A thermally-stable enzyme detection assay that amplifies signal autonomously in water without assistance from biological reagents

Article abstract of DOI:10.1039/c2cc36861g

This Communication describes a thermally-stable small molecule and a corresponding assay strategy that autonomously amplifies a colorimetric signal when a specific enzyme biomarker is detected. This journal is

Full text of DOI:10.1039/c2cc36861g

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
A thermally-stable enzyme detection assay that
amplifies signal autonomously in water without
assistance from biological reagents†
Cite this: Chem. Commun.,
2013, 49, 394
Received 20th September 2012,
Accepted 1st November 2012
Kimy Yeung, Kyle M. Schmid and Scott T. Phillips*
DOI: 10.1039/c2cc36861g
www.rsc.org/chemcomm
This Communication describes a thermally-stable small molecule signal for specific enzyme biomarkers. Both of these reagents
and a corresponding assay strategy that autonomously amplifies a operate cooperatively4a,b,5 and autonomously when a specific
colorimetric signal when a specific enzyme biomarker is detected. enzyme analyte is detected, thus offering an alternative to
traditional analyte detection and signal amplification reagents
The World Health Organization (WHO) has identified selectivity, and assays that employ thermally-unstable components
sensitivity, and thermal stability1 as three of seven key features (e.g., antibodies, enzymes, and/or nucleic acids).
that are needed in ideal point-of-care tests for use in the devel-
Our signal amplification reaction generates a colorimetric
oping world.2 Designing appropriate assays that incorporate readout in the form of 2-hydroxy-1,4-benzoquinone (4), which is
these three features, however, has proven to be a substantial an intense red-brown color (Fig. 1). During the assay, 2-hydroxy-
challenge, especially for assays and reagents that must be trans- 1,4-benzoquinone (4) is obtained from the auto-oxidation of
ported to remote villages without refrigeration. Of particular 1,2,4-trihydroxybenzene (3),6 which itself is released from both
difficulty in this context is the design of thermally-stable signal the enzyme detection reagent (2) and the signal amplification
amplification reagents that enable trace-level detection.3 In this reagent (1). This auto-oxidation reaction generates hydrogen
Communication, we describe the first-generation design of a new peroxide, which serves as a ‘‘signal transduction reagent’’ in the
thermally-stable small molecule reagent (1) for amplifying signal tandem assay, thus enabling the detection event with 2 to initiate
in water without assistance from enzymes or other thermally- the signal amplification reaction with 1 via the highly selective
unstable reagents.4 We also describe an assay strategy that uses oxidative cleavage reaction of hydrogen peroxide with aryl boronic
this signal amplification reagent in combination with a second acids.7
reagent (a detection reagent; 2) (Fig. 1) to detect and amplify
This reaction between hydrogen peroxide and the aryl boronic
acid in 1 generates a phenol intermediate (not shown), which then
undergoes two sequential quinone methide-mediated elimination
reactions to release two equivalents of 1,2,4-trihydroxybenzene (3)
(Fig. 1). Auto-oxidation of these two equivalents of 3 provides more
hydrogen peroxide (thus perpetuating the signal amplification
reaction via reaction with additional equivalents of 1) and produces
two equivalents of 2-hydroxy-1,4-benzoquinone (4) (the molecule
that provides the colorimetric readout). Multiple self-perpetuating
cycles of this reaction increase the quantity of 4 to levels where
the color can be seen easily by eye. Quantification is readily achieved
using a camera-equipped cellular phone in combination with
image processing software in a process referred to as Telemedicine.8
In principle, only a single molecule of 1,2,4-trihydroxybenzene (3)
must be released from 1 to perpetuate the signal amplification
reaction if a quantitative yield of hydrogen peroxide is obtained
from the auto-oxidation of 3. Because we suspected that the yield
of hydrogen peroxide would be less than 100%,9 we designed
1 to release two equivalents of 1,2,4-trihydroxybenzene to increase
the likelihood that at least one equivalent of hydrogen peroxide
would be obtained from each molecule of 1 that reacts.
