Copolythiophene-derived colorimetric and fluorometric sensor for visually supersensitive determination of lipopolysaccharide

Article abstract of DOI:10.1021/ja211570a

3-Phenylthiophene-based water-soluble copolythiophenes (CPT1) were designed for colorimetric and fluorometric detection of lipopolysaccharide (LPS). The sensor (CPT1-C) shows a high selectivity to LPS in the presence of other negatively charged bioanalyte

Full text of DOI:10.1021/ja211570a

Article
pubs.acs.org/JACS
Copolythiophene-Derived Colorimetric and Fluorometric Sensor for
Visually Supersensitive Determination of Lipopolysaccharide
Minhuan Lan,†,§ Jiasheng Wu,† Weimin Liu,† Wenjun Zhang,*,‡ Jiechao Ge,† Hongyan Zhang,†
Jiayu Sun,†,§ Wenwen Zhao,†,§ and Pengfei Wang*,†
†Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese
Academy of Sciences, Beijing, 100190, People’s Republic of China
‡Center of Super-Diamond and Advanced Films (COSDAF) and Department of Physics and Materials Science, City University of
Hong Kong, Hong Kong SAR, P.R. China
§Graduate School of the Chinese Academy of Sciences, Beijing, 100049, China
S
* Supporting Information
ABSTRACT: 3-Phenylthiophene-based water-soluble copoly-
thiophenes (CPT1) were designed for colorimetric and
fluorometric detection of lipopolysaccharide (LPS). The sensor
(CPT1-C) shows a high selectivity to LPS in the presence of
other negatively charged bioanalytes as well an extreme
sensitivity with the detection limit at picomolar level, which is
the lowest ever achieved among all synthetic LPS sensors
available thus far. Significantly, the sensing interaction can be
apparently observed by the naked eyes, which presents a great
advantage for its practical applications. The appealing perform-
ance of sensor was demonstrated to originate from the multiple
electrostatic and hydrophobic cooperative interactions, syner-
getic with signal amplification via the conformational change of the 3-phenylthiophene-based copolymer main chain. As a
straightforward application, CPT1-C is capable of rapidly discriminating the Gram-negative bacteria (with LPS in the membrane)
from Gram-positive bacteria (without LPS).
polydiacetylene liposome acting as a fluorescent turn-on sensor
for LPS.7 Although the detection sensitivity for LPS has been
improved from millimolar to submicromolar level thus far, it is
still a great challenge to meet the requirement to detect LPS at
toxic concentration of picomolar level.
In recent years, water-soluble conjugated polythiophenes
with charged substituents and steroidal groups for sensing
biological relevant targets, such as DNA, proteins, ATP, and
folic acid have received considerable attention.9−11 The
homopolythiophene-based sensors generally show good
sensitivity due to amplification by a collective system response,
and thus offer a great superiority over the sensors based on
small molecules.12 However, they often display a low selectivity
to analytes,13 and show single signal response to binding events,
as a result, sensing may be affected by some uncertain factors
such as the instrumental efficiency, environment condition, and
molecular concentration.14
INTRODUCTION
■
Lipopolysaccharide (LPS) is a structural component in the
outer cell membranes of all Gram-negative bacteria (also
named endotoxin).1 LPS is highly toxic and biologically active
even at a concentration as low as pg/mL range.2 Sepsis and
septic shocks, arising from the massive release of LPS, cause
about 150 000 casualties annually in the United States.3 Due to
its high toxicity at a low concentration, the specific and sensitive
determination of LPS is of a particular interest. Currently,
enzymatic limulus amebocyte lysate (LAL) assay is clinically
used for the determination of endotoxin via the gel formation
between LAL and endotoxin.4 However, the LAL assay is highly
susceptible to changes in temperature and pH. Moreover,
carbohydrate derivatives other than LPS, such as β-glucans, also
respond positively to LAL. Thus, many efforts have been
devoted to develop highly sensitive and selective sensors for
LPS detection in the recent years.5−8 For instance, Basu et al.
reported a colorimetric sensor for LPS based on functionalized
polydiacetylene liposome,5 which could function at a high LPS
concentration above 100 μM. Brock et al. developed a FRET-
based sensor consisting of a CD14-derived LPS-binding peptide
terminally labeled with organic fluorophores that can detect
LPS at a concentration of micromolar range.6 Very recently,
Carsten Schmuck et al. reported a peptide-functionalized
Along with our continuing efforts in the exploration of
fluorescent chemsensors for bioactive molecules including LPS,
heparin and antibiotics,15 we herein employ a new design
strategy by means of copolymerization of 3-phenylthiophene
Received: December 11, 2011
Published: March 27, 2012
© 2012 American Chemical Society
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Scheme 1. Synthetic Routes of CPT1-A, CPT1-B, and CPT1-C and Model Polymers PT1 and PT2
4500 fluorescence spectrometers. The water was purified by Millipore
bearing quaternary ammonium and DABCO group substituted
thiophene to overcome the above setbacks. The phenyl as a
rigid group is attached to the 3-position of thiophene to tune
the conformation of copolymer chain, resulting in the signal
amplification;12 and DABCO moiety is introduced by means of
its strong affinity to phosphate groups in LPS.16 This appealing
sensing platform is expected to show the following advantages:
i) good stability and water solubility, ii) multiple electrostatic
and hydrophobic cooperative interactions for highly selective
detection toward LPS, iii) high signal-to-noise ratio and the
capacity for signal amplification, and iv) significant red-shift in
both the absorption and fluorescence wavelength upon
interaction with LPS. This approach enables us to design new
colorimetric and fluorometric sensors for LPS with high
selectivity and a detection limit down to the picomolar level.
Furthermore, this design strategy is straightforwardly adaptable
to a variety of sensors for highly sensitive and selective
detection of various biologically important polyanions by
introducing different polymerized units and recognized groups.
To illustrate the importance of copolymerization applied in our
design for highly selective and sensitive determination of LPS,
two corresponding homopolymers (PT1 and PT2) were also
synthesized as model polymers (Scheme 1). The results show
that neither PT1 nor PT2 display decent signal responses to
LPS because of the lack of cooperative interactions as shown by
CPT1-C.
