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In-depth electrochemical investigation of surface attachment chemistry via carbodiimide coupling Marsilea Adela Booth, Karthik Kannappan, Ali Hosseini, and Ashton Partridge Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01863 • Publication Date (Web): 24 Jun 2015 Downloaded from http://pubs.acs.org on July 6, 2015

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In-depth electrochemical investigation of surface attachment chemistry via carbodiimide coupling Marsilea Adela Booth,§ Karthik Kannappan,§ Ali Hosseini,§,¥,* and Ashton Partridge§,¥,* §

Digital sensing Limited, 16 Beatrice Tinsley Crescent, Albany, Auckland 0632, New Zealand,

¥

Department of Chemical and Materials Engineering, The University of Auckland, Private Bag

92019, Auckland, New Zealand KEYWORDS Carbodiimide coupling, EDC and NHS, aminoferrocene, probe density.

ABSTRACT

Aminoferrocene is used as an electroactive indicator to investigate carbodiimide coupling reactions on a carboxylic acid functionalized self-assembled monolayer (SAM). The commonly used attachment chemistry with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is used for surface activation. A number of conditions are investigated including EDC and NHS concentration, buffer solutions, incubation timing and aminoferrocene concentration. Ferrocene is a well-documented electroactive species and the number of surface-bound ferrocene species can be calculated using electrochemical methods. This capability allows determination of optimal conditions, as well as providing a method for comparing and investigating novel carboxylated surfaces. An EDC-mediated procedure with ~5

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mM EDC and NHS (1:1) made in water, with a full acid monolayer, with 250 µM aminoferrocene for 40 min was found to give the highest ferrocene attachment. An application of this is demonstrated for preparing a probe-DNA coated surface for DNA sensing. By backfilling with aminoferrocene a differential quantification of the amount of probe DNA available for sensing can be obtained. This provides an elegant method to monitor an important aspect, namely probe surface characterization which will be highly useful for biosensing purposes.

INTRODUCTION. The controlled, reproducible construction of functionalized surfaces is vitally important for the development of cheap, reliable biosensors.1 The term biosensor denotes a sensing device which incorporates three main elements, a biological capture probe (supporting information SI2 Fig. S1) which is selective for a particular target, a recognition event between the probe and target, and a transduction mechanism to convey this recognition. Commonly utilized recognition events include recognition between antibodies and antigens, between complementary DNA strands and between synthetic probes and targets.2-5 Generally the ‘probe’ species requires anchoring to the biosensor surface, a key element in the fabrication of a successful biosensor. Using a convergent method (separating the surface modification step from probe attachment) imparts diversity and simplicity. Surface modification of gold electrodes is often performed using self-assembled monolayers (SAMs) of alkanethiols6, 7 with reactive terminations (amines, carboxylic acids and azides). The monolayers are readily formed and provide a well-defined, liquid crystalline, stable anchor to electrode surfaces. As a result they are well studied and are routinely used in biosensor


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technology.8-13 Subsequent covalent attachment of biological species is often based on the carbodiimide coupling between carboxylic acids and amines, commonly utilizing 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrcholoride (EDC) and N-hydroxy succinimide (NHS) in aqueous solutions, and under physiological conditions. The extensive use of this reaction in biosensor technology, as well as other areas, is due to the number of advantages that this coupling methodology offers. The ease and simplicity of the process, it’s versatility, ‘biocompatability’ and applicability in aqueous solutions, the lack of any chemical length addition during functionalization (only an amide bond is formed) and finally the ease of the synthetic functionalization of probe species with amines or acids (when necessary) while imparting little effect on bioactivity all add to this method’s allure. Surprisingly, despite all the advantages that this reaction provides and its extensive use, not all the factors affecting the efficiency of the reaction and its optimization14 are understood or explained; variability exists in coupling efficiencies15 and a large amount of ‘folklore’ is associated with it. Even though there is growing research into alternate techniques such as azidealkyne cycloaddition and azide-triarylphosphine reaction chemistry,16-18 carbodiimide coupling remains a popular method for biosensor surface preparation.14 Analysis of surface reaction is inherently difficult and as a result a number of techniques have been used to study carbodiimide coupling on surfaces, including SPR, FT-IR,1, 17-20 electrochemistry,9, 14, 21 and more recently polarization-modulation infrared reflection–absorption spectroscopy (PM-IRRAS) and time-offlight secondary ion mass spectroscopy (ToF-SIMS).15 Meanwhile, the use of electrochemistry provides both sensitivity and simplicity, and in addition allows for in-situ measurement. Liu, Xia and coworkers studied surface EDC/NHS coupling by monitoring changes in the blocking of electron transfer, utilizing the redox couple [Fe(II)(CN)6]4-


