Alex Stephenson-Browna, Hui-Chen Wangbc, Parvez Iqbala, Jon A. Preecec, Yitao Longd, John S. Fossey*c, Tony D. James*b and Paula M. Mendes*a
aSchool of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. E-mail: p.m.mendes@bham.ac.uk
bSchool of Chemistry, University of Bath, Bath, BA2 7AY, UK. E-mail: t.d.james@bath.ac.uk
cSchool of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. E-mail: j.s.fossey@bham.ac.uk
dShanghai Key Laboratory of Functional Materials Chemistry & Department of Chemistry, East China University of Science and Technology, Shanghai 200237, China
First published on 22nd October 2013
Saccharides – a versatile class of biologically important molecules – are involved in a variety of physiological and pathological processes, but their detection and quantification is challenging. Herein, surface plasmon resonance and self-assembled monolayers on gold generated from bis-boronic acid bearing a thioctic acid moiety, whose intramolecular distance between the boronic acid moieties is well defined, are shown to detect D-glucose with high selectivity, demonstrating a higher affinity than other saccharides probed, namely D-galactose, D-fructose and D-mannose.
Despite quantitative analysis and detection of saccharides and saccharides-containing biomolecules being of paramount importance, reliable and accurate non-enzymatic sensors are not widely available.5 The development of convenient, rapid and precise glucose monitoring systems has been studied extensively. The majority of clinically applicable glucose sensors used today are enzyme based and utilise glucose oxidase to catalyse the transformation of glucose to gluconolactone, producing H2O2 as a co-product.6 This reaction has been exploited by several strategies, including electrochemical peroxide measurement where, at a constant voltage, the current generated across the electrochemical cell is proportional to the concentration of hydrogen peroxide, which is in turn proportional to the glucose.7 Other methods used have included monitoring changes in mechanical and optical properties of polyelectrolyte gels induced by glucose oxidation, and subsequent changes produced in the gels ionic environment.8
Despite their widespread use, enzyme-based sensor systems for glucose often suffer from a number of limitations. Notably, they result in the consumption of the analyte of interest from samples, can be dependent on local oxygen concentrations and, like all proteins, are poorly heat stable and prone to reduced activity over time owing to loss of functional enzyme due to denaturation.9 Due to these limitations, there is a drive towards non-protein dependent systems,10 which harbour the potential for vast improvements to current glucose monitoring technologies.
One group of compounds which is well suited to this challenge is boronic acids, which are able to readily and reversibly form cyclic boronate esters with diols in aqueous basic media.11 Since saccharides contain 1,2- and 1,3-diol units they provide an ideal structural framework for binding to boronic acids.12 It is this property which, in recent years, has led to a growing interest in the development of detection and sensor systems that employ boronic acid groups as “synthetic lectins”.5,7,11b,13 The most favoured class of boronic acid-based sensors utilise an amine group proximal to a phenylboronic acid group, in which the Lewis acid–Lewis base interaction between the boronic acid and the tertiary amine enables the formation of boronate esters to proceed at neutral pH.7,11c,11d However, interactions between phenylboronic acids and saccharides are generally of limited selectivity and typically they display a high affinity for fructose over other monosaccharides.11f,14
By employing two intramolecular phenylboronic acid receptor units selectivity in favour of glucose may be achieved.11c,12 By using a six carbon linker unit, single molecules of D-glucose were shown to bind to bis-boronic acid (bis-BA) binding motifs using two sets of diols, thus forming stable, cyclic 1:1 complexes with a higher stability than other saccharides such as D-fructose, D-galactose and D-mannose.11c Although the selective binding of glucose has been demonstrated in solution, surface-based sensing platforms comprising these novel bis-BA derivatives still need to be developed as only then can their potential be fully exploited for highly sensitive, robust, and selective saccharide sensors.
This work forms part of a dual submission, a novel glucose selective receptor, designed for surface functionalisation, was synthesised and subsequently has been employed in a variety of settings. In this paper, a surface plasmon resonance (SPR) detection regime is used to probe the saccharide binding, whilst the partner paper uses electrochemical responses of a bis-boronic acid functionalised surface.15
With this proviso mind, we report the design and fabrication of a glucose selective surface sensor, using the principle of self-assembly to form the sensor surface and employing SPR optical technique to detect and measure the relative binding of analyte to the surface sensor. With its high sensitivity, and unique capacity for label-free and real time detection of molecular interactions,16 SPR provides an attractive sensing platform on which to build systems to monitor analytes, such as glucose, within physiological scenarios. Furthermore, recent developments towards easily transportable miniaturised SPR systems17 makes SPR a highly attractive platform for medical sensor applications, particularly with reference to application in near patient testing.
