Puchar A Md

Selleret L Md

Arfi A Md

Bendifallah S Md

Invasive and in situ cervical cancer associated with pregnancy: Analysis from the French cancer network (CALG: Cancer Associé à La Grossesse)

Keywords: Cervical cancer, Pregnancy, Pregnancy-associated cervical cancer, Prognosis, chemotherapy

Purpose: To describe the oncologic and obstetric outcomes of patients diagnosed with invasive cervical cancer (ICC) and in situ adenocarcinoma (ISA) during pregnancy or during the year following delivery. Methods: This retrospective observational study involved a cohort of 28 patients diagnosed with invasive cervical cancer (20 patients) or in situ adenocarcinoma (8 patients) during pregnancy or during the year following delivery who received expert opinion from physicians of the CALG (Cancer Associé à La Grossesse) network between 2005 and 2018. Descriptive results were expressed in median, range and interquartile range (IQR). Results: Between 2005 and 2018, 20 patients with ICC and eight with ISA received expert opinion from physicians of the CALG network. Both ICC and ISA were mostly diagnosed during pregnancy with a median term at diagnosis of 23.3 weeks of gestation (WG) for ICC and 7.3 WG for ISA. Overall, the median age at diagnosis for both ICC and ISAwas33 years. Most ICCs (n=9) had FIGO stage IB2 and five underwent neoadjuvant chemotherapy at a median term of 22.5 WG. Seventeen patients with ICC underwent surgery. Three patients had medical termination of the pregnancy. Two patients experienced recurrence and three died. Median time of follow-up was 59.3 months (IQR 30.5-129.2). Conclusion: Management of cervical cancer during pregnancy is challenging especially in terms of maternal outcomes with a relative poor prognosis requiring a multidisciplinary expert advice.

INTRODUCTION

Cervical cancer is defined as being associated with pregnancy when diagnosed during pregnancy or during the year following delivery. [START_REF] Petrek | Breast cancer during pregnancy[END_REF] Cervical cancer remains the fourth most common cancer in women of childbearing age after breast cancer, melanoma and thyroid cancer [START_REF] Mccormick | Cancer in Pregnancy[END_REF], even though prevention by Human Papillomavirus (HPV) vaccination and screening for cervical cancer has greatly improved in recent years [START_REF] Mitchell | Increasing HPV Vaccination Rates Through National Provider Partnerships[END_REF].

Although cervical cancer during pregnancy is rare, with an estimated incidence of 12 to 15 per 100.000 pregnancies [START_REF] Smith | Cancer associated with obstetric delivery: results of linkage with the California cancer registry[END_REF][START_REF] Eibye | Incidence of Pregnancy-Associated Cancer in Denmark, 1977-2006[END_REF], an increase in the number of cases has been observed during the last two decades particularly because of delayed childbearing [START_REF] Mathews | First Births to Older Women Continue to Rise[END_REF]. It constitutes a major medical challenge related to the impact of treatment on both maternal and fetal outcomes. In the absence of randomized trials guiding treatment, the management of cervical cancer during pregnancy is based on expert consensus taking into account gestational age, stage at diagnosis, the desire to continue the pregnancy, and the trade-off between optimal effective treatment and preservation of the health of the fetus. These women should thus be followed by an expert center.

The aim of the current study was to describe the oncologic and obstetric outcomes of patients diagnosed with invasive cervical cancer (ICC) and in situ adenocarcinoma (ISA) during pregnancy or during the year following delivery.

PATIENTS AND METHODS

The CALG network was created in France by funding from the Institut National du Cancer (INCa) mainly to optimize management of patients with cancer associated pregnancy. Data of women with histologically proven ICC and ISA recorded in the prospective database of the CALG network between June 2005 and July 2018 were retrospectively analyzed. We included all patients who were treated in Tenon University Hospital or whose treating physician had consulted the CALG network.

The extent of the pelvic tumor was assessed by clinical examination and magnetic resonance imaging (MRI). The 2018 FIGO classification based on MRI was used for ICC. [START_REF] Thomeer | Clinical examination versus magnetic resonance imaging in the pretreatment staging of cervical carcinoma: systematic review and meta-analysis[END_REF][START_REF] Mitchell | Early invasive cervical cancer: tumor delineation by magnetic resonance imaging, computed tomography, and clinical examination, verified by pathologic results, in the ACRIN 6651/GOG 183 Intergroup Study[END_REF][START_REF] Bhatla | Cancer of the cervix uteri[END_REF] The following data were recorded: epidemiologic data; circumstances of diagnosis; histologic data; and treatment details. The type of surgery, term and mode of delivery were also recorded as were modalities of NACT and adjuvant chemotherapy and adjuvant therapy.

The Ethical Review Committee (CEROG) approved this study (CEROG 2019-GYN-603). Written informed consent was obtained from all patients.

Statistical analysis was performed with R Studio version 3.5.2. Descriptive results were expressed in median, range and interquartile range (IQR).

RESULTS

3.1.Epidemiologic characteristics of the population

Between June 2005 and July 2018, 20 patients with ICC and eight with ISA were recorded in the database of the CALG network. Characteristics of the patients are summarized in Table 1.

3.2.Patients with ICC

Of the 20 patients, twelve were treated in Tenon University Hospital. Eighteen cases were diagnosed during pregnancy at a median term of 23. Eleven of the 20 ICCs were squamous-cell carcinomas, eight were invasive adenocarcinomas and one was an undifferentiated carcinoma. Histologic type and FIGO classification of the ICCs are shown in Table 2.Most of the patients (n=14) were stage IB2 at diagnosis. Four (22.2%) patients underwent a PET-scan, one during pregnancy at 28 WG.

Sixteen patients (80%) underwent MRI, 13 during pregnancy.

Medical and surgical management of patients with ICC

Five patients underwent NACT (Table 3) at a median term 22.0 WG [min18.7-max23.6; IQR (20.4; 22.8)].

Patient 1: The exact FIGO stage was not evaluable for this patient as the MRI at 22 WG was normal and no clinical exam details were available. This patient was diagnosed with invasive adenocarcinoma at 19 WG following a cervical smear test HSIL HPV 18+. She received four cycles of carboplatin and taxol. She had a cesarean delivery (C-section) at 37+1 WG and underwent radical hysterectomy. Histologic findings did not show any tumor residue. Eighteen patients (80%) with ICC underwent surgery (Table 3).One patient with IB1 stage adenocarcinoma underwent pelvic lymphadenectomy by laparoscopy during the second trimester of pregnancy. No details of the lymph node status or surgical and pregnancy outcomes were available.

One patient with a IIB stage squamous-cell carcinoma underwent a retroperitoneal para-aortic lymphadenectomy at 11+5 WG. None of the 18 lymph nodes removed were positive. She refused medical pregnancy termination and received concomitant radio-chemotherapy resulting in uteri fetal death at 17+4 WG. She underwent a completion radical hysterectomy 6 weeks after radiotherapy. Histology did not find tumor residue.

Three patients underwent medical termination of pregnancy: one at 30 WG because of myelomeningocele diagnosed in utero and the two others during the first trimester for FIGO IB2 and IIIC1.Of the 18 patients with ICC diagnosed during pregnancy, eight gave birth to healthy children by C-section. Median term at delivery was 37 WG (IQR 35.5; 37). Of the remaining 10 patients, three had a medical termination of pregnancy, and one had a fetal death in utero. No data on pregnancy outcomes were available for the six remaining patients treated out of Tenon Hospital.

