Cationic conjugated polymers for discrimination of microbial pathogens

Article abstract of DOI:10.1002/adma.201400636

A new cationic poly(phenylene vinylene) derivative (PPV-NMe ₃⁺) is synthesized, and it exhibits a differential binding ability to microbial cell walls with different components. By varying the ion strengths of the buffer solution, single PPV-NMe₃⁺ molecules can discriminate fungi, Gram-positive bacteria, and Gram-negative bacteria in a rapid and simple manner. Thus, cationic conjugated polymers exhibit high potential as a diagnostic material for the detection and discrimination of pathogens.

Full text of DOI:10.1002/adma.201400636

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Cationic Conjugated Polymers for Discrimination
of Microbial Pathogens
Huanxiang Yuan, Zhang Liu, Libing Liu,* Fengting Lv, Yilin Wang,* and Shu Wang*
With the increase of pathogen infections in clinical practice,
it becomes a public health problem which has aroused global
attention. Fungi and bacteria are a major cause of pathogen
infections which have resulted in extremely severe conse-
quences, including expensive medical costs, fatal diseases and
growing deathrate of patients.[1] Fungal infections (such as
C. albicans) are important causes of morbidity and mortality
in immunocompromised patients. Fungal biofilms formed
on the surface of implantable medical devices are mainly
responsible for hospital acquired infections.[2–4] Pathogenic
bacteria are a major cause of human disease and death, and
also cause infections (such as syphilis, foodborne illness and
tuberculosis). For example, E. coli is responsible for half of
infections.[5–7] The bacteria include Gram-negative and Gram-
positive ones which possess different components on cell
envelope. The pathogenic capability of bacteria is often asso-
ciated with cell envelope components.[8] In clinical practice,
multimicrobial infections resulted from multiple pathogens are
prevalent among patients.[9] Cultures of blood samples are usu-
ally utilized to discriminate and determine various pathogens
in clinical detection. The time-consuming, generally several
days, restricts its efficiency in early diagnosis.[10,11] Detections
using DNA-based techniques (such as PCR) are more used
due to their speed and specificity in comparison to culture-
based methods.[12] However, these methods typically depend
on sophisticated instrumentation and expensive dye-labeled
primers, which limits their extensively clinical applications.
Conjugated polymers have been extensively used for chem-
ical and biological detections because of their significant light
harvesting and optical signal amplification effects.[13–21] The
detection systems based on conjugated polymers for patho-
genic microorganism rely on the synthesis of carbohydrate-
functionalized polymers.[22–25] However, they cannot selectively
discriminate different microbial pathogens. Recently, charged
conjugated polymers have been used for detection and identifi-
cation of various bacteria mainly based on the analyte-induced
displacement of fluorophore with the aid of linear discrimi-
nant analysis (LDA).[26–29] The sensing elements in these sys-
tems must include one charged conjugated polymer (donors)
and several (three to five) gold nanoparticles or dye-labeled
DNAs (acceptors). It will be ideal to develop new methods only
based on single conjugated polymer to discriminate multiple
pathogens rapidly and simply. In this work, we prepared a new
+
cationic poly(phenylene vinylene) derivative (PPV-NMe3 , see
Scheme 1 for its structure and synthesis). Through the specific
+
interactions of PPV-NMe3 to microbial cell envelopes with dif-
+
ferent components, it is demonstrated that single PPV-NMe3
molecule could discriminate fungi, Gram-positive bacteria and
Gram-negative bacteria under fluorescence microscope only
via varying the ion strengths of the buffer solution. Although
it requires microbial culture, the new method is much more
rapid and only takes less than three hours to complete the assay
process in comparison to typical culture-based methods that
usually take several days.
+
The synthesis of PPV-NMe3 is outlined in Scheme 1. Reac-
tion of compound 1 with 1,6-dibromohexane provides com-
pound 2 in a 87 % yield. Reaction of compound 3 with vinyl
tributyltin provides compound 4 in a 51 % yield. The PPV-Br
was then prepared by copolymerization of compound 2 and
compound 4 through Heck coupling reaction in a 32% yield.
