Conformational Changes of Enzymes and Aptamers Immobilized on

Review pubs.acs.org/bc

Conformational Changes of Enzymes and Aptamers Immobilized on Electrodes Alejandra Tello,† Roberto Cao,*,§,# María José Marchant,§ and Humberto Gomez§ †

Universidad Andres Bello, Bionanotechnology and Microbiology Lab, Center for Bioinformatics and Integrative Biology (CBIB), Facultad de Ciencias Biológicas, República 239, Santiago, Chile § Instituto de Química, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330, Curauma, Valparaíso, Chile ABSTRACT: Conformation constitutes a vital property of biomolecules, especially in the cases of enzymes and aptamers, and is essential in defining their molecular recognition ability. When biomolecules are immobilized on electrode surfaces, it is very important to have a control on all the possible conformational changes that may occur, either upon the recognition of their targets or by undesired alterations. Both enzymes and aptamers immobilized on electrodes are susceptible to conformational changes as a response to the nature of the charge of the surface and of the surrounding environment (pH, temperature, ionic strength, etc.). The main goal of this review is to analyze how the conformational changes of enzymes and aptamers immobilized on electrode surfaces have been treated in reports on biosensors and biofuel cells. This topic was selected due to insufficient information found on the actual conformational changes involved in the function of these bioelectrochemical devices despite its importance.

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INTRODUCTION

as represented in Figure 1, with an unavoidable alteration in the conformation. Though several reviews regarding the conformational changes of immobilized enzymes have been published during this decade, none has specifically addressed the immobilization on electrode surfaces.2−7 Additionally, insufficient attention to the conformational changes of aptamers has been received,

Conformation constitutes an essential property of biomolecules, especially in the cases of enzymes and aptamers. The conformation of these biomolecules defines their molecular recognition ability. However, the conformations of both enzymes and aptamers are extremely sensitive to any small changes in their environment (pH, temperature, ionic strength, etc.). Changes in conformation involve folding and unfolding processes, which differ in enzymes and aptamers. Nevertheless, from a kinetics point of view, both types of biomolecules can behave similarly in the folding process when it comes to those that are relatively small. In this case, it has been assumed that both small enzymes and aptamers undergo the controversial two-step transition. This result was achieved from an experimental comparison between a 96-residue protein and a 38-base aptamer. The similarity was attributed to the poor hydrophobic interactions within such a small protein.1 When an enzyme or aptamer is immobilized on a naked electrode surface, significant conformational changes are to be expected. The unavoidable influence of the electrostatic interactions with the electrode surface is the first factor that must be considered. The simple deposition (adsorption) of these biomolecules on a charged electrode affects their conformation. For example, enzymes with both positive and negative charges distributed along their surfaces will be attracted or repelled according to the charge of the electrode, © 2016 American Chemical Society

Figure 1. Schematic representation of folded enzymes (in green) that are attracted by a negatively charged electrode and that finally unfold upon electrostatic interactions (in red). Received: September 23, 2016 Revised: October 14, 2016 Published: October 17, 2016 2581

DOI: 10.1021/acs.bioconjchem.6b00553 Bioconjugate Chem. 2016, 27, 2581−2591

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Bioconjugate Chemistry

Table 1. Reports on Electrochemical Aptasensors in Which Conformational Changes Are Defined in Order to Interpret How the Device Worksa electrodeb Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold Gold SPCE SPCE SPCE-AuNPs GCE-Au GCE-AuNPs GCE-AuNFs GCE-graphene-AuNPs GCE-graphene-AuNPs GCE-graphene-AuNPs Indium tin oxide Si-boron-doped diamond

spacer arm HS(CH2)3HSCH2CH(OH)CH(OH)CH2SH HS(CH2)6HS(CH2)6HS(CH2)6HS(CH2)8HS(CH2)6H2N(CH2)6HS(CH2)6HS(CH2)6HS(CH2)6HS(CH2)6HS(CH2)6HS(CH2)6HS(CH2)6HS(CH2)6HSHSHSHSHS-(four base pairs)HS(CH2)6-; HS(CH2)8-; HS(CH2)9H2N−PEG−COOH H2N(CH2)6HS(CH2)6H2N-(CH2)6H2N−Ph−C(O)−NHS(CH2)6HS(CH2)6HSHS(CH2)6(HBPE-CA)-NH2NH2−PEG−COOH

