_REF] Scherer | Connexin32-null mice develop demyelinating peripheral neuropathy[END_REF][START_REF] Tan | Novel mutations in the connexin 32 gene associated with X-linked Charcot-Marie tooth disease[END_REF][START_REF] Lewis | The challenge of CMTX and connexin 32 mutations[END_REF] and requires further exploration.

Misdiagnosis

The atypical clinical and electrophysiologic patterns observed in female CMTX patients, such as asymmetric clinical and electrophysiologic patterns associated with the presence of motor conduction blocks, can lead to misdiagnosis. In our cohort, four women received immunosuppressive therapy before being diagnosed with CMTX1.

CON CLUS IONS

In conclusion, our cohort of 137 women with CMTX1 showed that female patients have a heterogeneous phenotype. Fifty-five percent of women after the age of 48 years were as severely impaired as male patients, while the remaining 45% remained asymptomatic for a prolonged period. We also demonstrated that women with CMTX1 may exhibit atypical clinical presentations or motor conduction results that can lead to diagnostic wandering and misdiagnosis. Therefore, in women presenting with chronic neuropathy, the presence of clinical asymmetry, heterogeneous MNCV, and/or motor conduction blocks should raise suspicion for X-linked CMT, particularly CMTX1, and be considered in the differential diagnosis.

  We retrospectively reviewed clinical, neurophysiological, and genetic data of adult patients with CMT associated with GJB1 mutations in France. Patients were assessed between 2000 and 2022 (mean 2020) in 11 neuromuscular reference centers (Marseille, Lyon, Paris Pitié-Salpêtrière, Paris Kremlin Bicêtre, Strasbourg, Angers, Saint-Etienne, Nantes, La Réunion, Limoges, and Toulouse).

  Continuous variables were described by their mean, standard deviation, minimum and maximum value, or by their median and first and third quartiles. Categorical variables were described by their number and percentage. Continuous variables were compared by the nonparametric Wilcoxon rank sum test, depending on the conditions of the application. Categorical variables were compared by the chisquare test or Fisher's test, depending on the application conditions. For multivariate analyses, linear regressions were performed for continuous variables. Pearson correlation analysis was used to assess the correlation between age and clinical score and between the age at disease onset and clinical scores. The tests were performed in a two-sided situation and were considered statistically significant for p ≤ 0.05. Statistical analysis was performed with RStudio software (version 4.1.1).

F I G U R E 1

 1 Schematic diagram of gap junction protein beta 1 and GJB variants in our cohort, from Bone et al. [47]. All reported missense, nonsense, and frameshift GJB1 mutation are indicated with a colored circle. Novel variants are indicated with a green circle. New variants not related in the figures: three mutations located in the 5´-untranslated region (c.-16-459G > A, c.-16-529 T > C, c.-16-581G > A). One whole gene deletion (c.1_852del) is not indicated. [Colour figure can be viewed at wileyonlinelibrary.com] or 9/117) were also observed, as well as tremors (6% or 7/117) or scoliosis (3.4% or 4/117) as a mode of onset of the disease. Of note, 2.6% (4/117) started with another symptom such as subacute motor deficit or balance disorder.

F I G U R E 3 F I G U R E 4

 34 Charcot-Marie-Tooth Examination Score (CMTES) version 2 score in relation to age at the clinical visit. The square shows patients aged over 48 years with CMTES score ≤5. Phenotype of women with Charcot-Marie-Tooth disease type 1 (CMTX) in relation to the age of onset. Nineteen years old is the median of age of onset for women. (a) Mean Charcot-Marie-Tooth Examination Score (CMTES) score in relation to the age of onset, with standard deviation. (b) Mean Overall Neuropathy Limitations Scale (ONLS) score in relation to age of onset, with standard deviation.

F I G U R E 5

 5 Charcot-Marie-Tooth Examination score (CMTES) version 2 score in relation to the age of onset. For women, the Pearson correlation coefficient was ρ = -0.47, p < 0.01. For men, ρ = 0, and there is no linear relationship between the CMTES score and the age of onset.

Table 2

 2

). The disease mostly started in women with foot deformities (30.8% or 36/117), recurrent ankle sprains (20.5% or 24/117), or progressive motor deficits of the lower limbs (23.1% or 27/117). However, cramps (14.5% or 17/117), paresthesias (9.4% or 11/117), and progressive upper limbs deficit (7.8%

  123 patients, 16.3%) and scoliosis (13/123 patients, 10.6%). Additionally, 3/123 patients experienced sensorineural deafness, and 1/123 had optic nerve atrophy, while none of the patients exhibited cognitive disorders. Clinical characterization by gender.

	TA B L E 1 Characteristic	Women (N = 137)	Men (N = 126)	P-value
	Age at the evaluation, median (IQR)	48 (33-60)	40 (30-56)	0.060 a
	Age of onset, median (IQR) (NA)	19 (10-32) (9)	12 (7-18) (8)	<0.001 a
	CMTES, mean [±SD]	7.5 [±5.3]	11.0 [±4.5]	<0.001 a
	Sensory symptoms, mean [±SD] (NA)	0.6 [±0.9] (10)	0.6 [±1.0] (8)	0.671 a
	Motor symptoms, legs mean [±SD] (NA)	1.3 [±1.0] (10)	1.8 [±0.8] (8)	<0.001 a
	Motor symptoms arms, mean [±SD] (NA)	0.7 [±0.9] (10)	1.2 [±1.1] (8)	<0.001 a
	Pinprick sensibility, mean [±SD] (NA)	0.9 [±1.1] (10)	1.5 [±1.1] (8)	<0.001 a
	Vibration, mean [±SD] (NA)	1.5 [±1.2] (10)	2.1 [±1.1] (8)	<0.001 a
	Strength (legs), mean [±SD] (NA)	1.5 [±1.2] (10)	2.4 [±1.0] (8)	<0.001 a
	Strength (arms), mean [±SD] (NA)	1.0 [±0.9] (10)	1.6 [±1.0] (8)	<0.001 a
	Phenotype			<0.001 b
	Mild (CMTES 0-7), % (n)	51.8 (71)	21.4 (27)
	Moderate (CMTES 8-16), % (n)	41.6 (57)	69.0 (87)
	Severe (CMTES >16), % (n)	6.6 (9)	9.5 (12)
	ONLS, mean [±SD]	2.3 [±1.8]	3.5 [±1.7]	<0.001 a
	ONLS-upper limb, mean [±SD]	0.9 [±1.0]	1.6 [±1.1]	<0.001 a
	ONLS-lower limb, mean [±SD]	1.3 [±1.0]	1.9 [±0.8]	<0.001 a
	Walking aids (cane or walker), % (n)	10 (13/127)	16 (18/118)
	Wheelchairs, % (n)	0 (0/127)	3 (3/118)
	Orthosis, % (n)	37.0 (47/127)	64.4 (76/118)	<0.001 b
	Orthopedic surgery of the lower limbs, % (n)	15.0 (19/127)	26.3 (31/118)	0.028 b
	Treatment for neuropathic pain, % (n)	19.2 (23/120)	12.6 (14/111)	0.175 b
	Asymmetric, % (n)	15.0 (18/120)	3.6 (4/111)	0.003 b
	Scoliosis, % (n)	10.6 (13/123)	11.0 (13/118)	0.911 b
	Tremor, % (n)	16.3 (20/123)	23.7 (28/118)	0.147 b
	Deafness, % (n)	2.4 (3/123)	5.9 (7/118)	0.209 c
	Optic nerve atrophy, % (n)	0.8 (1/124)	0.8 (1/118)	>0.999 c
	Stroke-like syndrome, % (n)	2.4 (3/123)	0.8 (1/118)	0.622 c
	Cognitive impairment, % (n)	0 (0/123)	1.7 (2/118)	0.239 c

Abbreviations: CMTES, Charcot-Marie-Tooth Examination Score version 2; IQR, interquartile range; NA, not available; ONLS, Overall Neuropathy Limitation Score; SD, standard deviation. a Wilcoxon rank sum test. b Pearson's chi-squared test. c Fisher's exact test.

