A magnetic gram stain for bacterial detection

Article abstract of DOI:10.1002/anie.201202982

Magnetizing: Bacteria are often classified into Gram-positive and Gram-negative strains by staining with crystal violet (CV). The described bioorthogonal modification of CV with trans-cyclooctene (TCO) can be used to render Gram-positive bacteria magnetic

Full text of DOI:10.1002/anie.201202982

Angewandte
Chemie
DOI: 10.1002/anie.201202982
Biosensors
A Magnetic Gram Stain for Bacterial Detection**
Ghyslain Budin, Hyun Jung Chung, Hakho Lee, and Ralph Weissleder*
Bacterial cell walls are made up of peptidoglycans (polysac-
charides crosslinked by unusual peptides) in addition to other
components.[1] Bacteria are often classified into Gram-pos-
itive and Gram-negative strains by their visual staining
properties using crystal violet (CV), a triarylmethane dye.[2]
Herein we show that bioorthogonal modification of crystal
violet with trans-cyclooctene can be used to render Gram-
positive bacteria magnetic. This modification allows for class-
specific automated magnetic detection, magnetic separation,
or other magnetic manipulations.
The Gram stain is one of the most commonly used tools
for detecting and differentiating bacteria. The method is
routinely used for clinical diagnostic purposes, as well as
detecting bacteria in environmental samples. The procedure
involves staining bacterial samples with crystal violet, which
binds to the peptidoglycan layer of Gram-positive and Gram-
negative bacteria (Figure 1). Subsequent treatment with
iodine solution results in crystal violet to form an insoluble
complex. Gram-positive bacteria have a thick peptidoglycan
layer, whereas Gram-negative bacteria only have a thin
peptidoglycan layer covered by lipopolysaccharides and
lipoproteins. Upon decolorization with alcohol or acetone,
only Gram-positive bacteria remain purple, while Gram-
negative bacteria loose the purple color.[3–5] Despite the
simplicity and robustness of the staining procedure, the final
detection still relies on optical microscopy, which is often
susceptible to user-dependent sampling error. Strategies for
quantitative and automated detection are highly desirable,
especially for the diagnosis of infectious pathogens.
Magnetic, rather than optical, labeling and detection are
advantageous because of their high sensitivity and ability to
diagnose crude specimens without major purification.[6] For
example, one could envision rapid and sensitive detection of
bacterial samples in point-of-care settings by using a mini-
aturized micro nuclear magnetic resonance (mNMR)
device.[7,8] Direct bacterial detection by mNMR is a sensitive
diagnostic method[9] and potentially allows the exclusion of
culturing steps and thus minimizes the time required for
Figure 1. A) Chemical structure of crystal violet (left) and the new
bioorthogonal crystal violet TCO (right). B) General composition of
Gram-positive and Gram-negative cell walls.
diagnosis. Alternative magnetic detection devices include
giant magnetoresistance,[10] or Hall sensors.[11] Furthermore,
rendering bacteria magnetic also has implications for mag-
netic separation,[11,12] cell sorting,[13] magnetic force micros-
copy[14] or micromanipulation and force measurements using
magnetic tweezers.[15]
We hypothesized that orthogonal triarylmethane-dye
derivatives could be used as affinity ligands to bioorthogo-
nally couple magnetic nanomaterials onto Gram-positive
bacteria. We thus developed a crystal violet modified with
trans-cyclooctene (CV-TCO). We show that this reagent can
be used for staining Gram-positive bacteria similar to the
native crystal violet. Importantly, the CV-TCO can also serve
as an anchor to attach tetrazine (Tz)-modified magnetic
nanoparticles (or other Tz-derivatized reporters). The devel-
oped magnetic Gram stain method was then used to enable
highly sensitive detection of Gram-positive pathogens by
mNMR.
Crystal violet (CV; 4,4’,4’’-dimethylaminotriphenyl-
methane) is a deep purple dye. We sought to develop
a chromophore derivative where one of the anilino moieties
is modified with a trans-cyclooctene (TCO) orthogonal group.
We started the synthesis by the condensation of two
equivalents of dimethylaniline with para-nitrobenzaldehyde
under microwave (MW) irradiation at 908C for four minutes
in the presence of a catalytic amount of aniline (Scheme 1).[16]
The aromatic nitro group was then reduced quantitatively by
hydrogenolysis in presence of activated palladium affording
the free amine 2 (Scheme 1). However, the formed adduct
instantaneously oxidizes in presence of air, thus rendering
purification and further conjugation difficult. The oxidation
[*] Dr. G. Budin,[+] Dr. H. J. Chung,[+] Prof. H. Lee, Prof. R. Weissleder
Center for Systems Biology, Massachusetts General Hospital
185 Cambridge Street, Boston, MA 02114 (USA)
E-mail: rweissleder@mgh.harvard.edu
Prof. R. Weissleder
Department of Systems Biology, Harvard Medical School
200 Longwood Avenue, Boston, MA 02115 (USA)
[+] These authors contributed equally to this work.
