Pierre-Henry Leveau
email: pierre-henry.leveau@univ-angers.fr

Sandra Camus
email: sandra.camus@univ-angers.fr

INFLUENCE OF EMBODIMENT AND CHALLENGES ON CONSUMERS' BEHAVIORAL INTENTIONS DURING A VIRTUAL REALITY EXPERIENCE

Keywords: « virtual reality », « embodiment », « gamification », « challenges », « behavioral intentions », « experiential marketing »

The development of virtual reality (VR) applications provides users to physically interact in the virtual environment as in a real experience. This new experience offers for brands a new way to promote their offer to consumers, becoming the avatar and accomplish goals based on gamification mechanisms like video games. While actors from different professional fields are developing VR experiences (VRE), especially in the tourism industry, several works has focused on the role of VRE on enjoyment but not between the role played by gamification between the feeling of embodiment (resulting of the VRE sensorimotor interactions) and behavioral intentions. The research aims to measure the effects of the feeling of embodiment during a VR tourism experience on consumers' behavioral intentions and the role of the challenges level offered as moderator. Results of the experimental (n=200), where the user embodies a kayaker exploring Antarctica, confirm that the feeling of embodiment induces positive behavioral intentions towards the discovered tourist destination. More specifically, a greater intensity in the level of challenges offered to achieve the virtual experience moderates with greater intensity the relationship between feeling of embodiment and behavioral intentions. On a theoretical level, research enrich literature in marketing through the concept of embodiment, never used before. The study also makes recommendations to professionals and VR application developers on the positive effects of VRE directly related to the feeling of embodiment as well as the challenges offered. Finally, the study sets out certain limits to follow for further research in the field of marketing.

INTRODUCTION

Thanks to VR, the consumer can discover the offer before buying it [START_REF] Oh | Measuring Experience Economy Concepts: Tourism Applications[END_REF], explaining its increasing use by professionals to promote their offer [START_REF] Hashish | Les effets de l'expérience de téléprésence sur internet sur les émotions, les attitudes et les intentions comportementales des touristes : le cas du tourisme domestique en Égypte[END_REF], especially those of tourism [START_REF] Guerra | Virtual reality-shows a new vision for tourism and heritage[END_REF] take advantage that today, 26 million consumers own a VR headset [START_REF] Omdia | Omdia research reveals 12.5m consumer VR headsets sold in 2021 with content spend exceeding $2bn[END_REF] and that this figure will double by 2025 (ibid.).

Given the tremendous growth of VR, we believe it is essential to understand the role of VR on consumer attitudes and behavior. Indeed, as consumer behavior evolves with technological innovations [START_REF] Bargain | L'expérience : une approche conceptuelle au service du tourisme[END_REF], it is fundamental to "reconsider how to think and design experiences in virtual environments" (Gorisse, 2019, p.20) even though VR could, in the next few years, become accustomed in everyday uses [START_REF] Alcañiz | Virtual Reality in Marketing: A Framework, Review, and Research Agenda[END_REF].

The emergence of immersive technologies [START_REF] Milgram | A taxonomy of mixed reality visual displays[END_REF] offers the opportunity to discover extraordinary and memorable experiences for consumers [START_REF] Arnould | River Magic: Extraordinary Experience and the Extended Service Encounter[END_REF] in experiential marketing contexts [START_REF] Flavián | The influence of scent on virtual reality experiences: The role of aroma-content congruence[END_REF]. VR enhances interactivity with the offer, changing the modes of control as well as the engagement of the participant and his or her body [START_REF] Flavián | Integrating virtual reality devices into the body: effects of technological embodiment on customer engagement and behavioral intentions toward the destination[END_REF].

VR is a computer-based, interactive and multi-sensory environment that operates in real time and involves living or bringing to life a story that is focused on inducing emotions and engaging multiple human senses, and it thus stimulates greater mental imagination [START_REF] Henderson | Virtual reality in stroke rehabilitation: a systematic review of its effectiveness for upper limb motor recovery[END_REF][START_REF] Barnes | Understanding use continuance in virtual worlds: empirical test of a research model[END_REF]. The body does indeed take an important place when the participant performs VR activities [START_REF] Fuchs | Théorie de la réalité virtuelle[END_REF].

The rise of VR provides ideal ground for brands to place their offers by proposing playful, original, and pleasant experiences in which the consumer embodies the main actor's role and can interact with their whole body in the metaverse (shared virtual space, immersive, accessible in real time with sensorimotor interactions, in which it is possible to embody characters) [START_REF] Roth | Construction of the Virtual Embodiment Questionnaire (VEQ)[END_REF].

A significant amount of marketing research on VRE has recently emerged, particularly for tourism offerings [START_REF] Hopf | Exploring the impact of multisensory VR on travel recommendation: A presence perspective[END_REF][START_REF] Pasanen | Comparing Tablet and Virtual Reality Glasses for Watching Nature Tourism Videos[END_REF][START_REF] Marasco | Exploring the role of nextgeneration virtual technologies in destination marketing[END_REF]. These lasts ones show how important it is for destinations and brands to understand their customers to attract them and best promote their locations [START_REF] Pesonen | A Closer Look at Tourist Information Search Behaviour When Travelling Abroad: What Is the Role of Online Marketing in Choice of Destination?[END_REF].

In VRE, embodied avatars beyond "on real-life experience […] that often reached beyond real-life conditions" [START_REF] Nagy | My Avatar and Her Beloved Possession": Characteristics of Attachment to Virtual Objects[END_REF]Koles, 2014, p. 1130). When the VRE occurs, individuals reflect their personality traits on the avatar [START_REF] Belisle | Avatars as information: Perception of consumers based on their avatars in virtual worlds[END_REF] as if they were inhabiting him (Gee, 2008, p.258). What's more, with real sensorimotor interaction mechanisms [START_REF] Galvan-Debarba | Characterizing first and third person viewpoints and their alternation for embodied interaction in VR[END_REF]. Although this established knowledge exists, there is a lack of empirical research considering the impact and cognitive effects of embodiment on sensorimotor interactions and their mechanisms, as well as their subsequent impacts on consumer behavior. Here we set out to determine the extent to which sensorimotor interactions can create a feeling of embodiment that then has positive effects on consumer's behavior after the VRE. Furthermore, during VRE, these activities often incorporate gamification elements, such as challenges. Generally requiring the completion of a set of tasks, challenges are part of gamification mechanisms, aiming to create and stimulate iterative behavior on the part of the individual [START_REF] Salen | Rules of Play: Game Design Fundamentals[END_REF]. Challenges therefore have a central place in VRE. They underlie a psycho-motor [START_REF] Barnes | Understanding use continuance in virtual worlds: empirical test of a research model[END_REF] and cognitive (Hamdi-Kidar and Maubisson, 2012) engagement of the participant. However, the influence of challenges on the VRE has been little studied in the marketing literature [START_REF] Caroux | Presence in video games: A systematic review and meta-analysis of the effects of game design choices[END_REF][START_REF] De Simone | Is cheating a human function? The roles of presence, state hostility, and enjoyment in an unfair video game[END_REF].

To summarize, this research investigates the role of embodiment and level of challenge on tourist-consumer's behavior during a VRE. Therefore, beyond the technical configuration proposed to live the VRE, we ask what influences do the sense of embodiment and the challenges during VRE have on the user's behavioral intentions towards the discovered destination and activity?

Theoretically, the aims of this research are to study for the first time these effects in VRE experiences and in marketing, in this instance the positive role played by embodiment and challenges offered on behavioral intentions with a VR application. The study makes also recommendations to professionals and VR application developers on the positive effects of VRE directly related to the feeling of embodiment as well as the challenges offered. Finally, the study sets out certain limits and avenues of research to follow for further research in the field of marketing.

LITERATURE REVIEW

Considered the most immersive digital technology [START_REF] Fuchs | Théorie de la réalité virtuelle[END_REF], VR allows the user to feel as if they are becoming the protagonist of the virtual experience by interacting in real time [START_REF] Harris | Customer participation in retail service: Lessons from Brecht[END_REF], which tends to foster a sense of truly embodying the avatar within the virtual environment [START_REF] Roth | Construction of the Virtual Embodiment Questionnaire (VEQ)[END_REF][START_REF] Spanlang | How to build an embodiment lab: Achieving body representation illusions in virtual reality[END_REF]. In the literature, it is recognized that any effort to foster interaction with the offer and its environment is likely to improve the embodiment [START_REF] Krishna | Customer Sense. How the 5 Senses Influence Buying Behavior[END_REF][START_REF] Elder | The "visual depiction effect" in advertising: Facilitating embodied mental simulation through product orientation[END_REF]. While many studies have established that VRE have a positive influence on consumers' enjoyment and immersion as well as behavioral intentions [START_REF] Marasco | Exploring the role of nextgeneration virtual technologies in destination marketing[END_REF][START_REF] Pasanen | Comparing Tablet and Virtual Reality Glasses for Watching Nature Tourism Videos[END_REF], very few have investigated their determinants, while the relationship with the embodied virtual character, in front of the player's surrogate mind and body [START_REF] Gee | Video Games and Embodiment[END_REF] and in completing challenges [START_REF] Hamdi-Kidar | Les chemins d'accès à l'expérience de flow : le cas des jeux vidéo[END_REF], is central.

The feeling of embodiment

To embody literally means 'to enter into the flesh'. Embodiment is a concept currently mobilized in VR and user perception research [START_REF] Roth | Construction of the Virtual Embodiment Questionnaire (VEQ)[END_REF][START_REF] Galvan-Debarba | Characterizing first and third person viewpoints and their alternation for embodied interaction in VR[END_REF]. According to [START_REF] Biocca | The cyborg's dilemma: Progressive embodiment in virtual environments[END_REF], the self in the VE is composed of the individual's real (physical) body, their virtual body (visible or not in the experience), and their mental representation of the virtual body with their real body. [START_REF] Kilteni | The sense of embodiment in virtual reality[END_REF] assert that the user feels that they are in the virtual body when their motor properties are perceived as being identical to those of their real body. It is through this approach that we study the concept of embodiment, as the user in a VRE becomes an agent playing the lead role in the virtual persona's 'skin'. Embodiment corresponds to a fusion between the human and the virtual body, then forming the same entity [START_REF] Tussyadiah | Embodiment of wearable augmented reality technology in tourism experiences[END_REF][START_REF] Flavián | Impacts of technological embodiment through virtual reality on potential guests' emotions and engagement[END_REF].

