Jérome Dejeu
email: jerome.dejeu@univ-grenoble-alpes.fr

Hugues Bonnet

Nicolas Spinelli

Eric Defrancq

Liliane Coche-Guérente

Angéline Van Der Heyden
email: angeline.van-der-heyden@univ-grenoble-alpes.fr

P Labbé

Impact of Conformational Transition on SPR Signals -Theoretical Treatment and Application to Small Analytes/Aptamer Recognition

Surface plasmon resonance is a powerful technique for label-free and real-time characterization of molecular interactions at interfaces. However, the detection of small molecules still remains a challenge. Here, we report on the direct detection of a low molecular weight compound by its receptor presented as a monolayer. Moreover, the signal observed is more than twice the expected mass-weighted response. To establish the origin of the signal enhancement, we present herein a theoretical model that simulates the maximal SPR response by taking into account the aptamer conformational change. We demonstrated that the thickness layer variation is not the only parameter to be considered. We highlighted that the conformational transition of the aptamer also induces a deviation of the refractive index increment (RII) of the target /aptamer complex from the sum of the RII of individual entities. This non-additivity of the RII significantly contributes to the magnitude of the signal. We also propose the prediction of the maximal SPR response in function of the penetration depth, the ratio of the mass-weighted RII of the partners, the sensing layer thickness and the correction of the complex RII. This model provides new insights in parameters to be considered for analysis of SPR signals.

INTRODUCTION

Aptamers are short synthetic oligonucleotides, single-stranded DNA or RNA, which have unique three-dimensional structures to bind their targets with high affinity and specificity. Aptamers have been selected by SELEX method (Systematic Evolution of Ligands by EXponential enrichment) for a wide range of targets from ions to whole cells including small organic molecules and proteins. [START_REF] Song | Aptamer-Based Biosensors[END_REF][START_REF] Famulok | Aptamer Modules as Sensors and Detectors[END_REF] In comparison with antibodies, aptamers present advantageous properties such as their easy chemical synthetic accessibility, their thermal and chemical stabilities. Therefore, aptamers have already found applications in various domains such as diagnostics, [START_REF] Hong | Applications of Aptasensors in Clinical Diagnostics[END_REF] therapy 4 or drug delivery [START_REF] Tan | Molecular Aptamers for Drug Delivery[END_REF][START_REF] Wu | Aptamer-Modified Nanodrug Delivery Systems[END_REF] and have emerged as very attractive candidates for the design and development of biosensors especially for low molecular weight (LMW) analytes. [START_REF] Palchetti | Electrochemical Nanomaterial-Based Nucleic Acid Aptasensors[END_REF][START_REF] Tombelli | Analytical applications of aptamers[END_REF][START_REF] Iliuk | Aptamer in Bioanalytical Applications[END_REF] Indeed, LMW molecules represent a class of compounds with high level of interest since it includes toxins, environmental pollutants or endocrine disruptors. [START_REF] Nguyen | Aptamer-Based Environmental Biosensors for Small Molecule Contaminants[END_REF] Among the large choice of techniques for studying biomolecular interactions, SPR is a powerful tool enabling direct detection of binding events in real time and without labeling step. [START_REF] Gopinath | Biosensing Applications of Surface Plasmon Resonance-Based Biacore Technology[END_REF] Since SPR is based on the change of surface refractive index (RI), which is related to the molecular weight of the analyte, SPR detection of LMW molecules represents a challenge. The increase of the receptor density by its random 3D immobilization onto grafted hydrogels can overcome this limitation. In fact, this approach has been seldom employed for aptasensor [START_REF] Zhu | An Aptamer Based Surface Plasmon Resonance Biosensor for the Detection of Ochratoxin A in Wine and Peanut Oil[END_REF][START_REF] Sun | Aptamer Based Surface Plasmon Resonance Sensor for Aflatoxin B1[END_REF] while indirect strategies of detection are commonly reported. In these indirect assays, the main strategies are based on competitive [START_REF] Wang | Au NPs-Aptamer Conjugates as a Powerful Competitive Reagent for Ultrasensitive Detection of Small Molecules by Surface Plasmon Resonance Spectroscopy[END_REF][START_REF] Wang | Aptamer-Based Au Nanoparticles-Enhanced Surface Plasmon Resonance Detection of Small Molecules[END_REF] or sandwich approaches involving either aptakiss [START_REF] Durand | Riboswitches Based on Kissing Complexes for the Detection of Small Ligands[END_REF][START_REF] Durand | A Combinatorial Approach to the Repertoire of RNA Kissing Motifs; Towards Multiplex Detection by Switching Hairpin Aptamers[END_REF] or split aptamers where one subunit is grafted on the sensor and the complementary subunit is linked to metallic nanoparticles. [START_REF] Golub | Photoelectrochemical, and Surface Plasmon Resonance Detection of Cocaine Using Supramolecular Aptamer Complexes and Metallic or Semiconductor Nanoparticles[END_REF][START_REF] Wang | Surface Plasmon Resonance Detection of Small Molecule Using Split Aptamer Fragments[END_REF] However, it is well known, that due to the exponential decrease of the evanescent electric field intensity, the SPR detection is more sensitive for events occurring at a short distance from the gold layer surface. This property has been exploited in the present study that focuses on the feasibility of SPR direct detection of LMW analytes by a 2D aptamer platform. Our work describes as a proof of concept the recognition of L-tyrosinamide (L-Tym, MW 180.2 g.mol -1 ) by a 49-mer oligonucleotide aptamer immobilized as an oriented monolayer.

L-Tym was chosen as model since it is one of the lightest molecular weight molecules for which a highly selective aptamer has been selected. A significant SPR signal was observed allowing the possibility to quantitatively evaluate the L-Tym/aptamer interaction. More interestingly, the SPR response was almost twice the predicted mass-weighted response.

To investigate the origin of the signal enhancement, we have developed a theoretical model that takes into account the aptamer folding upon recognition. Indeed, the conformation transition of the aptamer induces a reduction of the aptamer layer as already demonstrated. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] SPR has been successfully used for monitoring conformational modification such as the thermal response of poly(N-isopropylacrylamide) brushes, [START_REF] Balamurugan | Thermal Response of Poly(N-isopropylacrylamide) Brushes Probed by Surface Plasmon Resonance[END_REF] the denaturation and renaturation of self-assembled yeast iso-1-cytochrome. [START_REF] Chah | Denaturation and Renaturation of Self-Assembled Yeast iso-1-Cytochrome C on Au[END_REF] , the conformational dynamics of adsorbed poly(acrylic acid), [START_REF] Sarkar | Conformational Dynamics of Poly(acrylic acid). A Study Using Surface Plasmon Resonance Spectroscopy[END_REF] or proteins conformational changes such as for RNase A and Lysozyme. [START_REF] Chen | Monitoring Conformational Changes of Immobilized RNase A and Lysozyme in Reductive Unfolding by Surface Plasmon Resonance[END_REF][START_REF] Nilebäck | Viscoelastic Sensing of Conformational Changes in Plasminogen Induced upon Binding of Low Molecular Weight Compounds[END_REF] However, the use of receptor conformational changes for the detection of LMW molecules by SPR has scarcely been reported in the literature. An interesting study reported by Pitner et al. [START_REF] Gestwicki | Using Receptor Conformational Change to Detect Low Molecular Weight Analytes by Surface Plasmon Resonance[END_REF] indicated how the target-induced conformational change of a protein allows the detection of LMW molecules with the example of maltose binding protein (MBP) and tissue transglutaminase (tTG). [START_REF] Gestwicki | Using Receptor Conformational Change to Detect Low Molecular Weight Analytes by Surface Plasmon Resonance[END_REF] It was demonstrated that target binding can trigger a change in the hydrodynamic radius of the receptor, which in turn induces a SPR detectable change of the sensing layer refractive index. Negative responses have been observed in case of decrease of hydrodynamic radius of MBP upon interaction with maltose whereas positive responses have been observed in case of increase of the hydrodynamic radius of tTG upon interaction with calcium. In both cases, the variation of the signal induced by maltose or calcium exceeded the response expected for a mass-weighted response. In complement, the SPR detection of the calcium induced conformational change for four proteins has been in depth explored by Koch and coworker. [START_REF] Dell'orco | Quantitative Detection of Conformational Transitions in a Calcium Sensor Protein by Surface Plasmon Resonance[END_REF][START_REF] Dell'orco | Dynamics of Conformational Ca 2+ -Switches in Signaling Networks Detected by a Planar Plasmonic Device[END_REF][START_REF] Sulmann | Conformational Changes in Calcium-Sensor Proteins under Molecular Crowding Conditions[END_REF][START_REF] Dell'orco | Fingerprints of Calcium-Binding Protein Conformational Dynamics Monitored by Surface Plasmon Resonance[END_REF] The authors demonstrated that the signal depends on the size variation of the protein as well as on variation of its hydration shells. With the theoretical model presented here, we proposed to evaluate the contribution of the various physical parameters of the system on the final response. Our model highlights that the reduction of the sensing layer thickness is not sufficient to explain the experimental signal and that modification of the optical properties of the L-Tym/aptamer complex has to be also considered. To the best of our knowledge, this is the first example demonstrating that SPR transduction can be efficiently used to detect directly LMW compounds with aptamers immobilized as 2-D platform. Based on this example, a theoretical model is proposed to evaluate the contribution of conformational transition on the SPR response.

