Boudjemaa Boudaa
email: boudjemaa.boudaa@univ-tiaret.dz

Slimane Hammoudi
email: slimane.hammoudi@eseo.fr

Sidi Mohamed Benslimane
email: s.benslimane@esi-sba.dz

Towards an Extensible Context Model for Mobile User in Smart Cities

In smarts cities environments, a recommender system (RS) has for goal to recommend relevant services to the user who is sometimes mobile. Thus, to be able to provide accurate personalized recommendations, the RS should be aware to the user's context (preferences, location, activities, environment, ...), thereby, it should be Context-Aware Recommender System (CARS, for short). Therefore, the context modeling becomes crucial for developing CARSs. Although there is a lack of context models in the RS literature, several ones have been proposed in pervasive computing eld. Nevertheless, most of them are dedicated for closed spaces and should be reviewed to be more suitable for open intelligent environments such as smart cities. This paper aims to propose an extensible ontology-based context model for representing contextual information within a smart city. The proposed context model would subsequently allow to design and develop CARSs.

1 Introduction

A recommender system [START_REF] Bobadilla | Recommender systems survey[END_REF] in smart cities [START_REF] Curry | Smart cities: enabling services and applications[END_REF][START_REF] Dustdar | In: Introduction to Smart Cities and a Vision of Cyber-Human Cities[END_REF] aims to propose a relevant service to a given user at a given instant, according to her prole, geolocation and surrounding environment. The real challenge here is to bring the services of a city closer to its citizens. The service in this context can be information about an event, a product to buy, a movie to watch, a web page to consult or a place to see, even a road to avoid. In order to be able to provide accurate personalized recommendations, the RS should be aware to the user's context, it should be Context-Aware Recommender System (CARS) [START_REF] Adomavicius | In: Context-Aware Recommender Systems[END_REF]. The context includes information about prole, preferences, mobilities, location of a user, even her emotions [START_REF] Zheng | Emotions in Context-Aware Recommender Systems[END_REF], and information about the surrounding environment (e.g., weather condition) in which the user evolves. Consequently, the context modeling [START_REF] Sheng | Contextuml: A uml-based modeling language for model-driven development of context-aware web services[END_REF] becomes crucial for developing CARSs.

While a lack of context models is found in the literature of recommender systems, several ones have been proposed in pervasive computing eld. However, most of them are dedicated for closed spaces and should be reviewed to be more suitable for CARSs in open intelligent environments such as smart cities.

By exploring the most popular context models of pervasive computing [START_REF] Alegre | Engineering context-aware systems and applications[END_REF][START_REF] Bettini | A survey of context modelling and reasoning techniques[END_REF][START_REF] Ye | Ontology-based models in pervasive computing systems[END_REF],

this paper aims to propose a generic context model for representing context information taking into account the user's mobility information within a smart city. This model is based on ontologies by beneting from their semantic expressiveness and reasoning capabilities which together enhance the prediction and accuracy to provide services recommendations.

Also, by using the ontologies for modeling, this context model can fulll to some requirements such as genericty and exibility as mentionned in [START_REF] Hammoudi | Model driven development of user-centred context aware services[END_REF].

After this introduction, some theoretical background regarding the scope of the paper are given in Section 2. Section 3 presents the proposed ontologybased context model for mobile user in a smart city that is instantiated for a tourism example, and related works are discussed in Section 4. Finally, Section 5 concludes this paper and indicates some future works.

Fundamental concepts

In order to be familiar with the concepts used in the present work, this section gives a theoretical background regarding the scope of the paper.

Context and Context-awareness

Context The context designates all information elements that can inuence the understanding of a particular situation. Despite its wide use in dierent research areas namely in Ubiquitous/Pervasive Computing, Ambient Intelligence and Intelligent Environments, we nd a variety of its denition in the literature [START_REF] Bazire | Understanding context before using it[END_REF] that does not reach to a consensus among them [START_REF] Alegre | Engineering context-aware systems and applications[END_REF]. However, we highlight that the denition proposed in [START_REF] Dey | Towards a better understanding of context and contextawareness[END_REF] is the most acknowledged one, considering it as any information that can be used to characterize the situation of an entity , where an entity can be a person, place, or object that is considered relevant to the interaction between a user and an application, including the user and application themselves . The authors give a general denition that can be used in a wide range of context-aware applications. To rene their denition, they identify four categories of context that they feel are more practically important than others.

These are location, identity (user), activity (state) and time [START_REF] Dey | A conceptual framework and a toolkit for supporting the rapid prototyping of context-aware applications[END_REF].

In our scope of context-aware recommender systems, we believe that this denition is quite sucient to identify the context change about a user and her various mobilities in a smart city compared to the application that remains unchangeable in most cases. For instance, imagine that a tourist takes the tramway (activity) to reach a tourist place (location), and that the trip will take a duration of 15 min (time). The recommender system in this case may suggest watching a video according to the user's preferences or about this touristic place (identity, location) whose duration is slightly less than 15 min.

Context-awareness

The characteristic context-aware is generally used to describe any type of system that benets from using context [START_REF] Alegre | Engineering context-aware systems and applications[END_REF]. Nowadays, computing applications operate in a variety of new settings; for example, embedded in cars or wearable devices. They use information about their context to respond and adapt to changes in the computing environment. These kind of applications are, in short, increasingly context-aware (sometimes called smart or intelligent) like as Google Now applications (https://www.google.co.uk/ landing/now/),

In the literature, there have been numerous attempts to dene contextawareness. In [START_REF] Dey | A conceptual framework and a toolkit for supporting the rapid prototyping of context-aware applications[END_REF], the authors also dened a system as context-aware if it uses context to provide relevant information and/or services to the user, where relevancy depends on the user's task . In our previous example (Subsection 2.1), the RS will be aware on the context information about user's preferences to recommend her a suitable set of videos.

Smart city

Owing to the fast and continual development of new technologies (e.g., Internet of Things) and infrastructures (e.g., smart administration, smart mobility, smart economy, ...) that have been (and/or will be) applied under the smart city concept, it is dicult to precise a unied denition of a smart city. Recent few years witnessed several evolutionist descriptions about smart cities according to these developed technologies/infrastructures. In [START_REF] Curry | Smart cities: enabling services and applications[END_REF], a Smart City can be viewed as an urban innovation and transformation that aims to harness physical infrastructures, Information and Communication Technologies (ICT), knowledge resources, and social infrastructures for economic regeneration, social cohesion, better city administration, and infrastructure management. The authors in [START_REF] Dustdar | In: Introduction to Smart Cities and a Vision of Cyber-Human Cities[END_REF] present the Smart City of today as a city planning/urban development methodology heavily relying on ICT to gather necessary input and make optimal engineering and planning decisions .

Briey, a smart city is a city that uses information and communication technologies (ICT) to improve the quality of urban services or reduce its costs.

The most distinguishing feature of the smart city concept is the welfare of city residents whom are not taking any active role in the development and daily management of the city and remain passive [START_REF] Dustdar | In: Introduction to Smart Cities and a Vision of Cyber-Human Cities[END_REF]. The main goal of smart cities is the transformation of life and work of city inhabitants for the best and make them (life and work) easier.

In this perspective, context-aware recommender systems nd their place in the smart city as intelligent applications to facilitate the professional and social lives of the user.

Context-Aware Recommender System

Recommender systems research [START_REF] Bobadilla | Recommender systems survey[END_REF] typically explores and develops techniques and applications for recommending various products or services or other items to individual users based on the knowledge of users' tastes and preferences as well as users' past activities (such as previous purchases).

Context-aware recommender systems (CARS) [START_REF] Adomavicius | In: Context-Aware Recommender Systems[END_REF][START_REF] Zheng | Emotions in Context-Aware Recommender Systems[END_REF] are an important subclass of recommender systems that take into account the context in which an item (e.g., service) will be consumed or experienced. In context-aware recommendation research, a number of contextual features have been identied as important in dierent recommendation applications [START_REF] Zheng | Emotions in Context-Aware Recommender Systems[END_REF]: such as companion in the movie and tourism domains, time and mood in the music domain, and weather or season in the travel domain. The context allow CARS to be more accurate in providing recommendations services using the prediction task into a multidimensional rating function R: Users Ö Items Ö Contexts Ratings [START_REF] Adomavicius | In: Context-Aware Recommender Systems[END_REF].

