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Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 631421 6 pageshttpdxdoiorg1011552013631421

Review ArticleFluorescent DNA Stabilized Silver Nanoclusters as Biosensors

Alfonso Latorre Romina Lorca and Aacutelvaro Somoza

Instituto Madrileno de Estudios Avanzados en Nanociencia (IMDEA Nanociencia)CNB-CSIC-IMDEA Nanociencia Associated Unit ldquoUnidad de Nanobiotecnologıardquo 28049 Madrid Spain

Correspondence should be addressed to Alfonso Latorre alfonsolatorreimdeaorg andAlvaro Somoza alvarosomozaimdeaorg

Received 26 April 2013 Accepted 18 June 2013

Academic Editor Eugen Stulz

Copyright copy 2013 Alfonso Latorre et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

DNA stabilized fluorescent silver nanoclusters are promising materials of which fluorescent properties can be exploited to developsensors Particularly the presence of a DNA strand in the structure has promoted the development of gene sensors where onepart of the sensor is able to recognize the target gene sequence Moreover since oligonucleotides can be designed to have bindingproperties (aptamers) a variety of sensors for proteins and cells have been developed using silver nanoclusters In this review theapplications of this material as sensors of different biomolecules are summarized

1 Introduction

The binding capabilities of oligonucleotides have allowedthe development of sensors for the detection of differ-ent molecules such as DNA or RNA through base com-plementarity interactions or proteins and small moleculesusing properly designed aptamers Different strategies havebeen developed depending on the detection method suchas electrochemistry [1 2] luminescence [3 4] or surfaceplasmon resonance [5ndash7] However one of the most usedmethodologies is fluorescence [8ndash10] due to the sensitivityand simplicity of this technique Despite these advantagesthis strategy has some drawbacks to be solved For instanceoligonucleotides have to be modified with a dye and some-times even with a quencher as inMolecular Beacons makingthe process more tedious and costly In addition the organicdyes employed are sensitive to photobleaching and some ofthem have significant background fluorescence which couldaffect the sensitivity

Fluorescent DNA stabilized silver nanoclusters [11 12](DNA-AgNC) have emerged as new powerful nanomaterialsin biology Their unique properties such as small size [13]biocompatibility great photostability and tunable fluores-cence [14ndash16] make these materials excellent candidates torival quantum dots [17] and organic dyes [18 19] in differentapplications such as biolabels and biosensors

The introduction of metallic silver in DNA has beenexplored by other approaches such as metalation of DNA[20] But the preparation of DNA AgNCs is quite sim-ple affording discrete number of atoms of silver on theDNA with fluorescent properties Typically a solution of anoligonucleotide is incubated with six equivalents of silvernitrate (AgNO

3) for 15min Then six equivalents of sodium

borohydride (NaBH4) are added vortexed and incubated in

the dark for several hours The stabilization of these clustersis mainly favored by the high affinity of Ag+ by N3 ofcytosines [21 22] Guanines are able to interact with Ag+through the N7 in less extent Although the influence of theoligonucleotide on the fluorescence properties of AgNC isnot clearly understood it can be easily modulated by theoligonucleotide sequence or length allowing the preparationof a wide palette of emitters from blue to near IR wavelengths[14] The fluorescent properties of this nanomaterial arealso affected by the environment [23] Thus changes in thesecondary structures of DNA-AgNC promoted by hybridiza-tion of a complementary sequence or complexation with amolecule may promote an emission wavelength shift or adrastic enhancement or decrease in the fluorescence intensityThis property has been exploited to develop sensors forions [24ndash27] small molecules [28ndash30] DNA RNA proteinsand cells In this article we have summarized the biologicalapplications of DNA-AgNCs

2 Journal of Chemistry

G T

G T

G richsequence

AgNC Complementary

WT gene Mutated gene

AgAgAg

AgAgAg Ag

AgAg

AgAg

Ag

regionsequence

Figure 1 Chameleon Nanocluster Beacon [32 33] Nanoclustersinteract with different regions of the G fragment depending on themutated baseThe different final structures obtained lead to differentcolors

2 Gene Detection

DNA-AgNCs have been successfully applied in the detectionof sequences of DNA and RNAThemost common approachrequires the preparation of an oligonucleotidewith twodiffer-ent domains One of them has a sequence complementary tothe DNA target and other domain is able to bind silver ionswhich will be reduced to give rise to fluorescent clusters Inthis sense Chang and coworkers were able to detect with highselectivity and sensitivity a gene associated with tyrosinemiatype I disease [31] They found that a C12 sequence directlyconnected to the probe sequence was able to generatethe corresponding fluorescent AgNC which emission wasstrongly enhanced after hybridization with the target geneInterestingly the system was able to discriminate betweenthe mutated and wild type gene because the fluorescenceintensity for the mutated one was almost 9 times biggerthan the wild type This difference could be due to duplexstructures obtained after hybridization where the most rigidcould protect better the AgNC from quenching