[**] This work was supported by the National Institutes of Health (NIH)
grant number 2P50CA086355. We thank Yoshi Iwamoto and Alex
Zaltsman for image processing.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202982.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
had e592 = 89146 LmolÀ1 cmÀ1.
These results suggest that the
TCO linker modification only
minimally affects the molar
absorptivity of the triaryl-
methane dye and that the bio-
orthogonal compound can like-
wise be used for Gram staining
(Figure S2 in the Supporting
Information). We then investi-
gated the cycloaddition of 6 with
a fluorescently labeled tetra-
zine,
fluorescein–tetrazine
(Fluo-Tz). After mixing the
two compounds (0.25 mm), stir-
ring for two minutes, the sample
was analyzed by high-perfor-
mance liquid chromatography–
mass spectrometry (HPLC–
MS). HPLC–MS spectra con-
firmed rapid and quantitative
conversion of Fluo-Tz to the
cycloaddition product without
any side products (Scheme 1B
and Figure S3 in the Supporting
Information).
We next evaluated the effi-
cacy of CV-TCO as a staining
agent for Gram-positive bacte-
ria. Three representative sam-
ples were prepared: Staphylo-
Scheme 1. A) Synthesis of the bioorthogonal CV-TCO (6). B) Bioorthogonal reaction between CV-TCO (6)
and tetrazine-conjugated probes (not all isomers shown). NHS=N-hydroxysuccinimide, DIPEA=diiso-
propylethylamine, Boc=tert-butyloxycarbonyl, TCQ=tetrachloroquinone, MFNP=magnetofluorescent
nanoparticle.
process is readily apparent since the oxidized compound has
an intense purple color. To avoid oxidation to the cationic
dye, the aniline was therefore derivatized twice to be stable
under oxygen. We thus explored the synthesis of the
disubstituted aniline 4 by using a multi-step one-pot synthetic
sequence. The nitro compound 1 was reduced by hydro-
genation and reaction progress was followed by LC–MS
(Figure S1 in the Supporting Information). After completion
of the reaction, the flask was purged with argon and the free
aniline was engaged in a reductive amination with Boc-2-
aminoacetaldehyde, sodium cyanoborohydride, and acetic
acid and stirred until completion. The secondary amine 3
underwent a classic reductive amination with acetaldehyde,
sodium cyanoborohydride, and acetic acid for 5 h yielding
compound 4 in 71% yield over three steps. Structure and
purity of 4 were confirmed by 1H NMR spectroscopy showing
the characteristic chemical shift of the methylene proton at
d = 5.30 ppm (see the Supporting Information). Compound 4
was then oxidized with tetrachloroquinone (TCQ) in ethyl
acetate that was heated to reflux, and the formation of an
intense blue indicated the formation of the cationic dye. After
removal of the Boc group under acidic conditions, compound
5 was isolated and purified on neutral alumina. Finally, the
free amine 5 was treated with TCO-NHS, thereby furnishing 6
(CV-TCO) with an overall yield of 17% over seven steps
(Scheme 1A).
coccus aureus (S. aureus; Gram-positive), Escherichia coli
(E. coli; Gram-negative), and the mixture of both bacterial
species. Bacterial smears on glass slides were stained with
a solution of CV-TCO (1 mm) or CV for three minutes,
followed by treatment with Gramꢀs iodine solution for one
minute, decolorization with 95% ethanol, and counterstain-
ing with red safranin solution. Microscopy revealed that only
Gram-positive S. aureus remained purple, while Gram-neg-
ative E. coli was decolorized owing to dissolution of the outer
membrane (Figure 2A). The specificity of CV-TCO was
further confirmed by UV/visible spectrometry; only Gram-
positive bacteria showed an intense absorption at 595 nm
(Figure S4 in the Supporting Information). Importantly, there
was excellent correlation between CV and CV-TCO staining
(r2 > 0.99; Figure 2B).
We further investigated if the bacteria stained with CV-
TCO could be magnetically labeled by using the TCO group.
