M.D. Gaëtan Poulen
email: poulen@chu-montpellier.fr

Jean-Rodolphe Vignes

Marine Le Corre

Hugues Loiseau

Luc Bauchet

WHO grade II meningioma: Epidemiology, survival and contribution of postoperative radiotherapy in a multicenter cohort of 88 patients

Keywords: Meningioma, radiotherapy, neuro-oncology, WHO classification

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

Résumé

Introduction. Les méningiomes sont les tumeurs primitives intracrâniennes les plus fréquentes représentant entre 13 et 36.6% de l'ensemble des tumeurs primitives du système nerveux central. Les méningiomes sont bénins dans environ 90% des cas. Les méningiomes de grade II de la classification OMS sont associés à un haut risque de récidive et à une survie plus faible que les méningiomes de grade I. Le traitement de référence est la chirurgie, aussi complète que possible. Actuellement, pour les méningiomes de grade II, il n'y a pas de recommandation pour un traitement adjuvant systématique comme la radiothérapie. Nous avons étudié une série homogène de méningiomes de grade II traités par chirurgie dans deux hôpitaux universitaires pour analyser l'utilisation de la radiothérapie et en évaluer son efficacité.

Méthodes. Nous avons analysé rétrospectivement les patients de notre base de données, opérés entre 2007 et 2010 d'un méningiome de grade II dans les hôpitaux universitaires de Montpellier et Bordeaux. Les données cliniques, les caractéristiques radiologiques, les traitements reçus ainsi que la survie ont été analysés.

Résultats. Quatre-vingt-huit patients ont été inclus. La survie globale à 5 ans pour les méningiomes de grade II était de 89.7%. Dix-neuf patients ont reçus un traitement par radiothérapie au cours du suivi. L'analyse de survie n'a pas montré de différence significative entre les patients irradiés et non-irradiés (p=0.27)

Conclusion.

A propos des méningiomes de grade II, il est actuellement difficile d'établir des recommandations claires concernant la radiothérapie. Nous étude est en accord avec la littérature en ce qui concerne l'absence de nécessité d'une radiothérapie post-opératoire précoce dans les cas d'exérèse macroscopiquement complète de méningiomes de grade II.

Méningiome, radiothérapie, neurooncologie, classification OMS

Introduction

Meningioma is the most common primary intracranial tumor, representing 13-36.6% of all primary central nervous system tumors. Meningiomas are benign in about 90% of cases [START_REF] Champeaux | World Health Organization Grade II Meningioma: A 10-Year Retrospective Study for Recurrence and Prognostic Factor Assessment[END_REF][START_REF] Ostrom | CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014[END_REF][START_REF] Baldi | Epidemiology of meningiomas[END_REF][START_REF] Zouaoui | Descriptive epidemiology of 13,038 newly diagnosed and histologically confirmed meningiomas in France[END_REF][START_REF] Goyal | Local control and overall survival in atypical meningioma: a retrospective study[END_REF].

World Health Organization (WHO) grade II meningioma is associated with a high rate of recurrence and poorer survival than in grade I [START_REF] Hug | Management of atypical and malignant meningiomas: role of high-dose, 3D-conformal radiation therapy[END_REF][START_REF] Rogers | Intracranial meningiomas of atypical (WHO grade II) histology[END_REF]. In comparison to the WHO classification of 2000 [START_REF]Pathology and genetics of tumours of the nervous system[END_REF], the rate of grade II increased in the 2007 classification ( 9) from 5% to 20-35%, conversely to the rate of grade I, since meningioma with cortical invasion came to be considered as grade II [START_REF] Louis | The 2007 WHO Classification of Tumours of the Central Nervous System[END_REF]. The rate of WHO grade III remained stable at 1-2% [START_REF] Willis | The accuracy of meningioma grading: a 10-year retrospective audit[END_REF][START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF][START_REF] Goldbrunner | EANO guidelines for the diagnosis and treatment of meningiomas[END_REF]. The more recent 2016 WHO classification of central nervous system tumors [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF] showed no significant difference in comparison to the 2007 classification [START_REF] Louis | The 2007 WHO Classification of Tumours of the Central Nervous System[END_REF].

An American study reported epidemiology of WHO grade II and III meningiomas between 2004 and 2010 using the CBTRUS (Central Brain Tumor Registry of United States) [START_REF] Kshettry | Descriptive epidemiology of World Health Organization grades II and III intracranial meningiomas in the United States[END_REF].

Incidence of grade II was approximately 0.30 per 100,000 person-years and incidence of grade III was about 0.06 [START_REF] Kshettry | Descriptive epidemiology of World Health Organization grades II and III intracranial meningiomas in the United States[END_REF]. A French study of all primary central nervous system tumors by Darlix et al. [START_REF] Darlix | Epidemiology for primary brain tumors: a nationwide population-based study[END_REF], using the French Brain Tumor Database (FBTDB), reported incidence of grade II meningioma, adjusted on the French population, of 0.43 per 100,000 person-years (0.37 per 100,000 person-years if adjusted on the American population) and 0.06 for grade III.

Treatment of meningioma is based on surgery, which should be as complete as possible [START_REF] Goldbrunner | EANO guidelines for the diagnosis and treatment of meningiomas[END_REF].

Several studies advocated systematic adjuvant radiotherapy [START_REF] Amsbaugh | Patterns of Care and Outcomes of Adjuvant Radiotherapy for Meningiomas: A Surveillance, Epidemiology, and End Results and Medicare Linked Analysis[END_REF][START_REF] Bagshaw | Adjuvant radiotherapy for atypical meningiomas[END_REF][START_REF] Lagman | Adjuvant Radiosurgery Versus Serial Surveillance Following Subtotal Resection of Atypical Meningioma: A Systematic Analysis[END_REF] while others favored classical surveillance [START_REF] Graffeo | Revisiting Adjuvant Radiotherapy After Gross Total Resection of World Health Organization Grade II Meningioma[END_REF]. The optimal approach in grade II meningioma is not well-defined. The aim of the present study was to analyze patients with grade II meningioma, operated on between 2007 and 2010, in two university medical centers, studying several characteristics: overall survival (OS), progression-free survival (PFS), the percentage of patients treated by radiotherapy and reasons for adjuvant radiotherapy and, finally, the efficacy of radiotherapy.

Methods

Selection of patients

A retrospective study included patients from our database (French Brain Tumor DataBase-FBTDB (4), or "RnhTPSNC: Recensement national histologique des Tumeurs Primitives du Système Nerveux Central " in French) with newly diagnosed and histologically confirmed grade II meningioma according to the 2007 WHO classification [START_REF] Louis | The 2007 WHO Classification of Tumours of the Central Nervous System[END_REF], operated on between January 1 st , 2007 and December 31 st , 2010. Patients without histological confirmation and patients who with prior history of meningioma surgery were excluded. Data were crossed between the RnhTPSNC and PMSI medical information system program ("Programme de Médicalisation des Systèmes d'Information") and the specialized Gironde regional registry, to increase the accuracy of inclusion. The study was approved according to French legislation (CCTIRS n°10.548; CNIL n°911013).

Clinical characteristics

Data concerning clinical characteristics (age, gender, medical history, neurological examination, Karnofsky performance status (KPS), WHO performance status), radiological characteristics (topography, tumor volume), treatments (resection quality on Simpson grade [START_REF] Simpson | The recurrence of intracranial meningiomas after surgical treatment[END_REF], adjuvant therapy or not) and survival were collected and analyzed. Radiotherapy was considered "early" when immediately postoperative, and "late" when performed after progression or recurrence.

Radiological analysis

Tumor topography was accurately analyzed on preoperative T1-weighted spoiled gradient echo MR images obtained with and without gadolinium. Preoperative tumor volume was calculated from the 3 largest diameters (D1, D2, and D3) on the 3 orthogonal planes (axial, sagittal, and coronal). Volume was estimated by ellipsoid approximation ([D1 × D2 × D3]/2), as previously reported for gliomas [START_REF] Mandonnet | Mathematical modeling of low-grade glioma[END_REF].

Radiological characteristics were analyzed on preoperative CT scan (presence or not of calcifications, cysts, bone erosion, enhancement after injection).

Follow-up screened for recurrence or progression of any residue on regular MRI.

Surgery

Quality of surgical resection was evaluated on Simpson grade, assessed on the postoperative CT scan or MRI. In cases without postoperative imaging, Simpson grade was evaluated only from the operative report. Dates of any revision surgeries for progression were noted.

Adjuvant therapy

Characteristics of the adjuvant therapy (radiotherapy or chemotherapy) were collected. For radiotherapy, type of radiation was specified: stereotaxic or conformational radiotherapy or tomotherapy. Radiation dose and duration were collected.

Statistical analysis

Statistical analysis was performed using SPSS (Statistical

RESULTS

Clinical characteristics

Eighty-eight patients (59 women and 29 men: sex ratio, 2.03) operated on for grade II meningioma between 2007 and 2010 in the 2 centers were included. Clinical characteristics of the population are summarized in Table I. Median age at diagnosis was 60.5 years. Presenting symptoms were most frequently headache (31.8%), seizure (29.5%), cognitive and/or behavioral disorder (27.3%) and sensory and/or motor deficit (28.4%) (Table I). Meningiomas were surgically resected based on the size of the tumor and/or the volumetric progression evaluated on successive MRI scans.

One patient had received brain irradiation during childhood after surgery for medulloblastoma.

Clinical examination before surgery

The majority of patients (n=55, 62.5%) had KPS of 80% or 90% and WHO performance status of 1 (n=40, 45.5%) (Table I). There was no statistical difference between the populations of the two centers for preoperative status (p=0.18 for KPS).

Radiographic characteristics

Tumor volume

Median tumor volume was 41.6 cm 3 , with no significant difference between the 2 centers (p=0.71).

Tumor location

The majority of meningiomas was located on the convexity (n=57, 67.8%) (Table I).

Surgical resection

In 59 cases, resection was assessed intraoperatively and on postoperative imaging, and in 30 cases only based on information from the operative reports.

For the majority of patients (n=47, 56.6%), resection of the meningioma and its base of implantation was complete, with Simpson grade 1. Only 7 patients (8.4%) had subtotal or partial resection with Simpson grade 4 (Table I). There was no significant difference in Simpson grade between centers (p=0.15).

Neuropathology

Eighty-eight patients had WHO grade II meningioma (93.6%), mostly atypical (n=76, 80.9%). Nine were chordoid (9.6%) and 3 were clear-cell meningioma (3.2%).

The majority of atypical meningiomas (57.3%) were so classified because of infiltration in the adjacent cerebral cortex despite low mitosis index. Thirty-two patients (42.7%) were classified as having atypical meningioma because of a high mitosis index (≥4 mitoses for 10 fields).

Postoperative clinical examination

The first clinical examination after discharge was between the second and the fourth month after surgery.

The majority of patients had KPS index 80 or 90 (n=54, 66.7%). There was global improvement at this stage of follow-up over preoperative evaluation. Results were similar for the WHO performance status, with a majority of patients PS=0 (n=38, 46.9%) (Table I).

There was no significant difference between centers (p=0.82 for KPS).

Follow-up, global survival and progression-free survival

Median follow-up was 69.2 months.

