Nanoparticle supracrystals and layered supracrystals as chemical amplifiers

Article abstract of DOI:10.1002/anie.201002295

Figure Presented Seeing is believing: Nanoparticle crystals and core-shell crystals detect and amplify the presence of chemical and enzymatic analytes. These crystals are made insoluble in water by cross-linking their surface with dithiols incorporating a

Full text of DOI:10.1002/anie.201002295

Angewandte
Chemie
DOI: 10.1002/anie.201002295
Nanotechnology
Nanoparticle Supracrystals and Layered Supracrystals as Chemical
Amplifiers**
Bartlomiej Kowalczyk, David A. Walker, Siowling Soh, and Bartosz A. Grzybowski*
One of the main applications of metal nanoparticles (NPs) is
in the detection of biologically important[1] or toxic substan-
ces,[2] usually by the spectral or surface plasmon resonance
(SPR) changes[3] accompanying particle aggregation[4] or
dispersion[2c,5] in the presence of an analyte. With the
exception of highly sensitive DNA-based methods,[6] how-
ever, most reported colorimetric detection schemes require
large excesses of analyte molecules per one NP (on the order
of one thousand,[2c,5] see the Supporting Information, Sec-
tion 1) to affect even small shifts in the nanoparticlesꢀ SPR
bands.[7] Improving the existing detection limits therefore
requires amplification of the assembly/disassembly process.
Unfortunately, the repertoire of amplification procedures
compatible with nanoparticle-based assays is currently lim-
ited to the catalytic deposition of silver on surface-immobi-
lized AuNPs.[8] Herein, we show that NP supracrystals and
previously unreported core–shell (CS) supracrystals
(Figure 1) stabilized by analyte-specific cross-linkers can
enhance dramatically (by over two orders of magnitude
compared to noncrystalline NP aggregates) the sensitivity of
NP-based detection. The cross-linked crystals are insoluble in
water and, owing to NP aggregation, do not exhibit SPR in the
visible regime. When, however, an analyte (an anion, a small
molecule, or an enzyme) is present, it cuts the cross-linkers so
that each crystal liberates, like a pinched “balloon” (Figure 2,
Figure 3), millions of individual NPs absorbing strongly in the
visible regime and effectively amplifying the molecular-scale
“cutting” events into pronounced color changes visible to a
naked eye (Figure 4). Moreover, in the CS supracrystals, the
“shell” and the “core” regions have different NP composi-
tions and are stabilized with different cross-linkers; conse-
quently, these crystals can dissolve in a step-wise fashion
under the action of two different analytes (Figure 5). This
property allows for spatially distributed sensing, whereby the
crystals release their cargo only if they travel through specific
concentration landscapes of the analytes.
We used AuNPs with an approximate diameter of (5.6 Æ
0.5) nm and AgNPs of diameter (5.6 Æ 1.2) nm coated with
self-assembled monolayers (SAMs) of either positively
charged N,N,N-trimethyl(11-mercaptoundecyl)ammonium
+
chloride (HS(CH2)11N(CH3)3 , TMA, ProChimia, Poland)
or negatively charged, deprotonated mercaptoundecanoic
acid (HS(CH2)10COOÀ, MUA, ProChimia; Figure 1a). The
AuNPs had a strong surface plasmon resonance (SPR) band
with maximum at lmax = 520 nm, and their aqueous solutions
appeared bright red; for AgNPs, lmax ꢀ 420 nm and particle
solutions appeared yellow. Supracrystals (henceforth, simply
“crystals”) were grown by slow evaporation of water from
DMSO/water mixtures containing equal numbers of oppo-
sitely charged NPs (see Ref. [9] and the Supporting Informa-
tion, Section 2 for details). The 1–2 mm crystals (Figure 1b)
thus assembled (ca. 2.2 ꢁ 107 crystals per mL) were held
together by electrostatic interactions between the NPs,
comprised several million nanoparticles each (ca. 2.5 ꢁ 106
NPs as determined by SEM imaging), and were soluble in
water. Upon exposure to alkane dithiols that cross-linked the
nearby NPs, however, the crystals became stable in water
(Figure 2a) and could be used as seeds or cores to epitaxially
deposit an additional shell of NPs. These shell NPs were again
deposited from an aqueous solution of oppositely charged
particles,[10] the material properties of which could be differ-
ent from those in the crystal core. In this way, core–shell
crystals (each up to ca. 2.5 mm across, Figure 1c, shell
thickness ca. 200–300 nm, Figure 5) were assembled and
could be stabilized and made insoluble in water by additional
dithiol cross-links. The presence of the core–shell architecture
was confirmed directly by EDX (energy dispersive X-ray
spectroscopy) scans of the crystalsꢀ composition (Figure 1c
and Figure 5) and indirectly by the fact that the shell regions
could be dissolved without destroying the crystalline core (see
Figure 5). Both the “regular” and the CS crystals slowly
(within hours) sedimented from solution and appeared as a
dark gray powder.