The quaternization of PPV-Br with trimethylamine afforded
+
PPV-NMe3 in a 78 % yield. Due to quaternized amine-termi-
+
nated groups, the cationic PPV-NMe3 is soluble in water. The
+
optical characterization of PPV-NMe3 was conducted in water
media (Figure S1). PPV-NMe3+ exhibits a maximum absorption
at 468 nm and a maximum emission at 593 nm with a fluores-
cence quantum yield of 1.5 %. The size of PPV-NMe3+ in water
media is 43 4 nm (Figure S2).
The proposed principle of the diverse interactions of cati-
+
onic PPV-NMe3 with different microbial pathogens in var-
ious concentrations of phosphate buffer saline (PBS) is illus-
trated in Scheme 2a. To demonstrate the concept, C. albicans
(fungi), B. subtilis (Gram-positive bacteria) and E. coli (Gram-
negative bacteria) are used as the analytes. The biggest differ-
ence between fungi and bacteria is the chemical composition
of their cell walls. Fungi (including C. albicans) have a typical
cell wall consisting largely of β-glucans, chitin and mannopro-
teins.[30] For Gram-negative and Gram-positive bacteria, the
structures of the cell walls are also significantly different. The
cell wall of Gram-negative bacteria (E. coli) is composed of an
outer membrane and a thin, intermittently cross-linked pepti-
doglycan network. However, Gram-positive bacteria (B. subtilis)
only have a thick layer of heavily cross-linked peptidoglycan
that lies outside the plasma membrane to make the cell wall
H. Yuan, Dr. L. Liu, Dr. F. Lv, Prof. S. Wang
Key Laboratory of Organic Solids
Interface and Chemical Thermodynamics
Institute of Chemistry
Chinese Academy of Sciences
Beijing 100190, P. R. China
Fax: (+86) 10-6263-6680
E-mail: liulibing@iccas.ac.cn; wangshu@iccas.ac.cn
Z. Liu, Prof. Y. Wang
Key Laboratory of Colloid
Interface and Chemical Thermodynamics
Institute of Chemistry
Chinese Academy of Sciences
Beijing 100190, P. R. China
E-mail: yilinwang@iccas.ac.cn
DOI: 10.1002/adma.201400636
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changes (ΔH) and the binding constant (K) indicate that the
+
binding of PPV-NMe3 to C. albicans and E. coli is dominated
by electrostatic interactions, while hydrophobic interactions for
+
PPV-NMe3 with B. subtilis. The electrostatic interactions are
strongly screened at higher buffer concentrations because of
the increase of the ionic strength in the systems. As shown in
Table S1, the K value for C. albicans is the largest among the
three species in lower PBS concentration (5 mM), which dem-
+
onstrates that PPV-NMe3 is more selectively bound to C. albi-
cans than E. coli and B. subtilis; while selectively bound to E. coli
in 20 mM and to B. subtilis in high PBS concentration (40 mM).
Thus, the diverse interactions of PPV-NMe3+ with fungi, Gram-
negative and Gram-positive bacteria can be regulated by varying
the buffer concentration.
The zeta potential (ζ) provides direct evidence of the selective
+
binding of PPV-NMe3 to different species (Figure S3). Upon
+
the addition of PPV-NMe3 , ζ potentials for E. coli become more
positive, while the ζ potentials of B. subtilis and C. albicans do
not change distinctly. Generally, B. subtilis have a comparatively
thick and poriferous cell wall in the range of 20∼80 nm and the
negatively charged teichoic acids which insert in the thick wall
+
would make B. subtilis negatively charged. Thereby PPV-NMe3
could bind to the negatively charged teichoic acids on the
membrane surface of B. subtilis and thereby PPV-NMe3+ is pro-
moted to intercalate into the poriferous cell wall. As the space
is sufficiently large to have a capacity for the whole polymer,
+
the cell wall almost hides PPV-NMe3 without exposure to the
surface. Therefore, no distinct change of the ζ potential was
observed for the Gram-positive B. subtilis.[35] For the case of C.
albicans, it is similar with B. subtilis. The components of glu-
cans, chitin and mannoproteins offer C. albicans approximately
+
a 25 nm thick cell wall for the intercalation of PPV-NMe3 , so
Scheme 1. Synthetic route of PPV-NMe3+ (m = n = 0.5).
