Chemiluminescence molecular probe with a linear chain reaction amplification mechanism

Article abstract of DOI:10.1039/C8OB03042A

A new signal amplification probe with a linear chain reaction amplification mechanism and distinct chemiluminescence output was developed. The probe is composed of a unique structural motif that combines a chemiexcitation mechanism with an intramolecular transesterification into a single molecular structure. As demonstrated with a probe designed to detect hydrogen peroxide, an auto-inductive chemiluminescence signal amplification was obtained through methanol release by an intramolecular transesterification of the generated 2-hydroxymethylbenzoate derivative. The methanol was then oxidized by alcohol oxidase to regenerate the analyte of interest, hydrogen peroxide. Our probe enabled direct measurement of light emission with a limit of detection of 2.5 μM, whilst the assay was rapidly completed within 14 to 150 minutes. Such molecular probes with chemiluminescence signal enhancement through analyte amplification could be used for the detection of other chemical and biological analytes.

Full text of DOI:10.1039/C8OB03042A

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DOI: 10.1039/C8OB03042A
Chemiluminescence Molecular Probe with a Linear Chain Reaction Amplification
Mechanism
Samer Gnaima and Doron Shabata*
aSchool of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv
University, Tel Aviv 69978 Israel.
*Corresponding Author:
Doron Shabat
Department of Organic Chemistry
School of Chemistry, Faculty of Exact Sciences
Tel Aviv University
Tel Aviv 69978, Israel
Tel: +972 (0) 3 640 8340
Fax: +972 (0) 3 640 9293
E-mail: chdoron@post.tau.ac.il
1

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Abstract: A new signal amplification probe with a linear chain reaction amplification
mechanism and distinct chemiluminescence output was developed. The probe is composed
of a unique structural motif that combines a chemiexcitation mechanism with an
intramolecular transesterification into a single molecular structure. As demonstrated with a
probe designed to detect hydrogen peroxide, an auto-inductive chemiluminescence signal
amplification was obtained through methanol release by an intramolecular transesterification
of the generated 2-hydroxymethylbenzoate derivative. The methanol was then oxidized by
alcohol oxidase to regenerate the analyte of interest, hydrogen peroxide. Our probe enabled
direct measurement of light emission with a limit of detection of 2.5 µM, whilst the assay was
rapidly completed within 14 to 150 minutes. Such molecular probes with chemiluminescence
signal enhancement through analyte amplification could be used for the detection of other
chemical and biological analytes.
Graphical Abstract
2

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Signal amplification techniques are widely used to enhance the detection sensitivity of
analytes and enzymes for diagnostic purposes.1, 2 The ability to detect certain analytes with
high sensitivity is of great importance in various scientific fields where rapid analyte detection
is required.3, 4 Thus, there is an unmet need for detection techniques with improved sensitivity
and selectivity via signal amplification modes.
Over the last 10 years, several new signal amplification approaches have been developed.5-8
One approach to obtain signal amplification relies on an auto-inductive mechanism that
produces a detectable optical output. Several groups, including ours, have demonstrated new
concepts to achieve auto-inductive signal amplification based on chemical reactions with
small molecules.9-13 We have developed a unique modular technique for exponential
amplification of a diagnostic signal; amplification is achieved through a dendritic chain
14-16
reaction, generated by the disassembly of self-immolative dendrimers.10,
A single
activation event initiated by the analyte of interest leads to a chain reaction that ultimately
results in complete disassembly of all probe molecules and release of many copies of a
reporter molecule. Taking advantage of the modular design of the probe, our group and others
have applied the dendritic chain reaction technique for the detection of various analytes.11, 14,
17-20 In order to follow the progress of the signal amplification, an optically detectable dye is
commonly used as a chromogenic reporter.
Fluorescence and visible-absorbance chromogens are among the most useful reporters for
optical sensing.8, 21-24 However, chemiluminescence is considered as the most sensitive
modality for detection due to the high signal-to-noise ratio obtained in such assays.25-35
Chemiluminescence is the conversion of chemical energy into the emission of visible light as
a result of a chemical reaction that leads to chemiexcitation. Very recently, we have developed
3