Nine patients died during follow-up. Five-year survival was 89.7%. PFS was 55.8 months. Analysis of the subgroups of irradiated and non-irradiated patients showed PFS of 39.1 months for the former (n=19), versus 60.4 months for the latter (p=0.02) PFS in the group with early adjuvant radiotherapy (n=7) was 69.3 months, versus 60.4 months without (n=81) (p=0.52).

Median PFS in the subgroup considered as atypical meningioma due to the number of mitoses (n=32) was 60 months, and 65.4 months (p=0.15) in the subgroup of atypical meningioma according to pathological characteristics such as infiltration of the adjacent cortex or low mitosis index (<4) (n=44).

Radiotherapy

Nineteen patients (21.6%) had adjuvant radiotherapy (Figure 1).

The majority had conformational radiotherapy, with a median dose of 56 Gray spread over approximately 20 sessions over 6 to 8 weeks. Four patients had tomotherapy, with a median dose of 52.8 Gray.

Survival and contribution of radiotherapy

Grade II meningioma

To treat WHO grade II meningioma after maximal surgical resection, two main options are described in literature: classical surveillance, and adjuvant radiation (radiotherapy or radiosurgery). The role of radiotherapy in grade II meningioma is controversial. There is no recommendation for systematic early adjuvant radiotherapy.

In the present cohort, the majority of patients had classical clinical and MRI monitoring after surgery to identify potential recurrence or progression of residue. Only 7 had early adjuvant radiotherapy; no clinical or neuropathological criteria underlay this choice. The 5-year survival rate was 89.7%, with no difference according to early radiotherapy or according to radiotherapy, whatever the timing versus non-irradiated meningioma.

Surveillance

Our findings are in agreement with the current literature. Graffeo et al. [START_REF] Graffeo | Revisiting Adjuvant Radiotherapy After Gross Total Resection of World Health Organization Grade II Meningioma[END_REF] analyzed 69 patients and showed that classical surveillance after complete surgical resection of atypical meningioma was not associated with higher risk of recurrence or increased mortality.

Jenkinson et al. [START_REF] Jenkinson | Early adjuvant radiotherapy in the treatment of atypical meningioma[END_REF] reported that early adjuvant radiotherapy had no influence on the progression of atypical meningioma after total surgical resection.

Champeaux et al. [START_REF] Champeaux | World Health Organization grade II meningiomas[END_REF], in 206 WHO grade II meningiomas, reported that radiotherapy did not increase overall survival even when resection was not complete. Macroscopically total resection, age <62 years, and absence of invasion of the adjacent cortex were independent factors associated with better outcome. 23) analyzed 224 patients and showed that, in case of recurrence of grade II meningioma, revision surgery was more effective than radiotherapy alone. This strategy was applied in our 2 centers in case of recurrence.

Champeaux et al. (

In a series of 90 patients, Lee et al. [START_REF] Lee | Atypical meningiomas: is postoperative radiotherapy indicated?[END_REF] reported that adjuvant radiotherapy was effective in lowering recurrence rates in patients after subtotal resection but did not provide significant benefit for patients after gross-total resection.

Adjuvant radiotherapy or radiosurgery

Conversely, Amsbaugh et al. [START_REF] Amsbaugh | Patterns of Care and Outcomes of Adjuvant Radiotherapy for Meningiomas: A Surveillance, Epidemiology, and End Results and Medicare Linked Analysis[END_REF] , in 92 patients with WHO grade II or III meningioma, showed that stereotactic radiosurgery decreased mortality in comparison with surgery alone; adjuvant therapy increased PFS in high-grade meningioma.

Bagshaw et al. [START_REF] Bagshaw | Adjuvant radiotherapy for atypical meningiomas[END_REF], in 59 patients, reported that adjuvant radiotherapy increased the rate of local control for atypical meningioma, whatever the Simpson grade. They concluded that initial aggressive treatment was important due to the difficulty of control of recurrence.

However, the present findings did not support this conclusion.

Lagman et al. [START_REF] Lagman | Adjuvant Radiosurgery Versus Serial Surveillance Following Subtotal Resection of Atypical Meningioma: A Systematic Analysis[END_REF], in 619 patients with subtotal resection of atypical meningioma, reported that potential benefit with adjuvant radiosurgery in comparison to classical surveillance.

A study retrospectively analyzing a cohort of 2,515 patients [START_REF] Wang | Overall survival benefit associated with adjuvant radiotherapy in WHO grade II meningioma[END_REF] reported that adjuvant radiotherapy for patients who underwent sub-total resection was associated with better overall survival than with no adjuvant radiotherapy. However, they confirmed that gross-total resection was associated with better OS than with subtotal resection.

In the present cohort, the significant difference in PFS between irradiated and nonirradiated patients was most likely due to the fact that radiotherapy was not radiated early in the postoperative period but implemented only when recurrence appeared on successive MRI scans.

To sum up, in WHO grade II meningioma, we did not find benefit with systematic adjuvant radiotherapy after surgery, even in case of residue. Radiotherapy and stereotactic radiosurgery seem to be adapted for progressive tumors after other surgical resections (Figure 4).

In some cases of grade II meningioma, when resection cannot be complete, early adjuvant radiotherapy on the residue can be indicated according to tumor growth-rate [START_REF] Apra | Current treatment options for meningioma[END_REF].

Adjuvant radiotherapy could be also justified in case of multiple successive surgeries.

Perspectives

The present study is limited by the size of the population, concerning only 2 centers.

Future study on a larger population is needed to evaluate more precisely the benefit of early adjuvant radiotherapy for WHO grade II meningioma. A future population study, at nationwide level, will assess the impact of postoperative radiotherapy, or the absence of postoperative radiotherapy, on overall survival and on quality of life at 10-years' follow-up.

The patients concerned are those with grade II or III meningioma operated on in France during the period 2007-2010.

Clinical trials could help to answer this question but, given the median survival, it is difficult to carry out.

A multicenter phase-3 clinical trial is ongoing, to evaluate Radiation versus Surveillance following surgical resection of Atypical Meningioma (a randomized controlled trial: ROAM trial, EORTC 1308). It will compare radiotherapy versus active monitoring in patients who have undergone gross total surgical resection (Simpson grade 1-3) of atypical (grade II) meningioma [START_REF] Jenkinson | The ROAM/EORTC-1308 trial: Radiation versus Observation following surgical resection of Atypical Meningioma: study protocol for a randomised controlled trial[END_REF][START_REF] Jenkinson | Radiotherapy versus Observation following surgical resection of Atypical Meningioma (the ROAM trial)[END_REF]. Forty of the 190 patients needed have already been included.

Another solution would be to retrospectively identify clinical, radiological and biological parameters in irradiated and non-irradiated patients, to shed light on prognosis.

Molecular profiles of WHO grade II meningiomas are one of the potential breakthrough points in future studies [START_REF] Peyre | Molecular genetics of meningiomas: Building the roadmap towards personalized therapy[END_REF]. Furthermore, it would be interesting to accurately describe the rate of progression of WHO grade I meningioma.

CONCLUSION

The classification of WHO grade II meningioma includes tumors with different progression profiles. However, therapeutic strategy is still under discussion. The present findings are consistent with current literature attesting that systematic early adjuvant radiotherapy after macroscopically complete surgical resection is not justified. Radiotherapy can follow multiple surgeries in case of recurrence. A further larger-scale multicenter prospective randomized controlled study and/or a study in a larger population may help to answer this question.

The issue is not whether we should be prescribing radiotherapy for every grade II meningioma, but rather to identify which meningiomas would benefit from adjuvant therapy following first or second surgery The majority of meningiomas were located on the convexity. There were no significant differences in population characteristics between the 2 centers.

A

  review of literature by Sun et al. (19) defined recommendations for WHO grade II and III meningiomas depending on the quality of the surgical resection. They recommended radiotherapy in both incomplete resection in grade II and complete resection with disturbing neuropathologic characteristics such as mitosis index >8. The recommendations by Goldbrunner et al. (12) for grade II meningioma with macroscopically total resection favor classical surveillance or adjuvant radiotherapy. For incomplete resection, they advised adjuvant radiotherapy (54-60 Gy in 1.8-2.0 Gy fractions; level of evidence III, recommendation level C).

  Package for the Social Sciences) software version 22 (IBM Corp.). The analysis included a descriptive part of initial data and the oncological treatments. Duration of follow-up, OS and PFS were analyzed. PFS, comparing early radiated and non-radiated meningiomas, concerned only WHO grade II meningiomas. Survival was estimated by the Kaplan-Meier method and defined as the time from first surgery (corresponding to histological diagnosis) to death or else censored at the date of last MRI follow-up. The log-rank test was used to compare survival curves between strata. The proportional hazards assumption was checked before comparing survival curve. A Cox proportional hazard regression model was performed to assess prognostic factors, including age, Simpson grade and use of postoperative radiotherapy.

Figure 1 .

 1 Figure 1. Treatment of WHO (World Health Organization) grade II meningioma.Nineteen patients with WHO grade II meningioma received radiotherapy.

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Survival curve: irradiated (blue) versus non-irradiated WHO grade II meningioma (red); p=0.27There was no significant difference between irradiated and non-irradiated WHO grade II meningiomas (log-rank test, p=0.27)

Table II .

 II Postoperative evaluation between 2 and 4 months after surgery highlighted global improvement in KPS and WHO Performance Status. Surgical resection was complete, with Simpson grade 1 for the majority of patients (n=47, 56.6%). Only 7 patients (8.4%) had subtotal or partial resection with Simpson grade 4. Multivariate analysis of overall survival. HR: Hazard Ratio; CI: Confidence Interval Table I. Clinical characteristics of the population (n=88), pre-and post-operative evaluation of Karnofsky Performance Status and WHO Performance Status and extent of resection on Simpson grade.

	* 4 missing data
	** 5 missing data CLINICAL
	CHARACTERISTICS *** 7 missing data Sex ratio (F/M)	2.03
	Median age at diagnosis	60.5
	FIRST SYMPTOMS
	Headache	28 (31.8%)
	Seizure	26 (29.5%)
	Cognitive and/or	24 (27.3%)
	behavioral disorder
	Sensory and/or motor	25 (28.4%)
	deficit
	Language disorder	15 (17%)
	Visual disorder	15 (17%)
	Intracranial hypertension	5 (5.7%)
	Asymptomatic	5 (5.7%)
	LOCATION*
	Convexity	57 (67.8%)
	Parasagittal	9 (10.7%)
	Ethmoid	4 (4.8%)
	Sphenoid / Spheno-	7 (8.3%)
	orbital
	Pterional	1 (1.2%)
	Cerebellum	4 (4.8%)
	Spinal	2 (2.4%)
	SIMPSON GRADE
	(N=83)**
	1	47 (56.6%)
	2	19 (22.9%)
	3	10 (12.1%)
	4	7 (8.4%)
	KARNOFSKY	PREOPERATIVE N=88	POSTOPERATIVE
	PERFORMANCE STATUS		N=81***
	100	6 (6.8%)	16 (19.7%)
	80-90	55 (62.5%)	54 (66.7%)
	60-70	25 (28.4%)	11 (13.6%)
	<60 The majority of patients (n=55, 62.5%) had a KPS 80% or 90% and WHO Performance 2 (2.3%) 0 WHO PERFORMANCE
	Status 1 (n=40, 45.5%) without significant difference between the 2 centers. STATUS
	0	31 (35.2%)	38 (46.9%)
	1	40 (45.5%)	36 (44.4%)
	2	16 (18.2%)	7 (8.6%)
	3	0	0
	4	1 (1.1%)	0

Table II .

