reviewarticle dna nanobiosensors: an outlook on signal...

Review ArticleDNA Nanobiosensors An Outlook on Signal Readout Strategies

Arun Richard Chandrasekaran

Confer Health Inc Charlestown MA USA

Correspondence should be addressed to Arun Richard Chandrasekaran arunrichardnyuedu

Received 18 January 2017 Revised 1 May 2017 Accepted 9 May 2017 Published 30 May 2017

Academic Editor Jorge Perez-Juste

Copyright copy 2017 Arun Richard Chandrasekaran This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A suite of functionalities and structural versatility makes DNA an apt material for biosensing applications DNA-based biosensorsare cost-effective and sensitive and have the potential to be used as point-of-care diagnostic tools Along with robustness andbiocompatibility these sensors also provide multiple readout strategies Depending on the functionality of DNA-based biosensorsa variety of output strategies have been reported fluorescence- and FRET-based readout nanoparticle-based colorimetryspectroscopy-based techniques electrochemical signaling gel electrophoresis and atomic force microscopy

1 Introduction

Biosensing is an area of research that has garnered attentiondue to its importance in medical diagnostics biomolecularanalysis and studies involving molecular pathways A varietyof nanomaterials such as quantum dots gold and silvernanoparticles metallic nanowires and carbon nanotubeshave been developed for sensing applications [1ndash3] Thesematerials provide unique optical electronic chemical andmechanical properties and contribute to both robust sensingand convenient and sensitive readout strategies DNA is oneother material that has found applications in biosensingOver the past three decades DNA has become a versatilematerial for bottom-upnanofabrication of two- [4] and three-dimensional lattices [5] nanoscale objects [6] and complexwireframe structures [7] with applications in biology [8]medicine [9] and materials science [10] The structuralaspects of DNAnanostructures combinedwith their ability torespond to stimuli have led to the creation of cost-effectiveand sensitive biosensors that are functional under a widerange of biologically relevant temperatures and conditions[11ndash14] The recognition event in such biosensing approachescan be translated by specific readout strategies which arebriefly discussed in this article in the following categories (i)fluorescence (ii) FRET (iii) nanoparticle-based color change(iv) electrochemical signaling (v) gel electrophoresis (vi)atomic force microscopy (AFM) and (vii) surface-enhancedRaman spectroscopy (SERS)

2 DNA Nanostructures for Biosensing

The field of DNA nanotechnology has seen the develop-ment of dynamic DNA machines [15] and devices [16] thatrespond to chemical or environmental stimuli This conceptis extended to DNA-based biosensors that rely on specificrecognition events between a substrate and the target analyte(eg nucleic acid and protein detection) or programmedconformational changes (eg pH sensing) DNA nanostruc-tures have some advantages for being used in biosensingThe intermolecular recognition of rationally designed DNAsequences allows highly precise design and construction ofDNA nanostructures The nanoscale dimensions of thesestructures provide large surface-to-volume ratios thus result-ing in large signal changes on target binding DNA sequencescan now be synthesized in large quantities in a cost-effectivemanner and can be chemically modified to enhance theirfunctionality

A majority of nucleic acid-based biosensors involvehybridization of a DNA or RNA strand to its complement(Figure 1(a)) or a complementary region in a stem loop(Figure 1(b)) Such stem-loop configurations can also be usedto monitor global environmental changes such as changesin temperature by using a fluorophore-quencher pair onthe ends of the strands Biosensors for other stimuli suchas pH changes are based on structures that involve triplex[17] (Figure 1(c)) i-motif formation [18] (Figure 1(d)) orpoly-dA helix formation (Figure 1(e)) [19] all of which are

HindawiJournal of NanomaterialsVolume 2017 Article ID 2820619 9 pageshttpsdoiorg10115520172820619

2 Journal of Nanomaterials

+

Duplex formation

(a)

+

Stem-loop configuration

(b)

pH 5pH 8

Triplex formation

(c)

pH 5pH 8

i-motif formation(d)

HClNaOH

poly dA helix

(e)

G-quadruplex formation

K+Na+

(f)

Aptamer-based recognition

Ligand

(g)

+

+

Toehold-based strand displacement

(h)

Figure 1 Concepts used in biosensing platforms (a b) Sequence-specific DNA hybridization in a duplex and stem-loop configuration (c)triplex formation under acidic conditions (d) i-motif formation under acidic conditions by C-rich DNA strands (e) poly-dA helix formationunder acidic conditions by poly-A strands (f) G-quadruplex formation in the presence of K+ or Na+ (g) aptamer reconfiguration in thepresence of specific ligands and (h) toehold-based strand displacement

sequence-specific and occur under acidic conditions G-quadruplex formation is another sequence-specific confor-mational change that forms under specific ionic conditionssuch as the presence of K+ or Na+ [20] (Figure 1(f)) In addi-tion aptamer-based recognition has also been widely used inbiosensors (Figure 1(g)) These are single-stranded oligonu-cleotides that can bind with high affinity to ions (eg K+Hg2+ and Pb2+) small organic molecules (eg ATP aminoacids and vitamins) peptides proteins (eg thrombin andgrowth factors) and even whole cells or microorganisms(eg bacteria) [21 22] resulting in a secondary struc-ture formation Toehold-based strand displacement [23](Figure 1(h)) has also been used for target readouts andsignal amplification in many DNA-based biosensing plat-forms

21 Fluorescence-Based Conformational changes in a DNAnanostructure can be observed by tagging the componentDNA strands with a fluorophore-quencher pair For exampleif a fluorophore and quencher are attached to two ends ofa single strand forming a stem loop the fluorescence isquenched in the stem-loop configuration due to the closeproximity (lt10ndash100 A) of the fluorophore to the quencher(Figure 2(a) top) Change in the stem-loop configurationmoves the fluorophore away from the quencher thus increas-ing the fluorescence This strategy has been used in a DNA-based beacon for the detection of antibodies and proteins[24] This molecular switch was composed of a stem-loopsystem comprising a long strand that contained the loopand two short complementary strands with single-strandedtails The ends of these tails were modified to contain an