Table 3

 3

	Eight of 66 (12%) women and 6/64 (9%) men had severe motor
	CB (p = 0.614). MNCV of the median nerve and ulnar nerve
	were significantly less slowed in women than in men (median
	nerve MNCV was 42.2 m/s [±10.2] for women, 35.6 m/s [±6.4]
	for men, p < 0.001). Women had axonal neuropathy in 64.6% of
	cases (42/65), while men had intermediate neuropathy in 72.6%
	of cases (45/65, p < 0.001). Median and ulnar nerve CMAP were
	significantly less reduced in women (median nerve CMAP was
	5.1 mV [±3.8] for women, 3.5 mV [±3.7] for men, p = 0.024). Distal
	latency and F wave latency were significantly less prolonged in
	women (p < 0.001 and p = 0.008, respectively).

). MNCV was more frequently asymmetric in women (13/43 patients, 30%) than in men (3/43 patients, 7%). The difference between gender was statically significant (p = 0.024). Motor CB in upper limbs was more frequent in women than in men (26/66 patients, 39.4% vs. 18/64 patients, 28.1%), but this difference was not significant (p = 0.175).

  Electroneurographic characteristics by gender.

	Characteristic	Women	Men	P-value
	Demyelinating (MNCV <25 m/s), % (n)	1.5 (1/65)	0 (0/62)	<0.001 c
	Intermediate (MNCV 25-38 m/s), % (n)	33.8 (22/65)	72.6 (45/62)
	Axonal (MNCV >38 m/s), % (n)	64.6 (42/65)	27.4 (17/62)
	MNVC median nerve, mean [±SD] (n)	42.2 [±10.2] (65)	35.6 [±6.4] (62)	<0.001 a
	MNCV median difference right/left, mean [±SD] (n)	5.9 [±4.7] (37)	3.2 [±2.4] (36)	0.007 a
	MNCV ulnar, mean [±SD] (n)	48.3 [±8.4] (60)	38.6 [±7.7] (63)	<0.001 a
	MNCV ulnar difference right/left, mean [±SD] (n)	5.6 [±6.1] (35)	3.7 [±4.5] (39)	0.047 a
	CMAP median, mean [±SD] (n)	5.1 [±3.8] (66)	3.5 [±3.7] (59)	0.013 a
	CMAP ulnar, mean [±SD] (n)	7.1 [±2.9] (61)	5.3 [±3.2] (59)	<0.001 a
	Asymmetrical MNCV (>10%), % (n)	30 (13/43)	7 (3/43)	0.024 b
	Distal latency >4 ms, % (n)	54.0 (34/63)	83.3 (45/54)	<0.001 b
	Distal latency >6 ms, % (n)	14.3 (9/63)	16.7 (9/54)	0.722 b
	Percentage of distal latency increase, mean [±SD]	135.8 [±37.8]	135.8 [±25.9]	0.998 a
	F wave latency >30 ms, % (n)	52.8 (19/36)	82.4 (28/34)	0.008 b
	F wave latency >45 ms, % (n)	0 (0/36)	17.6 (6/34)	0.01 c
	Percentage of F wave latency increase, mean [±SD]	119.9 [±11.2]	135.2 [±25.0]	<0.029 a
	Motor conduction block ≥30% and/or temporal dispersion, % (n)	43.9 (29/66)	42.2 (27/64)	0.84 b
	Motor conduction block ≥30%, % (n)	39.4 (26/66)	28.1 (18/64)	0.175 b
	Motor conduction block between 30% and 50%, % (n)	27.3 (18/66)	18.8 (12/64)	0.249 b
	Motor conduction block ≥50%, % (n)	12.1 (8/66)	9.4 (6/64)	0.614 a

TA B L E 3

Abbreviations: CMAP, compound muscle action potential (in mV); MNCV, motor nerve conduction velocity (in m/s); SD, standard deviation. a Wilcoxon rank sum test. b Pearson's chi-squared test.

  Conduction blocks, as observed in some of our patients, can lead to an inaccurate diagnosis favoring chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), as previously reported by Hauw et al.[START_REF] Hauw | Charcot-Marie-Tooth disease misdiagnosed as chronic inflammatory demyelinating polyradiculoneuropathy: an international multicentric retrospective study[END_REF]. Clinicians should bey cautious of certain red flags when diagnosing CIDP, such as young patient age, a family history of neuropathy, motor difficulties or foot deformities in childhood, scoliosis, and a lack of response to treatments like intravenous immunoglobulin (IVIG), steroids, and immunosuppressants.

c Fisher's exact test.

DATA AVA I L A B I L I T Y S TAT E M E N T

The data that support the findings of this study are available from the corresponding author upon reasonable request.

AUTH O R CO NTR I B UTI O N S

Luce Barbat du Closel: Conceptualization (equal), methodology (equal), data curation (equal), formal analysis (equal), investigation (equal), formal analysis (equal), project administration (equal),

CO N FLI C T O F I NTER E S T S TATEM ENT

The authors report no disclosures relevant to the article.


Méryem Mounaj

Florian Jean

Jeremy Dhainaut

Christian Courtois

Yannick Lorgouilloux

Préparation par fabrication additive de supports de catalyseurs pour la conversion de molécules biosourcées

Résumé : L'objectif de ces travaux est de préparer des supports de catalyseurs en céramique poreuse, telle que l'alumine ou la zircone, via l'utilisation de procédés de fabrication additive permettant d'élaborer des pièces par ajout de matière à partir de fichiers numériques. Dans un premier temps, la technique de mise en forme par stéréolithographie (SLA) a été utilisée afin d'obtenir des objets présentant une géométrie contrôlée et une microstructure optimale en termes de densité, porosité et surface spécifique. La SLA consiste à imprimer de fines couches successives d'un matériau durcissant lorsqu'il est exposé la lumière UV (Figure 1). La principale difficulté réside dans le contrôle du frittage des objets, afin d'assurer une cohésion d'ensemble sans détériorer la surface spécifique de manière trop importante. Ainsi, des traitements thermiques à diverses températures ont été appliqués aux objets imprimés de manière à obtenir des propriétés mécaniques satisfaisantes tout en maintenant une surface spécifique la plus élevée possible, comme illustré en Figure 2.