For

Context Modeling for User-centered Smart Cities

The context model is a centerpiece for the design of a context-aware recommender systems in mobile and intelligent environments [START_REF] Nicolas | A Framework for Context-Aware Service Recommendation for Mobile Users: A Focus on Mobility in Smart Cities[END_REF]. In the literature of context modeling [START_REF] Ye | Ontology-based models in pervasive computing systems[END_REF][START_REF] Bettini | A survey of context modelling and reasoning techniques[END_REF], we nd many modeling techniques of context (key-value, graphical, object-oriented, logic-based, ontology-based) [START_REF] Alegre | Engineering context-aware systems and applications[END_REF], although all techniques have their strong points and drawbacks, ontologies are the most widely adopted approaches [START_REF] Bettini | A survey of context modelling and reasoning techniques[END_REF]. They [START_REF] Boudaa | An aspectoriented model-driven approach for building adaptable context-aware service-based applications[END_REF] (i) represent an extensible large-scale formalism to model the context in which it is possible to add context elements (concepts, relationship, axioms, ...), delete or modify others in a exible way, (ii) oer more expressiveness and semantic richness to describe complex context information with a consensual conceptualization, (iii) are tted with inference capabilities enabling reasoning about the modeled context information in order to deduce other ones, (iv) make the possibility to share and/or reuse context among dierent information sources in intelligent environment, and (v) have fairly sophisticated tools available.

Once the context is modeled, it is possible to reason about the currently captured/sensed contextual information and to deduce other new knowledge.

The reasoning techniques can be divided into [START_REF] Perera | Context aware computing for the internet of things: A survey[END_REF]: supervised learning, unsupervised learning, rules, fuzzy logic, ontological, probabilistic. For this paper, we will based on ontologies for context modeling and SWRL rules for ontological reasoning.

Context Modeling Requirements for Intelligent Environments

In pervasive computing, several context models have been proposed. Nevertheless, most of them can not be more suitable for the CARS development, especially, in smart cities. The latter are tted abundantly by renew and smart technologies/infrastructures that enabling profound changes in the user's social life mode. For CARS in a smart city, the resident has not almost any role and remains a passive actor in the whole smart city process which allows her to receive pushed services recommendation.

In order to propose a generic context model, the authors in [START_REF] Hammoudi | Model driven development of user-centred context aware services[END_REF] specify some requirements that can be adopted for this work as follow:

be suciently general to be used by dierent user centered mobile applications in smart cities (such as, recommender systems).

be suciently specic to cover the main contextual entities proposed in the state of the art of context-aware mobile applications (such as, context-aware recommender systems).

be suciently exible to allow an extension and to take into account new entities specic to a given application domain in smart cities.

Also, we can specify the context more precisely in particular for recommendation services in smart cities. Therefore, the two following remarks are considered in our context model :

1. The user should not need to interact explicitly with the application to receive recommendations. Pushed recommendations could be very relevant for mobile users and the system could decide to recommend a user a given service because of her current context.

2. The notion of context is dened in a very general manner and would benet from being more precise. We argue that prole, Activity and the environment are three main entities of the context that should be precised, specically for context-aware recommender systems.

Ontology-based Context Model

Considering the previous requirements, Figure 1 depicts our ontology-based context model represented with Ontology Denition Meta-model (ODM) specication [START_REF] Gasevic | Model Driven Engineering and Ontology Development[END_REF] (for Model-Driven Engineering purpose).

Extending two works [START_REF] Nicolas | A Framework for Context-Aware Service Recommendation for Mobile Users: A Focus on Mobility in Smart Cities[END_REF][START_REF] Boudaa | An aspectoriented model-driven approach for building adaptable context-aware service-based applications[END_REF], it is structured on three sub-ontologies at three dierent abstraction levels : generic, domain and application ontologies.

They cover four main context dimensions that we believe contribute to develop context-aware recommender systems:

User (on which the CARS is centered): A person that has a prole and preferences. A user evolves in an environment, and her state can be mobile or static. The prole and preferences are strongly attached to the user and contains the information that describes her. For example, the goal of a tourist searching for a restaurant is to have dinner. A prole/preferences in this case can give information concerning the tourist's culinary preferences or whereabouts.

MobileDevice: It is the mobile computing system used by the user to invoke or receive services, and to capture contextual information from the environment. The device can obtain information concerning its type (e.g., tablet, laptop, smart-phone), the application, and the network.

Activity: The activity in which the user is involved may be a key to decide which service is relevant to her. It can be of professional, social or leisure type (e.g., working, meeting, visiting, playing, and so on.). However, recognizing users' activities is still a tough task.

Environment: It contains all the information that describes the surroundings of the user and its device, and that can be relevant for the recommender application. It includes dierent categories of information such as: spatial context information (e.g., Location, city, destination, speed), temporal context information (e.g., Time, date, season), Climatic context information (e.g., Temperature, type of weather).

The relationships between the User Entity and the others are respectively : uses-Device, involvedIn and evolvedIn. The dimensions are regrouped in the Generic ontology as top level abstraction entities.

The Domain ontology, on the other hand, allows covering all main contextual entities proposed in the literature of context modeling [START_REF] Ye | Ontology-based models in pervasive computing systems[END_REF][START_REF] Bettini | A survey of context modelling and reasoning techniques[END_REF][START_REF] Alegre | Engineering context-aware systems and applications[END_REF], and the Application ontology is to specify the context for a conned application space.

For a Tourism example, this Application ontology contains a set of specic context classes such as TouristProle and TouristPreferences for the User entity (i.e., the tourist), and TouristLocation as well as the TouristPlace related to the Environment entity. In practice, a CARS can recommend to a tourist to visit a place that it is closer to her location and according to his preferences.

In this proposed context model, the ontological attributes (OWLDataType-Property) is responsible to contain context information and can be modeled using also the OUP (Ontology UML Prole) notation proposed with ODM specication [START_REF] Gasevic | Model Driven Engineering and Ontology Development[END_REF].

The Domain and Application ontologies are suciently exible to enable storing all context entities involved in a contextual situation and adding each new entity that could be introduced as context information. In addition, other context information can be derived by using reasoning capabilities of ontologies to complete describing contextual situations.

Context Reasoning With SWRL

The context sources allowing individualizing described ontologies can be provided in two dierent ways: sensing or inferring methods. Sensing is used directly for basic and low-level context; whereas, inferring is used to provide a high-level context from low-level one using rule-based inference languages [START_REF] Bettini | A survey of context modelling and reasoning techniques[END_REF].

Semantic Web Rule Language (SWRL) [START_REF] Horrocks | SWRL: A Semantic Web Rule Language Combining OWL and RuleML[END_REF] allows to express reasoning rules that can be executed through a rule-based engine such as Java Expert System Shell (JESS).

As an example, if the Language of a tourist is only Arabic (as low-level context information sensed from her prole), we can infer that Arab Countries (as high-level context information) is among her Favorite tourist places, that is a useful information for Tourism Recommender Systems. This is expressed by the next SWRL rule: Tourist(?t) and hasLanguage(?t, Arabic) implies hasFa-voriteTouristPlaces(?t, ArabCountries ).

In the literature, the low-level and high-level context are also called atomic and composite context, respectively. The relationship between them can be represented by the UML composite design pattern (see Figure 2), in which, high-level context can be compose from others context information (whether low or high levels context).

Thereby, the low-level context can be provided by both types of context sources (Local or Remote). The rst is for local provisioning of context information that is available at client (MobileDevice ) or server (Recommender Application ) sides. For example, the location of a tourist is captured in LocationTourist, which is used to locate the tourist. The second source type is for extra context information that can be captured by a third-party (e.g., a Web service to provide the weather forecast for a tourist place to visit, ...). Based on the previous ontology, the same authors are presented another context ontology in [START_REF] Chen | Soupa: Standard ontology for ubiquitous and pervasive applications[END_REF]. In near future, we aim to extend much more our context model in order to be suciently generic and can be reused to build several context-aware recommendation systems for the dierent smart city applications, and to implement it taking into account how to create new context information attributes at run-time and integrate them to this ontological model in order to capture any new context entities could be appeared over time. Once, this is achieved, the design and the development of context-aware recommender system (CARS) can be undertook.