In another strategy employed to detect single strandedDNA the sensor developed uses two different single strandscomplementary to the target One strand has a poly Cfragment required for the generation of AgNC and thesecond one has a guanine rich fragment Upon hybridiza-tion with the target both fragments get close leading toan enhancement and shifting of the fluorescence emissionThis system was named NanoCluster Beacon [32] due tothe similarities with classical molecular beacons Based onthis work the same authors reported the preparation of asensor able to distinguish between the three possible singlepoint mutation of a KRAS gene in clinical samples In thisnew probe named Chameleon NanoCluster Beacon [33]the nanoclusters interact with different regions of the Grich fragment depending on the gene mutation (Figure 1)As a consequence of these interactions different colors areobtained with enough wavelength separation to be observedby the naked eye under UV irradiation

DNA duplexes are also able to stabilize AgNC and havebeen used to develop DNA sensors In this regard Wangand coworkers used hybridized duplexes to detect singlepoint mutations in sickle cell disease [34] In this workthe authors designed a complementary probe sequence toa fragment of the corresponding gene bearing a C6 looptwo nucleotides away from themutation After hybridizationAgNCs were generated in the C6 loop due to the bindingaffinity of cytosine to silver ions They observed a strongyellow emission when a match sequence is used On theother hand fluorescence was negligible when a mismatchis present The effect of the distance between the mutationand the loop as well as the nature of the mismatch wasevaluated highlighting again the sensitivity of AgNC to theenvironment In this sense they found that the presence of amutation four nucleotides away from the loop does not affectthe emission parameters Also it was found that a T or Amismatch produced a higher quenching of the fluorescencethan the C or G

It has also been reported the use of DNA duplexes withabasic sites [35] where the nucleobases are missing leavingthe deoxyribose residue in the strand in the preparation ofAgNCs Shao and coworkers reported the high affinity ofsilver ions by the unpaired bases opposite to an abasic sitewhich are able to accommodate few Ag+ [36] This findingwas applied in the preparation of a sensor for the p53 genewhich is involved in cancer suppression processes [37] Aprobe complementary to a fragment of the correspondinggene with an abasic site opposite to the nucleotide respon-sible for the mutation was employed Hybridization exper-iments with the wild type fragment and the three possiblemutations and subsequent incubationreduction procedurewith Ag+ and NaBH

4gave rise to the corresponding silver

nanoclusters The emitters formed with the mutant bases(A G and T) opposite to the abasic site showed similarweak intensities However when the wild type fragment wasused an increase in fluorescence of 18-fold was observedindicating the selectivity of the process The differencesobserved are due to the affinity of the cytosine to silver whichis present in the wild type example

3 MicroRNA Detection

MicroRNAs (miRNAs) are single stranded ribonucleotidesof around 22 nucleotides long They can be found in plantsanimals and humans and are involved in gene silenceprocesses avoiding the expression of certain proteins Thedetection and quantification of those structures are of greatimportance since they are involved in the initiation as well asthe generation of cancers [38]

DNA-AgNC has been successfully used in miRNA detec-tion as demonstrated by Yang and Vosch [39] They wereable to detect the miR160 which is a miRNA that targets aplant gene involved in hormone signaling They designed aprobe composed of two parts a complementary sequence tothe miR160 (part A) and directly grafted another fragmentable to stabilized red emitting AgNC (part B) The incuba-tion of this probe with AgNO

3and subsequent reduction

Journal of Chemistry 3

miRNA

Fluorescent AgNC No fluorescent AgNC

Ag AgAg

AgAgAg

probe

Figure 2 miRNA sensor based in DNA-AgNCs [39 40] Fluores-cence of DNA-AgNC is quenched upon miRNA target binding

with NaBH4afforded the corresponding highly fluorescent

material The incubation of these brighter clusters with thetarget afforded a drastic decrease of the fluorescence allowingits detection (Figure 2) This system has been applied to thedetection of the miR160 in total RNA extracts of plants Morerecently slight variations on the system described have led tothe same authors detect themiR172 at 82 nMof concentration[40] Also the system showed high selectivity since theexperiments with four random miRNA did not produce acomparable fluorescence quench

