A two-component small molecule system for activity-based detection and signal amplification: Application to the visual detection of threshold levels of Pd(II)

Article abstract of DOI:10.1021/ja108347d

A detection and signal amplification strategy aimed toward threshold diagnostic assays for use in resource-limited settings is described. The strategy employs two small molecule reagents that work in tandem. One reagent detects a specific analyte, while the second amplifies a colorimetric readout autocatalytically. The strategy is demonstrated using palladium(II) as a model analyte.

Full text of DOI:10.1021/ja108347d

COMMUNICATION
pubs.acs.org/JACS
A Two-Component Small Molecule System for Activity-Based
Detection and Signal Amplification: Application to the Visual
Detection of Threshold Levels of Pd(II)
Matthew S. Baker and Scott T. Phillips*
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
S Supporting Information
b
ABSTRACT: A detection and signal amplification strategy
aimed toward threshold diagnostic assays for use in re-
source-limited settings is described. The strategy employs
two small molecule reagents that work in tandem. One
reagent detects a specific analyte, while the second amplifies
a colorimetric readout autocatalytically. The strategy is
demonstrated using palladium(II) as a model analyte.
iagnostic assays are a crucial component of medical care in
resource-limited settings, yet the reagents used in most
D
point-of-care assays lack the thermal stability needed for envi-
ronments encountered in the developing world.1 Currently, the
most sensitive and selective diagnostic assays rely on antibodies,
enzymes, silver(I) salts, or a combination of these reagents to
achieve sensitivity and selectivity. These reagents, however, are
not stable for prolonged periods when stored above 0 °C. To
circumvent this limitation, point-of-care diagnostics typically
contain additives and/or are refrigerated to help maintain the
activity of the reagents. Even with these measures in place,
sensitive and selective assays like enzyme-linked immunosorbent
assays (ELISA),2 PCR,3 biobarcode assays,4 and other similar
assays are not easily translated to resource-limited environ-
ments.1 Given the dearth of temperature-stable reagents, other
analyte detection and signal amplification reagents must be
developed. This deficiency also provides the opportunity to
develop basic science that leads to new, general strategies for
detection and signal amplification.
Figure 1. Design of a thermally stable, two-component, small molecule
system for detection and signal amplification. The detection reagent reacts
specifically with an analyte and releases a signal transduction reagent
(e.g., Fꢀ). The signal amplification reagent then reacts with this signal
transduction reagent in an autocatalytic process to produce amplified
quantities of a colorimetric indicator (compounds 2ꢀ4).
that can be used to detect a variety of analytes in resource-limited
environments. Ideally, the system should be readily accessible,
thermally stable, specific in its response to an analyte, and com-
patible with complex biological fluids (i.e., not susceptible to
background reactions). Furthermore, the system should (i) amplify
the signal quickly (minutes to hours), (ii) amplify the signal to
levels that rival or exceed standard analytical methods (e.g.,
ELISA assays or PCR), (iii) provide a clear visual readout that
does not require spectrophotometers or electronics for detec-
tion, (iv) be easily reconfigurable to detect trace levels of different
analytes, and (v) provide qualitative and quantitative readouts.
This communication describes a new detection and signal
amplification strategy that we hope will provide the starting point
for realizing these goals. Our strategy (Figure 1) employs two
thermally stable small molecule reagents that work in tandem to
provide selective, trace-level detection of specific analytes. The
first reagent in this pair is an activity-based detection reagent that
reacts with the analyte (e.g., Pd(II)) and releases a specific
chemical signal (e.g., fluoride). The signal amplification reagent
then responds to this released signal (fluoride) in an autocatalytic
degradation reaction that releases amplified quantities of a yellow
indicator (e.g., compounds 2ꢀ4, discussed below) until the
A handful of research groups have described small molecule
reagents that avoid, to some extent, the thermal instability
problems described above.5 Each of these seminal efforts has
provided a reagent that specifically detects a single analyte and
amplifies the resulting signal. For example, Anslyn and Koide both
have developed catalytic systems for detecting Pd and Pd2þ 6
.