Journal of Nanomaterials 3

Quencher

Fluorophore

Quencher Fluorophore

Protein

Recognitionelement

Δ

(a)

Excitation

pH 73pH 5

Closed High FRET

Open state Low FRET

h]h]

(b)

Individual NPs(Red)

Aggregates(Blue)

Pb(II)

(c)

TargetDNA

Reporter

Avidin-HRP

Sens

ing

elem

ent

Tran

sduc

er

Sign

al p

roce

ssor

Analyte

Signaleminus

TMBRe

TMBOx

H2O2

H2O

(d)

OffOn

Detectors

Target

Off

On

Duplex DNA

+

minus

(e)

pH 82pH 56

i-binder

i-motif

CantileverDetector

Piezo

Laser

(f)

SpectographLaserSERS

Ramansubstrate Target biomolecule

+ATP

LaserSERS

Laser No SERS

h]

(g)

Figure 2 Readout strategies for DNA nanostructure-based biosensors (a) Fluorescence-based readout the example shown demonstratesthe detection of proteins resulting in stem-loop reconfiguration leading to a fluorescent signal [24] (b) FRET-based readout The exampleshows a DNA nanodevice containing a FRET pair on opposite ends of a nicked duplex The C-rich single-stranded extensions on either endof the duplex can form an i-motif at low pH resulting in a FRET signal [18] (c) NP-based color change nanoparticles aggregated via DNAstrands and a DNAzyme are blue presence or addition of Pb2+ ions causes cleavage of the DNAzyme resulting in nanoparticle disaggregationand a change in color to red [25] (d) Electrochemical readout DNA tetrahedra with single-stranded pendants can bind partially to targetDNA The remainder of the target DNA strand can bind a reporter strand that produces a HRP-based electrochemical readout [26] (e)Gel electrophoresis a DNA nanoswitch containing two single-stranded overhangs that are partially complementary to target DNA Bindingof target DNA to the two detectors causes the linear ldquooffrdquo state to change into a looped ldquoonrdquo state The two states of the nanoswitch migratedifferently on a gel thus providing a digital on-off signal [27] (f) AFM-based readout DNAorigami levers that containC-rich single-strandedextensions can act as pH sensors In acidic pH the single-stranded extensions on each half of the lever can form an intermolecular i-motifcausing a conformational change that can be visualized on an AFM [28] (g) SERS-based readout an ATP-binding aptamer is bound to asingle-stranded probe on a gold surfaceThe presence of ATP triggers conformational change of the aptamer causing it to dissociate from theprobe resulting in a loss of the SERS signal [29]

appropriate target-specific recognition element (eg digox-igenin) The stem region contained a fluorophorequencherpair and the fluorescent signal is quenched as long as thestem remains closed (Figure 2(a) bottom) Binding of adig-specific antibody to both recognition elements (bivalentbinding) pushes them away thus opening the stem region andin turn causing enhanced fluorescence This sensor was used

to detect a variety of antibodies and protein targets includingthe HIV biomarker anti-p17 antibody

Fluorescence-based biosensors have also been used forpH detection One such example involves C-rich DNAstrands containing a fluorophore on one end attached toa gold surface [30] At acidic pH the strands form anintramolecular i-motif bringing the fluorophore closer to the


gold surface and essentially quenching it At basic pH thesingle strands can bind to a complementary strand pushingthe fluorophore away from the gold surface thereby enhanc-ing the fluorescence A similar example used a graphenesurface instead of gold and worked on the basis of pH-dependent triplex formation [31] In addition solution-basedtriplex-forming nanoswitches have also been developed forpH detection [17] This switch was designed so that thefluorophore-quencher pair remains closer when the switchforms a triplex acting as an indicator of the pH rangeAnother example of fluorescence-based readout was DNA-tweezer nanostructures that were designed to contain restric-tion sites specific to endonucleases [32] The presence ofthese endonucleases causes cleavage of component strandsresulting in an increased fluorescent signal

22 FRET-Based Structural transitions in DNA nanostruc-tures have been analyzed using Fluorescence ResonanceEnergy Transfer (FRET) in which fluorescence signals aregenerated for molecular association and separation in the1ndash10 nm range (Figure 2(b) top) [33] One such example isa DNA nanomachine based on an intramolecular i-motifthat has been used as a pH sensor inside living cells [18]The basis of this machine was a conformational change froman open linear structure under physiological conditions (pH73 low FRET) to a closed triangular structure under acidicconditions (pH 50 high FRET) (Figure 2(b) bottom) Thisswitch was effective in pH ranges 55 to 68 and was usedto map spatial and temporal pH changes associated withendosome maturation in Drosophila hemocytes [18] as wellas inside a multicellular organism (Caenorhabditis elegans)[34] A similar strategy was also used to simultaneouslymap the pH gradients along two different but intersectingendocytic pathways inside the same cell [35] In anotherexample the edges of a DNA tetrahedron were designed tocontain dynamic sequences that are specific to adenosinetriphosphate (ATP) [36]These regions undergo a conforma-tional change in the presence of ATP and were used to detectintracellular ATP via a FRET signal

