Nucleic Acid Fluorescent Probes for Biological Sensing

focal point review

XIN SU,XIANJIN XIAO,CHEN ZHANG,MEIPING ZHAO BEIJING NATIONAL LABORATORY FOR MOLECULAR SCIENCES MOE KEY LABORATORY OF BIOORGANIC CHEMISTRY AND MOLECULAR ENGINEERING COLLEGE OF CHEMISTRY AND MOLECULAR ENGINEERING PEKING UNIVERSITY BEIJING 100871, CHINA Nucleic Acid Fluorescent Probes for Biological Sensing

Nucleic acid fluorescent probes are playing INTRODUCTION events (hybridization, target binding, or increasingly important roles in biological enzyme digestion) into fluorescence ucleic acid fluorescent probes sensing in recent years. In addition to the signals. Some fluorophores change their are oligonucleotides (DNA or conventional functions of single-stranded fluorescence properties when they are in DNA/RNA to hybridize with their comple- N RNA) modified by covalent close proximity to certain nucleobases. mentary strands, affinity nucleic acids (ap- attachment of fluorophores or both For example, most fluorophores can be tamers) with specific target binding fluorophores and quenchers. The unique properties have also been developed, which efficiently quenched by guanines scaffold of nucleic acids offers versatile through the photoinduced electron trans- has greatly broadened the application of molecular recognition capabilities. First, nucleic acid fluorescent probes to the detec- fer mechanism.14–16 Amorewidely tion of a large variety of analytes, including single-stranded DNA (ssDNA) or RNA employed signal transduction mecha- small molecules, proteins, ions, and even can be readily used to detect their nism is Fo¨rster (sometimes called fluo- whole cells. Another chemical property of complementary strands by hybridiza- 1,2 rescence) resonance energy transfer nucleic acids is to act as substrates for tion. In the past two decades, nucleic (FRET) between a donor and an accep- various nucleic acid enzymes. This property acids with target binding properties tor. In addition, pyrene has also been can be utilized not only to detect those (called aptamers), which show high enzymes and screen their inhibitors, but also used in nucleic acid fluorescent probes affinity and specificity to a diverse range as in close proximity an excited-state employed to develop effective signal amplifi- of analytes beyond DNA and RNA,3–5 cation systems, which implies extensive ap- pyrene monomer and a ground-state plications. This review mainly covers the have also been evolved. This has greatly pyrene monomer can form an excimer biosensing methods based on the above three broadened the application of nucleic that has a substantially longer emission types of nucleic acid fluorescent probes. The acid fluorescent probes to detect a large wavelength and longer fluorescence most widely used intensity-based biosensing variety of analytes, including small lifetime in comparison with the pyrene assays are covered first, including nucleic molecules, proteins, ions, and even monomer.17,18 acid probe-based signal amplification meth- intact cells. Another chemical property Fluorescence techniques have proved ods. Then fluorescence lifetime, fluorescence of nucleic acids is to act as substrates for to be valuable tools in the development anisotropy, and fluorescence correlation spec- various nucleic acid enzymes. This troscopy assays are introduced, respectively. of biosensors due to their distinct As a rapidly developing field, fluorescence property can be utilized not only to advantages in using the different prop- imaging approaches are also briefly summa- detect those enzymes and screen their erties of fluorophores. In addition to the rized. inhibitors,2,6,7 but also employed to most widely used fluorescence-intensity- develop powerful signal amplification based measurement, fluorescence life- systems, finding extensive applica- time and fluorescence anisotropy/polar- tions.8–13 In addition to these versatile ization are also excellent information functions of DNA structure, nucleic acid carriers. Additional techniques include fluorescent probes also benefit from fluorescence correlation spectroscopy Received 24 July 2012; accepted 13 August excellent water solubility and ease of and fluorescence imaging through the 2012. synthesis and modification. microscope. Moreover, nucleic acid * Author to whom correspondence should be sent. E-mail: [email protected]. The fluorescent labels act as trans- fluorescent probes have also proved to DOI: 10.1366/12-06803 ducers that transfer biorecognition be useful tools for signal amplification,