Anslyn reported two additional signal amplification systems: one
for detecting Cu2þ, and the other for Pb2þ 5,6
.
Mirkin described a
supramolecular system for detecting the combination of Clꢀ and
CO,7 as well as another reagent that responds to the combination
of acetate and CO,7 and Shabat recently reported a reagent for
detecting thiols.8 Polymer-based reagents for detection and signal
amplification also have been described; examples include Swager’s
versatile conjugated polymers for fluorescent and/or electrical
readouts9 and the degradable dendrimer reagents developed by
McGrath, de Groot, and Shabat.10
Despite these advances, there remains a need for a general
small molecule (or polymer) detection and amplification system
Received: September 15, 2010
Published: March 22, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Amplification Reagent 1
amplification reagent (1) is consumed. 2ꢀ4 can be detected
visually, thus enabling detection of threshold levels of an analyte.
Because two separate reagents control the detection and
amplification processes, this system can be easily reconfigured
to detect and amplify a variety of analytes, which is a feature that
is not common in other single molecule detection and signal
amplification reagents. Using our design, the amplification
reagent can be used in any assay, while the reactive portion on
the activity-based detection reagent (i.e., “substrate for analyte”
in Figure 1) can be modified to detect a variety of analytes.
To evaluate the efficacy of this detection and amplification
strategy, we first prepared and studied the amplification reagent
(compound 1). Reagent 1 was obtained via an efficient (40%
overall yield), three-step synthesis (Scheme 1).
Figure 2. Signal transduction reagent (fluoride) is amplified as ampli-
fication reagent 1 is consumed. The graph shows the sigmoidal increase
in the concentration of fluoride (expressed in terms of fluorescence
intensity (I) of 7-hydroxycoumarin) as 1 is consumed. Imax is the
maximum fluorescent signal (obtained upon complete consumption
of 1), and Iexp is the measured fluorescent signal at any time point in the
amplification reaction. Each line corresponds to a different number of
equivalents of added fluoride relative to 1. The experiments using 1.0,
0.1, 0.001, 0.0001, and 0 equiv of fluoride were repeated three times, and
those using 0.01 and 0.0005 equiv were repeated twice; the averages of
each experiment are shown on the graph. The amplification reactions
were conducted at 23 °C in MeOH/pyridine/H2O (18:1:2, 105 μL); the
concentration of 1 in the starting solution was 0.12 M.
The amplification process using 1 is initiated by fluoride-
induced cleavage of the tert-butyldimethylsilyl-protected phenol
on reagent 1, which results in the formation of quinone methide and
azaquinone methide. In addition, two new equivalents of fluoride
are released, and these ions react with additional equivalents of 1
to propagate the amplification reaction. As this iterative process
continues, the quantity of fluoride at the end of each cycle, in theory,
will be equal to 2n, where n is the number of cycles. As the
amplification reaction progresses, increasing numbers of indicators
2ꢀ4 are released, thus strengthening the colorimetric readout.
We tested the behavior of 1 by dissolving it in MeOH and
pyridine11 and then exposing it to a stoichiometric amount of
fluoride (CsF) dissolved in water (final solution: [1] = 0.12 M in
18:1:2 MeOH/pyridine/H2O, 105 μL). Under these conditions,
1 is consumed completely within 120 min. The quinone methide
intermediate arising from fluoride-induced decomposition of 1reacts
with 4-aminobenzaldehyde (2, the product arising from release
of fluoride) to produce colored byproducts 3 and 4 (Figure 1).