conformational change

LOD

ref

ssDNA to dsDNA From dual-hairpin Dual-hairpin to single-hairpin dsDNA to ssDNA Hairpin to open loop Hairpin to open loop dsDNA to hairpin Hairpin to G-quadruplex ssDNA to Hairpin Hairpin to G-quadruplex Hairpin to double helix Unfolded to antiparallel G-quadruplex ssDNA to G-quadruplex Random-coil to G-quadruplex dsDNA to hairpin Linear to hairpin Hairpin to linear Linear to pseudoknot To dsDNA From hairpin Hairpin to linear dsDNA to hairpin or linear

1.5 fM 1.4 nM 50 nM (DNA), 3 pM (thrombin), 30 nM (ATP) 1.4 nM 10−8 to 10−6 M 10 pM 10 pM 0.062 μA.mL.μg−1.cm−2 1.6 nM 0.054 nM 0.08 nM 0.315 nM 200 nM 10 pg/mL 10 nM 41 fM 11.7 pM 0.6 × 10−4 ppt 0.4 pg/mL 0.3/0.6 nM 1 pM

123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 29

To G-quadruplex Random coil to G-quadruplex dsDNA to hairpin dsDNA to ssDNA Hairpin to dsDNA From hairpin To G-quadruplex Hairpin to dsDNA Linear to hairpin ssDNA to G-quadruplex Random coil to G-quadruplex

0,12 ng/L 0.1 μg/L 60 pM 0.2 nM ∼ 1 pM 0.33 fg/mL 5 nM 3.3 fM 0.064 pM 0.9 fM ng/L

144 145 81 146 147 148 149 150 151 152 153

a Most of the conformations quoted in column 3 correspond to those represented in Figure 3. bSPCE: Screen Printed Carbon Electrode; GCE: Glassy Carbon Electrode; HBPE-CA: Hyperbranched polyester microspheres-COOH; AuNFs: gold nanoflowers.

that reason, the use of this method has been limited in the immobilization of biomolecules on electrodes, and mainly to aptamer-based biosensors.24 With the supramolecular association and covalent conjugation methods, the biomolecule is submitted to chemical reactions that must be carried out under the mildest conditions possible in order to minimize unavoidable conformational changes. In this sense it is important to mention that when the formation of covalent bonds between the biomolecule and the electrode surface is achieved enzymatically the conformation is practically unaffected.20 For this purpose transglutaminase has been mainly used,20 and also sortase A, horseradish peroxidase, and ligases, among others.25 When a biomolecule is entrapped within a polymer, presumable conformational changes could occur, resulting in random orientations. On the other hand, physical methods generally provoke significant conformational changes in the immobilized biomolecule, especially when it is based on electrostatic attraction (Figure 1). For a covalent conjugation, the presence of an amino, carboxylate, thiolate, or hydroxyl group in the biomolecule is required.17,26 In the case of enzymes, the mentioned functional

despite the fact that this process plays a determining role in the detection of a specific target molecule. In this sense, and as an exception, different reports of Plaxco and co-workers on this topic have to be mentioned, including two reviews.8,9 Other reviews, that only partially consider the conformational variations in immobilized aptamers, were published mainly around 2009.10−16 This picture is the reason that the present review will be centered mainly on reports published since 2010. Biomolecules can be immobilized on electrode surfaces using different procedures,17,18 such as supramolecular interactions,19,20 covalent conjugation, entrapment within a polymer (mainly an organic conductor),21 and physical immobilization.19,22 Nevertheless, covalent conjugation is the method that permits the most stable immobilization of both enzymes and aptamers. A specific and important supramolecular immobilization method corresponds to the avidin (or streptavidin)-biotin complex formation. This method has the advantage that it involves the formation of a highly stable supramolecular associate (KD = 1015 M−1).23 Its inconvenience is due to the complexity of the system, since it involves a previous double conjugation, one for each component (avidin and biotin).7 For 2582