APPLIED SPECTROSCOPY 1249 focal point review enhancing the detection signals of both din-biotin interaction, the fluorescent Werner and co-workers found that the DNA targets and other target mole- sensor allows on-chip detection of red fluorescence of DNA–AgNCs can be cules.8–13 cocaine with high sensitivity and low enhanced 500-fold when placed in This review mainly covers the bio- sample consumption. Krull and co- proximity to guanine-rich DNA se- sensing methods based on various workers demonstrated a solid-phase quences.30 Based on this new phenom- nucleic acid fluorescent probes with strategy for the transduction of nucleic enon, the authors have designed a DNA different fluorescence transduction ap- acid hybridization using immobilized detection probe (NanoCluster Beacon, proaches. The most widely used inten- QDs and FRET in an electrokinetically NCB) that ‘‘lights up’’ upon target sity-based biosensing methods are controlled microfluidic chip.26 The as- binding. Since NCBs do not rely on covered first, including nucleic acid say enabled the detection of as little as 5 Fo¨rster energy transfer for quenching, probe-based signal amplification meth- fmol of target nucleic acids and the the assay achieved very high (.100) ods. Then fluorescence lifetime, fluores- assay time was reduced from hours to signal-to-background ratios (S/B ratios) cence anisotropy, and fluorescence minutes. It is noteworthy that a novel upon target binding. In this separation- correlation spectroscopy assays are in- spatial-based method was applied by the free assay, the authors demonstrate NCB troduced, respectively. As a rapidly authors for quantification of the target detection of an influenza target with an developing field, fluorescence imaging concentration by measurement of the S/B ratio of 175, a factor of five better approaches are also briefly summarized. spatial length of coverage by the target than a conventional molecular beacon along the microfluidic channel. The (MB) probe. DNA–AgNCs were also FLUORESCENCE-INTENSITY- channel length coverage is determined utilized for the development of turn-on BASED BIOSENSING from a point where the Cy3 acceptor aptamer sensors of small molecules Novel Fluorescent Labels for In- fluorescence has decayed to 50% of the through target-induced split probe com- 31 tensity Measurements. Many organic initial intensity for the tested concentra- bination. Moreover, DNA–AgNCs fluorophores have been widely used as tion. Such a quantitative transduction were developed for building a selective the labels for nucleic acid fluorescent approach may offer some advantages in ‘‘turn-off’’ indicator of metal ions based probes. However, these conventional terms of simplicity and high reproduc- on the fluorescence quenching of DNA– 32 fluorophores have some inherent defi- ibility in comparison with the conven- AgNCs by copper ions. ciencies, such as low quantum yield and tional QD-FRET assays based solely on Upconversion Nanomaterials. Up- poor photostability, which put limita- fluorescence intensities for quantifica- conversion is a process wherein low tions on their application in biological tion. energy light, usually near-infrared (NIR) studies.19,20 In recent years, a tremen- By employing the size-dependent or infrared (IR), is converted to higher dous number of novel fluorescent nano- emission property of QDs, Willner and energy light (visible) through multiple materials have been developed, which co-workers used QD-labeled aptamers to photon absorptions or energy trans- hold great promise in comparison to perform multiplex target analysis by fers.33 To overcome autofluorescence organic fluorophores in terms of bright- chemiluminescent resonance energy of biological tissues, two-photon fluo- ness, photostability, and size-tunable transfer (CRET).27 The self assembly rophores and upconversion nanomateri- fluorescence spectrum. of QD–aptamer and hemin/G-quadru- als were developed as promising Quantum Dots. Semiconductor quan- plex DNAzyme is beneficial for exclu- alternative fluorescent labels. Because tum dots (QDs) have a number of sion of chemiluminescent background the two-photon fluorophores usually advantages over conventional organic signals due to diffusional hemin and show poor aqueous solubility and low fluorophores, most notably a broad enables the performance of multiplex quantum yield, upconversion nanomate- absorption spectrum and narrow emis- analyses in variable sample composi- rials are more widely used for construc- sion peaks, allowing the simultaneous tions by using different sized QDs with a tion of nucleic acid fluorescent probes. excitation of different QDs at a single common internal light source. Up-converting rare-earth nanocrystals wavelength for multiplex detection.21,22 Silver Nanoclusters. To simplify the (UCNCs) has attracted much attention QDs also offer higher quantum yield, labeling process for nucleic acid probes, due to their unique luminescence prop- longer fluorescence lifetime, and greater a novel strategy has been developed erties, such as sharp absorption and photostability (up to 100 times) than using Ag nanoclusters (AgNCs). For the emission lines, high quantum yield, long organic fluorophores,23 allowing for building of DNA-templated AgNCs, lifetime, and superior photostability.34 single-molecule observation over an oligonucleotides serve as a scaffold The anti-Stokes emission and NIR- extended period of time.24 and the silver ions can be easily reduced excitation nature of UCNCs make them ZhangandJohnsondevelopeda by NaBH4 in situ to form stable nano- a promising energy donor for FRET single QD-based aptamer sensor for clusters.28,29 DNA-templated silver assays in complex biological samples. In cocaine detection25 by taking advantage nanoclusters (DNA/AgNCs) are superb comparison with downconversion fluo- of single-molecule detection and a bi- fluorescence emitters that offer facile rescent materials, upconversion materi- FRET system between 605QD/Cy5 and synthesis, outstanding spectral and pho- als have advantages in biological Iowa Black RQ. With the QD-labeled tophysical properties, high photostabili- applications, such as noninvasive and aptamers immobilized on the surface of ty, and smaller size than semiconductor deep penetration of NIR radiation, the a microfluidic chip by the streptavi- QDs. absence of autofluorescence of biologi-