23 Nanoparticle-Based Metallic nanoparticles (NPs) havebeen shown to exhibit defined color changes between individ-ual nanoparticles and aggregated clusters [37 38] This char-acteristic has been used in colorimetric assays based onDNA-functionalized gold nanoparticles (AuNPs) that provide anoptical readout (visual color change) (Figure 2(c) top) Onesuch example is the DNAzyme-mediated self-assembly ofAuNPs that was used in the detection of Pb(II) ions [25] TheDNAzyme used in this case consists of an enzyme strand anda substrate strandThe substrate strand was extended on bothends with sequences complementary to the single strands onthe AuNPs Hybridization of these two DNA strands resultsin aggregation of the AuNPs (blue color) On addition ofPb(II) the enzyme strand catalyzes cleavage of the substratestrand resulting in disaggregation of the AuNPs therebyresulting in a color change to red (Figure 2(c) bottom) Thisvisual color change acts as an indicator of the presence orabsence of Pb(II) ions Another example that uses NP-basedvisual color change is a lateral flow nucleic acid biosensor for

detecting nucleic acid sequences [39] In this case AuNPswere modified to contain biotin-tagged DNA strands thatwere complementary to a target DNA strand Target-boundDNA-AuNP conjugates get accumulated on a streptavidincoated test line thus resulting in a color change (red) of thetest line

24 Electrochemical Readout DNA-based electrochemicalsensors use nanoscale interactions between the target and arecognition layer and the signal is transduced (eg by enzymeactivity) via a solid electrode surface (Figure 2(d) top) DNAtetrahedron-based biosensors combined with surface-basedassays have been used for electrochemical detection of nucleicacids [26] In this design the bottom three vertices of thetetrahedral DNA probe were bound to a gold electrode sur-face via thiol modifications The fourth vertex was designedto contain a DNA strand that is complementary to part ofthe target (Figure 2(d) bottom) When part of the targetbinds to the probe a biotinylated reporter probe binds tothe remaining part of the target This hybridization eventis then transduced into electrochemical signals through thespecific binding of an avidin-HRP (horseradish peroxidase)conjugate to the biotin leading to enzyme turnover-basedsignal transduction A similar strategy combined withmulti-branched hybridization chain reaction (mHCR) for improvedsensitivity was used for cancer cell detection [40] Moreoverby conjugating the DNA tetrahedral probe to an antibody(eg tumor necrosis factor alpha) the strategy has beenredesigned for immunological sensing as well [41] In anotherexample electrochemical sensing ofHIVDNAhas been doneusing long-range self-assembled DNA constructs [42] In thiscase the output signal was based on the accumulation of hex-aammineruthenium(III) chloride (RuHex) on the negativelycharged phosphate backbone of the DNA via electrostaticforces

Electrochemical aptamer-based sensors have been devel-oped for the detection of proteins small molecules andinorganic ions [43] In this case an aptamer probe containingan electrochemical redox reporter molecule is attached ontoa gold electrode Target binding induces a conformationalchange of the aptamer thus altering the position of thereporter relative to the electrode yielding a measurable cur-rent change Such a strategy has been used to construct a real-time biosensor capable of continuously tracking doxorubicin(a chemotherapeutic) and kanamycin (an antibiotic) in liverats and in humanwhole blood [44 45]This strategy has alsobeen used to detect specific proteins [46] and antibodies [47]directly in undiluted blood serum

25 Gel Electrophoresis-Based Gel electrophoresis is themost ubiquitous technique in a biology or biochemicallaboratory (Figure 2(e) top) DNA nanoswitches have beendesigned for analysis of biomolecular interactions such asbiotin-streptavidin antibody-antigen peptide ligation andrestriction enzyme cleavage [48] These events result in aconformational change of the nanoswitch that can be ana-lyzed through gel electrophoresis This strategy was recentlyadapted for the detection of specific nucleic acid sequences[27]The off state of the nanoswitch is a linear duplex formed


by a single-stranded M13 scaffold and a set of staple strandsTwo of the staple strands were modified to contain single-stranded extensions (detectors) each of which binds to partsof the target Hybridization of the target oligonucleotideto the detectors reconfigures the switch to form a loopthus changing it to the on state (Figure 2(e) bottom) Theldquooffrdquo and ldquoonrdquo states of the DNA nanoswitches migratedifferently on an agarose gel Gel-shift assays are routinelyused in laboratories and this strategy provides a relativelyeasy and one-step method to detect target nucleic acids bythe appearance of the ldquoonrdquo band

26 AFM-Based Atomic force microscopy (AFM) is fre-quently used to analyze two-dimensional constructs madefrom DNA (Figure 2(f) top) The DNA origami technique[49] provides a convenient route to the assembly of suchtwo-dimensional platforms that allow the arrangement offunctional moieties For example DNA origami sheets con-taining single-stranded DNA probes complementary to atarget sequence can act as molecular chips for detecting thepresence of the target oligonucleotide [50] Hybridizationof the probe tiles to the target in solution was detectedusing AFM based on the difference in elastic propertiesof single-stranded (probes without target) and double-stranded DNA (probes bound to target) AFM readout ismore pronounced in structures that change conformation orlead to a visual marker on target interaction In one suchexample DNA origami was used to construct a ldquonanoplierrdquocontaining C-rich sequences (i-binders) on each lever of theplier [28] Under acidic conditions these sequences forman intermolecular i-motif thereby bringing the two leverstogether (Figure 2(f) bottom) This structural transitioncan be visualized using AFM In another example DNAorigami tiles with specific topological markers were used todetect single nucleotide polymorphisms (SNPs) producing adirect visual readout of the target nucleotide contained inthe probe sequence [51] The platform contained graphicalrepresentations of the four nucleotides A T G and C andthe symbol containing the test nucleotide identity disappearsin the presence of the target

27 SERS-Based Surface-enhanced Raman scattering(SERS) a variation of standardRaman spectroscopy providesa significantly enhanced Raman signal through electromag-netic interaction between the analyte molecules and metalsurface [52 53] In typical SERS assays Raman reporters areattached to the surface of metallic nanoparticles (the SERSsubstrates) and covered by a protective shell (Figure 2(g)top) that prevents leaching out of the Raman reportersand improves water solubility and stability [54 55] In onesuch example an aptamer-based biosensor was designed todetect ATP using SERS [29]This study used malachite greenisothiocyanate as the Raman reporter which was sandwichedbetween a gold nanostar core and a silica shell The aptamerprobe specific to ATP was immobilized on a gold surface byhybridization to complementary single-stranded DNA that isattached to the gold surface (Figure 2(g) bottom) Binding ofATP to the aptamer causes it to fold thereby detaching fromits complementary strand This duplex dissociation causes

a reduction in the SERS signal thus acting as a detectionmechanism for ATP molecules

