Sensor Based on Aptamer Folding to Detect Low-Molecular Weight

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A sensor based on aptamer folding to detect low-molecular-weight analytes Alina Osypova, Dhruv Thakar, Jerome Dejeu, Hugues Bonnet, Angéline Van der Heyden, Galina V Dubacheva, Ralf Peter Richter, Eric Defrancq, Nicolas Spinelli, Liliane Coche-Guerente, and Pierre Labbé Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01736 • Publication Date (Web): 30 Jun 2015 Downloaded from http://pubs.acs.org on July 4, 2015

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Analytical Chemistry

A sensor based on aptamer folding to detect low-molecularweight analytes Alina Osypova,†¤ Dhruv Thakar,† Jérôme Dejeu,† Hugues Bonnet,† Angéline Van der Heyden,† Galina V. Dubacheva,‡ Ralf P. Richter,†,‡,§ Eric Defrancq,† Nicolas Spinelli,† Liliane Coche-Guérente†* and Pierre Labbé† † Université Grenoble Alpes, DCM UMR 5250, F-38000 Grenoble, France † CNRS, DCM UMR 5250, F-38000 Grenoble, France ‡ CIC biomaGUNE, 20009 Donostia - San Sebastian, Spain § Max-Planck-Institute for Intelligent Systems, 70569 Stuttgart, Germany * E-mail: [email protected] ABSTRACT

Aptamers have emerged as promising biorecognition elements in the development of biosensors. The present work focuses on the application of Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) for the enantioselective detection of a low molecular weight target molecule (less than 200 Da) by aptamer-based sensors. While QCM-D is a powerful technique for label-free, real-time characterization and quantification of molecular interactions at interfaces; the detection of small molecules interacting with immobilized receptors still remains a challenge. In the present study, we take advantage of the aptamer conformational changes upon the target binding that induces displacement of water acoustically coupled to the sensing layer. As a consequence, this phenomenon leads to a significant enhancement of the detection signal. The methodology is exemplified with the enantioselective recognition of a low molecular weight model compound, L-Tyrosinamide (L-Tym). QCM-D monitoring of L-Tym interaction with the aptamer monolayer leads to an appreciable signal that can be further exploited for analytical purposes or thermodynamics studies. Furthermore, in situ combination of QCM-D with spectroscopic ellipsometry unambiguously demonstrates that the conformational change induces a nanometric decrease of the aptamer monolayer thickness. Since QCM-D is sensitive to the whole mass of the sensing layer including water that is acoustically coupled, a decrease in thickness of the highly hydrated aptamer layer induces a sizeable release of water that can be easily detected by QCM-D.

KEYWORDS: aptasensor, low molecular weight compound detection, conformational change, oligonucleotide, biomolecular interaction, combined QCM-D/SE

INTRODUCTION Nucleic acid aptamers are single-stranded oligonucleotides that fold into well-defined three-dimensional structures and that recognize and bind to their targets (including small molecules, proteins and whole cells) with high affinity and selectivity.1,2 They are selected from a combinatorial library of sequences using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. Due to their remarkable

recognition properties, aptamers have emerged as promising biorecognition elements in the development of biosensors (i.e.“aptasensors” for aptamer-based biosensors).1,3,4 One of their major advantage (in comparison with antibodies) is that they can be chemically prepared by automated synthesis and thus multiple functional groups (e.g., a fluorescent label, a surface grafting function) can be easily and regioselectively introduced to confer them additional properties.5,6 As a consequence, they represent an alternative to antibodies and show promising applications for diagnosis and therapeutics.7 A

