Electrochemiluminescence Bioassays with a Water-Soluble Luminol Derivative Can Outperform Fluorescence Assays

Article abstract of DOI:10.1002/anie.201708630

The most efficient and commonly used electrochemiluminescence (ECL) emitters are luminol, [Ru(bpy)₃]²⁺, and derivatives thereof. Luminol stands out due to its low excitation potential, but applications are limited by its insolubility under physiological conditions. The water-soluble m-carboxy luminol was synthesized in 15 % yield and exhibited high solubility under physiological conditions and afforded a four-fold ECL signal increase (vs. luminol). Entrapment in DNA-tagged liposomes enabled a DNA assay with a detection limit of 3.2 pmol L^?1, which is 150 times lower than the corresponding fluorescence approach. This remarkable sensitivity gain and the low excitation potential establish m-carboxy luminol as a superior ECL probe with direct relevance to chemiluminescence and enzymatic bioanalytical approaches.

Full text of DOI:10.1002/anie.201708630

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Accepted Article
Title: Electrochemiluminescence Bioassays can outperform
Fluorescence Assays using a New Water-soluble Luminol
Derivative
Authors: Michael Mayer, Shigehiko Takegami, Michael Neumeier,
Simone Rink, Axel Jacobi von Wangelin, Silja Schulte, Moritz
Vollmer, Axel Georg Griesbeck, Axel Duerkop, and Antje Jutta
Baeumner
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201708630
Angew. Chem. 10.1002/ange.201708630
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http://dx.doi.org/10.1002/ange.201708630