[*] Dr. B. Kowalczyk, D. A. Walker, S. Soh, Prof. B. A. Grzybowski
Department of Chemical and Biological Engineering
Northwestern University
2145 Sheridan Rd., Evanston, IL 60208 (USA)
Fax: (+1)847-491-3728
E-mail: grzybor@northwestern.edu
The crux of the amplified detection (Figure 2a) was to
prepare crystals stabilized by dithiols incorporating groups
that can be cleaved by desired analytes. We considered three
such dithiols (Figure 2b; see the Supporting Information,
Section 2 for synthetic details): 11-mercaptoundecyl-11-mer-
captoundecanoate (1) containing an ester moiety prone to
base hydrolysis; 11-mercapto-N-(pyridin-2-yl)undecanamide
held together by coordination of CuII to two pyridine and two
carbonyl groups (2), which cleaves upon addition of a stronger
chelating agent such as ethylenediaminetetraacetic acid
(EDTA), and N-(6-amino-1-(11-mercaptoundecylamino)-1-
Homepage: http://dysa.northwestern.edu
Dr. B. Kowalczyk, Prof. B. A. Grzybowski
Department of Chemistry, Northwestern University
2145 Sheridan Rd., Evanston, IL 60208 (USA)
[**] This work was supported by the Non-equilibrium Energy Research
Center (NERC), which is an Energy Frontier Research Center funded
by the U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences under Award Number DE-SC0000989.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201002295.
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Figure 1. Nanoparticles, crystals, and core–shell crystals. a) Scheme of Au and Ag nanoparticles and of the charged MUA and TMA ligands
coating them. Arrows indicate the pairs of oppositely charged NPs that can form supracrystals. b) Scheme of a NP supracrystal (here made of
AuTMA and AuMUA NPs); gray-black arcs indicate dithiol cross-links bridging proximal NPs at the crystal’s surface. The SEM image shows a
typical crystal composed of AuTMA and AuMUA NPs (scale bar=500 nm); the inset shows individual, ordered NPs on the crystal’s surface (scale
bar=20 nm). The two rightmost images have compositional maps of an AuTMA/AuMUA crystal recorded on STEM (scanning transmission
electron microscope) equipped with dual Thermo-Scientific EDS (energy dispersive X-ray spectroscopy) detectors (color coding: Au=red;
Ag=yellow). These maps confirm that the crystal is composed solely of Au particles. c) A scheme, SEM image, and compositional maps of a
core–shell crystal. Here, the core of the crystal is made from AuTMA and AuMUA NPs and is stabilized with cross-linker 3 (gray-black arcs). A
mixture of AgTMA and AuMUA NPs is then deposited onto this cross-linked core, and the entire layered structure is stabilized with cross-linker 1
(also see Figure 5).
oxohexan-2-yl)-11-mercaptounde-
canamide (3), incorporating a lysine
amino acid and cleaving in the
presence of proteases such as trypsin
or proteinase K (but not papain). In
all cases, the stability of NP crystals
in water increased with increasing
dithiol concentration CDT (see Fig-
ure S1a in the Supporting Informa-
tion). On the other hand, if too
much dithiol was used, the tightly
knit crystals were hard to dissolve
upon exposure to dithiol-cutting
analytes, and the sensitivity of detec-
tion was poor. Consequently, the
crystals we used were cross-linked
with the minimal concentration of
min
DT
dithiols (C ꢀ 2 mm, 6–8 h soak-
ing) necessary to stabilize these
crystals in water such that no indi-
vidual NPs dissolved in solution
could be detected by either UV/Vis
spectroscopy, DLS (dynamic light
scattering), or TEM (transmission
electron microscopy).