Figure 1 .

 1 Figure 1. An Ontology-based Context Model

Figure 2 .

 2 Figure 2. Low and High Levels Context

5

  Conclusion and future work In this paper, we have proposed an extensible context model based on ontologies and beneting from their expressiveness and reasoning capabilities. It combine three levels of ontologies, Generic, Domain ontologies for popular and shared concepts used by context-aware applications developers and Application ontology for a specic description of context (e.g., Tourism domain) in wide space such as smart city.

  example, a traditional RS can give us a list of recommended movies to watch without taking into account any appropriate place/day or hour, but CARS can give this list if time and location permit it (e.g., at cinema/weekend or during a trip in train). Here, the context suggests the best time/location to watch the preferred movies.

  The ontology of Standard Ontology for Ubiquitous and Per-Platforms and Services. This ontology has been designed with the aim of solving many challenges, such as application adaptation, automatic code generation, code mobility, and generation of device-specic user interfaces for allow interoperability in an Ambient Intelligence environment.Strang et al.[START_REF] Strang | Cool: A context ontology language to enable contextual interoperability[END_REF] describe a context modeling approach using ontologies as a formal foundation. They introduce their Aspect-Scale-Context (ASC) model and show how it is related to other models. A Context Ontology Language (CoOL) is derived from the model, which is used to enable context-awareness and contextual interoperability during service discovery and execution in a distributed architecture.4 http://www.foaf-project.org/ 5 http://distrinet.cs.kuleuven.be/projects/CoDAMoS/ All proposed context models are specic for closed environment (e.g., a smart home), and can not be applied for wide intelligent spaces. While ours is extensible to new appeared context information in opened environments such as smart cities.

	vasive Applications(SOUPA) provides knowledge sharing, reasoning on context
	and interoperability in a pervasive and ubiquitous environment. SOUPA deals
	with more areas of pervasive computing than CoBra-Ont and many of its
	concepts to concepts of existing common ontologies like friend-of-friend (FOAF) 4
	ontology to enable interoperability by using ontology mapping constructs OWL
	standard (owl: equivallentClass and owl: equivallentProperty). It is constituted of
	two sets of ontologies called SOUPA-Core and SOUPA-Extensions. SOUPA-Core
	denes a generic vocabulary which is universal for dierent pervasive computing
	applications (e.g., person, agent, policy, time, space), while SOUPA-Extensions
	dene additional ontologies to support specic kind of applications and provide
	examples for extending future ontologies (e.g., home, oce, attraction).
	The CONtext ONtology (CONON) [21] is a context OWL ontology for mod-
	eling context in pervasive computing environment and supporting logic based
	In their project called Context-Driven Adaptation of Mobile Services (Co-
	DAMoS) 5 and suiting some requirements of mobile computing, Prenveneers at
	al., have presented an adaptable and extensible context ontology for creating
	context-aware computing infrastructures, ranging from small embedded devices
	to high-end service platforms [23]. It is dened around four modeling entities
	Users, Environment,

context reasoning.

Wang et al.

have proposed an upper context ontology that captures general concepts about basic context such as location, activity, person or computational entity, and a domain specic ontologies for detaining these general concepts and their features in each sub-domain covered in a hierarchical manner. Also and based on this context ontology, the authors have studied the use of logic reasoning to check the consistency of context information, and to reason over low-level, explicit context to derive high-level, implicit context. The notions of upper ontology and domain-specic ontologies are discusses in similar work

[START_REF] Gu | An ontology-based context model in intelligent environments[END_REF]

for presenting Service-Oriented Context-Aware Middleware (SOCAM).


Sébastien Choinière

Eric H Frost

Jan J Dubowski
email: jan.j.dubowski@usherbrooke.ca

J Jan

Dubowski

Binding strategies for capturing and growing Escherichia coli on surfaces of biosensing devices

Keywords: Biosensor, Bacteria detection, Bacterial growth, Escherichia coli, Antibiotic resistance, Surface capture, Antibody

Antibiotic resistant bacteria have become a threat to world health. An advanced method of detection, based on the matrix assisted laser desorption ionisation time-of-flight mass spectroscopy can identify bacteria relatively rapidly, but it is not suitable to measure bacterial antibiotic resistance. Biosensors may be able to detect resistance by monitoring growth after capture on sensor surfaces but this option has not been addressed adequately. We have evaluated the growth of Escherichia coli after capture in 96 well microplates and observed that growth/capture efficiency was relatively similar for antibody-based techniques, but non-specific capture varied considerably. We confirm that neutravidin binds E. coli non-specifically, which limited its use with biotinylated antibodies or aptamers. Centrifugation enhanced bacterial growth/capture considerably, indicating that procedures enhancing the interaction between bacteria and surface-bound antibody have the potential to improve growth efficiency. Capture and growth required larger numbers of bacteria than capture and detection on biosensor surfaces. Previously, we reported that the minimum concentration of live E. coli required for initiating growth on a GaAs/AlGaAs biosensor was ~ 10 5 CFU/mL [Nazemi et al., Talanta 178 (2018) 69-77], and we speculated that this could be related to the poisonous effect of Ga-and As-ions released during dark corrosion of the biosensor, however in the present report we observed that the same minimum concentration of E. coli was required for growth in an ELISA plate. Thus, we argue that this limitation was related rather to bacterial inhibition by the capture antibodies. Indeed, antibodies at titres designed to capture bacteria inhibited bacterial growth when the bacteria were added to growth medium at titres less than 10 5 CFU/mL, indicating that antibodies may be responsible for the higher limits of sensitivity due to their potential to restrict bacterial growth. However, we did not observe E. coli release after 6 h following the capture indicating that these bacteria did not degrade antibodies.