Agency and self-localization are the dimensions that compose the feeling of embodiment [START_REF] Roth | Construction of the Virtual Embodiment Questionnaire (VEQ)[END_REF]. Agency corresponds to the coordination between the intention expected by the individual through their bodily actions and the effective responses that manifest in the VE.

The more that movements performed in the real sphere correspond to those observed and reproduced in the virtual sphere, the greater the agency experienced by user [START_REF] Caspar | The relationship between human agency and embodiment[END_REF]. Ownership corresponds to the user attributing the virtual body to their own body and can even stretch as far as feeling real bodily sensations in response to what happens to the virtual body in the VE [START_REF] Tsakiris | My body in the brain: A neurocognitive model of body ownership[END_REF]. Ownership is like a mental transfer where the person becomes aware of the bodily reactions resulting from sensory stimulation.

With VR, users no longer just see the offering, "but interact with it and experience it" (ibid., p. 1464). This "experience with products in the virtual world (without interacting with their physical counterparts) plays an increasingly important role" (Łysik and Łopaciński, 2019, p. 42). Interacting even virtually with the offer strengthens satisfaction with the experience and then leads to consumer commitment and purchase intention towards the offer (ibid.).

Compared to a website, VR allows interactions as they would be possible in a real environment (Luangrath et al., 2021;[START_REF] Alcañiz | Virtual Reality in Marketing: A Framework, Review, and Research Agenda[END_REF]. Having interactive contacts with the offer becomes scripted in experiences involving the person physically. Indeed, it is no longer a matter of orienting or zooming using the keyboard and mouse, for example, but of using all or part of one's body and performing the same gestures in the same way as they would in a real environment [START_REF] Kilteni | The sense of embodiment in virtual reality[END_REF]. This phenomenon thus corresponds to embodiment (ibid.; [START_REF] Flavián | Integrating virtual reality devices into the body: effects of technological embodiment on customer engagement and behavioral intentions toward the destination[END_REF]. According to Tsakiris et al. (2006), haptic and ad hoc interactions in VR lead to the feeling of embodiment. Indeed, the "digital representation of the product's touch" (Luangrath et al. 2021, p. 5) can lead to perceiving the virtual experience as identical to the actual lived experience and consider the virtual body as one's own body (ibid.). To this end, the power of VR is superior to less immersive media (Łysik and Łopaciński, 2019). [START_REF] Elder | The "visual depiction effect" in advertising: Facilitating embodied mental simulation through product orientation[END_REF] were the first in marketing to study the role that body involvement, and more specifically the embodiment of a virtual body, has on an individual's behavioral intentions following interaction with an offer. These authors have shown that embodiment experienced through motor stimulation leads to positive behavioral intentions of the user towards the offer (ibid.). Thus, we formulate the following hypothesis:

H 1 : For a VRE, the embodiment felt has a positive effect on behavioral intentions.

More specifically: For a VRE, the embodiment felt has a positive effect on the intentions to discover and visit the destination for real (H 1a ) and to the intention to (re)do the sport activity for real (H 1b ).

The role of challenges

Gamification mechanisms [START_REF] Harwood | An Investigation into Gamification as a Customer Engagement Experience Environment[END_REF] "better engages end users" (Liu et al., 2011, p. 1). We therefore assume that challenges, as an experiential condition, will influence the sense of embodiment through a mechanism of mental and physical transfer of goals from the embodied avatar to one's own person [START_REF] Gee | Video Games and Embodiment[END_REF] to complete these challenges and that the efforts undertaken to complete them (Hamdi-Kidar and Maubisson, 2012) will engage the skills of the individual [START_REF] Gee | Video Games and Embodiment[END_REF]1996). Several studies have shown that sensorimotor interactions positively impact feelings of embodiment [START_REF] Roth | Construction of the Virtual Embodiment Questionnaire (VEQ)[END_REF][START_REF] Galvan-Debarba | Characterizing first and third person viewpoints and their alternation for embodied interaction in VR[END_REF]. The pseudo-natural interactions enabled with VR make it easier to accomplish challenges [START_REF] Foloppe | Évaluation et entraînement des activités de la vie quotidienne dans la maladie d'Alzheimer : Intérêt de la réalité virtuelle[END_REF]. Indeed, the techniques developed in VR to interact in virtual environments are often similar to those in real environments [START_REF] Sternberger | Interaction en réalité virtuelle[END_REF]. This allows, on the one hand, to facilitate the understanding of interaction systems in the virtual environment, then transposed to the identical of the real space [START_REF] Foloppe | Évaluation et entraînement des activités de la vie quotidienne dans la maladie d'Alzheimer : Intérêt de la réalité virtuelle[END_REF] and, on the other hand, to affect the perception of actually being the virtual character thanks to a natural behavior that does not require prior learning [START_REF] Fuchs | Le Traité de la réalité virtuelle[END_REF]. This feature improves the degree of perceived realism during sensorimotor interactions performed to carry out activities in the virtual environment [START_REF] Foloppe | Évaluation et entraînement des activités de la vie quotidienne dans la maladie d'Alzheimer : Intérêt de la réalité virtuelle[END_REF]. There is then an increased credibility on the mental illusion resulting from sensory realism [START_REF] Kilteni | The sense of embodiment in virtual reality[END_REF]. Thus, it therefore reinforces the bodily properties of the virtual body with those of the real body (ibid., [START_REF] Fuchs | Le Traité de la réalité virtuelle[END_REF]. Furthermore, the motor interactions required to complete the challenges will enhance the sense of agentivity [START_REF] Grodal | Stories for Eye, Ear, and Muscles: Video Games, Media, and Embodied Experiences[END_REF] and possession [START_REF] Klein | Réalité virtuelle et santé mentale: La fin d'un art de faire ?[END_REF] and lead to the sense of embodiment [START_REF] Harwood | An Investigation into Gamification as a Customer Engagement Experience Environment[END_REF].

Moreover, when interactions with the offer engage the individual bodily, behavioral intentions are better [START_REF] Krishna | Sensory marketing, embodiment, and grounded cognition: A review and introduction[END_REF]. The work of [START_REF] Lupinek | Congruity of Virtual Reality In-Game Advertising[END_REF] also concludes the positive influence of interaction on behavioral intentions during VR consumption experiences, highlighting in particular the role of the user's body involvement. That's why, we consider the level of challenges as a moderator between embodiment and consumer behavior.

Indeed, according to [START_REF] Salen | Rules of Play: Game Design Fundamentals[END_REF], goal attainment is closely related to the appreciation of the quality of playability. The higher the participant's goal attainment, the more engaged he or she will feel [START_REF] Liu | Gamifying intelligent environments[END_REF] and, consequently, the more he or she will feel that the virtual environment and the real environment are merging [START_REF] Gee | Video Games and Embodiment[END_REF]. This leads us to formulate the following hypothesis:

METHODOLOGY

To answer the research question, we carried out an experiment with participants during a university event that took place in Angers in 2022. Two hundred participants characterized by a varied profile (individuals of different ages, socio-professional categories, lifestyles, etc.which corresponds to the population targeted by our research) carried out the proposed VR experience. Participants were randomly assigned to one of our two experimental units (two levels of challenge: low vs. high) taking care that the two samples (low vs. high) are homogeneous in terms of gender, occupation, age and education. We have followed a between-subjects approach. The resulting convenience sample (n=200 after data cleaning) was focused on individuals who were old enough to travel and to finance the offer with their own income in this case, a journey. We paid particular attention to obtain a normal distribution for each experimental cell. The following table describes the sample of respondents in detail. The experiment is produced by National Geographic and available on the Oculus Store1 .

During this one, the participant is immersed for about ten minutes in a kayak ride to discover the virtualized Antarctic. Sensorimotor interactions allow exploration of the virtual environment. For the weak challenge, the user simply goes from point A to point B. The strong challenge requires following a specific path and take a picture of two animals.

Next, we performed a principal component analysis (PCA) and a confirmatory factor analysis (CFA) under IBM SPSS (v26) of the measurement instrument related to embodiment. We selected the scale of [START_REF] Roth | Construction of the Virtual Embodiment Questionnaire (VEQ)[END_REF]. We selected the one of [START_REF] Bigné | Tourism image, evaluation variables and after purchase behaviour: Inter-relationship[END_REF] to measure the intention to visit and discover the destination for real; and the one of [START_REF] Supphellen | Drivers of intention to revisit the websites of well -known companies[END_REF] to determine the intention to (re)do the sport activity for real.

The items of our scale (post-PCA) and the calculated indices of the CFA were controlled. The PCA allows us to rule on the representativeness of our construct according to the Kaiser,

RESULTS

The experiment (n=200) conducted with the Meta Quest 2 VR headset allows us to analyze these relationships using a partial least square's (PLS) approach. The PLS results show both that the feeling of embodiment positively influences behavioral intentions, and that the presence of challenges positively moderates this same relationship. Therefore, hypotheses H 1 and H 2 are validated. Next tables (Tab. 3 and 4) describe the meanings of the regression analysis realized.