Reaction-diffusion
modeling
Figure 2. Amplified chemical sensing using nanoparticle supracrystals. a) Crystals, each containing
several million NPs, self-assemble from oppositely charged NPs (i) and are soluble in water (ii).
These crystals gain permanence and become water-insoluble when they are cross-linked with dithiols
containing cleavable groups (iii). When a specific analyte is added, the cross-links are chemically cut,
and the crystals disintegrate into individual nanoparticles (iv). Scale bars are 250 nm in the SEM
images of crystals and 20 nm in the TEM images of free NPs. b) Chemical structures of the dithiol
cross-linkers 1–3 and the analytes that cut them.
accounting for the diffusion of the
dithiols into the nanometer-sized[9a]
pores of the crystals and for the
displacement of the TMA/MUA
thiols from the NPs estimates the
thickness of the cross-linked layer to
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be on the order of L ꢀ 15–30 nm from the crystalꢀs surface
(see the Supporting Information, Section 4 for details). In
other words, only the “skin” of the crystals is cross-linked, and
when it is ruptured, the crystals should liberate large numbers
of the interior, un-cross-linked NPs.
lowest analyte concentrations used—gave mostly individual
NPs rather than nanoparticle clusters in solution. This
behavior was in sharp contrast to that observed in control
experiments, where we studied the dissolution of cross-linked
but disordered NP aggregates (Figure 3c, violet and gray
This, indeed, is what was observed in experiments. We first
discuss the results for the regular (i.e., not CS) crystals. When
an analyte specific to the cleavable dithiols was added, the
crystals dissolved, thus coloring the solution brightly (Fig-
ure 3a). SEM imaging (Figure 3b) indicates that crystals
histograms). Such aggregates not only required a much higher
min
concentration of dithiols to be stable in water (C @ 10 mm)
DT
but also dissolved slowly and gradually—first into smaller,
colorless aggregates and only then into individual NPs. As a
result, the sensitivity of detection was much lower upon the
dissolution of disordered aggregates than NP crystals.
These trends are quantified in Figure 4a–c, which plots
the solutionꢀs absorbance Abs (proportional to the concen-
tration of dissolved NPs) as a function of the concentration of
the added analyte CA (here, EDTA cutting cross-links of 2).
Figure 3. Dissolution of cross-linked crystals. a) Optical images illus-
trating dissolution of the crystals (here, cross-linked with 1) upon
addition of (CH3)4N+OHÀ base (10 mm, 5 mL). b) Gradual dissolution
of the crystals from (a) monitored using SEM. The concentrations of
the base added are indicated in the upper-right corners of the images.
The hazy regions around the crystal in the left image (indicated by
white arrows) correspond to individual NPs “spilling out” (see the
Supporting Information, Section 3 for high-resolution images). Scale
bars=1 mm. c) Particle size distributions recorded by DLS on a
Malvern ZetaSizer Nano-ZS instrument (see the Supporting Informa-
tion, Section 2.2). Black: undissolved NP crystals cross-linked with 1.
Red: particles from the dissolution of the crystals (upon addition of
10 mL 10 mm (CH3)4N+OHÀ). Gray: cross-linked, disordered NPs
(cross-linker 1). Violet: aggregates from the dissolution of the disor-
dered aggregates (after 24 h; cutting agent: 10 mL 50 mm
Figure 4. Dissolution of NP crystals and of disordered NP aggregates.
a) UV/Vis spectra indicate dissolution of crystals (stabilized by 2) upon
gradual addition of EDTA. The corresponding images in the right column
illustrate the changes in the solution color; the red color is due to
individual AuNPs from the dissolved crystals. b) Control experiment in
(CH3)4N+OH).