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/ [Fe(III)(CN)6]3- and cyclic voltammetry.14 Although this approach is effective at monitoring surface changes, it provides little information on the actual yield of desired species on the surface, as blocking experiments involve diffusion limited processes and are not heterogeneous. Subsequently, they immobilized electroactive biomolecules (dopamine, amyloid-β-Cu2+ and laccase) on a surface under different conditions, and used cyclic voltammetry to compare the biomolecule coverage.21 This method is able to provide information on the attachment of electroactive biomolecules, however it is not suitable for monitoring the attachment of nonelectroactive species and is only used for comparison, not quantitation. In order to visualize and quantify surface attachment, we use the covalent attachment of a redoxactive chemical with amine functionality, aminoferrocene (FcAm). Ferrocene (Fc) as an electroactive indicator is known to be relatively cheap and stable, highly sensitive (imparted by the electroactive properties of Fc) and has been widely studied.22 The redox potential of Fc can impart information on local environment, including whether the Fc species are immobilized or in solution (minimizing false positives), including the nature of immobilization whether covalent or simply surface adsorption Fc attachment has been used to monitor azide-alkyne cycloaddition reactions16, 23, 24, 25 and for characterizing SAMs.26 Synthesized Fc derivatives containing primary amines in particular have been used to monitor coupling efficiency to N-hydrosuccinimidylterminated SAMs by Wang and co-workers.9 We aim to extend the understanding of the surface carbodiimide reaction specifically to cater for biosensor applications, and also to introduce an elegant method for quantifying attached biological probes. In this approach, detailed analysis of cyclic voltammetry is used to provide quantitative information on the covalent attachment of Fc, and therefore the surface carbodiimide reaction. In turn, this information is used to compare conditions for surface attachment and investigate new


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surfaces in terms of –COOH functionality and accessibility.27 Furthermore, this technique is extended to investigate the amount of probe DNA attached to a surface. This demonstrates a highly useful application of the approach to aid in the design, and optimization of the conditions employed in capture agent attachment for biosensor devices.*

MATERIALS AND METHODS. 2.1. Chemicals and materials 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), mercaptoundecanol (MUOH), mercaptodecane (C10 alkane), mercaptohexanol (MHOH) and 11-mercaptoundecanoic acid (MUA) were purchased from Sigma. Aminoferrocene (FcAm) was purchased from TCI America. Phosphate buffered saline buffer (PBS, pH 7.4) was made from tablets purchased from Sigma, 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.4) buffer was purchased from Sigma, while HBS-EP buffer (containing 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), pH 7.4) was purchased from GE Life Sciences. DNA was synthesized by Gene Link, Inc. (USA), with an amine functionality and the sequence 5'[AmMC6]AAACCCAGCAG-3'. All reagents were used as purchased. Solutions were prepared with deionized water (pH 6.2 ± 0.3). 2.2 Procedures

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FcAm attachment can be used as a guide for attaching biologicals, however, the diffusion rates, sizes and solution behavior of biologicals may differ. Fc is able to provide important information for experiment optimization, surface characterization as well as testing reproducibility.