Direct effects with the partial least square (PLS) approach

First, we carried out a regression analysis according to structural equations model (SEM) methodology based on the PLS approach [START_REF] Wold | Partial Least Squares[END_REF]. The estimation component approach offered by PLS is particularly suitable for formative factors [START_REF] Hair | Multivariate data analysis[END_REF]. We highlight the relationship between embodiment and behavioral intentions the path coefficient (β) which measures the direction (positive or negative) and strength of the relationship of the independent variable on the dependent variable. We observe that embodiment significatively (p<.05) and positively influence (β>0) the intention to visit the destination (H 1a ) and the intention to (re)do the sport activity for real (H1 b ).

Impact of challenges as moderator variable

We realized a multigroup analysis (MGA) [START_REF] Henseler | The Use of Partial Least Squares Path Modeling[END_REF] to measure the effect of the qualitative moderating variable (challenges). MGA has the advantage of being simple and robust [START_REF] Chin | A permutation procedure for multi-group comparison of PLS models[END_REF][START_REF] Hair | Advanced Issues in Partial Least Squares Structural Equation Modeling (PLS-SEM)[END_REF] and giving more reliable results than other comparison tests when the sample size is small (ibid.). The MGA is both simple and robust (ibid.) and is known to be more reliable than other tests when the sample size is relatively small (ibid.). MGA show that challenge levels significatively (p<.05) modulates the relationship of embodiment with intention to visit the destination (H 2a ) and intention to (re)do the sport activity for real (H 2b ). The direction of effect (≠ β high-level vs β low-level > 0) confirms that a higher level of challenge offers a greater effect on individual especially in the relationship between cognitive effects and resulting behavioral decisions.

CONCLUSION

As a reminder, the research question investigates the role that the sense of embodiment and the level of challenges have on the user's behavioral intentions towards the destination and the activity discovered following the virtual experience.

This research contributes to demonstrate for the first time in marketing the positive role played by embodiment on behavioral intentions with a VR application. This is a major theoretical contribution, as the latest generation of VR applications systematically include these mechanisms valued by consumers. This confirms the tendency for consumers to want to actively live their consumption experiences [START_REF] Durand | Le storytelling : le guide pratique pour raconter efficacement votre marque[END_REF].

Moreover, Krishna and Schwarz (2014, p. 1) showed that sensory and motor stimulation of the body are like "a language" and are determinants in the "judgment and decision-making" of consumers. Our study demonstrates that beyond sensory and motor stimulation, the feeling of embodiment (feeling like the virtual body was our body, feeling like we were controlling the movements of the virtual body, etc.) have a positive influence on behavioral intentions of consumers: intention to discover and visit the destination for real, and intention to (re)do the sport activity for real. The positive effects of the embodiment (in terms of feeling) on behavioral intentions confirm that VR enables a "try-before-you-buy" service (Tussyadiah et al., 2018, p. 141) that engages the consumer in the offer [START_REF] Huang | Exploring the implications of virtual reality technology in tourism marketing: An integrated research framework[END_REF]. It supports recent findings on the influence of technological embodiment on behavioral engagement [START_REF] Flavián | The influence of scent on virtual reality experiences: The role of aroma-content congruence[END_REF] and of embodied cognition on purchasing behavior [START_REF] Wen | Virtual wine tours and wine tasting: The influence of offline and online embodiment integration on wine purchase decisions[END_REF] but focusing, this time, on embodiment felt by individuals.

Additionally, our research shows that more engaging challenges in the experience lead to a higher sense of embodiment. This has not been the focus of previous research in marketing, where only a few studies have demonstrated the influence of challenges on consumer attitudes and behavior (e.g., [START_REF] Hamdi-Kidar | Les chemins d'accès à l'expérience de flow : le cas des jeux vidéo[END_REF]. The same is true in computer sciences, where few works have studied the links between challenges and user engagement in an embodiment context (e.g., [START_REF] Gee | Video Games and Embodiment[END_REF]. Our research shows that for a relatively high level of challenge (compared to a relatively simple level of challenge) the stronger the feeling of embodiment, the more favorable the intentions towards the proposed offer will be.

We can however note that the challenges and their levels are not easy to implement for professionals because they depend on many parameters, in particular individual parameters such as the skill level of the users. Completing a challenge induces a high level of pleasure and identification with the character but only if the difficulty is not too high given the user's skills [START_REF] Hamdi-Kidar | Les chemins d'accès à l'expérience de flow : le cas des jeux vidéo[END_REF]. Therefore, further research would be needed to determine, depending on the situation, the optimal level of challenge to reinforce behavioral intentions in the case of embodiment.

At the managerial level, the research provides insights into the experiential conditions, reinforcing consumer behavioral intentions during VRE. Thus, giving consumers the opportunity to interact with the offer during VRE by being in front of the actor is a source of positive and superior perception for the individual than a non-interactive media (Łysik and Łopaciński, 2019). We therefore advise VR professionals and managers operating or wishing to operate VR, with their customers, to integrate gamification mechanisms to enrich their virtual consumer experiences, leading to embodiment according to the storyliving principle [START_REF] Durand | Le storytelling : le guide pratique pour raconter efficacement votre marque[END_REF]. Indeed, it is important to give the sensation of physically experiencing the offer to consumers [START_REF] Papagiannidis | Modelling the determinants of a simulated experience in a virtual retail store and users' product purchasing intentions[END_REF] a true "try-before-you-buy" (Tussyadiah et al., 2018, p. 141).

In view of the increasing evolution of the number of people owning VR headsets and the development of applications and marketplaces in the metaverse, it seems crucial to be able to guide managers to offer immersive and pleasurable experiences and to know the conditions to offer an optimal level of embodiment [START_REF] Marasco | Exploring the role of nextgeneration virtual technologies in destination marketing[END_REF][START_REF] Alcañiz | Virtual Reality in Marketing: A Framework, Review, and Research Agenda[END_REF]. Our study is a first step showing the importance for strategic issues of considering and evaluating the level of challenge in immersive virtual experiences. The following step could be to deepen the study of the influence of challenge levels according to the level of embodiment felt by the consumer, but also according to individual variables such as the consumer's skills and uses in terms of digital and virtual devices, or the psychological and mental state of the consumer at the time of the virtual reality experience.

Finally, our results relating to the role of action on embodiment would deserve further research, by integrating, for example, variables relating to sensory perception, proprioception or physiological sensations [START_REF] Flavián | The influence of scent on virtual reality experiences: The role of aroma-content congruence[END_REF][START_REF] Hopf | Exploring the impact of multisensory VR on travel recommendation: A presence perspective[END_REF] induced by the virtual environment such as "cybersickness", like motion sickness [START_REF] Sevinc | 0 Psychometric evaluation of Simulator Sickness Questionnaire and its variants as a measure of cybersickness in consumer virtual environments[END_REF]. This opens the way to new research perspectives mobilizing virtual reality experiences.

H 2 :

 2 A high (vs. low) level of challenge positively moderates the effect of embodiment on behavioral intentions. More precisely: A high (vs. low) level of challenge positively moderates the effect of embodiment on the intentions to discover and visit the destination for real (H 2a ) and to the intention to (re)do the sport activity for real (H 2b )The following figure presents the representation of the theoretical model.

Figure 1 .

 1 Figure 1. Theoretical model of the proposed research.

Table 1 . Sample description and subsamples comparison

 1

		High-level	Low-level	Total	Chi 2 (F)	ddl	p
		100	100	200
	Gender
	Female	57.0%	61,0%	59,0%	1.979 a	1	.159
	Male	43.0%	39,0%	41.0%
	Occupation Employee	23.6%	11.5%	19.3%	5.753 a	7	.569
	Blue-collar worker	7.3%	0.0%	4.6%
	Entrepreneur	1.8%	1.9%	1.8%
	Retired	1.8%	0.0%	1.2%
	Unemployed	1.8%	0.0%	0.3%
	Executive	18.2%	23.1%	21.3%
	Middle manager	23.6%	30.8%	19.2%
	Students	21.8%	32.7%	32.3%
	Age	37.0	32.1	34.5	(.117)	1	.050
	Education High school diploma or less	34.5%	15.4%	28.7%	6.936 a	3	.733
	Bachelor degree	34.5%	36.5%	36.9%
	Master degree	25.5%	28.8%	26.8%
	> Master degree	5.5%	19.2%	7.6%

Table 2 . The validation scale (SPSS, v26; Xlstat, v2018)

 2

	Measurement scale	λ	KMO	Sig.	α	ρ	AVE
	Embodiment
	Emb1	,664	.829	.000	.855	.894	.699
	Emb2	,671
	Emb3	,746
	Emb4	,721
	Emb5	,842
	Emb6	,811
	Emb7	,577
	Emb8	,660

Legend: λ: Loadings; KMO: Kaiser-Meyer-Olkin Index; Sig.: Bartlett sphericity test significance; α: Cronbach's Alpha; ρ: Dillon-Goldstein's (DG's) rho; AVE: average variance extracted

Table 3 . PLS results (Xlstat, v2018)

 3

	Effects (Hypotheses)	R	2	β	SE	t	Pr > |t|	f 2	Result
	Embodiment → Visit intention (H 1a )	.039 .196 .07 2.82	.005(**) .040 Supported
	Embodiment → Do the activity (H 1b ) .025 .158 .07 2.25	.026(*)	.026 Supported
	Legend: R	2 : Pearson's correlation coefficient; β: regression coefficients (path coefficient); SE: standard error; t:
	Student test; Pr > |t|: significant at: ***p<0.001; **p<0.01; and *p<0.05

Table 4 . Moderation effects results (Xlstat, v2018)

 4

	Body position effect on:	p-value	β high vs β low	≠	Result
	Embodiment & Intention to visit (H 2a )	.043(*)	.253 vs .203	.050	Supported
	Embodiment & Do the activity (H 2b )	***	.229 vs .130	.099	Supported
	Legend: p-value significant at: ***p<0.001; **p<0.01; *p<0.05; β: regression coefficients (path coefficient)

Official VR app store for Meta Quest head mounted devices owned by Meta (formerly Facebook).


