C Español-Espinel

S P G Madabhushi

S K Haigh

C N Abadie

D M Xu

J E Go

P R J Morrison

Seismic Response of Large Diameter Monopiles for Offshore Wind Turbines in Liquefiable Soils

Offshore wind energy is rapidly expanding to earthquake prone regions, with design to seismic loading becoming a primary concern. This is especially relevant to monopiles, which are the preferred option in shallow to medium water depths at present. This paper presents centrifuge tests of a monopile foundation and turbine tower subjected to low and high frequency earthquakes. The experiment reproduces calm air conditions, meaning that no lateral wind load was considered. Low frequency earthquakes were found to cause excitation of the topmost part of the superstructure, whereas high frequency earthquakes generated strong resonance effects at the base. Further data analysis suggests that high frequency earthquake shaking can trigger liquefaction of the soil surrounding the monopile, which may in turn lead to differential settlement of the turbine. This is translated into significant rotation of the structure that can potentially exceed the allowable SLS rotation recommended by the technical guidelines.

Introduction

Offshore wind energy has crossed the borders of Europe to other regions of the world such as Southeast Asia and the West coast of the U.S. These areas are expected to accommodate the highest growth in the offshore wind sector over the coming years, though they involve a significant risk due to high seismicity. Hence, earthquake loading is increasingly becoming a primary concern for the design and construction of Offshore Wind Turbines (OWTs). This is especially relevant to foundations, which are meant to support the tower and rotornacelle assembly during earthquakes. The monopile is the preferred foundation type for OWTs representing 81.9% of the substructures in Europe [START_REF] Walsh | Offshore Wind in Europe -Key Trends and statistics[END_REF]. This trend will probably prevail in future developments located in earthquake prone areas with appropriate water depth and ground conditions, given the expertise gained in monopile installation across Europe.

Earthquakes generate specific effects on OWTs based on their intrinsic characteristics. This research distinguishes between two types of OWT structural responses depending on the frequency of shaking. Natural frequencies (fn) of current OWTs range between 0.17Hz and 0.35Hz approximately [START_REF] Arany | Closed form solution of Eigen frequency of monopile supported offshore wind turbines in deeper waters incorporating stiffness of substructure and SSI[END_REF]. Earthquake frequencies that fall within this range, are likely to excite the OWT superstructure significantly. These are called low frequency earthquakes.

Earthquake shaking may as well lead to the development of excess pore pressure (Uexcess) in saturated sand. The effective stress of sandy soils is progressively reduced as Uexcess develops. Sufficient accumulation of Uexcess may cause the complete loss of effective stress, meaning that the soil has fully liquefied. However, this phenomenon is only triggered by high frequency earthquakes, which contain frequencies ranging between 1Hz and 5Hz [START_REF] Madabhushi | How Well Do We Understand Earthquake Induced Liquefaction?[END_REF]. These are relatively far from typical OWT natural frequencies (fn). Hence, high frequency earthquakes are not expected to cause strong dynamic effects on the OWT superstructure. Liquefaction though, would still represent a major hazard arising from these earthquakes.

In traditional long-slender piles, the complete loss of effective stress due to full liquefaction translates into a substantial reduction of shaft friction and end bearing. Thus, the piled foundation may no longer be capable of supporting its self-weight and that of the superstructure. As a result, the entire structure keeps settling into the soil until enough shaft friction and end bearing are recovered to withstand the selfweight again [START_REF] Madabhushi | Design of pile foundations in liquefiable soils[END_REF]. A similar behaviour could potentially be attributed to monopiles. However, their reduced aspect ratios and larg-er diameters could lead to a different failure mechanism. This being the case, the subsequent settlement could widely differ from that of long-slender piles subjected to the same vertical load. In addition, the serviceability of OWTs could be severely compromised by differential settlements and the resulting rotation. Hence, current design standards restrict the allowable tilt angle to 0.5° [START_REF]Support structures for wind turbines[END_REF]DNV-RP-0585, 2021). This paper investigates the effects of both, low and high frequency earthquakes on OWTs supported by monopiles in liquefiable soils. Earthquake interaction with the soil and the structure has been reproduced using centrifuge modelling. The study sought to represent calm air conditions, and therefore, the effects of lateral load are not addressed. Nevertheless, future work is in progress to study the combined influence of lateral wind load and seismic loading on monopiles. The research by [START_REF] Esfeh | Earthquake response of monopiles and caissons for Offshore Wind Turbines founded in liquefiable soil[END_REF], is one of the few currently published studies that assesses the impact of liquefaction on offshore wind monopiles under no wind load. Esfeh and Kaynia (2020) modelled a 25 m long and 6 m diameter monopile utilising a 3D Finite Difference code. Their findings suggest limited vertical displacement and rotation such that the operational limit states of the OWT would not be compromised. It is noted, however, that the monopile was modelled wished in place and therefore, soil stresses generated during installation were not considered. The dynamic response was also excluded from the analysis since the superstructure was modelled as a lumped mass on top of the monopile. Furthermore, numerical results are in general, subjected to the behaviour described by the constitutive model. Although these constitute a very reasonable approximation to reality, validation against centrifuge data is required. [START_REF] Seong | Dynamic and monotonic response of monopile foundations for offshore wind turbines using centrifuge testing[END_REF] conducted one of the first published studies on centrifuge testing of monopile supported OWTs subjected to seismic loading. The experiments determined the first excitation mode or natural frequency (fn) of the system. Following this, the turbines were subjected to the first, second and third mode frequency earthquakes. The dynamic response of the superstructure was measured combining dry and saturated soil profiles. Strong dynamic responses were detected, especially for the first mode earthquake. The latter also excited the second and third modes of the OWT, although only to a minor extent. It is noted that the study involved small diameter piles of aspect ratio relevant to the offshore wind industry, but of restricted overall dimensions. Additionally, fn of the structure was 0.45Hz, far from typical OWT natural frequency values.

Test Design and Methodology

Realistic soil stresses and structural response have been achieved using centrifuge modelling at 70g. The soil sample included a 7m loose sand layer with relative density (Dr) ≈ 40% overlying a 14m dense sand layer with Dr ≈ 90%. The sand was pluviated inside the Equivalent Shear Beam (ESB) model container using the automatic sand pourer at the University of Cambridge [START_REF] Madabhushi | A new automatic sand pourer for model preparation at the University of Cambridge[END_REF]. The ESB container is designed to minimize boundary effects and to simulate the progressive increase in soil stiffness with depth [START_REF] Brennan | Comparing laminar and equivalent shear beam (ESB) containers for dynamic centrifuge modelling[END_REF]. Both sand layers were subsequently saturated with a viscous methylcellulose solution by means of the Cam-Sat system [START_REF] Stringer | Novel computercontrolled saturation of dynamic centrifuge models using high viscosity fluids[END_REF]. In this paper all dimensions are expressed in prototype scale. The centrifuge scaling laws are described in [START_REF] Madabhushi | Centrifuge modelling for civil engineers[END_REF]. The monopile diameter was 7.11m, whereas the embedded length was 17.5m, resulting in an aspect ratio of 2.5. Driving of the monopile into the soil sample was conducted at 1g using a hydraulic press. Following this, the superstructure was assembled on top of the monopile (Figure 1). The OWT tower model was designed so that (fn) at prototype scale was 0.2Hz. This is within the range of realistic OWT towers [START_REF] Arany | Closed form solution of Eigen frequency of monopile supported offshore wind turbines in deeper waters incorporating stiffness of substructure and SSI[END_REF]. The monopile foundation and superstructure were tested against one low frequency (EQ1) and two high frequency earthquakes, EQ2 and EQ3. Acceler-ation and frequency of EQ2 and EQ3 were specifically selected to generate substantial amounts of Uexcess. These were 10 cycles sinusoidal waves with a prototype frequency of 1Hz and a Peak Ground Acceleration (PGA) of 0.11g and 0.21g for EQ2 and EQ3 respectively (Table 1). The earthquake motions are transferred to the model utilising a hydraulic shaker [START_REF] Madabhushi | Development of a servo-hydraulic earthquake actuator for the Cambridge Turner beam centrifuge[END_REF] which is mounted on the beam centrifuge. A broad range of sensors were included in the model to record data on the OWT response and soil behaviour during the test. Uexcess accumulation with depth was recorded by a vertical array of four PPTs (Pore Pressure Transducers) located at one monopile diameter distance from the right side of the foundation. This setup allowed for comparison between Uexcess generated within the loose and dense sand layers. The settlement experienced by the OWT was recorded using LVDTs (Linear Variable Differential Transformers) mounted on top of the circular base of the OWT tower. Two LVDTs, one installed on each side of the tower base provided a measure of the rotation of the monopile and tower (Figure 1). Measurement of the rotation was conducted in the direc-tion of shaking. In addition, acceleration of the earthquake as it propagated across the superstructure was determined using MEMS (Micro Electro-Mechanical Systems), installed at the top and base of the OWT tower. Finally, two piezo-accelerometers were attached to the base of the model box to record the input acceleration.