Follow-up and recurrence in patients with ICC

The median time of follow-up was 51.2months (IQR 24.5; 156.4). Two patients experienced recurrence and three died of the disease. In the first case, recurrence occurred 4 years after diagnosis of an adenocarcinoma on the Troisier lymph node; she died 6.5 years after the first diagnosis. In the second case, the patient was diagnosed at 33+2 WG with welldifferentiated IB1 stage squamous-cell carcinoma. She underwent radical hysterectomy and pelvic lymphadenectomy 4 weeks after C-section at 37 WG. Out of the 10 pelvic lymph nodes removed, one was positive. The patient underwent para-aortic lymphadenectomy one month later removing 18 negative lymph nodes. Final FIGO stage was IIIC1. She received adjuvant radiotherapy and chemotherapy with a carboplatin-taxol regimen followed by paclitaxel with cisplatin. She relapsed at the iliac level 3 months after radical hysterectomy and pelvic lymphadenectomy and died 10 months after initial diagnosis.

The third patient underwent NACT for a differentiated IB3 squamous cell carcinoma at 21 WG. She had two cycles of NACT by taxol and carboplatin. Reassessment showed local progression and the patient underwent a C-section at 28 WG with pelvic and para-aortic lymphadenectomy. The patient continued to progress despite treatment and died 4 months after diagnosis.

Patients with ISA

Of the eight patients, four were treated in Tenon Hospital. Five were diagnosed on biopsy and the three others on conization because of suspicion of cervical intraepithelial neoplasia (CIN) 2 or 3 on biopsy. Four were diagnosed during pregnancy with a median term at diagnosis of 7.3 WG [min 6.1-max 8; IQR (6.7; 7.7)]; four were diagnosed in the year following delivery. None were HIV positive. Median age at diagnosis was 33 years old [min 26-max 43; IQR (30.3; 34.5)]. One patient was a current smoker. All the patients underwent surgery. Two of these had a hysterectomy: one after conization followed by trachelectomy, and the second in a 43-year-old patient who refused fertility sparing surgery. The six remaining patients underwent a conization including one during pregnancy at a term of 10+6 WG. Table 4 summarizes the surgical management of the women with ISA. Median term of follow-up was 79.00 months (IQR 63.1; 103.7) without recurrence.

DISCUSSION

The present study reports the experience of the CALG network on the management of cervical cancer associated with pregnancy and confirms the possibility of maintaining the pregnancy in spite of a relatively high rate of recurrence and death.

In our population, 55% had squamous-cell carcinoma and 40% had adenocarcinoma. This distribution of histologic types is in agreement with those of the meta-analysis of Halaska et al [START_REF] Halaska | Characteristics of patients with cervical cancer during pregnancy: a multicenter matched cohort study. An initiative from the International Network on Cancer, Infertility and Pregnancy[END_REF] but differs from those reported in a literature review by Song et al [START_REF] Song | Efficacy of neoadjuvant platinum-based chemotherapy during the second and third trimester of pregnancy in women with cervical cancer: an updated systematic review and meta-analysis[END_REF]who reported that 73.8 % of the women (45 of 61) were diagnosed with squalors-cell carcinoma and 14.8 %with invasive adenocarcinoma. Similarly, Kärrberg et al [START_REF] Kärrberg | Support for down-staging of pregnancyassociated cervical cancer[END_REF]reported rates of 76.6 % (36 of 47) and 17% ( 8) for squamous-cell carcinoma and adenocarcinoma, respectively. This apparent discrepancy cannot be related to differences in the epidemiologic characteristics: our population was similar to those of previous studies with most patients being Caucasian [START_REF] Halaska | Characteristics of patients with cervical cancer during pregnancy: a multicenter matched cohort study. An initiative from the International Network on Cancer, Infertility and Pregnancy[END_REF][START_REF] Song | Efficacy of neoadjuvant platinum-based chemotherapy during the second and third trimester of pregnancy in women with cervical cancer: an updated systematic review and meta-analysis[END_REF] but by difficulties in detecting adenocarcinoma. Several publications suggest that screening is less effective for adenocarcinoma than for squamous-cell carcinoma of the cervix [START_REF] Pettersson | Cervical cancer in the screening era: who fell victim in spite of successful screening programs?[END_REF][START_REF] Zappa | Lower protection of cytological screening for adenocarcinomas and shorter protection for younger women: the results of a casecontrol study in Florence[END_REF][START_REF] Castanon | Is cervical screening preventing adenocarcinoma and adenosquamous carcinoma of the cervix?[END_REF][START_REF] Mancini | Incidence and survival trends of cervical adenocarcinoma in Italy: Cytology screening has become more effective in downstaging the disease but not in detecting its precursors[END_REF]. This is because glandular lesions are mainly located in the cervical canal making sampling difficult and thus reducing the chance of early detection [START_REF] Castanon | Is cervical screening preventing adenocarcinoma and adenosquamous carcinoma of the cervix?[END_REF][START_REF] Davy | Cervical cancer: effect of glandular cell type on prognosis, treatment, and survival[END_REF][START_REF] Pirog | Cervical Adenocarcinoma: Diagnosis of Human Papillomavirus-Positive and Human Papillomavirus-Negative Tumors[END_REF]. Moreover, several authors reported a lower accuracy of colposcopy in pregnant than in nonpregnant women [START_REF] Morimura | Cervical cytology during pregnancy _ comparison with non-pregnant women and management of pregnant women with abnormal cytology[END_REF][START_REF] Russa | Invasive cervical cancer in pregnancy[END_REF]. Finally, Ciavattini et al observed a better reliability of colposcopy in the two first trimesters and especially before 20 WG while the median term at diagnosis was 22.3 WG in the current study.

In contrast to ISA, which was often diagnosed during the first trimester of pregnancy, most of the patients with ICC had a FIGO stage IB2 and were mainly diagnosed during the second half of the second trimester of pregnancy. Halaska et al [START_REF] Halaska | Characteristics of patients with cervical cancer during pregnancy: a multicenter matched cohort study. An initiative from the International Network on Cancer, Infertility and Pregnancy[END_REF] recently published a multicenter matched cohort that gathered data from six European centers between 1990 and 2012 with a total of 132 pregnant patients. Their data were similar to our study with a median age at diagnosis of 34 years but a median gestational age at diagnosis slightly earlier at 18.4 WG, and with most patients (80.3 %) being diagnosed with early stage disease. However, FIGO staging was essentially based on clinical examination, with a risk of misdiagnosis as previously demonstrated [START_REF]Clinical examination versus magnetic resonance imaging in the pretreatment staging of cervical carcinoma: systematic review and meta-analysis[END_REF][START_REF] Mitchell | Early Invasive Cervical Cancer: Tumor Delineation by Magnetic Resonance Imaging, Computed Tomography, and Clinical Examination, Verified by Pathologic Results, in the ACRIN 6651/GOG 183 Intergroup Study[END_REF], while in the current study MRI was often performed [START_REF] Song | Efficacy of neoadjuvant platinum-based chemotherapy during the second and third trimester of pregnancy in women with cervical cancer: an updated systematic review and meta-analysis[END_REF]. In 2018, the FIGO classification was revised to take into account imaging and pathologic findings [START_REF] Bhatla | Cancer of the cervix uteri[END_REF]. The contribution of imaging in this new classification is a major asset in the staging of cervical cancer associated with pregnancy. Physiologic changes, especially the presence of cervical edema related to pregnancy, might alter the clinical assessment of tumor size [START_REF] Michael | Pregnancy-related changes: a retrospective review of 278 cervical smears[END_REF]and MRI can be safely performed during pregnancy not only to evaluate the initial FIGO stage but also to determine response to chemotherapy and hence to adapt therapy [START_REF]Clinical examination versus magnetic resonance imaging in the pretreatment staging of cervical carcinoma: systematic review and meta-analysis[END_REF][START_REF] Mitchell | Early Invasive Cervical Cancer: Tumor Delineation by Magnetic Resonance Imaging, Computed Tomography, and Clinical Examination, Verified by Pathologic Results, in the ACRIN 6651/GOG 183 Intergroup Study[END_REF]. Moreover, patients with lesions under 2 cm are eligible for conization or simple trachelectomy (used in 21% of our population) which appears to be a safe and effective treatment during pregnancy [START_REF] Salvo | Simple trachelectomy with pelvic lymphadenectomy as a viable treatment option in pregnant patients with stage IB1 (≥2 cm) cervical cancer: Bridging the gap to fetal viability[END_REF][START_REF] Umemoto | Obstetrical prognosis of patients who underwent vaginal radical trachelectomy during pregnancy[END_REF][START_REF] Yoshihara | The Safety and Effectiveness of Abdominal Radical Trachelectomy for Early-Stage Cervical Cancer During Pregnancy[END_REF].