En perspectives à cette première étude, une phase catalytiquement active de type MOF (metal-organic framework) sera déposée à la surface des objets par enduction, afin d'évaluer ensuite leur application en catalytique hétérogène. De plus, ces solides catalytiques seront comparés à des solides préparés directement par robocasting, à partir d'une pâte formulée contenant le MOF.

Figure 1 :

 1 Figure 1 : Schéma de stéréolithographie (SLA) : technologie d'impression 3D de céramique

Fabrication additive de supports de catalyseurs pour la conversion de molécules biosourcées/ Oral.

	Auteurs : Meryem MOUNAJ, Florian JEAN, Jérémy DHAINAUT, Yannick LORGOUILLOUX,
	Christian COURTOIS
	Affiliation : Université Polytechnique Hauts-de-France, CERAMATHS -DMP (Département
	Matériaux et Procédés)


Habdias A Silva-Neto

Gerson F Duarte-Junior

Danielly S Rocha

Fethi Bedioui

Anne Varenne

Professor Wendell K T Coltro
email: wendell@ufg.br

Recycling 3D printed residues for the development of disposable paperbased electrochemical sensors

Keywords: Complex thermoplastic waste, 3D printing, stencil printing, green chemical principles, carbon-based electrodes, S-nitroso-cysteine

Here, we propose a recyclable approach using acrylonitrile-butadiene-styrene (ABS) residues from additive manufacture in combination with low-cost and accessible graphite flakes as a novel and potential mixture for creating conductive paste. The graphite particles were successfully incorporated in the recycled thermoplastic composite when solubilized with acetone and the mixture demonstrated greater adherence to different microchip substrates, among which cellulose-based substrate making possible the construction of paper-based electrochemical sensor (PES). The morphological, structural and electrochemical characterization of the recycled electrode material were demonstrated similar to the ones of the traditional carbon-based surfaces. Faradaic responses based on redox probe activity ([Fe(CN) 6 ] 3-/4-) exhibited well-definite peak currents, diffusional mass-transfer, as a quasi-reversible system (96 ± 5 mV) with a fast heterogeneous rate constant value of 2 x 10 -3 cm s -1 . To improve the electrode electrochemical properties, both the PES and the classical 3D-printed electrode surfaces were modified with a combination of multi-walled carbon nanotubes (MWCNT), graphene oxide (GO) and copper. Both electrode surfaces demonstrated the suitable oxidation of nitrite at 0.6 and 0.5 V vs Ag, respectively. The calculated analytical sensitivity for PES and 3D-printed electrodes were 0.005 and 0.002 µA/[µmol L -1 ], respectively.

The proposed PES was applied for the indirect amperometric analysis of S-nitroso-cysteine (CysNO) in serum samples via nitrite quantitation, demonstrating a limit of detection of 4.1 µmol L - 1 ., with statistically similar values when compared to quantitative analysis of the same samples by spectrophotometry (paired t-test, 95% confidence limit). The evaluated electroanalytical approach exhibited linear nitrite concentrate range between 10 and 125 µmol L -1 , that is suitable for realizing clinical diagnosis involving Parkinson's disease, for example. This proof of concept shows the great promise of this recyclable strategy combining ABS residues and conductive particles in the context of green chemical protocols for constructing disposable sensors.

Introduction

Additive manufacturing approach also nominated as 3D printing has been providing an infinite of notorious contributions, including to the civil construction, medicine and dental industry, aerospace market, automobile technology, consumer goods and electrochemical sensors. [START_REF] Furet | 3D Printing for Construction Based on a Complex Wall of Polymer-Foam and Concrete[END_REF][START_REF] Mirdamadi | FRESH 3D Bioprinting a Full-Size Model of the Human Heart[END_REF][START_REF] Ngo | Additive Manufacturing (3D Printing): A Review of Materials, Methods, Applications and Challenges[END_REF][START_REF] Ambrosi | Electrochemically Driven Multi-Material 3D-Printing[END_REF] Recently, many strategies have been proposed in the domain of printable industry for constructing individual protective equipment that can be devoted to COVID-19 for example (a reference from you?) leading to an increase in the 3D printing market. As a result and according to GlobeNewswire, [START_REF]Global 3D Printing Filament Market is Anticipated to Grow at a CAGR of 31.10% during the Forecast Period from 2021 to 2030[END_REF] the materials demand involving thermoplastic filaments was proximally USD 968 million in 2021, indicating that the 3D printing technology is extremely versatile and accessible in outbreak cases.

The most usual 3D printers are based on fused depositing modeling (FDM) combined with thermoplastic filaments such as polylactic acid (PLA), polyethylene terephthalate (PET), acrylonitrile-butadiene-styrene (ABS) for successfully constructing many groundbreaking plastic objects for multiple finalities from medicine assay to chemical analysis. [START_REF] O'neil | Toward Single-Step Production of Functional Electrochemical Devices Using 3D Printing: Progress, Challenges, and Opportunities[END_REF][START_REF] Kalsoom | Current and Future Impact of 3D Printing on the Separation Sciences[END_REF][START_REF] Ambrosi | 3D-Printing Technologies for Electrochemical Applications[END_REF][START_REF] Cardoso | Additive-Manufactured (3D-Printed) Electrochemical Sensors: A Critical Review[END_REF] Notably, these moldable devices have piqued the interest of laboratories due to some peculiarities, including low-cost, accessibility, versatility, chemical stability, mechanical resistance, hydrophobicity and most important superior quality and long utility span. [START_REF] Nasir | Emerging Mono-Elemental 2D Nanomaterials for Electrochemical Sensing Applications: From Borophene to Bismuthene[END_REF][START_REF] Hamzah | 3D Printable Conductive Materials for the Fabrication of Electrochemical Sensors: A Mini Review[END_REF][START_REF] Katseli | A Novel All-3D-Printed Cell-on-a-Chip Device as a Useful Electroanalytical Tool: Application to the Simultaneous Voltammetric Determination of Caffeine and Paracetamol[END_REF] Among the previously cited filaments, PLA allows the construction of well-defined objects and is bio-degradable, however it exhibits low mechanical and thermal resistance. [START_REF] Mikula | 3D Printing Filament as a Second Life of Waste Plastics-a Review[END_REF][START_REF] Gkartzou | Production and 3D Printing Processing of Bio-Based Thermoplastic Filament[END_REF] On the other hand, PET and ABS are more efficient in terms of mechanical, chemical, and thermal resistance. Furthermore, ABS is considered as toxic with a low degradability. [START_REF] Mikula | 3D Printing Filament as a Second Life of Waste Plastics-a Review[END_REF][START_REF] Ligon | Polymers for 3D Printing and Customized Additive Manufacturing[END_REF] For this reason, it is important to propose strategies to recycle those materials following the green chemical principles to reduce or eliminate the use or generation of hazardous compounds. [START_REF] Anastas | Green Chemistry : Principles and Practice[END_REF] Highlights papers in the literature revealed that the thermoplastic materials can be reusable by numerous ways, (i) recycling carbon fibers and using them as additive option in the fused filament fabrication (FFF) process of PLA, [START_REF] Tian | Recycling and Remanufacturing of 3D Printed Continuous Carbon Fiber Reinforced PLA Composites[END_REF] and (ii) reusing glass fiber obtained from turbine blades to reinforce the mechanical properties of PLA material. [START_REF] Rahimizadeh | Recycling of Fiberglass Wind Turbine Blades into Reinforced Filaments for Use in Additive Manufacturing[END_REF] . However, to the best of our knowledge, the aforementioned studies do not explore the residues of thermoplastic as binder structures for manufacturing disposable electrochemical sensors, which are of high potential in the modern analytical chemistry field, including forensic, [START_REF] Khorablou | Recent Advances in Developing Optical and Electrochemical Sensors for Analysis of Methamphetamine: A Review[END_REF][START_REF] De Araujo | Portable Analytical Platforms for Forensic Chemistry: A Review[END_REF] pharmaceutical, [START_REF] Katseli | A Novel All-3D-Printed Cell-on-a-Chip Device as a Useful Electroanalytical Tool: Application to the Simultaneous Voltammetric Determination of Caffeine and Paracetamol[END_REF] environmental, [START_REF] Cate | Multiplexed Paper Analytical Device for Quantification of Metals Using Distance-Based Detection[END_REF][START_REF] Silva-Neto | Fully 3D Printing of Carbon Black-Thermoplastic Hybrid Materials and Fast Activation for Development of Highly Stable Electrochemical Sensors[END_REF] food and biological ones [START_REF] Bao | Toward a Highly Selective Artificial Saliva Sensor Using Printed Hybrid Field Effect Transistors[END_REF][START_REF] Song | Mini-Pillar Microarray for Individually Electrochemical Sensing in Microdroplets[END_REF][START_REF] Noviana | Electrochemical Paper-Based Devices: Sensing Approaches and Progress toward Practical Applications[END_REF] .