1
filtration system. H NMR (400 MHz) and 13C NMR (100 MHz)
spectra were collected on a Bruker Advance-400 spectrometer with
tetramethylsilane as an internal standard. Electron impact (EI) mass
spectroscopy was carried out on a Waters GCT Premier mass
spectrometer. Matrix-assisted laser desorption ionization-time-of-flight
(MALDI-TOF) mass spectra were obtained on a Bruker Microflex
mass spectrometer and electrospray ionization (ESI) mass spectra on a
Shimadzu LC-MS 2010 instrument. The gel-permeation chromatog-
raphy was performed using gelatin as the standard, and the CH3CN−
water mixture solution containing NaNO3 (0.2 M) and CH3COOH
(0.5 M) under pH 5 was employed as eluent. Transmission electron
microscopy (TEM) images were taken on a JEOL JEM-2100F
operated at an acceleration voltage of 150 kV. Atomic force
microscopy (AFM) measurements were conducted using the DI
Multimode SPM from Veeco Systems and the images were obtained
with the tapping mode. Zeta potentials were recorded on Zetasize
3000 HS (Malvern, UK). Dynamic light scattering was performed on
Dybapro Nanostar from Wyatt Technology Corporation.
4-Bromobenzyl bromide, N,N-dimethyldodecylamine, thiophene-3-
boronic acid, tetrakis(triphenylphosphine)palladium(0), 3-thiophene-
acetic acid, 6-bromo-1-hexanol, 4-(dimethylamino)pyridine, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1,4-
diazabicyclo[2.2.2]octane were purchased form Alfa Aesar. ctDNA,
RNA, γ-globulins, NAD+, adenosine, AMP, ADP, ATP, guanine,
adenine, glucose, BSA, phosphatidylcholine, LPA, lipid A, EDTA,
malic acid, citric acid, chondroitin 4-sulfate sodium salt, heparin,
hyaluronic acid, and acetate were purchased from Sigma. Lip-
opolysaccharide (LPS) was purchased from Sigma, which was obtained
from Escherichia coli 055:B5. Note that the molecular weight of
commercial LPS varies between 3 and 20 KDa, and we assume a
molecular weight of 10 KDa in this work. Lipoteichoic acid (LTA),
obtained from Staphylococcus aureus, was purchased from Sigma. Other
reagents were purchased from Beijing Chemical Regent Co. All
EXPERIMENTAL SECTION
■
General Materials and Methods. All UV−vis and fluorescence
spectra in this work were recorded in Hitachi U3010 and Hitachi F-
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reagents and chemicals were AR grade and used without further
purification unless otherwise noted. CH2Cl2 and CHCl3 were distilled
from CaH2 under nitrogen. Escherichia coli ATCC 25922, S. aureus
ATCC 6538, Staphylococcus epidermidis ATCC 12228, Klebsiella
pneumoniae AS1.1736, and Pseudomonas aeruginosa AS1.2031 were
purchased from Antibacterial Material Testing Center of Technique
Institute of Physics and Chemistry, Chinese Academy of Sciences.
Syntheses of Sensor Probes and Model Polymers. The
synthetic routes of CPT1-A, CPT1-B, CPT1-C, and model polymers
PT1 and PT2 were outlined in Scheme 1, and the details were
described below.
residue was purified by column chromatography (eluent: CH2Cl2/
CH3OH = 12:1) to give compound 4 (0.3 g, yield 75%) as a colorless
solid. 1H NMR (400 MHz, CDCl3, TMS, ppm): δ 0.86−0.89 (t, 3H),
1.23−1.33 (m, 18H), 1.80 (m, 2H), 3.31 (s, 6H), 3.51−3.56 (m, 2H),
5.14 (s, 2H), 7.35−7.36 (d, J = 5 Hz, 1H), 7.39−7.41 (d, J = 8 Hz,
1H), 7.49 (s, 1H), 7.61−7.63 (d, J = 8 Hz, 2H), 7.69−7.70 (d, J = 8
Hz, 2H). 13C NMR (100 MHz, CDCl3, TMS, ppm): δ 14.1, 22.7, 23.0,
26.4, 29.3, 29.5, 29.6, 31.9, 49.6, 63.8, 67.2, 121.7, 126.0, 126.8, 126.9,
133.9, 137.9, 140.8. MALDI-TOF Mass spectrum m/z: Calculated:
386.29; Found: 386.13.
General Syntheses of Polythiophenes. All polymers in this
paper were prepared via an oxidative polymerization under nitrogen in
the presence of FeCl3. The general method for preparation of
polythiophenes was carried out as follows: 4 equiv of FeCl3 was
dissolved in 30 mL of dry CHCl3 under nitrogen, and then 1 equiv of
corresponding monomers dissolved in 20 mL of CHCl3 was added
dropwise. The reaction mixture was stirred at room temperature for 2
days. The resulting precipitate was collected, washed with methanol,
and finally dried under vacuum to give the desired polymers as a dark
red solid. For the copolymerization, monomer 4 and 2 with different
mole ratios (1/1, 1/2.5, and 2.5/1) were used to give the
corresponding copolythiophenes (CPT1-A, CPT1-B, and CPT1-C,
respectively). The polymerization of monomer 2 and 4 gives the
corresponding homopolythiophenes PT1 and PT2, respectively.
CPT1-A (Yield: 22%) Gel-Permeation Chromatography Analysis
(GPC). Mn = 9.789 × 104, polydispersity index (PDI = 1.100) 1H NMR
(400 Mz, CD3CN-D2O (v/v = 1/1), TMS, ppm) δ 0.68 (s, br), 1.07
(s, br), 1.28 (s, br), 1.59 (br), 2.85 (s, br), 2.99 (s, br), 3.24 (dbr), 3.46
(br), 3.53 (br), 3.84 (s, br), 4.23 (br), 4.44 (br), 6.80 (s, br), 7.22 (br),
7.36 (s, br). 7.49 (s, br).
6-Bromohexyl-2-(thiophen-3-yl)acetate (Compound 1). 6-
bromo-1-hexanol (0.362 g, 2 mmol) was added slowly with syringe to
a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (0.5 g, 2.6 mmol), 4-(dimethylamino)pyridine (70 mg, 0.6
mmol) and 3-thiopheneacetic acid (0.284 g 2 mmol) in 40 mL of dry
CH2Cl2 under nitrogen. The mixture was stirred at room temperature
for 12 h to complete the reaction. The solution was washed with H2O
(3 × 20 mL) and the organic layer was dried with anhydrous MgSO4.