The functional sensing surface was fabricated by formation of a two-component, mixed self-assembled monolayer (SAM) on a gold surface (Fig. 1). One of the components of the SAM is the previously described bis-BA derivative (separated by a six-carbon linker),12 which now bears a thioctic acid appended unit for binding to the gold surface. A tri(ethylene glycol)-terminated thiol (TEGT), described previously,16a was used as the second SAM component to ensure adequate separation between adjacent bis-BA on the surface, and eliminate the possibility of neighbouring boronic acid groups inhibiting saccharide binding or selectivity. In complex biological conditions, the presence of the TEGT on the surface could also serve as a shielding component to prevent non-specific protein adsorption.18 A control surface was also produced, in the same manner as described above but with a non-active compound (diamine) in place of the bis-BA (Fig. 1). The control molecule was structurally similar to the bis-BA molecule except for the absence of the phenylboronic acid moieties.
Fig. 1 Two-component, mixed SAM from a bis-BA derivative and a TEGT-terminated thiol. The control two-component mixed SAM, diamine:TEGT SAM, lacks the phenylboronic acid moieties. |
SAM | Contact angle (°) | Thickness (nm) | ||
---|---|---|---|---|
Advancing | Receding | Theoreticala | Experimental | |
a Theoretical thickness was determined using ChemBio 3D Ultra 11.0. | ||||
Bis-BA | 90.0 ± 2.5 | 83.3 ± 5.6 | 3.61 | 2.12 ± 0.12 |
Diamine | 85.4 ± 2.1 | 79.1 ± 3.2 | 3.61 | 2.35 ± 0.23 |
TEGT | 50.3 ± 1.8 | 47.1 ± 2.1 | 1.66 | 1.17 ± 0.11 |
1:1 bis-BA:TEGT | 77.8 ± 1.5 | 64.2 ± 3.9 | — | 2.17 ± 0.35 |
1:1 diamine:TEGT | 69.4 ± 0.7 | 60.4 ± 3.0 | — | 2.10 ± 0.34 |
Following contact angle and ellipsometric characterisation of the pure SAMs, studies were conducted to optimise a bis-BA:TEGT SAM ratio of 1:1 on the gold surface. This optimum ratio should enable maximum sugar binding capacity while avoiding steric hindrance from neighbouring bis-BA molecules in the SAM. As reported in previous literature,14,21 when producing mixed SAMs, the ratio of two-components in solution are rarely identical to those observed in the SAM, due to the preferential adsorption of one of the components. Thus, systematic studies were carried out in order to understand how the ratios of SAM components in solution diverge from the ratios in the formed SAM. A simple method of quantifying this was to use the relationship proposed by Cassie22 which relates the contact angle of a surface of mixed composition to those of pure SAMs (eqn (1)).
cosθAdv = xcosθAdv1 + ycosθAdv2 | (1) |
As shown in Table 1, and consistent with a mixed monolayer, the 1:1 bis-BA:TEGT and 1:1 diamine:TEGT SAMs exhibited contact angle and thickness values between those of the pure monolayers. Furthermore, the heterogeneity of the surface due to the presence of both molecules, either bis-BA and TEGT or diamine and TEGT, has led to a greater contact angle hysteresis on the 1:1 mixed surfaces than on the pure monolayers.
XPS confirmed the formation of pure and mixed SAMs, showing signals from C (1s), O (1s) and S (2p). High-resolution scans of the N (1s) and B (1s) regions (Fig. 2) show the presence of nitrogen and boron on the pure bis-BA SAMs and bis-BA:TEGT mixed SAMs, whereas, as expected, no boron peaks were observed in the mixed diamine:TEGT SAMs. XPS also confirmed the absence of nitrogen and boron on the pure TEGT SAM. For both a pure bis-BA SAM and bis-BA:TEGT mixed SAM, the B (1s) spectra display a peak at 192 eV, in good agreement with the values reported for other boronic acid derivatives.23 The N (1s) spectra can be deconvoluted into two peaks, the first one, centred at 400.2 eV, is characteristic of amide and amine moieties, while the second peak, centered at 402.0 eV, is attributed to protonated amino groups.16a,24 This finding is not surprising given the structure of the bis-BA molecule; previous studies have observed that the pyrene group is able to promote the protonation of the adjacent nitrogen groups.25 In addition, the mildly acidic nature of the methanol used as a SAM solvent could facilitate the protonation of the bis-BA and diamine molecules observed.