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two types of materials have been reported: gold (also platinum) and carbon-based materials (glassy carbon, graphite, graphene, and nanotubes).31,32 It is necessary to mention that gold constitutes the main type of electrode used for aptamer immobilization (see Table 1).33 A combination of both materials has also served as an electrode for enzyme and aptamer immobilization, especially when gold nanoparticles have been used to coat carbon-based materials.31 The method selected for the immobilization of biomolecules strongly depends on the type of material selected as the electrode. From a commercial point of view, graphite and pencil graphite are preferred due to their low cost.32 Gold electrodes have insignificant adsorption properties and are easily modified through the formation of compact and highly ordered chemisorbed self-assembled monolayers (SAMs).34,35 The modification (coating) of gold electrodes by SAMs is relatively simple and just requires dissolved thiolated (or other sulfur-containing) species within a mM−μM concentration range. Linear long-chain (>12 atoms) thiolates produce the most compact and stable SAMs. Such a SAM permits a flexible immobilization of biomolecules on its surface. However, long-chain aliphatic SAMs affect the electron transfer (ET) process.35 The formation of a SAM on gold should be practically complete after 12−18 h of immersion of the electrode in the solution. This way, a surface coverage within the pmol/cm2 order can be achieved.34 The nature of the terminal moiety of the SAM defines its hydrophobic characteristics, a property that must be in accordance with the predominant nature of the immobilized enzyme. The presence of terminal functional groups in the SAM, such as carboxylic and amino, permits the covalent conjugation of the required biomolecule and/or redox mediator (for enzymes). As can be seen further on, for aptasensors the aptamer is previously thiolated in order to form SAMs on gold electrodes, for which a low surface coverage is necessary. In order to avoid the previously mentioned steric hindrance with the use of spacer arms, the latter should not be much shorter that the coating SAM. The relative length of the spacer arm depends on the nature of the immobilized biomolecule.36 Carbon-based electrodes, contrary to the gold ones, can easily adsorb biomolecules, a method that is simple but does not guarantee stable immobilization of biomolecules or selective adsorption. A compromising and convenient alternative consists of the adsorption of an organic conducting polymer able to entrap the biomolecules, a method used for enzyme-based biosensors. Under such conditions, a redox mediator may not be necessary.21 Carbon-based electrodes can be chemically modified in order to introduce functional groups on their surfaces. By a simple chemical or electrochemical modification, carboxylic groups can be introduced in order to permit the covalent conjugation of the biomolecule. This method presents the inconvenience that a relatively low surface coverage is achieved, a situation that actually favors the necessary conformational stability of the immobilized biomolecule, especially in the case of aptamers.37 Carbon nanotubes and graphene-based electrodes, characterized by high electric conductivity and hydrophobic adsorptivity, can be chemically modified in different ways, all of which permit the introduction of different types of useful functional groups.31 The high absorptivity of graphene is strongly enhanced by its extremely high surface area, for which it can stably immobilize hydrophobic enzymes without any previous modification.38