Siyabonga Mange

Cédric Dever

Hank De Bruyn

Marianne Gaborieau

Patrice Castignolles

Robert G Gilbert
email: b.gilbert@uq.edu.au

Cnafs / Lcafs

Grafting of oligosaccharides onto synthetic polymer colloids

come

Introduction

Polymer colloids which are stabilized by glycopolymers have many potential applications, and also can provide models for the roles of polysaccharides on and in the membrane of living cells. The properties are strongly dependent on the nature of the comonomers used: for instance, biodegradable nanoparticles coated with polysaccharides can be used as biomimetic drug carriers for medical applications. The objective of the present paper is to graft oligosaccharides from debranched starch onto synthetic polymer colloids. Because the colloidal stabilizers of living cells often include polysaccharides, it is also of interest to examine the colloidal stability of cells stabilized only by a steric stabilizer comprising a simple polysaccharide: the linear -1,4-glucan which results from debranching starch.

A review of work done on surface modification of nanoparticles with poly-or oligosaccharides has been given by Lemarchand et al. 1 Well-defined glycopolymers have been synthesized by several polymerization techniques (e.g. 2,3 ). This process enables the preparation of amphipathic copolymers composed of a hydrophobic block and a hydrophilic polysaccharide block, which have the potential to then be used as stabilizers of polymer colloids. Passirani et al. 4 were able to prepare nonbiodegradable poly(methyl methacrylate) (PMMA) particles coated with either dextran or heparin. Similarly, Vauthier et al. 5,6 obtained biodegradable poly(isobutylcyanoacrylate) particles coated with dextran or heparin. The polymerization was initiated by polysaccharide radicals produced via the oxidation of the polysaccharide by Ce(IV) ions. This technique produces glycopolymer colloids with versatile properties, which depend on the nature, length and structure of the two different blocks.

Whole starch is often used as a stabilizer; for example, the common practice of stabilizing emulsions with natural starch relies on the ability of starch to impart some viscosity to the system, thereby impeding the breakdown of the emulsion. In order for starch to function purely as a (steric or polymeric) stabilizer, further modification is necessary: for example, attaching a hydrophobic group to the starch that renders it surface-active. [START_REF] Trubiano | Modified Starches: Properties and Uses[END_REF] Here we look at a particular way of using starch as a stabilizer: where the starch is chemically grafted to the latex particle.

Particles colloidally stabilized by a grafted water-soluble polymer (functioning as a steric stabilizer) are often referred to as "hairy particles". The present paper uses a new approach to create hairy particles sterically stabilized by oligosaccharides. The new method is as follows. A well characterized polymer colloid is prepared by conventional emulsion polymerization with an electrostatic stabilizer using standard techniques. [START_REF] Fitch | Polymer Colloids : A Comprehensive Introduction[END_REF][START_REF] Gilbert | Emulsion Polymerization: A Mechanistic Approach[END_REF] The resulting latex is then swollen with monomer, as is common for the first step of seeded emulsion polymerizations. Rather than adding an initiator of the type conventionally employed for such seeded emulsion polymerizations, instead oligosaccharides and an oxidizer, Ce(IV), are added in the process presented here. These oligosaccharide radicals are then expected to start to copolymerize with the small amount of monomer present in the water phase. These entities will continue to polymerize with aqueous-phase monomer until the radical end becomes sufficiently hydrophobic to adsorb onto the surface of a latex particle; this is effectively the mechanism by which radical entry into particles occurs in conventional emulsion polymerization. [START_REF] Maxwell | [END_REF]11 The radical end is now in a hydrophobic, monomer-rich environment and subsequent propagation with monomer will be rapid. The result is a grafted steric stabilizer, formed by a process analogous to what happens in conventional emulsion polymerizations in the presence of a water-soluble monomer such as acrylic acid to form electrosterically stabilized particles. 12,13 The electrostatic stabilizers which colloidally stabilized the parent seed latex are then removed by dialysis, giving colloidal particles coated with grafted oligosaccharides and which thus provide the only means of colloidal stabilization.

The oligosaccharide used in the present process comprises short linear starch chains obtained by enzymatically debranching amylopectin, 14 which is that variety of starch which has a very high molecular weight (up to 10 9 ) and is extremely highly branched, with the individual branches having a relatively low average degree of polymerization (the number-average degree of polymerization -X n ~ 30 for typical amylopectins). In the case of the waxy maize starch used herein (Shimuzi mochi amioca), the weightaverage degree of polymerization -X w was ~ 38, with a relatively broad distribution of degrees of polymerization.

Materials and methods

Chemicals High purity water (MilliQ) was used for all processes. 2,2'-Azobis(2methylpropionamidine) dihydrochloride (V-50, Aldrich) was recrystallized from 50 % w/w water/acetone before use. Maltoheptaose (Aldrich), sodium acetate (Univar), ethanol (95 %, Aldrich), sodium hydroxide (Aldrich), acetic acid (Aldrich), cerium(IV) ammonium nitrate (CAN, Aldrich), nitric acid (70 %, Riedel-deHaen) and dodecyltrimethylammonium bromide (DTAB, Aldrich) were used as received.

Styrene (Synthetic Resins, stabilized with 4-methoxyphenol inhibitor) was first passed through a column of basic alumina and then distilled under reduced pressure (first and last 10 % discarded), before being stored at 0 °C. MMA (Mitsubishi Rayon, stabilized with 4-methoxyphenol inhibitor) was passed through a column of basic alumina and distilled under reduced pressure (first and last 10 % discarded) to remove the inhibitor. The purified monomer was stored at 0 °C for no longer than 2 weeks before use. n-Butyl acrylate (Aldrich, >99 %, stabilized with methyl-hydroquinone) was passed through a de-inhibiting column packed with a methyl-hydroquinone inhibitor remover and used immediately.

A waxy maize starch, Amioca powder, was obtained from The National Starch and Chemical Company and debranched with isoamylase. Isoamylase (Pseudomonas sp.) was obtained from Megazyme and used as supplied: 250 UmL -1 stored in 3.2 M ammonium sulfate plus 0.02 % sodium azide. Pullulan standards (from Shodex) with narrow polydispersity index and weight-average molar masses ranging from 5,900 to 788,000 g.mol -1 were used to calibrate the size exclusion chromatography (SEC) columns.

A Total Starch Analysis Kit from Megazyme was used following the Total Starch Assay Procedure AOAC Method 996.11. The kit consists of thermostable -amylase (10 mL, 3000 UmL -1 ), amyloglucosidase (10 mL, 200 UmL -1 ), glucose oxidase / peroxidase reagent (GOPOD, high purity), glucose reagent buffer (concentrate), glucose standard solution and regular maize starch.

Preparation of debranched starch Linear oligosaccharides were prepared by debranching starch using the method described by Batey and Curtin, 15 with the modification developed by Lisle et al. 16 A typical procedure is as follows: a sample of starch (100 mg) was dispersed with ethanol (0.5 mL), gelatinized with sodium hydroxide (2 mL, 0.25 M) and then refluxed at 150 ºC for 10 min. The ethanol was allowed to evaporate. Water (4 mL), sodium acetate buffer (pH 4.13, 2 mL) and acetic acid (64 μL) were added to the gelatinized starch. The debranching enzyme, isoamylase (0.1 mL), was added to the mixture and incubated (40 °C, overnight). For SEC analysis, the debranched mixture was then boiled, then the mixture was desalted (1 h) with mixed bed resin (Biorad, 1 mg), and then centrifuged (10 500 g, 10 min).

The supernatant was retained for analysis.

1 H liquid-state NMR was used to determine the completeness of debranching of the starch by isoamylase. When the α- (1,6) branch point could no longer be detected the debranching was considered to be complete. A sample of the debranched starch was freeze-dried and then 2 mg were dissolved overnight in 0.5 mL of DMSO-d6/D2O (80/20 v/v) at 80 °C. Quantitative 1 H NMR spectra were recorded on a Bruker DPX300 at 90 °C at a Larmor frequency of 300.13 MHz. 64 transients were recorded with a 7.9-s 90° pulse and a 5-s relaxation delay. 17 The chemical-shift scale was calibrated with a trisilylpropanoic acid internal standard at 0 ppm. The temperature was calibrated with an ethylene glycol standard. 18 SEC analysis of debranched starch Molecular weight distributions were determined using a size exclusion chromatography (SEC) set-up comprising a Shimadzu LC-10ATVP pump, a Shimadzu DGU-14AVP degasser, a Shimadzu SIL-10ADVP autoinjector, a Shimadzu CT10ACVP column oven set at 60 °C, Waters Ultrahydrogel 250 and 120 columns fitted with a Ultrahydrogel guard column, and a Wyatt Optilab DSP interferometric refractometer. The system was controlled and data were recorded using Astra software. The eluent used was 0.05 M ammonium acetate (pH 5.2) with 0.05 % sodium azide at a flow rate of 0.5 mLmin -1 . The run time was 80 min. These conditions gave good SEC separation of the oligosaccharides obtained by debranching. 19,20 To obtain the molecular weight distribution, pullulan standards were used for calibration and molecular weights obtained by universal calibration with the Mark-Houwink-Sakurada parameters reported for pullulan and linear starch by Castro et al. 21 No corrections were made for band broadening.

Preparation of an electrostatically stabilized poly(methyl methacrylate) seed latex Ce(IV) ions formed a complex with the commoner anionically stabilized latex type due to the opposing charges which then led to coagulation of the system. To overcome this problem, a cationic PMMA latex was prepared using a procedure described in the literature. 22 Water (570 g) and the surfactant (DTAB, 4.57 g) were poured into a Mettler-Toledo RC1e calorimetric reactor, and the solution was heated to 90 °C. The reaction vessel was purged with high-purity nitrogen to remove oxygen.