Isothermal exponential amplification [41 42] in conjunc-tion with AgNC has been used to develop an attomolarmiRNA sensor [43] In such system a single stranded DNAwith different fragments acts as template of miRNA recogni-tion and replicator of a reporter DNAmoleculeThe reporteris obtained upon the miRNA hybridization and it is used togenerate the corresponding AgNC which fluorescence worksas positive signal indicatorMiR141 overexpressed in prostatecancer was the miRNA selected for this study This targetwas detected in real cell lysates samples in concentrationsof 10 aM representing one of the most sensitive methods inmiRNA sensing In order to check the specificity of the sensortwo miRNA of the target family and two random miRNAwere used as controls As expected just the familiar RNAgavea substantial fluorescent signal but it was lower comparedwith the fluorescence obtained with the target

4 Protein Detection

Oligonucleotides can be designed to bind a wide variety ofmolecules with high selectivity Those oligonucleotides withsuch binding capabilities known as aptamers have been usedcoupled with DNA-AgNC to develop protein sensors Thosesensors have some advantages with respect to previouslyreported [44] since a covalent substitution with a reportermolecule (dye electrochemical etc) is not necessary makingthe process more efficient and cost effective

The first example of a protein sensor using aptamerfunctionalized DNA-AgNC was reported by Martinez andcoworkers [45] In this example a DNA probe composed of athrombin aptamer and a C rich fragment was employed todetect such protein with a detection limit of 1 nM Due tothe silver binding capability of cytosines fluorescent AgNCcould be generated selectively on the C rich fragment Thoseclusters showed good photostability and stability in bioassayconditions but in the presence of thrombin the fluorescencewas quenched This system showed a good specificity asdeduced by the results obtained with other four proteins used

Thrombin

Aptamer 1 Aptamer 2

Hybridization region

G rich region

AgNC AgAg

Ag

Figure 3 Principle of binding-induced fluorescence turn-on assayfor protein detection [46] After aptamerrsquos binding to human 120572-thrombin the hybridization of complementary sequences takesplace bringing together the G rich fragments and AgNCs Thisinteraction leads to an enhancement of the fluorescence of AgNCs

as controls where any quenching effect was detected Themechanism of quenching was also studied and attributed to achange in the structure of AgNC induced by protein bindingto the aptamer

The same protein was also detected using a turn-onstrategy In this case the authors manage to detect thrombinat 1 nM by the fluorescence amplification of AgNC when a Grich protein is in close proximity [46] The design involvestwo different aptamers modified with two complementarysequences In addition one of them has a terminal fragmentable to form AgNC and the other one has a terminal Grich fragment Once the two aptamers bind the thrombinthe complementary sequences are close enough to hybridizeapproaching theG rich fragment toAgNCAs a consequencethe fluorescence is turned from a weak green emission tohighly red one (Figure 3) The specificity of the method wastested comparing the target with other four proteins Inall cases the fluorescence enhancement was lower comparedwith the thrombin even when the concentration of controlswas 10 times higher

Another original turn-on strategy based on fluorescentsilver nanoclusters was developed to detect the PDGF-BBprotein [47] a protein involved in some human cancersIn this case two-folded oligonucleotides are required Thefirst one is an aptamer which conformation changes uponbinding of its target This structural rearrangement leads tothe exposition of a domain of the aptamer which is ableto bind and unfold the other oligonucleotide Then theunfolded oligonucleotide is able to stabilize AgNCs afterthe addition of AgNO

3and NaBH

4(Figure 4) The limit of

detection of this protein using this approachwas 037 nMThesensor specificity was demonstrated using different proteinssuch as PDGF-AB a dimeric form with 60 of the aminoacids identical to the target Furthermore the sensor was ableto detect the protein in saliva samples at 5 nM concentration


PDGF-BB

Aptamer

Folded DNA

AgAg Ag

NaBH4

AgNO3

Figure 4 In the absence of PDGF-BB both aptamer and DNAprobes are folded [47] In this state fluorescent AgNCs cannotbe formed However in the presence of PDGF-BB the nucleationsequence is released and AgNCs can be formed By monitoring theincrease in fluorescence intensity the protein was detectedwith highsensitivity

CCRF-CEM cells

C rich sequence

A spacer Aptamer

AgAgAg

AgAgAgAgAgAg

AgNO3

Figure 5 Illustration of the preparation and use of an AgNC-aptamer as specific sensor for the detection of tumor cells [48 49]

without any interference showing the capability of the sensorto be used in real samples

5 Cell Labeling

Aptamers functionalized with silver nanocluster have beenalso commonly employed for surface cell labeling nucleistaining and tumor cell sensing due to their ease of synthesisspecific binding capability and photochemical properties

