A live bacteria SERS platform for the: In situ monitoring of nitric oxide release from a single MRSA

Article abstract of DOI:10.1039/c8cc02855a

A simple and unique surface-enhanced Raman spectroscopy (SERS) platform is developed for the precise and sensitive in situ monitoring of nitric oxide (NO) release from an individual bacterium. Using this live bacteria SERS platform, NO release from MRSA u

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A live bacteria SERS platform for in situ monitoring of nitric oxide
release from single MRSA
Zhijun Zhang,a Xuemei Han,a Zhimin Wang,a Zhe Yang,a Wenmin Zhang,a,b Juan Li,b Huanghao Yang,b
Xing Yi Ling,a and Bengang Xing*a,b
04
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
simple and unique surface-enhanced Raman spectroscopy fluorescence is the most promising approach for NO bioanalysis.9
(SERS) platform is developed for precise and sensitive in situ Numerous fluorescence probes have been designed for NO analysis
monitoring of nitric oxide (NO) release from individual bacteria. in tissues, in living cells or even at the subcellular level.10 These
Using this live bacteria SERS platform, NO released from MRSA efforts have greatly promoted our understanding of NO generation in
under the stress of antibiotics and co-infected bacteria was different types of cells and provided valuable insights on
evaluated, respectively.
physiological and pathophysiological processes in a living system.
However, so far, most of the studies mainly focused on mammalian
cells, the investigations that concentrate on the detailed
understanding of bacterial NO generation has lagged far behind. In
comparison to mammalian cells, bacteria are over three orders of
magnitude smaller in volume, but more than ten times higher in the
surface to volume ratio. Hence, the drawbacks of fluorescence
technique such as the background noise from probe diffusion and
autofluorescence, photobleaching and phototoxicity could be
dramatically enlarged for bioanalysis in bacteria. Toward this end, to
meet the demands of precise and sensitive monitoring NO generation
in bacteria, new strategies must be developed.
Since it was first identified as the endotheliumꢀderived relaxation
factor in 1987,1 nitric oxide (NO) has been recognized as a
ubiquitous intraꢀ and intercellular messenger involved in diverse
physiological and pathophysiological processes.2 In mammals, NO is
generated by three isoforms of NO synthase (NOS) to benefit the
regulation of nervous, cardiovascular and immune systems.3 In
contrast, aberrant NO induction is closely related to several diseases
such as neurodegenerative, septic shock, endothelial dysfunction or
cancer development.4 Very recently, certain Gramꢀpositive bacteria,
notably Staphylococcus and Bacillus species were discovered to
encode a bacterial NO synthase (bNOS) gene similar to eukaryotic
NOSs.5 Among these NOSꢀproducing bacterial species, methicillinꢀ
resistant Staphylococcus aureus (MRSA), which is a growing threat
to public health as they cause increased resistance to antibiotic
therapies, has drawn much attention.6 There is increasing evidence
that bNOSꢀderived NO plays a crucial role in MRSA antibiotic
resistance, and many other diverse aspects of bacterial physiology,
including bacterial virulence, oxidative stress tolerance, and biofilm
formation.7 Despite these significant achievements, the physiological
functions and mechanisms of NO in bacterial physiological
processes are still far from full interpretation. For better
understanding the roles of NO in bacterial physiology to guide the
design of bNOS targeted antibacterial strategy, the methods that can
precisely and sensitively probe bacterial NO are urgently demanded.
Surfaceꢀenhanced Raman spectroscopy (SERS) that benefits from
the localized surface plasmon resonance (LSPR) of plasmonic metal
nanostructures has emerged as a robust technique for various in vitro
and in vivo bioanalytical applications.11 SERS technique displays
many superiorities for bioanalysis, including extreme sensitivity, low
background, and high resolution with both spectra and imaging
analysis capability.12 SERS also offers unique advantages in
resistance to photobleaching and phototoxicity over conventional
fluorescence technique.13 These outstanding characteristics render
SERS a highly competitive alternative to satisfy the demanding
needs of NO biosensing. Indeed, very recently, several SERS
platforms have been designed for highly sensitive and selective
monitoring NO in living mammalian cells.14 Unfortunately, relevant
NO sensing strategies suitable for bacterial bioanalysis remain under
investigations, mostly due to the huge differences in cellular
structure.