In the current study, excluding the patient with a myelomeningocele diagnosed in utero, only two patients (11%) underwent a medical termination for advanced stage disease (IIB). This is a lower rate than that noted in Halaska et al study(26.5%) [START_REF] Halaska | Characteristics of patients with cervical cancer during pregnancy: a multicenter matched cohort study. An initiative from the International Network on Cancer, Infertility and Pregnancy[END_REF] and suggests that continued pregnancy is possible for most women with cervical cancer associated with pregnancy. Moreover, Perrone et al [START_REF] Perrone | Cervical cancer in pregnancy: Analysis of the literature and innovative approaches[END_REF] reported that termination of pregnancy was refused by two-thirds of patients even in the case of advanced cervical cancer. This raises the issue of NACT during pregnancy. After exclusion of the three patients undergoing pregnancy termination and the two patients diagnosed in the postpartum period, NACT was used in onethird of our population. Song et al [START_REF] Song | Efficacy of neoadjuvant platinum-based chemotherapy during the second and third trimester of pregnancy in women with cervical cancer: an updated systematic review and meta-analysis[END_REF]published a meta-analysis including 39 studies published between 1997 and 2018 including 88 patients with cervical cancer associated with pregnancy treated by platinum-based chemotherapy. In their meta-analysis, 87.5 % of the patients were diagnosed with early stage (I and IIA -FIGO 2009) and 12.5% with advanced stage (IIB, III and IV) disease. Eighty-eight babies were delivered including 71 completely healthy neonates. In 2018, the ESGO-ESTRO-ESP [START_REF] Cibula | The European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology Guidelines for the Management of Patients With Cervical Cancer[END_REF]guidelines point out that, in view of the low incidence of cervical cancer during pregnancy and the lack of high level of evidence, patients should be exclusively managed in expert centers with the advice of a multidisciplinary committee taking into account gestational age, FIGO stage, the maternal and fetal risk-benefit ratio of each option, and the desire of the patient to maintain the pregnancy.

No cancer treatment or surgical staging is proposed before 12 WG [START_REF] Cardonick | Use of chemotherapy during human pregnancy[END_REF][START_REF] Köhler | How much platinum passes the placental barrier? Analysis of platinum applications in 21 patients with cervical cancer during pregnancy[END_REF][START_REF] Pereg | Cancer in pregnancy: gaps, challenges and solutions[END_REF]. Over this term, surgery is recommended for early stage disease (Stage Ia-Ib1) and NACT for advanced stages (Ib2-IIa) [START_REF] Perrone | Cervical cancer in pregnancy: Analysis of the literature and innovative approaches[END_REF].

Nevertheless, it should not be forgotten that cervical cancer associated with pregnancy is a severe disease. In the present study, two recurrences and three deaths were observed.

Song et al [START_REF] Song | Efficacy of neoadjuvant platinum-based chemotherapy during the second and third trimester of pregnancy in women with cervical cancer: an updated systematic review and meta-analysis[END_REF]reported recurrence in 19.8 % of patients, 90 % of whom died from the disease.

Similarly, Halaska et al [START_REF] Halaska | Characteristics of patients with cervical cancer during pregnancy: a multicenter matched cohort study. An initiative from the International Network on Cancer, Infertility and Pregnancy[END_REF] reported a recurrence rate of 20% and a death rate of 14% among pregnant women similar to that observed for non-pregnant women (21% of recurrences and 18 % of deaths).

In our study, the rate of surgery during pregnancy, NACT, expectant management till delivery, and premature delivery before treatment were 59%, 29%, 12% and 12%, respectively. Our data are partially in contrast with those of Halaska et al [START_REF] Halaska | Characteristics of patients with cervical cancer during pregnancy: a multicenter matched cohort study. An initiative from the International Network on Cancer, Infertility and Pregnancy[END_REF]reporting a 17.4% rate of surgery, 16.7% of NACT, 26.5% of expectant management, and 12.9% of premature delivery [START_REF] Halaska | Characteristics of patients with cervical cancer during pregnancy: a multicenter matched cohort study. An initiative from the International Network on Cancer, Infertility and Pregnancy[END_REF]. This apparent discrepancy can be explained by the long study period of Halaskaet al's cohort and variations according to countries. Some limits of the current study deserve to be underlined. First, the retrospective nature cannot exclude all biases. Second, the low sample size linked to the low incidence of the disease could be a source of misinterpretation. However, it is clear that the lower incidence of pregnancy termination and higher number of NACT administered compared to previous studies confirms the trend for a more conservative management of pregnancy which is also probably linked to delayed pregnancy in western countries. Finally, although all the women included were followed either by the CALG network or by physicians who sought advice from the network, some physicians contributed to analysis bias by failing to report some data especially about treatment options and follow-up.

CONCLUSION

This study reports the experience of the CALG network on the management of cervical cancer associated with pregnancy and supports a more conservative management of cervical cancer associated with pregnancy.

  3 weeks of gestation (WG) [min7-max34.3; IQR (17.1; 28.0)] and two during the year following delivery (8 weeks post-partum in both cases). The median age at diagnosis was 33 years old [min26-max42; IQR (29.0; 36.8)]. Five patients were current smokers, none were HIV positive.