After removal of the solvent under reduced pressure, the residue was
purified by column chromatography (eluent: petroleum ether/ethyl
acetate = 5:1) to give compound 1 (0.45 g, yield 74%) as a colorless
1
liquid. H NMR (400 MHz, CDCl3, TMS, ppm): δ 1.34−1.36 (m,
2H), 1.43−1.46 (m, 2H), 1.60−1.66 (m, 2H), 1.83−1.86 (m, 2H),
3.37−3.41 (t, J = 13 Hz, 2H), 3.65 (s, 2H), 4.09−4.12 (t, J = 13 Hz,
2H), 7.03−7.05 (d, J = 6 Hz, 1H), 7.14−7.15 (t, 1H), 7.26−7.29 (m, J
= 13 Hz, 1H). 13C NMR (100 MHz, CDCl3, TMS, ppm): δ 25.1, 27.8,
28.5, 32.6, 33.7, 36.0, 64.8, 122.8, 125.7, 128.5, 133.8, 171.2. EI Mass
spectrum m/z: Calculated: 306.01 (100%), 304.01 (98.2%); Found:
306.01, 304.01.
1
CPT1-B (Yield: 33%) GPC. Mn = 1.003 × 105 (PDI = 1.007). H
1-(6-(2-(Thiophen-3-yl)acetoxy)hexyl)-4-aza-1-azonia-
bicyclo[2.2.2]octane Bromide (Compound 2). 1,4-Diazabicyclo-
[2.2.2]octane (0.2 g, 1.8 mmol) was dissolved in 50 mL of ethyl
acetate with the subsequent addition of intermediate 1 (0.3 g, 1
mmol). The mixture was stirred at room temperature for 36 h to
complete the reaction. The resulting white precipitation was collected,
washed with ethyl acetate, and dried in vacuum to give compound 2 as
a white solid (0.25 g, yield 60%). 1H NMR (400 MHz, CD3OD TMS,
ppm): δ 1.35−1.46 (m, 4H), 1.64−1.70 (m, 2H), 1.71−1.77 (m, 2H),
3.18−3.25 (m, 8H), 3.30−3.36 (m, 6H), 3.67 (s, 2H), 4.11−4.14 (t, J
= 13 Hz, 2H), 7.03−7.05 (d, J = 5 Hz, 1H), 7.22−7.23 (m, 1H), 7.36−
7.38 (d, J = 8 Hz, 1H). 13C NMR (100 MHz, CD3OD, TMS, ppm): δ
22.7, 26.4, 26.9, 29.3, 36.4, 46.1, 53.4, 65.5, 65.7, 123.9, 126.7, 129.7,
135.4, 173.0. MALDI-TOF Mass spectrum m/z: Calculated: 337.19;
Found: 337.03.
N-(4-Bromobenzyl)-N,N-dimethyldodecan-1-aminium Bro-
mide (Compound 3). 4-Bromobenzyl bromide (0.25 g, 1 mmol)
was dissolved in 20 mL of CH2Cl2/CH3OH (v/v = 3/2) with the
subsequent addition of N,N-dimethyldodecylamine (0.4 mL, 1.3
mmol). The mixture was stirred at room temperature for 12 h. After
the reaction was completed, the reaction solution was concentrated to
5 mL. The residue was poured into 200 mL of absolute diethyl ether
under stirring and then filtered. The precipitate was filtered, washed
with absolute diethyl ether and dried to give compound 3 (0.43 g,
yield 92%) as a white solid. 1H NMR (400 MHz, CDCl3, TMS, ppm):
δ 0.82−0.87 (t, 3H), 1.21−1.26 (m, 18H), 1.75 (m, 2H), 3.24−3.26
(s, 6H), 3.48−3.52 (m, 2H), 5.17−5.21 (s, 2H), 7.49−7.51(d, J = 8
Hz, 2H), 7.58−7.60 (d, J = 8 Hz, 2H). 13C NMR (100 MHz, CDCl3,
TMS, ppm): δ 13.8, 22.4, 22.7, 26.1, 29.0, 29.1, 29.3, 31.6, 49.2, 63.7,
66.1, 125.2, 126.3, 132.1, 134.7. MALDI-TOF Mass spectrum m/z:
Calculated: 382.21 (100%), 384.20 (97.4%); Found: 382.27, 384.29.
N,N-Dimethyl-N′-(4-(thiophen-3-yl)benzyl)dodecan-1-ami-
nium Bromide (Compound 4). Deionized water (10 mL) was
added with syringes to a mixture of compound 3 (0.4 g, 0.86 mmol),
Na2CO3 (0.5 g, 4.7 mmol), Pd(PPh3)4 (200 mg, 0.17 mmol), and
thiophene-3-boronic acid (0.128 g, 1 mmol) in EtOH (20 mL) under
nitrogen. After refluxing at 90 °C for 6 h, EtOH was removed under
reduced pressure. The residue was extracted with CH2Cl2 (3 × 20 mL)
and the resulting organic layer was collected and dried with anhydrous
MgSO4. After removal of the solvent under reduced pressure, the
NMR (400 Mz, CD3CN-D2O (v/v = 1/1), TMS, ppm) δ 0.69 (s, br),
1.08 (s, br), 1.30 (s, br), 1.57−1.68 (dbr), 2.99 (s, br), 3.36 (s, br),
3.54 (s, br), 3.66−3.67 (dbr), 4.03 (br), 4.32 (br), 6.99 (s, br), 7.18 (s,
br), 7.34 (s, br).
1
CPT1-C (Yield: 28%) GPC. Mn = 1.061 × 105 (PDI = 1.160). H
NMR (400 Mz, CD3CN-D2O (v/v = 1/1), TMS, ppm) δ 0.68 (s, br),
1.05 (s, br), 1.59 (s, br), 2.85 (s, br), 2.99 (s, br), 3.29 (s, br), 3.50−
3.59 (dbr), 4.23 (s, br), 4.45 (s, br), 6.80 (s, br), 7.35 (s, br), 7.49 (s,
br).