In 2011 Sun and coworkers used an aptamer-DNA-AgNCconjugate to stain specifically the nuclei of CCRF-CEM cells[48] The probe was synthesized by adding a C8 tail to 51015840end of the aptamer sgc8c followed by nanocluster generationThis aptamer was able to bind the human protein tyrosinekinase 7 (PTK7) presented in the membrane of CCRF-CEMcells internalize into the cells and finally reach the nucleias observed by fluorescence microscopy The specificity ofthis probe was demonstrated by the incubation of the sameaptamer with HeLa cells lacking the protein PTK7 In thiscase no detectable fluorescent signal was observed

In a similar report Yin and coworkers developed in2012 a sensor of CCRF-CEM cells using the sgc8c aptamerand a poly C tail connected by an A6 spacer (Figure 5)

(a)

(b)

(c)

Figure 6 Confocal laser scanning microscopy [50] of (a) MCF-7human breast cancer cells (b) NIH-3T3 mouse fibroblast cells and(c) MCF-10A human normal mammary epithelial cells incubatedwith aptamer-AgNC (1) bright-field images (2)AgNCsfluorescenceimages (red) Under the same procedure there were no detectableemissions from the control cells Reprinted with permission from Liet al [50] Copyright (2012) American Chemical Society

[49] They found that the fluorescence intensity and pho-tostability of DNA-AgNC generated using this spacer werebetter compared with others and much better than probeslacking a spacerThen the specific sensing capability of CEMcells was demonstrated using flow cytometry and confocalmicroscopy Furthermore the same strategy has allowed thedetection of Ramos cells just replacing the proper aptamersequence

MCF-7 nuclei were specifically detected using anotheraptamer-AgNC conjugate [50] In this case AS1411 nucleolinantiproliferative aptamer was conjugated to a poly C (12nt)through a 5T spacer The presence of this spacer was crucialto get high-emission intensities of the AgNC generatedThe specificity was demonstrated after the incubation ofthe aptamer-AgNC conjugate with MCF-7 NIH-3T3 mousefibroblast andMCF-10A human normalmammary epithelialcells Only in the case of MCF-7 cells fluorescence wasdetected due to the strong affinity of AS1411 by the nucleolinpresented in the membrane (Figure 6) Furthermore theantiproliferative function of AS1411 was retained as well andthe efficiency of growth inhibition unexpectedly improved


Proteins and antibodies have been also employed withsuccess for membrane cell targeting as Yu and coworkersreported in 2008 [51] In this work the authors synthe-sized an avidin DNA-AgNC conjugate using an orthogonalreaction between a thiolated DNA oligonucleotide and amaleimide group incorporated in the protein Then themodified oligonucleotidewas incubatedwith biotinylated livecells which showed a diffuse fluorescence and some silveraggregates which were not observed in nonbiotinylated cellsThis result evidenced the interaction between the avidin andbiotin signaled by the fluorescence of silver nanoclustersOn the other hand antibodies conjugated to DNA-AgNCwith heparin sulfate polysaccharide (SH receptor) affinitywere used to stain the cell membrane and nuclei of livecells Thus when the antibody conjugate was incubated at4∘C during 15min fluorescence was detected only in the cellmembrane due to the interaction of the antibody with the SHreceptor However after incubation at 37∘C the fluorescencewas centered in the nuclei suggesting an endocytosis processTherefore not only cell surface but internalization processescan be investigated using these materials

6 Conclusion and Outlook

Fluorescent properties ofDNA-AgNCare far to be completelyunderstood The influence of several parameters such asDNA sequence environment and structural changes on theemission of these materials represents a challenge to besolved In spite of this drawback the easy preparation and thelack of interferences with the probe targeting properties havepromoted the development of DNA-AgNC-based biosensorsThe use of DNA-AgNC for biosensing might suppose a con-siderable advantage since fluorescent and electrochemicaltags as well as the use of nanoparticles can be avoidedIn addition the sensibility and specificity of the biosensorsreported open the door to further investigations whichmightrender the discovery of optimized or new sensors withgeneral applications

Acknowledgments

Spanish Ministry of Science and Innovation (Grant SAF2010-15440) and IMDEA Nanociencia are acknowledged forfinancial support

References

[1] H Chang Y Wang and J Li ldquoElectrochemical DNA sensorsfrom nanoconstruction to biosensingrdquo Current Organic Chem-istry vol 15 no 4 pp 506ndash517 2011