 II Multivariate analysis of overall survival

		HR (95% CI)	p
	Age (≤65 vs >65)	1.09 (0.12-0.87)	0.02
	Postoperative radiotherapy	1.96 (0.47-8.2)	0.234
	Simpson Grade (1-2 vs 3-4)	0.498 (0.06-4.02)	0.168

One patient had stereotactic radiosurgery in the postoperative period, then a second surgery followed by conformational radiotherapy for recurrence.

Survival according to treatment

Survival curves of irradiated and non-irradiated patients showed no significant difference (log-rank test, p=0.27; Figure 2). Survival curves for patients with early adjuvant radiotherapy versus all others showed no significant difference (log-rank test p=0.93; Figure 3). Multivariate analysis for overall survival identified age as an independent prognostic factor for survival (HR, 1.09; 95% CI, 0.12-0.87; p=0.02). Simpson grade and postoperative radiotherapy were not independent factors for survival in the cohort as a whole (HR, 0.498; 95% CI, 0.06-4.02; p=0.168; and HR, 1.96; 95% CI, 0.47-8.2; p=0.234, respectively) (Table II)

DISCUSSION

Five-year survival in WHO grade II meningioma was 89.7%. This is consistent with the literature. Durand et al. [START_REF] Durand | WHO grade II and III meningiomas: a study of prognostic factors[END_REF] reported 78.4% 5-year survival for grade II meningioma patients operated on between 1990 and 2004. In a more recent study including 194 patients with grade II meningioma [START_REF] Champeaux | WHO grade II meningioma: a retrospective study for outcome and prognostic factor assessment[END_REF], 5-year survival was 83.2%. Hammouche et al. analyzed 79 patients with atypical meningioma and showed 5-year survival of 81% [START_REF] Hammouche | Long-term survival analysis of atypical meningiomas: survival rates, prognostic factors, operative and radiotherapy treatment[END_REF].

Consequences of the WHO 2007 and 2016 classifications of tumors of the central nervous system

In the WHO 2007 classification, meningiomas with otherwise grade I histological features but with microscopic brain invasion were moved to grade II [START_REF] Louis | The 2007 WHO Classification of Tumours of the Central Nervous System[END_REF]. The WHO 2016 classification confirmed the addition of brain invasion as a criterion for atypical meningioma [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF]. These classifications resulted in an increase in the number of meningioma classified as grade II, but did not answer the controversial question of postoperative radiotherapy for grade II meningioma.


Jantima Upan

Philippe Banet
email: philippe.banet@cyu.fr

Pierre-Henri Aubert

Kontad Ounnunkad

Jaroon Jakmunee
email: jaroon.jakmunee@cmu.ac.th

Sequential injection-differential pulse voltammetric immunosensor for hepatitis B surface antigen using the modified screen-printed carbon electrode

Keywords: Reagentless electrochemical immunosensor, Hepatitis B surface antigen, Sequential injection, Silver nanoparticles probes, Gold nanoparticles/CNT composite

A label-free electrochemical immunosensor was developed for the determination of hepatitis B surface antigen (HBsAg), a biomarker of hepatitis B virus (HBV). It is based on the specific binding of antigen-antibody on the electrode surface, which hindered the oxidation of silver nanoparticles (AgNPs) used as redox probe and hence reduced its oxidation current. The screen-printed carbon electrode (SPCE) was modified with carbon nanotube decorated with gold nanoparticles (AuNPs-CNT) and silver nanoparticles. The

Introduction

General research demonstrated that hepatitis B surface antigen (HBsAg) is a biomarker of hepatitis B viruses (HBV) infection [START_REF] Qiu | Biocompatible and label-free amperometric immunosensor for hepatitis B surface antigen using a sensing film composed of poly(allylamine)-branched ferrocene and gold nanoparticles[END_REF], which is one of the major causes of acute and chronic viral hepatitis. The diagnosis of hepatitis B virus infection is based on the presence of HBsAg in the blood which allows the detection during the incubation period.

Several methods have been reported for detection of HBsAg including enzyme-linked immunosorbent assay (ELISA) [START_REF] Kim | ELISA for Quantitative Determination of Hepatitis B Virus Surface Antigen[END_REF], chemiluminescent immunoassay [START_REF] Takeda | Highly sensitive detection of hepatitis B virus surface antigen by use of a semiautomated immune complex transfer chemiluminescence enzyme immunoassay[END_REF], and fluoroimmunoassay [START_REF] Ren | A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum[END_REF]. Although these methods provide good sensitivity, precision, and selectivity, they are complicated, have long analysis time and high cost. Therefore, a simple, rapid, and sensitive method for the detection of HBsAg is demanded for clinical diagnosis.

Electrochemical immunosensor has attracted more interest due to its high performance in terms of sensitivity, selectivity, rapidity and cost-effectiveness for detection of various biological species [START_REF] Luppa | Immunosensors-principles and applications to clinical chemistry[END_REF][START_REF] Amani | Electrochemical immunosensor for the breast cancer marker CA 15-3 based on the catalytic activity of a CuS/reduced graphene oxide nanocomposite towards the electrooxidation of catechol[END_REF][START_REF] Kaushik | A sensitive electrochemical immunosensor for label-free detection of Zika-virus protein[END_REF][START_REF] Xiang | A redox cycling-amplified electrochemical immunosensor for alpha-fetoprotein sensitive detection via polydopamine nanolabels[END_REF]. Recently, a direct type label-free electrochemical immunosensor has been being popular as compared to the sandwich-type one because it is lower cost and easier in handling as it avoids the use of the secondary antibody [START_REF] Zhang | Label-free electrochemical immunosensor based on enhanced signal amplification between Au@Pd and CoFe2O4/graphene nanohybrid[END_REF]. Normally, the solution-based redox probes such as ferricyanide [START_REF] Zhou | A simple immunosensor for alpha-fetoprotein determination based on gold nanoparticlesdextran-reduced graphene oxide[END_REF][START_REF] Haji-Hashemi | Simple and effective label free electrochemical immunosensor for Fig mosaic virus detection[END_REF], hydrogen peroxide [START_REF] Yan | Label-free immunosensors based on a novel multi-amplification signal strategy of TiO2-NGO/Au@Pd hetero-nanostructures[END_REF], and hydroquinone [START_REF] Wang | Label-free electrochemical immunosensor for prostate-specific antigen based on silver hybridized mesoporous silica nanoparticles[END_REF] were used in the direct detection. It would cause a high consumption of reagent, electrode contamination, low stability, and inconvenient procedure, so an immobilized redox probe attached on the electrode surface is preferred [START_REF] Qiu | A label-free amperometric immunosensor based on biocompatible conductive redox chitosan-ferrocene/gold nanoparticles matrix[END_REF]. Currently, AgNPs are one of the interested probes because they are not only high surface area, extremely strong conductivity, and good biocompatibility, but also have good electrochemical reactivity [START_REF] Karadeniz | Electrochemical sensing of silver tags labelled DNA immobilized onto disposable graphite electrodes[END_REF][START_REF] Wu | Silver-graphene oxide nanocomposites as redox probes for electrochemical determination of α-1-fetoprotein[END_REF][START_REF] Song | Fabrication of streptavidin functionalized silver nanoparticle decorated graphene and its application in disposable electrochemical sensor for immunoglobulin E[END_REF].

To obtain highly sensitive immunosensor, various nanomaterials such as CNT [START_REF] Lv | The label-free immunosensor based on rhodium@palladium nanodendrites/sulfo group functionalized multi-walled carbon nanotubes for the sensitive analysis of carcino embryonic antigen[END_REF][START_REF] Viet | Development of highly sensitive electrochemical immunosensor based on single-walled carbon nanotube modified screen-printed carbon electrode[END_REF], graphene oxide [START_REF] Borisova | Disposable amperometric immunosensor for Saccharomyces cerevisiae based on carboxylated graphene oxide-modified electrodes[END_REF] and AuNPs [START_REF] Sun | Investigate electrochemical immunosensor of cortisol based on gold nanoparticles/magnetic functionalized reduced graphene oxide[END_REF][START_REF] Suresh | Development of an electrochemical immunosensor based on gold nanoparticles incorporated chitosan biopolymer nanocomposite film for the detection of prostate cancer using PSA as biomarker[END_REF] were used to modify on the electrode surface. Furthermore, AuNPs have been widely employed in the fabrication of electrochemical immunosensors [START_REF] Huang | Disposable immunoassay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate[END_REF], because they are good biocompatibility and can also increase the amount of biomolecules loading on the electrode.

Although batch analysis has high sensitivity, accuracy, and reproducibility, it still has some drawbacks, for example, a high amount of chemical and sample consumption, long analysis time and low sample throughput. Therefore, flow analysis system has been employed in many applications for solving these problems [START_REF] Thunkhamrak | Sequential injection system with amperometric immunosensor for sensitive determination of human immunoglobulin G[END_REF][START_REF] Ayaz | Flow injection amperometric determination of hydrazine based on its electrocatalytic oxidation at pyrocatechol violet modified pencil graphite electrode[END_REF][START_REF] Tabrizi | Flow injection amperometric sandwich-type electrochemical aptasensor for the determination of adenocarcinoma gastric cancer cell using aptamer-Au@Ag nanoparticles as labeled aptamer[END_REF].

In this study, we developed an electrochemical immunosensor based on the specificity of antigen-antibody interactions for detection of HBsAg. Screen Printed Carbon Electrodes (SPCE) was modified with nanomaterials (CNT, AuNPs-CNT and PtNPs-CNT) to increase surface area, electrochemical activity of the electrode and enhance more target molecules immobilization. AgNPs were then deposited on the modified electrode for use as a redox probe of the detection system with a high current response and low oxidation potential. The effect of the nanomaterials on oxidation current of AgNPs was examined, and the best combination, which gives the highest analytical signal was selected. Oxidation peak currents decreased with increasing of HBsAg concentration because the formation of the antibodyantigen complex on the electrode surface acts as an insulator and hinders the electron transfer that allows label-free direct detection of the HBsAg. Furthermore, an effective detection system of sequential injection analysis (SIA) incorporated with differential pulse voltammetry (DPV) was proposed. The system provided cost-effective, simple, low reagent and sample consumption, high sample throughput, high sensitivity, selectivity, and reproducibility for the detection of HBsAg.

Experimental

Chemicals and materials

All chemical reagents were analytical reagent grade, purchased from Sigma-Aldrich and used without any further purification. Deionized water was obtained from a system of Milli-Q, Millipore, Sweden. All nanomaterials except AgNPs were obtained from Nanomaterial Research Unit, Chiang Mai University, Thailand. The TEM image of AuNPs-CNT is shown in Fig. S1. Hepatitis B surface antibody (HBsAb) and Hepatitis B surface antigen (HBsAg) were purchased from Abcam. Phosphate buffered saline (PBS, 10 mM, pH 7.4) with 137 mM NaCl and 2.7 mM KCl was prepared using deionized water.