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an effective molecular building block that intrinsically integrates a chemiluminescence turn-
ON phenomenon into a self-immolative domino-like fragmentation mechanism.36, 37 This type
of self-immolative chemiluminescence building block can execute the dual functions of
quinone-methide elimination and chemically initiated electron exchange luminescence. Using
this building block, a new signal amplification system with an auto-inductive mode of action
and distinct chemiluminescence output was also demonstrated.38 Understanding the
relationship between structure and function can assist in design of new amplification systems
with improved sensitivity and selectivity. Here we describe the use of a new type of molecular
building block for design and synthesis of a chemiluminescence probe with a linear chain
reaction (LCR) amplification mechanism.
It is well known that 2-hydroxymethylbenzoate esters are unstable under neutral and basic
aqueous conditions due to an intramolecular trans-esterification reaction between the
benzylic alcohol and the alkyl benzoate ester.39, 40 This reaction occurs through intramolecular
nucleophilic attack of the benzylic alcohol on the ortho-neighboring alkyl benzoate group and
affords the corresponding phthalide while releasing an alkoxy moiety. As presented in Figure
1A, the byproduct of the chemiexcitation process of Schaap’s 1,2-dioxetane I, is the alkyl
benzoate ester III. We therefore sought a molecular design that would generate a 2-
hydroxymethylbenzoate derivative from Schaap’s 1,2-dioxetane. We reasoned that this could
be implemented through a benzylic alcohol substituent on the original Schaap’s 1,2-dioxetane
(Figure 1B). The triggering of dioxetane V leads to the generation of 2-hydroxymethylbenzoate
VIII, which can then undergo an intramolecular cyclization reaction to release an alkoxy group
and simultaneously generate phthalide IX. The release of the alcohol molecule occurs by
intramolecular cyclization, and this reaction can be harnessed to generate a reagent with
4