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A detailed methods section can be found in the supporting information, section SI1. Working electrode surfaces were cleaned electrochemically using 0.5 M H2SO4 by continuous cycling between -0.35 and 1.65 V at 250 mVs-1 until gold reduction peaks appeared stable. Electrodes were then immediately washed with water and immersed in SAM solutions containing specified values of MUA and diluent 1:1 (either C10 alkane, MUOH or MHOH as specified) with the total SAM concentration a constant of 5 mM in ethanol, for 20 min. The electrode was then thoroughly washed with ethanol, water and PBS before baseline CVs were taken in PBS to ensure a stable monolayer had formed. EDC/NHS solutions were formed by mixing a 1:1 mole ratio and vortexing. Each solution was made fresh. Incubations were for 40 min or as specified. For DNA probe attachment experiments, DNA solutions were diluted to the desired concentration with PBS buffer and mixed with EDC/NHS solution (final concentration 5 mM, 1:1 EDC:NHS). FcAm solution was a 9:1 solution of PBS:DMSO, with a specified concentration of FcAm (stock solution made in DMSO), and incubations were performed for 10 min or as specified. FcAm solutions were made freshly as the ferrocene was seen to degrade over time, SI2 Fig. S2B. This degradation is thought to be a combination of the decomposition of the ferrocene moiety as well as the hydrolytic cleavage of the ester linkage.28 Thorough washing followed before re-measurement in PBS solution. 2.3 Electrochemical measurements Electrochemical measurements were performed on a CompactStat potentiostat from Ivium Technologies (the Netherlands). Cyclic voltammograms (CVs) were collected using a threeelectrode cell set-up with a platinum wire counter electrode and Ag/AgCl (3 M NaCl, +0.197 V


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vs. SHE) reference electrode. Working electrodes were flat gold sputter-coated substrates, with the working area isolated using an O-ring (3/8 inches I.D.) and with Teflon wells (9 mm I.D.) above. Measurements were performed in PBS buffer. The majority of scans were performed between -0.1 and 0.3V at 1000 mVs-1. 2.4 Data analysis Data was analysed using a combination of IviumSoft (Ivium Technologies) and Origin (OriginLab). A background CV scan in PBS was taken of each electrode prior to FcAm exposure, and a subsequent CV was taken after FcAm incubation. The peaks arising from the oxidation and reduction of surface bound Fc (confirmed as not being solution/non-adsorbed Fc, supporting information SI2 Fig. S2A) were then integrated (detailed analysis SI1.2). Controls show no significant binding of Fc in any of the conditions tested SI2 Fig S3. Integration of the Fc peak allows calculation of the number of Fc molecules/cm2, in-depth information can be found in the supporting information, section S2.3. Briefly, the peaks allow calculation of the charge arising from the redox activity of Fc, providing Fc molecular concentration which together with surface area can give a value of Fc molecules/cm2. Meanwhile, together with the theoretical value of an alkanethiol monolayer28, 29 a rough estimate of percentage Fc coverage can be calculated (with accompanying assumptions), section S2.3. A typical cyclic voltammogram series is displayed in Fig. 1, where no redox peak is observed in the monolayer before FcAm exposure, nor is any observed when no surface activation is performed, however an oxidation peak at approximately 116 ± 12 mV (n = 10), and a reduction peak at 77 ± 8 are observed under these conditions. A shift in potential is observed in comparison to literature Fc peaks16 however we believe this to be an effect of both the electrolyte (in our case


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PBS buffer) as well as the attachment chemistry and local environment of the Fc molecules, as other references have found all are important for Fc potential.8, 29-31 The peak current displayed a linear dependence on scan rate, see inset Fig. 1.30 The oxidation and reduction peaks were seen to be of similar integrals, indicating a fairly reversible process, and also suggesting covalently bound ferrocene. The width of the current peaks is strongly dependent on the distribution of individual ferrocenes.23 For non-interacting ferrocenes the entropically determined, ideal full width at halfmaximum (fwhm) is 90 mV.23, 29 Amide-bound ferrocene peaks had a fwhm of 134 ± 15 mV, therefore suggesting there may be some ferrocene interactions, or ferrocenes existing in a range of environments, more likely being the latter due to the low coverages observed.26, 28, 29