groups correspond to surface amino acid moieties (Lys, Cys, Asp, Glu, Ser, and Thr). For the covalent immobilization of aptamers, a previous modification is necessary in order to introduce the functional group required for the conjugation. Therefore, there is a difference between the covalent immobilization of enzymes and aptamers. Evidently, the latter can only be immobilized through the previously modified 3′ or 5′ terminal position. On the contrary, the point of covalent conjugation of an enzyme can be any of the numerous surface functional groups. Additionally, more than one conjugation point can generally participate in the covalent immobilization of an enzyme. As a result, the relative position achieved by the immobilized enzyme is random. For the covalent conjugation of a biomolecule, water-soluble 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), assisted by N-hydroxysulfosuccinimide (NHS), is generally used. In this sense, it is important to mention that EDC has been reported to affect the conformation of RNase A upon chemical conjugation, a fact that must be considered when selecting the covalent method.27 The effect of EDC on the conformation of RNase was attributed to the introduction of additional positive charges presumably due to an adduct formation of the enzyme with EDC. The formation of the adduct should have neutralized the negatively charged carboxylate group and couples it to a positively charged tertiary amine.27 For an effective use of immobilized biomolecules, a compromise between dense surface coverage and steric accessibility of the target molecule must be achieved.17,28 On one hand, a high surface coverage should arithmetically favor a high sensitivity of any device containing immobilized biomolecules. On the other hand, the access of the corresponding target molecule to such a crowded surface could be sterically hindered. Additionally, a high surface coverage can easily provoke conformational changes in the immobilized biomolecules, mainly due to both electrostatic interactions and reduced mobility. A way to attenuate the mentioned steric hindrance is by using an adequate spacer arm (tether or linker) between the surface and the biomolecule, which should offer the necessary flexible and effective immobilization, something very important to retain the conformation.29 Nevertheless, spacer arms cannot always solve this problem and not affect the electrochemical response. Therefore, a strict control on the concentration of the biomolecules used in the immobilization process is unavoidable. Circular dichroism is the most appropriate and powerful technique to follow conformational changes in biomolecules, a technique that has been limited to liquid samples. Nevertheless, more recently the chiroptical spectrophotometer (UCS) system for solid state determinations has permitted this limitation to be overcome.30 Other techniques offer valuable indirect information on the conformation of the immobilized biomolecule.6 In the cases in which the biomolecules are immobilized on electrode surfaces to be used as biosensors or biofuel cells, reusability and storage stability (lifetime) are a direct expression of any possible conformational change. The scarce information on these properties in papers on biosensors and biofuel cells is surprising, due to their importance in commercial applications.

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ELECTRODES A diversity of materials has been used as electrodes to immobilize biomolecules. For enzymes and aptamers, mainly 2583


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ENZYMES Enzymes differ from the conformational behavior of aptamers upon the recognition of the target molecule. The conformational change involved in the necessary preorganization process that takes place at the active site of the enzyme involves a variation in only a few angstroms,39 just the amount necessary for access of the substrate, as established by Koshland in his “Induced Fit Theory”.40 Enzymes, as other proteins, possess a defined threedimensional conformation, which depends on their amino acid sequence and noncovalent interactions (hydrogen bonds, and ionic, hydrophobic, and van der Waals interactions). Upon changes in their environment, enzymes unfold and lose their native conformation. This process can be reversible or not. In the latter case, the enzyme is said to be denaturized. The unfolded and folded states interchange reversibly very easily (ΔG = 20 to 65 kJ/mol).41 The composition of aqueous solutions has a strong influence on the conformation of an enzyme since entropically favored hydration, which induces intramolecular hydrophobic interactions, is considered the driving force of protein folding.42 This means that when an enzyme is hydrated, the hydrophobic surface areas tend to interact among each other, a process that leads to a stable folding process. As a result, the hydrophobic areas tend to end buried. The hydrophilic areas of an enzyme do not only depend on the simple presence of polar amino acid moieties. α-Helices can also develop a considerable dipole moment that is able to stabilize the folded conformation of an enzyme.6 The influence of the hydrophobic interactions in the folding process of a protein determines that there is a difference in the mechanism followed by small (single domain) and large biomolecules. In the folding transition states significant amounts (40−96%) of hydrophobic surface areas are buried.1 These factors indicate that when enzymes are immobilized in a crowd, the hydration process is directly affected and so are their conformations.42 When an enzyme is immobilized on an electrode in a biosensor or biofuel cell, it is important that its active center is oriented toward the solid surface in order to achieve a high electron transfer. Under such conditions, the participation of a redox mediator may not be indispensable.21,43 With an adequate orientation of the immobilized enzyme the modified electrode can work more efficiently, as schematically represented in Figure 2.44 However, when the active center of the enzyme points toward the electrode surface, the access of the substrate can be sterically restricted, while the contrary occurs when it is positioned in the opposite direction.