1250 Volume 66, Number 11, 2012 cal tissues, and feasibility of multiple on FRET from upconverting phosphors conditions, the target labels can be labeling by UCNCs with different emis- to carbon nanoparticles.37 The high selected in a wide emission range. sions under the same excitation. Al- sensitivity of the sensor (0.18 nM) Moreover, the number of codes can be thoughtheUCNCsarealways allowed monitoring of thrombin levels increased because the code emission hydrophobic, there are some modifica- in human plasmon. A limitation of the range has been widened greatly. This tion methods to improve the solubility assay is the relatively long response time kind of novel barcode material can be such as encapsulation of hydrophobic (2 h). Stucky and co-workers developed used for rapid and sensitive analysis of nanocrystals24 and coating of amphi- a nucleic acid encoding system by using genome sequences. philic copolymers.35 Huang and co- fluorescent upconversion microbarco- Cationic Conjugated Polymers. Nu- workers developed a simple and versa- des.38 The emission of UCNCs ranging cleic acids are negatively charged poly- tile strategy for converting hydrophobic from green to red was easily controlled electrolytes at neutral pH. Cationic UCNCs into water-soluble and carbox- by adjusting the Ho3þ concentration conjugated polymers (CCPs) offer a ylic-acid-functionalized analogs by di- doped in the NaYF4 : Yb,Ho,Tm@ convenient tool to interface with nega- rectly oxidizing oleic acid ligands with NaYF4 core/shell nanocrystals. These tively charged DNA probes. CCPs the Lemieux-von Rudloff reagent.29,36 UCNCs offered a better platform for feature a delocalized electronic struc- These surface-modified UCNCs were encoding nucleic acids than organic ture. Excitation energy along the poly- successfully conjugated and applied to dyes and QDs. Because UCNCs have mer backbone can transfer to an acceptor DNA detection through FRET between no optical cross-interference between by electron transfer or FRET, resulting UCNCs and organic dyes. Pang et al. the upconversion optical code and any in the superquenching of polymers or developed an aptamer biosensor based reporter dyes under different excitation the amplification of the fluorescence

FIG.1. Chemical structures of (A) polyﬂuorene derivatives, (B) polythiophene derivatives, (C) LNA, (D) PNA, (E) 20-O-methyl nucleic acids, and (F) a tetra-pyrene ODF sensor for esterases [adapted from Refs. 40, 106, 107, 109, and 7].