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Figure 1. Cyclic voltammograms of the monolayer before (blue solid line) and after derivitization by aminoferrocene (red solid line), as well as a control where no surface activation occurred before FcAm exposure (black dashed line). Scans were performed in PBS solution with a scan rate of 1 V/s (vs Ag/AgCl reference) and arrows indicate scan direction. The inset shows the plot of anodic peak current of Fc vs scan rate.


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Surface area for the electrode can be calculated in two ways – either by mathematical calculation of the gold surface exposed, Ageometric, or using the reduction peak arising from gold reduction in sulfuric acid, Aelectrochem.5, 33, 34 Methods for calculation are discussed in the supporting information, SI1.3. The former gives a physical value, however it assumes the substrate is an atomically flat plane and does not account for slight differences in surface area for example arising from differing O-rings or pressure. The latter is an indication of surface roughness and gives individual electrode information, but is an estimate due to the way it has been calculated with underlying assumptions.33, 34 We used Aelectrochem (SI1.3 and SI2 Fig. S4) as a measure of electrode surface area with the understanding that the areas are being used for comparative purposes,34 and that our surface areas may be overestimated using this method, hence our Fc coverages may be underestimated. The surface roughness factor (calculated by Aelectrochem/Ageometric) of our electrodes was on average 1.32 ± 0.14. RESULTS AND DISCUSSION. The mechanism for EDC NHS activation is well established,1, 14, 15, 35, 36 along with the possible side-reactions. Scheme 1 illustrates the number of possible pathways to both activation and deactivation of surface carboxylic acids. It is desirable to minimize the deactivation pathways in order to maximize the surface coverage of desired probe.1 Essentially, successful carbodiimide coupling can be broken down into two principal steps; surface activation followed by amide coupling. Surface ‘activation’ involves the generation of a succinimidyl ester-terminated surface layer, in this case from a carboxylated SAM. Subsequent ‘amide coupling’ of an aminecontaining probe species occurs to yield a surface with covalently bound probe.


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With this work we aim to gain fundamental understanding of what factors affect the efficiency of the surface carbodiimide coupling reaction. This is achieved by monitoring surface attachment with variations in the EDC/NHS concentrations, buffer solutions used for the activation of carboxylic acid and subsequent amide coupling, a one-step (EDC/NHS and amine-species) vs. two-step (EDC/NHS and then amine-species) reaction process, EDC/NHS incubation time, acid to diluent ratio used in the SAM, variations in the diluent, FcAm concentration and incubation time with FcAm. Although EDC alone can be used for surface activation, the importance of NHS addition has been proven.1, 37, 38 The scope of this work does not cover the effect of the variations in the pH of the solution on surface activation and the subsequent amide coupling efficiency as this has previously been demonstrated.14, 21, 35, 37, 39 Furthermore, the choice of the pH is highly dependent on the experimental conditions required; as most biological systems require solutions close to physiological pHs. However, pHs between 4.5-7.5 are commonly used with success,14, 21, 37

in this work we chose either deionized water (~pH 6.2) or buffered physiological pHs for

EDC/NHS activation, and physiological pHs (~7.4) for amide coupling.