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myriad of detection strategies have been developed for the design of aptasensors, in combination with various transducers, such as electrochemical,4 optical8 and microgravimetric9 methods. The designed aptasensors showed good reliability, reproducibility and high regeneration ability. Among the large choice of techniques for studying interactions, Quartz Crystal Microbalance (QCM) is a powerful acoustic tool enabling direct detection of binding events in real time and without labeling. Indeed, QCM is a mass sensitive technique able to detect changes in areal mass densities around 1 ng/cm2. QCM has been widely used in the context of biochemical analysis for the characterization of biomolecular interactions with label-free and real-time measurements of interfacial binding reactions. In the context of aptasensors, QCM has been mainly applied for protein detection.9 In fact, the QCM signal is directly related to the mass uptake due to the analyte interacting with the aptamer layer grafted onto the surface so that QCM detection of low molecular weight (LMW) compounds still represents a challenge. To improve the detection, signal amplification strategies have been adopted such as the use of mass enhancers (i.e. gold nanoparticles) through a sandwich structure.10 However this approach complicates the detection assembly and makes the devices less robust. Among various technical setups commercially available, QCM with dissipation monitoring (QCM-D) as described by Rodahl et al.,11 records the changes in resonance frequency ∆f (∆f is related to the change in mass) and variations in energy dissipation ∆D of the shear oscillation (∆D is related to the viscoelastic properties of the oscillating mass). ∆D is equivalent to the resonance bandwidth measured by QCM systems based on impedance analysis.12 As QCM-D provides information on the viscoelastic properties of the adsorbed layer bound to the sensor crystal, it affords reliable and direct detection of ligand-triggered conformational rearrangements of a soft layer upon analyte binding. Recently, Özalp13,14 reported the application of a QCM-D based aptasensor for sensitive and specific detections of adenosine-5’-triphosphate (ATP) (507 Da) and adenosine-5’ monophosphate (AMP) (382 Da), demonstrating that this technique is sensitive enough to detect low mass increases and has potential as a sensor for small molecules. In addition, as the mass uptake measured by QCM-D includes the water hydrodynamically coupled to the adsorbed biomolecules, this transduction method provides information about the hydration variations of the adsorbed layer upon interaction with the analyte. Thanks to this property, it has been shown that the sensitivity of the QCM-D is significantly higher than that of optical techniques (those based on detection of changes in interfacial refractive index) when the water content coupled to the biomolecular layer varies significantly during the recognition event.15 The characterization of conformational changes and variation of hydration has been recently shown using QCM-D in the case of cationic polyamines interacting with DNA layers where condensation of the DNA films with a decrease of film thickness and an expulsion of water was observed.16,17 The present paper focuses on the feasibility of QCM-D direct detection of LMW analytes via aptamer recognition by taking advantage of their binding-induced conformational changes and variation of hydration of the sensing layer. Our study describes the enantioselective recognition of Ltyrosinamide (L-Tym-scheme S-1) by a 49-mer oligonucleotide aptamer (Apt49) receptor organized as a monolayer on a

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gold surface. The L-Tym/Apt49 couple is used as a model system to demonstrate the concept of a sensor based on an aptamer folding to detect LMW analytes. In situ coupling of QCM-D with Spectroscopic Ellipsometry (SE) allows step by step characterization of the assembly build-up. Thickness, refractive index, optical mass, acoustic mass and hydration ratio of the aptamer layer could be simultaneously measured. While very small changes in Ψ and ∆ ellipsometric angles are observed upon injections of L-Tym, sensitive and selective QCM-D signals were obtained enabling the quantification of the interaction. It is concluded that L-Tym binding induces a conformational change of immobilized oligonucleotides which results in a decrease in the aptamer monolayer thickness and the concomitant release of hydrodynamically coupled water. The high sensitivity of the QCM-D detection is exploited to quantify the affinity of the L-Tym / aptamer interaction. EXPERIMENTAL AND METHODS SECTION Materials and reagents: Tris(hydroxymethyl)aminomethane (Tris), (11-Mercaptoundecyl)tetra(ethylene glycol) (HS(CH2)11-EG4-OH), MgCl2, NaCl, L-tyrosinamide (L-Tym) and streptavidin (SA), were purchased from Sigma-Aldrich. Polyoxyethelene sorbitan monolaurate (Tween ®20) was purchased from Euromedex, ethanol grade for analysis was purchased from Acros, 11-Mercaptoundecyl)hexa(ethylene glycol) biotinamide HS-(CH2)11-EG6-biotin was purchased from Prochimia (Poland). SPR bare gold sensor chips (SIA Kit AU) were from GE Healtcare. Solutions: All aqueous solutions were prepared with ultrapure water (Purelab UHQ Elga). Tris buffer (20 mM Trizma base, 50 mM NaCl, 5 mM MgCl2, Tween 20, 2% v/v, pH=7.5 in ultrapure water), was used as buffer in each experiment. 1mM thiol solutions of HS-(CH2)11-EG4-OH and HS(CH2)11-EG6-biotin were prepared in absolute ethanol. Sequences of oligonucleotides were dissolved in water, aliquoted and stored at -20°C. Before each measurement, solutions of aptamers or random sequence were prepared in Tris buffer, heated at 90°C and left to reach room temperature overnight. Oligonucleotides synthesis: 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; Apt49), similar to the one selected by Vianini et al,18 and a random sequence (5’TGA TCA GAT GAG CGT TCC CAG CAC TTC AGC CGA CGA TGC AAC CAG TTT T X3’) were synthesized on a 3'-BiotinTEG CPG resin (Eurogentec, Belgium) at 0.2 µmol scale using standard β-cyanoethyl phosphoramidite chemistry on a DNA synthesizer (ABI 3400). After elongation, oligonucleotides were cleaved from the solid support and released into solution by treatment with 28% ammonia (1.5 mL) for 2 h and finally deprotected by keeping in ammonia solution for 16 h at 55 °C. Purifications were carried out by denaturing polyacrylamide gel electrophoresis (PAGE) and oligonucleotides were desalted by size exclusion chromatography (SEC) on NAP-10 columns (GE Healthcare). The calculated molecular weights are 15 831.33 and 15 586.23 g.mol-1 for the aptamer and the random sequence, respectively. Quantifications were performed at 260 nm using a CARY 400 Scan UV-Visible Spectrometer (Apt49: 26 nmoles, 13 %, ε260nm= 463600 M-1.cm-1; random sequence, 17 nmoles, 8 %, ε260nm = 463300 M-1.cm-1); ε were estimated according to the nearest neighbour model. Transducer surface functionalization: QCM-D sensors with gold-coating (dAu = 100 nm) (QSX301) were purchased from