PT1 (yield: 55.6%) GPC. Mn = 1.643 × 105 (PDI = 1.233). 1H NMR
(400 Mz, CD3CN-D2O (v/v = 1/1), TMS, ppm) δ 1.14 (br), 1.30 (s,
br), 1.59−1.69 (dbr), 3.46 (s,br), 4.24 (s, br), 7.21, (s, br).
PT2 (yield: 50.0%) GPC. Mn = 6.655 × 104 (PDI = 1.161). 1H NMR
(400 Mz, CD3CN-D2O (v/v = 1/1), TMS, ppm) δ 0.67−0.69 (br),
1.06 (s,br), 1.60 (s, br), 2.85 (s, br), 3.00 (s, br), 4.56 (s, br), 6.81 (s,
br), 7.35−7.37 (dbr), 7.48−7.50 (dbr).
Fluorescent Spectra of CPT1-C with Different Bacteria. Five
kinds of freshly diluted bacteria (C = 108 cfu/mL) were incubated with
CPT1-C (70 μM, TBS, pH 7.4) for 5 min, and then the bacterial
suspensions were used for fluorescence test in a Hitachi F-4500
fluorescence spectrometer with an excitation at 460 nm.
Bacteria Imaging. Fluorescence imaging of bacteria (E. coli, K.
pneumoniae, and P. aeruginosa) was performed with an NIKON-SIM
confocal laser scanning microscopy using the light source at 561 nm
for excitation. A 100-oil-immersion objective lens was used. Freshly
diluted E. coli, K. pneumoniae, and P.aeruginosa were cultured in the
media in the presence of CPT1-C at 70 μM, (TBS buffer solution, pH
7.4). The bacteria were collected by centrifugation at 9,000 rap for 10
min, rinsed with TBS (pH 7.4), and then resuspended in TBS solution
(1 mL). The bacteria suspensions were dropped into a cover glass for
fluorescence imaging.
RESULTS AND DISCUSSION
■
Molecular Structures of LPS and LTA. LPS and LTA are
major constituents of the cell membranes of Gram-negative
bacteria and Gram-positive bacteria, respectively. To discrim-
inate the Gram-negative bacteria from Gram-positive bacteria, it
is necessary to clearly understand the molecular structures of
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Scheme 2. (A) Molecular Structures of LPS, LTA, and sensor CPT1 and (B) Schematic Illustration of the Proposed Interaction
Mechanism of CPT1 with LPS Utilizing the Electrostatic Interaction, the Hydrophobic Interaction, and the Conformational
Change
LPS and LTA. As shown in Scheme 2A, the primary toxic
component of LPS is the lipid A core which is linked with two
2-keto-3-deoxyoctonate units.17 LPS is highly negative charged
due to the polysaccharide formed with two phosphorylated
glucosamine sugars in the lipid A part and two 2-keto-3-
deoxyoctonate units. Moreover, six hydrophobic chains in one
structural unit make the overall molecule highly amphiphilic. As
a result, the highly hydrophobic and negatively charged LPS
would automatically arrange in a definite order in aqueous
solution. When the concentration of LPS reaches to a certain
extent, the phospholipid bilayers will be formed in aqueous
solution.18 In contrast, LTA consists of teichoic acids and long
chains of ribitol phosphate, and it is anchored to the lipid
bilayer via glyceride. The major difference between LPS and
LTA is that over 6 hydrophobic chains exist in one structural
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Figure 1. (A) Absorbance titration spectra of CPT1-C (50 μM) in 10 mM HEPES buffer solution (1% CH3CN, pH 7.4) upon gradual addition of
LPS from 0 to 5 μM. The inset shows the color change of the resulting solution (0.5 mM CPT1-C in the absence and presence of 50 μM LPS). (B)
Fluorescence titration spectra of CPT1-C upon addition of gradual addition of LPS from 0 to 5 μM with an excitation at 460 nm. The inset shows
the corresponding fluorescent color change. The fluorescence intensity ratio (I600/I566) of CPT1-C vs the concentrations of LPS (0−5 μM) (C) and
(0−30 nM) (D), respectively.
unit of LPS, while only two hydrophobic chains exist in that of
LTA.
room temperature by absorption and emission spectroscopy.
The UV−vis spectrum of CPT1-C in HEPES solution displays
a characteristic absorption band of polythiophene at 420 nm
(Figure 1A). Upon addition of LPS, the absorption peak at 420
nm decreased gradually, a new absorption band at around 540
nm emerged with an isosbestic point at 460 nm, and the color
changed from yellow to red. Their corresponding changes in
the fluorescence spectra are shown in Figure 1B. In the absence
of LPS, CPT1-C showed a characteristic polythiophene
emission at 566 nm. Upon addition of LPS, the emission
peak was red-shifted to 600 nm. Such significant changes in the
absorption and fluorescence spectra could be well explained by
the LPS-induced conformational change of conjugated back-
bone through the electrostatic and hydrophobic cooperative
interactions between CPT1-C and LPS. Particularly, the
corresponding effect could be evaluated quantitatively by
analyzing the dependence of the fluorescence intensity ratio
(I600/I566) on the concentration of LPS. As shown in Figure 1C,
the I600/I566 ratio increased gradually upon addition of LPS,
saturated at [LPS] = 2 μM. Further addition of LPS could not
Design of Copolythiophenes for LPS Detection.
Bearing the detailed structural information in mind (vide
supra), we designed a water-soluble copolymer (CPT1-C) for
LPS sensing on the basis of the interactive and complementary
interaction. CPT1-C is expected to discriminate LPS from
LTA, and thus discriminate the Gram-negative bacteria from
Gram-positive bacteria. As shown in Scheme 2A, the
ammonium and quaternary diazabicyclooctane (DABCO)
substituents in CPT1-C not only improve its water solubility
but also provide two positive centers to bind with the negatively
charged LPS via electrostatic interaction. In particular, the
DABCO moiety has been demonstrated to have a very strong
affinity to phosphate groups, as reported in previous work.16
The ammonium moiety with a C-12 alkyl chain and the
DABCO moiety with a C-6 alkyl chain in CPT1-C are expected
to increase its hydrophobic interaction with LPS in aqueous
solution. In addition, the phenyl group introduced to the 3-
position of the thiophene ring can be used as a blocking unit to
force the polythiophene backbone to adopt a twist
conformation.19 CPT1-C will form a quasi-sphere-shaped self-
assembly in aqueous solution (vide infra). Upon complexation
with LPS, the electrostatic and hydrophobic cooperative
interactions between CPT1-C and LPS can induce the
conformational change of copolythiophene backbone (Scheme
2B). Thus, the steric hindrance from the phenyl group is
decreased and the copolythiophene backbone becomes more
coplanar. Such a conformational change results in significant
red-shifts in both the absorption and fluorescence wavelengths
of CPT1-C, which has also been observed in other
polythiophenes.20 It should be mentioned that different
monomer ratios in the copolymer can be controlled to tune
the sensitivity and selectivity to LPS as well.