In this study we propose for the first time a recyclable approach using residues of ABS as binder to prepare carbon-based conductive ink and create disposable paper-based electrochemical sensor (PES). First the proof of concept of integration of the recycled ABS mixed with graphite particle as a material for electrodes on different microchip materials was demonstrated, highlighting the efficient electrode integration on paper-based materials thanks to morphological, structural and electrochemical characterizations. To improve the faradaic response of the sensor, we developed two simple strategies: (i) removing the insulating ABS structures based on fast electrochemical oxidation; (ii) improving the catalytic electrode performance for nitrogene-based groups through modification of the electrode surface with a mixture of multi-walled carbon nanotubes, graphene oxide and copper, as well-known reported in the literature for the oxidation of nitrite. [START_REF] Bagheri | Composite of Cu Metal Nanoparticles-Multiwall Carbon Nanotubes-Reduced Graphene Oxide as a Novel and High Performance Platform of the Electrochemical Sensor for Simultaneous Determination of Nitrite and Nitrate[END_REF] As a matter of comparison, fully 3D-printed carbon-black electrodes were also fabricated and tested in the same experimental conditions. As a further proof of concept, this disposable PES was applied for the indirect detection by chronoamperometry of S-nitroso-cysteine (CysNO) in human serum samples. This biomolecule from the S-nitrosothiols (RSNOs) group can play an essential role in several physiological and physiopathological processes, actuating as biomarker and NO-donors. [START_REF] Williams | The Chemistry of S-Nitrosothiols[END_REF] This new recyclable strategy combining ABS residues and conductive particles was finally compared to to classical spectrophotometry analysis of these human serum samples.

Experimental

Materials and reagents

Potassium ferrocyanide, potassium ferricyanide, potassium chloride, acetic acid, acetonitrile, copper sulfate pentahydrate, sulfuric acid, sulfanilamide, N-(1-naphthyl)ethylenediamine (NED), multi-walled carbon nanotubes (MWCNT) (OD: 6-9 nm, length: 5 μm), graphene oxide (GO), sodium sulfate, iron sulfate hexahydrate, uric acid, hydrochloric acid, and cysteine were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Acetone, sodium nitrite, sodium nitrate and hydrogen peroxide were acquired from Neon (Suzano, SP, Brazil). All reagents were analytical grade and used as received. The stock and standard solutions were prepared in ultrapure water processed through a purification system (Direct-Q 3, Millipore, Darmstadt, Germany) with a resistivity equal to 18.2 MΩ cm.

Vegetal paper also nonmined as waterproof (weight = 180 g m -2 ), thermosensitive polyester films (thickness = 250 μm) and nail polish were obtained from Ecrisam (Goiânia, GO, Brazil), Yidu Group Co., (Hsi-Chih, TPE, Taiwan) and RISQUÈ ® (São Paulo, SP, Brazil), respectively. Graphite powder (ø ≤ 50 µm), silver ink model PC-9070/1 and adhesive vinyl film were provided by Synth (Diadema, SP, Brazil), Joint Metal (São Paulo, SP, Brazil) and Imprimax (São Paulo, SP, Brazil), respectively. Thermoplastic filaments (ø = 1.75 mm) as acrylonitrile butadiene styrene (ABS) and polylactic acid/carbon black (PLA-CB) composite filament (brand name ProtoPasta) were supplied by 3D Fila (Belo Horizonte, MG, Brazil). Whatman ® grade 1 chromatography paper (thickness: 0.18 mm) was received from Sigma-Aldrich.

Instruments

The stencil masks were designed and constructed using a cutting printer Cameo (Silhouette, Belo Horizonte, MG, Brazil) and managed by Silhouette Studio ® software (version 4.1). The thermal laminator model 101118415 was acquired from Gazela (Divinópois, MG, Brasil). The layout of 3D printed electrode was designed in SolidWorks software and manufactured using an Original Prusa i3 mk2/S 3D printer (Prusa Research, Prague, Czech Republic). A portable cabin integrated with UV LED (7 W) purchased from Kiss NY Pro (New York, USA) was used to degrade CysNO samples. The electrochemical measurements were performed through a portable bipotentiostat/galvanostat, model μStat 400 (DropSens S.L., Oviedo, Spain) and monitored by DropView ® software.

Collection of ABS residues, preparation of conductive ink and electrochemical sensor

ABS residues were collected from two sources: (i) during the outbreak of COVID-19 when our group successfully fabricated face shield and magnetic holder to donations in local hospitals;

(ii) from microfabrication researcher involving microfluidic devices. ABS leftovers were crushed using an industrial processor (Mondial, São Paulo, Brazil) and stored in plastic bottles (see in Figure S1 available in electronic supporting information).

Next, the preparation of conductive ink based on combination of graphite flakes and ABS residues, and the fabrication of electrodes via stencil printing technique were performed in five steps. (i) The geometry of the device (auxiliary, AE; reference, RE; and working, WE, Ø = 4 mm) was designed using CorelDraw software. (ii) The stencil masks were constructed by combining the optimized substrate with polyester film (heat press at 120 °C for 5 s) followed by two layers of selfadhesive vinyl upon optimized substrate (vegetal paper). The plastic mask was performed by a reductive printing process via a cutting plotter. (iii) For realizing the synthesis of conductive ink, a mixture of 1.5 g oABS residue and 15 mL acetone were prepared and exposed to a continuous stirring process (700 rpm) for 30 min. 1.5 g graphite powder was then added to mixture and left again under stirring (700 rpm; 15 min). (iv) The conductive ink was dispersed over the stencil mold/substrate using a spatula and after drying overnight (25 ± 2 ºC) the vinyl masks were removed. (v) Additionally, steps involving the construction of a pseudo RE, a hydrophobic barrier, and an electrochemical cleaning process were realized through painting of RE with silver ink, delimitating the target geometric area using nail polish, adding 50 µL acetic acid (6 mol L -1 ) on the electrode surface and applying a potential of 1.8 V vs Ag for 100 s.

