A DNAzyme Catalytic Beacon Sensor for Paramagnetic Cu2+Ions in Aqueous
Solution with High Sensitivity and Selectivity
Juewen Liu and Yi Lu*
Department of Chemistry,Uni V ersity of Illinois at Urbana s Champaign,Urbana,Illinois61801
Received March12,2007;E-mail:yi-lu@uiuc.edu
Design of fluorescent metal nsors has recently become one of
the most active rearch areas becau the nsors can provide in
situ and real-time information for a number of applications including
environment monitoring,industrial process control,metalloneuro-
chemistry,and biomedical diagnostics.1A widely ud strategy is
to link the metal recognition portion cloly with a signal generation
moiety such as a fluorophore.While quite successful in designing
nsors for diamagnetic metal ions such as Pb2+,Hg2+,Zn2+,and
Cu+,2this method has been applied to paramagnetic metal ions such
as Cu2+with only limited success,due to their intrinsic fluorescence
quenching properties.3,4Most Cu2+nsors showed decread
emission upon Cu2+binding,3which was undesirable for analytical
purpos.First,the room for signal change was at most1-fold.
Second,such“turn-off”nsors may give fal positive results by
quenchers in real samples.Among the reported“turn-on”Cu2+
nsors,4few have nanomolar nsitivity,4a,d,f,g with high lectivity,4a,d
and free of organic solvents.4a One way to circumvent this
quenching problem is to spatially parate the metal recognition
part from the fluorescent signaling moiety so that they are
independent of each other.A significant challenge then is to
transduce metal binding to signal enhancement when the two parts
are well-parated.We have previously reported a novel metal
nsing platform with DNAzyme catalytic beacons that spatially
parated the two parts by rigid double-stranded DNA,5,6and
nsors for diamagnetic metal ions such as Pb2+and UO22+have
been demonstrated.7,8Herein,we apply this method to turn-on
nsing of paramagnetic Cu2+with high nsitivity and lectivity.
Copper is a widely ud metal that can leak into the environment
through various routes.Low concentration of copper is an esntial
nutrient.However,exposure to high level of copper even for a short
period of time can cau gastrointestinal disturbance,while long-
term exposure caus liver or kidney damage.9The U.S.Environ-
mental Protection Agency(EPA)has t the limit of copper in
drinking water to be1.3ppm(∼20µM).
We cho a Cu2+-dependent DNA-cleaving DNAzyme reported by Breaker et al.as a basis for the nsor design.10-12On the basis of the original DNAzyme quences,we rationally designed a Cu2+ nsor as shown in Figure1A.The nsor contained two DNA strands that formed a complex.T
he substrate(in black)was labeled with a FAM fluorophore(6-carboxyfluorescein)at the3′-end and a quencher(Iowa Black FQ)at the5′-end,while the enzyme(in blue)contained a5′-quencher.Such a dual-quencher approach was employed to suppress background signals.13The substrate and enzyme associate through two ba-pairing regions.The5′-portion of the enzyme binds the substrate via Watson-Crick ba pairs and the3′-region through formation of a DNA triplex.Initially, the FAM emission was quenched by the nearby quenchers.In the prence of Cu2+,the substrate was irreversibly cleaved at the cleavage site(the guanine in red).Following cleavage,we hypothesized that the cleaved pieces were relead due to decread affinities to the enzyme,leading to incread fluorescence(Figure 1B).This hypothesis was supported by the obrvation that the FAM emission incread by∼13-fold after addition of Cu2+(Figure1C). Such a signal generation method was termed catalytic beacon becau the involvement of catalytic reactions.7,8The nsor system also contained50µM ascorbate becau it can significantly enhance the reaction rate(Figure S9,Supporting Information).10-12Ascorbate was also uful for suppressing quenching.For example,FAM quenching was<15%with50µM Cu2+(Figure S8).
To test nsitivity,the kinetics of fluorescence increa at520 nm in the prence of varying concentrations of Cu2+were monitored.As shown in Figure2A,fluorescence enhancement rates were
higher with increasing levels of Cu2+.The rates in the time window of2-4min were plotted in Figure2B.A detection limit of35nM(2.3ppb)was determined,which reprents one of the most nsitive turn-on Cu2+nsors.4a,d,f,g The nsor has a dynamic range up to20µM,which is uful for detecting Cu2+in drinking water becau the U.S.EPA has defined a maximum contamination level of20µM.In addition to being highly nsitive and
posssing Figure1.(A)The condary structure of the Cu2+nsor DNAzyme.F and Q denote fluorophore and quencher,respectively.The cleavage site is indicated by an arrow.(B)Signal generation scheme of the Cu2+catalytic beacon.(C)Fluorescence spectra of the nsor before and10min after addition of20µM Cu2+
.