As already mentioned, the surface of an enzyme contains both hydrophilic and hydrophobic moieties.6 Any interaction of these moieties with the electrode surface may affect the conformation of the immobilized enzyme, especially under extreme conditions. An adequate coating of the electrode, and the use of suitable spacer arms, can avoid hydrophilic−hydrophobic interactions that could affect the conformation of the immobilized enzyme. For example, a hydrophobic surface can provoke a loss of αhelical structure with the corresponding gain of β-sheet. Unfortunately, hydrophilic−hydrophobic interactions in immobilized enzymes are complex processes and the information published is still scarce, for which it is not possible to offer general rules in this sense.6 The problem is that the hydrophilic−hydrophobic interactions mentioned also involve the nature of the electrode surface and of the immobilized enzyme itself. Nevertheless, it can be said that apparently mild hydrophilic surfaces are the ones with less effect on the folded conformation of an immobilized enzyme. Enzymes are highly sensitive to any kind of variation in their environment. The simple extraction of an enzyme from a cell can affect its conformation.2 Variations in pH, ionic strength, and temperature, among other factors, affect the conformation of a manipulated enzyme. Once immobilized, the enzyme is expected to achieve a higher stability, but with an implicit loss of activity and conformational flexibility. Such sensitivity of enzymes to the slightest changes is expressed in short-term storage and reusability when utilized in biosensors and biofuel cells.45,46 Precisely, this signifies that an increase in both previously mentioned parameters is a problem that still remains unsolved and requires additional attention. The most widely studied and commercialized electrochemical enzyme-based biosensors are the home glucometers based on disposable, screen-printed enzyme electrode test strips.47,48 A crucial characteristic of these devices is the cheap strips that contain the enzyme (glucose oxidase or glucose dehydrogenase), which are stable at room temperature storage within a relatively wide interval of temperatures (5−40 °C), though this has not been achieved in all cases. The specific characteristics of the available commercial home glucometers that determine their high storage stabilities are protected by patents. Nevertheless, several factors apparently play important roles in this sense. One is the capillary chamber in which the enzyme is retained together with the redox mediator and an enzyme stabilizer (monosodium glutamate, trehalose, bovine serum albumin, etc., and buffer).48 A surfactant to reduce the filling time may also be part of the composition. All of these components are stored in dry form in the chamber and may cover the working electrode.48 This electrode is generally covered by a film or mesh, which should also play a protective role. The nature of the electrode itself also seems to be important, since it is generally a dry carbon ink with high adsorptivity and no net charge. As already expressed, a conformational invariance of an enzyme immobilized on an electrode surface can be considered a synonym of stability. Only a few reports regarding enzymes immobilized on electrode surfaces with a storage stability (at 4 °C) higher than 30 days have been found.49 Even fewer cases have been reported with a storage stability above 100 days. For example, a biofuel cell based on glucose oxidase was reported to retain its activity for up to 120 days.50 Lactate oxidase immobilized in a mucin/albumin hydrogel matrix over a platinum electrode, and covered with a Nafion membrane, was

Figure 2. Schematic representation of how the orientation of cytochrome c adsorbed on ITO can affect the ET process. Reprinted with permission from ref 30. Copyright 2016 American Chemical Society. 2584