For realizing the comparative experiments, fully 3D-printed electrodes were manufactured, and the surface activated according to a recent study. [START_REF] Silva-Neto | Fully 3D Printing of Carbon Black-Thermoplastic Hybrid Materials and Fast Activation for Development of Highly Stable Electrochemical Sensors[END_REF]

Preparation of modified sensor surface

The WE surface modification by MWCNT/GO was realized based on three steps, as reported in a recent study. [START_REF] Bagheri | Composite of Cu Metal Nanoparticles-Multiwall Carbon Nanotubes-Reduced Graphene Oxide as a Novel and High Performance Platform of the Electrochemical Sensor for Simultaneous Determination of Nitrite and Nitrate[END_REF] (i) The MWCNT/GO composite was synthesized by dispersing 1.0 mg GO into 5.0 mL water and sonicating for one hour. After, 1.0 mg MWCNT was added and sonicated for one hour [11]. An aliquot of 10 µL of the MWCNT/GO dispersion was dropped on the electrode surface and dried by overnight (25 ± 2 ºC). (ii) The electrode was electrochemically reduced under a potential of -1.2 V vs Ag for 140 s in the presence of 0.1 mol L -1 H 2 SO 4 , as supporting electrolyte. (iii) A solution composed of 10 mmol L -1 Cu(II) prepared in 0.1 mol L -1 H 2 SO 4 was dropped on the MWCNT/GO surface and a potential of -0.7 V vs Ag was applied for 200 s, for the reduction of Cu(II) ion to Cu(0).

Synthesis and decomposition procedures of S-nitrosocysteine

The synthesis of CysNO was performed as described elsewhere by Peterson. [START_REF] Peterson | Decomposition of S -Nitrosocysteine via Sto N -Transnitrosation[END_REF] Briefly, an equimolar amount of cysteine and sodium nitrite (5 mmol L -1 ) were mixed in a dark flask (to prevent decomposition by the action of light), followed by the addition of 0.1 mol L -1 hydrochloric acid. After 5 minutes, 0.1 mol L -1 phosphate buffer (pH 7.4) prepared in 0.5 mmol L -1 EDTA was added to the target solution in order to neutralize and avoid decomposition through traces of contaminating metals. The concentration of CysNO was estimated by colorimetric characterization using an UV-Vis spectrophotometer model UV-M51 from BEL Photonics (Milan, Italy).

Absorption measurement was realized in a spectral scan ranging from 250 to 500 nm, highlighting a peak at a 335 nm wavelength with a ε = 719 L mol -1 cm -1 (Figure S2; available in the electronic supplementary information, SI). For the decomposition of CysNO, the solution was exposed for 15 minutes to UV-LEDs.

Electrochemical experiments

The proposed PESs were submitted to both cyclic voltammetry (CV) and amperometry analysis. CV experiments were performed in the presence of 1.0 mmol L -1 [Fe (CN) 6 ] 3-/4-prepared in 0.1 mol L -1 KCl, to optimize the substrate, characterize the electrochemical response and verify the repeatability and reproducibility of the raw recycled electrodes. The catalytic performance of modified electrodes in terms of redox activity, repeatability, and reproducibility was studied using the oxidation of nitrite (1.0 mmol L -1 nitrite prepared in 0.1 mol L -1 Na 2 SO 4 ), as a model reaction.

Finally, the amperometric approach was selected for quantitation of nitrite at 0.6 V vs Ag for 50 s.

As comparative experiments, electrochemical analysis was realized using 3D printed electrodes with detection conditions at 0.5 V vs Ag for 30 s. The selectivity study was also realized with a standard solution of 50 µmol L -1 nitrite dopped with different interfering agents (nitrate, uric acid, chloride, and Fe(II) prepared at 5 µmol L -1 each).

To prove the concept of the electroanalytical PES method, the electrodes were explored for analysis of CysNO in serum samples. Artificial serum was purchased from Doles (Goiânia, GO, Brazil) and the sample preparation and decomposition steps were adapted from Ismail et al. [START_REF] Ismail | Colorimetric Analysis of the Decomposition of S-Nitrosothiols on Paper-Based Microfluidic Devices[END_REF] Posteriorly, artificial serum samples were diluted in 0.1 mol L -1 Na 2 SO 4 (proportion 1:5; v/v) and the samples were doped with the CysNO degraded molecule at three different concentrations, i.e. 20, 40 and 80 µmol L -1 . Figure 1 shows a scheme of the sensor construction process and the indirect electrochemical detection of CysNO. All electrochemical measurements were realized at room temperature (25 ± 2 ºC).

Characterization

Electrical resistance (ER), scanning electron microscopy (SEM), Raman spectroscopy and electrochemical characterization were realized. ER of conductive trail was estimated by a digital multimeter model MD-360 (INSTRUTHERM, São Paulo, Brazil). SEM analysis was performed using a JEOL microscope model JSM 6610 (Waltham, MA, USA). Raman spectroscopy was performed with a confocal Raman spectrometer model LabRAM HR Evolution (Horiba, Kyoto, Japan). Electrochemical characterization was performed from CV measurements in the presence of 1.0 mmol L -1 [Fe (CN) 6 ] 3-/4-prepared in 0.1 mol L -1 KCl with a scan rate ranging from 10 to 100 mV s -1 at -0.6 to 0.5 V vs C. The procedure of water contact angle (WCA) analysis was realized in two steps, capturing the image via a digital camera followed by measuring the CA value through ImageJ software.

CysNO indirect measurements

For performing the reference measurements of CysNO, the colorimetric Griess method was adapted according to Peterson et al. [START_REF] Peterson | Decomposition of S -Nitrosocysteine via Sto N -Transnitrosation[END_REF] The Griess reagents were prepared by mixing 50 mmol L -1 sulfanilamide and 4 mmol L -1 NED (solubilized in citric acid) in a 1:1 (v/v) proportion. Then, aliquots of 1.5 mL (Griess solution) and 1.5 mL (target sample) were mixed. Analytical responses were performed using standard concentrations of nitrite ranging from 1 to 50 µmol L -1 and absorption detection at 510 nm. The serum sample was doped with CysNO at three concentrations (20, 40 and 80 µmol L-1) and recovery result was statistically compared to those values of CysNO measured by the proposed PES method.