A trace amount of bulk PMMA (1 wt% to monomer) was dissolved in the MMA monomer (86.52 g) prior to use and the polymer/monomer solution was then added to the reactor and emulsified by stirring at 450 rpm. The dissolution of a trace amount of polymer in monomer is thought to enhance radical capture efficiency in the early stages of the polymerization leading to a lower polydispersity of the latex particle size distribution. [23][24][25] After 5 min, the initiator (V-50, 0.288 g dissolved in 30 mL water) was added. Polymerization was allowed to proceed at 90 °C for 2 hours. The latex had a solids content of 10.9 wt% (measured by gravimetry after water evaporation).

The procedure for the synthesis of anionically stabilized polystyrene latex is described in the Supplementary Material. The cationically stabilized polystyrene latex was synthesized identically to the procedure described above for PMMA. 22 Grafting of oligosaccharides onto the MMA-swollen PMMA particles Preliminary grafting experiments were performed with styrene and n-butyl acrylate as swelling monomers. These syntheses are described in the Supplementary Material. This leads to the choice of MMA as the swelling monomer. The PMMA latex prepared previously (6.45 g) was diluted with deionized water (3.2 g) and stabilized further with DTAB (0.04 g) before swelling with MMA (0.77 g). The amount of added DTAB was such that it was below the critical micelle concentration (CMC) in the latex, to avoid secondary particle nucleation. The CMC in the latex was determined by measuring the surface tension of the latex as a function of DTAB concentration. The suspension of MMA in diluted PMMA latex solution was equilibrated under gentle stirring for 24 h at room temperature. The previously prepared solution of oligosaccharides (0.5 g debranched starch) was then added under gentle stirring and nitrogen bubbling at 40 °C. After 20 min, 4.0 mL of a solution of CAN (0.1 g) in nitric acid (0.2 M) was fed with a syringe pump over an hour under vigorous agitation. Nitrogen bubbling was maintained for 5 min and the reaction mixture was stirred gently overnight. The final latex was dialyzed against Milli-Q water for 7 days with daily changes of water.

Particle Size Analysis Determination of particle size distributions was carried out by hydrodynamic chromatography (HDC) using a Polymer Laboratories PSDA particle-size distribution analyzer and dynamic Light Scattering on a Malvern High Performance Particle Sizer (HPPS). The average particle size of the cationic samples was determined by dynamic light scattering on a Malvern Instruments Zetasizer.

Zeta potential Analysis Determination of the  potential of the colloids was carried out using a Malvern Zetasizer Nano Series operating at an angle of 177 ° coupled with an MPT-2 titrator. This employs the M3-PALS technique, which is a combination of laser Doppler velocimetry and phase analysis light scattering (PALS) in order to measure particle electrophoretic mobility (velocity of a particle in a unit electric field). The  potential is then related to the electrophoretic mobility by the Henry equation.

Transmission Electron Microscopy Analysis Information on particle morphologies was obtained by using a Biofilter Philips CM120 Transmission Electron Microscope at the Electron Microscope Unit, Sydney University, with a CCD camera and an accelerating voltage of 120 kV. Dilute dispersions of polymer particles (about 0.05 wt%) were embedded on a carbon-coated copper grid. After drying under vacuum, the grid containing the polymer particles was placed in the TEM. The length scale was calibrated with a standard sample of polystyrene latex particles.

Surface tension measurements

The surface tension of the PMMA seed latex was obtained using a standard ring measurement on a KSV Sigma 70 Surface Tensiometer. The sample, PMMA latex (40 mL), was added to the vessel. The standard ring was first heated to red hot on a Bunsen burner before being attached to the balance unit. The surface tension of the latex was measured as successive amounts of the surfactant (DTAB) were added until no change in the surface tension was observed.

13 C Solid-State NMR Characterization methods requiring complete solubility of all components are inapplicable for amphiphilic copolymers, as the copolymers are not completely soluble in any solvent. 26 13 C solid-state NMR was therefore chosen to quantify the amount of grafting. 13 C NMR spectra from a homogeneous copolymer gel swollen in either perdeuterated DMSO or a mixture of perdeuterated DMSO and perdeuterated pyridine was used by Gurruchaga et al. 27 to prove grafting for their acrylate graft copolymers without the need for complete solubility.

In the present study, solid-state 13 C NMR was used to confirm that grafting had occurred. The latex obtained from the grafting experiment was ultracentrifuged at 90 000 rpm and 25 ºC for 30 min. The centrifugate, which should contain the waterpage 10 insoluble fraction in the form of oligosaccharide-g-PMMA/polystyrene, was analyzed by 13 C solid-state NMR on a Bruker Avance 300 wide-bore spectrometer (Bruker Biospin Pty Ltd), operating at 75 MHz for 13 C, using a 4 mm magic-angle spinning (MAS) probe (Bruker Biospin Pty Ltd). The spectrometer was operated with XWIN-NMR software (Bruker Biospin GmbH). A 4 mm zirconia rotor with KelF cap was used and spun at 4.5 kHz MAS. A 13 C single pulse excitation (SPE) spectrum was recorded at ca. 50 °C with a 5.0 s 90° pulse and continuous wave decoupling (optimized on adamantane), and externally calibrated with respect to the resonance of adamantane at 29.5 ppm. The parameters for the acquisition of the data were as follows: 9306 transients were recorded, the duration of the FID was 0.16 s, the delay before a scan was 10 s and a total delay of 10.16 s between scans was applied.

1 H liquid state NMR 2 mg of the solids from drying the supernatant obtained from ultracentrifugation of the oligosaccharide-g-PMMA/polystyrene latex was dissolved in 0.5 mL DMSO-d6 at 80 °C overnight. Quantitative 1 H NMR spectra were recorded on a Bruker DPX300 at 90 °C at a Larmor frequency of 300.13 MHz. 64 transients were recorded with a 7.9 s 90° pulse and a 6 s relaxation delay.

Dialysis of the latexes

After grafting, the latexes were dialyzed to remove the electrostatic stabilizer used in seed synthesis. MEMBRA-CEL™ dialysis membrane MD 25-14 (Viskase) with a molecular weight cut off of 14 000 was conditioned by boiling in deionized water for 5 h prior to use. The conditioned membranes were filled with the latex samples, which were then put in beakers filled with deionized water under gentle stirring at room temperature with daily water exchanges.

Conductivities of the serum phase from dialysis became constant after ten days of dialysis (with the water changed daily), showing that all the labile electrostatic stabilizer had been removed.

Quantification of grafted starch

The dialyzed latex was ultracentrifuged at 90 000 rpm and 25 ºC for 30 min. The centrifugate, which should contain the oligosaccharide-g-PMMA, was dried in an oven at 60 ºC for 24 h. The amount of starch in 100 mg of the dry centrifugate was obtained with a standard assay procedure (AA/AMG) using the Total Starch Analysis Kit (Megazyme).

Results and discussion

Debranched starch The SEC molecular weight distribution of the debranched starch oligosaccharides is presented in Figure 1. All measurements were performed directly after the end of the debranching procedure to ensure that retrogradation of the oligosaccharide chains was minimized. 28 The detailed shape of this debranched chainlength distribution in amylopectin, with its characteristic shoulder, has been explained in detail by Castro et al. in terms of the enzymatic processes involved in starch biosynthesis. 21 For our purposes, the weight-average of the oligosaccharides from debranching was used in all calculations: -M w = 6150 g•mol -1 , i.e. a weight-average degree of polymerization ( -X w ) of 38 anhydroglucose units. The resulting oligosaccharides were used in seeded polymerization onto preformed polymer particles. 1 H NMR spectroscopy was used to check that debranching was complete. 29 An overlay of the 1 H NMR spectra for the native and debranched Amioca starch is shown in Figure 2. Complete debranching is demonstrated by the disappearance of the signal corresponding to -(1,6) linkages at 4.8 ppm, 30 as well as an increase in signals corresponding to  and  reducing ends at 5.15 and 4.4 ppm respectively, 30 The signal at 1.9 ppm present in the spectrum of debranched starch is from the acetic acid 31 introduced in the medium for the debranching step.

PMMA seed latex Preliminary work using an anionically stabilized polystyrene latex as a seed resulted in coagulation of the system on addition of the Ce(IV) solution (see Supplementary Material). The loss of stability was probably a result of the destabilizing effect of the Ce 4+ cation with the anionic latex, before any starch was added to the system. A cationically stabilized polystyrene latex was found to circumvent this problem (see Supplementary Material). Further improvement in the system was obtained when PMMA was used instead of polystyrene as the cationically stabilized seed latex, as discussed in detail above. The particle size of the PMMA seed is shown in Table 1.

Second-stage polymerization of seed

Preliminary grafting experiments were performed with styrene and n-butyl acrylate.

In the system where the anionically stabilized polystyrene seed latex was swollen with styrene, the conversion was about 48 %. 1 H NMR of the supernatant in DMSO-d6 showed a significant amount of polystyrene oligomers, Figure S.1 in the Supplementary Material. This was unexpected, since polystyrene is hydrophobic and therefore should precipitate from the aqueous phase. It was observed that addition of Ce 4+ to the monomer-swollen monomer latex without the oligosaccharides resulted in coagulation of the anionic latex but no coagulation for the cationic latex, as expected from double-layer theory. The water-soluble polystyrene oligomers are thought to be a result of the slow propagation of styrene monomer in the aqueous phase grafted onto an oligosaccharide chain leading to the propagating radical being terminated by another Ce(IV) or another propagating radical. In choosing a monomer which will minimize aqueous-phase termination, it is necessary to minimize the residence time of the growing radicals in the aqueous phase. This is encapsulated in the expression for initiator efficiency fentry:

10 fentry ≈       2 kd[I] kt,aq kp,aq[M] aq + 1 1-z ( 1
)
where z is the critical degree of polymerization for entry of a radical into a particle, k d is the initiator dissociation rate coefficient, [I] is the initiator concentration, [M]aq is the monomer concentration in the aqueous phase, and k p,aq and k t,aq the rate coefficients for propagation and termination in the aqueous phase (which for hydrophobic monomers can be assumed to be close to that in pure monomer, 32 denoted k p ). The monomer must have a high k p to ensure high initiator efficiency.