[2] A A Gorodetsky M C Buzzeo and J K Barton ldquoDNA-mediated electrochemistryrdquo Bioconjugate Chemistry vol 19 no12 pp 2285ndash2296 2008

[3] L L Li P Ge P R Selvin and Y Lu ldquoDirect detection ofadenosine in undiluted serum using a luminescent aptamersensor attached to a terbium complexrdquo Analatical Chemistryvol 84 no 18 pp 7852ndash7856 2012

[4] H Huang Y Tan J Shi G Liang and J Zhu ldquoDNA aptasensorfor the detection of ATP based on quantum dots electrochemi-luminescencerdquo Nanoscale vol 2 no 4 pp 606ndash612 2010

[5] J Spadavecchia A Barras J Lyskawa et al ldquoApproach forplasmonic based DNA sensing amplification of the wavelengthshift and simultaneous detection of the plasmon modes of goldnanostructuresrdquo Analytical Chemistry vol 85 no 6 pp 3288ndash3296 2013

[6] G Pelossof R Tel-Vered X Liu and I Willner ldquoAmplifiedsurface plasmon resonance based DNA biosensors aptasen-sors and Hg2+ sensors using heminG-quadruplexes and Aunanoparticlesrdquo Chemistry vol 17 no 32 pp 8904ndash8912 2011

[7] L Wang J Li S Song D Li and C Fan ldquoBiomolecular sensingvia coupling DNA-based recognition with gold nanoparticlesrdquoJournal of Physics D vol 42 no 20 Article ID 203001 2009

[8] D Huang C Niu X Wang X Lu and G Zeng ldquolsquoTurn-onrsquo fluorescent sensor for Hg2+ based on single-strandedDNA functionalized Mn CdSZnS quantum dots and goldnanoparticles by time-gated moderdquo Analytical Chemistry vol85 no 2 pp 1164ndash1170 2013

[9] T Tokunaga S Namiki K Yamada et al ldquoCell surface-anchored fluorescent aptamer sensor enables imaging of chem-ical transmitter dynamicsrdquo Journal of the American ChemistrySociety vol 134 no 23 pp 9561ndash9564 2012

[10] X J Xing X G Liu L Yue-He Q Y Luo H W Tang andD W Pang ldquoGraphene oxide based fluorescent aptasensorfor adenosine deaminase detection using adenosine as thesubstraterdquo Biosensors amp Bioelectronics vol 37 no 1 pp 61ndash672012

[11] A Latorre and A Somoza ldquoDNA-mediated silver nanoclusterssynthesis properties and applicationsrdquo ChemBioChem vol 13no 7 pp 951ndash958 2012

[12] B Han and E Wang ldquoDNA-templated fluorescent silver nan-oclustersrdquo Analytical and Bioanalytical Chemistry vol 402 no1 pp 129ndash138 2012

[13] J Zheng P R Nicovich and RM Dickson ldquoHighly fluorescentnoble-metal quantum dotsrdquo Annual Review of Physical Chem-istry vol 58 pp 409ndash431 2007

[14] J Sharma R C Rocha M L Phipps et al ldquoA DNA-templated fluorescent silver nanocluster with enhanced stabil-ityrdquo Nanoscale vol 4 no 14 pp 4107ndash4110 2012

[15] S Choi J Yu S A Patel Y Tzeng andRMDickson ldquoTailoringsilver nanodots for intracellular stainingrdquo Photochemical andPhotobiological Sciences vol 10 no 1 pp 109ndash115 2011

[16] B Sengupta C M Ritchie J G Buckman K R JohnsenP M Goodwin and J T Petty ldquoBase-directed formation offluorescent silver clustersrdquo Journal of Physical Chemistry C vol112 no 48 pp 18776ndash18782 2008

[17] H Mattoussi G Palui and H B Na ldquoLuminescent quantumdots as platforms for probing in vitro and in vivo biologicalprocessesrdquo Advanced Drug Delivery Reviews vol 64 no 2 pp138ndash166 2012

[18] F Wang W B Tan Y Zhang X Fan and M Wang ldquoLumi-nescent nanomaterials for biological labellingrdquoNanotechnologyvol 17 no 1 pp R1ndashR13 2006

[19] AWaggoner ldquoFluorescent labels for proteomics and genomicsrdquoCurrent Opinion in Chemical Biology vol 10 no 1 pp 62ndash662006

[20] C TWirges J TimperM Fischler et al ldquoControlled nucleationof DNA metallizationrdquo Angewandte Chemie vol 48 no 1 pp219ndash223 2009