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Biolin Scientific (Sweden). Prior to use, the sensors were exposed to a UV-ozone treatment for 10 min using a UVOzone cleaner (Jelight Company) and then immersed in ethanol under stirring for 20 min. The surfaces were then dried under nitrogen before dipping overnight in the mixture comprising 1 mM ethanolic solution of thiols (HS-(CH2)11-EG4OH:HS-(CH2)11-EG6-biotin, 8:2), then carefully rinsed with ethanol and dried with nitrogen. The sensor functionalized with a self-assembled monolayer of alkanethiolate (SAM) was then mounted in the QCM-D flow module and the formation of the biomolecular layer monitored with Tris buffer as the running buffer. The transducer surface was first exposed to a SA solution (10 µg.mL-1 in Tris buffer) and then to an oligonucleotide solution (100 nM in Tris buffer). The resulting functionalized surface was rinsed under flow until reaching the stabilization of the QCM-D signals. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D): QCM-D measurements were performed using QSense 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 provides structural information of biomolecular films via 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 are 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 E1E4 QCM-D platform and all solutions were stabilized to ensure stable operation at 24°C. All buffers were previously degassed in order to avoid bubble formation in the fluidic system. In the case of homogeneous, quasi-rigid films (for which ∆D/-∆f 0), the adlayer was modeled as a homogeneous layer of thickness dQCM and density ρ, using a continuum viscoelastic model.12 The shear elastic modulus G’ and the shear loss modulus G” represent the layer viscoelastic properties. The frequency dependence of the viscoelastic properties was approximated by power laws with exponents α’ and α”: α'

G' ( f ) = G0' ( f f0 )

,

α ''

G'' ( f ) = G0'' ( f f0 )

(3)

where G0’ and G0” are the shear storage and loss moduli at the (arbitrarily chosen) reference frequency f0 = 35 MHz. QCM-D data (at all available overtones) at selected time points were fitted with the software QTM (D. Johannsmann,

Technical University of Clausthal, Germany; http://www.pc.tu-clausthal.de/en/research/johannsmanngroup/qcm-modelling; option “small load approximation”).19,20 ρdQCM, ρG0’, ρG0”, α’ and α” were adjustable fitting parameters. The semi-infinite bulk solution was assumed to be Newtonian with a viscosity ηl = 0.89 mPa.s and a density of ρl = 1.0 g.cm-3. Initially the film density ρ was also fixed to 1.0 g.cm-3, without this affecting the generality of the modeling (see below). Details of the fitting procedure have been described previously,21 and the specified errors represent a confidence level of one standard deviation (68%). The viscoelastic model contains exclusively the product terms mQCM = ρdQCM, ρG0’, and ρG0”, and the fitting will therefore provide correct results for these product terms irrespective of the exact choice of ρ. Accurate values for dQCM, G0’ and G0” were calculated a posteriori from the product terms, with ρ refined with the help of the SE data (i.e. mSE) using:

mQCM

ρ

=

mSE

ρads

+

mQCM -mSE

ρl

(4)

where ρads is the density of the adsorbate (1.35 g.cm-3 for SA and 1.7 g.cm-3 for oligonucleotides15). This equation is readily derived when considering that the volume of the film is the sum of the volume occupied by the adsorbate and the volume occupied by solvent.15 Spectroscopic Ellipsometry (SE): In situ SE measurements were performed in combination with QCM-D, using the QSense E1 system with Ellipsometry Module (Biolin Scientific). A flow-through system was used for rapid sample injection (< 10 s) and buffer rinsing steps; sample incubation proceeded in still solution to ascertain homogeneous binding across the surface.22 Substrate functionalization with biotinylated monolayers was performed ex situ, before the measurement; all other surface functionalization steps proceeded in situ. Measurements were performed at 23°C. The Q-Sense E1 system was mounted on a spectroscopic rotating compensator ellipsometer (M2000V; Woollam, Lincoln, NE, USA) and ellipsometric data, ∆ and ψ, were acquired over a wavelength range of λ = 380 to 1000 nm at 65 degrees angle of incidence following an experimental procedure previously described.23 Bound SA and oligonucleotide areal mass densities were determined by fitting the ellipsometric data to a multilayer model, using the software CompleteEASE (Woollam). The model relates the measured ∆ and ψ as a function of λ to the optical properties of the sensor surface, the adsorbed film, and the surrounding solution. The opaque gold film covering the QCM-D sensor functionalized with the biotinylated mixed SAM was treated as a single homogeneous layer. Its effective optical properties were determined from data acquired in the presence of running buffer but in the absence of the SA film, by fitting the refractive index n(λ) and the extinction coefficient k(λ) over the accessible wavelength range using a Bspline algorithm implemented in CompleteEASE. The semiinfinite bulk solution was treated as a transparent Cauchy medium, with a refractive index n(sol, λ) = A(sol) + B(sol)/λ2. For the surrounding buffer solution, Asol =1.327 and Bsol =0.00332 µm2 were used. The solvated SA and aptamer layers were respectively treated as successive single layers, which we assumed to be transparent and homogeneous (Cauchy medium), with a given thickness (respectively dSE(SA) and dSE(Apt49), a wavelength-dependent refractive index (respectively n(SA, λ) = A(SA) + B(SA)/λ2 and n(Apt49, λ) = A(Apt49) + B(Apt49)/λ2 ), and a negligible extinction coefficient (k(SA)