Spatial control of plasmonic nanoparticles using rigidDNAnanostructures allows the creation of distinct structure-dependent optical features [56] One such example is the useof a DNA tetrahedron to control the positioning of AuNPs[57] By using thiol-modified DNA strands that can self-assemble into a tetrahedron the structure can be used torecruit 20 nm AuNPs on each of its four vertices In additionthe DNA tetrahedron was designed to contain Cy3 moleculea Raman active dye on one of its edges This structure wasfurther coated with silver to form Ag-Au nanoshells Thesystem was used to detect single-stranded DNA that wascomplementary to the component strands of the tetrahedronTarget addition causes formation of duplexes by hybridiza-tion of the component strands with the target resulting indisassembly of the NP cluster therefore causing a differentSERS signal for the duplexes Another example is a DNAorigami platform that was used to assemble 40 nm AuNPdimers with sub-5 nm gaps between them [58] The origamiplatform provides a strong plasmonic coupling between theNPs and this systemwas used to attain SERSmeasurements ofspecific single-stranded DNA molecules Depending on thesequences of the single-stranded DNA that is coated on thesurface of the NP dimers specific SERS spectral peaks areattained that can be used to detect or identify specific DNAsequences

3 Discussion and Outlook

Multidimensional DNA nanostructures have been shown tobe useful as frameworks for precisely programmed arrange-ment of functional molecules such as ligands enzymes andchemical groups [59 60] These strategies involve sequence-specific recognition of a DNA nanostructure [61] or covalentlinkage of a functional moiety on the DNA strand [62] Suchsite-specific positioning of biomolecules allows these struc-tures to be used as biosensing platforms for a variety of targetanalytes Moreover the ability to design triggered responsesto a variety of external chemical and biological stimuli makesDNA-based devices versatile for biosensing Such stimuliresponsive structures can be not only used as biosensors butalso configured to react to specific biomarkers and releasecargos from macromolecular containers [63] For example arecent enzyme-powered DNA-AuNP nanomachine was usedto release payloads while also serving as a biosensor fornucleic acid detection [64] Such nanostructures that can actaccording to stimuli can be used as ldquosense-and-treatrdquo devicesfor theranostic applications [65] Sensing capabilities can alsobe combinedwith computing platforms for this purpose [66]

The dynamic nature of such programmable DNA devicesplays a major role in the development of robust and sensitivemolecular sensing that is functional at the nanoscale whileproviding a convenient signal readout The potential useof a specific biosensing strategy is reliant on factors suchas assayreadout time skill required to perform the assaythe amount of sample required and the dynamic range andsensitivity and the cost of the method Considering thesefactors the use of AFM potentially limits the practicality


of such biosensors due to the equipment cost and therequirement of skilled personnel Furthermore AFM canonly be used to read out surface-based assays or thoseinvolving 2D DNA nanostructures and requires visualizingmultiple fields of the sample (usually deposited on a micasurface) to yield quantitative results The gel-based readoutsuch as the one using DNA nanoswitches provides a simpleassay for research laboratories to identify the presence of atarget nucleic acid without requiringmultiplemixing steps orenzymatic amplification This method is currently limited tolaboratory usage but can be extended to point-of-care testingby using bufferless gel systems and portable electrophoresisunits Moreover the cost of gel-based assays is much cheaperas it only requires already existing equipment in a labNanoparticle-based assays both optical and SERS have beenvery successful in developing point-of-care diagnostics witha relatively easier assay and quicker readout One notableplatform is the lateral flow assay which has been frequentlyused in clinical diagnostics with a simple visual readout (acolored test line) compared to a standard (a control line)These tests do not require any equipment and provide the enduser with a ldquoyes or nordquo answer

One limitation of current biosensing strategies is theuse of multiple steps for signal generation and amplificationwhich increases the time required for detection In additionDNA nanostructures used for sensing purposes have towithstand the different solution conditions while being intactduring detection of biomarkers in vivo [67] Specifically thesestructures are prone to degradation by nucleases in biologicalsystems Recent chemical strategies that provide a solution tothis problem include the use of a phosphorothioate backbone[68] locked nucleic acids (LNA) [69] L-DNA [70] 5101584031015840modifications including hexaethylene glycol (HEG) hexanediol (C6) and 51015840-phosphate (P) [71] and other xenonucleicacids [72] Previous research has shownDNA nanostructuresto be stable in cell lysates [73] and the integrity of thesestructures in tissue culture environment has been analyzed[74] It would be useful to analyze the stability of thesestructures in a variety of environments (eg different celltypes normal versus tumorous tissues) so that the biosensorscan be tailored for optimal responses Future work on DNA-based chemical and biological sensors will especially aid inthe characterization and development of these structures forin situ sensing With recent developments in DNA-PAINT(a variation of point accumulation for imaging in nanoscaletopography) [75 76] it is possible to create DNA nanostruc-tures that can signal the presence of specific biomarkers invivo Other recent developments in this front include DNAnanothermometers based on DNA clamp architectures thatare useful for temperatures in the range of 30∘C to 85∘C [77]In addition DNA origami structures have been combinedwith solid-state nanopores for detection of 120582-DNAmolecules[78] opening up a new route to single molecule detection ofbiomolecules

Self-assembly techniques especially DNA origami havemade the construction of nanoscale objects easier In addi-tion the cost involved in the preparation of DNA nanos-tructures has reduced in recent times with synthetic oligonu-cleotides being able to be simply ordered from a company