Patient 2 :

 2 This patient was diagnosed with squamous-cell carcinoma at 13 WG with a FIGO IB1 stage on MRI. She underwent a conization and cervical cerclage at 13+6 WG. Histologic findings showed a moderately differentiated squamous-cell carcinoma of 16x10x5 mm with embol. She began chemotherapy with carboplatin and taxol at 22 WG. She underwent a Csection at 32 WG with concomitant radical hysterectomy and pelvic lymphadenectomy. No tumor residue was found. None of the 17 lymph nodes removed were positive. Patient 3: In this patient a FIGO IB2 stage invasive adenocarcinoma was diagnosed at 20 WG. MRI revealed a lesion of 35 mm. She underwent NACT with carboplatin and paclitaxel at 23+4 WG. MRI reassessment found a tumor residue of 17 mm. She had a C-section at 37 WG and underwent radical hysterectomy with pelvic lymphadenectomy by laparoscopy 2weeks later. Histologic findings showed a 25x26x9 mm tumor residue of invasive adenocarcinoma p16 + p53 30%.Of the 24 lymph nodes removed, none were positive. Patient 4: The fourth patient was diagnosed with IB3 stage squamous-cell carcinoma at 21 WG. The MRI found a lesion of 65 mm. The patient refused medical pregnancy termination. She had two cycles of NACT by taxol and carboplatin. Reassessment showed local progression and revealed emergence of a dilatation of the right kidney cavities and bilateral external iliac lymph nodes. She underwent a C-section at 28 WG with pelvic and para-aortic lymphadenectomy. Out of the 15 para-aortic lymph nodes removed, five were positive. She received concomitant radio-chemotherapy. The patient continued to progress despite treatment and died 4 months after diagnosis. Patient 5: The last patient underwent NACT for aIB2 stage squamous-cell carcinoma diagnosed at 25+6 WG. Initial MRI found a lesion of 32x24x27 mm. She began chemotherapy with carboplatin and taxol. MRI reassessment found a tumor residue of 18x6x14 mm and positron emission tomography (PET) scan showed a partial metabolic response. She underwent radical hysterectomy, pelvic and para-aortic lymphadenectomy concomitant to a C-section. No data on final histology was available.

Funding:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability:

Data supporting the results reported in the article can be found by requested at the corresponding author.

Ethics approval and consent to participate:

The Ethical Review Committee (CEROG) approved this study (CEROG 2019-GYN-603).

Written informed consent was obtained from all patients.

The study was performed in accordance with the Declaration of Helsinki.

Consent for publication:

Our manuscript doesn't contain any individual person's data in any form (including individual details, images or videos).

Conflict of interest:

The authors declare no conflict of interest.

Authorship:

Puchar A made the analysis and wrote the article.

Boudy AS implement the database and helped write the manuscript.

Selleret L, Arfi A and Owen C implement the database.

Bendifallah S helped write the manuscript.

Darai E helped write the manuscript.


Tin- Shang-Yu Yu

Wei Tung

Yu-Chih Lee

Chang-Chiang Chen

∥,⊥ Hsiao-Wen Zan

Hsin-Fei Meng

Chia-Jung Lu

Olivier Soppera

Tin-Wei Tung

A Versatile Method to Enhance the Operational Current of Air-Stable Organic Gas Sensor for Monitoring of Breath Ammonia in Hemodialysis Patients

Keywords: Ammonia, breath, current enhancement, gas sensor, hemodialysis, POC device, stable performance

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

To enable the development of personalized medicine in the clouds and point-of-care (POC) applications, numerous research studies focused on developing non-invasive detecting methods to monitor disease related signals from human breath, saliva, tear, sweat, etc. [START_REF] Mascini | Biosensors for biomarkers in medical diagnostics[END_REF][START_REF] Tricoli | Miniaturized Bio-and Chemical-Sensors for Point-of-Care Monitoring of Chronic Kidney Diseases[END_REF] In particular, breath analysis is considered as a promising approach due to the easy availability of breath and its potential association with several human diseases. [START_REF] Kim | A review of breath analysis for diagnosis of human health[END_REF][START_REF] Arasaradnam | Review article: next generation diagnostic modalities in gastroenterology -gas phase volatile compound biomarker detection[END_REF] Human breath can contain many biomarkers associated with various diseases, typically in the form of volatile gas molecules. For example, nitric oxide (NO), acetone (ACE), carbon monoxide (CO) and ammonia in breath are regarded as biomarkers for asthma, diabetes, lung related conditions, and kidney diseases, respectively. [START_REF] Nagasaki | Using Exhaled Nitric Oxide and Serum Periostin as a Composite Marker to Identify, Severe/Steroid-Insensitive Asthma[END_REF][START_REF] Honkoop | Symptom-and fraction of exhaled nitric oxide-driven strategies for asthma control: A cluster-randomized trial in primary care[END_REF][START_REF] Deng | Determination of acetone in human breath by gas chromatographymass spectrometry and solid-phase microextraction with on-fiber derivatization[END_REF][START_REF] Turner | Breath acetone concentration decreases with blood glucose concentration in type I diabetes mellitus patients during hypoglycaemic clamps[END_REF][START_REF] Deveci | The measurement of exhaled carbon monoxide in healthy smokers and nonsmokers[END_REF][START_REF] Lee | A cross-sectional study of exhaled carbon monoxide as a biomarker of recent household air pollution exposure[END_REF][START_REF] Neri | Real-time monitoring of breath ammonia during haemodialysis: use of ion mobility spectrometry (IMS) and cavity ring-down spectroscopy (CRDS) techniques[END_REF][START_REF] Limeres | A breath ammonia analyser for monitoring patients with end-stage renal disease on haemodialysis[END_REF] Among these biomarkers, breath ammonia attracts lots of attention because of the prevalence of chronic kidney disease (CKD) which is 14.8 percent in the United States adult population, suggesting that about 30 million Americans may have CKD. [START_REF] Saran | US Renal Data System 2018 Annual Data Report: Epidemiology of Kidney Disease in the United States[END_REF] Besides, the economic burden of dialysis and CKD is increasing worldwide. [START_REF] Wang | The Economic Burden of Chronic Kidney Disease and End-Stage Renal Disease[END_REF] Ammonia, produced by the catabolism of protein, is converted into urea through the urea cycle in the liver. Then the urea is removed from the body by the kidneys. 15 Because of impairment of elimination of urea, CKD patients have urea nitrogen levels higher than normal range. Therefore, for patients with end stage renal disease, the removal of urea nitrogen mainly relies on dialysis treatment. Typically, to determine the level of nitrogen in the body and the adequacy of dialysis treatment, a blood test is performed to acquire blood urea nitrogen (BUN) level. [START_REF] Locatelli | Dialysis dose and frequency[END_REF] However, the fluctuation of the BUN level is related to the nutrition intake. Higher dietary protein intake may lead to an increase in BUN level that becomes harmful, while the insufficient protein intake could result in malnutrition condition. Therefore, a low-cost, easy-handled, and real-time monitoring device is needed to monitor the health condition in the CKD and dialysis patient.