The use of stoichiometric fluoride makes the autoinductive
properties of 1 difficult to observe. However, when 1 is exposed
to a substoichiometric quantity of CsF, a plot of fluoride con-
centration versus time produces a sigmoidal curve (Figure 2), as
would be expected for an autoinductive or autocatalytic process.12
We measured the concentration of fluoride arising from the
amplification reaction by exposing the amplification solution to
excess (relative to the maximum quantity of fluoride expected in
the solution) tert-butyldimethylsilyl (TBS)-protected 7-hydro-
xycoumarin. Fluoride reacts with TBS-protected 7-hydroxycou-
marin ∼60ꢁ faster than with 1. Under these conditions, the
fluoride is consumed rapidly, and 7-hydroxycoumarin, a fluo-
rescent indicator molecule, is produced.
Figure 3. Structures of the control compounds that were used to test
the cause of the background reaction shown in Figure 2.
them to the conditions used for amplification (i.e., 0.12 M
concentration of each control reagent in 18:1:2 MeOH/pyri-
dine/H2O, 105 μL, 23 °C). After 21 d, HPLC traces revealed that
none of the control reagents showed signs of decomposition (at
least within the detection limits of the HPLC).13 Thus, we
hypothesized that perhaps the background signal shown in
Figure 2 is the result of trace quantities of adventitious fluoride
either in 1 or in our solvents. Trace amounts of fluoride initially
would lead to slow, autocatalytic decomposition of 1 until the
quantity of fluoride in solution is sufficient to increase the rate of
the autocatalytic reaction to a measurable level. However,
because the background reaction is slow, even trace levels of
fluoride arising from a detection event often will be sufficient to
overcome the background reaction.
Reagent 1 is capable of amplifying fluoride to a concentration
much higher than the quantity of fluoride added to the system.
The amplification factor (R) forfluoride is described by (Iamplification
ꢀ
Ibackground)/Iinitial, where Iamplification is the intensity of the fluor-
escent signal produced by reaction with TBS-protected 7-hydro-
xycoumarin after amplification, Iinitial is the intensity without
amplification, and Ibackground is the signal arising from sponta-
neous breakdown of amplification reagent 1 (Ibackground is only
included in the calculation when 0.0001 equiv of Fꢀ is used, as
shown in Figure 2). We measured the amplification factor for five
concentrations of added fluoride. Figure S2 shows that as the
quantity of applied fluoride decreases the amplification factor
In the absence of applied fluoride (Figure 2, dark blue data), 1
is stable for 48 h in MeOH/pyridine/H2O (18:1:2) at 23 °C.
After 48 h, a slow breakdown leads to the release of fluoride,
initiating the autoinductive amplification reaction (Figure 2).
To determine the source of the slow background reaction with
1, we prepared three control reagents (Figure 3) and exposed
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Figure 4. Stability of amplification reagent 1 in the presence of
representative anions. In separate experiments, aqueous solutions of
CsF, NaCl, NaBr, NaI, and NaNO3 (0.5 equiv) were added to a solution
of 1 in MeOH and pyridine. The final concentration of 1 was 0.12 M in
18:1:2 MeOH/pyridine/H2O (105 μL), and the experiment was
conducted at 23 °C. Fluorescence measurements (Iexp) representing
the quantity of released fluoride were obtained after 4 h of amplification
using the procedure described in Scheme S1. Experiments were performed
in triplicate, and error bars reflect the standard deviations from the average
values. The fluorescence measurements were obtained by treating each
sample with TBS-protected 7-hydroxycoumarin. TBS-protected 7-hydro-
xycoumarin is weakly fluorescent, which gives rise to the same amount of
ꢀ
background signal seen with the control, Clꢀ, Brꢀ, Iꢀ, and NO3
.
increases rapidly to a maximum value of 2168 ( 35 for 0.0005 equiv
of added Fꢀ. This value of amplified fluoride corresponds to ∼11
cycles of the autoinductive amplification reaction shown in Figure 1.