MATERIALS AND METHODS

Chemicals. Tris(hydroxymethyl)aminomethane (Tris), (11-Mercaptoundecyl)tetra(ethylene glycol) HS-(CH 2 ) 11 -EG 4 -OH, MgCl 2 , NaCl, L-tyrosinamide and streptavidin, were purchased from Sigma-Aldrich. Polyoxyethylene sorbitan monolaurate (Tween 20 ® ) was purchased from Euromedex, absolute ethanol was purchased from Acros, (11-Mercaptoundecyl)hexa(ethylene glycol) biotinamide HS-(CH 2 ) 11 -EG 6 -biotin was purchased from Prochimia (Poland). All aqueous solutions were prepared by using ultrapure water (Purelab UHQ) filtered (0.2 µm pore size). Experiment are performed in Tris buffer (20 mM Tris, 50 mM NaCl, 5 mM MgCl 2 , 0.05% v/v Tween 20 ® , pH=7.5 in water). The oligonucleotides sequences were prepared as previously described. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] Their sequences are the following: L-Tym aptamer, 5'AAT TCG CTA GCT GGA GCT TGG ATT GAT GTG GTG TGT GAG TGC GGT GCC C X3', X represents the 3' biotin TEG; random DNA sequence, 5'TGA TCA GAT GAG CGT TCC CAG CAC TTC AGC CGA CGA TGC AAC CAG TTT T X3', X represents the 3' biotin TEG (for ease of reading, this latter sequence is named "Scramble" in the text). Aliquots of oligonucleotides were dissolved in water and stored at 20°C. Before each measurement, solutions of aptamer or random DNA sequence were prepared in Tris buffer, heated at 90°C and cooled overnight at room temperature. Surface Plasmon Resonance. All SPR measurements were performed at 25°C in a Biacore T200 instrument (GE Healthcare). SPR bare gold sensor chips (SIA Kit AU) were from GE Healtcare. The gold sensor surface was rinsed with water, dried in a nitrogen gas stream and cleaned by 10 min UV-Ozone irradiation (Model 42-220; Jelight Conpany Inc.). The chip was then immersed for 15 min in high purity ethanol under stirring and dried in a nitrogen gas stream.

The cleaned gold surface was then dipped overnight in a thiol solution (90% of 1mM HS-(CH 2 ) 11 -EG 4 -OH and 10% of 1mM HS-(CH 2 ) 11 -EG 6 -biotin) in ethanol. The surface was carefully cleaned with ethanol and dried with nitrogen. After thiol functionalization, the gold sensor chip was mounted inside the instrument and incubated in Tris buffer, used as running buffer (RB) in the whole experiment. The flow cells were modified by streptavidin injection (50 µg.mL -1 ) at a flow rate of 30 µL.min -1 during 15 min. Random DNA sequence and aptamer solutions were then injected respectively on the reference and active channels at a flow rate of 2 µL.min -1 during 15 min until saturation near 600 RU. L-Tym solutions were successively injected for 2 min at a flow rate 30 µL.min -1 at concentration of 10, 25, 50, 75, 100, 125, 150, 200, 300, 400, 500, 600, 700 and 1000 µM. Each L-Tym injection was followed by a 2 min running buffer rinsing step.

RESULTS

The L-Tym aptamer and a same size random DNA sequence monolayers (see sequences in the Materials and Methods part) were formed through streptavidin-biotin interactions. First, the gold surface was modified ex-situ by a mixed self-assembled monolayer (SAM) formed by coadsorption of two pegylated (PEG) alkane thiols, one exhibiting a biotin terminal group. PEG were introduced to shield the surface towards non-specific adsorption while a 10% molar ratio of biotinylated thiol was selected to allow the formation of a saturated streptavidin (SA) layer. [START_REF] Sandrin | Controlled Surface Density of RGD Ligands for Cell Adhesion: Evidence for Ligand Specificity by Using QCM-D[END_REF] Subsequently, the biotinylated aptamer and the random DNA sequence were anchored, respectively, on the sample and reference flow-cell until saturation. Finally, the recognition of L-Tym by the aptamer monolayer was evaluated by injection of L-Tym solutions at different concentrations on both flow-cells. Injection of L-Tym on the reference flow-cell led to square profiles (Figure S1A), the equilibrium response being linearly proportional to L-Tym concentration (see Figure SIB). Since L-Tym does not interact with the random DNA sequence, [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] this result suggests that L-Tym does not show nonspecific interaction with the underneath layer as well. Therefore, the signal measured on this reference flow-cell could be attributed exclusively to refractive index differences between the running buffer (RB) and injected L-Tym solutions that allows determination of the L-Tym RII (   ' A dn dc = 0.219 cm 3 .g -1 ). In contrast, curved profiles were recorded on the active flow-cell (Figure S2). Upon rinsing with RB, the signal returned to baseline meaning that the binding was completely reversible. The SPR signal responses related to the L-Tym specific interaction with the aptamer monolayer (figure 1A) were thus obtained after subtraction of the signals recorded on the reference flow-cell by applying a double referencing procedure. [START_REF] Rich | Advances in Surface Plasmon Resonance Biosensor Analysis[END_REF] It should be stressed that despite the LMW of L-Tym, its interaction with the aptamer 2D platform led to an unexpected intense signal that allowed determination of the equilibrium dissociation constant of the interaction, Figure 1B. The adsorption isotherm was fitted in agreement with the 1:1 interaction mode of this recognition system. [START_REF] Challier | Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3′versus 5′-End Sequence Modification[END_REF] The resulting equilibrium dissociation constant was K D =103 ± 5 µM and the maximal response R max was 17.9 ± 3.0 RU. In a satisfying way, similar values of R max and K D were obtained from kinetic data (see Figure S3 and Table S1). This K D value is in the same order of magnitude as the one previously determined for a similar configuration by QCM-D. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] It should be also noted that the K D value obtained in our heterogeneous binding assays is higher than K D values determined by other technics using homogeneous binding assays. [START_REF] Lin | Microcalorimetrics Studies of the Thermodynamics and Binding Mechanism Between L-Tyrosinamide and Aptamer[END_REF][START_REF] Ruta | Noncompetitive Fluorescence Polarization Aptamer-based Assay for Small Molecule Detection[END_REF][START_REF] Challier | Simple and Highly Enantioselective Electrochemical Aptamer-Based Binding Assay for Trace Detection of Chiral Compounds[END_REF] This difference could be explained by a surface crowding generated by the high concentration of the aptamer on the surface which hampers conformational transition that is necessary for the formation of the aptamer-target complex . Interestingly, the experimental maximal response recorded during the interaction (RU Amax =17.9 RU) appears to be higher than the one expected considering the low molecular weight of the target and the 2D presentation of the receptor. Indeed, the predicted response is only 7.9 RU according to Wilson formula (eq 1). 37

    max ' ' . . . A A A L L L L MW dn dc R RU V RU V MW dn dc      (1)
Where R Amax is the predicted maximum response, RU L is the amount of aptamer immobilized