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Figure 1. Combined measurements of QCM-D (top panel) and SE (bottom panel) performed during the build-up of the aptasensor on the initial biotinylated SAM (prepared ex-situ by overnight adsorption of thiols, the surfaces were mounted inside the QCM-D/SE liquid cell. SA (10 µg.mL-1) and biotinylated aptamer (100 nM) were successively injected until equilibration of the resulting QCM-D and SE responses). Top panel: QCM-D signals characterizing the adsorption of SA (left) and aptamer (right). The frequency shift and the dissipation shift are represented for overtones: i = 3 ( red line), 5 ( green line), 7 ( blue line) and 9 ( pink line). Bottom panel: areal mass density of SA and aptamer determined by SE. Start and duration of injection of SA (10 µg.mL-1) and aptamer (100 nM) are indicated by arrows. Before sample incubation, and after saturated binding, the surfaces were exposed to pure running buffer

= k(Apt49) = 0). dSE(SA), A(SA), dSE(Apt49) and A(Apt49) were treated as fitting parameters, assuming B(SA) = B(Apt49) = B(sol)l. The χ2 value for the best fit was typically below 2, indicating a good fit. The adsorbed streptavidin mass (mSE(SA)) and aptamer mass (mSE(Apt)) per unit area were determined using de Feijter’s equation with refractive index increments (dn/dc)SA = 0.182 cm3/g 24 and (dn/dc)Apt = 0.176 cm3/g taken as an average value from several published measurements.25-28 In applying de Feijter’s equation, we consider in a first approximation that the refractive index increments are concentration invariant. RESULTS AND DISCUSSION Build-up and characterization of the L-Tym aptasensor: monolayers of the anti-L-Tym aptamer and of the random oligonucleotide sequence (selected as a negative control) were formed through streptavidin-biotin interaction. First, the gold surface was functionalized ex situ by co-adsorption of two pegylated alkanethiols, one exhibiting a biotin terminal group. The pegylated function was introduced to shield the surface

towards non-specific adsorption while a 20% molar ratio of biotinylated thiol was selected to allow formation of a saturated streptavidin (SA) layer. The formation of the protein and oligonucleotide layers was characterized by coupling in situ QCM-D and SE (Fig. 1). The combined setup provides information about layer mechanical properties and morphology (through QCM-D) as a function of the surface density of adsorbed streptavidin and aptamer (determined through SE). Specifically, the ellipsometric angles ψ and ∆, measured by SE as a function of wavelength, can provide, by proper data treatment, quantitative information on the refractive index, thickness and optical areal mass density of thin films at planar interfaces. On the other hand, the shift in resonance frequency ∆f and in dissipation ∆D, measured by QCM-D at several overtones i, can provide, by proper data treatment, quantitative information on the acoustic areal mass density (which includes hydrodynamically coupled solvent), the thickness and the viscoelastic properties of thin films at planar interfaces. Table 1 presents these quantitative data for the sensing layer build-up.