Hydrolysis N

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Scheme 1. Mechanism1 indicating the pathways for activation reactions with carbodiimide coupling, A, a carboxylated thiol-SAM is formed on the gold surface, B, EDC addition forms an activated O-acylisourea surface (note, EDC is shown in neutral form, actually present in cationic form1). A number of different reactions can occur, dimerization to form C, formation of Nacylurea D (undesired reaction) or with NHS forming E, the succinimide intermediate (desired reaction). The anhydride C can also form the succinimide intermediate E with NHS, or can be hydrolysed back to the carboxyl groups A. Finally after addition of FcAm a Fc covered surface F is formed. 1. Effect of EDC and NHS concentrations and solutions, as well as one-step versus two-step processes on ferrocene coverage We initiated this work by looking at the effect of EDC and NHS concentrations. In literature there is a lack of consensus on the concentration of the EDC/NHS for optimal activation of surface carboxylic acids. Reported concentrations range from a few µM up to 0.4 M.1, 2, 14, 36 Even studies on the optimization of EDC/NHS concentration show differing results.1, 14 Therefore, we systematically changed the EDC/NHS concentration by an order of magnitude from 400 mM to 0.4 mM while keeping all other variables constant. The success of this coupling was evaluated by the integrated charge of the Fc redox reactions during cyclic voltammetry to give the number of covalently bound Fc molecules and % surface coverage. We observe that 4-5 mM EDC/NHS leads to the highest coverage of immobilized Fc on the surface, while high EDC/NHS concentrations (400 mM) imparted low Fc immobilization (Fig. 2) as well as carbon dioxide effervescence. This optimal concentration of EDC/NHS is consistent with previously reported work by Sam et al. using infrared spectroscopy to monitor the


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transformation of carboxylic acid groups to succinimidyl ester-terminated groups.1 This further supports the hypothesis that acquiring optimal EDC and NHS concentrations involves a balance between efficiently activating the majority of surface –COOH groups while preventing the formation of deactivated product N-acylurea.1, 15, 35 The EDC/NHS concentration of 4-5 mM appears a good compromise for our surface and was therefore used for remaining experiments.

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Figure 2. A) Bar graph indicating the coverage of ferrocene versus concentration of EDC and NHS used (1:1). B) Cyclic voltammograms of films incubated with different concentrations of EDC/NHS prior to FcAm incubation, i) 4 mM EDC/NHS, ii) 0.4 mM EDC/NHS, iii) 40 mM EDC/NHS and iv) 400 mM EDC/NHS. The apparent low coverages of ferrocene when compared to literature reports, for example 1 % coverage as compared to 50 % coverage,3, 16 may be in part due to the carbodiimide coupling process. Collman and coworkers used azide-alkyne cycloaddition of acetylene ferrocene to an azide-terminated monolayer, a reaction which has been shown to be high-yielding.16 Meanwhile, carbodiimide literature reports significant variation in the yield (10% to 90%) of activated carboxylic acids and subsequent amide coupling.3, 19, 36 We hypothesize that the combination of low efficiency of activation coupled with the overestimated surface area (section 2.4), and


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potential steric hindrance from the lack of linker between the amine group and ferrocene,9 leads to the observed low coverages.36 Where possible, we aimed to reproduce conditions compatible with the biological species needed for the fabrication of biosensors, for example using shorter incubation conditions (40 min as compared to 20 h),9 and aqueous media close to physiological pH for EDC/NHS incubation. We observed little influence of the chosen solution on the overall reaction yield, be it PBS, HBS, or MES buffer (SI2 Fig. S5A). DI water, however, gave the highest binding and therefore was used for further experiments. Carbodiimide coupling can be performed either as a one-step (EDC/NHS and aminecontaining species together) or a two-step process (EDC/NHS exposure then subsequently amine-conatining species).3 As observed by Walsh et al.3 and Nakajima et al.,35 a one-step process with our conditions gave marginally higher coverage of Fc (SI2 Fig. S5B). When only primary amines are present on the probe species a one-step covalent attachment is possible. However, for larger molecules with amine and carboxylic acid groups present a two-step process, where only one carboxylic-acid-containing component (in our case the surface) is exposed to EDC/NHS, can be advantageous to prevent cross-linking and self-conjugation between probe species e.g. with antibodies or proteins.40 The majority of the experiments performed here used a two-step attachment process. Another variable considered was EDC/NHS incubation time for the surface activation ranging from 5 to 120 min. Previous reports suggested the activation of carboxylic acid groups proceed within 20-30 min, while longer incubation times may result in lower amine coupling efficiency due to the competing hydrolysis of the ester in water.14, 15 Similarly, we also observed activation