(



is the ratio of the mass-weighted RII of the analyte versus the ligand (i.e. the ratio of the molar RII, see eqs S1 to S4) and V, the Valency i.e. the ratio number of analytes per number of ligands involved in the recognition (V=1 in the present system). Upon recognition, the aptamer undergoes a high structural rearrangement that triggers the decrease of the aptamer layer thickness from 5.2 nm to 3.8 nm as characterized by spectroscopic ellipsometry. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] The intensity of the SPR signal being function of the distance from the surface, this reduction of almost 27% of the sensing layer has certainly an impact on the response and is not taken into account by the Wilson formula. To enlarge the latter formula, we proposed a new relationship (eq 18) that quantifies the contribution of aptamer conformational transition on SPR signal. To this end, we started from the initial equations of the SPR signal. [START_REF] Jung | Quantitative Interpretation of the Response of Surface Plasmon Resonance Sensors to Adsorbed Films[END_REF][START_REF] Schoch | Nuclear Transport Receptor Binding Avidity Triggers a Self-Healing Collapse Transition in FG-Nucleoporin Molecular Brushes[END_REF]

DISCUSSION

General expression of the SPR signal. In Biacore T200 SPR technology, a lens is used to focus the incident p-polarized light beam onto the prism base. Within the focus, a variety of angles of incidence are covered, ranging a few degrees around the resonance angle  . Due to the presence of exponentially decreasing evanescent electric field intensity, SPR is sensitive to the effective refractive index of the medium in contact with the gold layer: [START_REF] Jung | Quantitative Interpretation of the Response of Surface Plasmon Resonance Sensors to Adsorbed Films[END_REF][START_REF] Schoch | Nuclear Transport Receptor Binding Avidity Triggers a Self-Healing Collapse Transition in FG-Nucleoporin Molecular Brushes[END_REF]

  0 1 exp eff p p z n n z dz d d           (2)
Where n(z) is the refractive index at a distance z perpendicular to the sensor surface and d p is the effective penetration depth of the SPR wave. Upon injection of L-Tym, the thickness of the aptamer layer decreases (figure 2B) whereas the one of the random DNA sequence remains unchanged (Figure 2A). [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] In details, according to L-Tym concentration and K D , only a part of the aptamer is folded (eqs S12 to S14). The physical parameters of the different layer are listed in Table 1. The SPR response, R A , attributed to the interaction of L-Tym with the aptamer layer is then defined as the difference (eq S9) between the effective refractive index of the active flow-cell ( sample eff n , eq S7) and the one of the reference flow-cell (

ref eff
n , eq S8). After integration, the response is given by eq 3:

        0 0 0 0 1 2 1
                (3)
The refractive indexes can be replaced by their expression using De Feijter's relationship [START_REF] Defeijter | Ellipsometry as a Tool to Study Adsorption Behaviorof Synthetic and Biopolymers at Air-Water Interface[END_REF] (eq S11) that takes into account the composition of each layer. The reference cell is composed of the random DNA sequence so the refractive index of the aptamer layer is:

0 ref Sc sc i Sc Sc dn nn dc d      (4)
The sample cell is composed of free aptamer (L) and of aptamer/L-Tym complex (LA). On the sample flow-cell, the refractive indexes of sublayers 1 and 2 are given by eqs 5 and 6 respectively:

1 LA L i LA L LA L dn dn nn dc d dc d                    (5)
                    (7)   0 2 1 L i L L dn nn dc d       (8)
After replacement of the different refractive index (eqs 4, 7 and 8) in eq 3, the response R A can be separated in two terms (eq 9), R struct (eq 10) and R int (eq 11).

(

) 9
                                                         (10)
                                                    (11) int A struc R R R 
The first term, R struc , is independent of  and refers only to the structure of both flow-cells before interaction. The second term, R int , is function of  and   LA dn dc , and refers to the aptamer/L-Tym interaction. Thanks to a careful adjustment of the reference flow-cell with the random DNA sequence, application of a double referencing procedure cancelled the term R struc 32 and the residual SPR response corresponds to the interaction of L-Tym with the aptamer (R int ) (eq 11).

Expression of the maximal SPR response. The maximal response R Amax is obtained when all the aptamers on the surface are in interaction with L-Tym i.e. for  = 1 (eq 12).

0 0 0 max 0 0 0 0 LA L p p p d d d d d d d d L L L L A LA L LA L LA L LA L dn dn dn dn R e e e dc d dc d dc d dc d                                         (12)
Considering the RII of the complex L-Tym/aptamer as the sum of the RII of the individual entities (eq S6), and by introducing the folding ratio of aptamer (

LA L dd  
) in eq 12, the influence of the variation of the sensing layer is evidenced (eq 13).

0 0 max 0 0 1 1 1 1 1 . L p p p L L p p d d d d d d d d L L A L L L d d A dn dn e e e e e V d dc d d R c                                                  (13)
The maximal response can be then estimated in function of the experimental SPR signal recorded during the aptamer immobilization (RU L ) since the areal molar density of the aptamer ( 0  L ) is linked to (RU L ) through the Jung's equation (eq S15). The expected SPR signal in the case of modification of layer thickness coupled to the target recognition is thus expressed in eq 14.

  max 11 . 1 1

1 L p L p d d AL d d e R RU V e                (14)
Based on eq 14, the expected maximal response that takes into account the target binding as well as the thickness variation of the aptamer layer is equal to 10.4 RU (with RU L = 560 RU, dp=175 nm, [START_REF] Schoch | Nuclear Transport Receptor Binding Avidity Triggers a Self-Healing Collapse Transition in FG-Nucleoporin Molecular Brushes[END_REF] d L =5.2nm [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] and ρ=0.73 [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] ). While reduction of the sensing layer contributes to the amplification of the SPR response, the theoretical response is still below the experimental maximal response (17.9 RU). In consequence, we hypothesized that modification of the tridimensional structure of the aptamer also induces a RII deviation of the L-Tym/aptamer complex from the sum of the RII of individual entities. Indeed, conformational changes of an immobilized receptor have an impact on the SPR detection of LMW molecules as seldom described in the literature. [START_REF] Gestwicki | Using Receptor Conformational Change to Detect Low Molecular Weight Analytes by Surface Plasmon Resonance[END_REF][START_REF] Dell'orco | Quantitative Detection of Conformational Transitions in a Calcium Sensor Protein by Surface Plasmon Resonance[END_REF][START_REF] Dell'orco | Dynamics of Conformational Ca 2+ -Switches in Signaling Networks Detected by a Planar Plasmonic Device[END_REF][START_REF] Sulmann | Conformational Changes in Calcium-Sensor Proteins under Molecular Crowding Conditions[END_REF][START_REF] Dell'orco | Fingerprints of Calcium-Binding Protein Conformational Dynamics Monitored by Surface Plasmon Resonance[END_REF] In the reported studies, change of the hydrodynamic radius of protein and of its hydration shell is defined as the origin of the amplification. In line with our hypothesis, an interesting study carried out by backscattering interferometry, recently reported by Bornhop et al, [START_REF] Bornhop | Origin and Prediction of Free-Solution Interaction Studies Performed Label-Free[END_REF] highlights that in recognition systems undergoing significant conformation or hydration changes, the RII value of the recognition complex is not equal to the sum of the RII values of two initial partners. Since the thickness decrease of the aptamer layer is not enough to originate the signal enhancement, we also assume a factor of correction of the refractive index increment of the aptamer/L-Tym complex.

Maximal SPR response in the case of thickness modification and of RII deviation.

Considering a deviation factor for the RII of the analyte/ligand complex, eq 14 is not valid anymore and a correction term has to be included. In consequence, the RII of the L-Tym/aptamer complex is expressed as a function of the aptamer one by introducing a correction factor, x (eq 15).

correction L dn dn dc x dc              (15)
Where   correction dn dc is the correction of the L-Tym/aptamer complex RII defined as a function of the RII of the aptamer. By adding this correction factor to the sum of the RII of the two entities separately considered (eq S6), we obtain eq 16.

  . 1 . (16) This new expression of the L-Tym/aptamer complex RII is then introduced in eq 12 to obtain a new expression of maximal SPR response (eq 17).