Apparatus

A µAutolab type II (Eco Chemie, Utrecht, Netherlands) and a GPES program were used for performing the cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). An electrochemical cell consisted of three electrodes including a homemade SPCE with a diameter of 3 mm as a working electrode (WE), a Ag/AgCl (3M KCl) as a reference electrode (RE) and a stainless steel rod electrode as a counter electrode (AE). The Sequential Injection -Differential Pulse Voltammetric (SI-DPV) system comprised a syringe pump (5.00 mL barrel, 6.00 cm plunger length, 48000 steps motor, Model XLP-6000, Cavro, USA), a 10-position selection valve (Valco Instrument, USA) and a 2.00 mL plastic vial modified to be a simple electrochemical cell (EC).

Preparation of the AgNPs

AgNPs were synthesized by reducing AgNO3 with sodium borohydride [START_REF] Mulfinger | Synthesis and Study of Silver Nanoparticles[END_REF]. Briefly, a 5.00 mL aliquot of 1.00 mM AgNO3 was added drop by drop to 15.00 mL of 2.00 mM NaBH4 with strong stirring the solution on a magnetic stirrer at 0 °C. After that, 60.00 mg of sodium citrate was added to the solution and stirred at room temperature. Finally, the solution was stored in a dark bottle. The UV-Vis spectrum and TEM image in Fig. S2 showed maximum absorption peak at a wavelength of 394 nm and an average size of about 14 nm, respectively.

Fabrication of the immunosensor

The fabrication procedure of the immunosensor is schematically illustrated in Scheme 1. Firstly, a home-made SPCE with 3.00 mm diameter was fabricated. Briefly, carbon ink was diluted in diethylene glycol monobutyl ether and it was screened on a polyvinyl chloride sheet substrate. Then, it was dried at 150 ºC for 30 min and the second layer of carbon ink was deposited on the first layer, and at 150 ºC again to obtain an SPCE. Secondly, an SPCE was treated in a chamber of a plasma cleaner (PDC-32G, Harrick, USA) for 1 min. Then, 10 μL of the AuNPs-CNT (2 mg mL -1 ) was dropped onto the surface of the electrode and dried in air at ambient temperature. After that, 10 μL of AgNPs (0.027 mg mL -1 ) was dropped on the nanomaterial modified SPCE and then allowed to dry at room temperature. For higher AgNPs volume, the previous step was repeated as often as needed. In order to activate the modified electrode, 20 μL of 0.4 M EDC/0.1 M NHS (1:1v) solution was dropped on the electrode and waited for 30 min. Then, the electrode was dipped into 400 ng mL -1 HBsAb solution overnight at 4 o C. Then, the unbounded antibodies were washed away with PBS. At last, the resulting electrode was immersed in 1.0 M ethanolamine solution at room temperature for 30 min to block the remaining active sites. After rinsing the modified electrode with PBS, the immunosensor was kept at 4 o C when not in use. Scheme 1.

Electrochemical measurement for detection of HBsAg

The SI-DPV system (in Fig. 1) was employed for recording the electrochemical responses of the immunosensor in 10 mM PBS pH 7.4 using the following procedure. The selection valve (SV) was connected to the holding coil (HC) at a center port, and the electrochemical cell (EC) at port 9. PBS solution (carrier) was pushed to fill the HC and flow through the EC for 1.00 mL. Then, and with optimized analysis conditions, a potential of 0.0 V vs Ag/AgCl reference electrode was applied for 15 s followed by scanning the potential to a positive direction from -0.1 V to +0.5 V. The oxidation current was recorded as a voltammogram and the DPV peak current before the incubation of biosensor with the antigen was noted as an initial peak current (I0). Then, the solution was discarded to waste at port 6 and the EC was washed with a PBS solution before analyzing the next sample. For sample analysis, EC was filled with 0.5 mL of analyte solution and the immunosensor was incubated at 37 o C for 30 min and then washed carefully with a PBS to remove the non-specific binding species. After that, the DPV response was measured using the same SI operation sequences.

The peak current of this step was noted as a current response after the immunointeraction (I).

The percentage of decreasing current was calculated as: Fig. 1 3. Results and discussion

The effect of the nature of nanomaterials

In this work, a homemade SPCE, which is low cost, disposable, and has high surface area was applied as a basement of a working electrode for immobilizing suitable nanomaterials, silver nanoparticles, and hepatitis B surface antibody for determination of HBsAg. The working electrode should have a high response to AgNPs oxidation serving as a redox probe to afford a highly sensitive HBsAg electrochemical immunosensor. For this purpose, voltammetric studies of AgNPs immobilized on SPCE unmodified or modified with various nanomaterials were carried out. For each test, 5 µL of 2 mg mL -1 nanomaterial suspension was firstly dropped cast on the SPCE, followed by dropping 5 µL of 0.027 mg mL -1 AgNPs and let it dry. Then, the electrode was tested by cyclic voltammetry in a 10 mM PBS pH 7.4. In all cases, oxidative sweep presented a sharp oxidation peak at 200 mV related to AgNPs oxidation (Fig. 2(a)). The maximum oxidation peak current was then recorded and is shown in Fig. 2(b) for the SPCE unmodified or modified with various nanomaterials. This figure highlights that SPCEs modified with CNT or AuNPs-CNT gave the higher anodic peak current of AgNPs, demonstrating that CNT and AuNPs-CNT can increase surface area and electron transfer from the AgNPs redox probe to the electrode. It should be noted that AuNPs facilitate the reduction of silver ions to deposit silver onto the electrode. This behavior could be linked to the catalytic effect of gold on silver ions precipitation [START_REF] Taton | Scanometric DNA Array Detection with Nanoparticle[END_REF][START_REF] Chu | An Electrochemical Stripping Metalloimmunoassay Based on Silver-Enhanced Gold Nanoparticle Label[END_REF]. The AuNPs-CNT/SPCE was chosen as a working electrode for further studies because it provided welldefined peaks of both oxidation and reduction of AgNPs (Fig 2(a)). Moreover, AuNPs also have good conductivity, biocompatibility with a biological component that can facilitate the immobilizing of the receptor antibody.

The effect of AuNPs-CNT amount, AgNPs amount, and scan rate

Cyclic voltammetry was employed to study the effect of the amount of AuNPs-CNT, and AgNPs as well as the influence of scan rate on the anodic current of AgNPs at the AuNPs-CNT/SPCE. The effect of AuNPs-CNT amount on the anodic current response of 5 µL of 0.027 mg mL -1 AgNPs was studied using AuNPs-CNT modified electrode prepared by dropping various volumes of AuNPs-CNT suspension from 2 to 30 µL. The results showed that the current response slightly increased with the increase of AuNPs-CNT from 2 to 30 µL as shown in Fig. 3(a) and 10 µL of the materials was selected to modify on SPCE surface. Subsequently, the effect of the amount of AgNPs being dropped on the electrode was investigated as it is the important parameter for sensitive detection of HBsAg. As the results shown in Fig. 3(b), peak currents increased linearly with the increasing amounts of AgNPs. However, 10 µL of AgNPs was chosen as a detection probe on the electrode because it provides the best compromise between high current and short preparation time.

Moreover, the effect of different scan rates from 10 to 250 mV s -1 were studied with the AgNPs/AuNPs-CNT/SPCE. It was found that anodic peak current (Ipa) of AgNPs linearly increased with the increment of a square root of scan rate with R 2 of 0.9995 as shown in Fig. S3, indicating the redox process of AgNPs at the modified electrode was diffusioncontrolled.

Fig. 3

Morphologies of the modified electrodes

Scanning electron microscope (SEM) was applied to characterize the morphologies of modified electrodes in each step. Fig. 4(a) shows that the bare SPCE displays uniform structure and high surface area. After modification with AgNPs/AuNPs-CNT (Fig. 4(b)), the electrode surface was well covered with AuNPs-CNT (diameter were about 10 and 30 nm, respectively, presented in Fig. S1) and AgNPs. When HBsAb and HBsAg were immobilized on the modified electrode, plentiful big clusters were obtained as shown in Fig. 4(c) and 4(d) that should be the adsorbed proteins, which are macro biomolecules. In addition, the EDS technique was also employed to characterize the element compositions on the modified electrodes. The EDS spectrum indicated that SPCE had carbon and oxygen as the main compositions as shown in Fig. 4(e), while AgNPs/AuNPs-CNT/SPCE (Fig. 4(f)) shows the composition of silver, gold, oxygen, and carbon, respectively. When AgNPs/AuNPs-CNT/SPCE was further modified with HBsAb and HBsAg, the presence of proteins was observed, as shown by the increase of the percentage of nitrogen content. The modified electrodes before and after immunointeraction with HBsAg provided 3.21% and 6.82% of nitrogen element, as shown in Fig. 4(g-h) indicating that HBsAb and HBsAg were successfully immobilized on the electrode surface. Fig. 4 10

Electrochemical characterization of the immunosensor

Cyclic voltammetry is widely used for probing the feature of modified electrode surface due to its effectiveness and convenient procedure. The cyclic voltammograms at different steps of electrode modification during the construction of the immunosensor were investigated, and the results are shown in Fig. 5. The AgNPs/AuNPs-CNT/SPCE shows a couple of well-defined redox peaks of AgNPs as mentioned previously (Fig. 5(a)). Anodic peak potential (Epa) and cathodic peak potential (Epc) of 208 and 30 mV, respectively, with ∆Ep of 178 mV were obtained. When the AgNPs/AuNPs-CNT/SPCE was incubated with HBsAb the peak current decreased (Fig. 5(b)) because HBsAb is a biomolecule and it can decrease the oxidation of AgNPS at the electrode surface. This result indicates that antibodies had been successfully immobilized on the electrode surface. The following step with adsorption of ethanolamine on the non-specific binding site resulted in a small decrease in peak current. Finally, after incubation with HBsAg, the anodic and cathodic peak currents decreased again (Fig. 5(c)) because of the immunocomplex between HBsAb and HBsAg, which constitutes a bigger proteinic adduct, further hinder the electron transfer of AgNPs at the electrode. This last trend confirms the oxidation current of AgNPs can be used as an analytical signal to directly detect the formation of antibody-antigen complex on the electrode surface. As shown in a cyclic voltammogram, AgNPs not only have good redox activity such as high current response, good stability, and sharp oxidation and reduction peak profile, but it can also oxidize and reduce at low potential minimizing the possibility of interferences.

Fig. 5

Electrochemical impedance spectroscopy (EIS) is also an effective method to investigate the change of the modified electrode surface in each step of fabrication. The electron-transfer resistance (Ret) is represented by the semicircle diameter in the Nyquist plot, while the diffusion-controlled process is represented by the straight line. After modification of SPCE with AgNPs and AuNPs-CNT, a small Ret was observed, as presented in Fig. 6(a) indicating that these nanomaterials are helpful to improve the electron transfer rate of the redox probe at the surface of the electrode. When HBsAb and HBsAg were immobilized, the Ret was obviously increased, as shown in Fig. 6(b) and 6(c), respectively. The results were in good agreement with the CV results, confirming the successful immobilization process of the immunosensor.

Optimization of experimental parameters

DPV technique was employed to measure the anodic current of AgNPs oxidation.

After the oxidation of AgNPs, a deposition step was necessary to reduce Ag + ion back to deposit on the electrode again. Therefore, a deposition potential was optimized in a range of -1.0 to +0.3 V for 60 s. The result demonstrated that anodic current increased with the increase of deposition potential and reached a maximum at 0.0 V hereafter, so, the deposition potential of 0.0 V was used for further experiments. Then, the deposition time was investigated in the range of 0-300 s. It was found that no significant difference was obtained, and a deposition time of 15 s was chosen.