Results

Uexcess generation within the soil

The loss of effective stress (𝜎 𝑣0 ′ ) due to generation of seismically induced Uexcess in the loose and dense sand layers has been assessed via the excess pore pressure ratio ru, defined as:

𝑟 𝑢 = 𝑈 𝑒𝑥𝑐𝑒𝑠𝑠 𝜎 𝑣0 ′ (1) 𝜎 𝑣i ′ = (1 -𝑟 𝑢 ) 𝜎 𝑣0 ′ (2)
Each cycle of the earthquake contributes to additional accumulation of Uexcess and the consequent reduction in 𝜎 𝑣0 ′ . Thus, ru ranges from zero, indicating the absence of Uexcess to one, where full liquefaction is reached. However, not all earthquakes are sufficiently strong to cause a complete loss of 𝜎 𝑣0 ′ within the soil, i.e. full liquefaction. As an example, EQ2 with a PGA of 0.11g produced a 46% reduction in 𝜎 𝑣0 ′ at 4.62m depth and a 33% loss at 6.44m depth, both points located within the loose sand layer (Figure 2). Uexcess generation also occurred within the dense sand layer, although to a minor extent, with ru values of 0.21 and 0.13 at 8.54m and 11.34m depth respectively. EQ3, with a PGA of 0.21g, was significantly stronger than EQ2 and generated a larger amount of Uexcess, especially within the loose sand layer. Liquefaction is evidenced at 4.62m depth with ru values up to one and the presence of suction cycles along the ru time history. Signs of dilation are also seen at 6.44m, where ru registered maximum values of around 0.87. In addition, the residual ru value at 4.62m depth reaches 0.8, whereas at 6.44m depth ru becomes 0.6. Thus, only 20% and 40% of the initial 𝜎 𝑣0

′ remains available at these depths towards the end of the earthquake. This trend also matches with that identified within the dense sand, where ru increased to almost 0.6 at 8.54m depth and to nearly 0.4 at 11.34m depth. The impact of the substantial loss of 𝜎 𝑣0 ′ within the loose and dense sand layers on the overall response of the OWT is described in Section 3.2.

Settlement and rotation of the OWT

The substantial loss of 𝜎 𝑣0 ′ due to the accumulation of Uexcess during earthquake shaking often translates into settlement of the OWT. Generally, the structure does not experience uniform settlement on both of its sides due to inherent eccentricities. This may lead to significant rotation of the monopile and tower. Settlements generated during EQ2 were minor and very similar on the left and right sides (Figure 4). Hence, rotation was very limited (<0.2°). In contrast, EQ3 caused strong rocking of the OWT tower, which oscillated ±0.5° during the first six cycles of the earthquake. The residual settlement was 167mm on the left side and 42mm on the right side, leading to a residual rotation of almost 1° in the anticlockwise direction (Figure 5). The results of this study suggest that the OWT that was tested in this research, rotated by more than 0.5°, exceeding the allowable limit recommended in the DNV-GL ( 2021) and the DNV-RP-0585 (2021) standards.

Dynamic response of the OWT tower

This section compares the dynamic response of OWT towers against low frequency and high frequency earthquakes. An earthquake with PGA of 0.02 and a frequency of 0.2Hz has been chosen as the low frequency motion (EQ1). Moreover, a sinusoidal wave with PGA of 0.21g and 1Hz has been selected to evaluate the high frequency response (EQ3). Accelerations recorded at the top and base of the tower have been used to assess the propagation of the earthquake across the superstructure. Both, top and base signals, together with the input acceler-ation, have been decomposed into their constituent frequencies using a Fast Fourier Transform [START_REF] Madabhushi | Centrifuge modelling for civil engineers[END_REF].

As observed in Figure 6, the low frequency input acceleration (EQ1) is magnified as it propagates across the structure. The acceleration recorded at the top is about two times larger than the input acceleration, whereas at the base this has barely amplified. The first excitation mode is detected at 0.2 Hz, where maximum excitation occurs at the top of the superstructure. Other secondary frequencies contained in the input signal (0.52Hz, 0.87Hz and 1.23Hz) lead to attenuation at the top of the structure and to slight amplification at the base.

Figure 7 illustrates the dynamic response of the OWT tower when subjected to a high frequency earthquake (EQ3). In this case, the main frequency of the earthquake, 1Hz, is far from the first mode of vibration of the OWT. Hence, the top of the structure does not experience amplification, but attenua-tion at this frequency. In contrast, the base of the tower is significantly more excited. Additionally, minor excitation of the top and base of the tower is evidenced at 2.9Hz, and at 0.2Hz at the top only. Overall, amplification is significantly larger at the base of the OWT tower rather than at the top. This is evidenced by the acceleration recorded at the base of the tower, which is approximately three times larger than the input acceleration. This paper addresses the response of OWTs supported by large diameter monopiles subjected to seismic loading. A full size OWT, including monopile foundation and superstructure, was replicated using centrifuge modelling at 70 g. The soil profile comprised a loose sand layer overlying a dense sand layer, both of which were saturated. The structure was assessed under calm air conditions, meaning that no lateral load was applied. The experiment covered the response of the monopile and superstructure under two well distinguished seismic loading scenarios. Firstly, earthquake shaking with frequencies similar to that of the OWT tower. These generally produce small amounts of Uexcess, meaning that 𝜎 𝑣0 ′ of the soil remains unaltered. Hence, shear waves can easily propagate through the soil, reach the monopile, and fully interact with the superstructure. EQ1, with a frequency of 0.2Hz, close to the first mode of the OWT, was specifically chosen to reproduce this scenario.

Secondly, high frequency earthquakes including two 1Hz frequency motions with 0.11g and 0.21g PGA were investigated. Both earthquakes were sinusoidal waves intended to generate enough Uexcess to liquefy the soil surrounding the foundation. This may distort shear wave propagation, given that shear waves cannot propagate across a liquid medium. But more importantly, the main frequency of the earthquakes selected, is considerably higher than typical OWT natural frequencies ranges. Hence, the superstructure should in theory be, less responsive to high frequency than to low frequency earthquakes.

Data gathered during EQ1 revealed that the acceleration at the top of the OWT was twice the input acceleration, meaning large amplification of the input motion at the top of the superstructure. The main excitation mode was detected at 0.2Hz, the natural frequency of the OWT tower.

EQ2 produced enough Uexcess within the soil to reduce 𝜎 𝑣0

′ by up to a half in the loose sand and by 20% or less in the dense sand only (Figure 2). Although the loss of 𝜎 𝑣0 ′ in the shallow loose sand layer was remarkable, the resulting settlement was minimal (Figure 4), and so was the rotation.

In EQ3, full liquefaction was nearly achieved at 6.44m and fully reached at 4.62m depth during shaking. Based on this, it is possible that 𝜎 𝑣0 ′ was completely lost within the entirety of the loose sand layer. In addition, the accumulation of Uexcess was significant within the dense sand layer as well. In fact, ru reached values of 0.57 and 0.37 at 8.54m and 11.34m depth respectively. Therefore, only 40% to 60% of the initial 𝜎 𝑣0 ′ was available towards the end of the earthquake (Figure 3). The substantial decay of 𝜎 𝑣0 ′ in the loose and dense layers translated into a reduction of the bearing capacity of the monopile. As a result, the OWT and foundation settled into the sand until enough shaft friction and end bearing were restored. The settlement towards the end of the earthquake, although not excessive, was around four times larger on the left side than on the right side of the OWT. Such a large variation in settlement, which may be explained by bias created by the first loading cycle of previous earthquakes, turned into a residual rotation of nearly 1°. This value exceeds the allowable threshold recommended by the technical guidelines (DNV-GL, 2021 and DNV-RP-0585, 2021) for the monopile investigated in this paper for shaking amplitudes of up to 0.2g. This research, therefore, suggests that OWTs could experience higher rotations than 0.5° where soil liquefaction is expected even though no lateral load was applied.

In addition to this, resonance effects were identified at the structure despite the earthquake frequency being far from the first excitation mode of the OWT tower. Substantial amplification was detected at the base of the superstructure, where the input acceleration was three times larger. The top and base of the superstructure experienced minor amplification at 2.9Hz. In addition, the topmost part of the tower was also excited at around 0.2Hz, although to a minor extent.