LA L A correction L A dn dn dn dn dn dn dc dc dc dc dc d V x V c                                         
   

                                               (17)
Noteworthy, the first term in eq 17 is related to the response generated by the aptamer folding and RII correction whereas the second term relies on target recognition. Again, using the Jung equation (eq S15), a new expression of the theoretically SPR signal taking into account the target recognition, the thickness layer variation and the correction of the L-Tym aptamer RII is obtained:

  max 11 1 1 1 L p L p d d A L d d e R RU V x e                       (18)
Based on eq 18 and taking into account the experimental maximal response (

max max 17.9 AA R RU RU 
), a variation in the RII of the aptamer equal to 1.48% is calculated meaning an RII increase from 0.176 cm 3 .g -1 to 0.179 cm 3 .g -1 . This RII variation is also in agreement to the one estimated from SE experiment (4%). [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] It should be noted that in absence of thickness modification (ρ=1) and of RII modification (x=0), eq 18 leads to the initial eq 1.

Equation 18 is thus applicable to a wide range of recognition system where different configuration could be considered. If the recognition layer doesn't undergo a thickness modification but requires a RII correction coupled to the target recognition, eq 18 becomes:

  max AL R RU V x     (19)
In a more general way, our approach could be extended to the prediction of the maximal SPR response for systems that undergo conformational transition with or without thickness modification and/or with or without RII deviation.

Prediction of SPR response to system undergoing conformation modification. Based on equ 18, the impact of the various parameters (d p , ρ d L ,  and x) on the maximal SPR response upon interaction can be evaluated under the dimensionless ratio R Amax /RU L . For fixed value of ρ, the penetration depth d p of the evanescent electric field has low influence on the maximal SPR response (Figure 3A) on the 100 to 250 nm range. In the present study, considering dp equals to 175 nm, [START_REF] Schoch | Nuclear Transport Receptor Binding Avidity Triggers a Self-Healing Collapse Transition in FG-Nucleoporin Molecular Brushes[END_REF] a d p variation of 10 nm induces an insignificant (less than 1%) variation of R Amax /RU L ratio (Figure S4). Interestingly, the R Amax /RU L ratio linearly decreases with ρ whatever is the thickness of the initial recognition layer (Figure 3B). However, the slope of this dependence increases with the layer thickness. Variation of the layer thickness leads to either significant increase of the R Amax /RU L ratio in case of folding or significant decrease in case of elongation of the sensing layer. This observation is in concordance with previous work relating the SPR recognition of LMW analytes by protein undergoing large conformation change. [START_REF] Gestwicki | Using Receptor Conformational Change to Detect Low Molecular Weight Analytes by Surface Plasmon Resonance[END_REF] It should be noted that this negative ratio is also obtained in absence of layer thickness variations simply by considering a negative correction of the RII of the recognition complexes as exemplified by Figure 3C. The modulation of the signal by x is of crucial importance in our recognition system ( =0.012, d L =5.7 nm), e.g. an increase of 2% of the RII of the complex leads to a 5 times enhancement. for different kind of system that undergo conformational transition and thus that do not conformed to a simple recognition as expressed by eq 1. Indeed, conformation transition are widely numerous in biological system and a more general equation taking into account the impact of such modification is of relevant interest.

CONCLUSION

In this paper, we demonstrate the direct SPR detection of a low molecular weight analyte by its receptor presented as a 2-D monolayer. Despite the low density of the receptor, significant SPR signals were obtained and quantification of the interaction was performed. The magnitude of the experimental signal being more than twice the one expected for a simple mass increase of the sensing layer, we further investigated the contribution of conformational transition of the aptamer on SPR signals. We propose a theoretical model to simulate the maximal SPR signal in order to quantify the contribution of target binding and of the conformation changes onto the final response. We highlight that the conformational transition of the aptamer contributes to the SPR signal via two phenomena i) a decrease of the sensing layer thickness and ii) a deviation of the analyte/receptor complex RII from the sum of the two initial partners RII. In a more general way, this model also allows prediction of the SPR signal for 2D recognition platforms and is applicable to a wide variety of biological recognition system where a conformation transition may occur. We proposed a new equation where the impact of each phenomenon could be evaluated: with or without thickness modification and/or with or without RII deviation. This approach opens new opportunity for using SPR in the physical chemistry domain.

Figure 1 .

 1 Figure 1. A) Double-reference subtracted sensorgrams recorded during the interaction of L-Tym (5 µM to 1 mM) with the aptamer monolayer in RB. T= 25°C. Flow rate: 30 µL/min. B) Adsorption isotherm (square) and fitting curve (line) using a 1:1 Langmuir interaction model.

Figure 2 BFigure 2 .

 22 Figure 2 depicts schematically the two surfaces corresponding to the reference and the sample

ΓLΓLΓ

  are linked by θ , the ratio of aptamer entities in interaction with L-Tym i.e. the ratio of aptamer/L-Tym complexes amount versus 0 the total areal density of aptamer immobilized (eqs S12 to S14). Introducing θ and 0 in eqs 5 and 6, we obtained eqs 7 and 8.

Figure 3 .

 3 Figure 3. Variation of the R Amax /RU L ratio as a function of A) ρ and d p B) ρ and dL C) ρ and x. The parameters are fixed to d p =175nm, d L =5.2 nm, ρ=0.73, = 0.012 and x=0.01369.

Figure 4 Figure 4 .

 44 Figure 4 presents the influence of x in line with variation of the ratio of the mass-weighted RII,

  560 RU), MW A and MW L are respectively the molecular weight of the analyte (L-Tym, 180.2 g.mol -1 ) and of the immobilized ligand (aptamer, 15 831 g.mol -1 ),  

									' dn dc and 	 dn dc are the '
											A		L
	mass refractive index increments (RII) for L-Tym (0.219 cm 3 .g -1 as calculated above) and for the
	aptamer (0.176 cm 3 .g -1 ) 38-41 respectively.		MW	.(	dn	) dc	'	MW	.(	dn	) dc	'
			A				A	L				L

Table 1 .

 1 Physical parameters involved in the recognition system.

		Refractive	Thickness Surface	Refractive index
		index			density	increment (RII)
		(RI)
	Scramble			d	Sc	Γ	0 Sc		dn dc	 sc
	Aptamer before interaction			d L	Γ	0 L		 dn dc L
	Free aptamer during interaction			d L	Γ	L		dn dc	
									L
	L-Tym/aptamer complex			d LA		LA		dn dc		LA
	Sublayer (SAM and SA)	n 0	d 0
	Scramble layer	n	ref Sc	d
					Sc
	Layer 1	n 1	d LA
	Layer 2	n 2	d L -d LA

ASSOCIATED CONTENT Supporting Information. List of abbreviations, SPR experimental data, details for the development of the model for the SPR response (eqs S1 to S15). Supplementary Figures S1 to S6. This material is available free of charge.

AUTHOR INFORMATION

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT


Hannah Macdonald

Hugues Bonnet

Angéline Van Der Heyden

Eric Defrancq

Nicolas Spinelli

Liliane Coche-Guérente
email: liliane.guerente@univ-grenoble-alpes.fr

Jérome Dejeu
email: jerome.dejeu@univ-grenoble-alpes.fr

Influence of Aptamer Surface Coverage on Small Target Recognition: a SPR and QCM-D Comparative Study

Aptamers have emerged as promising biorecognition elements for the development of biosensors. The present work focused on the direct detection, by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D), of a low molecular weight (LMW) compound (less than 200 Da) with an aptamer receptor presented as an oriented monolayer on surface. These techniques are powerful for label-free, real-time characterization and quantification of molecular interactions at interfaces. Herein, we analyzed the influence of aptamer surface density on the recognition properties. A decrease of the surface 2 concentration was shown to improve the affinity for the target due to a higher kinetic association constant that could be explained by a limitation of the steric hindrance of the aptamer on the surface. An aptamer folding is produced upon recognition of the LMW target that gives rise to the modification of the layer on the surface. This induces a displacement of water acoustically coupled to the sensing layer, a thickness layer variation and a deviation of the refractive index increment (RII) of the target/aptamer complex from the sum of the RII of individual entities. We also demonstrated that the recognition signal was still detectable for low aptamer density (lower than 1 pmol.cm -2 ).