Bacteria stained with CV-TCO were incubated with magneto-
fluorescent nanoparticles modified with tetrazine (MFNP-
Tz). Control samples were prepared by incubating unstained
bacteria with MFNP-Tz. The T2 relaxation values of samples
were measured using a miniaturized mNMR system. For
comparative analyses, the absorption (at 595 nm) of the same
samples was also measured. Cellular relaxivity (r2) was
obtained by normalizing the measured 1/T2 values with
bacterial concentration, and the r2 differences (Dr2) between
targeted and control samples were calculated. We observed
an excellent correlation (r2 > 0.9) between the extent of Gram
The molar extinction coefficient of CV-TCO (6) was e592
=
133013 LmolÀ1 cmÀ1 as compared to unmodified CV, which
2
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
sensitivity of the magnetic detector could be improved to the
level of single cells by 1) further miniaturizing the mNMR
detection coils, 2) implementing fluidic systems for bacterial
enrichment (e.g., membrane filters, magnetic separation
steps), and 3) employing different types of magnetic readers
(e.g., Hall-effect sensors, giant magnetoresistive sensors).[7–9]
Bioorthogonally labeled bacteria were also analyzed by
confocal microscopy using MFNP-Tz (Figure 4A). Bacteria
stained with CV-TCO showed uniform and high fluorescence
Figure 2. A) Gram staining of S. aureus (Gram-positive cocci), E. coli
(Gram-negative bacilli), and mixture of S. aureus and E. coli stained
with CV-TCO (left panels) or with CV (right panels; scale bar=10 mm).
B) Correlation of absorbance at 595 nm between bacteria stained with
CV and CV-TCO. C) Correlation between absorbance (595 nm) and
magnetic relaxivity values of bacterial cells stained with CV-TCO and
labeled with magnetic MFNP-Tz.
staining and the cellular relaxivity in Gram-positive species,
thereby confirming that CV-TCO on the bacterial surface was
accessible for reaction with MFNP-Tz.
The labeling strategy was further applied to a panel of
different bacterial species (Figure 3). Results showed that all
Gram-positive species tested showed significantly higher
cellular relaxivity values when compared to Gram-negative
bacteria. Such magnetic labeling enabled the performance of
highly sensitive and rapid detection of Gram-positive bac-
teria. Titration measurements with serially diluted bacterial
samples established that the detection limit with the current
experimental setup was approximately 4000 bacteria (Fig-
ure S5 in the Supporting Information). This detection method
is significantly better than standard UVabsorption detection,
which has a detection limit of approximately 105 bacteria
(Figure S6 in the Supporting Information). It is likely that the
Figure 4. A) Fluorescence confocal microscopy of S. aureus stained
with CV-TCO and labeled with MFNP-Tz. Left, middle, and right
images show images of red channel, green channel, and merged from
red and green, respectively (red: propidium iodide for nuclear staining;
green: MNFP-Tz staining; scale bar=10 mm). B) Transmission electron
microscopy of S. aureus stained with CV-TCO and labeled with AuNP-
Tz (left), AuNP-Tz alone (middle), and without any treatment (right;
scale bar=100 nm).
signals in the bacterial cell wall, while the control experiments
without CV-TCO showed no signal (Figure S7 in the Sup-
porting Information). Similarly, transmission electron micros-
copy was performed in bacteria treated with CV-TCO but
which were incubated with tetrazine-modified gold nano-
particles (AuNP-Tz). Gold nanoparticles were used instead of
magnetic nanoparticles to obtain higher contrast. Gold nano-
particles were found distributed throughout the bacterial
surface treated with CV-TCO, while bacteria without CV-
TCO labeling showed a smooth surface devoid of nano-
particles (Figure 4B).
By modifying the above procedure, the detection strategy
can be applied to detect both Gram-positive and Gram-
negative bacteria. Performing the staining without the
decolorization process would result in labeling both Gram-
positive and negative species, since the Gram-negative species
would also retain the CV-TCO (Figure S8A in the Supporting
Information). This coloring of both species in the first step is
in analogy to the conventional Gram stain where the first
staining step “colors” all bacteria and the second decoloriza-
tion step allows differentiation between the two Gram classes.
mNMR measurements showed that before decolorization,
both Gram-positive and negative bacteria could be magneti-
cally labeled and detected, while after decolorization, only
Figure 3. Magnetic detection by mNMR of different species of Gram-
positive and Gram-negative bacteria labeled using CV-TCO and MFNP-
Tz.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
Gram-positive species retained their signals (Figure S8B in
the Supporting Information). Through these sequential
measurements, it is thus possible to obtain total bacterial
counts (i.e. detection before decolorization) as well as their
Gram-negative and Gram-positive composition (i.e. detection
after decolorization).