10.1002/anie.201708630
Angewandte Chemie International Edition
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Electrochemiluminescence Bioassays with a Water-Soluble
Luminol Derivative can Outperform Fluorescence Assays
Michael Mayer[a]+, Shigehiko Takegami[b]+, Michael Neumeier[c], Simone Rink[a], Axel Jacobi von
Wangelin[c], Silja Schulte[d], Moritz Vollmer[d], Axel G. Griesbeck[d], Axel Duerkop[a], Antje J.
Baeumner[a]*
Abstract: The most efficient and commonly used electrochemilumi-
nescence (ECL) emitters are luminol, [Ru(bpy)3]2+ and derivatives
thereof. Luminol stands out due to its low excitation potential, yet
applications are limited by the insolubility under physiological
conditions. The water-soluble m-carboxy luminol was synthesized in
15% yield and exhibited high solubility under physiological conditions
amplification via nanovesicles such as liposomes[6] provides
untapped possibilities. A signal enhancement of up to three
orders of magnitude[7] can be obtained by the incorporation of a
multitude of reporters in the inner cavity and the introduction of
surficial recognition moieties. We have recently reported the
facile synthesis of a family of substituted luminol derivatives with
high chemiluminescence (CL) quantum yields,[8] however their
poor solubility in aqueous buffer systems does not enable their
use in bio/liposome assays. Alternative hydrophobic or
hydrophilic luminol derivatives, modified either at the amino
functionality or the benzene ring (see table S2) do not exhibit
any better performance in ECL (few were better in CL) than the
parent luminol 1.[9] In this work, we present the synthesis of a
new luminol derivative, with a carboxyl group modification in the
benzene-ring, to significantly enhance solubility in aqueous
buffers while additionally increasing ECL yields. The new
molecule shows favorable ECL capabilities in comparison to the
parent luminol. If encapsulated into liposomes, DNA could be
detected in a very simple manner yet at excessively low
concentrations (attomolar level) in a sandwich assay using ECL
signal transduction.
and afforded
a four-fold ECL signal increase (vs. luminol).
Entrapment in DNA-tagged liposomes enabled a DNA assay with a
detection limit of 3.2 pmol L-1 which is 150 times lower than the
corresponding fluorescence approach. This remarkable sensitivity
gain and the low excitation potential establish m-carboxy luminol as
a superior ECL probe with direct relevance to chemiluminescence
and enzymatic bioanalytical approaches.
Since its ascent with A.J. Bard’s work on [Ru(bpy)3]2+ based ECL
in 1972[1] and the discovery of its great benefits towards other
analytical techniques, ECL nowadays plays a major role in
highest-sensitivity (bio)detection applications.[2] Most analytical
applications employ only few ECL luminophores, focusing on
[Ru(bpy)3]2+ and luminol. Yet new nanomaterials such as
quantum dots and electrode coatings find recently considerable
attention.[3] Luminol-ECL was first described in 1928.[4] Due to its
very low excitation potential of +0.5 V (*vs. Ag/AgCl; on gold
electrodes) it is compatible with a large variety of working
electrode materials and offers benefits towards electrode fouling
resistance and long-term stability in contrast to [Ru(bpy)3]2+
-
ECL (+1.2 V, *). Also, transparent electrode materials like indium
tin oxide can be used allowing more flexibility in terms of
detection and easy combination with microfluidic systems. In
analytical chemistry, signal enhancement and improvement of
assay sensitivity remain a vital area of research[5] where the
combination of the essentially background-free ECL with signal
[a]
Michael Mayer, Simone Rink, PD Dr. Axel Duerkop and Prof. Dr.
Antje J. Baeumner
Institute of Analytical Chemistry / Chemo-and Biosensors
University of Regensburg
Universitätsstraße 31, 93053 Regensburg
E-mail: antje.baeumner@ur.de
[b]
[c]
[d]
+
Prof. Dr. Shigehiko Takegami
Department of Analytical Chemistry,
Kyoto Pharmaceutical University, Japan
Michael Neumeier, Prof. Dr. Axel Jacobi von Wangelin
Institute of Organic Chemistry
University of Regensburg
Silja Schulte, Moritz Vollmer, Prof. Dr. Axel G. Griesbeck
Institute of Organic Chemistry
Figure 1. Assay with the proposed luminol-ECL reaction and the nucleic-acid
sandwich structure on a streptavidin anchor and top-bound m-carboxy luminol
2 encapsulated liposome.
University of Cologne
equal contributors
We synthesized a hydrophilic luminol derivative by introduction
of a carboxylate-substituent at the benzene ring. As there is no
report of this strategy via late-stage aromatic derivatization, we
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201708630
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Table 1. Properties of the m-carboxy luminol-liposomes.
decided to build up the luminol scaffold from the suitably
decorated starting material trimellitic anhydride (Scheme 1).
Following a literature report,[10] a three-step sequence led to the
corresponding phthalimide, which was then subjected to
hydrazinolysis to give the desired m-carboxy luminol 2 in 15%
overall yield. This has the additional merit of providing an anchor
for bioorthogonal conjugation.
Liposome data
Value
Hydrodynamic diameter
Polydispersity index
Surface ζ potential
271 nm
0.22
-24 mV
Phospholipid content
Total lipid content
5.9 mmol L-1
10.9 mmol L-1
22 mmol L-1
∼ 14 %
Encapsulant concentration
Liposome volume fraction
Calculated concentration of 2 in
∼16.8 mmol L-1
liposomes
Encapsulation efficiency
77%
The new m-carboxy luminol-liposomes were then investigated
for the detection of DNA derived from pathogenic organisms in a
microtiter plate assay format. The target sequence was a DNA
derived from the C. parvum heat shock protein 70 (hsp70)
mRNA which we had used previously in the development of
microtiterplate, microfluidic and lateral-flow assays.[15] We
obtained a typical sigmoidal calibration curve (fig. 2) after minor
assay optimization.[16] The latter included the choice of detergent
used to release encapsulants from the liposomes which offers a
further way to enhance the ECL signal.[17]
Scheme 1. Synthesis of m-carboxy luminol 2; (i) KNO3, H2SO4, 120°C, 45 h;
(ii) 1,3-dimethylurea, 170°C, 30 min; (iii) H2, Pd/C, r.t., 5 h; (iv) hydrazine
hydrate, 120°C, 6 h.
Analytical characterization of the luminol derivative focused on
its ability to function as ECL emitter. Using the parent luminol
ECL conditions, a 4-fold higher signal intensity was obtained for