induce obvious changes in the spectrum. By plotting the I600
/
I566 ratio versus the concentration of LPS, a good linear
relationship (I600/I566 = 0.506 + 1.76 × 10−3 × [LPS], R2 =
0.999) was observed for a LPS concentration ranging from 0.3
to 30 nM (Figure 1D). Based on that, the limit of detection
(CLOD) was calculated to be 270 pM (Supporting Informa-
tion),21,22 which is the lowest value reported thus far (over at
least 3 orders of magnitude lower than the literature
reports).5−8
The zeta-potential analysis was carried out to provide direct
evidence for the effective binding of CPT1-C with LPS through
the electrostatic interaction. The zeta-potential of CPT1-C was
determined to be +50.2 mV even at low concentration (50
μM), indicating a mass of positive charges on its surface. Upon
addition of 5 μM of LPS, the zeta-potential of CPT1-C was
reduced to −2.12 mV, implying that the negatively charged LPS
did interact with CPT1-C through the electrostatic interaction.
LPS Sensing with CPT1-C. The interaction between
CPT1-C (50 μM, calculated on monomers basis) and LPS
was studied in HEPES buffer (10 mM, pH 7.4, 1% CH3CN) at
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Figure 2. TEM images of CPT1-C (50 μM) in 10 mM HEPES buffer solution (1% CH3CN, pH 7.4) in the absence (A) and presence (B) of 5 μM
LPS, respectively.
Figure 3. Absorbance and fluorescence titration spectra of CPT1-A (A and B) and CPT1-B (C and D) (50 μM) in 10 mM HEPES buffer solution
(1% CH3CN, pH 7.4) upon gradual addition of LPS with the concentration ranging from 0 to 10 μM, respectively. The exciting wavelength is 460
nm.
Transmission electron microscopy (TEM, Figure 2) and atomic
force microscope (AFM, Figure S1) observations of CPT1-C
also indicated that the copolymer with long alkyl chains and
highly positive charges can form a quasi-sphere-shaped self-
assembly in aqueous solution (Figure 2A). The structure was
destroyed upon addition of LPS due to the electrostatic and
hydrophobic cooperative interactions, and a strong supra-
molecular complex between LPS and CPT1-C with an obvious
conformational change was formed (Figure 2B). In addition,
the dynamic light scattering studies showed that the mean
radius of quasi-sphere-shaped structures of free CPT1-C was
118.3 nm, while it increased to 205.3 nm upon addition of LPS
(Figure S2), agreeing with TEM and AFM observations. The
change of particle structure from quasi-sphere to nonsphere is
the major reason for the increase of hydrodynamic radius.
Based on these results, it is reasonable to conclude that the
conformation of CPT1-C is changed from a coil to a more
extended structure. The similar conformational change was also
observed in homopolythiophenes.9−11 The above results are
well consistent with the spectral changes in Figure 1.
Effect of Different Monomer Ratios in Copolythio-
phenes. As discussed above, CPT1-C can effectively identify
LPS in a HEPES buffer solution through the ratiometric
changes in absorption and fluorescence spectra. To further
illustrate the role of phenyl group at 3-position of thiophene
ring in the sensing process, two copolymers (CPT1-A and
CPT1-B) with different monomer ratios (1/1 and 1/2.5,
respectively) were prepared. The interaction between CPT1-A
and LPS was similar to that of CPT1-C under the same
experimental conditions. For example, CPT1-A displayed a
characteristic absorption band of polythiophene at 415 nm, and
the band shifted to 540 nm with the addition of LPS (Figure
3A). Concomitantly, the emission peak was red-shifted from
566 to 600 nm upon addition of LPS (Figure 3B). The
observed color change from yellow to red is believed to be
related to a coil-to-rod transition of the conjugated back-
bone.19,20 In contrast, CPT1-B did not show a significant
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change in the absorption spectra upon addition of LPS (Figure
3C) due to fewer amount of rigid phenyl groups in the
copolymer chain, and the corresponding fluorescence spectra
displayed less ratiometric changes (Figure 3D).
To further compare the sensitivity of three copolymers to
LPS, we defined the signal-to-background ratio (S/B) using the
following equation:22
polymers (PT1 and PT2) for comparison. PT1 contains a C-6
alkyl chain with DABCO terminal, while PT2 contains a phenyl
and an ammonium with C-12 alkyl terminal. As depicted in
Figure 4B, PT1 showed a characteristic polythiophene emission
at 550 nm in the absence of LPS. Upon addition of LPS, the
fluorescence intensity at 550 nm was monotonously decreased
and the peak location maintained until the concentration of
LPS reached 4.5 μM. Meanwhile, there was a slight blue shift in
the absorption spectroscopy (Figure 4A). The nonregioregular
polythiophene derivative is considered to allow only the
formation of weak and localized conformation defects along the
backbone, leading to a continuous and monotonic decrease of
the fluorescence intensity upon complexation with analytes.23
PT2 is not able to interact effectively with LPS through the
electrostatic interaction because it lacks the DABCO group to
bind with the phosphate groups. In addition, the structure with
only one long chain also decreases its hydrophobic interaction
with LPS. As a result, the addition of LPS results in smaller red-
shifts both in the absorption and fluorescence wavelengths of
PT2 (Figure 4D). These results strongly support the suggestion
that the cooperative and complementary interaction can play
essential role in the molecular design, and each functional
substituent in CPT1-C is indispensable for highly sensitive
detection of LPS.
S/B = (I600/I
)
566 withLPS/(I600/I566 withoutLPS
)
where (I600/I566
)
and (I600/I566
)
represent the
withoutLPS
withLPS
I
600/I566 ratios in the presence and absence of LPS, respectively.