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Bioconjugate Chemistry reported to have a 5-month storage stability.51 A less drastic exception is the case of lipase immobilized on graphene oxide decorated with Fe3O4 nanoparticles, for which the reported reusability was of 85% after 56 days and 87% after 10 cycles, both at 30 °C.52 Another biosensor with high reusability (over 90%, 11 cycles) and storage stability (85%, 60 days) consisted of acetylcholinesterase encapsulated within hybrid mesoporous silica membranes deposited on a carbon electrode.53 Values not as high, although acceptable, have been reported.54−57 In contrast, a glucose biosensor with the enzyme immobilized on a poly(glycidyl methacrylate-co-vinylferrocene) film-coated pencil graphite electrode was reported to maintain a stable amperometric response only during the initial 8 days.58 NADH oxidase immobilized on functionalized SWNT only retained 60% to 70% of the initial activity after 10 uses.59 Other reported results are similar,60 or even with lower values than those mentioned above for biosensors.61,62 Similarly, poor results have also been achieved with biocatalysts.63,64 Several procedures have been reported to increase the stability of enzymes immobilized on electrode surfaces. A procedure widely used consists of capping the immobilized enzyme with Nafion, a permeable and highly stable sulfonated tetrafluoroethylene based fluoropolymer-copolymer.65 For example, Nafion has been observed to stabilize the secondary structure of glucose oxidase (GOx) at pH 7.4, while decreasing the content of unordered structural conformations, according to chiroptical CD determinations.50 A proper selection of the type of modification to apply on the electrode surface and the use of covalent conjugation of the enzyme offer a high stability to the immobilized protein.22 Contrary to the covalent conjugation, the entrapment of an enzyme within a polymer could not result in a stable immobilization, especially if it is predominantly hydrophilic. In such cases, the loaded enzyme can be continuously liberated into the solution.66 When a multipoint immobilization of an enzyme occurs, either supramolecularly20,67 or covalently,68 the retention of the native conformation is highly favored. The multipoint immobilization is able to anchor the biomolecule on the electrode surface through different positions, restricting the undesirable unfolding of the enzyme since the entropy involved is reduced. The majority of enzymes immobilized on electrode surfaces are oxidoreductases. The nature of this type of enzyme and the distance of the active center to the electrode surface define the efficiency of the ET process involved. When the ET is inefficient, no electrochemical signal can be observed.43 Enzymes containing prosthetic groups in deeply buried active sites tend to present inefficient ET processes. Nevertheless, the factor that mainly determines an efficient ET is the distance between the active site and the electrode surface, which should be less than 1.5 nm.21 In order to improve the ET process, redox mediators are used. However, a third generation of enzyme-based biosensors is based on designs in which the proximity of active site is near enough to the electrode surface so the use of a dissolved mediator is unnecessary.21 Hemoproteins immobilized on electrode surfaces are highly responsive to applied potentials. The electrochemical expression associated with a loss of conformation of an immobilized hemoprotein can be the observance, either of weak electrochemical signals or, in the best case, a shift of the redox peak or the appearance of new peak(s). For example, for cytochrome c oxidase immobilized on an electrode surface, a

new non-native heme species was detected with the redox peaks shifted toward more negative potential values due to the axial loss of one histidine ligand.69 A redox peak shift was also observed for cellobiose dehydrogenase immobilized on silver electrodes and was attributed to changes in the secondary structure of the enzyme according to electrochemistry surfaceenhanced resonance Raman (SERR) and surface-enhanced infrared absorption (SEIRA) spectroscopies (1630−1695 cm−1 region).70 Enzymes immobilized on electrode surfaces generally require storage at 4 °C, which is a strong limitation for outdoor uses. In order to overcome this limitation, enzymes obtained from thermophilic microorganisms, which can support wider temperature variations and longer storages, are starting to be studied.31 These enzymes, denominated hyperthermophilic or thermophilic, are singular proteins able to support temperatures above 100 °C. More than a difference in composition, the amino acid residue distribution and their interactions are more relevant factors in the stability of the thermophilic enzymes. The extremely high stability of the thermophilic enzymes has been mainly attributed to a combination of different factors among which α helix stability, disulfide bridges, and hydrophobic and aromatic interactions seem to play the major roles. Apparently, the thermostability of each type of thermophilic enzyme is mainly due to one of the mentioned factors and no generalization is possible. What does seem to be a general rule is that in the thermophilic enzymes both hydrophobic interactions and disulfide bridges are deeply buried in the protein structure.41

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APTAMERS An aptamer is an artificial single-stranded DNA (ssDNA) or RNA oligonucleotide that is able to bind a specific molecular target with high affinity, generally by means of a strong variation in conformation.71 The most studied application of aptamers immobilized on electrode surfaces is as biosensors (aptasensors).72−75 As oligonucleotides, ssDNA aptamers are negatively charged species due to the numerous (one per base) anionic phosphate groups contained. An aptamer can reversibly adopt a folded or unfolded conformation depending on its environment. When the negative charges of the phosphate moieties are able to express a strong repulsion, the conformation of the aptamer tends to be linear (unfolded). On the contrary, when negative charges of an aptamer are neutralized it can form intramolecular H-bonds and adopt a folded conformation, generally characterized by the presence of different types of loops and/ or knots. The folded conformations of an aptamer76 can vary to or from hairpin (also termed stem-loop),76−78 G-quadruplex,79,80 or pseudoknot structures,81 which are schematically represented in Figure 3.