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after 20-30 min, SI2 Fig. S6, however, our results showed maximum binding after approximately 40 min of activation, therefore this incubation time was used. 2. Comparing ferrocene coverage while changing diluent type and acid to diluent ratios The next variable to be investigated was the composition of the underlying monolayer. A number of studies exist which have examined diluent choice and length in monolayers.8, 9, 29, 30, 41 The choice of diluents in biosensor may be influenced by the biological probe or species being attached. For example when attaching a DNA strand to a surface, the negative charges arising from OH functionalities under basic conditions may be beneficial to the DNA strand’s orientation,41 or alkane diluents may aid in tighter monolayer packing42 for a different surface. Here, three diluents were chosen to examine their effect on Fc surface coverage. These were an alkane diluent with 10 carbons (C10 alkane) – chosen for its neutral and hydrophobic nature, an alcohol terminated diluent with 11 carbons (MUOH) – chosen as a negative diluent often used in biosensor monolayers43 and a similar alcohol terminated diluent with 6 carbons (MHOH) – chosen for its shorter length. The monolayers were formed in ethanol solution containing a 1:1 ratio of mercaptoundecanoic acid (MUA) with diluent. Subsequent attachment of Fc showed the diluent used had little influence on the surface coverage of the attached Fc molecules, Fig. 3A and B. A slight increase in binding is observed for the C6 alcohol, which may be due to the fact that the difference in height between the diluent (C6) and the functional group (C11) allows greater access for the FcAm, with less steric hindrance.9, 23 There is, however, a slight broadening as well as a shift in potential of the Fc redox peaks with MHOH diluent as compared to the MUOH diluent. It is known that the length and polarity of the diluent group may affect the local environment the Fc resides in.8, 30


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We hypothesize that the positive shift in potential when MHOH is used as diluent compared with MUOH, arises from the presence of defects within this film, that is due to its shorter chain.31 These defects in turn may also cause the attached ferrocene to exist in a range of environments, giving rise to the broadened peak. The broad redox peak observed with C10 alkane diluent, Efwhm of 151 ± 8 mV compared to 134 ± 15 mV for MUOH films, can also be attributed to the range of environments in which bound Fc resides.29

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Figure 3. A) A bar graph indicating the coverage of ferrocene with a 1:1 ratio of mercaptoundecanoic acid and three diluents, a C11 alcohol, C10 alkane and C6 alcohol. B) Cyclic voltammograms after Fc attachment for a film with a 1:1 ratio of mercaptoundecanoic acid and i) C6 alcohol, ii) C11 alcohol and iii) C10 alkane. C) A scatter plot depicting the relationship between the fraction of acid within the monolayer (remainder is diluent MUOH),