Full liquefaction occurred in the shallow loose sand layer only, and did not have an influence in shear wave propagation to the OWT. Share stress was still fully transferred from the dense sand to the monopile, which in turn propagated to the superstructure.

Based on the above, high frequency earthquakes may not only be capable of generating liquefaction, but also resonance effects at the base of the superstructure. Moreover, low frequency earthquakes with predominant frequencies similar to that of the first mode of the OWT or fn, generate significant excitation of the top of the superstructure.

5 Conclusions EQ2, although relatively small, could generate enough 𝑈 𝑒𝑥𝑐𝑒𝑠𝑠 to reduce 𝜎 𝑣0 ′ within the loose sand layer up to half of the initial value. However, the dense sand layer was barely affected. Settlement and rotation experienced by the OWT were negligible in this scenario. In contrast, the stronger high frequency earthquake (EQ3) triggered full liquefaction of the loose sand layer. Moreover, a significant amount of 𝑈 𝑒𝑥𝑐𝑒𝑠𝑠 accumulated within the dense sand layer, whose 𝜎 𝑣0 ′ was reduced between 40% and 60%. As a consequence, the monopile experienced substantial differential settlement that translated into rotation of nearly 1°. Thus, earthquake induced rotation in OWTs could exceed 0.5° (DNV-GL, 2021 and DNV-RP-0585, 2021) even if no lateral load due to wind and waves is considered.

The dynamic response of the OWT tower against low and high frequency earthquakes was also addressed in this paper. EQ1 with a predominant frequency similar to fn of the OWT, led to excitation of the topmost part of the superstructure. In contrast with this, input motion EQ3 with a main frequency of 1Hz, produced larger amplification of the input acceleration at the base of the OWT rather than at the top.

The results presented in this paper show that dynamic centrifuge modelling can be successfully employed to investigate OWT foundations subjected to seismic loading when the seabed is vulnerable to liquefaction.

Figure 1 .

 1 Figure 1. Cross section of the experimental setup (dimensions shown at prototype scale at 70g)

Figure 2 :

 2 Figure 2: ru time history recorded during EQ2

Figure 4 .

 4 Figure 4. Settlement and rotation experienced by the prototype OWT during EQ2

Figure 6 .Figure 7 .

 67 Figure 6. Dynamic response of the OWT tower relative to a input acceleration with frequency equal to fn of the OWT (EQ1)

Table 1 .

 1 Earthquake motions selected for the experiment

	Earthquake	EQ1	EQ2	EQ3
	No.
	Type	Low fre-	High fre-	High fre-
		quency	quency	quency
	Peak Ground	0.02	0.11	0.21
	Acceleration
	(PGA) [g]
	Frequency	0.2 (fn)	1.0	1.0
	[Hz]
	No. of cycles	10	10	10

Acknowledgements

The authors are grateful to EPSRC iCASE (20000012) and ARUP for funding and supporting this work. We are also thankful to the technicians of the Schofield Centre for their support during preparation and running of this centrifuge experiment, as well as the continuous help of Diarmid Xu.


Stuart Haigh

Chisom Ifeobu

Christelle Abadie

Cyclic response of

Keywords: onshore wind, shallow foundation, cyclic loading, centrifuge modelling, lifetime performance

INTRODUCTION

The world requires about 7 TW of wind energy to achieve net zero emissions by 2050 (IEA, 2022). A significant proportion of this capacity will be provided by onshore wind, with Sub-Saharan Africa playing a major role in the upcoming developments. Shallow raft foundations are the most popular solution to support onshore wind turbines, as they can resist very large overturning moment loads and are relatively straightforward to construct. They typically consist of a 15 -20 m diameter circular reinforced concrete slab, embedded 2 -3 m into the ground. These massive structures require colossal quantities of concrete (900 to 1500 m 3 ) and steel reinforcement (25 tonnes), leading to elevated construction costs and carbon emissions that can be prohibitive for future sustainable development of onshore wind, in particular for developing countries. As a result, cost-effective methods for foundation design that are less material-intensive are needed. This requires rigorous understanding of the lifetime performance of this type of shallow foundation to enable future optimisation.

The design of wind turbine foundations is governed by bearing capacity and tolerance on global rotation. In sand, the constraint on rotation can impose strict restrictions on geometry because of the accumulation of permanent tilt over the lifetime of the turbine. The IEC 61400-6 provides general guidelines for the design of shallow foundations for onshore wind turbines. For ultimate limit state design (ULS), foundation failure is when the maximum load carrying resistance is reached for bearing failure, sliding and overturning as well as considerable settlements or displacements occur under monotonic loading.

Serviceability limit state (SLS) specifies strict allowable settlements and rotations of 25 mm and 0.17 degrees respectively, but little consideration of the response to cyclic wind loading and how to address it is actually provided. It is acknowledged that the accumulation of rotation and settlement caused by cyclic loading depends on the number of cycles and cyclic load magnitude [START_REF] Gajan | Centrifuge modeling of load-deformation behavior of rocking shallow foundations[END_REF] but this is typically not considered in the current design guidelines.

Current research on the cyclic lateral response of shallow foundations is mostly focused on rocking behaviour under lateral cyclic loading of very few cycle number, relevant to earthquake design. [START_REF] Gajan | Centrifuge modeling of load-deformation behavior of rocking shallow foundations[END_REF] performed centrifuge tests involving slow and dynamic cyclic loads on a shallow foundation and demonstrated that permanent settlement of a foundation resting on the soil surface increases with the number of displacement cycles applied, while the rate of settlement accumulation decreases as the footing embeds itself. [START_REF] Gajan | Capacity, settlement, and energy dissipation of shallow footings subjected to rocking[END_REF] found that rotational stiffness depended on the displacement applied with a stiffer response at lower levels of rotation and stiffness degradation at higher rotation levels. However, this work only involves three loading packets with three cycles each and the experiment was displacement controlled. This provides limited data for extension to long-term cyclic loading of very many cycles, typically needed for wind energy applications. In addition, the work does not provide sufficient insight on the failure mechanism.

More recently, [START_REF] Moradi | Investigation of rocking mechanism of shallow foundations on sand via PIV technique[END_REF] studied the rocking mechanism of shallow foundations under displacement controlled two-way cyclic loads at 1g, and using Particle Image Velocimetry to evidence the failure mechanism. They evidenced the deformation mechanisms in the loading portion of the loading cycle. This consists of soil densification at a depth, involving a wedge mechanism, which causes soil to be pushed away from the slab. This dominates under low rotation conditions and is superseded by a scoop mechanism, which occurs when there are substantial foundation rotations and a considerable gap between the footing and the soil. However, the tests were performed at 1g and do not involve very many cycles (6 cycles).

As a result, there is limited understanding of the foundation response to long-term cyclic moment and lateral loading. To date, this uncertainty in the design has been addressed by using large safety factors, leading to over-engineered foundations. To remedy this problem, an in-depth understanding of the response to long-term wind loading is required.

The present paper investigates the response of a raft foundation in dense sand for a 2 MW onshore wind turbine to cyclic lateral loads at height using centrifuge modelling. The aim is to quantify the accumulation of settlement and rotation of a conventional wind turbine foundation simply resting on dry sand, and subjected to a series of long-term lateral cyclic loads of increasing magnitude. This study provides a worst-case scenario to which current and future optimised designs can be benchmarked against.

EXPERIMENTAL METHODOLOGY

Test set-up

The centrifuge test presented in this paper was performed at an enhanced gravity of 100g on the Turner beam centrifuge at the Schofield Centre of the University of Cambridge. The cross-section of the model, with all relevant dimensions, is shown in Figure 1(a), with a photo of the model loaded onto the centrifuge beam in Figure 1(b). Centrifuge modelling enables reduced scale model tests to be performed while preserving stress-strain behaviour of the soil. This is achieved by scaling up the gravity field by a factor of N, with N = 100 in this paper. A set of scaling factors relates the model to the prototype response, according to the framework from [START_REF] Schofield | Cambridge Geotechnical Centrifuge Operations[END_REF].

2D model strip footing

The test models a strip foundation resting on dry dense sand, with dimensions representative of a 2 MW turbine foundation, scaled to plane-strain conditions. A 2 MW turbine is typically supported by a reinforced truncated-conical concrete slab of 15.4 m in diameter and 3 m height. The 2D strip footing selected here, and shown in Figure 2, therefore has a width B = 9 m, length L = 17.8 m and a height h = 2 m at prototype scale. Because the length of the footing is fixed by the plane strain container, the width was chosen so that the prototype would have a similar area of contact as a representative foundation. The height was chosen so that the weight of the overall structure was not excessive compared with real turbines.