Introduction

The rise in multi-resistant microorganisms represents a major threat for public health worldwide [START_REF] Escmid | Antibiotic Armageddon in UK and Europe by[END_REF][START_REF] Fleming-Dutra | Prevalence of Inappropriate Antibiotic Prescriptions Among US Ambulatory Care Visits, 2010-2011[END_REF][START_REF] Nathan | Antibiotic Resistance -Problems, Progress, and Prospects[END_REF]. Microorganisms also cause problems for water, food, and pharmaceutical industries [START_REF] Nathan | Antibiotic Resistance -Problems, Progress, and Prospects[END_REF][START_REF] Ishii | Escherichia coli in the Environment: Implications for Water Quality and Human Health[END_REF][START_REF] Mellata | Human and avian extraintestinal pathogenic Escherichia coli: infections, zoonotic risks, and antibiotic resistance trends[END_REF][START_REF] Sikora | Prevalence of Legionella pneumophila in water distribution systems in hospitals and public buildings of the Lublin region of eastern Poland[END_REF]. Current methods of microbial detection, like culture and detection in an automated instrument, polymerase chain reaction (PCR) and immunology-based methods such as enzyme-linked immunosorbent assays (ELISA), are time consuming (24-48 h), complex, require trained personnel and may require enrichment steps to detect low concentrations [START_REF] Barreiros Dos Santos | Highly sensitive detection of pathogen Escherichia coli O157: H7 by electrochemical impedance spectroscopy[END_REF][START_REF] Jayamohan | Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads[END_REF]. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has emerged as a promising method for rapid, sensitive and cost-effective bacterial identification, but provides only limited determination of antibiotic resistance [START_REF] Singhal | MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis[END_REF][START_REF] Doern | Emerging and Future Applications of Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry in the Clinical Microbiology Laboratory[END_REF]. Even though these techniques are reliable, the recent increase in antibiotic resistance of bacteria [START_REF] Escmid | Antibiotic Armageddon in UK and Europe by[END_REF][START_REF] Fleming-Dutra | Prevalence of Inappropriate Antibiotic Prescriptions Among US Ambulatory Care Visits, 2010-2011[END_REF][START_REF] Nathan | Antibiotic Resistance -Problems, Progress, and Prospects[END_REF][START_REF] Martz | Funding faster diagnostics[END_REF][START_REF] Mediavilla | Colistin-and Carbapenem-Resistant Escherichia coli Harboring mcr-1 and bla NDM-5 , Causing a Complicated Urinary Tract Infection in a Patient from the United States[END_REF] has pushed health organizations to request alternative, fast, specific and sensitive methods for detection, identification and determination of antibiotic resistance of bacteria [START_REF]The White House Administration, National Action Plan for Combating Antibiotic-Resistant Bacteria[END_REF]. Biosensors [START_REF] Lazcka | Pathogen detection: A perspective of traditional methods and biosensors[END_REF] have been increasing in popularity as an alternative method due to their simplicity, speed, sensitivity, real-time monitoring, portability of the device and, potentially, low cost of testing [START_REF] Barreiros Dos Santos | Highly sensitive detection of pathogen Escherichia coli O157: H7 by electrochemical impedance spectroscopy[END_REF][START_REF] Jayamohan | Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads[END_REF]. Among numerous types of biosensors, the most frequently investigated appear to be those based on optical [START_REF] Wang | Monitoring of Escherichia coli O157:H7 in food samples using lectin based surface plasmon resonance biosensor[END_REF][START_REF] Vinay | Phagebased fluorescent biosensor prototypes to specifically detect enteric bacteria such as E. coli and Salmonella enterica Typhimurium[END_REF][START_REF] Hu | A novel biosensor for Escherichia coli O157: H7 based on fluorescein-releasable biolabels[END_REF] and electrochemical [START_REF] Barreiros Dos Santos | Highly sensitive detection of pathogen Escherichia coli O157: H7 by electrochemical impedance spectroscopy[END_REF][START_REF] Jayamohan | Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads[END_REF] methods. Surface plasmon resonance (SPR)-based and electrochemical impedancebased sensors using gold (Au) surfaces have shown attractive sensitivity for bacterial detection [START_REF] Barreiros Dos Santos | Highly sensitive detection of pathogen Escherichia coli O157: H7 by electrochemical impedance spectroscopy[END_REF][START_REF] Wang | Monitoring of Escherichia coli O157:H7 in food samples using lectin based surface plasmon resonance biosensor[END_REF], but have rarely been used to measure antibiotic sensitivity [START_REF] Chiang | Innovative antimicrobial susceptibility testing method using surface plasmon resonance[END_REF].

Photoluminescence-based biosensors that usually employ GaAs surfaces have been used for detection of some bacteria [START_REF] Duplan | A photoluminescence-based quantum semiconductor biosensor for rapid in situ detection of Escherichia coli[END_REF][START_REF] Nazemi | GaAs/AlGaAs heterostructure based photonic biosensor for rapid detection of Escherichia coli in phosphate buffered saline solution[END_REF][START_REF] Aziziyan | Photonic biosensor based on photocorrosion of GaAs/AlGaAs quantum heterostructures for detection of Legionella pneumophila[END_REF][START_REF] Lee | Plasmon-induced photoluminescence immunoassay for tuberculosis monitoring using goldnanoparticle-decorated graphene[END_REF][START_REF] Patel | A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection[END_REF][START_REF] Nazemi | Monitoring growth and antibiotic susceptibility of Escherichia coli with photoluminescence of GaAs/AlGaAs quantum well microstructures[END_REF] allowing quantification down to 10 3 colony forming units of bacteria (CFU) per ml and even lower with chemotaxis [START_REF] Hassen | Chemotaxis for enhanced immobilization of Escherichia coli and Legionella pneumophila on biofunctionalized surfaces of GaAs[END_REF].

Photoluminescence-based biosensors have also been employed to determine antibiotic resistance of E. coli, but only with relatively low limits of detection [START_REF] Nazemi | Monitoring growth and antibiotic susceptibility of Escherichia coli with photoluminescence of GaAs/AlGaAs quantum well microstructures[END_REF]. The response of these biosensors depends on the efficiency of capturing target biomolecules with targetspecific ligands [START_REF] Mustafa | Multifunctional nanotechnologyenabled sensors for rapid capture and detection of pathogens[END_REF], such as antibodies [START_REF] Jayamohan | Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads[END_REF][START_REF] Hu | A novel biosensor for Escherichia coli O157: H7 based on fluorescein-releasable biolabels[END_REF], aptamers [START_REF] Wu | A sensitive lateral flow biosensor for Escherichia coli O157: H7 detection based on aptamer mediated strand displacement amplification[END_REF], antimicrobial peptides [START_REF] Li | Impedance based detection of pathogenic E. coli O157: H7 using a ferroceneantimicrobial peptide modified biosensor[END_REF], lectins [START_REF] Wang | Monitoring of Escherichia coli O157:H7 in food samples using lectin based surface plasmon resonance biosensor[END_REF], phages [START_REF] Vinay | Phagebased fluorescent biosensor prototypes to specifically detect enteric bacteria such as E. coli and Salmonella enterica Typhimurium[END_REF] or molecular imprints [START_REF] Idil | Whole cell based microcontact imprinted capacitive biosensor for the detection of Escherichia coli[END_REF] as opposed to non-specific binding to Au surface after coating with poly-lysine [START_REF] Chiang | Innovative antimicrobial susceptibility testing method using surface plasmon resonance[END_REF].

The present study focuses on determining the best capture efficiency that can be employed on a biosensor surface and understanding why capture and growth is so much less efficient than capture and detection. The subsequent growth of the captured bacteria is an important factor to consider in order to monitor bacterial growth for antibiotic sensitivity tests in further studies. Typically, Au-based surfaces have been employed by numerous biosensors, although a large variety of biosensors employ GaAs, Si, and oxide coated surfaces. The purpose of this work was to investigate different bacterial capture strategies while working with plastic surfaces of 96 well plates, which allowed testing different binding structures in a short period of time. To this end, we have investigated passively adsorbed goat and chicken antibodies that are the recognized reference method for antigen capture on ELISA plates [START_REF] Lequin | Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)[END_REF]. We have compared them with strategies more amenable to GaAs or Au surfaces such as goat antibodies covalently linked to a plate with a carboxyl group and biotinylated goat antibodies or biotinylated aptamers linked via passively adsorbed neutravidin. Since low numbers of bacteria would not grow after capture, we employed high concentrations of bacteria as would be found in suspended bacterial colonies to be identified by MALDI-TOF. Although centrifugation is more challenging to input in a fully automated portable device, it can be considered as a strategy to investigate enhanced surface capture [START_REF] Czilwik | Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation[END_REF], e.g., as a proxy for other strategies such as chemotaxis or electrophoresis that might possibly overcome electrostatic repulsion between bacteria and surfaces. Centrifugation at less than 5000 G was not expected to disrupt surface integrity of bacteria [START_REF] Pembrey | Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?[END_REF] and it is easy to integrate into 96 well protocols. By growing bacteria captured by antibodies, we have investigated the ability of bacteria to degrade the antibodies and release themselves from capture. coli, both biotinylated and non-biotinylated, were from ViroStat (Portland, ME, USA), chicken Ab directed against E. coli from Immune Biosolutions (Sherbrooke, QC, CAN), biotinylated aptamers (Apt) (E2, EcO3R and EcO4R) [START_REF] Kim | Isolation and characterization of DNA aptamers against Escherichia coli using a bacterial cell-systematic evolution of ligands by exponential enrichment approach[END_REF][START_REF] Bruno | A novel screening method for competitive FRET-aptamers applied to E. coli assay development[END_REF] were synthesized by Integrated DNA Technologies (IDT) (Coralville, IA, USA), neutravidin (NA), Salmon sperm DNA and Tris-HCl were from Invitrogen (Burlington, ON, CAN). Tryptone, nutrient broth (NB) and agar were from Becton, Dickinson and Company (BD) (Mississauga, ON, CAN) and glucose from J. T. Baker (Phillipsburg, NJ, USA).