the unchanged ζ potentials were observed.[26] As for the Gram-
negative E. coli, PPV-NMe3+ is constrained by the relatively thin
cell wall and the outer membrane to remain on the bacterial
surface, which makes the ζ potential of Gram-negative E. coli
more positive. Thus, the different zeta potential changes of
these three species are related with the structure characteristics
of diverse microbes. The thermodynamic and the zeta poten-
tial results provide a deep insight into the interactions of PPV-
NMe3+ with microorganisms at a micro level.
porous (Scheme 2b).[31,32] The cell walls of fungi and bacteria
+
are both negatively charged. PPV-NMe3 was designed to bear
quaternary ammonium groups at the side chain, which could
regulate the diverse interactions with fungi and bacteria pos-
sessing different surface structures in different concentrations
of buffer.
To acquire further evidence for understanding the mecha-
+
nism of the interactions between PPV-NMe3 and pathogens,
In order to directly visualize the different interactions
+
isothermal titration microcalorimetry (ITC)[33,34] was employed
to investigate the thermodynamical changes of the microor-
between PPV-NMe3 and microbes, the adsorption of polymer
onto the surfaces of microorganisms was studied by fluo-
+
ganisms upon adding PPV-NMe3 in different concentrations
rescence microscopy. As shown in Figure 2a, all of the three
+
of PBS. As shown in Figure 1, the tendency of the enthalpy
variations of Gram-positive bacteria B. subtilis with the addi-
species were stained with PPV-NMe3 (5 µM in repeated units
(RUs)) in different concentrations of phosphate buffer saline
(5 ∼ 40 mM). With the increase of the buffer concentration,
the imaging differences are observed obviously. For the case
of E. coli, there is a sharp decrease of the fluorescence inten-
sity with increasing buffer concentration and E. coli cannot be
stained in 40 mM of PBS. Similar trend was observed for fungi
C. albicans. The fluorescence intensity of the stained B. subtilis
was nearly unchanged in the buffer in the range of 5 ∼ 20 mM,
while there is a slow decrease of the fluorescence intensity in
the buffer of 20 ∼ 40 mM. Therefore, the discrimination and
identification of these three species of microorganism can be
realized through monitoring the fluorescence intensity changes
+
tion of PPV-NMe3 is almost unchanged in various concentra-
tions of PBS systems, which indicates that the interaction of
+
PPV-NMe3 with B. subtilis is not influenced obviously by the
buffer concentration. As for fungi C. albicans and Gram- nega-
tive E. coli, significant changes of the enthalpy are induced
+
upon adding PPV-NMe3 by increasing the buffer concentra-
+
tion, which suggests that the binding ability of PPV-NMe3 to
C. albicans and E. coli is highly affected by the concentration
of PBS. Quantitative interaction thermodynamic parameters
were calculated from the ITC fitting curves as summarized in
Table S1. The binding number (N) (the number of polymer
repeat unit associated with a single microorganism), enthalpy
+
of PPV-NMe3 in various concentrations of buffer. Quantitative
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Scheme 2. (a) Schematic illustration of PPV-NMe3+ for discrimination of different microbial pathogens. (b) Illustration of the different surface struc-
tures of the Gram-negative bacteria, Gram-positive bacteria and fungi.
fluorescence intensity values are calculated by the softwares
(DVCView and Microsoft Excel) for the direct comparison of
different pathogens. Followed by testifying that PPV-NMe3
to B. subtilis is the strongest. It is noted that the fluorescence
intensity of the mixture of C. albicans and B. subtilis is the
highest among all the mixtures. For the case of the blend of
three species, the fluorescence intensity is lower than the mix-
ture of C. albicans and B. subtilis but higher than the mixture of
E. coli and B. subtilis, while the fluorescence of the mixture of E.
coli and C. albicans is the weakest because of the relatively weak
binding ability of PPV-NMe3 to E. coli and C. albicans. Thus,
it is expected to identify and differentiate various mixtures of
microorganism in different concentrations of PBS.