[21] A Ono S Cao H Togashi et al ldquoSpecific interactions betweensilver(i) ions and cytosine-cytosine pairs in DNA duplexesrdquoChemical Communications no 39 pp 4825ndash4827 2008

[22] D A Megger and J Muller ldquoSilver(I)-mediated cytosine self-pairing is preferred over hoogsteen-type base pairs with theartificial nucleobase 13-dideaza-6-nitropurinerdquo NucleosidesNucleotides and Nucleic Acids vol 29 no 1 pp 27ndash38 2010

[23] B Sengupta K Springer J G Buckman et al ldquoDNA templatesfor fluorescent silver clusters and i-motif foldingrdquo Journal ofPhysical Chemistry C vol 113 no 45 pp 19518ndash19524 2009

[24] L Deng Z Zhou J Li T Li and S Dong ldquoFluorescentsilver nanoclusters in hybridized DNA duplexes for the turn-on detection of Hg2+ ionsrdquo Chemical Communications vol 47no 39 pp 11065ndash11067 2011

[25] W Chen G Lan and H Chang ldquoUse of fluorescent DNA-templated goldsilver nanoclusters for the detection of sulfideionsrdquo Analytical Chemistry vol 83 no 24 pp 9450ndash9455 2011

[26] G Lan W Chen and H Chang ldquoCharacterization and appli-cation to the detection of single-stranded DNA binding pro-tein of fluorescent DNA-templated coppersilver nanoclustersrdquoAnalyst vol 136 no 18 pp 3623ndash3628 2011

[27] G Lan W Chen and H Chang ldquoControl of synthesis andoptical properties of DNA templated silver nanoclusters byvarying DNA length and sequencerdquo RSC Advances vol 1 no5 pp 802ndash807 2011

[28] X Guo L Deng and JWang ldquoOligonucleotide-stabilized silvernanoclusters as fluorescent probes for sensitive detection ofhydroquinonerdquo RSC Advances vol 3 no 2 pp 401ndash407 2013

[29] S Han S Zhu Z Liu L Hu S Parveen and G XuldquoOligonucleotide-stabilized fluorescent silver nanoclusters forturn-on detection of melaminerdquo Biosensors and Bioelectronicsvol 36 no 1 pp 267ndash270 2012

[30] K Ma Q Cui Y Shao F Wu S Xu and G Liu ldquoEmissionmodulation of DNA-templated fluorescent silver nanoclustersby divalent magnesium ionrdquo Journal of Nanoscience and Nan-otechnology vol 12 no 2 pp 861ndash869 2012

[31] G Lan W Chen and H Chang ldquoOne-pot synthesis of fluores-cent oligonucleotide Ag nanoclusters for specific and sensitivedetection of DNArdquo Biosensors and Bioelectronics vol 26 no 5pp 2431ndash2435 2011

[32] H Yeh J Sharma J J Han J S Martinez and J H Werner ldquoADNA-silver nanocluster probe that fluoresces upon hybridiza-tionrdquo Nano Letters vol 10 no 8 pp 3106ndash3110 2010

[33] H C Yeh J Sharma I M Shih D M Vu J S Martinez andJ H Werner ldquoA fluorescence light-up Ag nanocluster probethat discriminates single-nucleotide variants by emission colorrdquoJournal of the American Chemical Society vol 134 no 28 pp11550ndash11558 2012

[34] W Guo J Yuan Q Dong and E Wang ldquoHighly sequence-dependent formation of fluorescent silver nanoclusters inhybridized DNA duplexes for single nucleotide mutation iden-tificationrdquo Journal of the AmericanChemical Society vol 132 no3 pp 932ndash934 2010

[35] J Lhomme J F Constant and M Demeunynck ldquoAbasic DNAstructure reactivity and recognitionrdquo Biopolymers vol 52 no2 pp 65ndash83 1999

[36] K Ma Y Shao Q Cui F Wu S Xu and G Liu ldquoBase-stacking-determined fluorescence emission of DNA abasic site-templated silver nanoclustersrdquo Langmuir vol 28 no 43 pp15313ndash15322 2012

[37] K Ma Q Cui G Liu F Wu S Xu and Y Shao ldquoDNA abasicsite-directed formation of fluorescent silver nanoclusters forselective nucleobase recognitionrdquo Nanotechnology vol 22 no30 Article ID 305502 2011

[38] C M Croce ldquoCauses and consequences of microRNA dysreg-ulation in cancerrdquo Nature Reviews Genetics vol 10 no 10 pp704ndash714 2009

[39] S W Yang and T Vosch ldquoRapid detection of microRNA by asilver nanocluster DNA proberdquo Analytical Chemistry vol 83no 18 pp 6935ndash6939 2011