Fig. 1 A one-pot, tandem assay strategy that uses two thermally-stable small
molecules (1 and 2) to selectively detect an enzyme biomarker and
autonomously amplify a colorimetric output signal (4).
The Pennsylvania State University, 104 Chemistry Bldg., University Park, PA 16802,
USA. E-mail: sphillips@psu.edu; Fax: +1 814 865 5235; Tel: +1 814 867 2502
† Electronic supplementary information (ESI) available: Synthetic procedures,
compound characterization data, supporting figures, and tables of data. See DOI:
10.1039/c2cc36861g
c
394 Chem. Commun., 2013, 49, 394--396
This journal is The Royal Society of Chemistry 2013

View Article Online
Communication
ChemComm
consumed, even when only 0.1 equivalents of hydrogen peroxide
is present (Fig. 2a). This result is consistent with the expectation
that 1 is capable of self-perpetuating the signal amplification
reaction by regenerating hydrogen peroxide via auto-oxidation of
1,2,4-trihydroxybenzene. In the absence of hydrogen peroxide, a
background reaction is observed eventually, which we believe
arises from slow hydrolysis of 1,2,4-trihydroxybenzene from 1,
followed by auto-oxidation to generate hydrogen peroxide.
Two control experiments support this hypothesis: First, we
pre-treated the water used in the experiment with catalase
(480 U mLÀ1) for 15 min to remove traces of hydrogen peroxide
in the water that might initiate the background reaction. After
removing the catalase by filter centrifugation, we dissolved 1 in
this purified water and monitored the consumption of 1 over time
(Fig. 2a). The rate of consumption of 1 in the purified water was
nearly identical to the rate with untreated water, indicating that
trace levels of hydrogen peroxide that may be present in the water
are not a significant contributor to the background signal. Second,
when catalase (64 mg, 480 U mLÀ1) was included with 1 (2.5 mM in
0.1 M phosphate buffer containing 2% MeCN, pH 7.3, 34 1C), only
a gradual loss of 1 (10% after 15 h) was observed, which is
consistent with the notion that trace background hydrolysis of 1
to release 1,2,4-trihydroxybenzene is the major cause of the back-
ground reaction in the catalase-free reaction.11
Scheme 1 Synthesis of signal amplification reagent 1.
A graph similar to Fig. 2a was obtained when the intensity of
the amplified color (rather than the consumption of 1) was
monitored over time when 1 was exposed to varying equivalents
of hydrogen peroxide (Fig. S1a, ESI†). In this experiment, the
color of the signal amplification reaction was obtained by
photographing the samples and then quantifying the intensity
of the images using AdobesPhotoshops. Fig. 2b shows an
example of the intense red-brown color that is generated when
1 (2.5 mM in 0.1 M phosphate buffer containing 1.5% DMF,
pH 7.3, 23 1C) is exposed to 0.5 equivalents hydrogen peroxide.
The colorimetric response shown in Fig. S1a (ESI†) sug-
gested that a quantitative assay might be possible. Indeed, a
fixed assay time of 30 min provides a predictable dose response
curve (Fig. S2, ESI†) for detecting hydrogen peroxide. Fig. S2b
(ESI†) highlights the linear region of the 30 min assay, which
has a limit of detection of 54 mM hydrogen peroxide.
This data illustrates that hydrogen peroxide should serve as an
effective signal transduction reagent in the type of enzyme-detection
assay depicted in Fig. 1. To demonstrate this concept, we designed a
one-pot assay that uses reagent 1 as the signal amplification reagent
and reagent 2 to detect b-D-galactosidase12 (Fig. 3). The colorimetric
signal for this tandem reaction (using 1 and 2) is larger and
increases more quickly (once the assay has proceeded for 6 h) than
the signal obtained when only 2 is exposed to the enzyme. Back-
ground signal for 2 is minimal in the absence of the analyte.