APPLIED SPECTROSCOPY 1251 focal point review signal of the acceptor. CCPs can sensitively transduce the hybridization event between a single-stranded probe and target DNA to fluorescent signal by FRET.39,40 Two typical CCPs, poly- fluorene (Fig. 1A) and polythiophene (Fig. 1B) derivatives, initiated by the Bazan group41 and the Leclerc group,42 respectively, have been widely used in DNA sensing. Due to the ssDNA binding ability and the high quantum yield in aqueous medium, polyfluorenes have been successfully used to develop sensing approaches for detection of DNA methylation, DNA damage, single nucleotide polymorphism (SNP), and real-time monitoring of nucleases.40 Very recently, by modulating the interaction between CCP and the G-quadruplex-forming MB aptamer, Kim et al. developed a highly sensitive and selective assay for potassium ion detection against excess sodium ions in water.43 A detection limit of ~1.5 nM was achieved þ FIG.2. Schematic illustration of the principles for detection of mRNA and measurement of for the K assays in the presence of 100 nuclease’s activity by using DNA-AuNPs and nanoflare [adapted from Refs. 52 and 53]. mM Naþ ions, which is approximately three orders of magnitude lower than those reported previously. separated from the AuNP, resulting in through p–p interactions. Both of them Novel Quenchers Used for Intensity fluorescence emission. Melvin et al. have been used as effective quenchers Measurements. In the design of nucleic developed a fluorescent competitive for nucleic acid probes. acid fluorescent probes, additional assay for DNA identification based on In 2003, Zhang et al. reported that quenchers are necessary in most cases FRET between QDs and AuNPs. In the ssDNA may form stable complexes with to provide low background. Dimethyl- absence of the target sequence, the individual single-walled carbon nano- aminophenylazobenzoic acid (DABC- fluorescence of the CdSe QDs was tubes (SWNTs), wrapping around them YL) and Black Hole are commonly quenched by AuNPs assembled with by means of p-stacking interactions used quenchers for many fluoro- them through short cDNA strands. The between the nucleotide bases and the phores.1,44 However, these quenchers presence of the targeted complementary SWNT sidewalls.54 This close proximity share the common drawback of low oligonucleotides then displaced and results in complete quenching of the quenching efficiency when the distance released the AuNP from the QD-DNA, fluorophores labeled at the ssDNA between fluorophore and quencher is resulting in QD fluorescence restora- through energy or electron transfer.55,56 relatively large. Also, covalent attach- tion.48 Fan and his co-workers demon- Based on these properties of SWNTs, a ment of an organic quencher to nucleic strated multicolor fluorescent AuNP- series of sensing systems has been acid increases assay cost and the organic based MBs to detect multiple target developed for nucleic acid analysis55 structure is susceptible to cleavage by molecules.49 Mirkin et al. have fabricat- and protein recognition.57,58 nonspecific enzymes in biological applied a series of fluorescent sensors by Graphene oxides offer even better cations. using polyvalent DNA-functionalized fluorescence quenching properties than Gold Nanoparticles. Gold nanoparti- AuNPs and single-fluorophore-labeled SWNTs due to their planar structure.59 cles (AuNPs) are excellent fluorescence short oligonucleotides (called nano- Reduced graphene oxide (rGO) can quenchers for FRET-based assays due to flares) (Fig. 2). These sensors have been form a complex with acridine orange their extraordinarily high molar molar utilized for detection of intracellular (AO) and effectively quench the fluo- absorptivities and broad energy band- mRNA or DNA and measurement of rescence of AO, but the AO–rGO width.45 By labeling MBs with AuNPs46 the nuclease’s activity.50–53 complex can be reversibly destroyed or functionalizing AuNPs with fluoro- Carbon Nanomaterials. Carbon by a G-quadruplex structure aptamer phore-labeled single-stranded oligonu- nanotubes (CNTs) and graphene oxides (PS2.M). On the basis of this phenom- cleotide,47 the fluorophore is initially (GOs) contain highly delocalized p- enon, Li and co-workers designed a quenched by AuNP due to close donor electrons. Their surfaces can be easily DNA rGO-based fluorescent sensor for and acceptor distance. Upon binding functionalized with compounds that detection of hemin. Initially, PS2.M with the target DNA, the fluorophore is possess a p-electron-rich structure captures AO from rGO and gives out