Results and discussion

Fabrication of electrochemical paper-based sensor

In order to obtain the better interconnection between conductive ink and the insulating surface, different substrates have been explored, and materials such as cellulose-based (vegetal and chromatographic papers) and polystyrene films were carefully evaluated. The electrodes were manufactured via a stencil printed approach. Then, the morphological and electrochemical analysis were carefully performed. Morphological analysis by SEM involving cross-sectional views are exhibited in Figures 2A andS3 and the thickness values are estimated as 314, 226 and 206 µm for conductive ink deposited on vegetal paper, chromatographic material, and polystyrene film, respectively. While the polystyrene used as substrate exhibited poor adhesion with the conductive ink, the low thickness of carbon ink observed on chromatographic paper can be justified by the large porosities of these cellulose-based fibers (1 to 10 µm) in which the target ink disperses. On the other hand, the better thickness values of carbon ink measured on vegetal paper as insulating substrate can be due to a suitable interconnect between ABS and paraffin structures incorporated under cellulose fibers, and the fact that waterproof paper does not have large porosities, as demonstrated in recent studies. [START_REF] Camargo | Talanta Waterproof Paper as a New Substrate to Construct a Disposable Sensor for the Electrochemical Determination of Paracetamol and Melatonin[END_REF][START_REF] Habdias | Plug-and-Play Assembly of Paper-Based Colorimetric and Electrochemical Devices for Multiplexed Detection of Metals †[END_REF] So as to go deeper in this characterization, an electrochemical cleaning of the electrodes was performed on these three devices. Aiming at removing the residual and non-conductive material from the electrode surface, a cleaning process was performed upon the freshly manufactured electrode. This approach was adapted from recent study (cit) involving electrochemical oxidation in the presence of acetic acid solution (6.0 mol L -1 ). Figure S4 presents the electrochemical response of the redox mediator [Fe(CN) 6 ] 3-/4-that is extremely sensitive to chemical surface, on the noncleaned and cleaned electrodes. The electrochemical parameters evaluated were peak-to-peak separation (ΔEp) and anodic peak current (i pa ).

Through CV measurements for electrodes constructed with chromatographic and polyester substrates , large ΔEp (170 to 180 mV) were observed probably due to the low conductivity of the electrode material. [START_REF] Santhiago | Flexible and Foldable Fully-Printed Carbon Black Conductive Nanostructures on Paper for High-Performance Electronic, Electrochemical, and Wearable Devices[END_REF][START_REF] Silva | Use of Pyrolyzed Paper as Disposable Substrates for Voltammetric Determination of Trace Metals[END_REF] However, the carbon-based ink decorated on vegetal paper revealed better redox parameters with a ΔEp of 101 mV and an increase of target current signal by 1.6 times, when compared with the electrodes manufactured on chromatographic and polyester substrates. Examples of binders that are gaining prominence in literature are mineral oil, [START_REF] Mettakoonpitak | AgNP/Bi/Nafion-Modified Disposable Electrodes for Sensitive Zn(II), Cd(II), and Pb(II) Detection in Aerosol Samples[END_REF] polystyrene, [START_REF] Rymansaib | All-Polystyrene 3D-Printed Electrochemical Device with Embedded Carbon Nanofiber-Graphite-Polystyrene Composite Conductor[END_REF] glass varnish, [START_REF] Habdias | Plug-and-Play Assembly of Paper-Based Colorimetric and Electrochemical Devices for Multiplexed Detection of Metals †[END_REF][START_REF] Pradela-Filho | Glass Varnish-Based Carbon Conductive Ink: A New Way to Produce Disposable Electrochemical Sensors[END_REF] nail polish [START_REF] Pradela-Filho | Nail Polish and Carbon Powder : An Attractive Mixture to Prepare Paper-Based Electrodes[END_REF] , starch (cit), native ABS(cit) and PDMS [START_REF] Sameenoi | Dimethylsiloxane) Cross-Linked Carbon Paste Electrodes for Microfluidic Electrochemical Sensing[END_REF] . However, the carbon-based ink proposed herein makes possible the use of thermoplastic residues from 3D printed objects, showing the potential of this materials as low-cost binders, and most important the application of green chemistry's principles as preventing wastage and utilization of residues.

Electrochemical paper-based sensor characterization

As mentioned above the better results obtained in terms of ink thickness and electrochemical response were observed with the recycled conductive paste deposited on vegetal paper. In this way, the electrical resistivity, as well as SEM, Raman and electrochemical analyses of the conductive surface were characterized in detail on this vegetal paper. The measured value of electrical resistivity of 68 ± 10 Ω cm is coherent with the traditional macroscopic information found in literature reported (≤ 1000 Ω cm). [START_REF] Silva | Use of Pyrolyzed Paper as Disposable Substrates for Voltammetric Determination of Trace Metals[END_REF][START_REF] Noviana | Electrochemical Paper-Based Devices: Sensing Approaches and Progress Toward Practical Applications[END_REF][START_REF] Rocha | Sandpaper-Based Electrochemical Devices Assembled on a Reusable 3D-Printed Holder to Detect Date Rape Drug in Beverages[END_REF][START_REF] Stefano | Different Approaches for Fabrication of Low-Cost Electrochemical Sensors[END_REF][START_REF] Petroni | Development of Highly Sensitive Electrochemical Sensor Using New Graphite/Acrylonitrile Butadiene Styrene Conductive Composite and 3D Printing-Based Alternative Fabrication Protocol[END_REF] This behavior can be associated to the intrinsic properties of graphite-based surfaces such as micrometer-size flakes, high-specific contact area, pore depth and greater electron accessibility, for example. It can also be linked to the elevated proportion of the conductive particles (ø ≤ 50 µm) on ABS binder (1:1; w/w) upon the vegetal paper.

Figure 2 presents SEM results in terms of thickness of the carbon-based composite (Figure 2A), interconnection between substrate and conductive ink (Figure 2B), and material surface before (Figure 2C) and after (Figure 2D) the electrochemical cleaning process. The images allow to estimate a thickness of 314 m, the well-defined interconnection between the vegetal paper and the conductive material, and also the surface with partial removal of insulating ABS residues weakly adhering on the conductive trail. [START_REF] Silva-Neto | Fully 3D Printing of Carbon Black-Thermoplastic Hybrid Materials and Fast Activation for Development of Highly Stable Electrochemical Sensors[END_REF][START_REF] Richter | Complete Additively Manufactured (3D-Printed) Electrochemical Sensing Platform[END_REF] Figure 2E present the Raman measurements ranging from 1200 to 1800 cm -1 demonstrating distinct peak intensities at 1346 cm -1 and 1578 cm -1 as D and G bands, respectively. [START_REF] Caņado | General Equation for the Determination of the Crystallite Size La of Nanographite by Raman Spectroscopy[END_REF] These peaks can be attributed to sp 2 and sp 3 atoms hybridization in carbon-based composites and indicate that the calculated ratio of I D /I G = 0.142 is indeed in agreement with graphite-based inks prepared in well-known binder materials. [START_REF] Noviana | Electrochemical Paper-Based Devices: Sensing Approaches and Progress toward Practical Applications[END_REF] Figures 2F and 2G summarize the cyclic voltammograms at different scan rates (10 to 100 mV s -1 associated with the redox behavior of [Fe(CN) 6 ] 3-/4-exhibiting a value of ΔEp (96 ± 5 mV at 10 mV s -1 ) that is characteristic of a quasi-reversible system. [START_REF] Mccreery | Advanced Carbon Electrode Materials for Molecular Electrochemistry[END_REF][START_REF] Nicholson | Theory of Stationary Electrode Polarography Single Scan and Cyclic Methods Applied to Reversible , Irreversible , and Kinetic Systems[END_REF] A linear regression between scan rate and peak current (R 2 = 0.99) confirms that the electron mass-transfer is diffusional. Then, the calculated heterogeneous rate constant value was 2.0 (±0.1) x 10 -3 cm s -1 , based on the Nicholson method (cit). Therefore, the proposed surface can be suitable as sensing material, similar to the other reported studies (cit).