Likewise, when 1 is exposed to the enzyme, the signal is
negligible for 10 h, which, when compared to the more sub-
stantial background signal observed in the absence of surfactant
(Fig. S1a, ESI†), suggests that the surfactant in the assay slows
the rate of the signal amplification reaction relative to assays in
the absence of surfactant. Kinetics experiments (Fig. S1b, ESI†)
in the presence of surfactant (but absence of b-D-galactosidase)
support this hypothesis.13
Fig. 2 Self-perpetuating response of 1 when exposed to hydrogen peroxide.
(a) Consumption of 1 over time for experiments using 2.5 mM 1 and different
quantities of hydrogen peroxide. The percentage of 1 consumed during the
reaction was measured at 254 nm using an LC-MS. ‘‘Treated water’’ refers to
water that was treated with catalase to remove trace quantities of hydrogen
peroxide. The ‘‘plus catalase’’ label refers to an experiment in which catalase was
included in the assay. (b) Photographs over time after addition of 0.5 equivalents
of hydrogen peroxide to a 2.5 mM solution of 1 in 0.1 M phosphate buffer
containing 1.5% DMF (pH 7.3) at 23 1C.
We prepared amplification reagent 1 using the route shown
in Scheme 1, which includes a late-stage introduction of the
aryl boronic acid to avoid self-perpetuating decomposition
reactions that could otherwise hinder the preparation of this
autoinductive reagent. When stored dry at 40 1C, 1 is stable for
at least 21 days.10
When exposed to hydrogen peroxide, all of 1 (2.5 mM in 0.1 M
phosphate buffer containing 2% MeCN, pH 7.3, 34 1C) was
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 394--396 395

View Article Online
ChemComm
Communication
In conclusion, this communication describes a first-generation,
thermally-stable signal amplification reagent (1) that amplifies
signal for enzymatic detection events in water. Both the
selectivity and sensitivity of assays using 1 (in combination
with an activity-based detection reagent, such as 2) are achieved
without using thermally-unstable biological reagents, thus
pointing to a new strategy for realizing three of the seven
criteria put forth by the WHO for ideal diagnostic assays
for use in the developing world. Future efforts will focus on
increasing the rate of the signal amplification reaction
and decreasing the background signal. Success in these efforts
should enable the creation of highly developed assays for
enzyme biomarkers associated with infectious disease, water
quality, as well as other pressing diagnostic problems.
Fig. 3 Normalized intensity of color for assays exposed to 194 nM
b-D-galactosidase. I corresponds to the intensity of the colorimetric response and
I0 refers to the colorimetric signal at t = 0. The concentration of 1 and 2 were
2.5 mM in 0.1 M phosphate buffer (pH 7.3) containing 2.5% DMF and 0.5%
Tween-20 (v/v); the assays were conducted at 23 1C. The surfactant in these
assays slowed the rate of the signal amplification reaction as well as the back-
ground reaction with 1 in comparison to the conditions described in Fig. S1a (ESI†).
This work was supported by the Arnold and Mabel Beckman
Foundation, the Camille and Henry Dreyfus Foundation, and
Louis Martarano. S.T.P acknowledges support from the Alfred
P. Sloan Research Fellows program. We thank Landy K. Blasdel
for assistance with the manuscript.
Notes and references
1 The exact terminology used by the WHO is ‘‘robust’’.
2 M. Urdea, L. A. Penny, S. S. Olmsted, M. Y. Giovanni, P. Kaspar,
A. Sheperd, P. Wilson, C. A. Dahl, S. Buchsbaum, G. Moeller and
D. C. H. Burgess, Nature, 2006, 444(Suppl. 1), 73.
3 P. Scrimin and L. J. Prins, Chem. Soc. Rev., 2011, 40, 4488;
R. Bonomi, A. Cazzolaro, A. Sansone, P. Scrimin and L. J. Prins,
Angew. Chem., Int. Ed., 2011, 50, 2307; S. W. Thomas III, G. D. Joly
and T. M. Swager, Chem. Rev., 2007, 107, 1339; M. S. Masar III,
N. C. Gianneschi, C. G. Oliveri, C. L. Stern, S. T. Nguyen and
C. A. Mirkin, J. Am. Chem. Soc., 2007, 129, 10149; H. J. Yoon and
C. A. Mirkin, J. Am. Chem. Soc., 2008, 130, 11590; R. Perry-
Feigenbaum, E. Sella and D. Shabat, Chem. Eur. J., 2011,
17, 12123; N. C. Gianneschi, S. T. Nguyen and C. A. Mirkin, J. Am.