Recent research has shown that the cost of synthetic DNAcan be reduced further to as low as $0001 per base pair[79] Moreover custom-tailored DNA scaffolds now allowthe construction of DNA origami structures of different sizesand are not limited to the frequently used M13 single strand[80] With the aid of suitable purification methods [81ndash85]these nanostructures can be prepared in pure forms thatprovide enhanced sensitivity DNA being a biomolecule alsoprovides an advantage of being biocompatible [86] and canbe useful for biosensing in combination with biomimeticapproaches Thus designed DNA architectures provide aroute to the creation of highly sensitive biosensors withminimal cost and high assembly efficiency with a selectionof output strategies for varying purposes

Conflicts of Interest

The author declares that there are no conflicts of interestregarding the publication of this paper

References

[1] R T Ahuja and D Kumar ldquoRecent progress in the developmentof nano-structured conducting polymersnanocomposites forsensor applicationsrdquo Sens Actuators B vol 136 pp 275ndash2862009

[2] J Lei andH Ju ldquoSignal amplification using functional nanoma-terials for biosensingrdquo Chem Soc Rev vol 41 pp 2122ndash21342012

[3] Y Zhang Y Guo Y Xianyu W Chen Y Zhao and X JiangldquoNanomaterials for ultrasensitive protein detectionrdquo AdvMater vol 25 pp 3802ndash3819 2013

[4] W Wang T Lin S Zhang T Bai Y Mi and B Wei ldquoSelf-assembly of fully addressable DNA nanostructures from doublecrossover tilesrdquoNucleic Acids Research vol 44 no 16 pp 7989ndash7996 2016

[5] J Zheng J J Birktoft Y Chen et al ldquoFrom molecular tomacroscopic via the rational design of a self-assembled 3DDNAcrystalrdquo Nature vol 461 pp 74ndash77 2009

[6] D Bhatia S Arumugam M Nasilowski et al ldquoQuantum dot-loadedmonofunctionalized DNA icosahedra for single-particletracking of endocytic pathwaysrdquoNat Nanotech vol 11 pp 1112ndash1119 2016

[7] F Zhang S Jiang S Wu et al ldquoComplex wireframe DNAorigami nanostructures with multi-arm junction verticesrdquo NatNanotech vol 10 pp 779ndash784 2015

[8] A Czogalla H G Franquelim and P Schwille ldquoDNA Nanos-tructures on Membranes as Tools for Synthetic Biologyrdquo Bio-physical Journal vol 110 pp 1698ndash1707 2016

[9] R Chhabra J Sharma Y Liu S Rinker and H Yan ldquoDNA self-assembly for nanomedicinerdquo Adv Drug Deliv Rev vol 62 pp617ndash625 2010

[10] C M Niemeyer ldquoNanoparticles proteins and nucleic acidsbiotechnology meets materials sciencerdquo Angewandte ChemieInternational Edition vol 40 no 22 pp 4128ndash4158 2001

[11] J Chao D Zhu Y Zhang L Wang and C Fan ldquoDNA nano-technology-enabled biosensorsrdquo Biosens Bioelectron vol 76 pp68ndash79 2016


[12] H M Meng H Liu H Kuai R Peng L Moa and X BZhang ldquoAptamer-integrated DNA nanostructures for biosens-ing bioimaging and cancer therapyrdquoChem Soc Rev vol 45 no9 pp 2583ndash2602 2016

[13] A R Chandrasekaran H Wady and H K K SubramanianldquoNucleic acid nanostructures for chemical and biological sens-ingrdquo Small vol 12 pp 2689ndash2700 2016

[14] H Pei X Zuo D Pan J Shi Q Huang and C Fan ldquoScaffoldedbiosensors with designed DNA nanostructuresrdquo NPG AsiaMaterials vol 5 p e51 2013

[15] J Bath and A J Turberfield ldquoDNA nanomachinesrdquo Nat Nan-otech vol 2 pp 275ndash284 2007

[16] F C Simmel andWU Dittmer ldquoDNAnanodevicesrdquo Small vol1 pp 284ndash299 2005

[17] A Idili A Vallee-Belisle and F Ricci ldquoProgrammable pH-triggered DNA nanoswitchesrdquo J Am Chem Soc vol 136 pp5836ndash5839 2014

[18] S Modi M G Swetha D Goswami G D Gupta S Mayorand Y Krishnan ldquoA DNA nanomachine that maps spatial andtemporal pH changes inside living cellsrdquo Nature Nanotech vol4 pp 325ndash330 2009

[19] S Chakraborty S Sharma P K Maiti and Y Krishnan ldquoThepoly dA helix a new structural motif for high performanceDNA-based molecular switchesrdquo Nucl Acids Res vol 37 pp2810ndash2817 2009

[20] S Burge G N Parkinson P Hazel A K Todd and S NeidleldquoQuadruplex DNA sequence topology and structurerdquo NuclAcids Res vol 34 pp 5402ndash5415 2006

[21] S Song L Wang J Li C Fan and J Zhao ldquoAptamer-basedbiosensorsrdquo Trends Anal Chem vol 27 pp 108ndash117 2008

[22] S Tombelli M Minunni and M Mascini ldquoAnalytical applica-tions of aptamersrdquo Biosens Bioelectron vol 20 pp 2424ndash24342005

[23] B Yurke A J Turberfield A P Mills Jr F C Simmel and J LNeumann ldquoA DNA-fuelled molecular machine made of DNArdquoNature vol 406 pp 605ndash608 2000

[24] S Ranallo M Rossetti K W Plaxco A Vallee-Belisle and FRicci ldquoA modular DNA-based beacon for single-step fluores-cence detection of antibodies and other proteinsrdquoAngew ChemInt vol 54 pp 13214ndash13218 2015

[25] J Liu and Y Lu ldquoA colorimetric lead biosensor usingDNAzyme-directed assembly of gold nanoparticlesrdquo J AmChem Soc vol 125 pp 6642-6643 2003

