Bio-inspired Maillard-Like reactions enable a simple and sensitive assay for colorimetric detection of methylglyoxal

Article abstract of DOI:10.1039/c5cc02590g

A simple and selective assay for detecting methylglyoxal (MGO), a metabolite associated with diabetes, was developed by combining a bio-inspired chemical reaction with the anti-aggregation of gold nanoparticles. This assay could detect MGO at as low as 1 μM by the naked eye and 0.05 μM by UV/vis spectrometry, within the clinical range marking oxidative stress in diabetes, and demonstrated high selectivity over other physiologically relevant ketones and aldehydes.

Full text of DOI:10.1039/c5cc02590g

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Bio-inspired Maillard-Like reactions enable a
simple and sensitive assay for colorimetric
Cite this:DOI: 10.1039/c5cc02590g
detection of methylglyoxal†
Received 30th March 2015,
Accepted 2nd June 2015
Shih-Ting Wang, Yiyang Lin, Christopher D. Spicer and Molly M. Stevens*
DOI: 10.1039/c5cc02590g
www.rsc.org/chemcomm
A simple and selective assay for detecting methylglyoxal (MGO), a
metabolite associated with diabetes, was developed by combining a
bio-inspired chemical reaction with the anti-aggregation of gold
nanoparticles. This assay could detect MGO at as low as 1 lM by the
naked eye and 0.05 lM by UV/vis spectrometry, within the clinical
range marking oxidative stress in diabetes, and demonstrated high
selectivity over other physiologically relevant ketones and aldehydes.
Chronic hyperglycemia has been implicated in several diabetes-
specific microvascular and macrovascular pathologies. One of
the major pathways through which this occurs is the formation
of advanced glycation end products (AGEs), which accumulate
1
on long-lived proteins and cause tissue damage. Glucose-
derived dicarbonyls, including methylglyoxal (MGO), glyoxal,
and 3-deoxyglucosone have been shown to generate AGEs at
orders of magnitude higher rates than glucose owing to their
high reactivity towards amino acids (dominantly cysteine, lysine
and arginine). As such, reactive dicarbonyls are considered
important markers for monitoring diabetes complications.
Among these, MGO has been found to be particularly reactive
2
Scheme 1 (a) The Maillard reaction between methylglyoxal (MGO) and
protein lysine residues generates Schiff bases and MGO-lysine dimers
(
MOLD). (b) The reaction of o-phenylenediamine (OPD) and MGO to form
-methylquinoxaline (2-MQ). (c) Schematic view of the anti-aggregation
2
towards lysine through the formation of reversible Schiff base mechanism applied for MGO detection. In the absence of MGO, OPD
structures or stable imidazolium products (Scheme 1a) known as triggers the aggregation of citrate-capped AuNPs via bidentate binding to
3
gold. The presence of MGO consumes OPD in solution which ultimately
inhibits AuNP aggregation.
MGO-lysine dimers (MOLD). MGO is generated during the
metabolism of glucose by the fragmentation of glyceraldehyde-3-
phosphate and dihydroxyacetone phosphate. Its level increases in
the urine and plasma of diabetic individuals with clinical rele-
vance. MGO also exists in beverages and fermented food.
Development of reliable, sensitive and selective methods for chromatography-mass spectrometry (LC-MS), and gas chromato-
Traditional methods for MGO detection usually rely on
4
5
6
high performance liquid chromatography (HPLC), liquid
7
8
determining MGO concentration are therefore important to not graphy (GC). These methods require tedious sample prepara-
only monitor diabetes-associated pathologies but also for investi- tion, time-consuming testing cycles, expensive equipment, and
gating its reactivity and hypothetical toxicity during food proces- highly-trained personnel. There is therefore a clear need for
sing, cooking and prolonged storage.
simple, quick, and low-cost assays to sensitively and selectively
detect MGO at clinically relevant levels. Great progress in the
development of nanotechnology has offered new opportunities
Department of Materials, Department of Bioengineering and Institute for Biomedical
Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
E-mail: m.stevens@imperial.ac.uk
9
for diagnostics and sensing. In particular, gold nanoparticles
(
AuNPs) possess unique physical and chemical properties
†
Electronic supplementary information (ESI) available: TEM, DLS, DF-STEM,
10
through the variation of shape, size and chemical environment.
NMR and UV/vis spectra of AuNPs with or without OPD, the reaction kinetics
between OPD and MGO. See DOI: 10.1039/c5cc02590g
The interparticle surface plasmonic coupling induced by AuNP
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aggregation results in a colour change from red to blue, providing
a simple and practical platform for the colorimetric sensing of
various target analytes such as small molecules, enzymes, and
9,11
oligonucleotides.
While colorimetric assays have been devel-
oped for the detection of glucose in diabetic patients, to date no
such assay has been developed for the detection of reactive
dicarbonyls.