[1] J. Van Heijenoort, Glycobiology 2001, 11, 25R – 36R.
[2] R. Coico, Curr. Protocols Immunol. 2001, 23, A.3O.1 – A.3O.2.
[3] T. J. Beveridge, Biotech. Histochem. 2001, 76, 111 – 118.
[4] J. W. Bartholomew, T. Mittwer, Bacteriol. Rev. 1952, 16, 1 – 29.
[5] E. J. Bottone, Lab. Med. 1988, 19, 288 – 291.
[6] D. Issadore, H. Shao, J. Chung, A. Newton, M. Pittet, R.
Weissleder, H. Lee, Lab Chip 2011, 11, 147 – 151.
In summary, we show that an orthogonal CV can be used
to detect and broadly classify bacteria in biological samples.
Staining bacteria with CV-TCO using the standard Gram
stain procedure, followed by labeling with MFNP-Tz allows
the detection and characterization of bacteria both by mNMR
as well as by optical imaging. The “magnetic Gram stain”
could be potentially implemented into automated point-of-
care diagnostics, bacterial enrichment for subsequent analysis,
as well as into therapeutic applications that utilize the
antibacterial, antifungal, and antihelminthic properties of
CV. The method could also be used to label bacteria in vivo
for various imaging applications.[9] Moreover, the staining
strategy presented could be further extended to other small
molecule affinity ligands (e.g., bioorthogonal carbol fuchsin
or trehalose for mycobacterial species) to enable either
universal or specific detection of other bacterial targets. This
ability will not only facilitate the clinical diagnosis of a range
of bacterial infections but will also promote advances in basic
microbiological research.
[7] H. J. Chung, T. Reiner, G. Budin, M. Liong, D. Issadore, H. Lee,
R. Weissleder, ACS Nano 2011, 5, 8834 – 8841.
[8] M. Liong, M. Fernandez-Suarez, D. Issadore, C. Min, C. Tassa, T.
Reiner, S. M. Fortune, M. Toner, H. Lee, R. Weissleder,
Bioconjugate Chem. 2011, 22, 2390 – 2394.
[9] P. Panizzi, M. Nahrendorf, J-L. Figueiredo, J. Panizzi, B.
Marinelli, Y. Iwamoto, E. Keliher, A. A. Maddur, P. Waterman,
H. K. Kroh, F. Leuschner, E. Aikawa, F. K. Swirski, M. J. Pittet,
T. M. Hackeng, P. Fuentes-Prior, O. Schneewind, P. E. Bock, R.
Weissleder, Nat. Med. 2011, 17, 1142 – 1146.
[10] Y. Li, M. Castro, H. Im, X. Yao, S.-H. Oh, W.-S. Hu, J.-P. Wang, J.
Med. Devices 2008, 2, 027529.
[11] P. Lçnnbro, P. Nordenfelt, H. Tapper, BMC Cell Biol. 2008, 9, 35.
[12] K. Yang, D. M. Jenkins, W. W. Su, J. Microbiol. Methods 2011,
86, 69 – 77.
[13] D. Guillebault, M. Laghdass, P. Catala, I. Obernosterer, P.
Lebaron, Appl. Environ. Microbiol. 2010, 76, 7352.
[14] Y. Zhang, M. Yang, M. Ozkan, C. S. Ozkan, Biotechnol. Prog.
2009, 25, 923.
[15] R. C. Chaves, D. Bensimon, P. P. Freitas, J. Appl. Phys. 2011, 109,
064702.
[16] D. Guzmꢁn-Lucero, J. Guzmꢁn, D. Likhatchev, R. Martꢂnez-
Palou, Tetrahedron Lett. 2005, 46, 1119 – 1122.
Received: April 18, 2012
Published online: && &&, &&&&
Keywords: bacteria · biosensors · cycloaddition ·
.
imaging agents · nanoparticles
4
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Biosensors
G. Budin, H. J. Chung, H. Lee,
R. Weissleder*
&&&& — &&&&
A Magnetic Gram Stain for Bacterial
Detection
Magnetizing: Bacteria are often classified
into Gram-positive and Gram-negative
strains by staining with crystal violet
(CV). The described bioorthogonal
modification of CV with trans-cyclooctene
(TCO) can be used to render Gram-
positive bacteria magnetic with tetrazine-
functionalized magnetic nanoparticles
(MNP-Tz). This method allows class-
specific automated magnetic detection
and magnetic separation.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!