In the past few years, enormous efforts have been made to develop
different advanced techniques for NO detection.8 In particular, using
Herein, we report a simple and unique plasmonic nanostructureꢀ
based live bacteria SERS platform for precise and sensitive in situ
monitoring of nitric oxide release from single bacteria. Using this
platform, NO release from MRSA strain (e.g. ATCC BAAꢀ44) under
the stress of different antibiotics has been fully investigated.
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Meanwhile, for the first time, the doseꢀeffect relationship between and element mapping results indicated the successful fabrication of
antibiotics and NO generation in MRSA was analyzed. Moreover, the plasmonic platform (Fig. 1). Typically, bare MRSA was found to
we realized the precise SERS imaging of NO release in a be shriveled during drying (Fig 1a). After coating with the silica
polymicrobial infection model at the singleꢀbacterium level.
layer, nearly spherical shape with a smooth surface was presented
(Fig 1b). Through the electrostatic interaction, more than two
hundreds of AgNPs were found to be decorated on each MRSA (Fig.
1c and 1d). Such high density and uniform modification would help
to generate a strong and homogeneous SERS signal for effective
bioanalysis. The TEM image, UVꢀvis spectra and zeta potential
analysis further confirmed the successful fabrication of the SERS
platform (Fig. S1ꢀS4, ESI †). The silica layer was found to be
uniformly encapsulated around the bacteria with a thickness of ~ 50
nm (Fig. S2, ESI†). The pore diameter of the mSi was about 7 nm
from the N2 absorptionꢀdesorption isotherms (Fig. S5, ESI†), which
will facilitate the fast mass transportation. Based on the elemental
composition from the ICPꢀOES analysis, the molar ratio between the
probe and silver nanoparticle was further estimated to be over ten
thousand. Considering in situ monitoring NO generation in live
bacteria, the effect of AgNPs and probe immobilization on MRSA
viability was then investigated. Through live/dead staining with
standard indicators, fluorescein diacetate (FDA) ꢀ propidium iodide
(PI), the surface modified MRSA was demonstrated to remain a high
validity with over 90% of survival rate (Fig. S6, ESI † ). This
signified that the modification of AgNPs and the probe would cause
minimal perturbation on the bioactivity of bacteria (Fig. S7, ESI†).
Besides, the growth curves further revealed that the modified MRSA
remain in an unamplification status for at least 14 h (Fig. S8, ESI†),
which provides the feasibility to collect a reliable and stable SERS
signal for effective detection.
The working principle of the SERS platform was schematically
depicted in scheme 1. As shown in the scheme, silver nanoparticles
(AgNPs) were modified on bacteria surface acted as the plasmonic
antenna to generate enhanced Raman signals, and a novel SERS
reporter 2,2'ꢀdisulfanediylbis(Nꢀ(2ꢀaminophenyl) acetamide) (OPD-
dTGA) that anchored on AgNPs surface was designed to take charge
of NO recognition. In order to achieve facile AgNPs electrostatic
modification and prevent bacterial activity perturbation induced by
direct contacting with AgNPs, prior to AgNPs modification, the
bacteria biomineralization was first carried out with a thin aminoꢀ
functionalized mesoporous silica (mSi) layer through our method
reported previously.15 Typically, two functional groups are involved
in such wellꢀdefined SERS probe. Firstly, the disulfide group was
rationally designed for anchoring the probe onto the surface of
AgNPs through the stable AgꢀS bond. Secondly, the oꢀ
phenylenediamine group, a highly selective and efficient NOꢀ
responsive motif,16 was conjugated for NO recognition and SERS
signal generation. Specifically, NO generated from MRSA upon an
external stimulation could cleave the SERS probe to form free
benzotriazole and carboxyl groups, which thus leads to a strong
SERS variation for effective bioꢀsensing.
The reactivity of the probe towards NO was then verified
through HPLC analysis. The probe exhibited a single pure peak
around 5 min (in magenta color) in HPLC profile (Fig. 2a). In the
presence of NO, a decrease in peak area, together with a new peak
around 3.5 min (in cyan color) was observed, ascribed to the product
formation of benzotriazole (Fig. S9, ESI†). The increased NO could
lead to a further decrease in the probe peak and a corresponding area
increase in benzotriazole peak, revealing that the probe could be
cleaved in a NO concentrationꢀdependent manner.
Scheme 1 Illustration of the working principle of the live bacteria
SERS platform.