The movement of the foundation with cycle number was measured in terms of rotation and settlement, as illustrated in Figure 3.

Sand sample

The model was prepared in a 790 mm x 180 mm x 450 mm container in plane-strain conditions to enable the use of Particle Image Velocimetry (PIV) and further understand the failure mechanism caused by cyclic loads. The sample was prepared using Hostun sand (Table 1), poured at a relative density of DR=76%. Sand layers were prepared with air pluviation using an automatic sand pourer [START_REF] Zhao | Calibration and use of a new automatic sand pourer[END_REF].

Instrumentation

The soil sample was instrumented using accelerometers to verify the uniformity of the soil stiffness profile, and a cone penetrometer was used to verify the relative density of the sample. A load cell with 250 N capacity (2.5 MN at prototype scale) was used to verify and control the load. Three linear variable displacement transducers (LVDTs) were used to measure the rotation and settlement of the foundation (Figure 1(c). The structure was also instrumented with accelerometers to obtain the rotation. Figure 2 shows a schematic of the key variables used in the following to quantify the foundation movement.

A Canon Powershot G10 Camera with a 14.7 megapixel unit was used to capture soil particles displacements and infer the failure mechanism within the soil through Particle Image Velocimetry (PIV). For this purpose, one third of the sand was died using black ink to increase contrast amongst sand particles. The images were acquired at a frequency of 0.25 Hz. Sand particles displacements were calculated from the recorded images using GeoPIV-RG (Stanier et al., 2015).

Loading

The footing was subjected to cyclic lateral loads, applied at the top of an aluminum tower at an eccentricity of 63.5 m above the soil surface at prototype scale. The load was applied using a 2D actuator, which enabled to accommodate for vertical settlement during swing-up while applying a cyclic load at the desired eccentricity. A feedback loop, controlled via a load-cell, was implemented to permit repeated cyclic load control of the actuator in the lateral direction. A photo of the model and actuator is provided in Figure 1(b). The cyclic loading sequence was characterized in terms of load magnitude and amplitude, via the parameters 𝜁 𝑏 and 𝜁 𝑐 , originally defined by [START_REF] Leblanc | Response of stiff piles to random two-way lateral loading[END_REF].

𝜁 𝑏 = 𝑀 𝑚𝑎𝑥 𝑀 𝑢𝑙𝑡 (1) 𝜁 𝑐 = 𝑀 𝑚𝑖𝑛 𝑀 𝑚𝑎𝑥 (2)
where 𝑀 𝑚𝑎𝑥 and 𝑀 𝑚𝑖𝑛 are the maximum and minimum moment within a load packet respectively, and 𝑀 𝑢𝑙𝑡 is the ultimate moment capacity of the footing. The ultimate moment capacity was calculated analytically and represents the limit at which a slab of the same geometry and resting on a hard surface would topple over. This permitted to obtain an immutable value, independent of the soil properties, that could be used to predict the load sequence prior to testing. This simple definition gives the following expression for the ultimate capacity, calculated from a cantilever:

𝑀 𝑢𝑙𝑡 = 𝑊 𝑠𝑡𝑟𝑢𝑐 𝐵 2 (3)
where 𝑊 𝑠𝑡𝑟𝑢𝑐 is the total weight of the model, including the super-structure and the slab. This was calculated to be 66.4 MNm at prototype scale.

Typical design loads for a 2 MW onshore wind turbine foundation is shown in Table 2. The wind loads are based on specifications for the Gamesa G80 2 MW turbine with a hub height of 67 m (The WindPower, 2023). SLS loads were calculated using the rated wind speed of 15 m/s while the ULS loads were calcualted based on the cut out wind speed of 25 m/s. These informed the design of the wind turbine model and the cyclic loading sequence applied.

The wind turbine model weighed 14.7 MN at prototype scale and was similar to the typical vertical design load at full scale. Appropriate values of 𝜁 𝑏 were selected so that the experiment would reflect realistic loading conditions encompassing both the SLS and ULS loading cases.

The loading sequence was selected to apply partial one-way load packets (see definition in Table 2) of increasing load magnitude, representative of severe wind loading on a wind turbine (Table 3, see also later Figure 4(a)). The load magnitude of CY4 is representative of loading at ULS, according to design guidelines. The first and last two cycles of each packet were performed at a much slower rate to permit sufficient sampling for Particle Image Velocimetry. This change in rate is here unlikely to affect the results, as inertial effects are negligible for this type of soil conditions.

RESULTS

General Response

Figure 4 shows the moment-rotation curve for the entire loading sequence. The graph illustrates an accumulation of permanent deformation with cycle number, which becomes more prominent as load magnitude increases, due to the increased nonlinearity in the soil response. This ratcheting behaviour is commonly observed when studying the cyclic loading response of foundations in dry sand (e.g. [START_REF] Leblanc | Response of stiff piles to random two-way lateral loading[END_REF][START_REF] References Abadie | Rigid pile response to cyclic lateral loading: laboratory tests[END_REF][START_REF] Richards | Monopile rotation under complex cyclic lateral loading in sand[END_REF][START_REF] Frick | An experimental study on the parameters affecting the cyclic lateral response of monopiles for offshore wind turbines in sand[END_REF]. c) show the evolution of settlement and residual rotation with cycle number with respect to the cyclic moment sequence applied on top of the foundation (Figure 5(a)). The residual rotation is defined as the rotation at minimum load within a cycle after the foundation has effectively been unloaded. The rate of increase in settlement and rotation with cycle number decreases with the number of cycles within a loading packet and across the entire loading sequence.

The results obtained for this test are compared with the maximum allowable settlement (25 mm) and rotation (0.17 deg.) under Serviceability Limit State (SLS) proposed by the IEC 64100 international standard. The Figures show that these requirements are met at low load levels, close to the elastic regime, but once more severe yielding occurs, the rotation criteria is exceeded and the settlement is close to the tolerated limit. This proves that the design of shallow foundations for onshore wind applications requires careful consideration of the number of cycles and load levels that the slab is likely to experience over its lifetime. Further research is also needed to provide cyclic stability diagrams [START_REF] Poulos | Pile behaviour-theory and application[END_REF][START_REF] Jardine | Field axial cyclic loading experiments on piles driven in sand[END_REF], adapted for shallow foundation design.

Figure 6 shows the average footing settlement against its rotation in which positive vertical axis denotes uplift. The results highlight that settlement and rotation are accumulated concurrently, with large settlement and limited rotation accumulated during the cycles of low magnitude, but much larger rotations and more limited settlement generated by the much larger cycles (CY4 onwards). The rate of deformation accumulation is similar from CY1 to CY3, where the cycles are close to the elastic regime (see next section and Figure 5(d)). However, further yielding generated by CY4, generates more severe rotation, accumulated at a faster rate than in the previous load packets.

Stiffness

Figure 5(d) compares the initial (tangential) stiffness to the secant rotational stiffness, obtained by measuring the slope from minimum to maximum points within each moment-rotation loading cycle. When both stiffness are super-imposed, this shows that the response is quasi-elastic, which is the case for CY1 to CY3. Beyond this, the soil exhibits a much more pronounced non-linear response, with much greater unrecoverable deformation (Figure 5(b, c), Figure 6). Performing cyclic loads at much smaller amplitudes following a load packet of large amplitude (e.g. CY4 to CY5) typically brings the response back within the yield surface, towards a quasi-elastic response.

Comparing Figure 5(b) and 5(d), there is a marked difference between the rate of increase in settlement for the near-elastic regime (CY1 to CY3, CY5, CY7) and the yielding regime (CY4 and beyond). The rate of settlement and rotation accumulation is unsurprisingly higher after large nonlinear behaviour has been observed.

Deformation Mechanisms

Particle Image Velocimetry was finally used to evidence the deformation mechanism of the soil under the slab during each loading packet. Figure 7 shows the soil displacement vectors and maximum shear strain at the end of the most critical loading packet, CY4.

Large shear strains were observed around the loaded edge. Both a scoop and wedge mechanisms were observed, which concurs with the observations from [START_REF] Moradi | Investigation of rocking mechanism of shallow foundations on sand via PIV technique[END_REF], obtained at 1g. The soil particles are pushed away from the loaded edge forming the wedge mechanism. Additionally, substantial foundation rotation and gapping beneath the footing produces a scoop mechanism as soil flows to fill the gap formed. Figure 7 also identifies the location of the rotation point, situated at the intersection of the two mechanisms.