Thus n-butyl acrylate was chosen to replace styrene because of its high propagation rate coefficient. 33,34 In the system swollen with n-butyl acrylate, there was a significant amount of coagulation during the swelling stage (this problem can be solvent using a cationic seed) but the characterization of the latex was also complicated by the observation of three phases upon ultracentrifugation. The phase on the top of the centrifugation tube was unstable: it disintegrated and made the supernatant, which was clear after centrifugation, become cloudy when the supernatant was extracted with a syringe, making quantification of the process impossible.

The grafting experiments with styrene and n-butyl acrylate as monomers onto a cationically stabilized polystyrene seed gave products with coagulum of about 20 wt% to monomer and polymer. There are competing effects with regard to the water solubility of the monomer: it must be sufficiently soluble so that its aqueous-phase concentration will give a sufficiently high rate of propagation (k p [M aq ]), but sufficiently hydrophobic so that surface activity of the block copolymer will be obtained with fewer monomer units. MMA could be acceptable for the present purpose. It has a relatively high k p , 35 and an intermediate water solubility (0.15 M at 50 ºC 36 ), and therefore is expected to have a relatively high entry efficiency. [START_REF] Maxwell | [END_REF]11,37 With MMA as monomer the coagulum was about 1.5 wt% to monomer and polymer instead of 20 % with styrene and n-butyl acrylate. Styrene and n-butyl acrylate were therefore not used further as monomers; the MMA monomer was used to swell the seed latex in all subsequent experiments, for which the results are discussed in the next section. Following second-stage polymerization, the electrostatic stabilizer (DTAB) used in seed preparation and swelling stages was removed by dialysis.

page 14

It has been demonstrated that Ce(IV) complexes with diols, cleaving the carboncarbon bond between the hydroxyls in polysaccharides to yield an aldehyde or ketone and a free radical (see Scheme 1). [38][39][40] The Ce(IV) mainly creates radical sites on oligosaccharides. 41 The hemiacetal group at the reducing end of a cellulosic chain has been shown to be much more reactive towards Ce(IV) than the mid-chain glycol groups. [42][43][44] It is therefore likely that a radical is relatively easily created at the reducing end of most of the oligosaccharide chains. Moreover, as the oligosaccharides and the cerium were added in equimolar amounts, the formation of only one radical per oligosaccharide is favored. The oligosaccharide radical then propagates with MMA in the aqueous phase until it becomes sufficiently surface-active to irreversibly enter a monomer-swollen particle, analogous to the radical entry process in conventional emulsion polymerization. [START_REF] Maxwell | [END_REF]11 The polymerization would then be expected to continue within the swollen particles, forming a diblock poly(anhydroglucose-block-MMA) with the PMMA block anchored to the particle.

This procedure should thus optimize grafting of the oligosaccharides to the surface of the particles.

Characterization of the oligosaccharide-g-polyPMMA/polystyrene latex The particle size distribution of the oligosaccharide-grafted latex was obtained using hydrodynamic chromatography. HDC suggested a monomodal population for the oligosaccharide-g-PMMA/polystyrene latex, as shown in Figure 3. Although HDC may not be accurate for cationic latexes, it is reasonable to assume that the monomodality is not an artifact. However, the apparent small increase in particle size and broadness of the distribution may or may not be an artifact of the use of HDC for a cationic latex. The presence of the MMA oligomers in the supernatant due to aqueous phase termination was confirmed by liquid-state 1 H NMR of the supernatant in DMSO-d6, as seen in Figure 4.

The proof that the starch was indeed grafted was obtained using 13 C solid-state NMR. In Figure 5, a resonance peak at 62.8 ppm due to carbon number 6 in the glucose unit was observed. 45 The complete chemical shift assignment in given in the supplementary materials. The data are not quantitative, as the relaxation delay was not optimized, but do indicate significant grafting. Analysis of the supernatant with 1 H NMR in DMSO-d6 showed a significant amount of water-soluble PMMA oligomer fraction and a small amount of starch (Figure 4). Since starch is soluble in DMSO, 46,47 this confirms that most of the starch precipitated with the particles during ultracentrifugation.

Extensive dialysis of the seed and grafted latexes was performed to remove electrostatic stabilizers. It was expected that on removal of the electrostatic stabilizer, the oligosaccharide-g-PMMA/polystyrene latex would remain stable, sterically stabilized by the grafted oligosaccharide chains, whereas the seed latex, stabilized only electrostatically, would coagulate. Two latexes were synthesized with theoretically different surface coverages; the one with higher surface coverage (32 nm 2 per oligosaccharide chain) was stable after dialysis whereas the one with lower coverage (91 nm 2 per oligosaccharide chain) coagulated. The synthesis at the higher surface coverage is discussed below.

Characterization of the oligosaccharide-g-PMMA On swelling of the seed latex with monomer, precipitation of polymer was observed after some time. This loss of stability was presumed to be due to insufficient surfactant for adequate coverage of the swollen polymer particles. More DTAB surfactant was added to stabilize the monomer swollen latex particles, the amount of surfactant being chosen to be just below the latex CMC to avoid secondary nucleation. The CMC was determined by measuring the surface tension of the PMMA seed latex as a function of the amount of DTAB added (Figure 6). The latex obtained using the optimized conditions, i.e.: cationic PMMA swollen with MMA in the presence of additional DTAB and the increased starch concentration to give a surface area of 32 nm 2 per oligosaccharide chain, was extensively characterized. The z-average diameter of the oligosaccharideg-PMMA latex particles, 103 nm, was slightly larger than that of the starting seed of page 16

99 nm (see Table 1). (The z-average, commonly used in light scattering size measurements, is significantly higher for polydisperse distributions than, say, a volume average). Particle size distribution curves for the PMMA seed and the oligosaccharide-g-PMMA latexes measured with HDC as shown in Figure 7, also show an apparent slight increase in particle size after the grafting experiment, but as discussed above this could be an artifact. The latex particles were also examined using TEM, as shown in Figure 8. Unlike the PMMA seed latex, the oligosaccharide-g-PMMA latex particles do not merge under the electron beam: the grafted particles in the TEM images are more spherical and better defined than those of the PMMA seed latex particles.

The oligosaccharide-g-PMMA latex was dialyzed for over a week and the latex was still stable afterwards. The degree of grafting was determined by enzymatic digestion of the centrifugate from the dialyzed latex to glucose using the Total Starch Analysis Kit. UV-VIS spectrometry was used to quantify the amount of glucose. The amount of starch that was grafted was found to be at least 7.7 wt% to total solids, giving a grafting efficiency of at least 33 % based on the amount of the initial starch used in the formulation. This measurement may underestimate the amount of grafted starch because of possible limitations of the Total Starch Analysis Kit on starch grafted onto latex particles. When the Total Starch Analysis Kit was applied to pure natural starch, a recovery of 94 % starch was achieved; this recovery is taken into account in the calculation of the grafting efficiency but for grafted starch the recovery may be lower in the presence of synthetic polymer. Some starch may be embedded inside the particles and is then not taken into account at all in the Total Starch Analysis Kit.

Previously, solid-state 13 C NMR on a centrifugate of the grafted latex showed the presence of starch, Figure 5. Since ultracentrifugation may result in the breaking of grafted starch chains and also precipitation of adsorbed starch, the measurement with solid-state 13 C NMR after ultracentrifugation cannot be relied upon to be quantitative.

Dialysis is able to remove adsorbed starch and should be sufficiently gentle so as not page 17

to break grafted oligosaccharide chains. Thus the dialysis of the grafted latex followed by subjecting the centrifugate to the Total Starch Analysis Kit can be regarded as the best quantitative analytical method. The observed latex stability is thought to be from an increased particle surface coverage by the oligosaccharide chains.

The theoretical number of chains per particle for the syntheses described in the Supplementary Material was 200, whereas the higher-coverage synthesis described in this paper with the cationic PMMA seed that led to a stable latex was 820 oligosaccharide chains per particle. If grafting efficiency were 100 %, then there would be 820 oligosaccharide chains per particle; hence the experimental grafting efficiency is 33 %, with the actual number of chains per particle being 270. The number of oligosaccharides chain per particle was calculated by dividing the number of oligosaccharides chains by the number of particles. The number of particles per unit volume, NP is calculated as the ratio of the total polymer mass to the mass of one particle:

Np = m p 0 4 3  ru 3 d p (2)
where m p 0 is the initial mass of polymer (as the dry weight of seed latex) per unit volume of the aqueous phase, ru is the weight-average unswollen particle radius and d p is the polymer density.

The number of oligosaccharide chains given above was determined from the number average molecular weight ( -M n = 2500 g.mol -1 ) obtained from the SEC molecular weight distribution. This -M n value may be inaccurate because (i) the number average molecular weight suffers from low accuracy for most samples exhibiting a broad molecular weight distribution 48 (ii) aqueous SEC of starch suffers from incomplete recovery. 29 Capillary electrophoresis (CE) is a technique that can used to determine absolute molecular weight data, but was unavailable for the present study. To obtain an estimate of the area occupied per chain, the number average molecular weight data obtained from the SEC for the Amioca starch used here was page 18 compared to that of various starch types obtained with CE 49 assuming that the debranched Amioca sample had the same -X n as that of each of the debranched starches for which CE data are available. The results are shown in Table 2. The theoretical surface area per oligosaccharide chain using the CE data shown in Table 2 was determined with the assumption that the grafting efficiency with all the starch types would be 33 % under grafting conditions similar to those used for the Amioca starch. The surface area per oligosaccharide chain for the Amioca starch lies within the range of those assuming the same -X n as the rice, maize and wheat starches (103 ± 10 nm 2 ).