The effect of incubation time of the electrode in the sample solution was studied from 10-60 min in 50 ng mL -1 HBsAg solution at a temperature of 37 o C. The result in Fig. 7(a) shows that %decreasing current increased with the incubation time up to 30 min and then slightly decreased because it is insufficient for immunointeraction at short incubation time. Thus, 30 min was selected as the optimum incubation time. Moreover, the suitable incubation temperature was also evaluated in the range of 25-60 o C. Fig. 7(b) presents that the highest %decreasing current was observed at 37 o C, and then it decreased at a higher temperature.

Therefore, the incubation temperature of 37 o C was used for the next experiments.

Performance of the immunosensor

Under the optimum conditions, the SI-DPV system was employed to investigate the immunoreaction between HBsAb and HBsAg. As presented in Fig. 8(a), the percentages of decreasing of DPV peak currents increased dramatically with the increment of HBsAg concentration because the increasing amount of immunocomplex between antigen-antibody inhibited more and more the electron transfer from the redox probe to the electrode. The %decreasing current reached a plateau after HBsAg concentration of 80 ng mL -1 due to the saturation of HBsAg on the electrode surface. The proposed system provides a linear range of 1 to 40 ng mL -1 with a detection limit of 0.86 ng mL -1 (3SD/slope), as shown in Fig. 8(b).

The presence of HBsAg in serum indicates that the patient has contracted HBV infection [START_REF] Kaul | Chapter 9.17 -Hepatitis, The Immunoassay Handbook[END_REF]. Furthermore, numerous studies have presented that inactive hepatitis B virus carriers usually have serum HBsAg levels <1,000 IU mL -1 ( < few µg mL -1 ) [START_REF] Chan | Hepatitis B surface antigen quantification: why and how to use it in 2011-a core group report[END_REF]. Therefore, the detection limit of the developed method is useful for diagnosis of HBV both active and inactive carriers. It's also close to the usual cutoff of licensed HBsAg Assays [START_REF] Hollinger | Hepatitis B Virus Infection and Transfusion Medicine: Science and the Occult[END_REF]. The dilution level used to fall in the dynamic range can be used to discriminate between the two categories. A comparison of these results with other electrochemical immunosensors using different detection methods was summarized in Table 1. In the work of entry ( 1 4) used a redox probe immobilized on the electrode as similar to this work. The detection limit obtained in this work, 0.86 ng mL -1 is close to the best values 0.5 ng mL -1 , entry (2), and 0.42 ng mL -1 , entry (4) obtained. Although the narrower linear range obtained from this work, the immunosensor developed here is competitive with other electrochemical immunosensors with other redox probes immobilized on the electrode or labeled with an enzyme as it enables to quantify the lowest value.

Furthermore, this immunosensor provided more advantages such as working electrode is low cost, and the operation is semi-automatic.

Conclusion

In this work, a label-free electrochemical immunosensor was developed for the determination of HBsAg using the SI-DPV system with HBsAb/AgNPs/AuNPs-CNT/SPCE as a working electrode. The AgNPs on the modified SPCE does not only work as an electrochemical probe, but also intrinsically increase surface area, conductivity, biocompatibility, and increasing amount of the immobilized HBsAb loading on the electrode.

This leads to improving on sensitivity of the immunosensors. Moreover, the developed system is simple, cost-effective, low chemical consumption, convenient, and useful for the determination of HBsAg in clinical samples.

(Grant # RSA6080007) and the Franco-Thai Mobility Program (SIAM n° 35834SD) are also acknowledged. We appreciate partial support from Chiang Mai University.

Fig. 2

 2 Fig.2

Fig. 6

 6 Fig. 6

Fig. 7

 7 Fig. 7

  ) a potentiometric technique based on the shift of open circuit potential upon the formation of the immunocomplex is used. The other methods are amperometric technique, entry (2) used a HBsAb labeled with HRP, whereas entry (3) and (

Fig. 8

 8 Fig.8

Fig. 1

 1 Fig. 1 SI-DPV system for the determination of HBsAg; Carrier = 10 mM PBS pH 7.4, SP =

Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 A

 345678 Fig. 3 The effect of AuNPs-CNT (a) and AgNPs (b) amounts on peak current of AgNPs

Table 1

 1 Comparison of different electrochemical immunosensors for determination of HBsAg preparation time of immunosensor was calculated from the modification steps until the incubation step of blocking agent. can confirm that the proposed immunosensor could be applied for the determination of HBsAg in real samples.

	Entry	Modified electrode	Technique	Applied	*Preparation time	Linear	Detection	Reference
				potential	of immunosensor	range (ng	limit (ng
				(V)	(h)	mL -1 )	mL -1 )
	(1)	Nafion/HBsAb/AuNPs/gelatin/Pt	potentiometry -	~35	4-800	1.3	[33]
		electrode
	(2)	HRP-HBsAb/nanogold/RTIL-	CV	-0.8 to 0.2 ~20	1.5-400	0.5	[34]
		chitosan/GCE
	(3)	HBsAb/(AuNPs/Co(bpy)3 3+ )6/	Amperometry -0.35	~25	50-4500	5.0	[35]
		Nafion/Pt electrode
	(4)	HBsAb/AuNPs/Prussian Blue/GCE CV	-	~14	2-300	0.42	[36]
	(5)	HBsAb/AgNPs/AuNPs-CNT/SPCE DPV	-0.1 to 0.5 ~13	1-40	0.86	This work
	Pt = platinum; HRP = horseradish peroxidase; RTIL = Room temperature ionic liquid; GCE = glassy carbon electrode; Co(bpy)3 3+ = tris(2,2'-
	bipyridyl)cobalt(III)

*

Acknowledgements

The authors would like to thank Science Achievement Scholarship of Thailand (SAST), the Graduate School and Department of Chemistry, Faculty of Science, Chiang Mai, University and Laboratory of Physicochemistry of Polymers and Interfaces (LPPI, EA 2528), CY Cergy Paris Université for all supports. The financial support from the Thailand Research Fund

Reproducibility and selectivity

Different AgNPs/AuNPs-CNT/SPCEs were prepared to study the repeatability and reproducibility of the current response of AgNPs. The relative standard deviation (RSD) was 0.43% for 11 replicate measurements on one electrode. This stability of the initial peak current is vital for the accuracy of the sensor as the measurement is based on a decreasing percentage of the peak The reproducibility of 5 modified electrodes was 4.96% RSD when measurements were performed before the incubation with the antibody-antigen showing that the preparation of modified electrode had good repeatability and reproducibility.

Then, the reproducibility of the prepared immunosensor was investigated from the response of five different immunosensors to 40.00 ng mL -1 HBsAg. The result showed 7.19% RSD, as shown in Fig. S4(a) which exhibits good reproducibility.

Selectivity was investigated by measuring current responses of the immunosensor to three possible interfering substances, including 40 ng mL -1 of bovine serum albumin (BSA), immunoglobulin G (IgG) and prostate-specific antigen (PSA). It was found that no remarkable change of current response for these substances as compared to the result obtained from 40 ng mL -1 HBsAg, as presented in Fig. S4(b), showing a good selectivity of the immunosensor.

Application of the immunosensor

The SI-DPV immunosensor system was used for the detection of HBsAg in real samples to evaluate the feasibility of the immunosensor using the calibration method. Three normal human serum samples were diluted 100 times with PBS solution and then spiked with a certain concentration of HBsAg. After that, diluted samples were incubated with the prepared immunosensors to investigate the percentages of decreasing current. The recoveries were obtained in the range from 80.7% to 91.4%, as shown in Table 1. From the results, it Scheme 1. The stepwise fabrication process of the immunosensor


P Afanasiev
email: pavel.afanasiev@ircelyon.univ-lyon1.fr

Transfer of stored electrons between TiO2 polymorphs during photocatalytic H2 production in methanol-water medium

Keywords: TiO2 polymorphs, Pt/TiO2 polymorphs, electron transfer, hydrogen production, methanol-water medium

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

Introduction

Titanium dioxide is by far the most studied semiconductor material in photo catalysis, reported in thousands of original papers and reviewed many times [i, ii, iii]. One of the most promising applications is photo catalytic hydrogen evolution reaction (PHER) using the 2 energy of solar light. As bare titania has low PHER activity, a co-catalyst is usually added, in order to accelerate hydrogen release. PHER using methanol or water -methanol-mixtures and TiO2 has been studied with a wide range of co-catalysts, the noble metals (Pt, Pd, Au) appearing the most active [iv]. Great effort had been devoted to the engineering of optimal PHER catalysts containing titania and noble or non-noble metals [v, vi].

The overall PHER process is complex, its rate depending on many elementary steps, from light absorption to the release of hydrogen molecules. The rates of these steps depend on the crystallites size [vii, viii], exposed crystallographic facets [ix], particles morphology [x], energy levels matching, lattice defectness [xi] and many other properties. Charge mobility and charge transfer between semiconductor and co-catalyst phases play an important role.

If two or more semiconductors are present in a photocatalytic system, then exchange of photo generated charge carries becomes possible between the conduction bands (for electrons) and valence bands (for holes) of the corresponding solids, provided that the energy bands alignment allows it. It has been shown that combinations of semiconductors such as CdS/TiO2 lead to an enhanced rate of photocatalytic processes [xii].

Heterojunctions (solid state boundaries between different semiconductors) are supposed to improve charge separation and lead to enhanced PHER activity [xiii]. Among these, interactions between titania anatase (A), rutile (R) and somewhat less common brookite (B) are intensely studied. The reasons of apparent non-additivity between the catalytic properties in the mixttures of different TiO2 polymorphs of is still actively debated [xiv, xv, xvi]. Mixed phase A-R [xvii,xviii] or R-B [xix] systems with enhanced activity have been reported.

The mismatch between short lifetimes of bulk free charge carriers and slow kinetics of the interfacial transfer and surface chemical reactions was deemed to be one of the main obstacles to design efficient PHER systems [xx, xxi] . The characteristic times of charges separation and free carriers lifetimes are at the femtosecond to nanosecond scale [xxii, xxiii, xxiv]. The trapped holes react very quickly, during nanoseconds [xxv] whereas trapped electrons may survive for much longer times. In the illuminated TiO2 long-live trapped electrons are formed [xxvi, xxvii]. Electron accumulation is induced by photo generated electron trapping within the TiO2 lattice, where it is apparently located at a metal ion (Ti 3+ species) combined with an oxygen vacancy [xxviii, xxix, xxx, xxxi]. The catalytic role of such trapped states has been previously demonstrated [xxxii]. Accumulated electrons were used for electron transfer, to carry out organics hydrogenation [xxxiii] or nitrogen fixation [xxxiv]. Note however that beside increasing lifetimes of the charge carriers, the trapped states can act as recombination centers [xxxv].

If in the solid-solid TiO2 polymorphs heterojunctions the directionality of charge separation seems well established, less is known on the interactions of accumulated electrons stored on particles of different polymorphs in the liquid phase. Such interactions might obviously strongly differ from the solid-solid grain boundaries. As an example of exchange of active species between different polymorphs in liquid phase, Z-scheme water splitting was implemented by iodine/iodate pair between platinum-loaded A and bare R [xxxvi]. However, for the direct transfer of such active species between the particles of different polymorphs, neither the directionality nor the potential impact on PHER was ever clearly demonstrated.