+
can selectively recognize and image different microorganisms
individually in various concentrations of buffer, the blends of
two or three species of microbe imaging experiments by PPV-
+
NMe3 were performed to afford more insight into the com-
+
plicated situation of the existence of more than one species of
pathogen. As displayed in Figure S4, all of the blends stained
by PPV-NMe3+ have certain extent of fluorescence, but there are
significant disparities to distinguish the fungi, Gram-positive
and Gram-negative bacteria. The fluorescence of the mixtures
including B. subtilis is much brighter than the mixture in the
Similarly, the quantitative values of the fluorescence inten-
sity is calculated which could directly compare and identify dif-
ferent samples in different concentrations. We summarized the
data and made a definition of the evaluation standard according
+
absence of B. subtilis because the binding ability of PPV-NMe3
+
Figure 1. Fitting curves of observed enthalpy changes ΔHobs against the polymer/microorganism molar ratio by titrating 30 µM in RUs of PPV-NMe3
into B. subtilis solutions (a), C. albicans solutions (b), and E. coli solutions (c) (OD600 = 1.0) in different concentrations of PBS (5 mM, 20 mM and
40 mM, respectively). ΔHobs values are expressed in kJ/mol of polymer. The dilution enthalpy of the polymer has been deducted.
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Figure 2. (a) The adsorption of PPV-NMe3+ to the surfaces of E. coli, B. subtilis and C. albicans in various concentrations of PBS. [PPV-NMe3+] = 5 µM
in RUs. The exposure time for PPV-NMe3+ channel is 300 ms. The numbers on the images that stand for the values of the fluorescence intensity are
calculated by DVCView and Microsoft Excel softwares. (b) The evaluation criterion of the fluorescence intensity of the stained microorganisms in
10 mM and 5 mM PBS according to Figure 2a and Figure S4. Definition: the symbols +++++, ++++, +++, ++, + and − stand for the intensity values
higher than 2500, between 2000 and 2500, between 1500 and 2000, between 1000 and 1500, between 500 and 1000, and lower than 500, respectively.
E: E. coli, B: B. subtilis, C: C. albicans.
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to the calculated fluorescence intensity values for under-
applied to complicated situations in which the targets consist of
two or three classes of microorganisms (fungi, Gram-positive
bacteria, and Gram-negative bacteria). Thus cationic conjugated
polymers exhibit high potential as diagnostic materials for the
detection and discrimination of pathogens.
+
standing the different associations of PPV-NMe3 with diverse
+
species of microorganism and for the application of PPV-NMe3
to discriminate different microorganisms (Figure 2b). The plus
sign and minus sign were used to describe the fluorescence
intensity. Five plus signs (+++++) represent the strongest fluo-
rescence and one minus sign (−) stands for the weakest inten-
sity. As shown in Figure 2b, the fluorescence intensity values
for E (−), B and E+B (+++), C and E+C (+), C+B (+++++), and
E+C+B (++++) are in different ranges in 10 mM according to
the evaluation criterion. In single species zone, E. coli, B. sub-
tilis and C. albicans could be clearly discriminated. In mixed
species zone, different mixes of E. coli, B. subtilis and C. albi-
cans could also be clearly discriminated. While for the samples
of B in single species zone and E+B in mixed species zone, as
well as C and E+C, they could not be discriminated each other
in 10 mM PBS, however, they could be clearly discriminated in
5 mM PBS. Thus, it is convenient to discriminate all the single
and multimicrobial species in the 5 mM and 10 mM PBS.
Experimental Section
Synthesis of PPV-NMe3+: To a solution of polymer PPV (21 mg,
0.033 mmol) in THF (5 mL) was added a solution of 33% trimethylamine
in methanol (0.8 mL). The mixture was refluxed at 70 °C for 3 d. After
cooling to room temperature, the solvent and excess trimethylamine
were removed under vacuum. The residue was dissolved in doubly
distilled water, filtered, and the solution was dialyzed using a membrane
with a 3500 cut-off for 2 d. The water was removed under vacuum to
afford a dark red solid (18 mg, 78%).1H-NMR (400 MHz, DMSO, ppm):
7.3–7.6(br, 4H), 4.0–4.1(m, 2H), 3.9(s, 3H), 3.3–3.4(m, 2H), 3.1 (s, 9H),
1.2–1.9 (m, 8H).