Assuming that the experimental grafting efficiency for both the polystyrene and PMMA particles to be 33 %, then the surface area per oligosaccharide chain becomes 275 nm 2 and 97 nm 2 respectively. These two oligosaccharide-grafted particles were synthesized with starting amounts of oligosaccharide that would have given surface areas of 91 and 32 nm 2 respectively if grafting efficiency had been 100 %. The area of 97 nm 2 per oligosaccharide chain is 4 -8 times higher than values in the literature when polyethylene oxide macromonomer is used as a steric stabilizer. 50,51 Specifically, Kawaguchi et al. describe a critical point at which sterically stabilized particles are formed and coalescence between similar sized particles stops. 50 At this point the maximum surface area (S crit ) covered by undeformed polymer coils is calculated as the maximum surface area occupied by a single stabilizer chain, which in their work is 12.6 nm 2 . At 100 % conversion the surface area per chain is drastically reduced to 0.59 nm 2 . In the work of Wu et al. on PMMA and PS latexes stabilized by polyethylene oxide (PEO) macromonomer, stable latexes were obtained with the PEO macromonomer covering 25 nm 2 with 0.2 and 0.1 wt% PEO grafted onto particles. 51 In order to ascertain that there were no other contributory stabilizers in the system in this work, the  potential of the dialyzed grafted latex was measured in deionized water and found to be 7.2 mV. This amount of charge is too low to contribute to the stability of the latex and hence it can be concluded that the observed stability is due to the ability of the oligosaccharide chains to act as steric stabilizers. This result shows that sufficiently high particle surface area coverage by the oligosaccharides is able to render the particles colloidally stable through steric stabilization. When the dialyzed latex was diluted in salt solutions with a sodium chloride concentration range of 0.1 -3 M, the solutions became cloudy and coagulated over time.

In an ionic environment, the neutral oligosaccharide chains change conformation, which we postulate is the cause for the loss in stability. Theories of colloidal stability of sterically stabilized particles, such as that given by Vincent and co-workers, 52 require (through the Flory-Huggins  parameter) a knowledge of the (hairy-layer) oligomer/water interaction, and how this changes with ionic strength. There is no quantitative information on this in the literature; although there are interesting observations that amylopectin increases in size with higher ionic strengths, 53 that could well be due to effects of ionic strength on the conformation of the branches as much as on the conformation of the component oligomeric branches. Colloidal stability studies with particles stabilized by monodisperse polysaccharides may provide an answer to this interesting in the future.

Conclusions

A new method has been successfully developed which allows grafting of debranched natural starch onto monomer-swollen electrostatically stabilized PMMA particles. The process involves the in-situ grafting by a redox-initiated radical polymerization process between water-soluble linear oligosaccharide chains and electrostatically stabilized seed latex particles swollen with an appropriate hydrophobic synthetic monomer, chosen to minimize undesirable events such as inefficient grafting. The oligosaccharide which was used to form grafted latexes was obtained by enzymatically debranching amylopectin, yielding a broad distribution of linear oligosaccharides of -X w ~ 38. The initiating radicals are created on page 20 oligosaccharide chains by Ce(IV). These water-soluble chains then undergo polymerization with the small amount of monomer present in the water phase until the radical end becomes sufficiently hydrophobic to enter the monomer-swollen polymer particles. This results in rapid polymerization within the monomer-swollen particles to form poly(anhydroglucose-b-synthetic monomer) block copolymers, with the polymeric hydrophobic component irreversibly buried in the particle. MMA proved to be an appropriate monomer for the grafting experiments (more suitable than styrene and butyl acrylate) because it has a relatively high k p and intermediate water solubility, which allowed a rapid aqueous-phase propagation promoting high entry efficiency into the particles and thus ensuring that aqueous-phase termination is minimized, whence a high proportion of initiated chains actually become grafted to the particle.

The MMA-based system was analyzed as follows. The polymer colloids formed in the supposed grafting process precipitated under ultracentrifugation, giving a distinct separation between the supernatant phase and the centrifugate. Solid-state 13 C NMR and analysis of the amount of starch present in the centrifugate proved that significant grafting of the oligosaccharides onto the synthetic polymer seed latex particles had indeed occurred. After dialysis to remove the ionic surfactant used to make the seed latex, the latexes resulting from various syntheses were examined for colloidal stability. Both the seed and the oligosaccharide-grafted latexes with low coverage per oligosaccharide (275 nm 2 surface area per chain) coagulated after 7 days of dialysis, while the latex with higher surface coverage (97 nm 2 ) was stable for about a week after 7-day dialysis. It is surmised that the improved stability is due to increased coverage of the latex particle surface by the linear oligosaccharide chains.

The area per oligosaccharide stabilizer, 97 nm 2 per oligosaccharide chain, is very sparse compared to synthetic steric stabilizers. This result would make debranched starch a very efficient stabilizer in terms of the number of starch molecules required to stabilize a particle, though it should also be noted that its effectiveness is markedly page 21 reduced in the presence of strong electrolyte. Since polysaccharides (from different sizes and structures) are one of the colloidal stabilizers used in biological cells, this high stabilizing efficiency in the case of linear oligosaccharides at least can be seen to be advantageous from an evolutionary point of view, because it frees up considerable space on a cell surface for the large number of other components on the surface needed for cell viability (ion pumps, cholesterol, etc.).

The procedures developed here enable biomimetic polymer colloids to be synthesized with potential applications in biomedicine, including as model colloids at low electrolyte concentrations which share some of the same colloidal stabilization mechanisms as cells.

Schemes, Figures, Tables

Supplementary material

Methods

Preparation of anionically stabilized polystyrene seed latex A polystyrene latex was prepared according to a procedure described in the literature 1 . Water (90 g), the surfactant AMA-80 (1.97 g) and a buffer (NaHCO 3 , 0.21 g) were poured into a 250 mL flask and the solution was heated to 90 °C. The initiator (potassium persulfate, KPS, 0.22 g) was dissolved in 10 mL water. The reaction vessel was purged with high-purity nitrogen for 1 hour to remove oxygen. The monomer (15.33 g) was added without prior heating and the resulting solution was stirred vigorously to effect emulsification. After 5 minutes, the initiator (KPS, 0.22 g dissolved in 10 mL water) was added. Polymerization was allowed to proceed at 90 °C for 2 hours. The solids content for the latex was 32 wt%.

Grafting of oligosaccharides onto anionic polystyrene particles swollen with styrene or n-butyl acrylate

The polystyrene latex previously prepared (23.1 g) was diluted with water (25 mL) and then swollen with styrene (4 g) or n-butyl acrylate (4.92 g). The amount of added styrene was such that it was below the equilibrium solubility of styrene in polystyrene particles (~5.4 M) 2 , ensuring that no monomer droplets were present after equilibration, and hence that all polymerization was in "Interval 3" (absence of any polymerization in droplets). The suspension of styrene and latex solution was equilibrated for 24 h at room temperature. A solution of oligosaccharides containing 100 mg of starch was then added under gentle stirring and nitrogen bubbling at 40 °C. After 20 min, a solution of cerium ammonium nitrate (89 mg in 14 mL of 0.2 M HNO 3 ) was added under vigorous agitation. Nitrogen bubbling was maintained for 5 min and gentle stirring maintained overnight.

Preparation of cationically stabilized polystyrene seed latex A polystyrene latex

was prepared according to a procedure described in the literature. 3 Water (570 g) and the surfactant dodecyltrimethylammonium bromide, DTAB (4.57 g) were poured into a Mettler-Toledo calorimetric reactor, RC1e, and the solution was heated to 90 °C.

The reaction vessel was purged with high-purity nitrogen to remove oxygen. A trace amount of bulk polystyrene (1 wt% to monomer) was dissolved in the styrene monomer prior to use and the polymer/monomer solution was then added to the reactor and emulsified by stirring at 450 rpm. After 5 minutes, the initiator 2,2'azobis(2-methylpropionamidine) dihydrochloride (V-50, 0.288 g dissolved in 30 mL water) was added. Polymerization was allowed to proceed at 90 °C for 2 hours. The solids content for the latex was 6.8 wt%.

Grafting of oligosaccharides onto cationic polystyrene latex particles swollen

with n-butyl acrylate or MMA The cationic polystyrene latex prepared previously (51.6 g) was swollen with either n-butyl acrylate (4.95 g) or MMA (3.85 g). The suspension of monomer and polystyrene latex was equilibrated for 24 h at room temperature. A solution of previously prepared debranched starch (12 mg starch) was then added under gentle stirring and nitrogen bubbling at 40 °C. After 20 min, a solution of cerium ammonium nitrate (2.6 mg in 0.4 mL of 0.2 M HNO 3 ) was fed with a syringe pump over an hour under vigorous agitation. The reaction was allowed to run overnight under gentle stirring.

Determination of the critical micellar concentration of the cationic polystyrene latex

In order to maintain the stability of the latex particles during the swelling stage and minimize the amount of coagulum formed, it was necessary to add surfactant to just below the CMC of the latex (see Materials and methods in main text). The surface tension of the latex was measured as a function of the amount of surfactant DTAB added.

Grafting of oligosaccharides onto the MMA-swollen cationic polystyrene latex particles The cationic polystyrene latex prepared previously (51.6 g) was stabilized further with DTAB (0.3 g) before swelling with methyl methacrylate (2.0 g). The amount of added DTAB was such that it was below the CMC of the latex, to avoid secondary particle nucleation. A suspension of MMA and polystyrene latex solution was equilibrated for 24 h at room temperature. A solution of previously prepared debranched starch (12 mg starch) was then added under gentle stirring and nitrogen bubbling at 40 °C. After 20 min, a solution of cerium ammonium nitrate (2.6 mg in 0.4 mL of 0.2 M HNO 3 ) was fed with a syringe pump over an hour under vigorous agitation. MMA (1.9 g) was also fed simultaneously over half an hour. Nitrogen bubbling was maintained for 5 min and the reaction mixture was stirred gently overnight. These conditions were found to minimize the amount of coagulum formed.

