Ratiometric electrochemical detection of β-galactosidase

Article abstract of DOI:10.1039/c7ob01593c

A novel ferrocene-based substrate for the ratiometric electrochemical detection of β-galactosidase was designed and synthesised. It was demonstrated to be an excellent electrochemical substrate for β-Gal detection with sensitivity as low as 0.1 U mL^-1.

Full text of DOI:10.1039/c7ob01593c

Organic &
Biomolecular Chemistry
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Ratiometric electrochemical detection of
β-galactosidase†
Cite this: DOI: 10.1039/c7ob01593c
Received 30th June 2017,
Accepted 9th August 2017
Sam A. Spring,
Sean Goggins * and Christopher G. Frost
DOI: 10.1039/c7ob01593c
rsc.li/obc
A novel ferrocene-based substrate for the ratiometric electro- electrochemical probes,11,12 with ferrocene derivatives widely
chemical detection of β-galactosidase was designed and syn- used in biological systems due to their stability in aerobic and
thesised. It was demonstrated to be an excellent electrochemical aqueous environments.13 The development of ratiometric
substrate for β-Gal detection with sensitivity as low as 0.1 U mL−1
.
probes has overcome the issues of reproducibility, by the
ability to obtain direct conversions, minimising both sampling
errors and systematic errors from instrument variation.14
Ferrocene-based ratiometric chemodosimeters for enzyme
detection have previously been reported for alkaline phospha-
tase,15 and glucose oxidase,16 but none currently for β-Gal.
Ferrocene-based electrochemical sensing is a continuing
interest within our research group as it enables an inexpensive
and convenient way to monitor enzyme activity.17 Utilising
trigger–linker–effector methodologies,18 we designed ferro-
β-Galactosidase (β-Gal, EC 3.2.1.23) is a prominent enzyme
used biologically as a reporter gene as it has been well charac-
terised and demonstrates excellent stability.1 The low level of
background substrate hydrolysis and ready availability makes
β-Gal an attractive enzyme label within enzyme-linked
immunosorbent assays (ELISAs).2 It can also be used in heavy-
metal ion detection,3 and rapid enzyme assays have been used
in the detection of coliform and E. coli in waste water treat-
ment.4 β-Gal is typically detected either chromogenically, using
ortho-nitrophenyl-β-galactoside (ONPG),3 or 5-bromo-4-chloro-
3-indoyl-β-D-galactopyranoside (X-Gal),5 or is detected fluoro-
metrically using fluorescein-di-β-D-galactopyranoside (FDG).6
Lanthanide based coumarins have also been utilised as a lumi-
nescent probe in the detection of β-Gal.7 Optical substrates
however, are limited by the use of expensive equipment, non-
linear fluorescence, and potentially high levels of background
signal.1 The development of electrochemical enzyme sub-
strates allows for the direct conversion of a biochemical reco-
gnition event into an electrical signal enabling facile biosensor
integration within a handheld device.8 4-Aminophenyl-β-D-
galactosidase (PAPG) is standardly used as an electrochemical
substrate for β-Gal, but high background signals prevents accu-
rate analysis at low enzyme concentrations.9 A modified PAPG-
style substrate, 4-methoxyphenyl-β-D-galactosidase (4-MPGal)
has a negligible background signal, but the unfavourable use
of modified graphene oxide electrodes is required.10
cenylcarbamoylphenyl-β-D-galactosidase
1 as a ratiometric
electrochemical substrate for β-Gal. Previously, ferrocenyl-
amine 3 has been shown to be oxidised at a lower potential
than carbamate derivatives.12 Substrate 1 would have a higher
oxidation potential than 3 making them electrochemically dis-
tinguishable, allowing for the ratiometric electrochemical ana-
lysis of β-Gal activity. We propose that in the presence of β-Gal,
hydrolysis at the anomeric position would afford an unstable
phenolate intermediate 2. 1,6-Elimination would follow releas-
ing ferrocenylamine 3, quinone methide and CO2 (Scheme 2).
The synthesis of substrate 1 (Scheme 1) started from the
commercially available D-galactose pentaacetate which was
converted to benzyl alcohol
5 (see ESI† for synthesis).