[26] H Pei N Lu Y Wen et al ldquoA DNA nanostructure-based biomolecular probe carrier platform for electrochemicalbiosensingrdquo Adv Mater vol 22 pp 4754ndash4758 2010

[27] A R Chandrasekaran J Zavala and K Halvorsen ldquoPro-grammable DNA Nanoswitches for Detection of Nucleic AcidSequencesrdquo ACS Sensors vol 1 no 2 pp 120ndash123 2015

[28] AKuzuya RWatanabe Y Yamanaka T TamakiMKaino andY Ohya ldquoNanomechanical DNA origami pH sensorsrdquo Sensorsvol 14 pp 19329ndash19335 2014

[29] M Li J Zhang S Suri L J Sooter D Ma and N Wu ldquoDetec-tion of adenosine triphosphatewith an aptamer biosensor basedon surface-enhancedRaman scatteringrdquoAnal Chem vol 84 pp2837ndash2842 2012

[30] D Liu A Bruckbauer C Abell et al ldquoA reversible pH-drivenDNA nanoswitch arrayrdquo J Am Chem Soc vol 128 pp 2067ndash2071 2006

[31] X M Li J Song T Cheng and P Y Fu ldquoA duplex-triplexnucleic acid nanomachine that probes pH changes inside living

cells during apoptosisrdquoAnal Bioanal Chem vol 405 pp 5993ndash5999 2013

[32] X Y Li J Huang H X Jiang Y C Du G M Hana and D MKong ldquoMolecular logic gates based on DNA tweezers respon-sive to multiplex restriction endonucleasesrdquo RSC Advances vol6 no 44 pp 38315ndash38320 2016

[33] E A Jares-Erijman and T M Jovin ldquoFRET imagingrdquo NatBiotech vol 21 pp 1387ndash1395 2003

[34] S Surana J M Bhat S P Koushika and Y Krishnan ldquoAnautonomous DNA nanomachine maps spatiotemporal pHchanges in a multicellular living organismrdquo Nat Commun vol2 p 340 2011

[35] S Modi C Nizak S Surana S Halder and Y Krishnan ldquoTwoDNA nanomachines map pH changes along intersecting endo-cytic pathways inside the same cellrdquo Nat Nanotech vol 8 pp459ndash467 2013

[36] H Pei L Liang G Yao J Li Q Huang and C Fan ldquoRecon-figurable three-dimensional DNA nanostructures for the con-struction of intracellular logic sensorsrdquo Angew Chem Int vol51 pp 9020ndash9024 2012

[37] J Y Kim and J S Lee ldquoSynthesis and thermally reversibleassembly of DNAgold nanoparticle cluster conjugatesrdquo NanoLett vol 9 pp 4564ndash4569 2009

[38] R Elghanian J J Storhoff R C Mucic R L Letsinger and CA Mirkin ldquoSelective colorimetric detection of polynucleotidesbased on the distance-dependent optical properties of goldnanoparticlesrdquo Science vol 277 pp 1078ndash1081 1997

[39] P Lie J Liu Z Fang B Dun and L Zeng ldquoA lateral flowbiosensor for detection of nucleic acids with high sensitivity andselectivityrdquo Chemical Communications vol 48 no 2 pp 236ndash238 2012

[40] G Zhou M Lin P Song X Chen et al ldquoMultivalent cap-ture and detection of cancer cells with DNA nanostructuredbiosensors and multibranched hybridization chain reactionamplificationrdquo Anal Chem vol 86 pp 7843ndash7848 2014

[41] H Pei Y Wan J Li et al ldquoRegenerable electrochemicalimmunological sensing at DNA nanostructure-decorated goldsurfacesrdquo Chem Commun vol 47 pp 6254ndash6256 2011

[42] X Chen C Y Hong Y H Lin J H Chen G N Chenand H H Yang ldquoEnzyme-free and label-free ultrasensitiveelectrochemical detection of human immunodeficiency virusDNA in biological samples based on long-range self-assembledDNAnanostructuresrdquoAnal Chem vol 84 pp 8277ndash8283 2012

[43] A A Lubin and K W Plaxco ldquoFolding-based electrochemicalbiosensors the case for responsive nucleic acid architecturesrdquoAcc Chem Res vol 43 pp 496ndash505 2010

[44] H Li N Arroyo-Curras D Kang F Ricci and K W PlaxcoldquoDual-Reporter drift correction to enhance the performance ofelectrochemical aptamer-based sensors inwhole bloodrdquo Journalof the American Chemical Society vol 138 no 49 pp 15809ndash15812 2016

[45] B S Ferguson D A Hoggarth D Maliniak et al ldquoReal-timeaptamer-based tracking of circulating therapeutic agents inliving animalsrdquo Science TranslationalMedicine vol 5 Article ID213ra165 2013

[46] A J Bonham N G Paden F Ricci and K W PlaxcoldquoDetection of IP-10 protein marker in undiluted blood serumvia an electrochemical E-DNA scaffold sensorrdquoAnalyst vol 138pp 5580ndash5583 2013

[47] A Vallee-Belisle F Ricci T Uzawa F Xia and K W PlaxcoldquoBioelectrochemical switches for the quantitative detection of


antibodies directly in whole bloodrdquo J Am Chem Soc vol 134pp 15197ndash15200 2012

[48] M A Koussa K Halvorsen A Ward and W P Wong ldquoDNAnanoswitches a quantitative platform for gel-based biomolecu-lar interaction analysisrdquoNat Methods vol 12 pp 123ndash126 2015

[49] P W K Rothemund ldquoFolding DNA to create nanoscale shapesand patternsrdquo Nature vol 440 pp 297ndash302 2006

[50] Y Ke S Lindsay Y Chang Y Liu and H Yan ldquoSelf-assem-bled water-soluble nucleic acid probe tiles for label-free RNAhybridization assaysrdquo Science vol 319 pp 180ndash183 2008