Many studies have revealed that the ammonia concentration in exhaled breath is highly related to the BUN level. [START_REF] Neri | Real-time monitoring of breath ammonia during haemodialysis: use of ion mobility spectrometry (IMS) and cavity ring-down spectroscopy (CRDS) techniques[END_REF][START_REF] Limeres | A breath ammonia analyser for monitoring patients with end-stage renal disease on haemodialysis[END_REF][START_REF] Wakabayashi | Measurement of the expiratory ammonia concentration and its clinical significance[END_REF][START_REF] Narasimhan | Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis[END_REF][START_REF] Endre | Breath ammonia and trimethylamine allow real-time monitoring of haemodialysis efficacy[END_REF][START_REF] Popa | Ethylene and ammonia traces measurements from the patients' breath with renal failure via LPAS method[END_REF][START_REF] Davies | Quantitative analysis of ammonia on the breath of patients in end-stage renal failure[END_REF] To date, several instruments for the detection of breath volatile organic compounds have been well established in the clinical trial, such as selected ion flow tube mass spectrometry, gas chromatography/mass spectrometry, and laser photoacoustic spectroscopy. [START_REF] Davies | Quantitative analysis of ammonia on the breath of patients in end-stage renal failure[END_REF][START_REF] Fan | Applications of Hadamard transform-gas chromatography/mass spectrometry to the detection of acetone in healthy human and diabetes mellitus patient breath[END_REF][START_REF] Hibbard | Breath Ammonia Analysis: Clinical Application and Measurement[END_REF] However, even with high precision and selectivity of these instruments, some limitations in terms of size and cost impede the integration of these techniques into portable POC devices. In the past few years, some researches (including our prior works) based on organic devices have been reported for breath ammonia (BA) detection. [START_REF] Li | High Performance Field-Effect Ammonia Sensors Based on a Structured Ultrathin Organic Semiconductor Film[END_REF][START_REF] Hibbard | Point of Care Monitoring of Hemodialysis Patients with a Breath Ammonia Measurement Device Based on Printed Polyaniline Nanoparticle Sensors[END_REF][START_REF] Setka | Nanostructured Polypyrrole-Based Ammonia and Volatile Organic Compound Sensors[END_REF][START_REF] Chuang | Organic Gas Sensor with an Improved Lifetime for Detecting Breath Ammonia in Hemodialysis Patients[END_REF][START_REF] Guntner | Sniffing Entrapped Humans with Sensor Arrays[END_REF] These studies demonstrated the possibility of organic-based sensors for low cost and miniaturized sensing devices. In 2013, Hibbard et al. have shown that a polyaniline nanoparticle-based sensor integrated with a breath sampling system was capable of measuring human BA in the hemodialysis (HD) patients, and obtained a good correlation coefficient of 0.96 between BA and BUN levels. [START_REF] Hibbard | Point of Care Monitoring of Hemodialysis Patients with a Breath Ammonia Measurement Device Based on Printed Polyaniline Nanoparticle Sensors[END_REF] However, despite all the advantages of organic sensors, most of them suffer from lifetime issue caused by degradation under air ambiance, which restricts the usage to the single-use disposable sensor. In 2017, our team reported a Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4-(N-(4sec-butylphenyl)diphenylamine)) (TFB) based sensor that exhibits a high sensitivity in part per billion (ppb) regime and a good storage lifetime in the air. The sensor was further utilized to measure the BA in HD patients. The correlation coefficient between natural logarithm of BA concentrations (ln(BA)) and natural logarithm of BUN level (ln(BUN)) was 0.82. [START_REF] Chuang | Organic Gas Sensor with an Improved Lifetime for Detecting Breath Ammonia in Hemodialysis Patients[END_REF] Although an organic sensor with improved lifetime was demonstrated, the low operational current as a few tens of nanoampere of the sensors caused by the intrinsically low carrier mobility of organic materials makes it hardly compatible with low-cost read-out circuitry. [START_REF] Tricoli | Miniaturized Bio-and Chemical-Sensors for Point-of-Care Monitoring of Chronic Kidney Diseases[END_REF] Thus, to achieve long lifetime and high operational current in organic based sensor still remain in challenge.

In this work, we propose a new double active layer (DL) strategy to enhance the operational current of the organic sensor. Combined with an acceptor-donor polymer Poly [3,4-b]thiophenediyl]] (PBDTTT-C-T) as a new sensing material, the DL sensor with a stable ammonia sensitivity and an enhanced operational current of microampere level is demonstrated. The sensor structure is based on numerous cylindrical nanopores. [START_REF] Chuang | Modulated gas sensor based on vertical organic diode with blended channel for ppb-regime detection[END_REF] The DL strategy exploited an ultra-thin layer of Poly(3hexylthiophene-2,5-diyl) (P3HT) to serve as a work function buffer for carrier transfer, and showed no substantial effect on sensing stability. The effect of this strategy was carefully investigated by several steps. The vertical composition profile was firstly confirmed by coupling UV-vis spectrophotometer and reactive ion etching. Then, a comparative study of the sensing performance and the surface morphology of different sensing layers were conducted to investigate the sensing behavior overtime. Remarkably, by replacing the PBDTTT-C-T with TFB, we also show the high compatibility of our strategy to different organic sensing material. Finally, we examined the selectivity of PBDTTT-C-T/P3HT DL sensor and utilized the sensors to conduct a long-term tracing of BA concentration in two HD patients. A good correlation coefficient of 0.96 between In(BA) and ln(BUN) was recorded, demonstrating an easy and feasible approach for integrating organic sensor in POC devices.

[[4,8- bis[5-(2-ethylhexyl)-2-thienyl]benzo[1,2-b:4,5-b']dithiophene- 2,6-diyl][2-(2-ethyl-1-oxohexyl)thieno

EXPERIMENTAL SECTION

Fabrication of the Ammonia Sensor. Figure 1(a) shows the concept of cylindrical nanopore architecture of ammonia sensors. The cylindrical nanopore were fabricated via the colloid lithography method. Firstly, a pre-cleaned indium tin oxide (ITO) glass substrate with designed pattern was treated with oxygen plasma (100 W 600 s) to increase surface energy and form hydrophilic surface. A cross-linkable poly(4vinylphenol) (PVP) layer (thickness ~ 300 nm) was then spin-coated on the ITO substrate and annealed at 200 °C for 1 h. After annealing, a thin layer of P3HT was spin-coated onto PVP and annealed at 200 °C for 10 min. This P3HT layer served as a surface modification layer to ensure a good adhesion of negatively charged polystyrene (PS) nano-spheres (Fluka, 200 nm diameter). [START_REF] Lin | Review of a solution-processed vertical organic transistor as a solid-state vacuum tube[END_REF] To form monolayer PS nanospheres which served as a shadow mask for Al electrode deposition, the substrate was immersed into PS solution (10 wt% in ethanol) for 90 s and dipped into boiling isopropanol (IPA) for 8 s to remove excess PS spheres. Then the substrate was dried immediately by nitrogen flow. After Al deposition, the PS nano-spheres were then peeled off with 3M Scotch Tape to obtain porous top electrode with the thickness of 60 nm. The porous top electrode further served as another shadow mask for subsequent oxygen plasma etching. During etching process, the PVP in the area not covered by the top electrode was removed, thus forming the cylindrical nanopore structure. Finally, three different types of active layer were deposited onto the structure and annealed at different temperatures, enabling the conduction of vertical current. For sidewall single layer (SL) sensors, PBDTTT-C-T (0.9 wt% in chlorobenzene), TFB (0.7 wt% toluene), or P3HT (1.5 wt% in chlorobenzene) was deposited on the structure by blade-coating (PBDTTT-C-T and TFB)or by spin-coating (P3HT) and annealed at 80°C for 10 min, 200°C for 10 min, and 180°C for 40 min, respectively. The molecular structure of PBDTTT-C-T and P3HT are shown in Figure 1(b). The thickness of PBDTTT-C-T and P3HT single sensing layer was around 41 nm and 35 nm, respectively. For sidewall double layer (DL) sensors, P3HT (0.5 wt% in chlorobenzene) was spin-coated onto the structure and annealed at 200°C for 10 min prior to the deposition of PBDTTT-C-T and TFB, forming a thin layer (thickness ~ 13 nm) as work function buffer. For blended layer sensors, the weight ratio between PBDTTT-C-T and P3HT was fixed at 5:1 (0.9 wt% in chlorobenzene). The blending layer was blade-coated on the structure and annealed at 80°C for 10 min. After fabrication, all sensors were stored under air atmosphere, covered by Al foils to prevent the photo-induced degradation. Figure 1(c) shows the cross-section image of PBDTTT-C-T DL sensors by scanning electron microscope (SEM). It can be observed that the active layer wraps well around the structure, increasing the surface to volume ratio to achieve ppb-range sensitivity. Thin Film Characterization. The properties of organic sensing layers were characterized by UV-vis spectrophotometer and the atomic force microscopy (AFM). The sensing materials were deposited on plain glass with the same conditions as those on cylindrical nanopore structure. The absorption spectra of organic thin films were recorded by UV-vis spectrophotometer (Agilent Technologies Cary 50). To further verify the vertical composition profile of the double layer film, the film was etched incrementally by reactive ion etching and subsequently recorded the absorption spectrum. The reactive ion etching (SYSKEY) with gas flow of 40 sccm and RF generator power of 30W was used. The thickness of films was confirmed by microfigure profilometer (Kosaka Laboratory Ltd., ET200) before and after each etching steps. The surface morphologies of organic thin films were investigated by using AFM (Picoplus 5500 Agilent) in resonant mode. Si 3 N 4 tips with a typical resonance frequency of 285 kHz (stiffness 42 N/m) were used. The thin film of sensing material was formed on the top of PVP film to provide a same surface condition as on cylindrical nanopore structure.