Fig. 2 a) HPLC analysis of the probe upon reaction with NO. The
probe peak was in magenta, and the peak of benzotriazole was in
cyan. b) SERS spectra of the probe with or without NO. c) The SERS
spectra of the probe under different concentrations of NO (0, 50,
200, 600, and 1000 nM). d) The plot of I1446/I960 as a function of NO
concentration.
Fig. 1 False-colour SEM images of a) bare MRSA, b) mSi coated
MRSA and c) AgNPs modified MRSA. d) SEM image and
corresponding elemental mappings of Ag, Si and O signals of the
AgNPs modified MRSA. Scale bar = 200 nm.
The SERS sensing platform was first fabricated and characterized
with various techniques. As proof of concept, the MRSA strain
(ATCC BAAꢀ44) was used as a model bacterium. The SEM imaging
Furthermore, the SERS spectra of the probe in the absence and
presence of NO were measured. As shown in Fig. 2b, the probe
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possessed abundant intense SERS signals. The main peaks at 1233, MRSA. The NO produced by the MRSA ranges from 100ꢀ600
1263, 1322 and 1349 cmꢀ1 were attributed to the amide III modes. nM upon ampicillin stimulation, and 300ꢀ800 nM for
The strongest peak at 1446 cmꢀ1 could be assigned to aromatic CꢀC vancomycin (Fig. S15, ESI
†). At the low concentration, NO
stretching modes. Meanwhile, a distinct band around 960 cmꢀ1 production was enhanced with increasing the antibiotics, while
assigned to multiꢀphonon scattering generated in Si substrate could the further increased antibiotics led to a decrease in NO
be observed. Incubating the probe with NO will lead to an apparent generation. The inflection points were found to be around the
decrease in all the characteristic peaks from the probe. The cleavage MICs of the antibiotics (e.g. ampicillin: ~ 32 ꢁg mlꢀ1;
of the aromatic group in the NO reporter was considered to be vancomycin: ~ 2 ꢁg mlꢀ1).17,18 This phenomenon is considered
responsible for the attenuation since there was no detectable SERS to be related to the impact of antibiotics on bacterial activity. In
signal from the dithiodiglycolic acid (Fig. S10, ESI†), which was the comparison, vancomycin triggered more NO production in
residual group of the reporter upon NO treatment.16 Remarkably, MRSA than ampicillin. Such differences were mostly caused
after NO treatment, there was almost no change for the band around by the different susceptibilities of MRSA against the two
960 cmꢀ1, which could act as a reference for ratiometric analysis. The antibiotics, as MRSA is known resistant to ampicillin but
optimal condition for probe immobilization was also studied. The susceptible to vancomycin.18 When in the presence of a NOS
strongest signal could be observed after incubation with 1 mM of the inhibitor NωꢀNitroꢀLꢀarginine methyl ester hydrochloride (Lꢀ
probe for 30 min (Fig. S11, ESI†). Moreover, it was found that NAME), the antibiotics induced NO production was greatly
SERS signal could reach the saturation within 30 min (Fig. S12, ESI suppressed (Fig. S16, ESI†), which demonstrated that
†), suggesting a fast response to NO in the sensing process.
antibiotics induced upregulation of NOS expression was
responsible for the NO generation. Notably, in the absence of
antibiotic, the inhibitor didn’t cause a noticeable different
SERS signal, revealing that there was almost no detectable
amount of NO released from MRSA in the tested condition
without stimulation. As control, the NO synthesis was also
measured in a Gramꢀnegative strain Escherichia coli Kꢀ12
without bNOS gene (Fig. S17 and S18, ESI†). As expected,
there was no evident NO generation in Kꢀ12 upon the
antibiotics treatment, indicating very different NO generation
from the MRSA.
We further examined the SERS signal variation under spiking
with a series of concentration of NO (Fig. 2c). As expected, the peak
intensity of the probe decreased gradually with the increasing of NO
concentration. A linear relationship between the ratiometric intensity
of I1446/I960 and the concentration of NO was identified with the
detection limit (triple signalꢀtoꢀnoise ratio) less than 100 nM (Fig.