It was found that the monomer ratios of three copolymers
calculated from the integrity area were associated with the mole
ratio of two added monomers and would signaly affect the S/B
value. As shown in Table 1, the signal-to-background ratio of
Table 1. Comparable Data of Three Copolythiophenes to
LPS
CPT1-A
CPT1-B
CPT1-C
a
b
monomer 4/monomer 2
1/1
1/2.5
1/2
2.5/1
5/1
monomer 4/monomer 2
1.07/1
0.825
1.605
1.945
c
(I600/I566
(I600/I566
)
)
0.753
0.855
1.135
0.51
withoutLPS
c
withLPS
2.781
5.453
d
S/B
Reactivity of CPT1-C with Other Biologically Impor-
tant Species. Selectivity is another essential parameter for the
practical application of CPT1-C. The selectivity of the sensor
a
b
The mole ratio obtained from the added monomers. The mole ratio
calculated from the integrity in 1H NMR spectrum (Supporting
Information). The ratio of the fluorescence intensity at 600 and 566
nm in the absence and presence of LPS (4.5 μM), respectively. The
c
d
for LPS was evaluated by the fluorescence intensity ratio (I600
/
signal-to-background ratio (S/B) of three copolythiophenes to LPS.
I566) response in the presence of various biologically important
species in buffer solution. As illustrated in Figure 5, nearly all of
the other biological molecules (except for LTA) do not give
rise to significant increase in I600/I566 ratio. LTA and LPS have
closely similar molecular structures; both of them contain
phosphates and long hydrophobic chains. LPS is higher
negative charged and more hydrophobic than LTA. However,
only LPS could induce change in the fluorescence intensity
ratio (I600/I566) by at least a factor of 3. The addition of LTA
gave rise to a less signal response compared to that of LPS. The
CPT1-C with 2.5/1 mol ratio is up to 5.453, which is much
higher than those of CPT1-A (1.945, 1/1) and CPT1-B (1.135,
1/2.5). The results coincide well with the spectral changes of
the copolythiophenes.
Interaction between Two Homopolythiophenes and
LPS. To verify that it is indeed the cooperative interaction
leading to the high snsitivity of CPT1-C to LPS, two
corresponding homopolythiophenes were prepared as model
Figure 4. Absorbance and fluorescence titration spectra of PT1 (A and B) and PT2 (C and D) (50 μM) in 10 mM HEPES buffer solution (1%
CH3CN, pH 7.4) upon gradual addition of LPS with the concentration ranging from 0 to 4.5 μM. The exciting wavelength is 460 nm.
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Figure 6. Effect of pH on the fluorescence intensity ratio (I600/I566) of
free CPT1-C (50 μM) (black squares) and CPT1-C/LPS (1.5 μM)
mixtures (red dots) at room temperature.
Figure 5. Selectivity of CPT1-C (50 μM) to LPS (1.2 μM) in 10 mM
HEPES buffer solution (1% CH3CN, pH 7.4) in the presence of some
coexisting biological species including lipoteichoic acid (LTA), lipid A,
ctDNA, RNA, γ-globulins, NAD+, adenosine, AMP, ADP, ATP,
guanine, adenine, glucose, BSA, Phosphatidylcholine, LPA, EDTA,
malic acid, citric acid, phosphate, pyrophosphate, chondroitin 4-sulfate
sodium salt (Chs), heparin, hyaluronic acid (HA), acetate. ctDNA,
RNA, γ-globulins, and BSA were 1 mg/L, LTA was 1.2 μM, and the
other anions or biological molecules were 2 μM. Blank refers to free
CPT1-C solution. Black pillars and red pillars refer to these analytes in
the absence and presence of 1.2 μM LPS, respectively.
staining, while Gram-negative bacteria, which cannot retain the
crystal violet stain, instead take up the counter-stain and appear
in red or pink.24 However, this method requires numerous
tedious procedures including pretreatment in harsh conditions.
Because LPS is the main constituent of the outer membrane of
Gram-negative bacteria and CPT1-C is highly selective and
sensitive to LPS, CPT1-C is thus expected to be able to
distinguish Gram-negative bacteria and Gram-positive bacteria.
In this work, three kinds of Gram-negative bacteria (E. coli, K.
pneumoniae, and P. aeruginosa) and two kinds of Gram-positive
bacteria (S. epidermidis and S. aureus) were used as example
targets. Freshly diluted bacteria (C = 108 cfu/mL) were
incubated with CPT1-C (70 μM, TBS, pH 7.4) for 5 min, and
then the bacterial suspensions were used for fluorescence test in
a Hitachi F-4500 fluorescence spectrometer with an excitation
at 460 nm. Figure 7A shows the photo- and fluorescent-images
of five kinds of bacteria after incubation with CPT1-C. Gram-
positive bacteria incubating with CPT1-C induced a colorless
solution (2 and 3) and the fluorescence was quenched. In
observation can be simply explained by the fact that a biological
species having only a single alkyl chain or a low negative charge
density cannot effectively bind with CPT1-C and cause
significant conformational changes. In contrast, CPT1-C
interacts with LPS by the multiple electrostatic and hydro-
phobic cooperative interactions, which results in a distinct
conformational change. Concomitantly, the UV−vis absorption
spectra of CPT1-C also varied significantly only in the presence
of LPS and induced obvious color change (Figure S3). It was
revealed that the lipid A itself only led to a slight increase in the
fluorescence intensity ratio (I600/I566) compared with LPS
(Figure 5). The reason could probably be attributed to more
electrostatic interaction sites (i.e., carboxyl) between LPS and
CPT1-C than that of lipid A itself in comparison of the
molecular structures of LPS with lipid A. Furthermore, CPT1-
C shows a distinct signaling increase to LPS in the presence of
the coexisting lipid A, demonstrating that CPT1-C has a
superior selectivity to LPS over lipid A. The results indicate that
the colorimetric and fluorometric sensor is highly selective to
LPS even in the presence of biologically competing species.
pH-Dependent Response. To further study the practical
applicability of this sensor, the pH effects of CPT1-C in the
presence of LPS were also investigated. Experimental results
indicated that the fluorescence intensity ratio (I600/I566) of
CPT1-C did not change obviously in a wide pH range from 4
to 11 (Figure 6). Upon addition of LPS, this intensity ratio
showed enhancements by about a factor of 3 and a distinct
platform between pH 4 and 11, demonstrating that CPT1-C
can be used to detect LPS throughout a wide pH range.