Here we present a novel colorimetric assay for MGO detec-
tion based on controllable AuNP aggregation modulated by an
MGO dependent chemical reaction (Scheme 1). In a similar
manner to the Maillard reaction (Scheme 1a) in biological
systems, MGO reacts quickly with o-phenylenediamine (OPD)
and leads to the cyclic product 2-methylquinoxaline (2-MQ) in
high yield (Scheme 1b). As shown in Scheme 1c, the bidentate
binding of OPD to Au surfaces effectively triggers AuNP aggre-
gation and causes a peak shift in the UV/vis spectrum which
changes the solution colour from red to blue. The presence of
MGO consumes OPD through chemical conjugation, reducing
the concentration of OPD in solution and inhibiting AuNP
aggregation. This results in a colorimetric response that can
be exploited to detect and quantify MGO.
As shown in Fig. 1a, the addition of OPD to citrate-capped
AuNPs (OD520 = 0.2, Fig. S1, ESI†) immediately generates
a colour change dependent on OPD concentration. A visible red-
to-blue colour change was noted at as low as 0.5 mM OPD. The
Fig. 1 (a) Photograph of AuNP aggregation with varying OPD concen-
UV/vis spectra showed a decreased absorbance at 520 nm and the tration at different pH values. (b) UV/vis spectra of AuNPs incubated with
OPD at various concentrations. (c) Absorbance ratio of 680 nm and
simultaneous appearance of an absorbance peak at 680 nm which
5
20 nm (A680/A520) of AuNP solutions incubated with OPD at pH 6.5,
.5 and 8.5. (d) A680/A520 of AuNP solutions incubated with various OPD
analogues including aniline, 2,3-diaminonaphthalene (DAN), 3,4-diaminotoluene
DAT) and 4,5-dimethyl-2-phenylenediamine (DMPD). (e) Dark field-
increased in intensity with OPD concentration (Fig. 1b). By plotting
the A680/A520 value against OPD concentration, the detection limit
of OPD was determined to be 0.25 mM (Fig. 1c). A similar
7
(
phenomenon of OPD-induced AuNP aggregation was observed at scanning transmission electron microscopy (DF-STEM) imaging of aggre-
gated AuNPs induced by 0.5 mM OPD, and the element mapping (f)
performed by line scanning across the aggregates (pink line in (e)),
different pH values (Fig. 1a and c). This solution colour change was
demonstrated to be due to the modulation of surface plasmonic
resonance (SPR) originating from the aggregation of AuNPs
confirming the presence of nitrogen correlating with the Au bands,
indicating the attachment of OPD to AuNPs.
through dynamic light scattering (DLS, Fig. S2, ESI†) and trans-
mission electron microscopy (TEM, Fig. S3a, ESI†). We also inves-
tigated a series of OPD analogues (aniline, 2,3-diaminonaphthalene, is due to an increase in surface hydrophobicity originating from
3
,4-diaminotoluene and 4,5-dimethyl-2-phenylenediamine) to better OPD binding to AuNPs via amine-gold affinity as proposed in
13,14
understand the mechanism of AuNP aggregation. Interestingly, the previous works.
The binding of OPD to Au surface was char-
analogues bearing two amino groups at ortho- positions exhibited acterised by energy-dispersive X-ray spectroscopy (EDX) (Fig. S3b,
similar abilities to induce AuNP aggregation (Fig. 1d), while aniline ESI†) and imaged through dark field-scanning transmission electron
with only a single amino group caused AuNP aggregation at only microscopy (DF-STEM). Nitrogen originating from OPD could be
much higher concentrations (410 mM).
detected in the large AuNP aggregates and correlated with the Au
According to the DLVO theory, colloidal particles in aqueous bands (Fig. 1e and f). This superior binding over citrate was due to
solution are stabilised by a combination of electrostatic repulsion the bidentate structure of OPD that coordinates to the metal. This
12
and steric effects. However, the AuNP aggregation induced by also explains why diamine analogues exhibit a much higher effi-
OPD seen here is not ascribed to the screening of electrostatic ciency in inducing AuNP aggregation than aniline (Fig. 1d). By
b
repulsion or the decrease of sterics. Since the pK s of OPD are 9.43 contrast, AuNP aggregation was not observed even at higher con-
and 13.2, only 1% of amine groups are protonated at pH 6.5. This centrations (1 mM) of OPD when the AuNP surface was capped with
implies that OPD induced Au aggregation is not due to the 3-mercaptopropionic acid (MPA), which prevented the coordination
screening of surface charges. It is hence reasonable that solution of diamine groups to the AuNPs (Fig. S4, ESI†). We conclude that the
pH does not affect the sensitivity of AuNP aggregation (Fig. 1c). binding of OPD to the Au surface through nitrogen–gold coordina-
The z-potential of aggregated AuNPs in the presence of OPD tion increases the surface hydrophobicity and hence induces AuNP
(
À25.7 mV) is also close to that in its absence (À29.4 mV) which aggregation.