CONCLUSIONS

This paper describes the long-term lateral cyclic behaviour of a shallow foundation resting on a dense sandy soil. The results show that large settlement and rotations can be accumulated during the lifetime of a foundation, which could potentially reach SLS Figure 6. Moment-rotation response of multiamplitude cyclic test design limits. Low amplitude cycles produce an increase in settlement caused by the quasi-elastic response of the slab to the moment loads, while larger magnitude cycle produce more significant rotation of the foundation, combined with moderate settlement accumulation.

Cyclic lateral and moment loading of the shallow foundation generate the formation of a wedge mechanism on the passive side of the foundation, while a scoop mechanism forms on the active side, where the slab is most likely to lose contact with the soil. Tracking the evolution of this mechanism can provide the impetus for further understanding of the transition in behaviour of the slab between lowamplitude and large-amplitude loads. This would provide grounds for the development of safety guidelines to ensure that shallow foundations do not exhibit excessive settlement nor rotation during the lifetime of onshore wind turbine, potentially also providing information for lifetime extension of existing structures.

Figure 1

 1 Figure 1. (a) Cross-section of centrifuge model. Dimensions at prototype scale. MEMS: Microelectromechanical systems accelerometer; Acc: Piezoelectric accelerometer, (b) Photo of centrifuge model

Figure 2 .

 2 Figure 2. Model strip footing as seen by the camera

Figure 4 .

 4 Figure 4. Moment-rotation response of multiamplitude cyclic test

  (a) Applied moment history and generated (b) settlement and (c) residual rotation with cycle number (prototype scale). (d) Evolution of the rotational stiffness with cycle number 3.2 Foundation Displacement Figure 5(b) and (

Table 1 :

 1 Hostun sand (HN31) properties (da Silva, 2017).

	Property	Symbol Units Value
	Coefficient of uniformity	Cu	-	1.43
	Average particle size	d50	mm	0.356
	Minimum void ratio	emin	-	0.555
	Maximum void ratio	emax	-	1.010
	Specific gravity	Gs	-	2.65
	Critical angle of friction	Фc	o

Table 2 :

 2 Typical design loads for a 2 MW turbine

		H (kN)	M (MNm)	𝜁 𝑏	V (MN)
	ULS	813	52.9	0.58	11.8
	SLS	293	19.0	0.21	11.8
	Table 3: Definition of Loading Asymmetry
		𝜁 𝑐 = 0		One-way loading
		𝜁 𝑐 = 1		Two-way loading
		0 < 𝜁 𝑐 < 1	Partial one-way loading
		-1 < 𝜁 𝑐 < 0	Partial two-way loading
	Table 4: Definition of Loading Asymmetry
	ID		𝜁 𝑏	𝜁 𝑐	N
	CY1		0.22	0.35	20
	CY2		0.30	0.43	20
	CY3		0.42	0.36	120
	CY4		0.56	0.32	50
	CY5		0.45	0.47	10
	CY6		0.65	0.25	2
	CY7		0.44	0.02	10
	CY8		0.57	0.10	10
	CY9		0.63	0.02	2
	CY10		0.63	-0.03	0.5
	Total Number of Cycles for the test:	244.5

Figure 7. Soil deformation mechanisms at the end of load packet CY4. Displacement field overlain on maximum shear strain contours. Note that shear strains are calculated relative to the beginning of each loading packet, dimensions are in prototype scale and displacement vectors are not to scale

ACKNOWLEDGEMENTS

The author is grateful for the generous support of the Cambridge-Africa Scholarship. We are also thankful for the help of the technicians of the Schofield Centre at the University of Cambridge (John Chandler, Kristian Pether, Mark Smith and Chris McGinnie) and the valuable guidance from Prof. Gopal Madabhushi, Dr Chuhan Deng and Douglas Morley.

, Phung (Edt).


Olivier Attrée
email: oattree@crssa.net

Valérie Guglielmo-Viret

Valérie Gros

Philippe Thullier

Development and comparison of two immunoassay formats for rapid detection of botulinum neurotoxin type A

Keywords: Botulinum neurotoxin, Rapid detection, Antibody-based assays, Immunochromatography, Immunoaffinity column BoNT/A, botulinum neurotoxin type A, ICT, strip immunochromatography, IAC, immunoaffinity column, LD50, 50% lethal dose, poly-HRP, polymerized horseradish peroxydase

We have evaluated two formats of immunoassays for the rapid detection of Clostridium botulinum neurotoxin type A (BoNT/A), in assay buffer and various matrices (human serum and nasal swabs, fresh milk, sugar, flour and talcum). The two formats, a verticalflow strip immunochromatography (ICT) and a small disposable immunoaffinity column (IAC), were selected because they are both rapid and readily usable in the field without sophisticated equipment. We utilised the same critical reagents to develop and optimise both assays, making it possible to compare the corresponding technologies on the same toxin preparations, without interference due to the properties of the antibodies. Results were interpreted using a standard statistical test (ANOVA) and showed little difference of sensitivity between matrices. Though both assays were completed in 40 min, the sensitivity of the IAC, evaluated at 0.45 pM (5 mouse LD50 units/ml), was 40 to 80 times better than that of the ICT. Furthermore, the sensitivity of the IAC assay was improved to 0.09 pM (1 mouse LD50 unit/ml) when performed on a 5-ml volume of human serum. Thus, the IAC appears to be one of the most sensitive and rapid assays for the detection of BoNT/A reported to date and, because it is also highly transportable, it is amongst the best suited for field diagnosis of BoNT/A poisoning.

Introduction

Natural human botulism occurring through accidental food poisoning or inoculation by drug abusers became an infrequent disease, though it is not disappearing [START_REF] Sobel | Foodborne botulism in the United States, 1990-2000[END_REF][START_REF] Lindstrom | Laboratory diagnostics of botulism[END_REF]. The main concern about botulinum neurotoxins (BoNTs) has now been focused on their potential use as bioweapons because they are relatively easy to produce, are highly potent and lethal, and the induced paralysis requires prolonged intensive care [START_REF] Arnon | Botulinum toxin as a biological weapon: medical and public health management[END_REF].

BoNTs are 150,000 Da proteins with zinc-endopeptidase activity, secreted by the bacterium Clostridium botulinum as complexes with non-toxic proteins. The neurotoxicity requires that the BoNT reaches the cytoplasm of presynaptic nerve endings where its enzymatic activity results in the cleavage of proteins involved in vesicle docking, thus inhibiting neurotransmitter release. There are seven defined serotypes (designated A to G) of BoNTs. Type A (BoNT/A) is one of the most frequent causes of natural human botulism, is implicated in the most severe cases, and is the serotype probably presenting the highest threat as a bioweapon [START_REF] Arnon | Botulinum toxin as a biological weapon: medical and public health management[END_REF].

BoNTs must be detected rapidly to enable timely administration of neutralizing antibodies to the exposed persons and to prevent additional exposures. A debated study recently analysed the risk of bioterrorism involving the release of BoNT into milk supplies [START_REF] Wein | Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk[END_REF]. The findings stressed the need for assays that could be used in the field and that allow rapid detection of BoNTs.

The reference method for detecting BoNTs continues to be the mouse lethality assay because it is a highly sensitive functional assay that directly measures the amount of toxic BoNT. Nevertheless, it has well-known limitations. Foremost, this assay usually requires 24 to 48 h to yield results. However, antibody therapy is most efficient when given before the onset of clinical signs of botulism, which can occur less than 12 h after exposure. Also, the necessity to maintain test mice makes it impractical outside reference laboratories and particularly in field conditions.

Various assays for BoNTs have been developed, aiming to equal the sensitivity of the mouse lethality assay while alleviating its limitations [START_REF] Doellgast | Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay[END_REF][START_REF] Ekong | Immunological detection of Clostridium botulinum toxin type A in therapeutic preparations[END_REF][START_REF] Szilagyi | Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes A and B[END_REF][START_REF] Schmidt | High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F[END_REF][START_REF] Chiao | Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B[END_REF][START_REF] Barr | Botulinum neurotoxin detection and differentiation by mass spectrometry[END_REF][START_REF] Gessler | Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test[END_REF][START_REF] Sapsford | Biosensor detection of botulinum toxoid A and staphylococcal enterotoxin B in food[END_REF][START_REF] Sharma | Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis[END_REF][START_REF] Sharma | Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies[END_REF][START_REF] Rivera | Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic electrochemiluminescence detection[END_REF]. Antibody-based assays have been the most studied. These differ in procedure time and sensitivity due to the assay design and the technology used to produce the detection signal. It is possible to compare the performances of various immunoassay formats only if the same antibodies are used for each, because the sensitivity of the assay depends largely on the affinity of the antibodies for the target antigen. We recently used this approach to compare ELISA and electrochemiluminescence immunoassay technologies for the detection of BoNT type B (BoNT/B) [START_REF] Guglielmo-Viret | Comparison of electrochemiluminescence assay and ELISA for the detection of Clostridium botulinum type B neurotoxin[END_REF].