[40] P Shah A Roslashrvig-Lund S B Chaabane et al ldquoDesign aspectsof bright red emissive silver nanoclustersDNA probes forMicroRNA detectionrdquo ACSNano vol 6 no 10 pp 8803ndash88142012

[41] W Xin-Ping Y Bin-Cheng W Ping and Y Bang-CeldquoHighly sensitive detection of microRNAs based on isothermalexponential amplification-assisted generation of catalytic G-quadruplexDNAzymerdquo Biosensors amp Bioelectronics vol 42 pp131ndash135 2013

[42] Y Zhang and C Zhang ldquoSensitive detection of microRNAwith isothermal amplification and a single-quantum-dot-basednanosensorrdquo Analytical Chemistry vol 84 no 1 pp 224ndash2312012

[43] Y-Q Liu M Zhang B C Yin and B C Ye ldquoAttomolarultrasensitive MicroRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal amplificationrdquo Analyti-cal Chemistry vol 84 no 12 pp 5165ndash5169 2012

[44] M Famulok and G Mayer ldquoAptamer modules as sensors anddetectorsrdquo Accounts of Chemical Research vol 44 no 12 pp1349ndash1358 2011

[45] J Sharma H Yeh H Yoo J H Werner and J S MartinezldquoSilver nanocluster aptamers in situ generation of intrinsicallyfluorescent recognition ligands for protein detectionrdquo ChemicalCommunications vol 47 no 8 pp 2294ndash2296 2011

[46] J Li X Zhong H Zhang X C Le and J J ZhuldquoBinding-induced fluorescence turn-on assay using aptamer-functionalized silver nanocluster DNA probesrdquo AnalyticalChemistry vol 84 no 12 pp 5170ndash5174 2012

[47] J J Liu X R Song Y W Wang A X Zheng G N Chen andH H Yang ldquoLabel-free and fluorescence turn-on aptasensorfor protein detection via target-induced silver nanoclustersformationrdquo Analytica Chimica Acta vol 749 pp 70ndash74 2012

[48] Z Sun Y Wang Y Wei et al ldquoAg cluster-aptamer hybridspecifically marking the nucleus of live cellsrdquo Chemical Com-munications vol 47 no 43 pp 11960ndash11962 2011

[49] J Yin X He K Wang et al ldquoOne-step engineering of silvernanoclusters-aptamer assemblies as luminescent labels to targettumor cellsrdquo Nanoscale vol 4 no 1 pp 110ndash112 2012

[50] J Li X Zhong F Cheng J Zhang L Jiang and J Zhu ldquoOne-pot synthesis of aptamer-functionalized silver nanoclusters forcell-type-specific imagingrdquo Analytical Chemistry vol 84 no 9pp 4140ndash4146 2012

[51] J Yu S Choi C I Richards YAntoku andRMDickson ldquoLivecell surface labelingwith fluorescent ag nanocluster conjugatesrdquoPhotochemistry and Photobiology vol 84 no 6 pp 1435ndash14392008

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CatalystsJournal of


G T

G T

G richsequence

AgNC Complementary

WT gene Mutated gene

AgAgAg

AgAgAg Ag

AgAg

AgAg

Ag

regionsequence

Figure 1 Chameleon Nanocluster Beacon [32 33] Nanoclustersinteract with different regions of the G fragment depending on themutated baseThe different final structures obtained lead to differentcolors

2 Gene Detection

DNA-AgNCs have been successfully applied in the detectionof sequences of DNA and RNAThemost common approachrequires the preparation of an oligonucleotidewith twodiffer-ent domains One of them has a sequence complementary tothe DNA target and other domain is able to bind silver ionswhich will be reduced to give rise to fluorescent clusters Inthis sense Chang and coworkers were able to detect with highselectivity and sensitivity a gene associated with tyrosinemiatype I disease [31] They found that a C12 sequence directlyconnected to the probe sequence was able to generatethe corresponding fluorescent AgNC which emission wasstrongly enhanced after hybridization with the target geneInterestingly the system was able to discriminate betweenthe mutated and wild type gene because the fluorescenceintensity for the mutated one was almost 9 times biggerthan the wild type This difference could be due to duplexstructures obtained after hybridization where the most rigidcould protect better the AgNC from quenching