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identical reactivity to that of the analyte of interest. Such a pathway will produce a sensor with
an auto-inductive amplification mechanism.
Figure 1: Molecular structure and activation pathway of (A) triggerable 1,2-dioxetane I and (B)
triggerable 1,2-dioxetane V; the latter reacts through an intramolecular trans-esterification
reaction.
Probe 1, designed to detect the analyte hydrogen peroxide, consists of phenoxy 1,2-dioxetane
as a chemiluminescent reporter and phenylboronic ester as a triggering substrate for
hydrogen peroxide (Figure 2). The LCR amplification cycle of the probe is illustrated in Figure
2B. Cleavage of the trigger of probe 1 by hydrogen peroxide under alkaline conditions leads
to the emission of chemiluminescent blue light and the generation of phenolate intermediate
1c. The latter undergoes intramolecular trans-esterification to release a methanol molecule.
The released methanol is then oxidized by alcohol oxidase (AOX) and molecular oxygen to
generate formaldehyde and hydrogen peroxide, which can then activate an additional probe
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molecule. The assimilation of both chemiexcitation and analyte regeneration processes in one
single molecular moiety is expected to provide an effective probe for a chemiluminescence
amplification cycle.
Figure 2: (A) Chemical structure and disassembly mechanism of probe 1. (B) Auto-inductive
disassembly and chemiluminescence emission of probe 1 triggered by hydrogen peroxide.
The synthesis of probe 1 was achieved as described in the Supporting Information section. The
amplification kinetic profiles of probe 1 (250 M) incubated with AOX (1 EU) and different
concentrations of hydrogen peroxide (0-250 M) in carbonate buffer (pH 9.5) was monitored
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by chemiluminescence emission at 499 nm (Figure 3A). Typical light emission profiles were
obtained for all solutions with signal increase to a maximum followed by decay to zero. In a
low concentration of hydrogen peroxide, a longer time period was required to achieve
maximum chemiluminescence intensity. When 1.0 equivalent (250 M) of hydrogen peroxide
was applied, the chemiluminescence system reached a maximum intensity within 14 minutes
compared to 23 minutes for 0.3 equiv., 36 minutes for 0.1 equiv., 50 minutes for 0.01 equiv.,
and 64 minutes in the absence of hydrogen peroxide. The total light emission values were
identical for all hydrogen peroxide concentrations tested (Figure 3B).
The light emission signal generated in the absence of hydrogen peroxide was also amplified
as a result of spontaneous side reactions. However, the observed background signal was
substantially lower than the signal obtained for a sample treated with 0.01 equiv. of hydrogen
peroxide. Within the time frame of the measurements (200 min), the background signal
reached a maximum of about half that of the signal generated in the presence of hydrogen
peroxide. After approximately 12 hours, the background signal reached maximum intensity
(data not shown). Given the noise generated by this background signal, the lowest detectable
hydrogen peroxide concentration was found to be 2.5 M.
For a control experiment, we synthesized probe 2; an analogues probe that cannot produce
an amplified signal. This probe has a pinacol-boron group that serves as a triggering substrate
for hydrogen peroxide. The benzylic alcohol is protected with a methyl group to prevent an
intramolecular trans-esterification reaction. Thus, probe 2 emits the chemiluminescent
reporter in an equal molar ratio to the applied concentration of the hydrogen peroxide
analyte. The increased sensitivity of the LCR technique with probe 1, in comparison to this
classic probe 2, was demonstrated when the background noise was subtracted from the
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measured signals and signal obtained were compared (Figure 3D). In the presence of 1.0
equivalent of hydrogen peroxide, probe 2 generated signal intensity identical to that of probe
1. However, in the presence of less hydrogen peroxide, the signals measured by probe 1 were
significantly larger than those obtained by probe 2. The ratio of the auto-inductive signal,
obtained by dividing the signal from probe 1 to that obtained from probe 2, increased with
decreasing analyte concentration. In the detection limit of our assay sensitivity, using 0.01
equivalents of hydrogen peroxide, the signal obtained by probe 1 was about 19-fold brighter
than that obtained using probe 2.
Figure 3: (A) Kinetics of chemiluminescent signal from probe 1 (250 µM) with various
concentrations of H2O2 (0, 2.5, 25, 83, and 250 M) in the presence of 1 EU AOX in buffer of
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pH 9.5. B) Total signal as a function of time from probe 1 with various concentrations of H2O2
(0, 2.5, 25, 83, and 250 M) in the presence of 1 EU AOX in buffer of pH 9.5. (C) Chemical
structures of probes 1 and 2. (D) Comparison of signals measured from probe 1 (LCR
amplification technique, blue) vs. signals measured using control probe 2 (green). The
background signal present at 72 min was subtracted from the values shown.
To test whether the amplification cycle indeed occurs as proposed in Figure 2, we monitored
the disassembly profile of probe 1 over time by a comparativereversed-phase HPLC technique.
Figure 4 shows three HPLC chromatograms recorded at different times after the addition of
probe 1 to hydrogen peroxide (0.01 equiv.) and AOX (1 EU) in buffer of pH 9.5. Before pinacol-
borane oxidation, the HPLC spectrum of probe 1 shows one characteristic peak with a
retention time of 20 minutes (Fig. 4A). At 130 minutes after addition of hydrogen peroxide,
the chromatogram shows three peaks (Figure 4B): probe 1, benzoate intermediate 1c
(retention time; 6.5 minutes) and phthalide 1d (retention time; 7.5 minutes). The complete
disassembly of probe 1 was achieved within 210 minutes (Fig. 4C). This time course of
disassembly is in good agreement with the kinetic profile obtained by monitoring of the
chemiluminescence (Fig. 3B). Furthermore, these results demonstrate that complete
disassembly of probe 1 is achieved in the presence of 0.01 equiv. of the hydrogen peroxide
analyte.
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Figure 4: The progression of the autocatalytic disassembly of probe 1 (250 μM) upon addition
of hydrogen peroxide (2.5 μM) in buffer (pH 9.5) monitored using RP-HPLC (λ = 270 nm).
Mechanistically, most of the previously reported chemiluminogenic probes of hydrogen
peroxide react with a single hydrogen peroxide molecule and release a single
chemiluminogenic probe molecule.41-43 Recently, auto-inductive signal amplification
techniques involving chemiluminescence have been reported. Turan’s group described the
attachment of Schaap’s dioxetane to an AB2 self-immolative dendron. Such a probe shows a
moderate chemiluminescence signal amplification (only 2-fold) with a detection-limit of the
analyte of around 240 µM.44 Akkaya and co-workers reported a self-immolative dendritic
probe for amplification of chemiluminescent signal based on dendritic chain reaction.35 This
self-immolative dendron was equipped with two dioxetane moieties as reporters, two fluoride
units attached through benzylic group, and a dimethyl-tertbutylsilyl group as a triggering
substrate for fluoride. Similarly, Hisamatsu et al. described a chemiluminescent amplification
cycle based on a two-component dendritic chain reaction system.28 The chemiluminescence
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molecular probe 1 described in this work enabled direct measurement of light emission with
a limit of detection of 2.5 µM. Furthermore, the assay was completed within 14 to 150
minutes.
The reported molecular design for a probe with chemiluminescence signal enhancement
through analyte amplification could be applicable for the detection of other chemical and
biological analytes. The latter could be achieved by introduction of various analyte-responsive
groups as triggering substrates and conversion the methoxy leaving group (here a methyl
ester) to the appropriate triggering analyte such as methyl thiol or fluoride.
In summary, we have developed a new signal amplification system with an LCR mode of action
and distinct chemiluminescence output. The molecular system combines a chemiexcitation
mechanism with an intramolecular transesterification into a single building block. As
demonstrated with the probe designed to detect hydrogen peroxide, LCR chemiluminescence
signal amplification was obtained through methanol release and in situ oxidation by an
intramolecular transesterification of the generated 2-hydroxymethylbenzoate derivative. A
kinetic experiment analyzed by RP-HPLC provided support for the proposed amplification
mechanism. This type of molecular probe with chemiluminescence signal enhancement
through analyte amplification could also be used for the detection of other analytes.
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