In the present study, we evaluated two formats of immunoassay for the rapid detection of BoNT/A. One was a vertical-flow strip immunochromatography (ICT) with colloidal gold particles, and the other was a small disposable immunoaffinity column (IAC) with a polymerized form of peroxidase as the detector. We selected these formats because they yield rapid results and can be used in the field without sophisticated equipment. Although they have been used previously for BoNT detection [START_REF] Chiao | Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B[END_REF][START_REF] Gessler | Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test[END_REF], the two formats have not been compared directly. Here, we used the same antibodies to develop and optimise both formats and compared their performance on the same toxin preparations, in assay buffer and various matrices. Development and use of point-of-care and field-portable assays are increasing, thus such a comparison should be of wide interest.

Materials and methods

Botulinum toxins

Purified BoNT complexes, derived from type A1 Hall, type B Okra, and type E Alaska strains were purchased from Metabiologics Inc. (Madison, WI, USA). The BoNT complex preparations were indicated by the manufacturer to be pure (by SDS-PAGE) and have specific activities of 2.2 × 10 7 mouse 50% lethal dose (mouse LD50 unit)/mg for BoNT/A, 1 × 10 7 mouse LD50 unit/mg for BoNT/B, and 3 × 10 7 mouse LD50 unit/mg for BoNT/E complexes. An average molecular weight of 500,000 Da was used to calculate the molar concentration of the BoNT/A complex [START_REF] Inoue | Molecular composition of Clostridium botulinum type A progenitor toxins[END_REF][START_REF] Smith | Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization[END_REF]. Aliquots of the preparations (0.5 mg/ml in 50 mM citrate buffer pH 5.5 containing 5% BSA) were stored at -80 °C, and used immediately after thawing. The mouse lethality assay was used as described [START_REF] Solomon | Botulinum toxins[END_REF] to verify the specific activity of one freshly thawed aliquot for each serotype. Briefly, Swiss (RjOrl) mice (Elevage Janvier, Le Genest Saint Isle, France) weighing 18 to 22 g were injected with serial two-fold dilutions of toxin, based on the specific activity values provided by Metabiologics, in gelatin-phosphate buffer (50 mM NaH 2 PO 4 , 0.2% gelatin, pH 6.2). A total of 0.5 ml was injected intraperitoneally into each of two mice for each dilution. Animals were monitored for 72 h post-injection and examined regularly according to institutional guidelines. The specific activities of the thawed aliquots were found to be identical to those determined by the manufacturer.

Antibodies

Two antibodies were used in this study. One was an affinity-purified rabbit polyclonal IgG (rabbit pAb) raised against purified BoNT/A (Metabiologics Inc.). The other one (mAb 11F7) was a monoclonal murine IgG 2a, derived by hybridoma technology from a mouse immunized against the 50 kDa C-terminal fragment of the BoNT/A (subtype A1) heavy chain, that was purified from a recombinant E. coli strain generously provided by Dr. G. Schiavo (ICRF, London, UK) [START_REF] Lalli | Functional characterisation of tetanus and botulinum neurotoxins binding domains[END_REF]. The 11F7 mAb was purified on a Hi-Trap protein A HP Column (GE Healthcare/Amersham Biosciences, Orsay, France), according to the manufacturer's instructions.

Vertical-flow strip immunochromatography test (ICT)

The principle and design of this assay have been described formerly [START_REF] Guglielmo-Viret | An immunochromatographic test for the diagnosis of ricin inhalational poisoning[END_REF]. An initial series of ICTs was made under fixed conditions. The capture antibody (0.5 μg in PBS/test) was sprayed at a rate of 1 μl/cm to form a capture line on polyesterbacked nitrocellulose membrane (MTP, Whatman, Kent, UK) with a dispenser (IsoFlow reagent dispenser, Biodot, Chichester, UK). A control line made of Goldline (British Biocell International, Cardiff, UK), which chemically reacts with gold, was applied 8 mm above the capture line. The detection antibody (0.1 μg/test) conjugated to anti-mouse or anti-rabbit IgG gold beads (40 nm diameter, British Biocell International) (5 μl beads at OD 520 = 10 per test) was sprayed onto a 1.7 mmwide conjugate pad (British Biocell International) at a flow rate of 15 μl/cm. After application of the reagent, the nitrocellulose membrane and conjugate pads were dried for 2 h at 37 °C, then assembled with the absorbent pad (Whatman) and the sample pad (British Biocell International) using the sticky backing of the MTP. The assembled strips were cut to a width of 5 mm using a Guillotine cutter (BioDot).

Under these conditions, we compared two set-ups using either the rabbit pAb for capture and 11F7 mAb for detection, or vice versa. The assays were run with 100 μl of BoNT/A complex (with concentrations from 10 to 0.1 μg/ml) spiked in PBS-Tween 0.5%-BSA 0.5%, and this volume practically represents a maximum for an adequate migration. The best set-up was chosen and various conditions were tested for further optimisation: the capture antibody (0.5 to 2 mg/ml) in PBS with BSA (0 to 2%) was sprayed in volumes from 0.5 to 2 μl/test. The detection antibody (5 to 50 μg/ml) was conjugated with various concentrations of gold beads (OD 520 = 1 to 10) before spraying. The assay buffer was composed of PBS, Tween (0 to 1%) and BSA (0 to 2%) and was used at a volume of 100 μl/test. Before use, strips were stored at 4 °C in a hermetic tube with a desiccant packet.

The limit of detection apparent by the naked eye was determined by trained personnel, as would be the case in the field. The strips were also read by a GelDoc XR imaging system (Biorad, Hercules, USA) and the associated software, Quantity One 4.6.0, to ensure reproducibility of detection and to quantify the signal. First, bands were automatically detected to confirm the naked eye detection. Second, bands were manually delineated and the intensity of the signals was calculated as the mean intensity of the pixels inside the surface of the band minus that of background (same surface manually defined in the vicinity of the band), using a fixed exposure time setting of 0.045 s.

Immunoaffinity column (IAC) assay

In this assay the sample and reagents are passed over a miniature disposable plastic column (65 mm × 7 mm) packed with a 5 mm × 5 mm polyethylene filter (average pore size of 20 μm) at the bottom. The filter, coated with a capture antibody, traps the analyte which is then detected by the binding of a biotinylated antibody, followed by streptavidin conjugated to a polymerized form of horseradish peroxydase (streptavidin-poly-HRP) (Fig. 1).

This technology is patented and commercialised under the name Abicap®, by Senova GmbH (Jena, Germany) which produced and optimized the test components. The signal produced in the column is directly quantified with a small (20 × 10 × 4 cm) dedicated photometer (Abicap reader, Senova). We decided to use the mAb 11F7 as the capture antibody because of the relatively large amounts requested by Senova for coating filters (10 mg). Briefly, filters were coated by adsorption with mAb 11F7 diluted in carbonate buffer (100 mM Na2CO3, 100 mM NaHCO3, pH 9.5) at a concentration of 15 μg/ml for 40 min at room temperature and then saturated with 750 μl PBS containing 1% (w/v) bovine serum albumin (BSA) and 0.05% 5-bromo-5-nitro-1,3-dioxane as a preservative. The columns were stored in plastic blisters under vacuum at room temperature. The rabbit pAb (detection antibody) was biotinylated with Sulfo-NHS-LC Biotin (Pierce, Perbio Science, Germany) at a biotin-to-antibody molar ratio of 20:1. Biotinylation was performed according to the manufacturer's instructions, which included the removal of unconjugated biotin by dialysis.

We modified and tested various parameters for optimisation of the procedure. These included the concentration of detection antibody (0.5-10 μg/ml), the sequential application of sample and detection antibody to the column or their pre-incubation, the time and temperature of this pre-incubation (10 min at room temperature or 1 h at 37 °C), the degree of polymerisation of the HRP conjugated to streptavidin (poly-HRP 40 or poly-HRP 80), number of washes and composition of wash buffer (PBS-BSA 0.2%, PBS-Tween 0.1%). The optimisation led to the selection of the following protocol. Reagents were added sequentially to each test column, and let flow through by gravity, in the following order : 750 μl wash buffer (WB: PBS-BSA 0.2%), a volume of sample as indicated, 500 μl WB, 500 μl detection antibody (2.5 μg/ml in PBS-casein 2.5%), 500 μl WB, 500 μl streptavidin-poly-HRP 40 (1 mg/ml), 500 μl WB, 500 μl substrate buffer (0.1 M Na-Acetate-Citrate-Buffer, pH 4.9), 500 μl precipitating HRP substrate (TMB), and again 500 μl substrate buffer. The absorbance was measured directly on the column. The sample, detection-antibody, poly-HRP and TMB steps each included a 6-min incubation. The whole procedure was performed at room temperature.