which EDTA is added to disordered, cross-linked aggregate. Even for high
concentrations of EDTA, only a small fraction of the aggregates dissolves
into free NPs, and the solutions are barely colored. c) Comparison of the
two experiments quantified by absorbance at lmax ꢀ530 nm. For a given
concentration of EDTA, the colorimetric response of the crystals is
approximately 100 times stronger than that of disordered aggregates.
d) Kinetics of dissolution of crystals cross-linked with 3 upon addition of
trypsin (red) and proteinase K (yellow), both at the same 5 mm enzyme
concentration, at T=378C for trypsin and 508C for proteinase K. Crystals
are stable against papain (violet), which does not cleave the cross-linker 3.
dissolved from one or few locations on their surfaces. These
locations were usually along crystalꢀs edges or near its
vertices, where the surface NPs were the least stable (i.e.,
corresponding to the highest free energies).[11] Once a small
hole was “pinched” in the crystalꢀs cross-linked “skin”, large
numbers of individual NPs spilled from the crystalꢀs interior
into the solution. DLS measurements (Figure 3c, black and
red histograms) confirmed that this process—even for the
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For a given value of CA, Abs measured for the crystals was
result compares favorably with conventional NP-based col-
orimetric methods,[2b,4a,5] which require approximately 1000 or
more analyte molecules to effect even minute spectral shifts
or changes detectable only using spectroscopic methods.
Qualitatively similar trends to those for 2/EDTA were
observed for the dithiol 1/OHÀ analyte pair, for which the
smallest visually detectable change corresponded to 43
analyte molecules per NP, and dissolution time was approx-
imately 90 s. In both cases, the crystals were not dissolved by
analytes not specific to the dithiol cross-linkers.
Crystals stabilized with dithiols containing enzyme-spe-
cific motifs were also used as selective enzymatic sensors. For
instance, crystals cross-linked with lysine-containing 3 were
dissolved readily by CA ꢀ 5 mm solutions of serine proteases
such as trypsin or proteinase K (Figure 4d), the active centers
approximately 100 times greater than the absorbance from
dissolving disordered aggregates (Figure 4c). In other words,
the use of cross-linked crystals offered an approximately two-
orders-of-magnitude improvement in sensitivity compared to
cross-linked but disordered NPs. The dissolution of crystals
was also much more rapid. A typical crystal sample (ca. 8.8 ꢁ
107 crystals or 2.2 ꢁ 1014 NPs in 4 mL solvent) exposed to an
analyte of concentration CA ꢀ 1 mm dissolved completely
within 60 s; for a disordered NP aggregate exposed to a
tenfold higher analyte concentration, the time to see even a
bleak color was on the order of tens of minutes. Lastly, for the
crystals, the smallest change in the solutionꢀs color discernible
to a naked eye (Abs ꢀ 0.3–0.4 in a 10 mm cuvette) was 14
EDTA molecules per one AuTMA/MUA nanoparticle. This
Figure 5. Sensing by core–shell crystals. When the core–shell crystals (middle column) are exposed first to the solution of an analyte cutting the outer cross-
links (here OHÀ cutting 1) and are then transferred into the solution containing analytes cutting the inner cross-links (here proteinase K cutting 3), both the
core and the shell dissolve completely into individual NPs (rightmost column). When, however, the crystals are first exposed to the enzyme and only then to
the base, only the shell dissolves, thus leaving behind intact crystal “cores” (leftmost column). The specific crystals used here have cores composed of
AuTMA and AuMUA NPs and shells made of AgTMA and AuMUA NPs. The second row from the top shows typical SEM images of the crystals (scale
bars=500 nm; 100 nm in the rightmost image). The third row shows the UV/Vis spectra and optical images of the solutions. Solutions are colorless if
crystals are not dissolved, orange if only the shells are dissolved, and dark red if both the shells and the cores are dissolved. The bottom row has typical
EDS composition scans across individual crystals. Intact core–shell crystals contain cAu ꢀ78% Au and cAg ꢀ22% Ag (composition averaged over scan
length); when the shell is dissolved, the composition of the core is pure Au. Note that near the ends of the scan of intact CS crystals, the composition of Ag
increases to approximately 50%, as predominantly the crystal’s Au/Ag shell is probed by the beam. Shell thickness xsh can be estimated from the elemental
composition near the middle of the crystal, cAu ꢀ85%, where the beam passes through two Au/Ag shells (each of thickness xsh; Au metal content 50%) and
the Au/Au core (thickness xcore; Au content 100%). For 2 mm crystals, the equations (100% xcore +250% xsh)/(xcore+2xsh)=0.85 and (xcore +2xsh)=2 mm give
an estimated xsh ꢀ300 nm.