Because specificity is critical in the context of our amplification
reaction, we examined whether amplification reagent 1 would
react with anions other than fluoride (Figure 4). Phenolic silyl
ethers are known to be quite unreactive to anions other than
fluoride (such as Clꢀ, Brꢀ, Iꢀ, NO3ꢀ, SO42ꢀ, SCNꢀ, and
PO43ꢀ),14 but we were concerned about the stability of the
carbamate and the benzylic difluoro-substituted carbon in 1. As
shown in Figure 4, representative anions such as Clꢀ, Brꢀ, Iꢀ,
and NO3ꢀ have no effect on 1, thus supporting the notion that 1
may be useful in the context of complex fluids. Furthermore, 1 is
stable in air at 37 °C for at least one month with no decomposition
orchange inreactivity (one month was the durationof our test). In
certain parts of the developing world, extreme temperatures may
surpass 37 °C, but the stability of the compound in these
preliminary tests suggests that this type of amplification reagent
may provide a suitable starting point for further development.
We next turned our attention to developing a complete
diagnostic system (Figure 1), and we chose Pd(II) as a model
analyte. Palladium is capable of cleaving allyloxycarbonyl (Alloc)
protecting groups catalytically, even in water; therefore, this
diagnostic system serves as a model of more advanced systems
that will be used to detect enzymes (experiments to link the
amplification reagent with an enzymatic detection event are in
progress). Moreover, palladium is an interesting analyte in its own
right: it is an environmental contaminant found in roadside soil
and on roadside plants, with quantities often exceeding 0.3 ppm
due tolossfrom catalytic converters.15 Palladium also can be found
within some pharmaceutical products (the government-regulated
threshold for palladium contamination in drugs is 10 ppm).16 A
handful of fluorescent and colorimetric sensors have been devel-
oped recently for detecting palladium,17 with sensitivities reaching
∼0.5 ppm palladium for colorimetric responses.6b
Figure 5. Detection of Pd(II) using a two-component reagent combi-
nation. (a) Schematic of the procedure for detecting Pd(II). (b)
Reflectance (R) (obtained using a camera and image processing soft-
ware) of the colorimetric output as a function of assay time. Total assay
time equals the time required for activity-based detection (6 h) plus the
time required for signal amplification. The y-axis reflects the colorimetric
response (Rexp), which is reported as the percentage of the maximum
possible colorimetric response (Rmax). Experiments were performed in
triplicate; all data are shown on the graph. (c) Photographs of colori-
metric response observed with amplification (reaction with 11 and 1)
and without amplification (reaction with 11 only) and in the presence
and absence of Pd(II).
catalytic reaction with Pd(0) (via reduction of Pd(II) in situ), as
well as 1 equiv of the colored byproduct 2.
When activity-based detection reagent 11 (1.0 mg in MeOH/
pyridine (20:1, 52.5 μL) and 400 μM tri-(2-furyl)phosphine) is
exposed to a solution of 36 ppm Pd(II) (NaPdCl4) inwater(10μL)
at 23 °C, the colorimetric response reaches a maximum signal
within 2 h (as determined by photographing the samples and
quantifying the reflectance using imaging processing software).
When 3.6 ppm Pd(II) is used, a colorimetric signal does not
develop, even after 33 h of incubation (Figure 5c). At such low
concentrations of Pd(II), activity-based detection is not suffi-
ciently sensitive to provide a colorimetric readout in this system,
and therefore, the activity-based detection reagent must be
coupled with a signal amplification reaction.