Statistics

All samples were tested in triplicate. We used oneway ANOVA followed by a multiple comparisons versus control group test (Holm-Sidak method) (SigmaStat 3.0; SPSS Inc., Chicago, IL, USA) for analysis of each IAC series of measurements. The limit of detection was defined as the smallest concentration of BoNT/A complex giving a signal significantly different (p b 0.05) from the negative control. For IAC and ICT, coefficients of variation (CVs) were calculated as ratios of the standard deviations to the means at the respective concentrations of BoNT. Standard curves were fitted by non-linear regression using a four-parameter logistic equation (Prism version 5, GraphPad Software Inc., San Diego, CA, USA).

Sample preparation in selected matrices

Human serum (pooled donors) was purchased from Sigma Chemical (St Louis, MO, USA). Fresh whole milk, sugar, flour and talcum were purchased from a local supermarket. Nasal sampling was performed on laboratory personal using a standard bacteriology swab (Eurotubo Collection swab, I.A.S.A., Rubi, Spain) per nostril, each washed in 1 ml of assay buffer. Flour, sugar and talcum were dissolved or suspended (5% w/v) in assay buffer. The two powders represent nutrient compounds while talcum mimics dust. Milk and serum were centrifuged at 13,000g for 15 min at 4 °C, then the lipid layer was discarded and the aqueous supernatants were spiked with the various concentrations of BoNTs. For ICT, but not for IAC, serum and milk samples were diluted with equal volumes of assay buffer prior to testing. For IAC on human serum, Super ChemiBlock heterophile blocking agent (Chemicon International Ltd, Chandlers Ford, UK) was added to the samples at a final concentration of 50 μg/ml.

Results

ICT

The ICT was best prepared with the rabbit pAb used for capture and the 11F7 mAb used for detection. Conditions for manufacturing were optimized as follows.

The capture antibody (1 mg/ml) in PBS was applied at a flow rate of 1 μl/cm membrane. The detection antibody (final concentration of 15 μg/ml) was incubated (2 h at 37 °C) with anti-mouse IgG gold beads (OD 520 = 7) before application at a flow rate of 15 μl/cm. After drying (2 h at 37 °C), the coated membranes were assembled with the absorbent pad and the sample pad, then cut in 5 mm-wide strips. Assay buffer was composed of PBS and Tween (0.5%) and was used at a volume of 100 μl/test. The test protocol, including drying, took about 40 min. Samples spiked with various concentrations of BoNT/A complex, from 2 to 2500 picomolar (pM), had detectable signals starting with 20 to 40 pM (220 to 440 mouse LD50 unit/ml), which corresponds with the limit of detection established both by the naked eye and the imaging system (Fig. 2). These measurements were repeated for three days, the intra-day CV (evaluating reproducibility) was 17.5% for the whole range of concentrations tested. The CV between days (evaluating repeatability) was 18.6%. Six months storage at 4 °C in the presence of a desiccant did not affect the detection limit of the strips.

No visible signal was obtained in samples of PBS-Tween 0.5% spiked with BoNT complex type B or type E at 200 pM.

The sensitivity of the ICT assay was also evaluated in matrices other than buffer (Fig. 3). For all tested matrices except serum and milk, concentrations of 40 pM gave readable signals while concentrations of 20 pM gave very weak signals. For serum and milk, BoNT/A complexes were spiked at 80 pM, then diluted prior testing, corresponding to a final concentration of 40 pM in the mobile phase as for the other matrices and this concentration gave readable signal. No false positive was observed in any of the tested matrices and all samples were assayed in triplicates. No significant loss of sensitivity was thus observed in the selected matrices, except for a two-fold loss in serum and milk, corresponding to the dilution in assay buffer necessary to ensure their migrations.

IAC assay

The selected protocol was completed in about 40 min for a sample of 750 μl, the volume recommended by the manufacturer. In assay buffer spiked with various concentrations of BoNT/A complex (0.09 to 900 pM) the limit of detection was at 0.45 pM (5 mouse LD50 unit/ml) ( p b 0.01) and the signals became higher with higher concentrations until a plateau was reached with about 80 pM BoNT/A complex (Fig. 4). The columns became clogged if 900 pM BoNT/A complex was used, presumably due to a large amount of TMB precipitating in the filter. These measurements were repeated for three days, the intra-day CV (evaluating reproducibility) was 6.3% for the whole range of concentrations tested. The CV between days (evaluating repeatability) was 14.3%.

We observed no cross reactivity for samples spiked with BoNT complex type B or type E at 200 pM. Storage for 12 months at room temperature did not affect the detection limit of the columns.

The sensitivity of the IAC assay was also evaluated in matrices other than buffer (Fig. 5). For sample volumes of 750 μl, the limit of detection for BoNT/A complex remained unaffected at 0.45 pM ( p b 0.01) in all tested matrices, except for nasal swab and fresh milk where it was at 0.91 pM ( p = 0.01) and 1.82 pM ( p = 0.03), respectively.

For human serum we tested whether the analysis of larger volumes of sample would lower the limit of detection and found that increasing the volume to 5 ml allowed the sensitivity to reach a value of 0.09 pM (1 mouse LD50 unit/ml) ( p b 0.01). In that case the procedure was longer, due to the larger volume of sample to flow through, but was completed in less than an hour.

Discussion

In this study we used commercially sourced BoNT/A complex as the analyte to compare the capability, especially sensitivity of two rapid immunoassay formats, ICT and IAC. We initially ran the assays in standard buffer to avoid effects from sample matrices and facilitate comparison with previously published results. We then evaluated the effect of various matrices on the sensitivity of both assays. We used the BoNT complex in this study because it is the most stable form of the botulinum neurotoxin and the form that would be encountered in most types of samples. Other reported assays have sometimes been evaluated with purified neurotoxin instead of BoNT complex. A ratio of one molecule of neurotoxin (MW 150,000 Da) per molecule of complex (average MW of 500,000 Da) can be assumed [START_REF] Inoue | Molecular composition of Clostridium botulinum type A progenitor toxins[END_REF], thus equivalences can be calculated. We expressed the limits of detection as molar concentrations of BoNT, both in our results and in the discussion below, again to facilitate comparison of values obtained with the two forms of toxin.

The limit of detection for the various assays must be compared to that of the mouse lethality assay, the gold standard, which is expressed in toxicity concentrations such as a number of mouse LD50 unit per ml of sample. However non-functional assays including immunoassays and endopeptidase-activity assays do not measure solely the amount of toxic BoNT. Part of the analyte detected in these assays might not be toxic for motorneurons, due for instance to degradation in regions of the toxin that are responsible for cell binding. Thus a relatively high limit of detection (poor sensitivity) could correspond to a small number of mouse LD50 unit/ml depending on the BoNT preparation used. Therefore it is essential, when evaluating the sensitivity of such assays, that the specific activity of the botulinum toxin preparation is indicated, typically in mouse LD50 unit/mg of toxin, or that reference toxin standards of known toxicity and stability are used [START_REF] Sesardic | Role for standards in assays of botulinum toxins: international collaborative study of three preparations of botulinum type A toxin[END_REF]. This is particularly important for tests of clinical interest or those that aim to replace the mouse lethality assay.

Both tests evaluated here needed the same amount of time for completion. While the procedure for the ICT is slightly simpler than that for the IAC, the IAC was 40 to 80 times more sensitive. If we had been able to use the affinity-purified rabbit pAb for capture, which we found to be the best set-up during the ICT optimisation, the sensitivity of the IAC might have been even better. The same antibodies were used in both tests, thus the enhanced sensitivity of the IAC is likely due in part to the larger volume of sample that can be analysed (a standard sample volume of 750 μl for the IAC versus 100 μl for the ICT, potentially representing 7.5 times greater sensitivity for the IAC) and to the signal amplification provided by the polymerized HRP.

The intra-day CV was smaller for the IAC (6.3%) than for the ICT (17.5%) but inter-day CVs, that best reflect the consistency of results in practical use, were equivalent (14.3% and 18.6%, respectively). No significant loss of sensitivity was observed in the various matrices except for a two-fold reduction in serum and milk with ICT, corresponding to the dilution necessary to ensure their migrations, and a two-and four-fold reduction in nasal swab and milk, respectively, with IAC. With IAC in serum, it was necessary to add an interfering-antibody blocking agent to keep the background signal at acceptable levels.