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of which contain the serine-histidine-aspartate catalytic
triad.[12a] The smallest change in the solution color detectable
by a naked eye corresponded to 56 molecules of trypsin or
proteinase K per one liberated nanoparticle. In contrast, the
same crystals remained stable in the presence of even high
(CA > 0.1 mm) concentrations of the protease papain (violet
curve in Figure 4d), whose catalytic triad is cysteine-histidine-
asparagine[12b] and which has low affinity to 3.[12c] Interest-
ingly, the rates of crystal dissolution were similar for trypsin
and for proteinase K, despite the fact that the rates of
proteolytic cleavage of free 3 by these enzymes are quite
different (tens of minutes for trypsin[13a] and a few minutes for
proteinase K[13b]). This observation suggests that crystal dis-
solution is limited by the slow diffusion of large enzymes into
the nanoscopic pores of the crystalsꢀ cross-linked “skin” layer.
This limited accessibility of the cross-linkersꢀ cleavable group
can also rationalize why the dissolution of the crystals was
markedly slower (15–30 min to reach Abs ꢀ 0.3–0.4) than in
the previously discussed 2/EDTA or 1/OHÀ systems. Still,
these times are commensurate with those for commercial
proteinase assays (typically, tens of minutes to hours[14]).
The core–shell crystals (see Figure 1c) enable additional
modes for amplified sensing, especially when the core and
shell regions are made of different NPs and are stabilized by
cross-linkers specific to different analytes. Under such con-
ditions, it is possible to dissolve the core and the shell
selectively and to liberate the NPs that compose these regions
sequentially. While in homogeneous solutions such sequential
sensing is probably of little advantage (one can always use two
types of “regular” crystals, each stabilized with different
cross-links), it can be of use in “stratified” media in which
different regions contain different types of analytes. This
situation is illustrated in Figure 5 for CS crystals comprising
Au cores stabilized with 3 and Au/Ag shells stabilized with 1.
When these crystals are first exposed to a medium containing
OHÀ analyte (cutting the outer 1 cross-links), the shell
dissolves; subsequent passage through a medium containing
proteinase K (cutting inner 3 cross-links) dissolves the
crystalꢀs core. Importantly, when the same crystals are
passed through the same media but in reverse order, only
shells dissolve, but the cores remain intact. In other words, the
crystals perform spatially distributed sensing, whereby they
report the path they traveled (note that this cannot be
achieved with mixtures of “regular” crystals of different types
stabilized by different cross-linkers). We suggest that this
property can be even more relevant to nanoparticle-based
delivery systems, whereby appropriately structured and cross-
linked CS crystals traveling through chemically stratified
media release their cargo sequentially in desired locations.
In summary, nanoparticle crystals of different internal
architectures, including unprecedented core–shell NP crys-
tals, and stabilized with chemically cleavable cross-links
effectively “amplify” the presence of cross-link-specific
analytes into pronounced color changes. This mode of sensing
can be generalized to different cross-link/analyte combina-
tions. An exciting opportunity for future research would be to
use supracrystals of medically relevant nanoparticles (e.g.,
antibacterial AgNPs, antifungal CuNPs, anticancer arsenic
NPs, MRI contrast-enhancing magnetite NPs) in biodelivery
applications, where the crystals, especially the CS ones, could
dissolve and liberate their contents sequentially upon reach-
ing specific intracellular targets.
Received: April 18, 2010
Published online: July 13, 2010
Keywords: amplification · nanoparticles · self-assembly ·
.
sensors
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[14] Examples of times needed to observe color change in different
commercial protease assays include QuantiCleave protease
assay kit: within 1 h, protease assay kit from G Biosciences:
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1 h.
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