Figure 3. Schematic representation of the main conformations of aptamers. 2585


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ferrocene,95,96 or including both,97 at the distal end (Figure 4). Depending on the nature of the conformational change, the

The nitrogen base sequence is the parameter that determines the molecular recognition capacity of an aptamer. Each aptamer, according to its sequence, is able to express a very high affinity for a specific target molecule. Upon binding the target, the aptamer can undergo structural changes in order to permit the maximum affinity.82 The conformation of an unfolded aptamer can be strongly, but reversibly, affected by the charge of the electrode on which it is immobilized. In contrast, immobilized aptamers with a folded conformation are able to significantly reduce the electrostatic interactions with charged electrodes. Negatively charged unfolded aptamers tend to be vertically oriented when immobilized on negatively charged electrodes.83 A similar situation is observed when large aptamers (from 15mer up) are adsorbed on graphene oxide, which does not occur with the dsDNA conformation. As expected, a decrease in pH and increase in ionic strength favors that adsorption,84,85 and therefore, these parameters must be strictly controlled. Additionally, the presence of unfolded aptamers can favor the desorption process through hybridization.86 The primary criterion for the immobilization of an aptamer is to not compromise its molecular recognition ability,87 which can involve strong conformational changes. The techniques mostly employed for aptamer immobilization are based on covalent interactions. For the covalent conjugation of aptamers, spacer arms are commonly used. These spacer arms should possess a thiol, carboxylate, or amino terminal group, among others, able to anchor on gold,88 aminated or carboxylated carbon surfaces, respectively.71,89,90 Large-scale immobilization of thiolated aptamers on a gold surface can produce thermodynamically stable and highly ordered self-assembled monolayers (SAMs).34,35 These SAMs should be compact and highly ordered, but able to permit the accessibility of the target molecules, and the necessary conformational changes that must occur in the recognition process.28 The use of mixed SAMs that combine thiolated aptamers and mercaptohexanol permit a more free conformational change of the immobilized aptamer, without electrostatic interactions with the surface of the electrode. Electrochemical atomic force microscopy has been used to reveal the conformations and dynamics of aptamer-based SAMs. Defects in this type of SAM were found to significantly perturb the conformations and adsorption/desorption kinetics of surface-tethered DNA.91 Entropic and electrostatic effects on the folding energy have been observed to induce the dissociation of an aptamer from the electrode surface, a process that is expressed at low ionic strengths (below 130 mM).77 The authors interpreted this behavior as the result of two competing mechanisms: excluded volume effects and electrostatic repulsions. While the excluded volume effects stabilize the folded conformation by reducing the entropy of this state, the negative charges of the aptamer provoke a strong repulsion from the charged surface. Aptasensors are mostly based on conformational changes of the immobilized aptamers upon their interaction with the target molecule.92 The conformational changes of immobilized aptamers can be electrochemically detected using different techniques, such as alternating current voltammetry, square wave voltammetry, cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy.72,93 The sensing mechanism can be based on a target-induced folding−unfolding of the immobilized aptamer containing a pendant redox reporter, typically methylene blue 94 or

Figure 4. Scheme of an aptasensor containing a redox reporter based on a conformational change that permits an approach of the latter to the electrode surface.

redox reporter may move closer to the electrode surface (“signal-on” sensor) or further away from it (“signal-off” sensor). Plaxco and collaborators have analyzed the binding-induced folding of aptamers, considering both signal-on and signal-off sensors (Figure 4).8,9 The work of this group has continued in the present decade with another review centered on the genetic detection of pathogens.98 The articles of Plaxco’s group on aptasensors during the present decade can be found in Table 1, and others have been expressly mentioned throughout this review.99 The articles on electrochemical aptasensors cited in Table 1 correspond to reports in which conformational changes are defined (or assumed) to explain the way the devices work. There are other reports in which the conformation is just mentioned, but not defined in detail.83,90,100−122 It can be observed that gold, including its nanoparticles (AuNPs), constitutes the surface that has been mainly used to associate the selected aptamer containing a thiol group, either directly tethered (−SH) or, more commonly, through a spacer arm containing that same terminal group. Aptamers, in contrast to enzymes, are thermostable which minimizes the storage stability problem. Aptamers exhibit high affinities for specific targets, which are expressed in the very low limit of detection (LOD) values determined for the developed aptasensors (column 4, Table 1). The conformation achieved upon target recognition can be reversed by simply rinsing with water, or using saline solutions,154−156 cycles of washing and drying with ethanol and nitrogen gas,157 changes of temperature,156,158 or pH,155,159,160 or using detergents.155,161,162 With these different regeneration methods, the sensor can be reused to a limited extent (10 to 25 cycles) for target detection. In other cases, the reusability of the aptasensor can be more complex involving interactions with other biomolecules.159,163,164 The reusability of an aptasensor involves the liberation of the associated target molecule, which is generally not totally completed in a single process. Therefore, the continuous regeneration of an aptasensor can imply an accumulation of associated target molecules, a fact that limits the quantity of useful cycles. Only robust aptasensors are able to endure repeated regeneration processes. For example, an increase in acidity can be used for the regeneration of an aptasensor based on a thiolated aptamer immobilized on a gold electrode due to the neutralization of the negative charges of the phosphate moieties that provokes a conformational change.155,159,160 This 2586