Several subtypes of BoNT/A, differing in amino acid sequence by up to 10%, have been recently described and may be recognised with differing affinities by antibodies [START_REF] Smith | Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization[END_REF][START_REF] Arndt | A structural perspective of the sequence variability within botulinum neurotoxin subtypes A1-A4[END_REF]. If it were the case for the antibodies used in our tests, their sensitivities would have to be evaluated for each subtype.

We evaluated how our assays compare to those reported previously for the detection of BoNT, though they did not use the same antibodies and toxin preparations. [START_REF] Chiao | Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B[END_REF] reported an ICT for the detection of BoNT/B with a limit of detection of 330 pM and that could be increased by silver enhancement to 0.33 pM. In our hands, this published protocol for silver enhancement produced high background, resulting in decreased sensitivity. The sensitivity of our ICT, 20 pM (220 mouse LD50 unit/ml), is similar to that reported recently for two commercial BoNT detection kits based on the same technology [START_REF] Sharma | Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis[END_REF], indicating that our optimisation was efficient.

With a limit of detection of 0.45 pM (5 mouse LD50 unit/ml), that can be lowered to 0.09 pM (1 mouse LD50 unit/ml) when testing 5 ml of serum, our IAC assay is one of the most sensitive rapid assays for detection of BoNT/A reported to date. It approaches the sensitivity of the mouse lethality assay which, for the number of animals usually assayed [START_REF] Solomon | Botulinum toxins[END_REF], has a practical limit of detection of 2 mouse LD50 unit/ml. A test based on similar IAC technology for detection of BoNT/C and BoNT/D has been reported [START_REF] Gessler | Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test[END_REF] with a sensitivity of 1 minimal lethal dose (defined as the minimal dose which kills all mice) per ml. This would correspond with 2 mouse LD50 unit/ml in our experience. Assuming the specific activities of these preparations (in mouse LD50 unit/mg of toxin) were similar to ours, the sensitivities of the two assays (expressed in molar concentrations of BoNT) would be equivalent, indicating that our optimisation was efficient. Our IAC was however more rapid than that of Gessler et al., which required over four h for such sensitivity. Overall, these two studies demonstrate the potential usefulness of the IAC format.

Several amplified ELISA with sensitivities approaching that of the mouse lethality assay for BoNT/A have been reported [START_REF] Doellgast | Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay[END_REF][START_REF] Ekong | Immunological detection of Clostridium botulinum toxin type A in therapeutic preparations[END_REF][START_REF] Szilagyi | Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes A and B[END_REF][START_REF] Sharma | Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies[END_REF]. Though faster than the mouse lethality assay, these ELISA procedures take about five h in addition to the plate-coating step to yield results. Our IAC assay also offers a favourable compromise of sensitivity and time when compared to other BoNT/A detection assays relying on more sophisticated equipment. These include electrochemiluminescence [START_REF] Rivera | Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic electrochemiluminescence detection[END_REF] (0.36 pM, 8 mouse LD50 unit/ml, 2 h), protease activity assays coupled with mass spectrometry [START_REF] Barr | Botulinum neurotoxin detection and differentiation by mass spectrometry[END_REF][START_REF] Kalb | The use of Endopep-MS for the detection of botulinum toxins A, B, E, and F in serum and stool samples[END_REF] (0.07 pM, 1.2 mouse LD50 unit/ml, 2 h) or coupled with fluorescence detection [START_REF] Schmidt | High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F[END_REF]) (4 pM, 3 h), or array immunosensors [START_REF] Sapsford | Biosensor detection of botulinum toxoid A and staphylococcal enterotoxin B in food[END_REF] (40 pM of toxoid, 30 min).

Because BoNTs are so toxic, assays for their detection must be highly sensitive if they are to be practical. On closer examination, the required sensitivity depends on the detection situation, thus our two tests, which differ in their sensitivities, may be theoretically of interest in different situations.

Maximum sensitivity is necessary for clinical serum samples because of the dilution factor in the human body. Arnon et al. reported a mildly affected patient (no mechanical ventilation required) whose serum contained 2 mouse LD50 unit of BoNT/A per ml [START_REF] Arnon | Botulinum toxin as a biological weapon: medical and public health management[END_REF]. The possibility to analyse large volumes of sample is an asset of the IAC technology, as illustrated by its limit of detection at 0.09 pM (1 mouse LD50 unit/ml) on 5 ml of human serum. Thus, the current sensitivity of the IAC may be sufficient even for the diagnosis of such a mild case.

Inhalation is thought to be the most threatening route for poisoning, at least for military troops as their drinking water and food can be controlled, but protective masks can be worn only for limited periods of time. Diagnosis made possible from analysis of nasal swab samples with one of our tests would be practical for such personnel. A substantial amount of toxin, estimated to be over 10% of the inhaled aerosolised dose [START_REF] Guglielmo-Viret | An immunochromatographic test for the diagnosis of ricin inhalational poisoning[END_REF], would be trapped in the anterior nasal passage. The lethal dose of BoNT for humans by inhalation is not known, but it has been determined to be over 300 mouse LD50 unit/kg in nonhuman primates [START_REF] Franz | Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism[END_REF]. The extrapolation for a 70-kg patient at this dose suggests that a sample concentration over 210 mouse LD50 unit/ml would be obtained, assuming a 10% recovery of the toxin trapped in the anterior nasal passage by a swab washed in one ml of buffer. The concentration of BoNT obtained by these calculations would be at the low end of the detection limit for the ICT, but could be detected by the IAC with a 20-fold safety margin.

Regarding the oral route, by which only a small fraction of the toxin is absorbed, the lethal dose for humans has been estimated to be 70 μg of BoNT/A (equivalent to 0.5 nmol) [START_REF] Arnon | Botulinum toxin as a biological weapon: medical and public health management[END_REF][START_REF] Wein | Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk[END_REF]. If a serving of food or beverage (around 500 ml, for example) contained such an amount of BoNT/A, it would be detected by the ICT, with a 25 to 50-fold safety margin for the given example. This amount of BoNT/A would be detected by the IAC with a 500 to 2000-fold safety margin for this example.

The IAC assay would be more useful than the ICT for the detection of analyte present in minute amounts, as shown in the present study. Although the IAC format is not presently convenient for the analysis of a large number of samples, it does combine essential assets of rapidity, high sensitivity, portability and ease of handling, which are of particular interest for field laboratories. Automation and multiplexing of this technology would be beneficial.

Fig. 1 .

 1 Fig.1. IAC assay set-up. The sample is passed over the column by gravity. A capture anti-BoNT/A antibody coated on the filter binds the toxin present in the sample. Captured toxin is detected by the sequential application of biotinylated anti-BoNT/A antibody, Streptavidin-poly-HRP and precipitating TMB, resulting in a blue coloration of the filter. Each step is followed by one wash as indicated in the text.

Fig. 2 .

 2 Fig. 2. ICT assay. (A) Calibration curves for BoNT/A complex in assay buffer. Points represent the mean signal intensities calculated as indicated in Materials and methods and expressed as a function of BoNT/A concentration, for triplicate strips on each day. The experiment was repeated on three days. Standard deviations are indicated as error bars. Where not visible, the error bars fall within the symbol itself. (B) Photograph of representative capture line signals for various concentrations of BoNT/A complex. The limit of detection is 20 to 40 pM.

Fig. 4 .

 4 Fig. 4. IAC assay. (A) Calibration curves for BoNT/A complex in assay buffer, with sample volumes of 750 μl. Points represent the mean absorbance values expressed as a function of BoNT/A concentration, for triplicate columns on each day. The experiment was repeated on three days. Standard deviations are indicated as error bars. Where not visible, the error bars fall within the symbol itself. (B) Representative columns for various concentrations of BoNT/A complex. The limit of detection by photometry is 0.45 pM and by the naked eye is 4.5 pM.

Fig. 5 .

 5 Fig. 5. Limits of detection with the IAC assay in various matrices. The matrices were spiked with the indicated concentrations of BoNT/A complex; the assays were realized in triplicates. Where not indicated, the sample volume was 750 μl. Error bars and asterisks indicate standard deviations and statistical significance ( ⁎ p b 0.05, ⁎⁎ p b 0.01), respectively.

O. Attrée et al. / Journal of Immunological Methods 325 (2007) 78-87

Acknowledgements

This work was supported by grant 3-d/LR/EMA from the French Ministère de la Défense.