Here we provide an experimental evidence of accumulated electrons transfer between the particles of different bare and Pt-loaded TiO2 polymorphs in water-methanol slurry, which strongly modify the observed PHER rates.

Experimental

Preparation of catalysts

Commercial PC500 anatase TiO2 and mixed 75 % A 25% R Degussa P25 were used as received. Pure R nanorods were obtained by modified procedure reported earlier [xxxvii]. In brief, first hydrous titania was prepared by means of TiOCl2 precipitation with an excess of aqueous ammonia. Then the precipitate was washed with distilled water to neutral pH and the absence of qualitative reaction for chloride anion. Next, the precipitate was peptized overnight with HClO4 at pH=0. Then hydrothermal reaction was carried out in the presence of ammonium sulfate which plays the role of structure and morphology -directing additive. To remove surface impurities of chloride and sulfate ions, the R nanorods were purified by ion exchange with 1M NH4NO3 at pH = 2, followed by calcination in air flow at 350 °C for 2h.

Pure Brookite (B) phase was prepared by hydrothermal treatment of hydrous titania at pH 12-13 as described elsewhere [xxxviii]. In brief, washed hydrous titania obtained from precipitation of TiOCl2 with ammoniac, was treated by NaOH to adjust equilibrium pH to 12.

Then the mixture was hydrothermally treated at 200 °C for 48 h, washed and cleaned by ion exchange as described for rutile, but at pH = 10. Then the material was heated in air flow at 350 °C.

Pt/TiO2 catalysts were prepared by incipient wetness impregnation of TiO2 with aqueous solution of Pt(NH3)4(NO3)2 (Sigma-Aldrich), followed by drying at 80 °C overnight, calcination in 50 mL/min air flow at 300 °C for 2h and then reduction in H2 50 mL/ flow at 300 °C for 2h (heating rate 2 °C/min). Pt loading was measured by inductively coupled plasma -optical emission spectroscopy (Activa, Horiba Jobin Yvon) after the samples were dissolved in an acid mixture at 300 °C. The target Pt loading was 0.5 wt% Pt. The actual loadings are close to the target value within the range 0.02 wt% (Table 1).

Characterizations

Transmission electron microscopy (TEM) was carried out on a JEOL 2010 device with an accelerating voltage 200 keV. The samples were dispersed in ethanol by ultrasound and put onto a lacey carbon on a copper grid. Low-temperature nitrogen adsorption was measured on a Micromeritics ASAP 2010 instrument. Specific surface area was determined using BET equation. Prior to measurements, the samples were heated in a secondary vacuum at 300 °C for 4h. The X-ray diffraction (XRD) patterns were obtained on a Bruker D8 Advance diffractometer with Cu-Kα emission and identified using standard JCPDS files. The metal content in the solids was determined, after dissolution in a HNO3/H2SO4 mixture, by plasmacoupled atomic emission spectroscopy (ICP-ES Activa Jobin Yvon). Hydrodynamic size distribution was measured using Dynamic Light Scattering (DLS) on a Wyatt Nanostar device. Aqueous suspensions containing about 0.2 g/L of solids were applied for the DLS measurements. Size distribution was obtained from averaging 10 series of 100 single measurements 2 s each. Autocorrelation function was cut below 1.5 and above 1500 nm.

Zeta-potential (ZP) was determined from electrophoretic mobility in aqueous suspensions on a CAD Zetacompact device. Weighted amounts of about 100 mg of were ultrasonically dispersed in 200 ml of 10 -2 M KCl solution. The pH values were adjusted with either HClO4 or KOH 0.1 M solutions. Electrophoretic migration rate was converted to zeta potential by the Helmholtz-Smoluchowski equation. To improve the accuracy at the pH near IEP, ZP was measured in several points outside the stationary position the curves of ZP vs. focal position were built.

Reflectance UV-visible spectra were collected and converted to Kubelka Munk function using a Perkin-Elmer Lambda 35 scanning spectrometer equipped with an integrating sphere over the spectral range 200-1000 nm. Pure BaSO4 was used as a reference. The band gap energies were determined by Shapiro [xxxix] and Tauc [xl] methods using Kubelka Munk function.

Catalytic tests.

A calculated volume of water-methanol slurry containing from 0.6 to 90 mg of catalyst was added to 30 ml of water-methanol reaction mixture containing 1% vol. of water, in a batch slurry photoreactor. A 125 W high-pressure mercury lamp was applied with the window of 21 cm 2 area. A quartz optical window and a Pyrex window cutting light below 300 nm were positioned between the lamp and the reaction mixture. The photoreactor had gas inlet and outlet used to purge the system with argon before the reaction and to sample the gases during the reaction. The gases were analyzed with Agilent 3000A micro gas chromatograph. The catalyst concentration in the slurry varied from 0.02 to 2.5 g L -1 . Before irradiation, the solution was stirred for 10 min and the reactor was purged with Ar flow. The PHER rate and methanol conversion were calculated from the integration of GC peaks during the reaction period of 3 h. To check the stability, the experiments were repeated for several times with the same reaction mixture, after refreshing methanol and Ar purge. The methanol conversion in all the experiments was below 0.1 %. No CO2, CO or other secondary methanol oxidation products were detected in agreement with the literature findings [xli, xlii], demonstrating that the primary reaction is dehydrogenation to formaldehyde described by the equation (1).

CH3OH → CH2O + H2

(1)

The apparent photonic efficiency (PE) was calculated by dividing the amount of released H2 by the integrated absorbable photon flux [xliii]:

=
where rH2 is the hydrogen formation rate and is the absorbable photon flux. In liquidphase conditions, the UV lamp is polychromatic (Fig. S1), with the flux of absorbable photons is = 1370 µmol/h.

Results and Discussion

3.1. Characterizations of bare TiO2 and Pt/TiO2 materials.

Titania polymorphs purity was attested by powder XRD (Fig. 1). Pure phases with no extraneous peaks of other polymorphs were obtained. Analysis of XRD peaks width using Scherer equation gives crystallite size of 6.5 nm for A (PC500), 119 nm for B and 41 nm for R. This difference of size is confirmed by TEM observation (Fig S2). The properties of commercial PC500 anatase have been extensively studied previously [xliv]. This solid consists of agglomerated primary nanoparticles of 5-10 nm size (Fig. S2a). Homemade B particles show shuttle-like morphology with size mostly between 200 and 300 nm (Fig S2b).

Hydrothermally prepared R contains single crystalline nanorods oriented along [110] direction with narrow size distribution (width: 20-30 nm, length: 200-300 nm) (Fig. S2c,d).

Phase purity has been confirmed by Raman spectroscopy. All Raman peaks in the corresponding spectra are attributed to A [xlv, xlvi], R [xlvii] and B [xlviii] single phases (Fig. S3). For PC500 TiO2 anatase at neutral pH, DLS showed the presence of agglomerates of 200-300 nm size and greater (Fig. S4). Agglomerate size depends on the slurry concentration and on the pH [xlix]. Detailed investigation of such effects is beyond the scope of this work. Rutile nanorods demonstrate hydrodynamic size corresponding to the largest dimension of the acicular individual particles (200-300 nm). Brookite has the largest individual particle size, but the particles are less agglomerated. As a result the observed DLS size for three polymorphs is comparable (Fig. S4, Table 1).

The suspensions were characterized by Zeta potential measurements. (5.8±0.3) ≈ R (5.7±0.3) > B (4.3±0.3), in a general agreement with our previous results [36].

Earlier, on the basis of valence bond principle the PZC values anatase and rutile were predicted by Hiemstra et al. to be close to 6 [l]. Since a Zeta potential of at least 20-30 mV is usually necessary to stabilize colloidal suspensions by electrostatic repulsion, at neutral pH applied for the catalytic tests, all TiO2 suspensions are prone to agglomerate. Indeed, all water-methanol suspensions fully decanted upon storage, after several hours for A and B and after two days for R. As the curves of Zeta potential vs. pH are similar, at neutral pH applied for the PHER experiments, the particles of all polymorphs should bear a slightly negative charge, i.e. there is presumably no specific attraction in the mixtures of A, R and B.

The specific surface areas (Ssp) of three TiO2 polymorphs change in the sequence A > R > B, in agreement with the XRD crystallite size (Table 1). After supporting Pt, no significant modifications of Ssp were observed as the treatment conditions are soft and the amount of introduced Pt is low (Table 1). 20) 19(4) 0.49(1) a values measure in the first 15 min after sonication; b from chemical analysis UV-visible spectra (Fig. 2) show the energy gap (Eg) sequence A > B > R, with Eg of 3.26(5) eV for anatase, 3.18(5) for brookite and 3.06(5) eV for rutile (Fig. S6, Table 1), in agreement with the literature [li]. Addition of platinum does not significantly modify the apparent Eg but adds a small absorption in the visible region [lii, liii].

TEM study of Pt-loaded materials reveals the presence of highly dispersed metal particles.

Size histograms of Pt particles show a significant size variance between 1 and 4 nm (Fig. S7).

Platinum dispersion changes in the sequence Pt/A > Pt/B > Pt/R (Table S1). It seems that Pt tends to from raft like particles on rutile (Fig. 2a), whereas on anatase and brookite isotropic particles are observed. Fine details of the Pt morphology and dispersion are beyond the scope of this work. TEM observation after the PHER tests (vide infra) shows that the Pt particles always remain supported on the polymorph onto which they have been initially supported by impregnation, i.e. no Pt scrambling was ever detected (Fig. 2). Post mortem TEM analysis of the catalysts always shows that A, R and B agglomerates are preserved as they were in the individual suspensions before test (Fig. 2). Overall, three pure TiO2 polymorphs were obtained and metallic Pt was supported onto these materials in a highly dispersed state. The properties of the oxides and the supported catalysts under study are typical for such Pt/TiO2 systems, widely reported in the literature.

Catalytic activity

Activity of bare TiO2 polymorphs and individual Pt/TiO2 samples

Bare titanias show low PHER activity (Table 2). The color of the initial bare TiO2 suspensions was white. After UV irradiation, the color of suspensions changed to gray-blue for bare anatase and rutile suspensions. No appreciable color change was observed for brookite. When the irradiation was stopped, the blue coloration persisted in the closed reactor.

Upon opening to air, blue coloration disappeared in few seconds for anatase, but rutile remained blue for a longer time (hours). The color change upon UV irradiation was observed to "accumulated" or "stored" electrons previously and attributed to Ti 3+ ions generated due to electrons accumulation, occurring simultaneously with holes consumption by methanol. Such sites may be exposed at the surface or to be localized in the bulk, which might explain their different stability in air. Earlier studies using operando EPR of frozen suspensions or photoacoustic technique confirmed the presence of Ti 3+ [liv, lv, lvi].

Platinum is a well-known co-catalyst which increases PHER rate by two orders of magnitude, whatever the polymorph (Table 2). For the purposes of this study, activity of bare polymorphs can be considered negligible as compared with the Pt-loaded catalysts. PHER activities of three Pt -loaded catalysts vs. slurry concentration are depicted in Fig. 3. The PHER activity changes in the sequence: Pt/A > Pt/R > Pt/B in the whole range of slurry concentrations studied (Fig. 3). The activity difference between polymorphs has been widely studied and attributed to many factors, such as the electrons mobility and lifetime in the solid, or the energy bands positions more or less favorable to the H2 formation [lvii, lviii]. The differences between polymorphs depend on the properties of specific samples, on the conditions and on the target reaction, but anatase often gives more active catalysts than rutile and brookite [lix, lx]. Detailed study of the differences between individual Pt/A, Pt/B and Pt/R catalysts is beyond the scope of this study.