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order to attain marketable outputs. Considering the prospective applications of graphene in the immobilization of biomolecules,85,38 graphite electrodes covered with graphene could become an acceptable alternative.

way, the target is liberated but, unfortunately, at the same time, the Au−S bond is weaken and can cause the dissociation of the aptamer.

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OUTLOOK The molecular recognition ability of enzymes and aptamers completely depends on their conformation. For this reason, we have tried to emphasize the importance of knowing and controlling the conformation, and its changes, of these biomolecules when immobilized on electrode surfaces. This necessary information can aid the researchers in designing efficient, stable, and reusable bioelectrochemical devices. Further studies on these topics must be conducted in the following years, especially those devoted to aptasensors, which have practically unlimited potentialities and with few, and only partial, conformational studies reported. Enzymes with higher thermostability than those already reported must be obtained, mainly by means of biotechnological engineering,165 especially if de novo computational design is used.3 Apparently, this constitutes the most prospective way to create bioelectrochemical devices with high reusability and storage stability. There are two main approaches in enzyme engineering, directed evolution and rational design, which are not exclusive. Both are based on the necessary gene mutagen in order to achieve an amino acid sequence able to improve the desired property of the enzyme, From a biosensor point of view, the enzyme must become more thermostable, such as thermophilic enzymes. In this case, the DNA of these enzymes is genetically modified by the introduction of an adequate amino acid sequence in order to increase the number of H-bonds, disulfide bridges, and so forth.165 Aptamers, as synthetic biomolecules, can be simpler designed (than enzymes) in order to achieve an optimum reusability when knowing the conformational changes involved in the molecular recognition processes. An adequate selection of the regeneration method to be used should contribute to achieving aptasensors with high reusability. In order to retain the necessary conformation of immobilized enzymes and aptamers more attention must be paid to the type of modification to be made on the electrode surface, mainly when using covalent conjugation. The coverage of enzymecontaining systems with Nafion and/or polymers/hydrogels should favor an increase in the stability of immobilized biomolecule.166,167 On the contrary, in aptasensors the immobilized aptamers must be able to freely vary their conformation, while the strength of the immobilization is actually the factor determinant in the stability of the device. Actually, the immobilization strategy used is important for both biomolecules.168 According to a recent publication, in the function of aptasensors (at least for guanine rich aptamers) much attention must be paid to the ionic strength. The report developed a strong influence of potassium on the conformation of PW17 (5′-GGGTAGGGCGGGTTGGG-3′). For example, for K+ concentrations less than 0.5 mM PW17 adopted a loose and unstable G-quadruplex conformation, while it became compact and stable for cation 7 mM concentrations or higher.169 Both biosensors and biofuel cells have high potential and are necessary in modern life. Therefore, we consider that future studies should be oriented toward the use of easily manipulated170 and low cost electrodes, such as graphite (especially in disposable screen printed carbon electrodes), in

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. # On leave from the University of Havana, Cuba.

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ACKNOWLEDGMENTS Prof. Cao thanks CONICYT (Chile) for PEI 2016 grant. Dr. A. Tello thanks the US Air Force, USAF AFLR SOARD for project: FA9550-15-1-0140-52015-20106. M. J. Marchant thanks CONICYT (Chile) for Doctoral Fellowship 21130616.

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