INTRODUCTION

Aptamers are single-stranded RNA or DNA oligonucleotides (with a length in the range of 10-100 nucleotides) that have been developed for the recognition of various targets including small molecules, proteins and even whole cells with high affinity and selectivity. [START_REF] Zhou | Aptamer-Based Biosensors for Biomedical Diagnostics[END_REF] They are usually selected from a combinatorial library of oligonucleotide using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. [START_REF] Zhou | Aptamer-Based Biosensors for Biomedical Diagnostics[END_REF] Due to their remarkable recognition properties, the aptamers have emerged as promising biorecognition elements in the development of biosensors (i.e."aptasensors" for aptamer-based biosensors), and are now considered as a valuable alternative to antibody-based sensors. [START_REF] Song | Aptamer-Based Biosensors[END_REF][START_REF] Han | Design Strategies for Aptamer-Based Biosensors[END_REF][START_REF] Ilgu | Aptamers in Analytics[END_REF] The grafting of the aptamer onto a solid support requires its functionalization by a suitable functional moiety depending on the immobilization procedure. This is a crucial point as the attachment method could have a great influence on the affinity of the aptamer for its target and consequently on the sensitivity of the biosensor. Indeed, non-specific interactions of the aptamer with the surface or limitation of the binding site accessibility due to proximity towards the surface [START_REF] Ruan | Molecular Dynamics Simulation of a RNA Aptasensor[END_REF][START_REF] Walter | Systematic Investigation of Optimal Aptamer Immobilization for Protein-Microarray Applications[END_REF][START_REF] Martin | Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers[END_REF] or steric hindrance induced by high surface density [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF][START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] can lead to a loss of affinity. It has been reported that the aptamer orientation, spacer length (between aptamer and anchoring moieties) and electrical charge of the surface have influence on aptamer microarray performance for the detection of fluorescently labeled compound, [START_REF] Walter | Systematic Investigation of Optimal Aptamer Immobilization for Protein-Microarray Applications[END_REF][START_REF] Martin | Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers[END_REF] as also suggested by dynamic molecular simulation. [START_REF] Ruan | Molecular Dynamics Simulation of a RNA Aptasensor[END_REF] Martin et al. demonstrated that an oligothymidylate linker is a consistent choice for linker across different types of aptamer/target pairs and approximately 10 nucleobases are required to increase the sensitivity of the response without affecting the affinity. [START_REF] Martin | Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers[END_REF] We recently reported the study of a new aptasensor, which binds enantioselectively a low molecular weight compound using saturated aptamer layer. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF][START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] In the surface plasmon resonance (SPR) study, a higher signal was obtained compared to the one expected for the target recognition as proposed by Wilson and David's formula. [START_REF] Davis | Determination of the Refractive Index Increments of Small Molecules for Correction of Surface Plasmon Resonance Data[END_REF] To explain this phenomenon, a new model was developed to identify the contribution of the aptamer conformational transition to the signal. In addition to the reduction of the sensing layer thickness, the model highlighted the non -additivity of refractive indexes increment (RII) for this recognition system: the RII value of the recognition complex is not equal to the sum of the RII values of two initial partners. [START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] This nonadditivity of the RII for the complex was already measured by Bornhop 11 et al. in solution but never revealed on surface. The recognition was also monitored using quartz crystal microbalance with energy dissipation (QCM-D) [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] and similar affinity constants were measured around 100 µM for both technics. However, these values are two orders of magnitude compared to affinity measured in solution (1-3 µM). [START_REF] Lin | Microcalorimetrics Studies of the Thermodynamics and Binding Mechanism Between L-Tyrosinamide and Aptamer[END_REF][START_REF] Ruta | Noncompetitive Fluorescence Polarization Aptamer-based Assay for Small Molecule Detection[END_REF][START_REF] Challier | Simple and Highly Enantioselective Electrochemical Aptamer-Based Binding Assay for Trace Detection of Chiral Compounds[END_REF] The immobilization being performed using saturated surface of biotinylated aptamer on a streptavidin platform, we hypothesized that the aptamers crowding should hamper the folding (necessary for recognition [START_REF] Ruta | Noncompetitive Fluorescence Polarization Aptamer-based Assay for Small Molecule Detection[END_REF] ) leading to a decrease of the affinity. The notion of surface probe density has been approach for the elaboration of electrochemical aptasensors via the direct coupling of aptamer through a thiolate or phosphoamidate anchoring function. [START_REF] Ceretti | Adenosine Reagentless Electrochemical Aptasensor Using a Phosphorothioate Immobilization Strategy[END_REF][START_REF] White | Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry[END_REF] Ceretti et al. reported an optimized surface densities for the signal generated by the capture of radiolabeled target. [START_REF] Ceretti | Adenosine Reagentless Electrochemical Aptasensor Using a Phosphorothioate Immobilization Strategy[END_REF] In the case of redox tagged aptamer, White et al. studied the effect of the surface density on the response of their electrochemical aptasensors. [START_REF] White | Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry[END_REF] However, to the best of our knowledge, no systematic study of the impact of aptamer surface densities on the affinity of the interaction has been reported to date. In the present article, we report on the influence of the aptamer surface coverage on its affinity for a small target and in particular its kinetic evaluation. We also confirmed that the non -additivity of refractive indexes increment (RII) for this recognition, previously observed for saturated aptamer density, was not induced by steric hindrance and was present whatever the aptamer density.

Materials and Methods

Chemicals

Tris(hydroxymethyl)aminomethane (Tris), (11-mercaptoundecyl)tetra(ethylene glycol) HS-(CH2)11-EG4-OH, MgCl2, NaCl, L-tyrosinamide and streptavidin, were purchased from Sigma-Aldrich. Polyoxyethylene sorbitan monolaurate (Tween 20®) was purchased from Euromedex, absolute ethanol was purchased from Acros, (11-Mercaptoundecyl)hexa(ethylene glycol) biotinamide HS-(CH 2 ) 11 -EG 6 -biotin was purchased from Prochimia (Poland). All aqueous solutions were prepared by using ultrapure water (Purelab UHQ) filtered (0.2 µm pore size).

Experiments were performed in Tris buffer (20 mM Tris, 50 mM NaCl, 5 mM MgCl 2 , 0.05% v/v Tween 20®, pH=7.5 in water). The oligonucleotides sequences were prepared as previously described.8 Their sequences are the following: L-Tym aptamer (Apt 49 ), 5' AAT TCG CTA GCT GGA GCT TGG ATT GAT GTG GTG TGT GAG TGC GGT GCC C X 3' and random DNA sequence, 5' TGA TCA GAT GAG CGT TCC CAG CAC TTC AGC CGA CGA TGC AAC CAG TTT T X 3' , in which X represents the 3' biotin triethylene glycol (TEG). Aliquots of oligonucleotides were dissolved in water and stored at -20°C. Before each measurement, the solutions of aptamer or random DNA sequence were prepared in Tris buffer, heated 5 minutes at 90°C and cooled overnight at room temperature.

Sensor Chip surface generation

Upon removal from storage in fridge, bare gold sensor chip was rinsed with ultrapure water, dried in a nitrogen gas stream and exposed for 10 min to UV-Ozone cleaning (Model 42-220; Jelight Conpany Inc.). The cleaned gold surface was then dipped overnight in the mixture solution of thiols (90% of 1mM HS-(CH 2 ) 11 -EG 4 -OH and 10% of 1mM HS-(CH 2 ) 11 -EG 6 -biotin) in ethanol. Next, the surface was carefully cleaned with ethanol and dried with gaseous nitrogen.

It has been demonstrated that 10% of biotinylated thiol is necessary to obtain a streptavidin layer with maximum surface coverage. [START_REF] Sandrin | Controlled surface Density of RGD Ligands for Cell Adhesion: Evidence for Ligand Specificity by Using QCM-D[END_REF] The functionalized gold sensor chip was mounted on the sample holder for immediate usage using QCM-D or SPR. All the experiments were performed with a saturated streptavidin layer and the aptamer density was controlled by the aptamer injection time and concentration. Indeed, this stratified system offers the selective immobilization of molecules (protein, DNA, …) with well-controlled orientation and density. 18- 20

Surface Plasmon Resonance Measurement

All SPR measurements were performed at 25°C in a four flow-cell Biacore T200 instrument (GE Healtcare). The experimental conditions were similar to the previous reported.10 Briefly, streptavidin (50 μg mL -1 ) was injected on the biotinylated surface at 30 μL.min -1 for 15 min.