The Pt/A sample is slightly more active than benchmark 0.5%wt. Pt/P25 catalyst (Table 2). Degussa P25 is not systematically studied here as it contains A and R phases mixture, not relevant for the goals of this work. However its activity is given in the SI, as it is an often studied reference. According to the literature reports [lxi], the PHER rate increases with Pt loading up to 0.1-0.2 % Pt wt. and then it has a plateau, where the PHER rate slowly changes with loading, up to the value of several wt.%, when it begins to decrease. The PHER rate vs.

Pt loading was not studied in this work. The unique mass loading of 0.5 %wt. Pt/TiO2 was applied for the impregnated samples, for which the PHER rate is supposed to be close to its maximal value. Note however, that when the 0.5% Pt/TiO2 samples were further mixed with bare TiO2, the formal Pt loading in the resulting composite catalysts varied in a wide range, from 0.005% to 0.5 wt. %. In all experiments, the rate of hydrogen release was constant in time and no deactivation of the catalysts was observed for several repetitive 3 h tests (Fig S8). The overall amount of H2 formed in one test was always lesser than 0.25 mmol. The corresponding methanol conversions are lesser than 0.03%, therefore pseudo zero order kinetics by methanol can be safely applied in all instances.

The literature values for similar Pt/TiO2 systems are strongly scattered, probably due to the variations of the experimental setup and details of preparation. In this study we obtain the PHER rates at the level of usual values reported so far for Pt/TiO2 catalysts [5, lxii, lxiii, lxiv, lxv]. The PHER rate for Pt/R is lower than for Pt/A and the PHER activity of Pt/B is the lowest, perhaps because of much lower specific surface area of this phase (Fig. 3, Table S2).

Mixtures of bare TiO2 and Pt/TiO2.

The main point of this work is non-additivity effect of PHER rate in the mixed suspensions of TiO2 and Pt/TiO2 particles of different polymorphs. Such effects exist probably due to the transfer of accumulated (stored) electrons between suspended particles, as discussed in the present section.

Note that when discussing further the "transfer of accumulated electrons" we make an obvious shortcut, because this work does not clarify in any way neither the structure of these species nor the precise mechanisms of their transfer. The "stored electrons" may have different physical nature, from bulk defects to surface adsorbed states involving Ti(III) ions.

By the same token, the term "transfer" by no means implies a direct CB-to-CB transfer between TiO2 phases, but probably involves a more complex exchange phenomenon. Herein a phenomenological description of the effects in the mixtures is provided, with minimal assumptions on chemistry and physics involved at the atomic level.

The PHER activity of Pt/TiO2 has much higher value than for any bare TiO2 (Table 2).

Therefore, if transfer of photo generated electrons in the suspension from one polymorph to another has a pronounced directionality and a sufficient intensity, then we should observe either an increase or a decrease of the PHER rate in the mixtures, depending on whether the transfer is directed toward a Pt-loaded TiO2 or outward from it. Namely, transfer of stored electrons from a bare TiO2 polymorph towards a Pt -loaded one should increase the PHER rate. Inversely, if the flow of accumulated electrons is directed from the Pt/TiO2 particles towards a bare TiO2, then the overall PHER rate should decrease. Both increase and symmetric decrease of PHER rate in different mixtures is equally important for a proper demonstration of the effect.

Release of H2 in the dark after Pt/TiO2 injection onto pre-illuminated TiO2

The experiments with the mixtures described in the previous sections strongly suggest the existence of a preferential transfer of stored electrons between the polymorphs. However, more direct and compelling evidence could be obtained from the experiments in which the stored electrons are first generated by illumination in bare TiO2 and then they are released as H2 in the dark, after addition of Pt/TiO2.

In Fig. 5 are shown the curves of hydrogen release from three bare polymorphs illuminated for 90 min, to which a smaller amount of Pt/TiO2 suspension (1:10 wt.) was added immediately after the moment when illumination was stopped. Bare A shows a rapid outburst of H2 production, of 42 μmol/g. Illumination of bare R under the same conditions followed by addition of Pt/A leads to a lesser H2 production (near 7 μmol/g) at a significantly slower rate.

For brookite, the amount of released H2 was negligible (Fig. 5). A mere observation of H2 release after addition of Pt/TiO2 to pre-irradiated bare TiO2 proves that stored electrons are transferred from TiO2 to Pt/TiO2. However it cannot be considered as compelling evidence of stored electrons transfer directionality between the TiO2 polymorphs.

Indeed, some of Pt particles located at the surface of added Pt/TiO2 can form contacts (albeit scant) with the surface of bare polymorph and cause the H2 release. Therefore not only the fact of H2 release is important but its relative rate and amount. The rates and the amounts of H2 release were therefore compared for different combinations of TiO2 -based solids and for the mixtures of TiO2 with reference 0.5% Pt/Al2O3 (Fig. 6). It suggests that transfer of stored electrons from A to Pt/R is by an order of magnitude faster than the reverse process (Fig. 6). Addition of Pt/Al2O3 to both pre-illuminated A and R leads to some H2 release. However, for the same amount of Pt added, injection of Pt/Al2O3 slurry to the pre-illuminated A or R leads to an order of magnitude lesser H2 release than addition of Pt/R (Fig. 6). It allows concluding that accumulated electrons in the Pt/TiO2 -TiO2 mixtures are mostly transferred due to the contacts between the TiO2 particles. Here we demonstrate that exchange of stored electrons between TiO2 particles has a pronounced directionality that plays an important role in the overall PHER activity.

Mixing of

The amounts of electrons that each sample is capable to store (42 µmol/g for A and 7 µmol/g for R) are in general agreement with the values reported in the literature determined from chemical reactivity and lying in the range from units to few tens of µmol/g (e.g. ~10 µmol/g [lxix]; ~50 µmol/g [55] ~10 µmol/g [lxx]; ~7 µmol/g [lxxi]). The intrinsic properties of such species should obviously depend on the polymorph and on the crystalline face exposed.

However the absolute amount of stored electrons revealed by the H2 release experiments (which obviously affects the rate of H2 release), changes in step with the specific surface area of three samples, A >R > B, thus suggesting that surface species are involved.

Mechanistic considerations.

In this section we present a simple phenomenological model that gives a coherent quantitative description of the observed PHER rate variations. Interaction between Pt/A and R is arbitrarily taken as an example. Other combinations of polymorphs could be described by essentially the same equations with different rate constants. The model contains several simplifications and assumptions. In order to focus on the observed effects of polymorphs mixing, only the mechanistic rate steps are considered that involve the accumulated electrons.

The concentrations of all stable chemical species (methanol, water) are supposed to be constant and hence do not explicitly participate in the differential rate equations but are encapsulated in the respective rate constants. Photo generated holes are supposed to be shortlive and to be rapidly consumed by excess of methanol, so their transfer between the polymorphs is not considered. Complex mechanisms of surface chemical reactions of protons reduction and methanol oxidation which follow Langmuir-Hinshelwood type kinetics [lxxii, lxxiii] are not considered here. Indeed, conversions in all experiments are low (< 0.01%) so the concentrations of organic reaction products are considered to be negligible. In all the experiments the rate of H2 production was stable in time, which allows applying steady state approximation to the reaction kinetics.

In the dark, the initial "ground state" particles i.e. non-excited by light platinum-bearing anatase (PtA) and bare rutile (R) particles are loaded in the slurry respectively at the concentrations [PtA]0 and [R]0. The particles can be excited by light to the states bearing an electron-hole pair, PtA * and R * respectively. These excited states have short lifetime and rapidly recombine, but in the presence of alcohol the holes are consumed and long-live electron -bearing particles are produced, PtA -and R -. These states can in turn be deactivated back to the ground state without producing hydrogen via recombination with (novel generations of) holes, reaction with residual oxygen or other processes. The equilibria between ground state and electron-bearing species are described by reversible equations (1,

2). The interaction between ground-state and electron -bearing particles may lead to the transfer of accumulated electron, as expressed by the equation ( 3).

The electron -bearing particles of both types PtA -and R -are capable of reducing protons and so producing hydrogen. However as observed in the experiment, PHER rate on any bare TiO2 is by two orders of magnitude lower than on any Pt/TiO2. Therefore only Pt-bearing particles are considered as hydrogen producers, for simplicity. Production of hydrogen consumes the accumulated electrons and irreversibly returns the particles to the ground state, according to the equation ( 4). The surface chemical HER mechanistic steps (dissociation of organic substrate, recombination of H atoms, etc.) with their complex kinetics are lumped here into one final step (4) with an effective constant k4.

⇔

⇔

(2)

+ ⇔ + (3) → (4)
The rate constants k1-k4 and (for the reversible processes 1-3) the equilibrium constants K1-K3 correspond to the mechanistic stages (1-4). The well-known two -electron character of the hydrogen release reaction is not revealed by the equations 1-4 as here we suppose pseudo-first order of the reaction rate by the amount of stored electrons and zero order by all other species present in a large excess (assumption always valid at sufficiently low conversions). The experimentally observed H2 release rate rPHER is constant, so the steady state equation is:

/ = = (5)
Or, equivalently,

d[PtA -]/dt = 0 (6)
Applying the steady state approximation to the system of differential equations corresponding to the steps (1-4) we reduce them to algebraic relationships and obtain the eq. ( 7):

! " # ! " = $ % & $ #' ( # $ #% & $ ' ( & $ ) (7)
If low relative concentration of PtA -and R -is assumed (most part of TiO2 is in the ground state), then the ground state concentrations are approximately equal to the initial values and we obtain:

! " # ! " * = $ % & $ #' ( # $ #% & $ ' ( * & $ ) (8)
From eq. 8 the rate expressions for two limiting cases of low and high [PtA]0/[R]0 can be immediately seen. Indeed, if [PtA]0 >> [R]0 and k4 is small (hydrogen release is the rateliming step), then expectedly:

rPHER = k4K1[PtA]0 (9)
as it obviously should be for pure PtA.

On the other hand, at a sufficiently large

[R]0, k3[R]0 >> {k-1, k4} and k-3[R -] >> k1, therefore rPHER = k4[PtA]0 $ #' ( # $ ' ( * = k4[PtA]0K3 -1 ( # ( * (10)
The physical meaning of the equation ( 9) is that at high excess of R particles relative to PtA ones, the PHER rate is controlled by excitation of R by light and by the equilibrium of stored electrons transfer between R to PtA. If R is in a large excess then the flow of electrons transfer from R -to PtA is negligible as compared with the flow of R own excitation and decay, and therefore R is in equilibrium with R -, i.e. approximately [R -] = k2[R]0. Supposing again k4 to be relatively small, we obtain the equation ( 11), depending on the rate constant k4 and the equilibrium constants K2 and K3:

rHER = k4K2K3 -1 [PtA]0, (11)
The last equation means that hydrogen is formed mostly due to the accumulated electrons, which are first generated by light on the (present in a large excess) bare R phase and then transferred to the active sites on PtA. Again, as in eq. ( 10) the dependence of PHER rate on

[PtA]0 is linear, but with a different proportionality coefficient. Note that in the eq. ( 11) the rPHER does not explicitly depend on the amount of added R, but the last is supposed to be large enough to define the resulting amount of accumulated electrons available for the reaction. In agreement with these equations, asymptotic PHER rate behavior is observed in the experiment vs. the relative amount of R.