Chem. Soc., 2005, 127, 1644.
4 Conceptually related designs for signal amplification reagents are
described in the following references: (a) M. S. Baker and
S. T. Phillips, J. Am. Chem. Soc., 2011, 133, 5170; (b) H. Mohapatra,
K. M. Schmid and S. T. Phillips, Chem. Commun., 2012, 48, 3018;
(c) M. S. Baker and S. T. Phillips, Org. Biomol. Chem., 2012, 10, 3595;
(d) E. Sella, R. Weinstain, R. Erez, N. Z. Burns, P. S. Baran and
D. Shabat, Chem. Commun., 2010, 46, 6575.
Fig. 4 Calibration curve for a 10 h assay in which 2.5 mM 1 and 2 in 0.1 M
phosphate buffer (pH 7.3) containing 2.5% DMF and 0.5% Tween-20 (v/v) were
exposed to various concentrations of b-D-galactosidase at 23 1C. The intensity of
color for each experiment was obtained using image analysis of photographs.
The data points represent the averages of three measurements. (b) Expanded
view of the linear region in (a).
5 E. Sella, A. Lubelski, J. Klafter and D. Shabat, J. Am. Chem. Soc., 2010,
132, 3945.
6 P. A. Clapp, N. Du and D. F. Evans, J. Chem. Soc., Faraday Trans.,
1990, 86, 2587.
7 A. R. Lippert, G. C. Van de Bittner and C. J. Chang, Acc. Chem. Res.,
2011, 44, 793.
8 A. Martinez, S. T. Phillips, S. W. Thomas, H. Sindi and
G. M. Whitesides, Anal. Chem., 2008, 80, 3699.
9 E. W. Miller, N. Taulet, C. S. Onak, E. J. New, J. K. Lanselle,
G. S. Smelick and C. J. Chang, J. Am. Chem. Soc., 2010, 132, 17071.
10 21 days was the duration of the experiment.
The combined results from Fig. 3 and Fig. S1b (ESI†) indicate
that (i) the assay provides selectivity for the detection event (via 2),
and (ii) the enzyme detection event is capable of initiating the signal
amplification reaction with 1. More importantly, the combination of
1 and 2 provides greater signal than the use of 2 alone.
Motivated by the results in Fig. 3, we next generated a
quantitative assay for b-D-galactosidase by imaging the intensity of
the colorimetric response (using a camera) after 10 h of exposure of
1 and 2 to solutions of the enzyme. The resulting calibration curve
(Fig. 4) provides a dynamic range between 12 nM and 150 nM
b-D-galactosidase and a limit of detection14 of 12 nM (Fig. 4b), thus
11 In this latter experiment, any hydrogen peroxide arising from
background hydrolysis should be consumed by catalase, thus
substantially slowing the consumption of 1.
12 The enzyme b-D-galactosidase is
a general marker of fecal
contamination in drinking water. See: C. M. Davies and S. C. Apte,
Environ. Toxicol., 1999, 14, 355.
confirming the idea that the one-pot detection and signal amplifica- 13 We will study the mechanistic role of the surfactant on the rate of
the signal amplification reaction in due course.
tion reaction shown in Fig. 1 is capable of providing quantitative
measurements of an enzyme biomarker using only thermally-stable
14 The limit of detection was calculated as 3 Â (sd/s) where sd is the
standard deviation at 0.0 mM b-D-galactosidase and s is the slope of
small molecules as reagents in the assay.
the calibration curve.
c
This journal is The Royal Society of Chemistry 2013
396 Chem. Commun., 2013, 49, 394--396