The final latex was dialyzed against Milli-Q water for 7 days with daily changes of water.

Results and discussion

Polystyrene seed latex An anionic latex with a monomodal particle size distribution was obtained with a weight average diameter of 118 nm. The solids content of the latex was 32 wt%. This latex was then used in the oligosaccharide grafting experiments discussed below with styrene as the monomer.

Grafting of oligosaccharides onto anionic polystyrene particles swollen with styrene or n-butyl acrylate

The grafting of oligosaccharides onto styrene swollen polystyrene latex produced a significant amount of coagulum on addition of the oligosaccharides and cerium ammonium nitrate solutions to the latex and monomer suspension. It was thought that the coagulation was a result of the high amount of starch that was being used hence the reduction in the amount of starch in subsequent formulations. This view was reinforced by work that was done on another project where large amounts of starch resulted in the formation of gels. 4 Further investigation showed that the coagulation was due to the affinity between the anionically stabilized polystyrene latex and the cationic Ce(IV) ions. Characterization of the supernatant This work is discussed in the main text.

The work done described above allowed the determination of the optimum conditions for the grafting of oligosaccharides onto monomer swollen synthetic polymer latex and were applied in the work discussed in the main text. These conditions include the use of a cationic seed latex, additional surfactant during the swelling stage to below the latex CMC, addition of cerium ammonium nitrate solution in nitric acid via a syringe pump to keep the ionic strength of the reaction solution low.

Figure Captions

1 Figure 1 : 8 Figure 2 :Figure 3 :Figure 4 : 1 H

 1182341 Scheme 1

ppmFigure 5 :

 5 Figure 5: 13 C solid-state NMR spectrum for the precipitate of an oligosaccharide grafted polystyrene/PMMA copolymer

Figure 6 :Figure 7 :Figure 8 :

 678 Figure 6: Surface tension measurement for a cationic PMMA latex (40 mL) with increasing additional DTAB. The linear fits on two different ranges are shown as solid lines.

with 1 H

 1 NMR in DMSO-d 6 showed a significant amount of styrene oligomers in the presence of a large amount of starch, see Figure S.1. The grafting of oligosaccharides onto the n-butyl acrylate swollen polystyrene latex particles showed an even higher amount of coagulum than the styrene based one. A gel-like substance formed on the sides of the reaction vessel. When the reaction products were ultracentrifuged, two solid phases were observed separated by the supernatant. Further characterization could not be done since the lighter solid phase got redispersed into the supernatant when being separated. Preparation of cationically stabilized polystyrene seed latex The particle size distribution obtained using HDC for the cationically stabilized polystyrene latex is shown in Figure S.2. The latex particles had a weight average diameter of 62.5 nm (see Table 1 in main text). TEM images of the seed latex also show a monomodal particle size distribution (see Figure S.3(a)).Grafting of oligosaccharides onto the cationic polystyrene particles swollenwith n-butyl acrylate or MMA When oligosaccharides were grafted onto the cationic polystyrene latex swollen with either n-butyl acrylate or MMA a coagulum of over 20 wt% was recovered in each case. Further optimization was necessary to reduce the amount of coagulum.Determination of the critical micellar concentration of the cationic polystyrene latexThe surface tension measured as the function of the amount of surfactant DTAB added is shown in Figure S.4. Further syntheses were done with additional DTAB added before the addition of monomer during the swelling stage to a concentration just below 0.021 M. Grafting of oligosaccharides onto the MMA-swollen cationic polystyrene latex particles The grafting of oligosaccharides onto the MMA swollen cationic polystyrene latex with the additional DTAB reduced the coagulum to less than 2 wt%. The particle size distribution of the latex determined by HDC is shown in Figure S.2 and displays a slight increase in particle size. A TEM micrograph of the oligosaccharide-g-PMMA/polystyrene latex particles is shown in Figure S.3(b). When the latex was dialyzed for a week it coagulated. Further work was done in order to obtain latex particles that were sterically stabilized by the grafted oligosaccharides.

Figure S. 1 : 1 HFigure S. 2 :Figure S. 3 :Figure S. 4 :

 11234 Figure S.1: 1 H NMR spectrum of the supernatant of oligosaccharide-g-polystyrene

Table 1 :

 1 Particle size distribution of the latexes by HDC and HPPS; * values determined by HPPS as the z-average

	Latex	Weight-average diameter (nm)	Weight-average width of distribution (nm)
	cationic polystyrene seed	62.5	8.9
	oligosaccharide-g-polystyrene/PMMA	64.5	11.5
	cationic PMMA seed	87.5 99.2*	11.8 21.7
	oligosaccharide-g-PMMA	90 102.6*	12.6 14.8

Table 2 :

 2 Number-average molecular weights and degrees of polymerization from molecular weight distributions from capillary electrophoresis data obtained for five types of debranched starch.49 These data were used to estimate the range of surface area per oligosaccharide chain on each latex particle for the present debranched Amioca sample. * Values using SEC data for the debranched Amioca starch used in the grafting experiments.

	Starch type	M n	X n	Surface area per oligosaccharide chain (nm 2 ) assuming the present Amioca system had the same -X n
	Potato	4200	26	163
	Barley	3278	20	127
	Rice	2806	17	109
	Maize	2786	17	108
	Wheat	2350	14	91
	Amioca*	2500	15	97

  Table:13 C chemical shift assignment of signals present on the13 C solid-state NMR spectrum of the precipitate of an oligosaccharide grafted polystyrene/PMMA copolymer (Figure5) -see database for polymers NMR spectra of the National Institute for Materials Science, (NIMS, Japan): http://polymer.nims.go.jp/NMR/top_eng.html;

		Tang,	H.;	Hills,	B.	P.
	Biomacromolecules 2003, 4, 1269.
	δ (ppm)	Assignment
	14	PMMA, backbone CH 3
	24	PMMA, backbone CH 3
	27	PMMA, backbone CH 3
	31	PMMA, backbone CH 3
	33	PMMA, backbone CH 3
	35 to 52	polystyrene, backbone; PMMA, backbone quaternary and OCH 3
	54	PMMA, backbone CH 2
	67	starch, C6
	75 to 85	starch C2, C3, C4, C5
	85 to 130	KelF from rotor cap
	100	starch, C1
	128	polystyrene, aromatic C not linked to backbone
	145	polystyrene, aromatic C linked to backbone
	176	PMMA, C=O

Acknowledgments

The partial support of a Lavoisier Fellowship for Cédric Dever is gratefully acknowledged, as is the support of a Discovery, LIEF and Linkage grants of the Australian Research Council. The help of Dr Binh Pham with the surface tension measurements, and of Matias Hernandez and Hollie Zondanos with the optimization of the debranching technique, are much appreciated. The support of Dow Chemical and BASF are also gratefully acknowledged, as well as the collaboration of the University of Sydney Electron Microscope Unit, and of Dr Duc Nguyen and Yanjun Wang with transmission electron microscopy. MG gratefully acknowledges the University of Queensland Centre for Magnetic Resonance and Prof. Andrew Whittaker for the use of the solid-state NMR equipment. The Key Centre for Polymer Colloids was established and supported by the Australian Research Council Research Centres program.


Christelle Ghazaly

Krystyna Biletska

Etienne Thevenot

Philippe Devillier

Emmanuel Naline

Stanislas Grassin-Delyle

Emmanuel Scorsone

Optimization of E-nose technology for detecting nonanal: a COVID-19 biomarker in exhaled breath

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A low-cost, rapid and non-invasive diagnosis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection is needed for the prevention and control of the pandemic. Coronavirus disease 2019 (COVID-19) mainly affects the respiratory tract and lungs. Therefore, analysis of exhaled breath could be an alternative scalable method for reliable SARS-CoV-2 screening. In this work, an experimental protocol using an electronic nose ("e-nose") for identifying a specific respiratory imprint in COVID-19 patients was optimized. The analytical performances of the Cyranose ® , a commercial e-nose device, were characterized by using a gas rig. In addition, the effect of various experimental conditions on its sensor array response was assessed, including relative humidity, sampling time and flow rate, aiming to select the optimal parameters. We also evaluated whether the Cyranose ® could distinguish between expired air from five healthy patients spiked or not with nonanal, identified as one putative COVID-19 biomarker. Electrical resistance variation of 32 sensors was recorded in real-time by using the PC-nose software during all tests. A statistical data analysis was applied to e-nose sensor response using a software called "Enair" developed on purpose and using a built-in optimized algorithm. Cyranose ® reveals a possible detection of low concentrations of nonanal (5 ppb) in breath and a significant discrimination from others Volatile Organic Compounds (VOCs) of healthy patients.  Cyranose ® sensor's detect low concentrations of nonanal in the presence of others VOCs of healthy patients.

Abstract Materials & Methods

Results

Conclusions & Perspectives

 Application of "Enair" software for a rapid statistical data processing and PCA plots visualization.

Conclusions

• Development of a strategy for analyzing exhaled breath via Cyranose ® .

• Detection of 5 ppb of nonanal and discrimination among other VOCs in the breath of 5 healthy donors. • Application of "Enair" software for statistical data processing.

Perspectives

• Evaluation of the Cyranose ® in the presence of other Covid-19 biomarkers (e.g. 2,4-octadiene). • Evaluation of measurement repeatability.

• Validation of the optimized analytical strategy in clinical trials.



  Possible detection of various concentrations of acetone or nonanal in the presence of a complexe mixture of VOCs.  Clear differentiation between the absence of acetone or nonanal in humidified nitrogen at 94%HR (reference test).  Discrimination between exhaled breath from one healthy donor spiked or not with acetone or nonanal. &c  Evaluation of exhaled breath of five healthy donors  Satisfactory discrimination before (ExB1 to ExB5) and after adding 5 ppb of nonanal (ExBN1 to ExBN5), a COVID-19 biomarker.