Random DNA sequence and aptamer solutions were then injected, into the reference and sample channels respectively, at a flow rate of 2 μL.min -1 . The injection time of the oligonucleotide sequences was adjusted to control their areal density. L-tyrosinamide solutions, with different concentration, were injected for 2 min at a flow rate 30 µL.min -1 and the flow-cells were then rinsed with the running buffer during 3 min. The value of the signal was measured 4s before the end of the L-tyrosinamide injection. The signal recorded on the scramble reference channel 1 was subtracted from the signal of the sample channel 2, 3 and 4 by applying a double-referencing procedure: correction of the device drift and of the refractive index difference between the RB and the injected solutions. [START_REF] Rich | Advances in Surface Plasmon Resonance Biosensor Analysis[END_REF] Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) Measurements QCM-D measurements were performed using Q-Sense E1 or E4 instruments (Biolin Scientific) equipped with one or four flow modules, respectively. Besides measurement of bound mass, which is provided from changes in the resonance frequency, f, of the sensor crystal, the QCM-D technique also provided structural information of biomolecular films via the changes in the energy dissipation, D, of the sensor crystal. f and D were measured at the fundamental resonance frequency (4.95 MHz) as well as at the third, fifth, seventh, ninth, eleventh, and thirteenth overtones (i = 3, 5, 7, 9, 11 and 13). Normalized frequency shifts f = fi/i and dissipation shifts D = Di were presented.

Experiments were conducted in a continuous flow of buffer with a flow rate of 50 µL.min -1 by using a peristaltic pump (ISM935C, Ismatec, Switzerland). The temperature of the QCM-D platform and all solutions were stabilized at 24°C to ensure stable operation. All buffers were previously degassed in order to avoid bubble formation in the fluidic system.

In the case of homogeneous, quasi-rigid films, the frequency shifts are proportional to the mass uptake per unit area (mQCM), which can be deduced from the Sauerbrey relationship:

22 QCM m = -C. f  (1)
where the mass sensitivity, C, is equal to 18 ng.cm -2 .Hz -1 at f 1 = 4.95 MHz. It should be kept in mind that if the film is solvated, the acoustic areal mass density of the film will be composed of the areal mass densities of the adsorbate, mads, and the hydrodynamically coupled solvent, m solvent :

QCM ads solvent m = m + m (2)
In the case of soft films (ΔD > 0), the adlayer was modeled as a homogeneous layer of thickness d QCM and density ρ, using a continuum viscoelastic model (See equation S1). 23

Results and Discussion

Quartz crystal microbalance (QCM-D) studies.

The anchoring of the oligonucleotides (i.e. L-Tym aptamer and scramble) was achieved through streptavidin-biotin interactions (see materials and methods). The self-assembled monolayer of pegylated alkane thiols bearing hydroxyl and biotin groups was first performed ex situ. The formation of the subsequent biomolecular layers was monitored using QCM-D. After equilibration of the QCM-D signals (f and D) in the running buffer, the streptavidin (SA) was injected in the measurement chamber until reaching saturation of the QCM-D signals. As previously reported, [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] the immobilization of SA led to a change in frequency closed to -22 Hz (Figure S1) whereas the change in dissipation was very low indicating the formation of a rigid layer. By using Sauerbrey equation, the shift in f is converted in hydrated mass mQCM(SA) = 396 ng.cm -2 . This value is as expected for the formation of rather dense protein monolayers. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] The biotinylated aptamer Apt 49 was adsorbed onto the SA platform for which the average molecular surface density was controlled by the time of injection. A roughly linear relationship between the molecular surface density Apt49 and the frequency shift was reported from QCM-D ellipsometry coupling experiment. A conversion factor of -5.95 Hz cm 2 .pmol -1 was obtained that allows the quantification of the surface density of the aptamer from QCM-D frequency shifts. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] Several functionalized surfaces were prepared with frequency shifts ranging from -4 to -33 Hz (Table S1) corresponding to aptamer molar surface densities of 0.7 to 5.5 pmol.cm -2 . The Apt 49 layer could not be considered as a rigid layer as the changes in energy dissipation varied from 0.7 to 2.7 10 -6 . The QCM-D signals measured at several overtones i, could provide quantitative information, in particular the acoustic areal mass density (mQCM which includes hydrodynamically coupled solvent) but the application of the viscoelastic modeling requires a sensing layer, which behaves as a homogenous film. At low surface coverage we could probably not make the hypothesis of a homogeneous film of aptamer because the oligonucleotide behaves as hydrated discrete particles distributed through the SA platform. [START_REF] Challier | Simple and Highly Enantioselective Electrochemical Aptamer-Based Binding Assay for Trace Detection of Chiral Compounds[END_REF] Nevertheless, we could make an estimation of the acoustic mass m QCM using the QTM software (See SI for results for modeling with QTM and Figure S2) and calculate the hydration ratio by the following relation: H% = (m QCM -m SE )×100/m QCM , (See Figure S3), m SE is the optical mass calculated from f by using the factor: -5.95Hz.cm 2 .pmol -1 previously reported and an aptamer molecular weight of 15 831 g mol -1 . [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] The calculation revealed a degree of hydration varying from 88% (for high surface coverage) to 93% (in the case of low surface coverage), such high hydration ratio have ever been reported for DNA films. [START_REF] Larsson | Characterization of DNA Immobilization and Subsequent Hybridization on a 2D Arrangement of Streptavidin on a Biotin-Modified Lipid Bilayer Supported on SiO2[END_REF][START_REF] Sun | Construction and Modeling of Concatemeric DNA Multilayers on a Planar Surface as Monitored by QCM-D and SPR[END_REF] The decrease of coupled water induced by lowering the surface coverage as sensed by QCM-D has been reported and modeled for globular proteins, virus particles and small unilamellar vesicles. [START_REF] Bingen | Solvation Effects in the Quartz Crystal Microbalance with Dissipation Monitoring Response to Biomolecular Adsorption. A Phenomenological Approach[END_REF] The authors showed that the hydration curves could be reproduced quantitatively using a model that ascribed a hydration coat (truncated pyramidshaped) to each adsorbed particle. We reported previously that the low thickness of the saturated aptamer layer suggests that the strands did not adopt an elongated structure but were presented in a coiled conformation. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] The surface area ranged from 30 nm² to 300 nm² per Apt 49 (Figure S4), corresponding to an average spacing from 19.5 nm to 5.5 nm (Figure S5), respectively (assuming packing in a square, circular polymer-like chain arrangement). The ligand spacing distance of 5.5

nm determined for the highest surface coverage is closed to the gyration radius of Apt49 (Rg = 4.7 nm calculated from the hydrodynamic radius of Apt49 Rh = 3.3 ± 0.1 nm per Apt49 measured by Dynamic Light Scattering, see SI). [START_REF] Thakar | A Quartz Crystal Microbalance Method to Study the Terminal Functionalization of Glycosaminoglycans[END_REF][START_REF] Heinen | Poly(glycidyl ether)-Based Monolayers on Gold Surfaces: Control of Grafting Density and Chain Conformation by Grafting Procedure, Surface Anchor, and Molecular Weight[END_REF] The recognition properties of the Apt 49 sensing layer have been analyzed by the transient exposure of the aptasensors to various concentrations of L-Tym ranging from 5 nM to 1 µM. We previously demonstrated the selective response of the Apt 49 sensing layer by the absence of QCM-D response of a random DNA sequence. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] The QCM-D responses of aptasensors bearing various surface coverages upon exposure with 1mM L-Tym are depicted in figures 1 and Figure S6. As previously reported, [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] the recognition of the target molecule is identified by a frequency increase and a dissipation decrease. These variations were specific of the recognition since signal variations were not observed upon target injection on streptavidin alone and on a random DNA sequence immobilized on the same SA platform. Moreover, the recognition is enantiomeric as no frequency and dissipation variations were recorded upon D-Tym injection. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] The QCM-D signals variation demonstrated a loss of mass (positive shift in f and negative shift in D), which was attributed to a dehydration of the aptamer layer associated to a change in the conformation of the oligonucleotide on the surface upon recognition of the target molecule. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] In the present study, the dehydration was found to be dependent of the Apt 49 surface coverage and consequently the positive shifts in f measured during the recognition event were also dependent on the Apt 49 surface coverage (See Figure S6).