Ammonia Measurement System. The schematic and picture of ammonia sensing system are shown in Figure 2(a),(b).

The system comprised a desiccation cylinder (sodium hydroxide (NaOH)) to provide a steady relative humidity (RH) around 10% and eliminate the interference from ambiance RH variation, a sensing chamber where the sensors are mounted, an electric source measurement unit (Keysight U2722A USB modular) to apply operational voltage on sensor and read out electric signal, a rotameter to control the flow at 500 cc/min, a pump to provide a constant air flow, and a humidity measurement unit to confirm the RH value during experiments. To evaluate the sensing performance, the target gas was introduced into the system automatically by step-motor. The concentration of target gas was determined by the injection speed of step-motor and background air flow. [START_REF] Chuang | Organic Gas Sensor with an Improved Lifetime for Detecting Breath Ammonia in Hemodialysis Patients[END_REF] However, the measuring time of each target gas was fixed at 30 s and the operational voltage of all sensors was fixed at 5V. Therefore, a comparable calibration curve between different sensors can be created by plotting response versus ammonia concentration.

The response was calculated by ΔI/I0 x 100. Measurement of Breath Ammonia in HD Patients. We recruited two stable patients on long-term HD treatment, three times per week for four to five hours per HD session. BA concentrations and BUN levels were checked before and after HD every one to two months. Total three times of pre-and post-hemodialysis measurements of BA and BUN levels were performed in HD center at National Taiwan University Hospital Hsin-Chu Branch. For breath sample collection, a sample bag (Taiwan ISO Biotec. Co.,Ltd.) that can seal by a gas valve was used. Firstly, after fasting for at least four hours, the patient blew directly into the sample bag and sealed the valve. Next, the breath sample was filtered through a NaOH tube to remove most water molecular. Finally, the breath sample was introduced into the measurement system with a fixed measuring time of 30 s. The whole collection and measurement process was completed within a few minutes. The BA of patients were examined pre-and post-hemodialysis by PBDTTT-C-T/P3HT DL sensors. The BUN measurements were performed immediately after BA measurements by the hospital laboratory.

RESULTS AND DISCUSSION

To study the mechanism of double active layer, the PBDTTT-C-T-based gas sensors were fabricated with SL and DL configuration of active layer. PBDTTT-C-T will be denoted as CT in the following part. Figure 3(a),(b) presents the real-time current measurements of CT-SL and CT/P3HT-DL sensors and their responses to different concentration of ammonia. For both configuration, three individual sensors were tested simultaneously. The cyan region represents the time under ammonia exposure, which was set to 30 s; in other cases, the sensors was purged with constant dry air. The ammonia concentrations were 500 ppb, 1500 ppb, 2500 ppb, and 5000 ppb. From the real-time current measurement, it can be noticed that the current level of CT/P3HT-DL sensor is roughly 1 order larger than CT-SL sensor. Moreover, in both sensors, the current drops significantly when the sensors are exposed to ammonia. This phenomenon is usually explained by a redox reaction. [START_REF] Tiwari | Poly-3-hexylthiophene based organic field-effect transistor: Detection of low concentration of ammonia[END_REF][START_REF] Zhou | An ammonia detecting mechanism for organic transistors as revealed by their recovery processes[END_REF] For CT, a p-type organic material, the hole is major carrier in conjugated chain. When CT sensor is exposed to ammonia, a reducing agent, part of the major carriers in CT are trapped by ammonia's lone electron pairs. The hole concentration in CT, thus, becomes lower and results in a decrease of current. By fixing the ammonia exposure time, the current variation ratio which is the current drop divided by the initial current can be defined as the sensing response. The average responses to different ammonia concentrations and standard deviation of three sensors were plotted in the lower part of figures. The sensing responses of CT-SL sensor to 500 ppb, 1500 ppb, 2500 ppb, and 5000 ppb were -25.8%, -49.2%, -58.9%, and -71.1%, respectively; In the other hand, those of CT/P3HT-DL sensor were -12.5%, -29.2%, -37.6%, -50.3%, respectively. Both sensors displayed a good sensitivity to ammonia. However, the CT/P3HT-DL sensors exhibited a lower sensing response compared to CT-SL. This result can be attributed to the elevation of current level in CT/P3Ht-DL sensor, which decrease the relative current variation upon ammonia exposure. Additionally, the increase of current level can be ascribed to the good matching of work function in DL configuration, as shown in Figure 3(c). The current was injected from the ITO electrode to the Al electrode. The work functions of ITO and Al are 4.8 eV and 4.3 eV, respectively. It is noted that the work function of ITO after oxygen plasma treatment can be slightly increased. [START_REF] He | Work function changes of plasma treated indium tin oxide[END_REF] The presence of P3HT with a HOMO level about 5 eV served as a buffer layer, favored the hole injection property between ITO and CT layer. An enlarged current level can, hence, be expected. To confirm this assumption, I-V characteristic measurement was performed on both sensors. The current versus voltage square was plotted to verify the SCLC dominated conduction, shown in Figure 3(d).

Although a linear dependence was observed in both curve, CT/P3HT-DL sensor showed a lower voltage requirement to reach SCLC regime. According to SCLC mechanism, this result indicated that the injection barrier of CT/P3HT-DL sensor is lower than those of CT-SL sensor, which was coincident with our assumption. [START_REF] Dacuna | Modeling space-charge-limited currents in organic semiconductors: Extracting trap density and mobility[END_REF][START_REF] Nicolai | Electron traps in semiconducting polymers: Exponential versus Gaussian trap distribution[END_REF] To further investigate the sustainability of current enhancement and the effect on sensing stability overtime, the electrical properties and response of sensors were followed in a period of 15 days. Figure S1(a),(b) in the supporting information shows the I-V characteristic of CT-SL and CT/P3HT-DL sensor on different days. The current values of both sensors at 5 V were extracted and plotted over storage day, as shown in Figure 4(a). A similar evolution trend of current was observed. In the first week, the current of both sensors declined to a certain extent and then became stable. However, CT/P3HT-DL sensor displayed a constantly superior current level around 1 order to SL sensor. Moreover, the current is sustained around microampere level, confirming that the DL strategy can provide a sustainable current enhancement. Next, the stability of the sensors was examined by the evolution of calibration curves overtime. The calibration curves were built up by plotting the response as a function of ammonia concentration. The calibration curves of CT-SL, CT/P3HT-DL and P3HT-SL on different days are displayed in Figure 4 ammonia. However, it is well known that P3HT degrade very fast under air environment. [START_REF] Sai | First principles study of photo-oxidation degradation mechanisms in P3HT for organic solar cells[END_REF][START_REF] Rashmi; Kapoor | Degradation process in organic thin film devices fabricated using P3HT[END_REF] The results above indicating that the utilization of DL strategy can not only provide a sustainable current enhancing but also retain a similar sensing stability of SL sensor.