the m-carboxy luminol vs. parent luminol in aqueous buffer
(figure S3). This is remarkable because quenching generally
becomes more prevalent in more polar solvents and commonly
decreases luminescence yields of probes by enhanced
radiationless decay. At the same time, the favorably low
excitation potential and the emission wavelength of 425 nm are
retained (figures S3 - S5) suggesting that 2 follows a similar ECL
pathway as described for 1.[11]
Subsequently, we utilized the m-carboxy luminol 2 in a bioassay.
Here, we took further advantage of the signal enhancement
strategy afforded by liposomes and developed
a DNA
hybridization assay. Liposomes were therefore synthesized
using the standard protocol[12] but with 22 mmol L-1 of 2 in 0.2
mol L-1 Hepes buffer (pH 8.6) as encapsulant solution.
Characterization of the liposomes revealed successful and
highly reproducible formation and typical data regarding size,
stability and composition (Tables 1 and S4). For comparison, we
aimed at the entrapment of 1 within liposomes by a literature
procedure.[13] However, the solubility of
1 under various
conditions (Table S3) was lower than published[13b] which
prevented its encapsulation in liposomes. Even though a broad
range of liposome syntheses was studied, none led to liposomes
containing sufficient amounts of parent luminol 1. Furthermore,
luminol leakage resulted in very limited stability and prohibited
characterization or bioassay application (Table S3). In contrast,
the m-carboxy luminol-liposomes were long-term stable upon
Figure 2. Calibration curve for target DNA sequence of C. parvum (hsp70).
°
storage at 10 C[14] which was in addition significantly increased
The limit of detection (3σ) was 3.2 pmol L-1, which corresponds
to 400 amol of target DNA per test. This is about 150 times
lower than previously demonstrated optimized limits of detection
with respect to earlier data.[13a]
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10.1002/anie.201708630
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using a fluorescence-based liposome assay and the same type
of liposome/lipid compositions.[12] With only 17 mmol L-1 m-
carboxy luminol entrapped in the liposomes vs. 150 mmol L-1
sulforhodamine B in the case of the fluorescent liposomes,[12] the
lower limit of detection afforded by the ECL liposomes is
therefore more than significant with a total enhancement factor
of almost 1500.
Acknowledgements
We are deeply indebted to Andrei Georgescu (CAD liposome
model), Vanessa Tomanek and Joachim Rewitzer (ICP-OES),
Florian Olbrich and the SensorikApplikationsZentrum at OTH
Regensburg (design and fabrication of 3D-printed ECL cells),
and the machine shop of the Faculty of Chemistry at the UR
(construction of the xyz stage) for their excellent technical
support. M.N. is a doctoral fellow of the Fond der Chemischen
Industrie (FCI).
Table 2. Comparison of matrix effects through real samples on 100 pmol L-1
DNA sequence.[18]
Sample
Signal recovery[a]
100 ± 5
Intensity integrals[b]
0.42 ± 0.02
Keywords: luminol • ECL • liposomes • biosensors •
luminescence
Reference
River water
Soil extract
Bovine serum
105 ± 23
0.44 ± 0.09
[1]
[2]
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Furthermore, the minimal matrix effects that were observed
upon spiking 100 pmol L-1 DNA sequences into various complex
matrices demonstrate the strength of the background-free ECL
assay principle (table 2). In summary, we synthesized and
characterized the new m-carboxy luminol 2 and demonstrated its
outstanding performance in bioassays.[19] Due to its high
solubility under physiological conditions, its low oxidation
potential, its high ECL yield, and the presence of a bioorthogonal
functional group, 2 is highly attractive for a variety of analytical
applications from which the parent luminol 1 is precluded. For
example, ECL can be easily miniaturized into microfluidic
systems.[20] Thus, one takes advantage of the absence of
scattered excitation light, its low inherent background, the highly
localized signal generation and minimized instrumental
hardware. These are all advantages especially in comparison to
miniaturized fluorescence detection. Therefore, the successful
combination of m-carboxy luminol with liposomes reported here
is a promising signal amplification tool for point-of-care and in-
field detection for DNA or RNA sequences. Further approaches
for bioassays can take advantage of the covalent coupling
capabilities through the carboxylate group. m-Carboxy luminol
enables studies under physiological conditions and provides
efficient signal enhancement and thus bears the potential to
replace luminol and [Ru(bpy)3]2+ in most of their bioanalytical
[6]
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applications.
This
opens
up
new
possibilities
in
chemiluminescence-based cellular imaging, in vivo detection of
biomarkers such as ROS, highly sensitive point-of-care sensing
and microarray technologies.
[14] See supplementary information, chapter 3.5
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[16] See supplementary information, chapter 3.7
[17] See supplementary information, chapter 3.6
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[18] See supplementary information, chapter 2.8.
[20] K. Kadimisetty, S. Malla, N. P. Sardesai, A. A. Joshi, R. C. Faria, N. H.
[19] Detection of DNA spiked into various matrices was chosen as powerful
example demonstrating assay stability and high sensitivity of the ECL
liposome approach. Detection of nucleic acids after their extraction from
a matrix or after their amplification through a molecular biological
approach results in cleaner matrices and is therefore significantly
simpler. The applicability to extracted and amplified RNA and DNA
sequences was shown previously [15b] through fluorescent liposomes.
Lee, J. F. Rusling, Anal. Chem. 2015, 87, 4472-4478.
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We present a newly synthesized,
highly water-soluble luminol derivative
(m-carboxy luminol) as efficient
luminescence probe and demonstrate
its superior performance in bioassays
using a DNA hybridization assay with
liposome signal enhancement.
M. Mayer, S. Takegami, M. Neumeier,
S. Rink, A. Jacobi von Wangelin, S.
Schulte, M. Vollmer, A. G. Griesbeck, A.
Duerkop, A. J. Baeumner*
Page No. – Page No.
Electrochemiluminescence
Bioassays with a Water-Soluble
Luminol Derivative can Outperform
Fluorescence Assays
This article is protected by copyright. All rights reserved.