Materials and Methods

Materials and reagents

A genetically modified strain of E. coli K12 BW25113 continually expressing the green fluorescent protein (GFP) was provided by Sébastien Rodrigue from the Département de Biologie of the Université de Sherbrooke, and referred to as E. coli GFP. The E. coli GFP was conserved at 4 °C on nutrient agar (NB + 15% agar) medium and refreshed each month. A fresh culture of E. coli GFP was used each day of tests, after incubation overnight (O/N) at 37 °C in NB medium. Tryptone glucose (1% tryptone, 1% glucose and 0.5% NaCl) (TG) was used for the capture tests to allow subsequent growth and reduce the fluorescent background compared with NB or Luria-Bertani medium.

Fluorescence was measured using a CytoFluor Series 4000 (Applied Biosystems, Foster City, CA, USA) microplate reader with excitation at 485 nm and emission at 535 nm.

Data were exported to Microsoft Excel and GraphPad Prism version 7.03 for Windows (GraphPad Software, La Jolla, California, USA, www.graphpad.com) for further analysis.

Standard curve for quantification of E. coli GFP

In order to quantify captured E. coli GFP, a standard curve was made with unbound E. coli GFP using 200 µL of known concentrations of E. coli GFP suspended in TG medium in a 96 well plate without Ab. Concentrations of 10 1.2 , 10 2.2 , 10 3.2 , 10 4.2 , 10 5.2 and 10 6.2 colony forming units per mL (CFU/mL) were monitored for 12 h with data taken each 15 or 30 min in the CytoFluor. Data points for each growth curve were plotted to identify the time when fluorescence increased above background levels, which was called the Time threshold (Tt).

Passive capture of antibodies.

For the capture of bacteria with Ab, 100 µL of goat or chicken Ab (concentrations tested included: 0; 0.5; 1; 2; 5; 10; 20; 50; 100 and 200 µg/mL) in carbonate buffer pH 9.6 were added to the wells of a 96 well plate and incubated O/N at 4 °C with agitation. The wells were washed 5 times with 300 µL of PBS + 0.05% Tween-20 (PBST) and 200 µL of blocking solution (PBST + 3% BSA) were added to the wells and incubated O/N at 4 °C with agitation.

Active capture of goat Ab with EDC/NHS

To test similar structures to those used with Au or GaAs surfaces functionalized with carboxyl-thiol linkers [START_REF] Bekir | Electrochemical impedance immunosensor for rapid detection of stressed pathogenic Staphylococcus aureus bacteria[END_REF], COOH plates were used with activators EDC/NHS to covalently link the carboxyl group of the wells with an amine group from the Ab. For this structure, 50 µL of fresh solutions of 800 mM EDC in water and 200 mM NHS in water were used to activate the carboxyl group in the wells for 1 h at room temperature (RT).

The EDC/NHS from Sigma-Aldrich and EDC/NHS from Biacore were tested separately for comparison. After washing the wells, 100 µL of Ab (concentrations tested: 0; 20; 100 and 200 µg/mL) in water were added and then incubated for 1 h at RT. The wells were washed between each step with PBST and blocked as mentioned above.

Capture of biotinylated antibodies or aptamers with neutravidin bound to surfaces

In order to simulate capture of biotinylated linkers to Au or GaAs surfaces (functionalized with biotin alkane thiols and NA) [START_REF] Nazemi | GaAs/AlGaAs heterostructure based photonic biosensor for rapid detection of Escherichia coli in phosphate buffered saline solution[END_REF], we bound NA directly to 96 well plates. 100 µL of NA in PBS (concentrations tested: 0; 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1; 2; 3; 4; 5; 10; 20; 50; 100 and 200 µg/mL) was added to the wells and incubated O/N at 4 °C with agitation. The wells were washed and, for the antibody before bacteria (AB) method, 100 µL of biotinylated Ab (concentrations tested: 0; 20; 100 and 200 µg/mL) in PBS or 100 µL of biotinylated aptamer in their respective binding buffers (BB) (BB E2: PBST + 1 % BSA + 0.1 mg/mL salmon sperm DNA, BB EcO3R and EcO4R: 10 mM Tris-HCl pH 7.5 + 0.5 M NaCl + 1 mM MgCl 2 ) [START_REF] Pembrey | Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?[END_REF][START_REF] Kim | Isolation and characterization of DNA aptamers against Escherichia coli using a bacterial cell-systematic evolution of ligands by exponential enrichment approach[END_REF] were added to the wells and incubated 1 h at 37 °C with agitation. The washing and blocking steps were done as mentioned above.

Alternatively, in the pre-incubation (PI) method, biotinylated Ab or Apt were preincubated with 100 µL of 10 8 CFU/mL of E. coli GFP in PBS or in the specific BB of each Apt in micro centrifuge tubes and incubated 1 h at 37 °C with agitation. The tubes were centrifuged 15 min at 2900 G and the supernatant was removed and the pellet washed with 100 µL of PBS or with the respective BB of each Apt. The content of the tubes was then transferred to the wells of the NA-coated plate and incubated 1 h at 37 °C with agitation. The blocking step was done as mentioned above and the steps of monitoring and analysis were performed as described below.

Capture of E. coli GFP with ligands bound to 96 well plates

After the blocking step, the wells were washed 5 times with PBST, then 200 µL of E. coli GFP (usually at 10 8 CFU/mL, unless otherwise mentioned) in PBS was added to the wells and incubated 1 h at 37 °C with agitation. The wells were washed 5 times with 300 µL of PBST including 100 mM EDTA prior to addition of 200 µL of TG medium to the wells. The plate was incubated in a CytoFluor microplate reader for 12 h at 37 °C with fluorescence intensity measured ( abs = 485 nm,  em = 535 nm) every 15 or 30 min. A quantification curve with unbound E. coli GFP was constructed in parallel with each capture test. The quantity of bacteria captured was estimated by comparison with the parallel standard curve.

Centrifugation

Using the protocol described for passively adsorbed goat Ab, different concentrations of bacteria (10 0.2 , 10 1.2 , 10 2.2 , 10 3.2 , 10 4.2 , 10 5.2 , 10 6.2 , 10 7.2 and 10 8.2 bacteria/mL) were added to the wells after the Ab adsorption and blocking steps. The plates were centrifuged 5 min at 250 G or 10 min at 550 G to pellet the bacteria gently [START_REF] Tan | Kinetically limited differential centrifugation as an inexpensive and readily available alternative to centrifugal elutriation[END_REF] followed by incubation for 1 h at 37 °C to allow the Ab bound to the plate to capture the bacteria. The plates were then washed with PBST + 100 mM EDTA and incubated in the fluorometer as described above.

Antibody inhibition of bacterial growth

In order to titrate Ab inhibition of E. coli GFP growth, 100 µL of goat anti-E. coli Ab at concentrations of 0.5; 1; 2; 5; 10; 20; 50 and 100 µg/mL in TG and 100 µL of E. coli GFP (concentrations tested: 10 0.5 , 10 1.5 , 10 2.5 , 10 3.5 , 10 4.5 , 10 5.5 and 10 6.5 CFU/mL) in TG were added to wells of a 96 well plate. The plate was incubated in the fluorometer with data taken every 15 min as above.

E. coli GFP degradation of captured antibodies

In order to ascertain if E. coli GFP could degrade Ab in the well after capture, we have investigated the growth of bacteria at 37° C in TG medium in uncoated and Ab functionalized wells. The number of bacteria was determined after washing designed to remove Ab released bacteria. E. coli GFP was captured on plates prepared with passively adsorbed goat Ab and unbound bacteria washed off as described above leaving attached bacteria in the well with 200 µL of TG. The plate was incubated in the fluorometer. After 0 to 6 h, some wells of the plate were washed with PBST + 100 mM EDTA and 200 µL of TG was added to the washed wells to quantify the bacteria still bound, and investigate if bacteria were still retained by the Ab or if the bacteria had degraded the Ab and been removed by the washing step.