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The QCM-D profile of Fig. 1 shows the overlapping of the frequencies for all the overtones and negligible shifts in dissipation during the adsorption of SA. These observations clearly indicate the formation of a rigid monolayer of SA molecules that are tightly coupled to the biotinylated SAM.29 The Sauerbrey relation (Eq. 1) can be applied for the determination of the hydrated film mass. The final shift in frequency ∆f(SA) = – 22 Hz corresponds to an acoustic areal mass density mQCM(SA) = 396 ng.cm-2. For comparison, the optical areal mass density determined by SE is mSE(SA) = 200 ng/cm2 (Fig. 1). Indeed, the mass uptake extracted from the QCM-D measurements includes the water that is hydrodynamically coupled to the adsorbed biomolecules whereas optical methods based on refractive index measurement are sensitive only to the mass of the adsorbed biomolecules. The hydration ratio, H, can be calculated from the relative difference between the acoustic mass and the optical mass, H = (mQCM – mSE) / mQCM, and thus H(SA) = 49.5% was obtained (Table 1). A surface density of SA molecules θ(SA) = 3.33 pmol cm-2 (MW = 60 kDa) and an effective thickness dQCM(SA) = 3.44 nm can also be estimated, which is in agreement with a densely packed 2D arrangement of SA.15-17 Incubation of 100 nM Apt49 solution leads to an initially fast and then gradually slowing decrease in frequency accompanied by a noticeable increase in dissipation (Fig. 1). After rinsing with pure buffer, the final shifts in frequency ∆f and dissipation ∆D were found to be ∆f(Apt49) = – 28 Hz and ∆D(Apt49) = 2.4 × 10-6 (for i = 7). The immobilization of the Apt49 sensing layer was reproducible since the relative variations of ∆f7/7 and ∆D7 (recorded for eleven experiments) were respectively less than 8.3% and 6.8%. The relatively high shift in dissipation together with the spread in frequency shifts for the different overtones indicate the formation of a soft aptamer layer, ruling out the application of the Sauerbrey equation for the quantification of acoustic areal mass density. Consequently, QCM-D data were fitted with a viscoelastic model using QTM software. The acoustic areal mass density of the aptamer film was estimated to be mQCM(Apt49) = 669 ng.cm-2. This value is consistent with those given by Larsson et al.15 and Sun et al.16,17 who reported mQCM values of 437 ng.cm-2 and 798 ng.cm-2 for a 30-mer and a 59-mer oligonucleotides, respectively, immobilized on a SA platform. The coupling of SE with QCM-D provided mSE(Apt49) = 79.8 ng cm-2, which corresponds to an aptamer surface density θ(Apt49) = 5.04 pmol cm-2. The molecular Apt49:SA ratio is thus ~1.5, significantly smaller than the intuitive value of 2 (i.e. assuming that two of the four biotin-binding sites of SA are exposed to solution in a planar 2D arrangement), which is in agreement with the literature.15-17 The hydration ratio H(Apt49) = 88 % confirms that a highly hydrated film is formed, as expected for a layer of endgrafted oligonucleotides.15-17 This high degree of hydration together with the large energy dissipation suggest that the aptamer strands adopt a flexible structure on top of the SA platform. However, we could not consider an elongated structure of the oligonucleotide strand due to the low thickness determined by SE (Table 1). Indeed, a contour length of 19.6 nm would be expected for Apt49 considering an elongation per base of 0.4 nm30. This is four times higher than the film thickness measured by using SE (Table 1). Such low aptamer layer thickness is in agreement with a closed-like conformation of the oligonucleotide attributed to the presence of magnesium that is known to affect the structure of single-strand DNA leading to a compact structure.30,31 An acoustic ratio ∆D/-∆f of 0.0857 10-6 Hz-1 can be calculated from the adsorption of Apt49 on SA platform (Fig. 1). The acoustic ratio was intro-

duced by Gizeli et al. as a tool for the characterization of surface protruding DNA conformation.32 The last value (calculated from the 7th overtone) is in perfect agreement with that reported in the literature for a 75 base pairs oligonucleotide with a closed-like structure in Tris/Mg2+ buffer.30 Table 1. Quantitative analysis of film properties from SE and QCM-D data. Standard errors or confidence intervals are calculated on the basis of data fitting with optical or viscoelastic models, respectively.

SA Aa,b

Apt49

Apt49 + L-Tym 1 mM

1.410±0.003 1.354 ± 0.002

1.366 ± 0.002

4.4 ± 0.2

5.2 ± 0.4

3.8 ± 0.4

mSE (ng.cm )

200 ± 0.5

79.8 ± 0.5

+ 4.2 ± 1g

θ (pmol.cm-2)

3.33 ± 0.01

5.04 ± 0.03

/

mQCMc (ng.cm-2)

396 ± 9

669 (+99 /-30)

495(+85/-17)

Hd (%)

49 ± 1

88.1(+1.4/-0.5)

83

1.15

1.05

1.07

3.44±0.09

6.37 (+0.98/-0.30)

4.61 (+0.84/-0.17)

b

dSE (nm) -2

e(

-3

ρ g.cm ) dQCM (nm) G0’f (MPa)

/

0.326 (+0.074/-0.150)

0.370 (+0.072/0.135)

G0”f (MPa)

/

0.545 (+0.015/-0.070)

0.636 (+0.029/0.131)

α’

/

0.359 (+0.179/-0.059)

0.334 (+0.094/0.096)

α”

/

0.959 (+0.041/-0.084)

1.000 (+0/-0.106)

a A corresponds to the first term in the Cauchy dispersion model of the refractive index: n = A + B/λ2 (see experimental section) b

Errors represent data deviation due to the noise of SE signals

c QCM masses were calculated either using Sauerbrey equation (SA layer) or viscoelastic modeling by QTM (Apt49, Apt49+LTym). In the latter case, the confidence intervals are given in brackets. d

The degree of hydration has been calculated by: (mQCM - mSE) / mQCM. e

The effective densities were obtained from equation (4).

f

G0’ and G0” have been obtained from viscoelastic modeling with f0 = 35 MHz g Mass increase due to L-Tym binding. This mass increase is calculated using de Feijter’s equation by assuming that the mass increase is only due to the binding of L-Tym and that the refractive index increment of Apt49 is concentration invariant and independent of aptamer conformational change

Furthermore, the acoustic thickness of the aptamer layer (dQCM(Apt49) = 6.37 nm) is in agreement with the published