In the most general case when the values of the constants and the amounts of [PtA]0 and [R]0 are comparable, the PHER rate as a function of the amount of added [R]0 has no simple analytic expression and should be obtained numerically. However, physically relevant and instructive is the case of a relatively rapid stored electrons exchange as compared with other steps (i.e. the electrons are supposed to be long-live and travel relatively long distances, before being consumed to form H2). In such case the step 3 is at equilibrium and

[R -] = K3 ! " # ( ! " ≈ , - ! " # ( * ! " * (12)
After substitution the expression ( 12) for [R -] in (7), some trivial algebraic transformations and normalization, the equation ( 7) can be reduced to a linear rational function, expressing the dependence of the relative PHER rate on the relative amount of added bare TiO2, [R]0 :

Conclusions

In summary, strong positive or negative non-additivity of PHER rate observed upon mixing of Pt-loaded and bare TiO2 polymorphs in the methanol slurries suggests existence of directional transfer of stored electrons. Hydrogen release in the dark after injection of Pt/TiO2 to the preirradiated bare TiO2 slurries demonstrates that stored electrons might migrate from the preirradiated TiO2 towards the particles of Pt-loaded polymorph. Depending on the polymorphs involved, the rates of H2 release in the dark differ by order of magnitude, which gives additional support to the preferential directionality of the stored electrons.

Establishing the exact nature and the relative energies of the long live stored electrons needs further investigations and remains outside the scope of this work. Recent results from the energy-resolved photo acoustic spectroscopy demonstrate that the energy of stored (or trapped) electrons has a relatively wide distribution that depends on the TiO2 individual sample properties [lxxiv, lxxv]. Unlike the band gaps of bulk TiO2 phases which are single values characteristic of the corresponding crystalline structures, the trapped states energy might depend on many factors besides polymorph type, such as chemical environment and crystalline facet exposed, or trapped states generation method. The energies of experimentally reported and calculated trapped levels are located near the CB edge of TiO2 in a wide energies range of 0.3 -1.8 eV [lxxvi, lxxvii, lxxviii]. At least two distinct long-live trapped states can exist in irradiated anatase [lxxix]. Theory models are also versatile and include step edges for anatase (101) [lxxx], polarons associated with surface hydroxyls [lxxxi], deeply trapped bulk species [lxxxii] and others [lxxxiii]. The diversity of such states does not allow attributing the observed effects to one or them. However particularly strong effects observed in this work for the high-surface area PC500 anatase allow speculating that stored species relevant to catalysis are located at the surface. As the particles surfaces are obviously solvated, the transfer probably occurs with assistance of OH groups, linked respectively to Ti(III) and Ti(IV) atoms (but participation of methanol molecules part cannot be excluded as well):

Ti(III)-OHA + Ti(IV)-OHR <=> Ti(IV)-OHA + Ti(III)-OHR

The observed directionality is in a seeming contradiction with the bands alignment in pure TiO2 phases. The electron affinity and the work function of anatase were reported to be greater than for rutile, and therefore the bottom of conduction band of rutile is higher by 0.4-0.1 eV, depending on the study [lxxxiv, lxxxv, lxxxvi]. Heterojunction electrons flow direction from rutile to anatase has been verified by measurement of the surface potential profile over the interface of TiO2 phase junction [lxxxvii]. However both the probed species and the time scales are obviously very different from our case. In the solid-state heterojunctions, the events on the time scales from ps up to few ns are involved. On the other hand, in the liquid-phase PHER experiments, transfer of long-live (for at least minutes) species occurs between the solvated surfaces. Obviously, the process at stake is not a direct electrons transfer between the lattices of two polymorphs, but an exchange between surface reduced species (in which participation of organic molecule could not be excluded).

The observed effects open new prospective for the design of novel simple and efficient experiments, both on the fundamental and applied sides. Thus, noble metals atoms economy 21 can be achieved if the directionality of stored electrons transfer is taken into account (in our case -when bare anatase PC500 is mixed with Pt-loaded rutile). Beside these specific catalysts, novel composite systems might be conceived on the basis of exchange of stored electrons between the nanoparticles in suspensions.

Fig. 1 .

 1 Fig. 1. (a) XRD patterns of three titania polymorphs. All peaks on the patterns correspond respectively to the ICDS cards of anatase (01-076-1940), rutile (01-089-0552) and brookite (01-076-1937). The intensity scale for rutile and brookite is changed for the presentation convenience; (b) UV-visible reflectance spectra of three phase-pure TiO2 polymorphs.

  Figure S5 shows Zeta potential for three titanias, measured in 1:1 water-methanol mixture in 0.001M KNO3. Zeta potential measurements in water-methanol suspensions show IEP values in the range between 4 and 6, usually observed in pure aqueous medium. The IEP value changed in the sequence A

Fig. 2 .

 2 Fig. 2. TEM images for the Pt/R + A (a), Pt/B +R (b) and Pt/A+R (c) mixtures after PHER catalytic tests. Three polymorphs are clearly distinguishable due to striking difference of their morphology. Platinum particles can be seen as dark spots.

Fig. 3 .

 3 Fig. 3. Specific PHER activity of the Pt/TiO2 catalysts vs. the polymorph type and the slurry concentration.

Figure 4

 4 Figure4shows variation of PHER rate for a fixed amount of Pt/TiO2, as a function of the relative amount of added bare polymorph. The experiments with slurry concentrations of 0.2 g/L are presented in the main text, but similar behavior was observed whatever the initial Pt/TiO2 concentration. Strong non-additivity of PHER properties, of both negative and positive sign, has been observed. The PHER rate for Pt/A always decreases after addition of bare TiO2 polymorphs. The strongest effect was observed due to rutile addition. Addition of A powder to Pt/A leads only to a slight PHER rate decrease (Fig.6a). Pt/B and Pt/R PHER performance is increased by addition of bare anatase: by a factor of 1.5 for Pt/R and by a factor of 3 for Pt/B (Fig.4 b, c). The effects of mixing of Pt/R or Pt/B and bare B or R are much weaker. Addition of B to Pt/B and of R to Pt/R leads to a slight decrease of PHER rate, probably due to the light absorption in the concentrated slurry which leads to lesser illumination (directed on the bottom of reactor) of the topmost layers and therefore to lower apparent specific PHER rate. Indeed, light absorption is an important factor explaining the decrease of PHER rate vs. the catalyst loading (Fig.3). We applied the total slurry loadings in the domain of approximately constant specific rate. Under this condition the relative activity values vs. the amount of added bare TiO2 obtained for different initial Pt/TiO2 concentrations merge to common continuous curves with a clear asymptotic (ascending or descending) behavior.

Fig. 4

 4 Fig. 4 (a-c) Normalized PHER rates in the mixtures of 0.5% wt. Pt/TiO2 catalysts with the powders of bare TiO2 polymorphs. Concentration of the initial Pt/TiO2 slurries is 0.25 g/L; (d) Specific PHER rates per Pt mass as a function of the mixtures composition at 0.25 g/L slurries concentration. Note that the compositions ranges in (a-c) and (d) overlap but are not the same. In all cases activity is normalized taking to unity the activity of the initial pure Pt/TiO2 catalysts.

Fig 5

 5 Fig 5 Release of H2 in the dark after injection of 10 mg of Pt-loaded TiO2 to the suspensions of 100 mg of bare TiO2 polymorphs, pre-illuminated for 90 min (only the last 18 min of this period represented in the figure). The amount of released hydrogen is given per gram of the initial (preirradiated) catalyst.

  Pt/TiO2 with pre-irradiated bare TiO2 of the same polymorph leads to H2 release due to the scrambling of stored electrons, by hopping between TiO2 particles towards Pt, in agreement with antenna mechanism [40]. However such scrambling in case of Pt/A + A mixture is obviously much slower than directional transfer in case of Pt/R + A or Pt/B + A (Fig. 6a). On the contrary, while the rate of scrambling for Pt/R + R and Pt/B + R mixtures is approximately the same, it is considerably slower for Pt/A + R (Fig 6b). Therefore transfer of stored electrons has a clear directionality from A to Pt/R (or from A to Pt/B).

Fig. 6

 6 Fig. 6 H2 release after injection of Pt/TiO2 and Pt/Al2O3 suspensions (1 mg each) to the slurries of 100 mg of pre-irradiated A (a) and R (b). The amount of released hydrogen is given per gram of the initial (pre-irradiated) catalyst.

  rate for Pt/A without addition of R, whereas a, b and c are some combinations of the rate constants. Depending on the values of the coefficients a, b and c, the PHER rate can be decreasing or increasing linear rational function of [R]0. In the general case of the amount of bare phase (here [R]0) close to zero, the slope of PHER rate vs. the amount of added base phase (here the derivative of rPHER by [R]0) is equal to b-ca, which might have a positive sign (which corresponds to addition of A to Pt/R or Pt/B) or negative sign (addition of R or B to Pt/A). In all cases the ensemble of experimental results, expressing the relative PHER rate vs. the amount of added bare polymorph could be fitted with a linear rational function at high figures of merit (Fig. S11-S13).

Table S1

 S1 Properties of the solids under study and Pt/P25 reference.

	Solid	Ssp m 2 /g Eg , eV	TiO2 XRD size	DLS size nm a	size Å Pt TEM	Pt, %wt. b
				nm
	Anatase (PC500) 338(15)	3.26(5)	6.5(2)	280(50)	ND	ND
	Rutile	88(5)	3.06(5)	41(4)	250(50)	ND	ND
	(nanorods)
	Brookite	47(5)	3.18(5)	119(8)	220(50)	ND	ND
	0.5%Pt/A	315(15)	3.28(5)	ND	290(50)	16(3)	0.52(1)
	0.5%Pt/R	80(5)	3.08(5)	ND	300(50)	23(6)	0.49(1)
	0.5%Pt/B	49(5)	3.17(5)	ND	280(50)	18(4)	0.48(1)
	0.5%Pt/P25	55(5)	3.30(5)	20(2)	90(

Table 2

 2 PHER rates, photon efficiency and experimental conditions for selected solids studied in this work and for literature references

	Co-catalyst	PHER	PHER	PHER	Lamp,	Slurry	PE, %
	Catalyst	rate,	rate,	rate,	mW	g/L
		µmol/h	µmol/g.h	mmol/gPt.h
	TiO2 rutile (R)	0.18	5.1	-	Hg 150	1.5	0.02
	TiO2 Brookite (B)	0.06	1.7	-	Hg 150	1.5	0.007
	TiO2 P25 (A+R)	1.1	33	-	Hg 150	1.5	0.08
	TiO2 PC500 (A)	1.3	35	-	Hg 150	1.5	0.09
	0.5%Pt/P25	57	10190	2038	Hg 150	0.25	4.6
	0.5%Pt/A 0.5%Pt/A+R 0.1%Pt a	66 10.0	11800 1787	2360 357	Hg 150 Hg 150	0.25 0.25	5.4 0.81
	0.5%Pt/A+R 0.025%Pt	9.1	1626	325	Hg 150	0.25	0.74

a total Pt content in the composite catalyst b approximately recalculated using lamp power ratio and curve of normalized PHER rate vs. slurry concentration (Fig

3

).

Table 2 )

 2 .

P. AfanasievGraphical abstract