Discrimination of Gram-Negative Bacteria from
Gram-Positive Bacteria. Bacterial contamination is a major
health hazard especially in the context of food safety,
environmental monitoring, and the pharmaceutical industry.
Thus, rapid differentiation of bacteria is imperative for clinical
diagnosis, food safety and therapeutic strategies. Gram staining
is a conventional method to differentiate bacterial species from
Gram-positive bacteria and Gram-negative bacteria based on
the distinct chemical and physical properties of their cell walls.
Gram-positive bacteria are stained dark blue or violet by Gram
Figure 7. (A) Photoimages and fluorescent-images for five kinds of
bacteria (108 cfu/mL) after incubation with CPT1-C for 5 min. The
numbers 1, 2, 3, 4, 5, 6 refer to free CPT1-C, S. aureus, S. epidermidis,
E. coli, K. pneumoniae, and P. aeruginosa incubate in CPT1-C solution,
respectively. (B) Fluorescence intensity ratio (I600/I566) vs five kinds of
bacteria (108 cfu/mL) after incubated with CPT1-C (70 μM). (C)
Confocal fluorescence image of E. coli (108 cfu/mL) after incubation
with CPT1-C in fresh medium solution (λex = 561 nm). (D) The
fluorescence intensity profile across the line is shown in (C).
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contrast, the Gram-negative bacteria which contain LPS
showed a visual color change from yellow to orange and the
fluorescence color changed from yellow to pink. The
observations are also consistent with the fluorescent spectra
obtain from five kinds of bacteria after incubation with CPT1-C
(Figure 7B). These results demonstrate that CPT1-C as a dual
colorimetric and fluorometric sensor for LPS is able to rapidly
distinguish Gram-negative bacteria from Gram-positive bac-
teria, which is very important and useful for practical
application. In order to demonstrate directly whether the
sensor is also capable of interacting with LPS on the surface of
bacteria, fluorescence imaging of bacteria (E. coli, K. pneumo-
niae, and P. aeruginosa) after incubated with CPT1-C was
performed with an NIKON-SIM confocal laser scanning
microscopy. As shown in Figure 7C, a strong red fluorescence
was observed outside the cells and the fluorescence intensity of
the outside cells was stronger than that of the middle (Figure
7D). Similar results were also obtained on K. pneumoniae and P.
aeruginosa after incubation with CPT1-C (Figure S4),
confirming that CPT1-C is capable of interacting with LPS
on the surface of bacteria.
Knowledge Innovation of Chinese Academy of Sciences, and
the National High Technology Research and Development
Program of China (863 Program; Grant No. 2009AA03Z318).
REFERENCES
■
(1) (a) Young, L. S.; Martin, W. J.; Meyer, R. D.; Weinstein, R. J.;
Anderson, E. T. Ann. Intern. Med. 1977, 86, 456−471. (b) Ulevitch, R.
J. Adv. Immunol. 1993, 53, 267−289. (c) Ulevitch, R. J.; Tobias, P. S.
Curr. Opin. Immunol. 1994, 6, 125−130. (d) Seltmann, G.; Holst, O.
The Bacterial Cell Wall; Springer: New York, 2002.
(2) (a) Beutler, B.; Rietschel, E. T. Nat. Rev. Immunol. 2003, 3, 169−
176. (b) Warren, H. S.; Fitting, C.; Hoff, E.; Adib-Conquy, M.;
Beasley-Topliffe, L.; Tesini, B.; Liang, X.; Valentine, C.; Hellman, J.;
Hayden, D.; Cavaillon, J. M. J. Infect. Dis. 2010, 201, 223−232.
(3) (a) Raetz, C. R. H. Annu. Rev. Biochem. 1990, 59, 129−170.
(b) Van Amersfoort, E. S.; Van Berkel, T. J. C.; Kuiper, J. Clin.
Microbiol. Rev. 2003, 16, 379−414.
(4) (a) Roslansky, P. F.; Novitsky, T. J. J. Clin. Microbiol. 1991, 29,
2477−2483. (b) Zhang, G.; Baek, L.; Nielsen, P. E.; Buchardt, O.;
Koch, C. J. Clin. Microbiol. 1994, 32, 416−422.
(5) Rangin, M.; Basu, A. J. Am. Chem. Soc. 2004, 126, 5038−5039.
(6) Voss, S.; Fischer, R.; Jung, G.; Wiesmuller, K. H.; Brock, R. J. Am.
̈
Chem. Soc. 2007, 129, 554−561.
CONCLUSIONS
(7) Wu, J.; Zawistowski, A.; Ehrmann, M.; Yi, T.; Schmuck, C. J. Am.
Chem. Soc. 2011, 133, 9720−9723.
■
In summary, we developed for the first time a copolythiophene
CPT1-C to function as a colorimetric and fluorometric sensor
with an ultrahigh sensitivity at the picomolar level toward LPS
in aqueous solution, which is by 3 orders of magnitude lower
than the most recently reported LPS sensors. To the best of our
knowledge, the CPT1-C sensor shows the lowest detection
limit among all synthetic LPS probes reported so far. The
sensor is also highly selective toward LPS in the presence of
other negatively charged analytes coexisting with LPS, and the
excellent selectivity has been illustrated to be due to the
multiple electrostatic and hydrophobic cooperative interactions.
As a practical example, we also demonstrate that CPT1-C is
able to distinguish Gram-negative bacteria from Gram-positive
bacteria even upon on visual observation. We believe that the
newly proposed design strategy based on the copolymerization
method can also be extended to design other sensors for highly
sensitive and selective sensing of various biologically important
polyanions.
(8) (a) Rustici, A.; Velucchi, M.; Faggioni, R.; Sironi, M.; Ghezzi, P.;
Quataert, S.; Green, B.; Porro, M. Science 1993, 259, 361−365. (b) Li,
C.; Budge, L. P.; Driscoll, C. D.; Willardson, B. M.; Allman, G. W.;
Savage, P. B. J. Am. Chem. Soc. 1999, 121, 931−940. (c) Chan, S.;
Horner, S. R.; Fauchet, P. M.; Miller, B. L. J. Am. Chem. Soc. 2001, 123,
11797−11798. (d) Hubbard, R. D.; Horner, S. R.; Miller, B. L. J. Am.