Figure2.(A)Kinetics of fluorescence increa over background at varying Cu2+levels.The arrow indicates the point of Cu2+addition.Int:respons at low Cu2+levels.(B)The rate of fluorescence enhancement plotted against Cu2+concentration.Int:rates at the low Cu2+region.(C)Sensor lectivity.The buffer contained1.5M NaCl,50mM HEPES,pH7.0,and 50µM ascorbate.Cu2+concentrations were labeled on the left side of each well,while others were on the right end(inµ
M).
Published on Web07/24/2007
98389J.AM.CHEM.SOC.2007,129,9838-983910.1021/ja0717358CCC:$37.00©2007American Chemical Society
turn-on signals,the nsor respon was also fast,and quantitative results can be obtained within veral minutes.
马王堆汉墓To test lectivity,16competing metal ions were assayed at three concentrations:1mM,100µM,and 10µM.The assay was performed in a 96-well plate,and emission intensities at 12min after addition of metal ions were compared.As shown in Figure 2C,besides Cu 2+,only the spots with 1mM Fe 2+and 1mM UO 22+lit up,and the intensities were lower than that with 0.5µM of Cu 2+.Therefore,the nsor lectivity for Cu 2+was at least 2000-fold higher than the two metals and >10000-fold higher than any other tested metal ions.The relatively high lectivity of Cu 2+over paramagnetic Fe 2+may be due to either lack of DNAzyme recognition of Fe 2+as the DNAzyme was in vitro lected for Cu 2+,
11,12or lack of H 2O 2in the nsor solution that is needed for Fe 2+to go through the Fenton chemistry for DNA cleavage.14In gel-bad assays,UO 22+did not produce well-defined cleavage bands (Figure S4).Therefore,the incread emission by UO 22+was attributed to DNA denaturation,although minor oxidative cleavage cannot be ruled out.15Cu +is unstable in water,and Cu +was tested using [Cu(MeCN)4](PF 6)in acetonitrile as a metal source.With ascorbate,the rate of fluorescence increa was similar to that with Cu 2+(Figure S9).In the abnce of ascorbate,both Cu 2+and Cu +can induce fluorescence increa,with the rate with Cu +being much faster.Therefore,it is likely that Cu 2+was reduced by ascorbate to Cu +,which subquently reacted with oxygen to oxidatively cleave DNA.Ag +was not tested becau the reaction buffer contained 1.5M NaCl,which can form insoluble AgCl.Au +was not tested becau it is unstable in the open air aqueous solution.For testing environment samples,such as detection of Cu 2+in drinking water,Cu +or Fe 2+is unlikely to interfere due to the oxidative aqueous environment.UO 22+is also unlikely to be prent in millimolar concentration in drinking water.Fe 3+with ascorbate can also cleave the DNAzyme.However,little fluorescence increa was obrved due to the slow reaction rate and the quenching effect of Fe 3+(Figures S6and S7).It needs to be pointed out that the previously reported lead and uranium nsors were bad on hydrolytic RNA cleavage.7,8In the current copper DNAzyme,the substrate was made completely of DNA,and the cleavage was oxidative.10
Finally,we constructed a nsor array as shown in Figure 3.The array contained three rows,and each row was loaded with a different DNAzyme-bad nsor.Eight metal mixtures were prepared with all the possible combinations among Cu 2+,Pb 2+,and UO 22+(1µM each).As can be obrved from Figure 3,the wells lit up only when the cognate metals were prent and the metal compositions can be read directly from the array.We can imagine that larger metal nsor arrays can be built with the isolation of more metal-specific DNAzymes.5
In summary,we have demonstrated a highly effective fluorescent nsor that showed strong fluorescence enhancement in the prence of a paramagnetic metal ion:Cu 2+.This result further demonstrated that the DNAzyme-bad metal nsing approach can be applied to a broad range of metal ions.
秋天是一个什么样的季节
Acknowledgment.This material is bad upon work supported by the U.S.Department of Energy (DE-FG02-01-ER63179),the NSF (CTS-0120978and DMI-0328162),and by the Illinois Waste Management and Rearch Center (WMRC).
狂躁症八个表现Supporting Information Available:Experimental procedures,gel-bad assays,controls and fluorescence quenching (PDF).This material is available free of charge via Internet at References
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包公案JA0717358
Figure 3.Respons of a DNAzyme nsor array to metal mixtures.
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