Ferrocenoyl azide 6 was synthesised according to a literature
procedure,19 and then coupled to the benzyl alcohol via a
Curtius rearrangement. Zemplén deacetylation of 7 afforded
the desired substrate 1 in a 16% overall yield. Once syn-
thesised, substrate 1 was found to be a bench stable orange
solid with no observable degradation over several months at
room temperature. Substrate 1 was also stable to hydrolysis in
Tris buffer (pH 7, 50 mM) solutions for several weeks at room
temperature (see ESI, Fig. S1†). The electrochemical behaviour
of substrate 1 was tested via differential pulse voltammetry
(DPV) and compared to ferrocenylamine 3.
Due to its facile oxidation potential and excellent synthetic
utility, ferrocene is implemented as the redox-active moiety in
Department of Chemistry, University of Bath, Bath, BA2 7AY, UK.
E-mail: s.goggins@bath.ac.uk; Fax: +44 (0)1225 386231; Tel: +44 (0)1225 386142
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, characterisation data, and copies of NMR spectra of synthesised com-
pounds. See DOI: 10.1039/c7ob01593c
As expected, substrate 1 had a higher oxidation potential
than ferrocenylamine 3, with the difference between the two
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Scheme 1 Synthesis of substrate 1.
Fig. 1 Differential pulse voltammogram obtained for substrate
(0.1 mM) and ferrocenylamine 3 (0.1 mM) in 50 mM pH 7 Tris buffer.
1
Substrate 1 (0.1 mM) was initially subjected to varying con-
centrations of β-Gal in Tris buffer (pH 9, 50 mM) at room
temperature (21 °C) and subjected to electrochemical analysis
every 3 minutes for 60 minutes (see ESI, Fig. S2†). The voltam-
mogram of each sample was then integrated and conversions
calculated using eqn (1). At high β-Gal concentrations, 5 and
Scheme 2 Structure of substrate 1 and the proposed mechanism of 10 U mL−1 20
,
quantitative conversion was observed within
18 minutes, and 60 minutes for 1 U mL−1. Pleasingly, no back-
ground substrate hydrolysis was observed in the absence of
β-Gal allowing for a β-Gal concentration as low as 0.1 U mL−1
to be detected within 60 minutes. Intriguingly, the presence of
a third peak was present in the voltammogram at 390 mV (see
ESI, Fig. S3†). This was confirmed to be 4-hydroxybenzyl
alcohol 10, formed when quinone methide 8, produced as a
by-product from the result of self-immolation, reacts with
water or hydroxide (Scheme 3).21 Despite being produced in an
equimolar concentration as ferrocenylamine 3, the peak
obtained was still significantly smaller than the ferrocene
β-Gal catalysed breakdown with subsequent release of ferrocenylamine 3.
peaks being approximately 250 mV and were completely
resolved which allowed for the peaks to be integrated indepen-
dently (Fig. 1). The conversion of substrate 1 was calculated
using eqn (1).
ꢀ
ð
ꢁ
ð
3
ꢀ
ð
ꢁ
Conversion ð%Þ ¼
ꢀ 100
ð1Þ
3 þ
1
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Scheme 3 Proposed formation of 4-hydroxybenzyl alcohol from
quinine methide.
peaks showing the clear benefit of using the organometallic
redox label compared with other commonly used organic
ones.
Next, the effect of pH was investigated (see ESI, Fig. S4†).
A β-Gal concentration of 1 U mL−1 was chosen as it allowed
for both positive and negative effects due to the pH to be
observed. At pH 8 there was a significant increase in the rate
Fig. 2 Conversion of the substrate 1 to the product after addition of
β-Gal (1 U mL−1) using different concentrations of the substrate at room
temperature in Tris buffer (pH 7, 50 mM). Error bars represent the stan-
dard deviation where n = 3.