In another strategy employed to detect single strandedDNA the sensor developed uses two different single strandscomplementary to the target One strand has a poly Cfragment required for the generation of AgNC and thesecond one has a guanine rich fragment Upon hybridiza-tion with the target both fragments get close leading toan enhancement and shifting of the fluorescence emissionThis system was named NanoCluster Beacon [32] due tothe similarities with classical molecular beacons Based onthis work the same authors reported the preparation of asensor able to distinguish between the three possible singlepoint mutation of a KRAS gene in clinical samples In thisnew probe named Chameleon NanoCluster Beacon [33]the nanoclusters interact with different regions of the Grich fragment depending on the gene mutation (Figure 1)As a consequence of these interactions different colors areobtained with enough wavelength separation to be observedby the naked eye under UV irradiation

DNA duplexes are also able to stabilize AgNC and havebeen used to develop DNA sensors In this regard Wangand coworkers used hybridized duplexes to detect singlepoint mutations in sickle cell disease [34] In this workthe authors designed a complementary probe sequence toa fragment of the corresponding gene bearing a C6 looptwo nucleotides away from themutation After hybridizationAgNCs were generated in the C6 loop due to the bindingaffinity of cytosine to silver ions They observed a strongyellow emission when a match sequence is used On theother hand fluorescence was negligible when a mismatchis present The effect of the distance between the mutationand the loop as well as the nature of the mismatch wasevaluated highlighting again the sensitivity of AgNC to theenvironment In this sense they found that the presence of amutation four nucleotides away from the loop does not affectthe emission parameters Also it was found that a T or Amismatch produced a higher quenching of the fluorescencethan the C or G

It has also been reported the use of DNA duplexes withabasic sites [35] where the nucleobases are missing leavingthe deoxyribose residue in the strand in the preparation ofAgNCs Shao and coworkers reported the high affinity ofsilver ions by the unpaired bases opposite to an abasic sitewhich are able to accommodate few Ag+ [36] This findingwas applied in the preparation of a sensor for the p53 genewhich is involved in cancer suppression processes [37] Aprobe complementary to a fragment of the correspondinggene with an abasic site opposite to the nucleotide respon-sible for the mutation was employed Hybridization exper-iments with the wild type fragment and the three possiblemutations and subsequent incubationreduction procedurewith Ag+ and NaBH

4gave rise to the corresponding silver

nanoclusters The emitters formed with the mutant bases(A G and T) opposite to the abasic site showed similarweak intensities However when the wild type fragment wasused an increase in fluorescence of 18-fold was observedindicating the selectivity of the process The differencesobserved are due to the affinity of the cytosine to silver whichis present in the wild type example

3 MicroRNA Detection

MicroRNAs (miRNAs) are single stranded ribonucleotidesof around 22 nucleotides long They can be found in plantsanimals and humans and are involved in gene silenceprocesses avoiding the expression of certain proteins Thedetection and quantification of those structures are of greatimportance since they are involved in the initiation as well asthe generation of cancers [38]

DNA-AgNC has been successfully used in miRNA detec-tion as demonstrated by Yang and Vosch [39] They wereable to detect the miR160 which is a miRNA that targets aplant gene involved in hormone signaling They designed aprobe composed of two parts a complementary sequence tothe miR160 (part A) and directly grafted another fragmentable to stabilized red emitting AgNC (part B) The incuba-tion of this probe with AgNO

3and subsequent reduction


miRNA


Ag AgAg

AgAgAg

probe





4 Protein Detection



Thrombin

Aptamer 1 Aptamer 2


G rich region

AgNC AgAg

Ag





3and NaBH




PDGF-BB

Aptamer

Folded DNA

AgAg Ag

NaBH4

AgNO3


CCRF-CEM cells

C rich sequence

A spacer Aptamer

AgAgAg

AgAgAgAgAgAg

AgNO3



5 Cell Labeling




(a)

(b)

(c)








Acknowledgments


References






























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


miRNA


Ag AgAg

AgAgAg

probe





4 Protein Detection



Thrombin

Aptamer 1 Aptamer 2


G rich region

AgNC AgAg

Ag





3and NaBH




PDGF-BB

Aptamer

Folded DNA

AgAg Ag

NaBH4

AgNO3


CCRF-CEM cells

C rich sequence

A spacer Aptamer

AgAgAg

AgAgAgAgAgAg

AgNO3



5 Cell Labeling




(a)

(b)

(c)








Acknowledgments


References






























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


PDGF-BB

Aptamer

Folded DNA

AgAg Ag

NaBH4

AgNO3


CCRF-CEM cells

C rich sequence

A spacer Aptamer

AgAgAg

AgAgAgAgAgAg

AgNO3



5 Cell Labeling




(a)

(b)

(c)








Acknowledgments


References






























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





Acknowledgments


References






























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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