Results

Quantification of E. coli GFP

Growth curves for E. coli GFP synthesis were analyzed to determine the time when fluorescence increased above background levels (Tt), either by identifying the abscissa crossing point of the tangent of the ascending portion of the curves between 25% and 75% of maximal fluorescence [START_REF] Guescini | A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition[END_REF], as shown in Fig. 1a, or using the maximum of the second derivative of the growth curve [START_REF] Luu-The | Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction[END_REF], as shown in Fig. 1b. Standard plots of Tt versus bacterial concentration were constructed for both of these methods as shown in Fig. 1c. Initial concentrations of bacteria could be estimated from these plots by measuring the Tt of unknown samples.

Capture of E. coli with passively bound antibodies

As a standard reference binding interface, Ab were adsorbed to 96 well ELISA plates.

The results, summarized in Table 1, indicate that E. coli GFP bacteria were captured with passively adsorbed goat Ab at 10 4.3 CFU/mL when 10 8 CFU/mL of these bacteria in PBS were added to wells. When chicken Ab were passively adsorbed, a comparable concentration of E. coli GFP at 10 4.0 CFU/mL was captured. In the absence of Ab, E. coli GFP were non-specifically captured by the structure up to 10 0.2 CFU/mL.

Table 1: Summary of capture methods

Method

Capture of E. coli with covalently bound goat IgG antibodies

Ab were linked with a COOH plate that had been activated with EDC/NHS to covalently link an amine group of the Ab. With EDC/NHS from Biacore, E. coli GFP were captured at 10 3.5 CFU/mL with goat Ab. With EDC/NHS from Sigma-Aldrich a comparable ligand-based binding was achieved at 10 3.7 CFU/mL, but this was also associated with a considerably more non-specific capture of bacteria at 10 2.2 CFU/mL compared to EDC/NHS from Biacore giving 10 0 CFU/mL (Table 1). The number of bacteria captured by the covalently bound Ab increased over 10-fold for the 4-fold increased concentration of Ab solutions (from 5 to 200 µg/mL), which is comparable to the increased number of bacteria captured on passively bound Ab for the same increase of the concentration of Abs (Table 2). Capture of E. coli was with PBST for the Passively Adsorbed Ab method and with PBS for the Covalently-Bound AB method. Sigma EDC/NHS was used for the Covalently-Bound AB method.

Capture of E. coli with biotinylated antibodies and neutravidin

Neutravidin was bound passively to an ELISA plate, then biotinylated Ab followed by bacteria (AB method) or bacteria preincubated with biotinylated Ab (PI method) were added. More efficient bacterial capture was observed with the PI method than with the AB method (10 4.9 CFU/mL versus 10 4.0 CFU/mL). E. coli GFP were non-specifically captured at 10 2.3 CFU/mL with 1 µg/mL of NA passively adsorbed on an ELISA plate without Ab (Table 1). The log difference between the specifically and non-specifically captured bacteria was 2.6 for NA employed to capture biotinylated Ab-bacteria complexes (PI method) versus 1.2 with the AB method.

Capture of E. coli with biotinylated aptamers and neutravidin

Neutravidin was also used to capture biotinylated Apt either before bacterial capture (AB method) or as aptamer-bacteria complexes (PI method). The AB method did not show an increase over non-specific binding with NA (Table 1). With the PI method, E. coli GFP were captured at 10 5.2 CFU/mL, versus 10 3.3 CFU/mL observed for non-specifically captured bacteria with NA and without added aptamer.

Increasing capture efficiency with centrifugation

It is assumed that bacterial capture on surfaces is hindered by electrostatic or other repulsive forces. Several strategies have been proposed to overcome this repulsion and improve contact with the surface such as centrifugation, electrophoresis and chemotaxis.

We attempted to improve capture by centrifuging bacteria onto the surface. With the addition of centrifugation at 250 G for 5 min, 10 7.0 CFU/mL E. coli GFP were captured from suspensions containing 10 8.2 CFU/mL compared to 10 4.3 CFU/mL captured without centrifugation (Table 3). When centrifugation was performed at 550 G for 10 min, 10 7.1 CFU/mL of E. coli GFP were captured. However, centrifugation also gave rise to higher amounts of non-specifically captured bacteria (on surfaces without Ab added) at 10 4.1 CFU/mL with centrifugation at 250 and 550 G (Table 3).

When capture was attempted on surfaces with passively absorbed goat Ab, without centrifugation, bacteria were captured at 10 4.3 and 10 3.4 CFU/mL from 10 8.2 and 10 7.2 CFU/mL suspensions, respectively, but no bacteria were captured from a 10 6.2 CFU/mL suspension. When centrifugation was employed at 250 G, bacteria were captured from suspensions containing 10 8.2 CFU/mL (10 7.0 CFU/mL), 10 7.2 CFU/mL (10 6.0 CFU/mL) and 10 6.2 CFU/mL (10 4.1 CFU/mL), and it also allowed detection down to 10 5.2 CFU/mL (10 2.2 CFU/mL) (Table 3). Similar results were observed with centrifugation at 550 G. * Bacteria at different concentrations were added to wells with or without passively adsorbed goat anti-E. coli Ab using 20 µg/mL of Ab. The plates were centrifuged at 250 G for 5 min or 550 G for 10 min, or not centrifuged, followed by 1 h incubation at 37 °C, and then bacteria titrated from the growth curves.

Inhibition of bacterial growth by antibodies

Tests showed that there was minimal (10 2.2 CFU/mL) bacterial capture when 10 5.2 CFU/mL or fewer bacteria were added to wells for capture with centrifugation, and none when fewer than 10 7.2 CFU/mL were added without centrifugation. In order to determine whether Ab could have contributed to this lower capture efficiency by inhibiting bacterial growth, different concentrations of bacteria were added to wells with different concentrations of Ab. Figure 2 demonstrates the effect of growth inhibition of bacteria by the increasing amount of Ab. Suppression of bacterial growth was much more evident with 25 and 50 µg/mL than with 10 µg/mL or less of Ab, and particularly for bacterial concentrations of 1.5 x 10 4 or less.

Bacterial degradation of antibodies and release from plates

In Fig. 3, we compared the number of bacteria captured in uncoated wells of a microtitre plate and in wells coated with Ab at 100 and 20 µg/mL. The number of bacteria that remained bound to those wells after up to 6 h of incubation in TG was measured following washing to remove released bacteria. A slightly greater concentration of bacteria was observed captured with Ab at 100 µg/mL, compared with Ab at 20 µg/mL, while a negligible number of bacteria was captured in wells without Ab. However, it can be seen that regardless of whether Ab at 20 µg/mL or 100 µg/mL had been employed, and despite washing, the number of retained bacteria increased with the incubation time, indicating capture of progeny rather than release due to Ab degradation. Clearly, under conditions of the current experiment, we could conclude that E. coli GFP did not degrade the Ab that captured it.

Fig. 3:

Concentration of E. coli GFP bacteria captured in wells of a microtitre plate coated with Ab at 100 or 20 µg/mL, washed, and then incubated at 37° C in TG for up to 6 h, followed by washing to remove released bacteria. Note that the concentration of bacteria captured on the uncoated surface of a well was negligible in comparison to that of bacteria captured on Ab coated well surfaces.

Discussion

We have quantified bacteria capable of growing after capture on surfaces by measuring the doubling time necessary to reach the minimum number of bacteria required to produce detectable GFP fluorescence (Fig. 1). This method resembles the strategy used for the quantitative polymerase chain reaction (qPCR) where DNA copies double until they reach a minimal detectable amount called the quantification cycle threshold (Ct). This is usually ascertained by identifying the first maximum value from the second derivative of the fluorescence curve [START_REF] Luu-The | Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction[END_REF][START_REF]Essentials of Real Time PCR About Sequence Detection Chemistries TaqMan Chemistry[END_REF]. Because this value may depend on amplification efficiency, it was proposed to identify the abscissa crossing point of the tangent of the inflection point of the Richard's curve obtained by non-linear regression of the raw fluorescence data [START_REF] Guescini | A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition[END_REF]. In our quantification method with E. coli GFP, the bacteria also doubled each 20 to 30 min and the Tt can be determined by this method as well. As it can be seen in Fig. 1c, both the second derivative fluorescence data and data obtained from the abscissa crossing points provided a comparable dependence of Tt on bacterial concentration.