[51] H K K Subramanian B Chakraborty R Sha and N CSeeman ldquoThe label-free unambiguous detection and symbolicdisplay of single nucleotide polymorphisms on DNA origamirdquoNano Lett vol 11 pp 910ndash913 2011

[52] K Kneipp YWangH Kneipp et al ldquoSinglemolecule detectionusing surface-enhanced raman scattering (SERS)rdquo Phys RevLett vol 78 pp 1667ndash1670 1997

[53] K L Kelly E Coronado L L Zhao and G C Schatz ldquoTheoptical properties of metal nanoparticles the influence of sizeshape and dielectric environmentrdquo J Phys Chem B vol 107no particles pp 668ndash677 2003

[54] B Kustner M Gellner M Schutz et al ldquoSERS labels forred laser excitation silica-encapsulated SAMs on tunablegoldsilver nanoshellsrdquoAngew Chem Int vol 48 pp 1950ndash19532009

[55] W E Doering and S Nie ldquoSpectroscopic tags using dye-embedded nanoparticles and surface-enhanced raman scatter-ingrdquo Anal Chem vol 75 pp 6171ndash6176 2003

[56] Y C Cao R Jin C S Thaxton and C A Mirkin ldquoA two-color-change nanoparticle-based method for DNA detectionrdquoTalanta vol 67 pp 449ndash455 2005

[57] J W Keum M Kim J M Park C Yoo N Huh and SC Park ldquoDNA-directed self-assembly of three-dimensionalplasmonic nanostructures for detection by surface-enhancedRaman scattering (SERS)rdquo Sensing and Bio-Sensing Researchvol 1 pp 21ndash25 2014

[58] V VThacker L O Herrmann D O Sigle et al ldquoDNA origamibased assembly of gold nanoparticle dimers for surface-enhanced Raman scatteringrdquoNat Commun vol 5 article 34482014

[59] O I Wilner and I Willner ldquoFunctionalized DNA nanostruc-turesrdquo Chem Rev vol 112 pp 2528ndash2556 2012

[60] A R Chandrasekaran ldquoProgrammable DNA scaffolds forspatially-ordered protein assemblyrdquoNanoscale vol 8 pp 4436ndash4446 2016

[61] D A Rusling A R Chandrasekaran Y P Ohayon et al ldquoFunc-tionalizing designer DNA crystals with a triple-helical veneerrdquoAngew Chem Int vol 53 pp 3979ndash3982 2014

[62] V Valsangkar A R Chandrasekaran R Wang et al ldquoClick-based functionalization of a 21015840-O-propargyl-modified branchedDNA nanostructurerdquo J Mater Chem B vol 5 no 11 pp 2074ndash2077 2017

[63] C H Lu and I Willner ldquoStimuli-responsive DNA-function-alized nano-microcontainers for switchable and controlledreleaserdquo Angew Chem Int vol 54 pp 12212ndash12235 2015

[64] X Yang Y Tang S D Mason J Chen and F Li ldquoEnzyme-powered three-dimensional DNA nanomachine for DNAwalk-ing payload release and biosensingrdquo ACS Nano vol 10 pp2324ndash2330 2016

[65] N Chen S Qin X Yang Q Wang J Huang and K WangldquoldquoSense-and-Treatrdquo DNAnanodevice for synergetic destruction

of circulating tumor cellsrdquo ACS Applied Materials amp Interfacesvol 8 no 40 pp 26552ndash26558 2016

[66] D LiW Cheng Y Li et al ldquoCatalytic hairpin assembly actuatedDNAnanotweezer for logic gate building and sensitive enzyme-free biosensing of microRNAsrdquo Analytical Chemistry vol 88no 15 pp 7500ndash7506 2016

[67] D S Lee H Qian C Y Tay and D T Leong ldquoCellularprocessing and destinies of artificial DNA nanostructuresrdquoChemical Society Reviews vol 45 no 15 pp 4199ndash4225 2016

[68] J J Fakhoury C K McLaughlin T W Edwardson J WConway andH F Sleiman ldquoDevelopment and characterizationof gene silencing DNA cagesrdquo Biomacromolecules vol 15 pp276ndash282 2014

[69] T Shimo K Tachibana K Saito et al ldquoDesign and evaluationof locked nucleic acid-based splice-switching oligonucleotidesin vitrordquo Nucl Acids Res pp 8174ndash8187 2014

[70] C Lin Y Ke Z Li J HWang Y Liu andH Yan ldquoMirror imageDNA nanostructures for chiral supramolecular assembliesrdquoNano Lett vol 9 pp 433ndash436 2009

[71] J W Conway C K McLaughlin K J Castor and H SleimanldquoDNAnanostructure serum stability greater than the sum of itspartsrdquo Chem Commun vol 49 pp 1172ndash1174 2013

[72] V B Pinheiro and P Holliger ldquoTowards XNA nanotechnologynewmaterials from synthetic genetic polymersrdquoTrends Biotech-nol vol 32 pp 321ndash328 2014

[73] QMei XWei F Su et al ldquoStability ofDNAorigami nanoarraysin cell lysaterdquo Nano Lett vol 11 pp 1477ndash1482 2011

[74] J Hahn S F J Wickham W M Shih and S D PerraultldquoAddressing the instability of DNA nanostructures in tissueculturerdquo ACS Nano vol 8 pp 8765ndash8775 2014

[75] C Lin R Jungmann A M Leifer et al ldquoSub-micrometer geo-metrically encoded fluorescent barcodes self-assembled fromDNArdquo Nat Chem vol 4 pp 832ndash839 2012

[76] B J Beliveau A N Boettiger M S Avendano et al ldquoSingle-molecule super-resolution imaging of chromosomes and in situhaplotype visualization using Oligopaint FISH probesrdquo NatCommun vol 6 article 7147 2015

[77] D Gareau A Desrosiers and A Vallee-Belisle ldquoProgrammablequantitative DNA nanothermometersrdquo Nano Lett vol 16 pp3976ndash3981 2016