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data for the 30-mer and 59-mer oligonucleotides adsorbed on a SA platform.15-17 From our measurements (Table 1), it appears that the fitted values of acoustic thickness dQCM are systematically slightly higher than the optical thickness dSE. Several reasons could be at the origin of this difference. First, the QCM-D always measures an areal mass density, not a geometrical thickness. The conversion from areal mass density to thickness requires the physical density as an independent input. The aptamer layer density has been estimated indirectly on the basis of equation (4). A second reason lies in the fact that the acoustic contrast is much higher than the optical contrast.33 As a consequence the acoustic thickness approaches the geometric thickness even for diluted functionalized layer much more rapidly than the optical thickness. Finally, during the fitting process of both QCM and ellipsometric data, the aptamer layer is treated as a single homogeneous layer with a given thickness (box profile). However, it is clear that a box profile does not correspond to the real geometry of the interface between the aptamer layer and the bulk liquid. Due to the difference in contrast of these two technics, one can thus expect that the modeling process will give different box thicknesses, the acoustic thickness being usually higher than the optical one. Although a certain uncertainty exists concerning the absolute values of acoustic and optical thickness of the aptamer layer, it is important to emphazise that both SE and QCM-D demonstrate unambiguously a decrease of these two parameters upon interaction with L-Tym. Fig. 2A illustrates the dependence of the frequency shifts measured by QCM-D during the adsorption of the Apt49 as a function of the molecular surface density θApt49 extracted from SE experiments (data from Fig. 1). The relationship is roughly linear. Once established, this calibration plot allows the quantification of the surface density of this aptamer from QCM-D frequency shifts for arbitrary coverage even without any parallel SE measurements. Fig. 2B presents in parallel the variation of the acoustic ratio ∆D/-∆f. This ratio can be used to calculate the elastic compliance J’(f) (a measure for film softness) via equation (5),34 and thus provides information on the mechanical properties of a soft layer without the need for modeling:32

J' =

1 ρ ∆D 4π iηl ρl ∆f

G' 2

G' +G''

2

strates that the simple calculation of J’ from ∆D/-∆f provides a reasonable estimation of J’, but that the value is somewhat overestimated (i.e. by about 1.5-fold) even for films with a few nanometers in thickness. This is in line with previous findings for slightly thicker films of grafted biopolymers.21

Figure 2. (A) Parametric plot of the frequency shifts (i = 7) (from QCM-D) vs Apt49 surface density (from SE) for the data from Fig. 1. The relationship can be approximated rather well by a straight line over the entire range of densities investigated, with a slope of – 5.95 Hz cm2.pmol-1 (R = 0.99899; red line). (B) Parametric plot of the acoustic ratio ∆D/-∆f (i = 7) (from QCM-D) as a function

(5)

where ηl = 0.89 mPa s and ρl = 1 g cm-3 are the viscosity and the density of the aqueous bulk solution, respectively, and ρ is the film density. Alternatively, J’(f) can be calculated from the storage modulus G’ and the loss modulus G” as extracted from viscoelastic modeling (Table 1), using equation (6):

J' =

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(6)

The graph presented in figure 2B shows a monotonic decrease of the ∆D/-∆f ratio (and thus film softness) with increasing Apt49 density until reaching a constant value close to 0.0896 ± 0.0012.10-6 s (in the case of the seventh overtone). We can compare the values of J’ derived from G’ and G” (where G’ (storage modulus) and G’’ (loss modulus) were extracted from the full viscoelastic modeling) with the estimated value of J’ derived from equation (5). J’ values in the same order of magnitude were obtained, 1.20 ± 0.03 MPa-1 as estimated from ∆D/-∆f and 0.770 (+0.078/-0.284) MPa-1 for the calculated J’ using G’ and G”. The comparison demon-

of the aptamer surface density (from SE). Data between 0 and 0.04 pmol/cm2 were affected by transient perturbations during sample injection, and are not shown.

Aptasensor response to analyte binding. Figure 3 illustrates the QCM-D responses of the Apt49 sensing layer during a transient exposure to 1 mM L-Tym. Fast association and dissociation steps were observed. The signal returned to its initial value after the rinsing step, confirming full reversibility of the recognition process. In contrast, L-Tym did not generate any QCM-D response on the layer of a random-sequence oligonucleotide, and no response was observed upon exposure of the Apt49 layer to 1mM of the D-Tym enantiomer (Fig. 3). These results prove the high specificity and enantioselectivity of the interaction in this heterogeneous binding assay, as it has been previously reported for homogeneous binding assays.35


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Figure 3. QCM-D signals recorded during injection of L-Tym and D-Tym onto the aptamer film (red) and the film of randomized oligonucleotide sequences (blue). The arrows represent the start and duration of injections. Frequency (bottom panel) and dissipation (top panel) variations for the 7th overtone are shown; T = 24°C, flow rate = 50 µL.min-1.