suggests that aggregation is not due to the decrease of charge In a similar manner to the Maillard reaction, MGO can react
density at the Au surface. Instead, we propose that AuNP aggregation with the ortho-amine group of OPD to form the stable adduct
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2-MQ. This reaction has been used previously to develop assays allowed detection of as low as 1 mM MGO in 30 v/v% serum
for the chromatographic sensing of MGO. Nuclear magnetic (Fig. S7, ESI†). Thus, this method could prove to be a reliable
resonance (NMR) spectroscopy was performed to confirm the means by which to discriminate the concentration of MGO in
formation of 2-MQ in a solution of OPD and MGO (Fig. S5, hyperalgesia in diabetic neuropathy, which is in general around
ESI†). Subsequently, this reaction was used to develop a colori- 2–4 fold higher in the plasma samples from diabetic indivi-
4
metric assay for MGO detection. Briefly, MGO at different duals. The anti-aggregation of AuNPs mediated by MGO was
concentrations was incubated with 0.5 mM OPD for 30 min also confirmed by DLS (Fig. S8, ESI†) and TEM (Fig. 2d–f), with
before the addition of citrate-capped AuNPs. Depending on the AuNP aggregation significantly inhibited by MGO, while large
concentration of MGO and thereby the remaining OPD in particle aggregates were found in its absence. Importantly, the
solution, the AuNP solution displays varied aggregation states, assay could be completed within 40 min. This compares favour-
leading to different surface plasmonic properties and solution ably with currently used techniques for MGO detection which
7
colours. In principle, higher concentrations of MGO result in typically take up to 5 h to obtain accurate quantification.
less OPD in solution and hence inhibit the aggregation of
The selectivity of this assay was examined by comparing the
AuNPs. As shown in Fig. 2a, the OPD–Au solution gives a detection of MGO with other physiologically relevant ketones and
blue-to-red transition above an MGO concentration of 1 mM. aldehydes, including glyoxal, 3-deoxyglucosone, 3-hydroxybutyrate,
The absorption spectra also showed a decrease in absorption acetylacetone, 4-oxo-nonenal, 4-hydroxynonenal, crotonaldehyde,
peak at 680 nm and simultaneous increase at 520 nm with and glutaraldehyde (Fig. 3). High selectivity towards MGO was
increasing MGO concentration (Fig. 2b). By plotting the A520/ observed among the carbonyls tested, including other glycation-
A680 against MGO concentration in Fig. 2c, it was observed that related dicarbonyls, such as glyoxal, and 3-deoxyglucosone. The
MGO could be detected by UV/vis spectroscopy at a concen- successful discrimination of MGO from the other reactive dicar-
tration as low as 0.05 mM. The effect of particle size and bonyls is due to the high reactivity of MGO with OPD in which a
concentration on this assay was studied (Fig. S6, ESI†), with a 78% conversion to 2-MQ is accomplished in 30 min (Fig. S9, ESI†).
reduction in AuNP concentration lowering the resultant signal By contrast, only 18% of the corresponding product is formed by
as expected. Interestingly, larger AuNPs (20 nm) showed the reaction of 3-deoxyglucosone and OPD in the same time, while
1
5
enhanced target signal likely due to stronger localised SPR.
the reaction of glyoxal shows an even lower reaction rate. There-
Importantly, MGO detection was also possible in pre-treated fore, the assay provides a selective strategy for detecting MGO, and
human serum. Due to the coating of proteins on the Au surface, a potential system for monitoring long-term diabetes development
the degree of AuNP aggregation is less in serum and therefore and the onset of AGE formation.
the MGO sensing was determined by plotting A520/A598 which
A new colorimetric assay was designed for the detection of MGO, a
diabetic marker, by employing a bio-inspired MGO–OPD reaction to
mediate AuNP aggregation. Bidentate-amino ligands such as OPD
were found to cause AuNP aggregation by increasing the surface
hydrophobicity of the particles. Depending on the concentration of
target MGO and the remaining concentration of OPD, the AuNP
solution displays different aggregation states, plasmonic spectra and
solution colours. This assay has been demonstrated to be able to
detect 1 mM MGO by the naked eye and 0.05 mM by UV/vis
spectroscopy, which is in the clinical range for diabetes diagnosis
and monitoring. Discrimination of MGO from other biologically
relevant ketones and aldehydes was observed to benefit from the
Fig. 2 (a) Photograph of OPD–AuNP solution in the presence of 10, 5, 1, and
0.5 mM MGO (from left to right). (b) UV/vis spectra and (c) absorbance ratio
(A520/A680) of the detection system with different concentrations of MGO at
a fixed OPD concentration of 0.5 mM. (d–f) TEM images showing AuNPs in the
presence of 0.5 mM OPD and (d) 5 mM, (e) 0.5 mM and (f) 0 mM MGO.
Fig. 3 Plot illustrating the selectivity of the MGO assay. A520/A680 was
plotted against different ketones and aldehydes at a concentration of 10 mM.
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different reaction rates of these compounds with OPD. This detection
system offers the advantages of simplicity, specificity, and selectivity
against other physiologically-related ketones and aldehydes. In addi-
tion, it offers significantly improved detection times over previously
reported methods. Such transduction of a chemical reaction into
plasmonic signals is also expected to be useful for the monitoring of
organic reactions or designing plasmon-based logic gates.
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