Chem. Soc. 2001, 123, 5810−5811. (e) Ganesh, V.; Bodewits, K.;
Bartholdson, S. J.; Natale, D.; Campopiano, D. J.; Mareque-Rivas, J. C.
Angew. Chem., Int. Ed. 2009, 48, 356−360.
(9) (a) Nilsson, K. P. R; Inganas, O. Nat. Mater. 2003, 2, 419−424.
̈
(b) Ho, H. A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168−
178.
(10) Ho, H. A.; Ber
11, 1718−1724.
́
a-Aber
́
em, M.; Leclerc, M. Chem.Eur. J. 2005,
(11) (a) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S. Angew. Chem.,
Int. Ed. 2005, 44, 6371−6374. (b) Li, C.; Numata, M.; Takeuchi, M.;
Shinkai, S. Chem. Asian J. 2006, 1, 95−101. (c) Yao, Z.; Li, C.; Shi, G.
Langmuir 2008, 24, 12829−12835. (d) Yao, Z.; Feng, X.; Hong, W.;
Li, C.; Shi, G. Chem. Commun. 2009, 4696−4698.
(12) (a) Zhou, Q.; Swager, T. M. J. Am. Chem. Soc. 1995, 117,
12593−12602. (b) Swager, T. M. Acc. Chem. Res. 1998, 31, 201−207.
(c) Heeger, P. S.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
12219−12221. (d) McQuade, D. T.; Pullen, A. E.; Swager, T. M.
Chem. Rev. 2000, 100, 2537−2574. (e) Thomas, S. W.; Joly, G. D.;
Swager, T. M. Chem. Rev. 2007, 107, 1339−1386. (f) Lee, K.; Povlich,
L. K.; Kim, J. Analyst 2010, 135, 2179−2189.
ASSOCIATED CONTENT
* Supporting Information
Details of the syntheses, spectroscopic characterization, UV−vis
spectra, DLS and AFM experiments, the calculation of the limit
of detection, confocal fluorescence imaging of K. pneumoniae
and P. aeruginosa after incubation with CPT1-C. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
(13) (a) Petrak, K. Polyelectrolytes: Science and Technology; Marcel
Dekker: New York, 1992; pp 265−297. (b) Song, Y.; Wei, W.; Qu, X.
Adv. Mater. 2011, 23, 4215−4236.
(14) (a) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303−308.
(b) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 17278−17283. (c) Han, Z.; Zhang,
X.; Zhou, L.; Gong, Y.; Wu, X.; Zhen, J.; He, C.; Jian, L.; Jing, Z.; Shen,
G.; Yu, R. Anal. Chem. 2010, 82, 3108−3113.
(15) (a) Zeng, L.; Wu, J.; Dai, Q.; Liu, W.; Wang, P.; Lee, C. S. Org.
Lett. 2010, 12, 4014−4017. (b) Zeng, L.; Liu, W.; Zhuang, X.; Wu, J.;
Wang, P.; Zhang, W. Chem. Commun. 2010, 46, 2435−2437. (c) Dai,
Q.; Liu, W.; Zhuang, X.; Wu, J.; Zhang, H.; Wang, P. Anal. Chem.
2011, 83, 6559−6564. (d) Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P.
Chem. Soc. Rev. 2011, 40, 3483−3495.
(16) (a) Tabushi, I.; Imuta, J. I.; Seko, N.; Kobuke, Y. J. Am. Chem.
Soc. 1978, 100, 6287−6288. (b) Giwa, C. O.; Hudson, M. J.;
Morenoreal, L.; Rodriguez-Castellon, E. J. Chem. Soc. Chem. Commun.
AUTHOR INFORMATION
Corresponding Author
wangpf@mail.ipc.ac.cn; apwjzh@cityu.edu.hk
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
M.L. thanks Engineers Jianchun Zhou and Zhihong Xue
(Nikon Instruments (Shanghai) Co. LTD. Beijing Branch) for
help with the confocal fluorescence imaging. This work was
supported by the NNSF of China (Grant Nos. 21073213,
60978034 and 20903110), the Main Direction Program of
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Journal of the American Chemical Society
Article
1987, 536−537. (c) Li, H.; Wang, P.; Wu, S. Acta Chim. Sinica 1999,
57, 149−154. (d) Singh, R.; J. D., Jr; Dutta, P. K. Micropor. Mesopor.
Mat. 2004, 71, 149−155.
(17) (a) Shands, J. W. Infec. Immun. 1971, 4, 167−172. (b) Morath,
S.; von Aulock, S.; Hartung, T. J. Endotoxin Res. 2005, 11, 348−356.
(18) Snyder, S.; Kim, D.; McIntosh, T. J. Biochemistry 1999, 38,
10758−10767.
́
(19) (a) Levesque, I.; Leclerc, M. J. Chem. Soc. Chem. Commun. 1995,
2293−2294. (b) Faïd, K.; Leclerc, M. Chem. Commun. 1996, 2761−
2762. (c) Chayer, M.; Faïd, K.; Leclerc, M. Chem. Mater. 1997, 9,
2902−2905.
(20) Feng, X.; Liu, L.; Wang, S.; Zhu, D. Chem. Soc. Rev. 2010, 39,
2411−2419.
(21) Zheng, J.; Li, J.; Gao, X.; Jin, J.; Wang, K.; Tan, W.; Yang, R.
Anal. Chem. 2010, 82, 3914−3921.
(22) Huang, J.; Zhu, Z.; Bamrungsap, S.; Zhu, G.; You, M.; He, X.;
Wang, K.; Tan, W. Anal. Chem. 2010, 82, 10158−10163.
(23) Xue, C.; Cai, F.; Liu, H. Chem.Eur. J. 2008, 14, 1648−1653.
(24) (a) Dolley, C. D. The Technology of Bacteria Investigation; explicit
directions for the study of bacteria: their culture, staining, mounting, etc.; S.
E. Cassino and Company: Boston, 1885. (b) Bergey, D. H.; John, G.
H.; Noel, R. K.; Peter, H. A. S. Bergey’s Manual of Determinative
Bacteriology, 9th ed.; Lippincott Williams & Wilkins: New York, 1994.
6694
dx.doi.org/10.1021/ja211570a | J. Am. Chem. Soc. 2012, 134, 6685−6694