Interestingly, even with the presence of P3HT in DL configuration, the response stability of CT/P3HT-DL sensor was not significantly affected. To understand this effect, the vertical composition profile of DL configuration was firstly investigated via UV-vis absorption spectroscopy coupled with incremental oxygen plasma etching. As shown in Figure 5(a), the pure P3HT film displays two absorption peaks at 540 and 610 nm. In the other hand, the pure CT film shows two absorption peaks at 656 and 707 nm. For DL film, the absorption spectrum corresponds to the superposition of P3HT and CT spectra. Following the intensity variation of these distinctive peaks enabled to verify the composition variation after oxygen plasma etching. The thickness of CT/P3HT-DL film was etched from 62 nm to 45 nm, and to 9 nm which is thinner than the original P3HT layer in the DL film. It can be observed that both P3HT and CT characteristic peaks presented in three conditions, indicating the presence of CT material even in the bottom part of the CT/P3HT-DL film. This results indicated that during the DL fabrication process, when the CT solution was blade-coated onto the thin P3HT layer, the P3HT layer was partially dissolved by the solution, forming a layer of the mixture of CT and P3HT. To confirm the composition of the upper part film, the CT absorption spectra was subtracted from the spectra of DL films, shown in Figure 5(b). The identical absorption spectra of both conditions revealed that the P3HT material in the DL film was intact during the first oxygen plasma etching treatment. According to the result above, the vertical composition profile of CT/P3HT-DL film can, therefore, be confirmed that the upper part of the film was enriched with CT material and the bottom part was consisted of the mixture of CT and P3HT. Since the sable sensing property of CT/P3HT-DL sensor may be attributed to the intrinsic stability of CT material or the mixture of CT/P3HT, we therefore tested the blended CT/P3HT materials as sensing layer in the same structure for comparison. The blended CT/P3HT sensor was fabricated by coating the CT/P3HT solution onto the cylindrical nanopore structure, and the responses to different ammonia concentration on different days were measured (Figure 6). As shown in Figure 6(a), the response of blended CT/P3HT sensor declined slightly in the first week and then became stable. The current level retained around 10 -7 during storage. As plotting the responses to different concentration of ammonia into the calibration curve, an evolution trend of calibration curve over storage days similar to that of the CT-SL sensor can be observed in Figure 6(b). This results demonstrate that the blended CT/P3HT sensor can also exhibit a stable sensing property, which can be explained by the increase of organic-organic interaction in the blended materials. [START_REF] Zhong | Air-Stable and High-Mobility n-Channel Organic Transistors Based on Small-Molecule/Polymer Semiconducting Blends[END_REF][START_REF] Huang | Electrical gas sensors based on structured organic ultra-thin films and nanocrystals on solid state substrates[END_REF][START_REF] Kolesov | Solution-based electrical doping of semiconducting polymer films over a limited depth[END_REF] However, it is noted that the sable sensing response can, indeed, be achieved in the blended CT/P3HT sensor, but due to the lack of progressive matching of work function as in DL configuration, the operational current enhancement was no longer seen in blending CT/P3HT sensor. Next, a morphological study of the organic thin films surfaces was performed via AFM to investigate its effect on sensing stability. Figure 7 presents the AFM topography images of fresh CT, blending CT/P3HT, and P3HT samples and those after 15 days storage, respectively. In fresh samples, two types of morphology were observed. The CT and blended CT/P3HT films displayed a larger grain feature, while that of P3HT films was smaller and denser. Besides, from the phase-mode AFM images in Figure S2 in the Supporting Information, we cannot observe any significant phase difference in the blending film, indicating that the two materials homogeneously distributed in the film at the nanoscale. This results revealed that the morphological property of the blended film was dominated by the property of CT material, which may contribute to the stable sensing response of the blending CT/P3HT sensor. Moreover, after 15 days storage, the surface roughness of all samples retained at same level, and no phase separation can be found in the blending film. We can thus confirm that the degradation of films is mainly through the route of chemical reaction instead of the structural modification in the polymer itself. [START_REF] Rashmi; Kapoor | Degradation process in organic thin film devices fabricated using P3HT[END_REF] Finally, considering that the human breath is composed of various gaseous compound which may affect the ammonia detection precision and the requirement of wide detection range of exhaled ammonia concentration, good selectivity and wide detection range of the sensor are of paramount importance. We, therefore, measured the selectivity of the CT/P3HT-DL sensor by testing several biomarker related gases, including CO, NO, and ACE, as shown in Figure 9(a). [START_REF] Nagasaki | Using Exhaled Nitric Oxide and Serum Periostin as a Composite Marker to Identify, Severe/Steroid-Insensitive Asthma[END_REF][START_REF] Deng | Determination of acetone in human breath by gas chromatographymass spectrometry and solid-phase microextraction with on-fiber derivatization[END_REF][START_REF] Deveci | The measurement of exhaled carbon monoxide in healthy smokers and nonsmokers[END_REF] The concentration of these gases was set at 500 ppb. Compared to ammonia, the sensor exhibited weaker response to CO (0.4%) and ACE (-0.9%). Although the sensor exhibited the response of 2.9% to 500 ppb NO, it is noted that the NO concentration in breath is normally lower than 30 ppb. For such concentration, the response is almost negligible in the CT/P3HT-DL sensor. [START_REF] Ratnawati; Morton | Exhaled breath condensate nitrite/nitrate and pH in relation to pediatric asthma control and exhaled nitric oxide[END_REF] Moreover, we also examined the detection range of the CT/P3HT-DL sensor. Figure 9(b) presents the calibration curve of the sensor with the detection range from 100 ppb to 5600 ppb. The response to 100 ppb, 500 ppb, 1000 ppb, 2800 ppb, and 5600 ppb ammonia was -0.52%, -6.74%, -11-21%, -23.81% and 31.81%, respectively. In general, the BA concentration in healthy individual and renal disease patient is ranging from 50 ppb to several ppm. [START_REF] Timmer | Ammonia sensors and their applications -a review[END_REF][START_REF] Broza | Synergy between nanomaterials and volatile organic compounds for non-invasive medical evaluation[END_REF] Therefore, the detection range of the CT/P3HT-DL sensor is suitable for BA detection. The good response stability, good selectivity, and wide detection range of the CT/P3HT-DL sensor make it reliable for the clinical experiment. After examined the properties of the CT/P3HT-DL sensor, we utilized the sensor with our sensing system as a POC device to conduct a long-term tracing of BA concentration in two HD patients and studied the correlation between BA concentrations and BUN levels. For the long-term tracing of HD patient, each patient had their BUN levels and BA concentrations measured before and after hemodialysis for three occasions. The interval between each occasion was one to two months. The BUN levels and the BA concentrations of each patient in each occasion were shown in Figure 10(a). Before HD, the BUN levels were relatively high (>60 mg/dL). Also, the BUN levels of one patient between each occasion were fluctuating. It might indicate that the protein intake and BA metabolism of the patient were different from time to time. After HD, a significant reduction of BUN levels to less than 30 mg/dL was seen. In the other hands, the same reduction can also be observed in BA concentrations following HD. The preand post-hemodialysis BA concentrations were ranging from 1277 ppb to 7084 ppb, and 50 ppb to 596 ppb, respectively, of which the ranges were reasonable as shown in previous stud-ies. [START_REF] Narasimhan | Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis[END_REF][START_REF] Endre | Breath ammonia and trimethylamine allow real-time monitoring of haemodialysis efficacy[END_REF][START_REF] Chuang | Organic Gas Sensor with an Improved Lifetime for Detecting Breath Ammonia in Hemodialysis Patients[END_REF] The sensor's lower limit of the BA concentration detection was defined as 50 ppb. Subsequently, the significant correlation between the natural logarithm of BA concentrations and the natural logarithm of BUN level is shown in [START_REF] Narasimhan | Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis[END_REF][START_REF] Hibbard | Point of Care Monitoring of Hemodialysis Patients with a Breath Ammonia Measurement Device Based on Printed Polyaniline Nanoparticle Sensors[END_REF] This result demonstrated that with the long-term tracing of patients, the high correlation coefficient could be achieved.