Interestingly, we noticed unexpected variations of the QCM-D signals upon interaction of L-Tym with Apt49: instead of conventional negative shifts in frequency (corresponding to an increase in mass) and positive shifts in dissipation for the binding of the analyte, positive shifts in frequency and negative shifts in dissipation were observed. Such behavior has also been reported in the literature in the case of the binding of calcium ions to calmodulin-functionalized surface.36 The authors interpreted these unusual shifts for the capture of an analyte by a conformational change of the protein. Considering that it can be originated from a loss of mass, we performed quantitative analysis of the QCM-D data through viscoelastic modeling. Indeed this treatment revealed a decrease in the acoustic mass by 174 (+26/-13) ng.cm-2, or equivalently of acoustic thickness by 1.76 nm, upon 1 mM L-Tym binding. We also performed the analysis of the Apt49 response to 1 mM L-Tym by SE. The responses were close to the detection limit. By fitting the full ellipsometric spectrum, a small increase in refractive index (by + 0.012 ± 0.002) and a small reduction in thickness (by - 1.4 ± 0.4 nm) were observed (Table 1). Using de Feijter’s equation, we calculated a low yet clearly measurable increase of the optical areal mass density, mSE = + 4.2 ± 1 ng/cm2, which was not found in the case of the 1 mM D-Tym injection negative control (Table 1 and Figure S-1). Taken together, these experimental data obtained by QCMD and SE are fully consistent with a net increase in the areal mass density upon L-Tym recognition accompanied by a conformational change of the aptamer receptor, which decreases the aptamer layer thickness. Conformational changes have already been reported in the literature for the Apt49 upon binding of L-Tym in homogeneous solution using fluorescence polarization37 and circular dichroism,38 and further corroborate our interpretation. The binding of L-Tym by the Apt49 layer induces a restructuration of the aptamer and this conformational change is attended with a decrease in film thickness (from a compact structure to a more compact one) and a release of water. The observed increase of refractive

index is a joint effect of accumulation of L-Tym in the film and increase of film compactness as a consequence of the LTym induced aptamer conformational change. The observed decrease in the acoustic areal mass density thus originates from the expulsion of water upon the aptamer conformational change. It is noteworthy that the expelled water mass is substantial, i.e. 25 ± 1% of the water initially coupled to the Apt49 sensing layer is released. These results highlight the superior sensitivity of QCM-D compared to SE (43 fold higher calculated from the ratio of changes in acoustic mass to optical mass). The significant variation of the water content proper to the present recognition event confers the higher sensitivity to the QCM-D technique. It should be emphasized that SE measurements related to L-Tym binding are close to the detection limit of this technique. Decrease of optical thickness and increase in refractive index is qualitatively demonstrated, but since these two parameters are covariant in the model fitting process a large uncertainty in the absolute value of these parameters must be considered. In contrast, the ellipsometric mass that is the product term of thickness and refractive index change compared to the surrounding solution (∆n × d) is rather constant for different couples of thickness and refractive index as obtained from the fitting process. Using de Feijter’s equation, an increase of Apt49 optical mass of 4.2 ng cm-2 is calculated upon L-Tym binding by assuming that the mass increase is only due to the binding of L-Tym and that the refractive index increment of Apt49 is concentration invariant and independent of aptamer conformational change. Taking in mind that initially the Apt49 optical mass is 79.8 ng cm-2, a 1:1 binding with L-Tym should result in a theoretical mass increase of about 0.9 ng cm-2. This means that in the present experiment, SE should be able to detect precisely a mass variation of about 1%, which is unrealistic under the present conditions. We propose that the larger areal mass density determined through de Feijter’s equation is apparent, and a consequence of the simplifying assumptions made about the invariance of refractive index increments. In the present study, SE unambiguously demonstrates an increase of ellipsometric mass (∆n × d) upon interaction with L-Tym, and the absence of response in the presence of D-Tym. The absolute variation of optical mass cannot be determined with precision since we are at the detection limit of the technique. The observed increase of refractive index is a joint effect of accumulation of L-Tym in the film and increase of film compactness (thickness decrease) due to the aptamer conformational change upon LTym binding. As a consequence, the local concentration of Apt49 in the film increases which is known to induce a variation of the refractive index increment.39 Moreover, the L-Tym induced conformational change of Apt49 is potentially able to induce a significant variation of the aptamer refractive index increment as already observed with other biomolecules.40 Each of these parameters should be taken into account before applying in a rigorous way de Feijter’s equation for quantifying the optical mass variation of the Apt49 detection layer. From these considerations, it is clear that SE is able to detect a small and significant variation of the ellipsometric mass (∆n × d) upon L-Tym binding, although near the detection limit. Converting this variation into a variation of optical areal mass is not possible in the present case because the respective contributions of L-Tym binding and Apt49 conformational change onto optical mass variation cannot be resolved. The QCM-D responses of the aptasensor for L-Tym concentrations ranging from 25 nM to 1 µM gave signals of increasing magnitude (Fig. S-2). By fitting the responses in ∆f and