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and Fc coverage. D) Cyclic voltammograms showing the increased Fc surface coverage with varying acid fraction within the monolayer (remainder is diluent MUOH). The fact that there was not a large increase in Fc coverage observed with a shortened diluent, suggested that steric hindrance is not the limiting factor in Fc attachment to the monolayers. We then investigated the relationship between observed coverage and changing the acid to diluent ratio. A slight potential shift was observed with increasing acid fraction within the monolayer, explained by the increased polarity of the acid groups relative to alcohol group diluents. Increasing amounts of acid within the monolayer were found to yield increased Fc surface coverage, with a maximum attachment from a full acid monolayer. A similar observation was found by Fabre et al. who showed increasing Fc surface coverage with increasing fraction of Fc binding moieties.26 Here, Fc coverage shows 2 % bound Fc, (equating to approx. 9.6 × 1012 molecules/cm2) which is about 10 nm2 per ferrocene (assuming distribution homogeneity). At this low coverage, steric hindrance from a neighbouring Fc is unlikely to have an influence. Furthermore, this suggests the limiting factor for Fc attachment in this system may be surface activation rather than the FcAm coupling step. Indeed, Touahir et al. report increased carbodiimide surface activation occurred with a 100 % acid film as compared to the use of mixed monolayers. They attribute this to a decreased fast exponential process (one of the two processes suggested to occur for EDC/NHS activation) when fewer carboxylic acid groups are present. This finding is supported by simulation of the fraction of acid sites able to react through the anhydride pathway (Scheme 1 pathway C to E) as a function of acid sites on the initial surface,20 whereby it follows that a higher acid concentration gives rise to more species undertaking the anhydride to succinimide pathway. 3. Influence of aminoferrocene concentration and reaction time on ferrocene coverage


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When fabricating biosensors the biological probes are often attached at relatively low concentrations, perhaps due to the cost of the probes or to prevent steric interferences on the surface. A wide range of solution probe concentrations is not commonly investigated during biosensor optimization; hence it was of interest here to investigate how changing the concentration of FcAm in solution affected surface coverage of Fc. Increasing FcAm concentration lead to increased Fc coverage which saturates at approximately 250 µM. Furthermore, the time dependency of the incubations with FcAm appeared to maximize the surface coverage of Fc after which saturation was achieved, Fig. 4B. Xia and coworkers found similar trends with dopamine attachment, whereby prior to saturation, higher dopamine binding was observed when higher concentrations of dopamine were used. It should be noted that while FcAm was used as a model compound here, the size and property differences between FcAm and other biological probes (e.g. antibodies) may afford differences in optimal conditions.36 This

6E12

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does, however, provide guidance for example conditions to be used for attaching other species.


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squares denote control experiments where no EDC/NHS was added to the surface prior to FcAm incubation. B) The dependence of Fc coverage on incubation time. 4. A novel quantification method to estimate ‘Probe’ coverage for a biosensing surface Of great importance for reproducible and reliable biosensor readings is the quantification and indication of surface bound probe species.44 Therefore, a number of methods have been developed to estimate the concentration of surface carboxylic acid groups or coverage of the biological probes.35, 45-47 Inherently these techniques, however, have many disadvantages, such as significant non-specific binding which requires rigorous control experiments, and expensive labelling for detection. The use of FcAm can be extended to differentially quantify surface coverage of the bound probe, in turn removing most of these obstacles. We tested this by immobilizing single-stranded DNA (probe DNA). As the surface coverage of DNA probes has been shown to produce a profound effect on the hybridization of DNA target;48 precise control and quantification of surface probe species holds great importance. The differential quantification method whereby two surfaces are compared is summarised in the scheme in Fig. 5A. A ‘maximum coverage’ measurement is performed using FcAm to indicate the capacity of the –COOH surface. The second surface (‘probe bound’ measurement) initially involves the binding of the desired probe species (in this case probe DNA), followed by subsequent exposure to FcAm to bind any remaining activated surface species. A comparison in the Fc coverage of these two surfaces can then quantify the surface coverage of the bound probe DNA species. As cautionary notes, the ‘maximum coverage’ experiment must possess identical conditions to the ‘probe bound’ experiment (particularly where length of time for incubation is concerned).


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Where longer incubations are required (e.g. overnight incubations) re-activation of –COOH groups with EDC/NHS may also be required (for both experiments) before FcAm incubation.