We previously reported that the K D value determined from the values of shifts in frequency was similar to that determined from the change in acoustic mass corresponding to a loss in water mass. [START_REF] Osypova | Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes[END_REF] In the present study, for low surface coverage, the calculation of the change in acoustic mass from the viscoelastic modeling was not relevant because of film inhomogeneity. The We noticed a quasi-exponential increase of the apparent KD as a function aptamer surface density. The affinity was thus improved when the surface coverage was decreasing, figure 2.A (i.e. increase of the interligand distance between Apt 49 receptors, figure 2.B). This observation was correlated with the steric hindrance induced by the saturation of the aptamer layer on the 2D platform of SA, which should hamper the conformational change of the aptamer upon L-Tym binding. Indeed, for high surface coverage, the KDapp values are about two orders of magnitude higher than those measured by homogeneous techniques such as isothermal calorimetry (1.75-3.2 µM), [START_REF] Lin | Microcalorimetrics Studies of the Thermodynamics and Binding Mechanism Between L-Tyrosinamide and Aptamer[END_REF] fluorescence polarization (1.7-2.2 µM) [START_REF] Ruta | Noncompetitive Fluorescence Polarization Aptamer-based Assay for Small Molecule Detection[END_REF] or electrochemistry (2.9 µM). [START_REF] Challier | Simple and Highly Enantioselective Electrochemical Aptamer-Based Binding Assay for Trace Detection of Chiral Compounds[END_REF] In contrast, for lower surface coverage the apparent K D values are higher of only one order of magnitude. We can assume that the folding of the aptamer upon recognition is less hampered thanks to a higher degree of freedom on the surface for low than for high coverage. These difference with experiments performed in solution could be explained by the limitation of the access of the target to the binding site in close proximity to the surface. [START_REF] Ruan | Molecular Dynamics Simulation of a RNA Aptasensor[END_REF][START_REF] Walter | Systematic Investigation of Optimal Aptamer Immobilization for Protein-Microarray Applications[END_REF][START_REF] Martin | Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers[END_REF] While some recognition systems led to similar affinity [START_REF] Pioszak | Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides[END_REF][START_REF] Grauschopf | The N-Terminal Fragment of Human Parathyroid Hormone Receptor 1 Constitutes a Hormone Binding Domain and Reveals a Distinct Disulfide Pattern[END_REF] analyzed from binding assays performed in homogeneous solution or from current immobilized ligand-based assays, several studies exhibited discrepancies between these methods and a lower affinity constant is usually measured by surface sensitive techniques because the affinity could be affected by the grafting of the ligand to the surface. . In our case, the transition between brush-like and mushroom-like with no overlap would occur around 9 nm, a value closed to 2 Rg = 9.4 nm. That demonstrates the influence of aptamer surface density on the Apt 49 /L-Tym interaction and emphasizes the necessity to preserve the aptamer flexibility. In addition, the aptamer surface density must be optimized for the development of aptasensors by taking into account the sensitivity of the transducing technique. Indeed, the decrease of aptamer surface coverage should be accompanied by a decrease of the QCM-D response, which is connected to a loss of water hydrodynamically coupled to the sensing layer. When the aptamer surface coverage is too low, the loss of water should reach a mass below the detection limit of the technique. The optimization of the aptamer surface density thus results from a compromise between a sufficiently high sensitivity of detection and a low overlapping of the oligonucleotides onto the functionalized surface.

Surface Plasmon Resonance Study.

To further analyze the influence of the aptamer surface density on the aptamer/L-Tym interaction SPR studies were also performed. This optical technique allows to sense directly the amount of target molecule trapped by the aptamer layer. The affinity of L-Tym for the aptamer immobilized with various surface coverages was explored using the same recognition platform as the one used for QCM-D study. The aptamer and random DNA sequence monolayers were immobilized through streptavidin-biotin interactions using a saturated SA layer formed on the 10% biotinylated self-assembled monolayer. The surface density of aptamers was controlled using different times of injection and correlated to the SPR signal variation, expressed in Response Unit (RU). [START_REF] Davis | Determination of the Refractive Index Increments of Small Molecules for Correction of Surface Plasmon Resonance Data[END_REF] This experimental signal could be converted in aptamer surface density with the Jung's relationship (Eq. S5). [START_REF] Jung | Quantitative Interpretation of the Response of Surface Plasmon Resonance Sensors to Adsorbed Films[END_REF] The recognition of L-Tym for the different aptamer density was evaluated at different surface densities of aptamers (R.U. values from 50 to 600 corresponding to 0.33 to 4 pmol.cm -2 ). L-Tym solutions ranging from 250 nM to 1mM were injected on the aptamer layers exhibiting various surface densities (see sensorgramms on Figure S8). The signals, obtained on the reference flowcell, were proportional to the analyte concentration and independent of the random DNA surface density. These variations were thus assigned to the refractive index variation of the injected target solutions. [START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] Moreover, no recognition signal was observed on the active flow-cell when the enantiomeric conformation of the target, D-Tym, was injected, whatever the aptamer density (Figure S9). Therefore, the double-reference subtracted signal was specific of the target recognition by the aptamer layer.

Sensorgrams were obtained after subtraction of the signals recorded on the random oligonucleotide layer from the one recorded on the aptamer layer by applying a double referencing procedure. As previously reported, [START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] a specific signal for the L-Tym recognition was obtained. The interaction was completely reversible and no regeneration step was necessary. On (green), 1.84 pmol.cm -2 (blue) and 2.86 pmol.cm -2 (blue cyan). T= 25°C, Flow rate: 30 µL.min -1 .

Time (s)

It is noteworthy that despite the low molecular weight of the target, its interaction with the aptamer 2D platforms led to measurable signals even for low aptamer densities. After doublereferencing procedure, the maximum signal (RUmax) recorded for injection of 1µM L-Tym solution varied from 1.7 RU (for an aptamer density of 0.33 pmol.cm -2 ) to 17 RU (for an aptamer surface density of 4 pmol.cm -2 ) as represented on Figure S11. For each aptamer density, the signal intensity evolved in a close-dependent manner with the L-Tym concentration in solution.

The obtained sensorgrams thus allowed the determination of the equilibrium dissociation constant (Figure S10) of the interaction from the responses obtained at the steady state (Req) or from the kinetic constants based on a 1:1 interaction model. For both methods, the determined KD values were similar for each aptamer surface coverage (Figure S10). K D values decreased from 120 to 20 µM as the interligand distance increased confirming the influence of steric hindrance. The values of KD obtained from both techniques (QCM-D and SPR) were found similar at low coverage.

The analysis of kinetic association and dissociation rates, k on and k off , respectively can give useful information on the recognition process on surface (Figure 4). No significant influence of the aptamer surface density on k off was observed (average value of 0.04 s-1), whereas the kinetic association rate (k on ) was found to increase with the interligand distance. Indeed at low coverage, k on value was close to 1500 M-1.s-1 and decreased 3.5 fold (around 400 M-1.s-1) at aptamer surface saturation. The affinity increase was thus due to a faster recognition of the L-Tym by the aptamer at high interligand distances. This process was favored by using a low aptamer density, which does not impede the aptamer folding. These results suggest that the critical step for the recognition of the L-Tym by the aptamer is the association step, which could be disrupted by an important aptamer coverage so when the strands hinder each other.

Non -additivity of the complex RII

We have previously reported that the maximum response obtained experimentally upon recognition at saturated aptamer coverage (17.9 RU) was higher than the theoretical one (7.9 RU) obtained from the Davis and Wilson's relationship 10 :

    max ' ' . . . A A A L L L L MW dn dc R RU V RU V MW dn dc      (3)
Where R Amax is the expected maximum response, RU L is the aptamer immobilization level, MW A and MW L are the molecular weights of the injected analyte (L-Tym, 180.2 g.mol -1 ) and of the immobilized ligand (aptamer, 15 831 g.mol -1 ), respectively.   ' A dn dc and   ' L dn dc are the refractive index increments (RII) for the analyte (L-Tym, 0.219 cm 3 .g -1 ) [START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] and for the ligand (aptamer, 0.176 cm 3 .g -1 ) [START_REF] Liang | In Vitro Non-Viral Gene Delivery With Nanofibrous Scaffolds[END_REF][START_REF] Lai | Monitoring DNA/poly-L-Lysine Polyplex Formation with Time-Resolved Multiangle Laser Light Scattering[END_REF][START_REF] Tumolo | Determination of the Refractive Index Increment (dn/dc) of Molecule and Macromolecule Solutions by Surface Plasmon Resonance[END_REF] respectively. ' ' ) .( ) .(

L L A A dc dn MW dc dn MW  
is the ratio of the mass-weighted RII of the analyte versus the ligand and the V, the valency i.e. the ratio number of analytes per number of ligands involved in the recognition (V=1 in the present system). This observation was confirmed for all surface coverages (see Figure S11 and Figure 5). We have demonstrated that the amplification of the signal (compared to the theoretical one) is attributed only partially to the sensing layer thickness decrease of the surface that occurs upon aptamer folding (around 25 %) and majorly to the aptamer-L-Tym complex RII deviation from the sum of the RII of the individual entities. [START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] This RII deviation, expressed with a correction factor x of the aptamer RII, could be triggered by the conformation transition of the aptamer upon recognition to the L-Tym. Such RII deviations were also reported from backscattering interferometry measurement, for recognition systems undergoing significant conformation or hydration changes. [START_REF] Bornhop | Origin and Prediction of Free-Solution Interaction Studies Performed Label-Free[END_REF] As demonstrated by QCM-D and QCM-D-SE experiments, upon L-Tym recognition, the aptamer sensing layer undergoes both a reduction of its thickness and a decrease of its hydration ratio.