CONCLUSION

A new double layer (DL) strategy to enhance the operational current of the organic sensor was proposed. By the effect of DL approach, the CT/P3HT-DL sensor exhibited a greater operational current than that of CT-SL sensor, while the stable sensing response overtime was still retained even with the presence of P3HT material. The effect of DL strategy was carefully examined with different aspect, including vertical composition profile examining, lifetime testing on different sensing layer, and morphological analysis. Moreover, the high versatility of this strategy was also demonstrated by TFB/P3HT-DL sensor. Finally, with a good sensing selectivity and a capability of measuring ammonia from 100 ppb to 5600 ppb, the CT/P3HT-DL sensor was utilized for long-term tracing of BA concentration in HD patients, leading to a good correlation coefficient of 0.96 between ln(BA) and ln(BUN). These finding demonstrate the potential of this DL strategy to be used to integrate organic sensor in POC devices.

Figure 1 .

 1 Figure 1. (a) The schematic drawing of device structure and concept of sidewall single layer and double layer sensors. (b) The molecular structure of PBDTTT-CT and P3HT. (c)SEM crosssection image of PBDTTT-CT DL sensor.

Figure 2 .

 2 Figure 2. Ammonia measurement system. (a) The schematic drawing of measurement system. The blue arrows illustrate the gas flow direction. (b) Picture of measurement system indicating relative position of all components.

Figure 3 .

 3 Figure 3. Real-time current measurement and corresponding ammonia sensing response of (a) CT-SL sensors and (b) CT/P3HT-DL sensor exposing to ammonia concentrations of 500 ppb, 1500 ppb, 2500 ppb, and 5000 ppb, sequentially. (c) Energy level diagram of CT/P3HT-DL sensor. (d) The dependence of current versus voltage square in both CT-SL and CT/P3HT-DL sensor.

  Figure 4. (a) The evolution trend of current value at 5 V over storage time for CT-SL sensors and CT/P3HT-DL sensor. The calibration curve of (b) CT-SL sensor, (c) CT/P3HT-DL sensor, and (d) P3HT sensor on different days, plotted as the sensing response as a function of ammonia concentration. All sensors were stored under air atmosphere and covered by Al foils.

Figure 5 .

 5 Figure 5. (a) UV absorption spectra of pure P3HT film, pure CT film, the pristine CT/P3HT-DL film and those etched to different thickness. (b) The absorption spectra of CT/P3HT-DL films with the subtraction of CT spectra from its original spectra; the insert shows the schematic drawing of vertical composition profile of CT/P3HT-DL film.

Figure 6 .

 6 Figure 6. (a) Responses of the blending CT/P3HT sensor to 500 ppb, 1500 ppb, 2500 ppb, and 5000 ppb ammonia concentration on different days. (b) The calibration curve of the blended CT/P3HT sensor on different days.

Figure 7 .

 7 Figure 7. AFM images of fresh (a) CT, (b) blended CT/P3HT, and (c) P3HT films on the top of PVP layer, and those after 15 days storage. The storage condition is as same as that of sensors. The obtained roughness values are mentioned in respective image.After studying the effect of DL configuration, the versatility of this DL strategy was further tested by replacing the CT sensing layer with TFB material. The molecular structure of TFB and the energy level diagram of TFB/P3HT-DL sensor are shown in FigureS3in the Supporting Information. The same effect of operational current enhancement was expected by work function matching. As shown in Figure8(a), the TFB/P3HT-DL sensor exhibits a greater operational current than that of TFB-SL sensor. The same diminution of sensing response was also observed, which is caused by the elevation of current level. The responses to different ammonia concentration and the current level on different days were displayed in Figure8(b). It was observed that the evolution trends of sensing response of the TFB/P3HT-DL sensor was similar to those of the CT/P3HT-DL sensor, and the operational current was also retained around microampere level during storage. This result demonstrates the potential versatility of the DL strategy to apply on different air stable sensing materials.

Figure 8 .

 8 Figure 8. (a) Real-time current measurement and corresponding ammonia sensing response of TFB-SL and TFB/P3HT-DL sensors exposed to ammonia concentrations of 500 ppb, 1500 ppb,

Figure 9 .

 9 Figure 9. (a) Responses of the CT/P3HT-DL sensor to 500 ppb CO, NO, ACE, and ammonia. (b) The calibration curve of the CT/P3HT-DL sensor with a detection range from 100 ppb to 5600 ppb.

  Figure 10(b). Pearson correlation coefficient (r) was 0.96, p <0.001. This value was comparable with the r obtained by Killard et al. (from 0.86 to 0.96 in 11 patients) and with r 2 of 0.95 obtained by Narasimhan et al.

Figure 10 .

 10 Figure 10. (a) Pre-and post-hemodialysis BUN levels and BA concentrations in two HD patients. (b) Correlation between BUN and BA concentrations in two HD patients. The Pearson's correlation coefficient (r) is 0.96 with p <0.001. ln BUN : natural logarithm of BUN level, ln(Breath NH3) : natural logarithm of BA concentration.

ACKNOWLEDGMENT

The authors would like to thank all the master students in NCTU organic semiconductor lab for their supports for the data collection in the clinical trial. This work was supported by funding from Ministry of Science and Technology, Taiwan (Project 107-2218-E-009-008).

ASSOCIATED CONTENT

Supporting Information. I-V characteristic of CT-SL and CT/P3HT-DL sensor on different days, the AFM phase images of fresh CT, blending CT/P3HT, and P3HT samples and those after 15 days storage, the molecular structure of TFB and energy level diagram of TFB/P3HT-DL sensor. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *E-mail: cck85515@yahoo.com.tw. *E-mail: hsiaowen@mail.nctu.edu.tw Fax:+886-3-5737681. *E-mail: meng@mail.nctu.edu.tw.

ORCID

Hsiao-Wen Zan: 0000-0002-7685-1245

Notes

The authors declare no competing financial interest.

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