Fluorescence Microscopy Measurements: Suspensions of C. albicans
(100 µL, OD600 = 2.0), E. coli (100 µL, OD600 = 1.0) or B. subtilis (300 µL,
OD600 = 1.0) in final volume of 500 µL different concentrations of PBS
(5 mM, 10 mM, 20 mM, 30 mM, and 40 mM, respectively) were incubated
+
The three species treated with PPV-NMe3 were studied by
+
without or with PPV-NMe3 ([PPV-NMe3+] = 5 µM in RUs) at 37 °C
laser scanning confocal microscope (LSCM) to better show
the interaction between polymer and microbes (Figure S5).
Obvious difference among these three kinds of microorgan-
isms could be observed and the results are consistent with the
images taken by fluorescence microscope. In order to further
prove the feasibility of this method, one more species of each
type of microorganism (P. aeruginosa represents Gram-nega-
tive bacteria, E. faecalis represents Gram-positive bacteria and
S. cerevisiae represents fungi) was selected to be treated with
for 20 min. The cultures were centrifuged (10000 rpm for 2 min) and
washed once using 500 µL of corresponding concentrations of PBS. The
supernatant was removed, and the pellets of the three kinds of microbes
were then resuspended in 100 µL corresponding concentrations of PBS.
Solutions (10 µL) of C. albicans, E. coli or B. subtilis incubated without
+
or with PPV-NMe3 were added to clean glass slides followed by slightly
covering coverslips for immobilization. Photographs were then taken. The
phase contrast images were taken and the fluorescence images were taken
via fluorescence microscopy with the exposure time of 300 ms for Channel
PPV-NMe3+. The false color was green for Channel PPV-NMe3+. The type
of light filter was D455/70 nm exciter, 500 nm beamsplitter, D525/30 nm
emitter. Magnification of the object lens was 100×. As for the mixtures
of C. albicans (100 µL, OD600 = 2.0) and E. coli (100 µL, OD600 = 1.0),
E. coli (100 µL, OD600 = 1.0) and B. subtilis (300 µL, OD600 = 1.0),
C. albicans (100 µL, OD600 = 2.0) and B. subtilis (300 µL, OD600 = 1.0) or
the three kinds of microbes, experimental conditions and operations were
totally the same as that of the individual microbe. The determination of
the intensity value of fluorescence images was calculated according to
the data which were read from the software DVCView.
+
PPV-NMe3 . The fluorescence images were taken with LSCM
(Figure S5), and the results were consistent with the situation
of each type of microorganism selected before. To determine
the detection limit, E.coli which is selected as the representative
with different concentrations (OD600 = 0.05−1.0) were incubated
with polymer and then were imaged by fluorescence micros-
copy. The fluorescence of the stained E. coli could be detected
with an OD as low as 0.05 (Figure S6). For real samples, the
samples need be cultured for 2−12 h, and then the pathogens
are harvested by centrifuging. The obtained pathogens were
suspended to dilute to an appropriate OD and analyzed by fluo-
rescence microscopy.
Supporting Information
In conclusion, a cationic poly(p-phenylene vinylene) (PPV-
NMe3 ) has been synthesized for rapid and simple discrimi-
Supporting Information is available from the Wiley Online Library or
from the author.
+
nation of fungi, Gram-positive, and Gram-negative bacteria.
The thermodynamical and the zeta potential measurements
exhibit that the binding of PPV-NMe3+ to C. albicans and E. coli
is dominated by electrostatic interactions, while hydrophobic
Acknowledgements
+
The authors are grateful to the National Natural Science Foundation of
China (Nos. 21033010, 21203213, TRR61) and the Major Research Plan
of China (No. 2011CB932302, 2012CB932600, 2011CB808400).
interactions for PPV-NMe3 and B. subtilis. It is demonstrated
that single self-luminous PPV-NMe3+ molecules could discrimi-
nate fungi, Gram-positive bacteria, and Gram-negative bac-
teria under a fluorescence microscope only via varying the ion
strengths of the buffer solution in a rapid and simple way. There
are several unique features for our new assay system. First, the
method is rapid. It only takes less than 3 hours to complete the
analysis including culturing, detecting, and discriminating the
pathogens. Second, the method is much simpler than other
assays (such as PCR). Direct discrimination by the fluores-
cence intensity under a fluorescence microscope is the most
important characteristic of the assay. Third, this method can be
Received: February 10, 2014
Revised: March 14, 2014
Published online: April 16, 2014
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