of conversion, with quantitative conversion observed in under were tested. Z-buffer exhibited significant background signal,
30 minutes. There was a marginal increase in rate from pH 8 potentially due to the thiols present in Z-buffer, that obscured
to pH 7, but importantly, at the lower pH the presence of the the peaks and prevented accurate electrochemical analysis. In
third peak was suppressed, presumably due to protonation of phosphate buffer the stability of ferrocenylamine 3 was dimin-
the electrochemically active phenolate ion 9. The suppression ished with a second peak forming at a higher oxidation poten-
of the peak produced a cleaner voltammogram, allowing for tial, assumed to be an electroactive by-product from ferroceny-
more accurate conversion to be calculated and as a result, pH lamine decomposition.23 Tris buffer (pH 7) was therefore
7 was used moving forward.
chosen as this maintained a low background and ensured fer-
According to previous literature, the optimum working rocenylamine stability, and the effect of buffer concentration
temperature for β-Gal is 37 °C.22 A lower β-Gal concentration was investigated using a β-Gal concentration of 1 U mL−1 (see
was chosen, specifically 0.1 U mL−1 in Tris buffer (pH 7, ESI, Fig. S6†). In unbuffered solution, the rate of conversion
50 mM) (see ESI, Fig. S5†), to allow for changes in the rate of was significantly improved compared to 50 mM Tris buffer
conversion to be noticeable. Interestingly, increasing the temp- solution, however, the voltammogram peaks were shifted to a
erature from room temperature had minimal effect on the rate lower oxidation potential. At this lower potential, the presence
of conversion, and above 37 °C the rate was retarded. At 57 °C of voltammogramatic artefacts prevented accurate detection.
negligible conversion was observed due to denaturing of the At 25 mM Tris buffer concentration, a comparable rate of con-
enzyme. Substrate 1, however, remained stable to hydrolysis version to 50 mM Tris buffer concentration with quantitative
even at elevated temperatures, exhibiting the high stability of conversion observed in 18 minutes. Further analysis showed
the substrate. With no improved rate of conversion, the assays that β-Gal was unstable at the lower buffer concentration, with
were continued to be conducted at room temperature (21 °C).
increases in conversions stopping after 18 minutes. Increasing
The concentration of substrate 1 in the assay was then the concentration of the Tris buffer above 50 mM prevented
screened, utilising a β-Gal concentration of 1 U mL−1 (Fig. 2). accurate ratiometric analysis as we suspect the reduced stabi-
Increasing the probe concentration to 0.25 mM from 0.1 mM lity of ferrocenylamine 3 resulted in unreliable peak inte-
had minimal effects on the rate of conversion with quantitative grations. Therefore, a Tris buffer (pH 7, 50 mM) was selected
conversion still observed within 24 minutes. However, increas- as the optimal buffer concentration.
ing the concentration further showed no discernible increase
With optimal conditions obtained, the sensitivity of β-Gal
in the rate of reaction. When the substrate concentration was was tested (Fig. 3). In the optimised conditions, there was no
decreased to 0.05 mM, the reduced current observed was sus- background hydrolysis observed, allowing for detection of low
ceptible to artefacts on the voltammogram affecting accurate β-Gal concentration of 0.1 U mL−1 within 60 minutes. Utilising
conversion calculations, which were unavoidable when using pseudo-first order kinetics (see ESI, Fig. S7†), a rate constant
disposable screen-printed carbon graphite electrode cells. At of 2.91 × 10−3 s−1 was calculated for a β-Gal concentration of
concentrations above 0.1 mM, additional sample manipu- 1 U mL−1. At higher concentrations of 10 and 5 U mL−1, quanti-
lation, via serial dilutions, was required before analysis, due to tative conversions were exhibited within just 6 minutes, and at
overloading of the electrodes, and therefore, an optimal sub- a low concentration of 0.25 U mL−1, a 73 5% conversion was
strate concentration of 0.1 mM was chosen.
achieved within 60 minutes, with an observed rate constant of
The final condition to be optimised was the buffer system 0.044 × 10−3 s−1. The small error bars afforded, indicate the
used. Other common β-Gal buffer systems such as potassium good reliability showing the benefit of using ratiometric
phosphate buffer (50 mM, pH 7),3 and Z-buffer (50 mM, pH 7), electrochemical analysis.
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Conflicts of interest
There are no conflicts to declare.
Notes and references
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Fig. 3 Conversion of the substrate 1 (0.1 mM) to the product after
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Fig. S8†). Comparatively, substrate 11, was significantly slower
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Fig. 4 Substrate 11.
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