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∆D for the six overtones, we extracted the decrease of acoustic areal mass density as a function of analyte concentration (Fig. 4). Assuming a 1:1 binding stoichiometry for the L-Tym / Apt49 interaction,37 an apparent dissociation constant KD,app = 181 ± 27 µM was determined by fitting the data on the basis of a Langmuir adsorption model. Although the quality of the fit was good, this value should be considered as an apparent KD as it was indirectly obtained from the quantification of the expelled water upon L-Tym binding. It is noteworthy that an apparent affinity constant can also be calculated directly from the shifts in frequency, considering in a first approximation that changes in frequency are related to changes in mass (Fig. S-3). The resulting affinity constant, extracted from fits to Langmuir isotherms and averaged over 6 overtones is 158 ± 12 µM, i.e. close to the value determined from the acoustic mass through viscoelastic modeling. It should be also noted that the KD value obtained in our heterogeneous binding assays is higher by about two orders of magnitude than KD values determined by other techniques using homogeneous binding assays.35,37,38 This difference could be explained by the steric hindrance, due to the coupling of the aptamer on the surface and/or the proximity of other aptamers at the employed high aptamer surface density, which should hamper the conformational change of the aptamer during L-Tym binding.

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Figure 5. Elastic compliance J’ (calculated from ∆D/-∆f measured at i = 7 and using Eq. (5) with a linear adjustment of the film density as a function of the concentration) of the aptamer layer as a function of L-Tym concentration. The error bars were calculated from the standard errors of ∆D and ∆f measurements performed from the data of figure S-2.

Figure 4. Decrease in acoustic areal mass density (−∆mQCM, obtained through viscoelastic modelling of the measurement shown in Fig. S-2, with data from all 6 overtones) as a function of LTym concentrations (black square), and fitted (red line) to a ligand binding curve (Langmuir isotherm). The error bars were calculated from the analysis of the QCM-D signals of two aptasensors.

L-Tym binding also appreciably affects the mechanical properties of the sensing layer, as shown in Fig. 5. Here, the elastic compliance J’, related to film softness, was calculated from the ∆D/-∆f acoustic ratio through Eq. (5). The decrease in compliance is characteristic of film stiffening, which agrees well with a local densification of the aptamer layer as a consequence of L-Tym induced aptamer folding and thickness decrease.

The reproducibility of the aptasensor response was examined by measuring the QCM-D responses upon repetitive injections of L-Tym solutions at 1 mM (Fig. S-4). This assay demonstrates the high reproducibility of the response with relative standard deviations of 3% (for ∆f) and 5.4% (for ∆D). The stability of the QCM-D response was also investigated by performing injections on the same sensor over two days. We observed a decrease of the L-Tym response of less than 10%. This experiment was conducted by maintaining the functionalized sensor crystal inside the QCM-D measurement chamber under flow (buffer solution) at 24°C. During this time, the QCM-D signals were monitored continuously in order to analyze the stability of f and D along the rest period. Weak drifts of 0.8 Hz for f and 0.06 × 10-6 for D over 24 hours were measured proving the stability of the signals. These results confirm the stability of the biomolecular assembly, the reversibility of the binding interaction and the reliability of the sensor response. In conclusion, we have demonstrated that, in contrast to an optical technique such as ellipsometry, QCM-D can be used as a highly sensitive and selective technique for the direct detection of low molecular weight compounds (less than 200 Da) in the case of conformational change. To the best of our knowledge this study represents the lowest molecular weight detected by a QCM-D aptasensor without any amplification process. The detection principle is based on the mass of water expelled from the film, due to the folding of the aptamer upon the interaction with the target. The high water content expelled from the sensing layer (25±1% of the initial hydrated mass) demonstrates the reliability of the transduction method. This rearrangement also induces a significant change in the viscoelastic properties of the sensing layer. The Apt49 functionalizedsensor revealed highly specific and enantioselective QCM-D


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response to the L-Tym target molecule. QCM-D represents a highly sensitive detection technique, the present method can be applied to the field of aptasensors for the detection of low molecular weight compounds when the capture of the target molecule induces a folding of the aptamer.

ASSOCIATED CONTENT Supporting Information. "This material is available free of charge via the Internet at http://pubs.acs.org." Scheme S-1, figures S-1, S-2, S-3 and S-4.

AUTHOR INFORMATION Corresponding Authors Liliane Coche-Guerente, † Université Grenoble Alpes, DCM UMR 5250, F-38000 Grenoble, CNRS, DCM UMR 5250, F-38000 Grenoble, France Fax: +33 456520814 E-mail: [email protected]

Present Address : ¤

Present address: Alina Osypova, Universite Catholique de Louvain, IMCN/ BSMA, Place Croix du Sud 1, Bte Boltzmann 2d floor, Louvain-la-Neuve, Wallonia, BE 1348

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” LabEx support (ANR11-LABX-0003-01), and the Joseph Fourier University by AGIR program (Caractérisation et quantification des interactions oligonucléotides-petites molecules). The authors acknowledge support from ICMG FR 2607, (Characterization of Interactions Platform and synthesis platform) of the Nanobio Program and by the Nanosciences Foundation. G. V. Dubacheva acknowledges Marie Curie Career Integration Grant CELLMULTIVINT (PCIG09-GA2011-293803).

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Sensor Based on Aptamer Folding to Detect Low-Molecular Weight

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