Our method is optimal for bacteria expressing fluorescent products like E. coli GFP, but methods based on optical density determination would be necessary for non-fluorescent bacteria. Interference with contaminating bacteria from the environment, however, could affect results, especially when few target bacteria are captured. When we followed E. coli growth with optical density at 600 nm, our observed Tt were similar (data not shown).

The qPCR technique could also be employed to quantify bacterial capture, but it would be more expensive and would probably not be able to quantify small numbers of bacteria captured as accurately as our procedure due to qPCR inherent lack of sensitivity with fewer than 10 bacteria [START_REF] Zhang | Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection[END_REF][START_REF] Botaro | Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction[END_REF][START_REF] Adams | Detection of Salmonella Enteritidis in Pooled Poultry Environmental Samples Using a Serotype-Specific Real-Time -Polymerase Chain Reaction Assay[END_REF] and with the requirement to use only part of the bacterial DNA extract from a well in small PCR reaction volumes.

Our procedure to quantify live E. coli based on growth is not as rapid as qPCR detection for low numbers of bacteria. Our experiments showed that the minimum concentration of live E. coli required for detection within 3 hours was at 10 5.2 CFU/mL with single bacteria detection requiring as much as 9 hours (Fig. 1).

It was striking that the number of input bacteria able to grow after capture on an ELISA plate by a passively adsorbed goat Ab decreased precipitously from 10 3.4 to 0 when the number of bacteria exposed to a surface decreased by only one order (10 7.2 to 10 6.2 ) (see Table 3 for data with no centrifugation). This implies that capture with growth does not proceed with one-hit kinetics, or alternatively, that growth is inhibited in a non-linear fashion. As results documenting capture and detection without growth have indicated linear relationships between number of bacteria captured and number presented [START_REF] Barreiros Dos Santos | Highly sensitive detection of pathogen Escherichia coli O157: H7 by electrochemical impedance spectroscopy[END_REF][START_REF] Bekir | Electrochemical impedance immunosensor for rapid detection of stressed pathogenic Staphylococcus aureus bacteria[END_REF][START_REF] Torun | Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria[END_REF],

it could be assumed that capture occurs by one-hit kinetics and probably implies that capture occurs when one Ab molecule interacts with one bacteria, and does not require several Ab with variable-binding energy to reach a total binding energy sufficient to oppose disruptive forces like electrostatic repulsion. Inhibition of growth must be suspected as the explanation for the rapid decline in growth potential after capture.

Previously, we observed that the growth of E. coli on GaAs or Au surfaces required a minimum of 10 5 CFU/mL [START_REF] Nazemi | Growth of Escherichia coli on the GaAs (001) surface[END_REF], while testing the sensitivity of E. coli strains to antibiotics with a photoluminescence-based biosensor was not possible for bacterial concentrations under 2 x 10 8 CFU/mL [START_REF] Nazemi | Monitoring growth and antibiotic susceptibility of Escherichia coli with photoluminescence of GaAs/AlGaAs quantum well microstructures[END_REF]. It has been suggested that the toxic effects of Ga or As ions released by GaAs-based biochips might be the cause of this requirement for high bacterial concentration necessary to allow growth. In the experiments reported here, Ga and As were not present, and so inhibition may have resulted from the Ab used to capture the bacteria on the surface. Indeed, Ab added to cultures of bacteria inhibited bacterial growth dependant on the concentration of the Ab and the concentration of the bacteria. It might be possible to overcome this inhibition if it were possible to find an Ab that did not inhibit bacterial growth, but did capture the bacteria, in an analogous fashion to Ab that neutralize viruses versus Ab that react with viruses but do not neutralize them [START_REF] Tan | Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection[END_REF]. It is generally considered that antibodies classically inactivate bacteria in conjunction with complement or by opsonisation to enhance phagocytosis [START_REF] Xie | Antibody-dependent cell-mediated cytotoxicity-and complement-dependent cytotoxicity-independent bactericidal activity of an IgG against Pseudomonas aeruginosa O6ad[END_REF]. A few studies have shown direct antibody killing of bacteria by interaction with membrane bound enzymes [START_REF] Storek | Monoclonal antibody targeting the β-barrel assembly machine of Escherichia coli is bactericidal[END_REF][START_REF] Cabral | Design of live attenuated bacterial vaccines based on D-glutamate auxotrophy[END_REF] or lipopolysaccharides [START_REF] Xie | Antibody-dependent cell-mediated cytotoxicity-and complement-dependent cytotoxicity-independent bactericidal activity of an IgG against Pseudomonas aeruginosa O6ad[END_REF][START_REF] Apicella | Bactericidal antibody response of normal human serum to the lipooligosaccharide of Neisseria gonorrhoeae[END_REF]. Chicken IgY antibodies have also been shown to directly kill E. coli. [START_REF] Sunwoo | Growth Inhibition of Escherichia coli 987P by Neutralizing IgY Antibodies[END_REF].

We employed centrifugation to overcome static repulsion between surfaces and bacteria and increase bacterial contact with Ab. Centrifugation at low speed was used so as not to affect the bacterial surface [START_REF] Pembrey | Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?[END_REF]. Chemotaxis and electrophoresis have also been shown to improve capture of bacteria, e.g., E. coli and Legionella pneumophila by attracting them to glucose generated at the surface from lactose cleavage by surface-bound galactosidase enzyme [START_REF] Hassen | Chemotaxis for enhanced immobilization of Escherichia coli and Legionella pneumophila on biofunctionalized surfaces of GaAs[END_REF]. Other authors have brought bacteria into contact with a surface by electrophoresis [START_REF] Balasubramanian | A microfluidic device for continuous capture and concentration of microorganisms from potable water[END_REF] or using alternating current electrokinetic effects [START_REF] Liao | Fast and sensitive detection of bacteria from a water droplet by means of electric field effects and micro-Raman spectroscopy[END_REF]. These methods have been shown to improve the efficiency of capture and merit further study to improve and simplify them.

Purified IgG goat and IgY chicken Ab bound passively to ELISA microplates gave similar crude growth/capture rates (Table 1). When goat Ab were bound covalently to microplates approximately 3-5 times fewer bacteria grew after capture than when using passively bound Ab. The lowest background values were observed with the EDC/NHS chemistry activated MHDA manufactured by Biacore. When NA was used to capture biotinylated Ab or Apt, it was found to bind E. coli resulting in high background levels for wells without Ab as has been reported previously [START_REF] Kulagina | Antimicrobial peptides for detection of bacteria in biosensor assays[END_REF]. Other bacteria may have less interaction with NA as it has been demonstrated previously for L. pneumophila [START_REF] Aziziyan | Photonic biosensor based on photocorrosion of GaAs/AlGaAs quantum heterostructures for detection of Legionella pneumophila[END_REF] and

Staphylococcus aureus [START_REF] Abbaspour | Aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of staphylococcus aureus[END_REF]. Preincubating biotinylated Ab or Apt with bacteria and then adding to wells with NA already bound resulted in better crude rates of growth/capture than when bacteria were added to wells with biotinylated Ab or Apt already bound to the wells with passively adsorbed NA. However, even with preincubation, growth/capture was still not as efficient as with passively or covalently bound Ab.

The diversity of the capture molecules tested in this study was limited to polyclonal goat, polyclonal chicken Ab and Apt, which act like monoclonal Ab. It could be interesting to compare the capture efficiency between polyclonal and monoclonal Ab. Some studies have shown efficiency of polyclonal or monoclonal Ab and even compared different structures [START_REF] Barreiros Dos Santos | Highly sensitive detection of pathogen Escherichia coli O157: H7 by electrochemical impedance spectroscopy[END_REF][START_REF] Jayamohan | Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads[END_REF][START_REF] Shen | A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles[END_REF], but none has compared the efficiency of polyclonal vs monoclonal Ab with the same structure. The use of Apt for capture and detection of bacteria has been demonstrated [START_REF] Pembrey | Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?[END_REF][START_REF] Bruno | Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold[END_REF][START_REF] Queirós | A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins[END_REF][START_REF] Wu | An Aptamer-Based Biosensor for Colorimetric Detection of Escherichia coli O157:H7[END_REF]. In this study, Apt from other studies were used for the capture of E. coli GFP, but the results showed only modest efficiency due to high background.