Fig. 2 XPS spectra of (a) B (1s) and (b) N (1s), from, pure bis-BA, pure TEGT, mixed bis-BA:TEGT and mixed diamine:TEGT and SAMs. |
With the XPS analysis, the ratio of bis-BA:TEGT and diamine:TEGT on the mixed SAM can be further calculated. By integrating the area of the S (2p) and N (1s) peaks for the mixed monolayers, a S:N ratio of 1:1 was obtained. Since both, the bis-BA molecule and the diamine compound, consist of 3 N atoms and 2 S atoms and TEGT has no N and 1 S atom only, a S:N ratio of 1:1 corresponds to a ratio of 1:1 of bis-BA:TEGT and 1:1 of diamine:TEGT on the mixed SAM. Thus, the surface ratio determined by XPS is in close agreement to that determined using the Cassie equation.
Each saccharide solution was injected over a mixed bis-BA:TEGT surface for 5 min to reach equilibrium, followed by a dissociation phase with only PBS buffer flowing over the chip (Fig. 3). It should be noted that for each saccharide all five curves presented in Fig. 3 were performed using the same SAM surface. After the dissociation phase for 2 min, the chip was regenerated for 2 min with an acidified (pH = 5) 3:1 (v/v) ethanol:PBS solution to ensure that all bound saccharide was removed from the surface. Regeneration was verified by a return to the baseline established prior to each run.
Fig. 3 SPR kinetic measurements showing the binding of D-glucose, D-galactose, D-fructose and D-mannose to 1:1 bis-BA:TEGT SAMs using different saccharides concentrations (0.6 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM and 20 mM). |
When considering the SPR data, all hexose sugars exhibited clear concentration dependent-responses, although the intensities differed among the individual sugars. Across all concentrations, glucose produced the largest change in SPR response. To derive affinity binding constants for the interaction between the immobilised boronic acid moieties and the different saccharides in solution, equilibrium analysis were chosen because they can avoid problems resulting from mass transport limitations.27 In order to correct for bulk refractive index contributions arising from the differing buffer composition and some possible nonspecific binding to the bis-BA:TEGT SAMs, SPR responses from the control mixed diamine:TEGT were subtracted from those obtained from the bis-BA:TEGT SAMs. The corrected SPR responses at equilibrium (Req) were plotted against the concentration of injected saccharide (CS) (Fig. 4) and fitted to a 1:1 steady-state affinity model. The model utilises a nonlinear least-squares regression method to fit data to the Langmuir adsorption isotherm (eqn (2)). KD is the dissociation constant of the BA–sugar complex and Rmax is the maximum response if all available BA binding sites are occupied. The calculated KD were inversed, to give the association constant, KA, to allow comparison with data obtained previously from solution.12 These values are presented in Table 2.
(2) |
Fig. 4 Calibration curve of control subtracted SPR response change for bis-BA sensor versus glucose (red), fructose (blue), galactose (black) and mannose (green) (0.3 mM, 0.6 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM and 20 mM). |
The KA results illustrate that the surfaces exhibit a higher affinity for glucose, with a comparatively reduced affinity to other hexose sugar isomers, including demonstrating over double the affinity for glucose when compared to fructose (Table 2). These results are comparable with stability constants (KOBS) previously observed in solution, producing the same orders of saccharide binding affinities as previously determined. While the absolute values differ this is likely to be caused by the different steric constraints imposed by the surface attachment.
Glucose affinity of the surface is comparable to some biological glucose ligands which have been previously investigated; bacterial binding proteins utilised in an SPR based sensor have been found to have similar affinities for glucose.28 Furthermore, the sensor produced here displays a sensitivity range which is useful to a clinical setting, unlike previously described sensors which have been only useful over much lower saccharide concentrations before the surface becomes saturated.
The results are also in the form of a calibration curve (Fig. 4), exhibiting a detection range over the clinically relevant concentrations of saccharides analysed. Although a response from the other hexose isomers is observed, the impact on glucose measurements in clinical samples would be minimal as glucose is by far the most prevalent saccharide found in blood and other bodily fluids; typically found in concentrations orders of magnitude greater than other saccharides.3
Footnotes |
† Electronic supplementary information (ESI) available: General experimental procedures and spectra are provided. See DOI: 10.1039/c3an01233f |
‡ Dedicated to Professor Seiji Shinkai to celebrate his 70th birthday. |
This journal is © The Royal Society of Chemistry 2013 |