Figure 5. A) Schematic of the method proposed for determining species coverage on a surface, here single-stranded probe DNA, i) a ‘maximum signal’ measurement is performed where the – COOH surface is carbodiimide activated after which incubation with FcAm occurs. The Fc coverage then can be calculated. In ii) the surface is activated and incubation with an aminefunctionalised probe DNA is performed (in this case surface activation and DNA incubation are performed as one step). Subsequently the surface is incubated with FcAm, giving a ‘probe bound’ measurement (overall process a two-step process is used). A comparison of the Fc coverages of the final surfaces in i) and ii) can then be compared providing quantification of bound species. B) A bar graph showing results from the technique described in A, with different concentrations of probe DNA incubations. In this case ‘0 µM’ corresponds to ‘maximum coverage’, while ‘0.5 µM’ and ‘25 µM’ refer to experiments where these concentrations of probe DNA are exposed to the activated surface. ‘Control’ is where no EDC/NHS activation step is performed, but 25 µM of probe DNA and subsequently FcAm exposure is still performed. Cyclic voltammograms can be found in the supplementary information (SI2 Fig. S7B).


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As seen in Fig. 5B, exposure to different concentrations of probe DNA results in different Fc coverages. The lower Fc coverage was confirmed as arising from probe DNA attachment by the use of fluorescently labelled target DNA (SI2 Fig. S7). Our differential method estimates a surface coverage of approximately 25 ± 5 ×1012 molecules of DNA/cm2 when the surface was incubated with 25 µM of probe DNA. This is consistent with a previous report where the surface densities of between 30 × 1012 and 40 × 1012 probes/cm2 were observed for shorter (less than 24 base pairs) probes.49 This coverage may be higher than the desired amount,46 however as presented, this variable can be controlled by the concentrations of probe DNA used. This differential technique provides a unique platform to compare the probe binding capacity of novel surfaces, as well as permitting comparison of the coverages of different probe species. In the field of biosensing, the possibilities for this technique are not limited to only investigate probe DNA attachment, but could be extended to investigate the attachment of amino acids, peptides and other biomolecules. In addition to giving information about the biosensor surface, the bound Fc can be used for direct biosensing either by using already functionalized probe species,44 post-binding attachment,9 or modification of the target species.22, 50

CONCLUSIONS. The methods described in this manuscript are able to achieve three main purposes; namely, suggest optimal conditions for the binding of aminated species to guide biosensor design, characterize and compare the binding capacity of novel –COOH containing surfaces, and indicate and quantify the binding of probe species on to a –COOH surface.


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In further detail, optimal conditions for the coupling of FcAm to the monolayer by EDC/NHS chemistry have been identified through investigating the influence of a number of variables on surface coverage of the bound Fc. These include EDC/NHS concentration, buffer solution used for the activation of carboxylic acid and subsequent amide coupling, a one-step vs. two-step reaction process, EDC/NHS incubation time, acid to diluent ratio used in the SAM, variations in the diluent, FcAm concentration and incubation time with FcAm. Fc coverages are calculated based on the integral of the current generated by the redox reactions of surface bound Fc during cyclic voltammetry. With optimized conditions a ‘maximum coverage’ experiment can be performed providing a reproducible method to calculate Fc attachment efficiency. This in turn can be used to obtain the binding capacity of different surfaces, as well as affording an ideal platform to differentially quantify the amount of surface bound ‘probe’ species. The highly important task of characterizing probe functionalized surfaces can thus be aided by using this elegant method. Although this study is aimed at the development of biosensing surfaces, the techniques discussed can be translated to any surface involving carbodiimide coupling and surface characterization. The ability to quantify surface-bound probe species is a powerful method which we believe may be well utilized by the biosensing community.

ASSOCIATED CONTENT Supporting Information. Detailed methods section describing the procedures used for CV integration to calculate ferrocene coverage and electrode surface area calculation, supplementary figures: biosensors, Ferrocene CVs under different conditions, control experiments, and


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ferrocene coverages under different experimental conditions. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *To whom correspondence should be addressed: E-mail [email protected], [email protected].

ACKNOWLEDGEMENTS The authors thank the Ministry of Business, Innovation and Employment for the financial support (UOAX1201).


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