The recycled electrodes were tested in different electrolytes of different pH values, i.e. 0.1 mol L -1 NaOH, KCl and H 2 SO 4 , as summarized in Figure S5. It is to be noted that the solid electrode does not show any faradaic activity (at potentials ranging from -0.5 V to 0.8 V vs C) involving contaminated species from conductive ink. The repeatability (number of cycles) and reproducibility (different PES microdevices) of the electrochemical response were then determined using [Fe(CN) 6 ] 3-/4-, as a model probe (see Figures 2H and2I). The calculated statistical results of relative standard deviation (RSD) exhibit values of 4.6% (repeatability experiment; n=10) and 7.1% (reproducibility analysis; n=5). In addition, a one-way ANOVA (n=5) was performed on reproducibility responses, showing no significant difference between all manufactured devices (Fvalue = 1.33; p-value = 0.29; F-critical = 3.88), which suggests the potential for high-throughput manufacturing of these recycled carbon-based electrodes. A-D). Raman spectra (E). Cyclic voltammograms (F) and respective plots peak current intensity vs scan rate (G). Reproducibility (H) and repeatability (I) analysis.

Modification and characterization of the recycled electrodes for improving PES performances

Pure and applied studies involving solid state electrodes have demonstrated that the surface chemistry of allotrope carbon such as MWCNT and GO are important electrocatalyst materials (cit).

Besides, the incorporation of nanostructured carbon with copper can improve the redox activities of nitrite, for example. [START_REF] Bagheri | Composite of Cu Metal Nanoparticles-Multiwall Carbon Nanotubes-Reduced Graphene Oxide as a Novel and High Performance Platform of the Electrochemical Sensor for Simultaneous Determination of Nitrite and Nitrate[END_REF] Therefore, the disposable recycled electrode surface was sequentially modified using a mixture of MWCNT/GO followed by electrochemical deposition of Cu. So as to demonstrate the interest of such surface modification, it was characterized in terms of (i) interfacial tensions of the electrode surface via water contact angle (WCA) measurements and (ii) catalytic properties of the electrodes before and after the modification with Cu/MWCNT/GO via CV electrochemical experiments on nitrite solutions. As a matter of comparison, similar studies were conducted using 3D printed carbon black sensor, including WCA (Figure S6) and catalytic responses (Figure 3). WCA measurement on bare PES and 3D-printed surface showed values of 88 ± 3 and 85 ± 3º, respectively, indicating that both heterogeneous interfaces exhibited equivalent hydrophilic character. When both electrodes are modified with Cu/MWCNT/GO structure, the WCA values shift to 60 ± 3 and 56 ± 3º . These values are still statistically indifferent (paired t-test; p = 0.05) as expected as the modification process and nature is the same. Furthermore, these WCA values lower than 90° indicate a compatibility of these electrodes for performing drop-cast experiment and stationary measurements, for example.

As observed in Figures 3A-B, the evaluated electrochemical behavior indicated that both devices showed well-defined peak and acceptable catalytic behavior for nitrite oxidation at 0.63 and 0.51 V vs Ag, respectively. In addition, the electrochemical signals of the modified exlectrodes are enhanced by 6.3 and 4.8 times for the proposed device and 3D-printed electrode, respectively.

These comparative experiments successfully demonstrate that both kinetic regimes are irreversible. [START_REF] Bagheri | Composite of Cu Metal Nanoparticles-Multiwall Carbon Nanotubes-Reduced Graphene Oxide as a Novel and High Performance Platform of the Electrochemical Sensor for Simultaneous Determination of Nitrite and Nitrate[END_REF][START_REF] Mccreery | Advanced Carbon Electrode Materials for Molecular Electrochemistry[END_REF][START_REF] Nicholson | Theory of Stationary Electrode Polarography Single Scan and Cyclic Methods Applied to Reversible , Irreversible , and Kinetic Systems[END_REF] Thus, the electrode surface covered with Cu/MWCNT/GO nanostructures shows excellent catalytic properties for the oxidation of nitrite when compared with responses obtained upon bare electrodes. Bagheri et al. [START_REF] Bagheri | Composite of Cu Metal Nanoparticles-Multiwall Carbon Nanotubes-Reduced Graphene Oxide as a Novel and High Performance Platform of the Electrochemical Sensor for Simultaneous Determination of Nitrite and Nitrate[END_REF] reported glass carbon electrode decorated with Cu/reduced GO/MWCNT for drastic improvement of the nitrite and nitrate oxidations. The authors showed that the best redox activities exhibited by Cu/reduced GO/MWCNT surface can be explained by the large surface area, action sites, and a high number of edges and defects.

As it can be seen in Figures 3C-D andS7, other comparative studies were performed using both modified electrodes with cyclic voltammograms of what? at scan rates ranging from 20 to 80 mV s -1 . The obtained plots in terms of peak current vs scan rate successfully indicate that the redox process is diffusion-controlled. [START_REF] Santos | Amperometric Sensor for Nitrite Using a Glassy Carbon Electrode Modified with Alternating Layers of Iron(III) Tetra-(N-Methyl-4-Pyridyl)-Porphyrin and Cobalt(II) Tetrasulfonated Phthalocyanine[END_REF] We also investigated the performance of the modified surface in terms of reproducibility and repeatability of the electrochemical signal using the oxidation of nitrite, as model reaction. These studies were realized via three different devices and through five measurements in sequence (Figure 4). The recorded electrochemical data when provide respective RSD values of 3.4 and 1.8% for repeatability and reproducibility analysis, demonstrating the suitable performance of the PES decorated with Cu/MWCNT/GO.

Recycled PES for sensing CySNO

As the electrode proposed herein exhibits satisfactory performance for the oxidation of nitrite, we explored those redox properties to achieve the indirect analysis of low molecular weight RSNOs. RSNO can be converted to nitrite through many strategies of decomposition, making possible the use of nitrite as indirect marker. [START_REF] Bedioui | Electrochemical Detection of Nitric Oxide and S-Nitrosothiols in Biological Systems : Past , Present & Future[END_REF] For this reason, chronoamperometry was selected for Under amperometric conditions, the linear regression associated to detection of nitrite and CySNO exhibited a good R 2 value (0.99) and similar analytical sensitivity with values 0.008 and 0.005 µA/[µmol L -1 ], indicating that the present electroanalytical method can be performed to indirect analysis of CySNO. As can be seen in Figure S7, the electrochemical performance with modified 3D-printed sensor showed values of R 2 = 0.99 and sensitivity of 0.002 µA/[µmol L -1 ] in the same order as those obtained for Cu/MWCNT/GO modified PES. Furthermore, quantitative analysis for the sample composed of decomposed CySNO in diluted serum samples (Figure 5C and D) indicated sensitivity level of 0.006 µA/[µmol L -1 ] that is similar to sensing properties observed on amperometric measurements in the absence of complex biological matrix. In another study, Rebecca et al. [START_REF] Hunter | S -Nitrosothiol Analysis via Photolysis and Amperometric Nitric Oxide Detection in a Micro Fl Uidic Device[END_REF] manufactured a microfluidic device combined with platinum electrode for amperometric analysis of RSNOs. For facilitating the photolysis of nitrosothiol groups and realizing the amperometric detection of nitrite, the authors reported a simple degradation through UV-LED and an analysis with a procedure time low than 2 min and LOD value of 73 nmol L -1 . Here, the proposed modified PES provides suitable detectability for CySNO and the estimated LOD is better or similar when compared to low-cost analytical tools (you must put referecnes then). Also, the herein reported linear concentration range (10-125 µmol L -1 ) can be useful to clinical application that involved Parkinson's disease. (cit)