To demonstrate the diagnostic system outlined in Figure 1, we
first prepared an activity-based detection reagent for Pd(II) (i.e.,
reagent 11, Figure 5a) that releases 2 equiv of fluoride for every
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This two-component system requires two steps: (i) detection
of the Pd(II) to create a fluoride signal, and (ii) amplification of
the fluoride with concomitant formation of colorimetric pro-
ducts. Initial experiments were performed with 10 ppm Pd(II)
(the upper limit of palladium that is allowed in drugs).16 When
activity-based detection reagent 11 is exposed to Pd(II), the
quantity of fluoride released from the reagent increases linearly
with time (Scheme S2). After 6 h of incubation,18 we diluted the
detection solution with amplification reagent 1 (42.5 μL, 0.29 M
in 16:1 MeOH/pyridine) and followed the development of
colorimetric products 2ꢀ4. The intensity of the yellow color
produced by 2ꢀ4 was quantified using reflectance measurements
via photographs and image processing software. Figure 5b shows
the results of this combined detection and signal amplification
reaction. The x-axis in Figure 5b reflects the total time for the
assay (i.e., the time required for detection and amplification), and
the y-axis reflects the percentage of colorimetric signal observed
relative to the maximum colorimetric signal possible under the
current reaction conditions.
’ AUTHOR INFORMATION
Corresponding Author
sphillips@psu.edu
’ ACKNOWLEDGMENT
This work was supported in part by the Bill and Melinda Gates
Foundation as a subcontract from Harvard University (Subcontract
No. 01270716-00), the Arnold and Mabel Beckman Foundation, the
Camille and Henry Dreyfus Foundation, 3M, Mr. Louis Martarano,
and The Pennsylvania State University. We appreciate the effort of
Kimy Yeung in preparing additional quantities of reagent 11.
’ REFERENCES
(1) Giljohann, D. A.; Mirkin, C. A. Nature 2009, 462, 461–464.
(2) Gosling, J. P. Clin. Chem. 1990, 36, 1408–1427.
(3) Blow, N. Nat. Methods 2007, 4, 869–875.
(4) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003,
301, 1884–1886.
(5) (a) Zhu, L.; Anslyn, E. V. Angew. Chem., Int. Ed. 2006, 45,
1190–1196. (b) Cho, D.-G.; Sessler, J. L. Chem. Soc. Rev. 2009, 38,
1647–1662.
(6) (a) Wu, Q.; Anslyn, E. V. J. Am. Chem. Soc. 2004, 126, 14682–
14683. (b) Houk, R. J. T.; Wallace, K. J.; Hewage, H. S.; Anslyn, E. V.
Tetrahedron 2008, 64, 8271–8278. (c) Song, F.; Garner, A. L.; Koide, K.
J. Am. Chem. Soc. 2007, 129, 12354–12355. (d) Garner, A. L.; Koide, K.
Chem. Commun. 2009, 86–88. (e) Garner, A. L.; Koide, K. J. Am. Chem.
Soc. 2008, 130, 16472–16473. (f) Garner, A. L.; Song, F.; Koide, K. J. Am.
Chem. Soc. 2009, 131, 5163–5171.
(7) (a) Yoon, H. J.; Mirkin, C. A. J. Am. Chem. Soc. 2008,
130, 11590–11591. (b) Masar, M. S., III; Gianneschi, N. C.; Oliveri,
C. G.; Stern, C. L.; Nguyen, S. T.; Mirkin, C. A. J. Am. Chem. Soc. 2007,
129, 10149–10158.
(8) Sella, E.; Weinstain, R.; Erez, R.; Burns, N. Z.; Baran, P. S.;
Shabat, D. Chem. Commun. 2010, 46, 6575–6577.
While the current detection and signal amplification reagents
provide signal slowly (ideal assays would require <1 h to obtain
results), this detection system is quite sensitive: Figure 5b reveals
that even 0.36 ppm palladium (ca. the quantity of palladium
found in roadside soil)15 can be detected by visual inspection.
This level of sensitivity compares well with the sensitivity of other
colorimetric sensors for palladium.6b
Yellow color is apparent at only 20% of the maximum
colorimetric signal (i.e., (Rexp/Rmax) ꢁ 100 = 20 in Figure 5b)
(see Figure 5c for the actual colors), so a threshold level of
palladium in a sample can be determined easily by eye. For
example, Figure 5c shows that concentrations of palladium that
are g0.36 ppm cause the solution to turn yellow within 29 h of
initiating the detection/amplification reaction. The time is short-
er for higher concentrations of Pd(II): e.g., if g10 ppm is present
in a sample, then the solution turns yellow within 6.5 h.