2d). The SERS response to other different ROS was subsequently
investigated. NO could trigger a significant SERS intensity change
(Fig. S13, ESI † ). In contrast, other ROS (e.g. ONOO−, ClO−,
1
H2O2, ·OH, O2, and O2− etc) could not induce apparent changes in
the SERS signal even at high concentration, clearly indicating the
Finally, the capacity of this SERS platform to give a direct
high selectivity of the probe toward NO. Moreover, the platform imaging view of NO generation in a model of polymicrobial
demonstrated impressive longꢀterm stability, as the SERS signal infection was also examined. In clinical settings, MRSA
remained almost unchanged even after oneꢀweek storage (Fig. S14, suffered patients are often coꢀinfected with other pathogens
ESI†).
such as Pseudomonas aeruginosa, one of the frequently
reported species to cause clinical infection.19 Under the
polymicrobial stimulation, the NOSs would be overexpressed in
MRSA to improve their survivability.19, 7c Hence, to this end,
we built a polymicrobial infection model by coꢀculture of the
modified MRSA with Pseudomonas aeruginosa (PA01) for
direct view of NO production. As shown in Fig. 4, the clear
SERS imaging at the singleꢀbacterium were observed by
monitoring the signal change at 1446 cmꢀ1 (Fig. 4). Typically,
the MRSA alone showed a high imaging signal from the NO
reporter (Fig. 4, left). However, after coꢀculture with PA01, the
imaging signal was found to be sharply reduced (Fig. 4, middle),
Fig. 3 SERS signal changes in the MRSA after exposure with different
concentrations of a) Ampicillin and b) Vancomycin. Significant
difference: *P<0.05, **P<0.01, ***P<0.001.
Consequently, the feasibility of in situ detection of bacterial suggesting that the coꢀcultured PA01 could trigger the NO
NO under the stress of antibiotics treatment was further synthesis in MRSA. The pyocyanin secreted from PA01 was
investigated. Generally, bNOSꢀderived NO generation closely considered to be responsible for stimulation of NO synthesis in
related to antibiotic resistance in MRSA, in which NO was MRSA,7c which was further confirmed by monitoring the UVꢀ
produced to engage in MRSA selfꢀprotection.7a To explore the Vis absorbance of the medium (Fig. S19, ESI
†). As control, the
doseꢀeffect relationship between antibiotics and NO generation coꢀculture group with NOS inhibitor (LꢀNAME) was performed.
in MRSA, we examined the NO release from MRSA under the Apparently, in the presence of LꢀNAME, an enhanced imaging
stress of different antibiotics with series concentrations. As signal was observed (Fig. 4, right), indicating that the
proof of concept, we chose ampicillin (a commonly used upregulation of NOS expression was responsible for PA01
penicillin betaꢀlactam antibiotic), and vancomycin (a induced NO synthesis in MRSA.
glycopeptide antibiotic as last resort against MRSA) as the two
In summary, we presented a simple and unique plasmonic
representative drug molecules.17 As presented in Fig. 3, both nanostructureꢀbased live bacteria SERS platform for precise
the two antibiotics could stimulate significant NO generation in and sensitive in situ monitoring nitric oxide release from
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8 (a) J. H. Kim, D. A. Heller, H. Jin, P. W. Barone, C. Song, J. Zhang,
L. J. Trudel, G. N. Wogan, S. R. Tannenbaum and M. S. Strano,
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individual bacteria. The SERS platform was fabricated by the
combination of a novel NO reporter with the plasmonic
nanostructure, which was demonstrated to be highly selective,
sensitive and fast responsive towards NO generation. Using this
platform, we elucidated that both the antibiotics ampicillin and
vancomycin could induce NO generation in MRSA in a
concentrationꢀdependent manner. Moreover, we realized the in
situ and precise SERS imaging of NO release at a single MRSA
level in a polymicrobial infection model. This work not only
provides more understanding of NO generation in bacterial
physiological processes but also affords a new idea to fabricate
SERS platform for various bacteria secretions sensing.
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Fig. 4 SERS images monitored at 1446 cm-1 of the MRSA after being
cultured in LB (the left), co-cultured with PA01 (the middle) and co-
cultured in the presence of the NOS inhibitor (the right). The
pictures in the bottom row showed their corresponding bright field
images. Scale bar = 2 μm.
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This work was partially supported by NTUꢀAITꢀMUV
NAM/16001, RG110/16 (S), (RG 11/13) and (RG 35/15),
NTUꢀJSPS JRP grant (M4082175.110) awarded in Nanyang
Technological University, Singapore and National Natural
Science Foundation of China (NSFC) (No. 51628201).
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Conflicts of interest
27
.
There are no conflicts to declare.
13 D. W. Li, L. L. Qu, K. Hu, Y. T. Long and H. Tian, Angew. Chem.
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