Selectivity of the recycled PES

Nitrite analysis in biological matrix can be susceptible to any interference, as biomolecules and ions are commonly found in biological fluids associated to sensing applications in terms of RSNOs-based analysis. [START_REF] Williams | The Chemistry of S-Nitrosothiols[END_REF][START_REF] Bedioui | Electrochemical Detection of Nitric Oxide and S-Nitrosothiols in Biological Systems : Past , Present & Future[END_REF][START_REF] Oliveira | Thermal Stability of Primary S -Nitrosothiols : Roles of Autocatalysis and Structural Effects on the Rate of Nitric Oxide Release[END_REF] The selectivity of Cu/MWCNT/GO/PES as nitrite sensor was investigated by amperommetric measurements of nitrite in the absence and presence of nitrate, uric acid, chloride and Fe(II) with a proportion of 1:10. The electrochemical responses before and after the addition of interfering compounds are presented in Figures 6A-B, with interference levels of -11, -2, -0.6 and 3% for nitrate, uric acid, chloride and Fe(II), respectively. We observed that the redox activity of nitrite is not drastically influenced by the presence of the above mentioned interference compounds, denoting the great potential of the modified electrodes to analysis of CysNO in complex biological compound.

Sample analysis

To demonstrate the feasibility of the manufactured electrochemical device as CysNO sensor in human serum samples, the Cu/MWCNT/GO/PES was tested in terms of recovery measurements through the addition of three different levels of decomposed CysNO into artificial serum samples, 20, 40 and 80 µmol L -1 , as indicated in Figures 6C-D. All obtained amperometric responses exhibited recovery values ranging from 90 to 105% (see summarized results in Table 1). The same experiments were performed using the Griess-colorimetric method, [START_REF] Peterson | Decomposition of S -Nitrosocysteine via Sto N -Transnitrosation[END_REF] with similar samples preparation step. Summarized responses under both instrumental systems showed detected levels of CysNO without statistical difference (paired t-test; p=0.05), indicating that the proposed recycled PES allows quantitation with equivalent performance as for reference method.

Conclusions

We demonstrated for the first time the performance of recycled electrodes for paper-based sensors, composed of ABS residues and graphite flakes, making it a potential valuable protocol for recycling thermoplastic waste from 3D-printing industry, according to the green chemical principles. We demonstrated the huge performance of the recycled composite as binder structure through: (i) Detailed macroscopic, microscopic, and electrochemical characterizations confirm that the recycled ABS is a great alternative for constructing conductive ink; (ii) The comparative study between the proposed sensor and a fully 3D-printed device has shown similar faradaic performance, indicating that the recycled and disposable electrode proposed herein can be explored to electrocatalysis; (iii) The proposed electrochemical cell when combined with electrochemical detector has successfully demonstrated huge potential to indirect analysis of S-nitrosothiol in diluted serum samples. Therefore, this cleaner approach for reusing complex thermoplastic materials to future fabrication of conductive ink is attractive in order to overcome the vast number of ABS residues. In addition, the proposed recyclable method for manufacturing paper-based sensor allows low-cost materials and green chemical principles, opening novel opportunities in the modern chemical sensor field dedicates to biological applications.

Figure 1 .

 1 Figure 1. Schematic representation of fabrication (i) and modification of electrode aiming for sensing of

Figure 2 .

 2 Figure 2. SEM measurements (A-D). Raman spectra (E). Cyclic voltammograms (F) and respective plots

Figure 3 .

 3 Figure 3. Electrochemical behavior of nitrite before and after the surface modification involving PES

Figure 4 .

 4 Figure 4. Catalytic performance of modified electrodes to oxidation of nitrite in term of reproducibility

  evaluating the possibility of indirect detection of CySNO in three different steps: (i) the use of standard nitrite to calibrate the electrode, (ii) the use of pre decomposed CySNO in supporting iii) the use of pre decomposed CySNO prepared in diluted serum samples and in supporting electrolyte (which rate? Wifh supporting electrolyte . All analytical responses were performed at a detection potential 0.6 V vs Ag for 50 s with nitrite concentrations ranging from 10 to 125 µmol L -1 . The obtained chronoamperograms and respective plots (current signal vs concentration) are presented in Figures5A-D. It is important to mention that we also realized parallel amperometric experiments through fully 3D printed electrodes with detection potential at 0.5 V for 30 s (see the analytical curve in FigureS8in the SI).

Figure 5 .

 5 Figure 5. Chronoamperogram of standard nitrite solutions (A), decomposed CySNO (B), decomposed

  The estimated limit of detection (LOD) values to indirect detection of CySNO in standard and serum samples were 4.4 and 4.1 µmol L -1 , respectively. (LOD was calculated using the 3.3*SD blank /slope of analytical curve). Duarte-Junior et al.[START_REF] Duarte-Junior | Integrated Microfluidic Device for the Separation, Decomposition and Detection of Low Molecular Weight S-Nitrosothiols[END_REF] proposed a microfluidic device with integrated electrochemical detector for separating and detecting RSNOs, after a decomposition to nitrite based on mercury with LODs of 20 µmol L -1 . Ismail et al.[START_REF] Ismail | Colorimetric Analysis of the Decomposition of S-Nitrosothiols on Paper-Based Microfluidic Devices[END_REF] further constructed a microfluidic paper-based microdevice for indirect colorimetric detection of RSNOs, with the same decomposition protocol with a LOD of 4 µmol L -1 .

Figure 6 .

 6 Figure 6. Electrochemical responses (A) and respective histogram (B) of the selectivity of PES.

Table 1 .

 1 Recovery experiment involving serum samples doped CysNO in different concentrationswith the PES microdevice and the Griess-method.

Acknowledgments

The authors would like to thank CAPES, CNPq (grants 426496/2018-3, 308140/2016-8, 307554/2020-1 and 142412/2020-1) and INCTBio (grant 465389/2014-7) for the financial support and granted scholarships. The authors acknowledge the Multi-user Laboratory of high-resolution microscopy (LabMic/UFG), PhD. Lucas C. Duarte and Professor Paulo Sergio for using their facilities during (SEM and Raman) measurements, technical supporting for solubilize the ABS residue and technical apport during the spectrophotometric analysis, respectively.