Monitoring bacterial sensitivity to antibiotics requires that the bacteria be growing. The present study indicates that Ab used to capture bacteria might contribute to the high level of bacterial concentrations required for studying reactions to antibiotics. It might be possible to reduce the impact of Ab on bacterial growth by choosing monoclonal Ab that capture the bacteria by targeting epitopes that do not inhibit bacterial growth. Our study

shows that E. coli K12 BW25113 didn't degrade the Ab used for capturea feature potentially attractive for biosensors designed for investigating resistance of these and similar bacteria to antibiotics. Other types of bacteria, particularly Streptococcus, may be capable of specifically degrading Ab [START_REF] Pawel-Rammingen | IdeS and SpeB: Immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes[END_REF], or produce extracellular proteases that will degrade Ab. In the absence of reducing agents, however, Ab are relatively resistant to other proteases [START_REF] Underdown | Studies on the structural and conformational basis for the relative resistance of serum and secretory immunoglobulin A to proteolysis[END_REF].

Conclusions

As a proxy for a biosensor surface, we have employed a 96 well ELISA plate surface in order to study different structures for binding and growth of bacteria. In comparison with reference passively adsorbed goat Ab, we have investigated passively adsorbed chicken Ab, covalently linked goat Ab to a plate with a carboxyl group and biotinylated goat Ab or biotinylated aptamers with passively adsorbed NA. With the GFP E.

coli K12 BW25113, we found that passively bound goat or chicken Ab was a better approach for tethering bacteria and allowing growth than a covalent binding strategy. Our experiments showed that the minimum concentration of live E. coli required for detection within 3 hours was at 10 5.2 CFU/mL. Only 10 3.4 CFU/mL of bacteria was captured on an ELISA plate with passively captured goat Ab from the initial concentration of bacteria at 10 7.2 CFU/mL, which represented a ~ 10 -4 fraction of the initial concentration.

Furthermore, no capture of bacteria was observed for the concentration reduced to 10 6.2 CFU/mL. Adding a centrifugation step considerably increased the capture of bacteria, but it also increased the background contribution. The use of NA resulted in increased nonspecific interactions, but better absolute capture performance and could be investigated further. Inhibition of bacterial growth with Ab in the growth medium could translate into inhibition of growth when capturing bacteria on a surface. In presence of Ab at 10 µg/mL, we observed a drastic decrease of bacterial growth in TG medium at 1.5 x 10 5 CFU/mL and at lower bacterial concentrations. The growth of bacteria was only minimally affected if exposed to Ab at less than 10 µg/mL. It might be possible to overcome the Ab growth inhibition of bacteria, if it were possible to find an Ab analogous to those that are known to neutralize viruses versus Ab that react with viruses but do not neutralize them [START_REF] Tan | Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection[END_REF]. Of particular importance to the development of future Ab-based immunosensors of bacterial reactions to antibiotics is that we have not observed degradation of anti-E.

coli K12 BW25113 Ab, even after 6 h of incubation.

  Tests were carried out with clear Corning 96 well plates 3590 (ELISA plate) from Fischer Scientific (Whitby, ON, CAN), and clear carboxylated 96 well plates (COOH plate) from BioWorld (Dublin, OH, USA). Common laboratory chemicals including NaH 2 PO 4 , Na 2 HPO 4 , NaCl, NaHCO 3 , Na 2 CO 3 , polyoxyethylene sorbitan monolaurate (Tween-20), and MgCl 2 were from Fisher Scientific (Fair Lawn, NJ, USA). Bovine serum albumin (BSA) was from Roche Diagnostics (Indianapolis, IN, USA), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), 1-ethyl-3-(3-diméthylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were from Sigma-Aldrich (St. Louis, MO, USA) or alternatively, EDC and NHS were from Biacore (GE Healthcare bio-science AB, Uppsala, Sweden). The purified IgG fraction of goat antibodies (Ab) directed against E.

Fig. 1 :

 1 Fig. 1: Time dependent fluorescence intensity from different bacterial dilutions grown in TG in microtitre plate wells (a). Tangents of the ascending portion of the curves between 25% and 75% of maximal fluorescence were drawn to allow estimation of Tt from the

  = Reference method by using passively adsorbed goat Ab Passive Chicken Ab = Method using passively adsorbed chicken Ab Covalent Biacore = Method using COOH-plate and EDC/NHS from Biacore with goat Ab Covalent Sigma = Method using COOH-plate and EDC/NHS from Sigma with goat Ab NA-Antibody PI = Pre-incubation Method using passively adsorbed NA and biotinylated goat Ab NA-Antibody AB = Antibodies before bacteria Method using passively adsorbed NA and biotinylated goat Ab NA-Aptamer PI = Pre-incubation Method using passively adsorbed NA and biotinylated Apt NA-Aptamer AB = Antibodies before bacteria Method using passively adsorbed NA and biotinylated Apt

Fig. 2 :

 2 Fig. 2: Comparison of time dependent fluorescence intensity from E. coli GFP bacterial dilutions between 1.5x10 and 1.5x10 6 CFU/mL grown in TG in microtitre plate wells without anti-E. coli Ab, or in the presence of anti-E. coli Ab at 5 µg/mL (a) and 50 µg/mL (b), and as a function of anti-E. coli Ab concentration after 6, 8.5 and 9.5 h of incubation (c).

Table 2 :

 2 Capture of bacteria by different amounts of passively adsorbed or covalently-

	bound antibodies.
	Ab conc.	Passively adsorbed Ab	Covalently bound Ab
	(µg/mL)	Log C (CFU/mL)	Log C (CFU/mL)
	200	3.9 ± 0.3	3.7 ± 1.1
	100	3.8 ± 0.3	3.5 ± 1.2
	50	3.7 ± 0.3	3.2 ± 1.3

* Different concentrations of Ab were passively adsorbed or covalently bound to surfaces.

Capture of E. coli was with PBST for the Passively Adsorbed Ab method and with PBS for the Covalently-Bound AB method. Sigma EDC/NHS was used for the Covalently-Bound AB method. * Different concentrations of Ab were passively adsorbed or covalently bound to surfaces.

Table 3 :

 3 Use of centrifugation to enhance capture of E. coli GFP on ELISA plates without Ab and with passively absorbed goat Ab.

	Centrifugation*	Log Bact.	Captured bacteria	Bacteria
		Conc.	With Ab	Without Ab	Net Capture
		(CFU/mL)	Log C (CFU/mL) Log C (CFU/mL)	Log (C Ab /C nAb )
		8.2	4.3 ± 0.8	0.1 ± 0.1	4.2
	No	7.2	3.4	0	3.4
		6.2	0	0	0
		8.2	7.0 ± 0.5	4.1 ± 0.7	2.9
		7.2	6.0 ± 0.1
	250 G
		6.2	4.1 ± 0.3
	5 min
		5.2	2.2 ± 0.4	1.5	0.7
		4.2	0
	550 G	8.2	7.1 ± 1.0	4.1 ± 0.1	3.0

Acknowledgement This research was supported by the Canada Research Chair in Quantum Semiconductors Program (Grant No 950-220304), the Fonds de Recherche Nature et Technologie du Québec (FRQNT) team research project No 2015 -PR-184056, and the Natural Sciences and Engineering Research Council of Canada Strategic Project (Grant No 494057).

Graphical Abstract

Highlights

 Capture and growth inhibition of E. coli investigated on GaAs substrates  Neutravidin (NA) binds E. coli nonspecifically  Antibody binding to the surface of E. coli inhibits growth  Minimum bacterial concentration determined by the antibody inhibition effect  GaAs dark corrosion negligible in comparison to the antibody inhibition effect  No degradation of anti-E. coli antibodies observed up to 6 h of incubation