[78] N A W Bell C R Engst M Ablay et al DNA OrigamiNanopores Nano Lett vol 12 pp 512ndash517 2012

[79] ANMarchi I Saaem BNVogen S Brown andTH LaBeanldquoToward larger DNAorigamirdquoNano Lett vol 14 pp 5740ndash57472014

[80] A R Chandrasekaran M Pushpanathan and K HalvorsenEvolution of DNA origami scaffolds Mat Lett vol 170 pp 221ndash224 2016

[81] C Lin S D Perrault M Kwak F Graf andWM Shih ldquoPurifi-cation of DNA-origami nanostructures by rate-zonal centrifu-gationrdquo Nucleic Acids Research vol 41 no 2 p e40 2013

[82] A Shaw E Benson and B Hogberg ldquoPurification of func-tionalized DNA origami nanostructuresrdquo ACS Nano vol 9 pp4968ndash4975 2015

[83] E Stahl T G Martin F Praetorius and H Dietz ldquoFacile andscalable preparation of pure and dense DNA origami solutionsrdquoAngew Chem Int vol 53 pp 12735ndash12740 2014

[84] G Bellot M A McClintock C Lin andWM Shih ldquoRecoveryof intact DNA nanostructures after agarose gel-based separa-tionrdquo Nat Methods vol 8 pp 192ndash194 2011


[85] K Halvorsen M Kizer X Wang A R Chandrasekaranand M Basanta Sanchez ldquoShear dependent LC purification ofan engineered DNA nanoswitch and implications for DNAorigamirdquo Anal Chem 2017

[86] A R Chandrasekaran N Anderson M Kizer K Halvorsenand X Wang ldquoBeyond the fold Emerging biological applica-tions of DNA origamirdquo ChemBioChem vol 17 pp 1081ndash10892016

Submit your manuscripts athttpswwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biomaterials


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CoatingsJournal of

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Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of



+

Duplex formation

(a)

+

Stem-loop configuration

(b)

pH 5pH 8

Triplex formation

(c)

pH 5pH 8

i-motif formation(d)

HClNaOH

poly dA helix

(e)

G-quadruplex formation

K+Na+

(f)

Aptamer-based recognition

Ligand

(g)

+

+

Toehold-based strand displacement

(h)

Figure 1 Concepts used in biosensing platforms (a b) Sequence-specific DNA hybridization in a duplex and stem-loop configuration (c)triplex formation under acidic conditions (d) i-motif formation under acidic conditions by C-rich DNA strands (e) poly-dA helix formationunder acidic conditions by poly-A strands (f) G-quadruplex formation in the presence of K+ or Na+ (g) aptamer reconfiguration in thepresence of specific ligands and (h) toehold-based strand displacement

sequence-specific and occur under acidic conditions G-quadruplex formation is another sequence-specific confor-mational change that forms under specific ionic conditionssuch as the presence of K+ or Na+ [20] (Figure 1(f)) In addi-tion aptamer-based recognition has also been widely used inbiosensors (Figure 1(g)) These are single-stranded oligonu-cleotides that can bind with high affinity to ions (eg K+Hg2+ and Pb2+) small organic molecules (eg ATP aminoacids and vitamins) peptides proteins (eg thrombin andgrowth factors) and even whole cells or microorganisms(eg bacteria) [21 22] resulting in a secondary struc-ture formation Toehold-based strand displacement [23](Figure 1(h)) has also been used for target readouts andsignal amplification in many DNA-based biosensing plat-forms

21 Fluorescence-Based Conformational changes in a DNAnanostructure can be observed by tagging the componentDNA strands with a fluorophore-quencher pair For exampleif a fluorophore and quencher are attached to two ends ofa single strand forming a stem loop the fluorescence isquenched in the stem-loop configuration due to the closeproximity (lt10ndash100 A) of the fluorophore to the quencher(Figure 2(a) top) Change in the stem-loop configurationmoves the fluorophore away from the quencher thus increas-ing the fluorescence This strategy has been used in a DNA-based beacon for the detection of antibodies and proteins[24] This molecular switch was composed of a stem-loopsystem comprising a long strand that contained the loopand two short complementary strands with single-strandedtails The ends of these tails were modified to contain an


Quencher

Fluorophore


Protein

Recognitionelement

Δ

(a)

Excitation

pH 73pH 5

Closed High FRET

Open state Low FRET

h]h]

(b)

Individual NPs(Red)

Aggregates(Blue)

Pb(II)

(c)

TargetDNA

Reporter

Avidin-HRP

Sens

ing

elem

ent

Tran

sduc

er

Sign

al p

roce

ssor

Analyte

Signaleminus

TMBRe

TMBOx

H2O2

H2O

(d)

OffOn

Detectors

Target

Off

On

Duplex DNA

+

minus

(e)

pH 82pH 56

i-binder

i-motif

CantileverDetector

Piezo

Laser

(f)



+ATP

LaserSERS

Laser No SERS

h]

(g)





























References




































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of



Quencher

Fluorophore


Protein

Recognitionelement

Δ

(a)

Excitation

pH 73pH 5

Closed High FRET

Open state Low FRET

h]h]

(b)

Individual NPs(Red)

Aggregates(Blue)

Pb(II)

(c)

TargetDNA

Reporter

Avidin-HRP

Sens

ing

elem

ent

Tran

sduc

er

Sign

al p

roce

ssor

Analyte

Signaleminus

TMBRe

TMBOx

H2O2

H2O

(d)

OffOn

Detectors

Target

Off

On

Duplex DNA

+

minus

(e)

pH 82pH 56

i-binder

i-motif

CantileverDetector

Piezo

Laser

(f)



+ATP

LaserSERS

Laser No SERS

h]

(g)





























References




































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


























References




































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


























































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


reviewarticle dna nanobiosensors: an outlook on signal...

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