While the RII value of the aptamer was 0.176 cm 3 .g -1 before the recognition, we have demonstrated that this parameter was increased by 1.48% during the recognition with L-Tym. [START_REF] Dejeu | Impact of Conformational Transitions on SPR Signals-Theoretical Treatment and Application in Small Analytes/Aptamer Recognition[END_REF] The SPR signal is influenced by i) the analyte binding, ii) the modification of the sensing layer thickness (corresponding to the folding of the aptamer) and iii) the RII deviation of the recognition complex. Considering the latter parameters, we have proposed a new evaluation of the theoretical maximal response using Dejeu et al.'s equation 9 :

  max 11 1 1 1 L p L p d d A L d d e RU RU V x e                       (4)
Where d p is the effective penetration depth of the SPR wave (175 nm), d L is the sensing layer thickness before the analyte recognition,

LA L dd  
is the thickness variation ratio of the sensing layer with d LA the sensing layer thickness during the analyte recognition, and x is the ligand RII correction factor due to its interaction with the analyte.

Based on this equation, the RII variation was calculated for each aptamer layer displaying different surface coverage. As suggested by the similar k off values calculated for the recognition occurring at various aptamer surface coverage (Figure 4B), we assumed that the folding ratio of the aptamer is constant to 0.73. Figure 5 highlights the contribution of the aptamer conformational transition to the SPR signal. It should be noted that for all surface coverages, the decrease of the sensing layer thickness induced by the aptamer folding increased the expected signal by a factor 1.3 and was not sufficient alone to fit with the experimental values. If we consider only the RII correction without aptamer folding, the expected signal increased twice, and was slightly inferior to experimental one. This observation confirms that, in our case, the aptamer folding is not the main source of the SPR signal enhancement. A better correlation between the experimental and theoretical values is observed when taking into account all these parameters (RII correction and folding). These experiments highlight that the major contribution of the SPR response is the target recognition and the RII correction and that this latter parameter should not be ignored when studying LMW target recognition. To date, signal enhancement have been seldom

observed and reported for proteins undergoing target-induced conformational change. The authors ascribed the amplification to modification of the protein hydrodynamic radius upon recognition. In the present study, we confirmed that in the case of low molecular weight analyte recognition by receptor undergoing conformational, signal amplification mainly originates from the non-additivity of RII. [START_REF] Gestwicki | Using Receptor Conformational Change to Detect Low Molecular Weight Analytes by Surface Plasmon Resonance[END_REF][START_REF] Dell'orco | Quantitative Detection of Conformational Transitions in a Calcium Sensor Protein by Surface Plasmon Resonance[END_REF]

Conclusion

In this paper, we studied the influence of the aptamer surface density on the recognition of a low molecular weight analyte by its receptor presented as a 2-D monolayer. Despite the low density of the ligand (from 5.5 to 0.7 pmol.cm -2 ) and low molecular weight of the analyte (180 Da), significant SPR and QCM-D signals were observed. Indeed, at the lower coverage, even if the signals did not exceed 2 RU for SPR and 0.5 Hz for QCM-D the quantification of the interaction could be performed. We observed that the equilibrium dissociation constant decreased when the aptamer surface coverage decreased. By SPR, we demonstrated that this is due to a higher kinetic association rate whereas the kinetic dissociation rate remained constant whatever the aptamer surface density was. The aptamer spacing improved the recognition until a minimal value of 9 nm. This minimal distant is necessary to not disturb the aptamer folding and the mass water expelling. The aptamer conformational transition induced an unexpected enhanced SPR signal compared to the one expected from the Wilson and Davies relation. The experimental SPR signal was found higher not only thanks to the folding of the aptamer but also thanks to a deviation of the analyte/receptor complex RII from the sum of the two initial partners RII. By taking into account these two parameters using the Dejeu's relationship, a good concordance was found between the experimental and the theoretical SPR values for all aptamer surface coverage and was not induced by steric hindrance (present whatever the aptamer density). A higher influence of the RII variation on the SPR signal was demonstrated compared to the increase due to the sensing layer thickness variation.

ASSOCIATED CONTENT

Supporting Information. QCM-D data analysis, table S1, calculation of hydration ratio, gyration radius, interligand distant and SPR signal conversion to areal molar density. Equation S1 to S5. Supplementary Figures S1 to S11. This material is available free of charge.

Figure 1 :

 1 Figure 1: QCM-D signals recorded during injection of L-Tym 1mM onto various sensing

  Figure S7). These K D values should be considered as apparent K D (noted K Dapp ) as they were

Figure 2 :

 2 Figure 2: Plot of apparent K D (determined by the shifts in frequency for concentrations in L-Tym

Figure 3 ,

 3 Figure 3, sensorgrams recorded for injection of L-Tym at 1mM are overlaid for several surface

Figure 3 :

 3 Figure 3: SPR double-reference subtracted sensorgrams recorded during the interaction of L-

Figure 4 :

 4 Figure 4: Variation of the kinetic parameters of the interaction as a function of the aptamer

Figure 5 :

 5 Figure 5: Comparison of the maximal response (green square) experimentally measured, RU Amax , (red ring) expected from the Davis and Wilson's relationship 10 and (pink star) expected from Dejeu et al. relationship 9 . The latter relationship, in addition to the target recognition takes into

  [START_REF] Ruan | Molecular Dynamics Simulation of a RNA Aptasensor[END_REF][START_REF] Martin | Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers[END_REF][START_REF] Lin | Studies of the Binding Mechanism Between Aptamers and Thrombin by Circular Dichroism, Surface Plasmon Resonance and Isothermal Titration Calorimetry[END_REF][START_REF] Chang | Kinetic and Equilibrium Binding Characterization of Aptamers to Small Molecules using a Label-Free, Sensitive, and Scalable Platform[END_REF][START_REF] Flores | Exploring the Sequence Space of a DNA Aptamer Using Microarrays[END_REF][START_REF] Collett | Production and Processing of Aptamer Microarrays[END_REF] In our case, similar results could probably be obtained if measurable signals were obtained at lower

	beginning of overlap with open space for	d		2	Rg	2 or no overlap for d  Rg	and mushroom-like with 2 d Rg 

aptamer density (< 0.7 pmol.cm

-2

). From figure

2

, we could observe a threshold value of aptamer density of about 2.5 pmol.cm -2 (ligand spacing of about 9 nm) below which, K Dapp values were constant (

26

± 8 µM). This interpretation is supported by the work of Heinen et al. who calculated the anchor distances, d, between two polymer chains on gold surface and the degree of chain overlap for poly(glycidyl ether) copolymers. They classified the polymers conformation through different regimes, brush-like with extensive chain overlap for d Rg  (Rg stands for gyration radius) or overlap without open space for

and CBH-EUR-GS (ANR-17-EURE-0003) and the University Grenoble Alpes. The authors wish to acknowledge the support from the ICMG (FR2607) Chemistry Nanobio Platform, Grenoble.

Prof. P. Labbé is acknowledged for fruitful discussions.

AUTHOR INFORMATION

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT

This work was partially supported by the French National Agency (ANR) under ECSTASE, Contract ANR-10-blan-1517, (Rational design of a sensitive and enantiospecific electrocatalytically-amplified aptasensor for amphetamine derivatives drugs), under ARCANE