The strategy outlined has several features making it a starting
point for further development of point-of-care diagnostic assays.
The results of the detection and amplification process are
colorimetric, so they are visually easy to read for qualitative
yes/no assays, the reagents are capable of detecting and signaling
the presence of analytes that originate in aqueous solutions, and
in theory, the activity-based detection reagent can be modified so
that it responds specifically to analytes other than Pd(II), which
would enable the detection of a variety of analytes using the same
diagnostic strategy.
The current detection and amplification system does have
some drawbacks; e.g., the strategy requires the use of organic
solvents (to dilute the aqueous sample) and pipettes (to supply
the sample), and the amplification reaction is slow. Our current
efforts are focused on overcoming these issues.
The key component of our new strategy is the signal ampli-
fication reagent that produces a colorimetric readout through an
autocatalytic process. The proof-of-concept systems described in
this communication represent a new approach to designing
nonenzymatic, thermally stable, easily accessible, and analyte-
specific amplification reagents that may be of broad use in the
context of point-of-care diagnostic assays.
(9) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. Rev. 2007,
107, 1339–1386.
(10) Avital-Shmilovici, M.; Shabat, D. Soft Matter 2010, 6, 1073–1080.
(11) This solvent system was chosen after a screen of other solvents.
Reagent 1 is not soluble in pure water, and therefore, we explored
mixtures of water and MeCN, DMF, DMSO, MeOH, and THF. We
chose the combination of solvents that gave the fastest signal amplifica-
tion reaction. The relative order of reactivity was MeOH > DMSO >
DMF > MeCN > THF. We included pyridine as a precaution to buffer
any HF that might develop during the signal amplification process.
(12) (a) Tjivikua, T.; Ballester, P.; Rebek, J., Jr. J. Am. Chem. Soc.
1990, 112, 1249–1250. (b) Severin, K.; Lee, D. H.; Martinez, J. A.; Vieth,
M.; Ghadiri, M. R. Angew. Chem., Int. Ed. 1998, 37, 126–128. (c) Todd,
M. H. Chem. Soc. Rev. 2002, 31, 211–222.
(13) Silyl ethers are known to hydrolyze slowly in water; therefore, it
remains a possibility that the background signal obtained with reagent 1
may be the consequence of a slow hydrolysis reaction followed by
autocatalytic amplification of the released fluoride.
(14) (a) Kim, S. Y.; Hong, J.-I. Org. Lett. 2007, 9, 3109–3112. (b)
Zhu, C.-Q.; Chen, J.-L.; Zheng, H.; Wu, Y.-Q.; Xu, J.-G. Anal. Chim. Acta
2005, 539, 311–316.
(15) Hodge, V. F.; Stallard, M. O. Environ. Sci. Technol. 1986,
20, 1058–1060.
(16) Carey, J. S.; Laffan, D.; Thomson, C.; Williams, M. T. Org.
Biomol. Chem. 2006, 4, 2337–2347.
(17) (a) Duan, L.; Xu, Y.; Qian, X. Chem. Commun. 2008, 6339–
6341. (b) Jun, M. E.; Ahn, K. H. Org. Lett. 2010, 12, 2790–2793. (c) Li,
H.; Fan, J.; Du, J.; Guo, K.; Sun, S.; Liu, X.; Peng, X. Chem. Commun.
2010, 46, 1079–1081.
’ ASSOCIATED CONTENT
(18) After 6 h, enough fluoride was present to provide an efficient
amplification process in the second step. See the SI for details.
S
Supporting Information. This material is available free
b
of charge via the Internet at http://pubs.acs.org.
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