1252 Volume 66, Number 11, 2012 strong fluorescence. Addition of hemin to the AO-PS2.M/rGO mixture results in specific binding of hemin with PS2.M and release of AO from PS2.M, which is subsequently quenched by rGO. The target hemin was detected sensitively and selectively, achieving a detection limit as low as 50 nM for hemin.60 Nucleic Acid Fluorescent Probe- Based Enzyme Biosensing. In addition to hybridization with complementary sequences and binding with specific targets, nucleic acids are also the substrates for various nucleic-acid-processing enzymes. Nucleic acid fluorescent probes can be utilized to detect these enzymes by coupling the enzymatic reaction with a signal transduction FIG.3. Singly labeled smart probes for real-time monitoring of the activity and kinetics of mechanism. With the typical stem-loop different DNA end-processing enzymes: (A) Exonuclease, (B) Polymerase, and (C) structure, MBs could be directly used to Polynucleotide kinase [adapted from Refs. 6, 61, and 63]. detect three different nucleases (S1 nuclease, DNase I, and mung bean 2 oligonucleotide probe with a fluoro- pendent amplification process that nuclease). The cleavage of the loop phore, the fluorescence was quenched substantially increases the sensitivity of sequence destabilizes the stem duplex by stacked guanines at the other end. DNA detection. Typically, upon binding and restores fluorescence. However, This free end base offers a flexible with the target DNA strands, the probe MBs cannot be directly used to react substrate for various DNA end-process- strands in the resultant probe–target with DNA end-processing enzymes ing enzymes. complexes are immediately recognized because of the hindrance of the two We have successfully detected the 50- and cleaved by a specific enzyme and labels at each end. For example, the 0 phosphorylation activity of T4 polynu- give out fluorescence signals. The re- labeled quencher at the 3 end of MB cleotide kinase,6 the DNA polymeriza- leased target DNA strands then quickly partially inhibits the hydrolytic reactions tion reaction,63 and DNA polymerase hybridize with another probe and pro- 61 of exonucleases. To study more so- fidelities64 with these smart probes. duce more fluorescence signals. This phisticated processes such as DNA Recently, we further improved the cycling process greatly enhances the ligation and phosphorylation, MBs are design of the probes and successfully yield of fluorescence signals per target used as the template to facilitate enzy- applied them for in situ, real-time molecule, resulting in a significant matic reactions and give out the fluores- monitoring of the 30-50 exonucleases increase of the sensitivity of DNA cent signals. secreted by living cells without the detection. There are a large number of DNA requirement for sample cleanup or Several different enzymatic reactions end-processing enzymes (such as poly- preconcentration.61 These singly labeled have been utilized to accomplish signal nucleotide kinases, phosphatases, exo- probes showed distinct low-cost advan- amplification (Fig. 4). Xie and co- nucleases, and polymerases) that play tages and are particularly suitable for workers used nicking endonuclease to decisive roles in biological processes high-throughput analysis of a large remove the molecular beacons bound to and have been considered to be attrac- variety of different enzymes from natu- the target DNA strand and achieved a tive targets for drug design and cancer ral and artificial libraries and screening detection limit of 20 pM. For further treatment.62 To establish a more generic of their inhibitors. enhancement of the sensitivity, they detection approach for these enzymes, Other DNA fluorescent probe-based coupled the nicking endonuclease am- our group proposed a new strategy for methods for the detection of enzyme plification with rolling circle amplifica- monitoring of the activity and kinetics of activities and related processes include tion and further reduced the detection different DNA end-processing enzymes the use of fluorescent natural base limit to 0.1 pM.8 Plaxco and co-workers in real time by using singly labeled analogs, multiple organic fluorophores, used exonuclease III (Exo III) to achieve smart probes (Fig. 3).6,61,63,64 These and affinity-based DNA fluorescent signal amplification. Exo III strongly probes take advantage of the excellent probes. Detailed designs and applica- prefers digestion of MBs bound to the electron-donating properties of the nat- tions of these probes have been exten- complementary strand, while the free ural base guanine, which can efficiently sively reviewed by Dai and Kool.7 MBs remain unaffected and may hy- quench the fluorescence of most of the Nucleic Acid Fluorescent Probes bridize with another target strand.9 The fluorescent dyes via the photo-induced for Signal Amplification. Nucleic acid method appears to be more generic than electron transfer mechanism. By label- fluorescent probe-based signal amplifi- the above-mentioned nicking endonu- ing one end of the self-complementary cation is an enzyme-driven target-de- clease signal amplification system,

APPLIED SPECTROSCOPY 1253 focal point review

high temperatures (50–60 8C) allows the method to selectively differentiate the signal of 1.0% perfectly matched target strand from single-base different sequences. Later, we further established a more fascinating method by making use of a unique property of the Endo IV and k exonuclease coupling system, in which the position of a mismatch in the probe DNA drastically affects the ampliﬁcation rates.12 A mismatch-directed selective ampliﬁcation system is developed for rapid detection of single- nucleotide polymorphisms and low- abundance mutations at physiological, isothermal conditions. The assay enables sensitive detection of 1.0 fmol of target strand and selective differentiation of 0.5% target strand from single-base different sequences at 37 8C without the need for temperature adjustment.

FLUORESCENCE-LIFETIME- BASED BIOSENSING FIG.4. Schematic principle of the enzymatic signal amplification system based on different enzymes. (A) Nicking enzyme. (B) Exo III. (C) DNAzyme. (D) Endonuclease IV Fluorescence intensity measurements [adapted from Refs. 8–11]. sometimes suffer from nonuniformities of illumination and fluorophore concentration. The measurement of fluores- which requires the presence of a specific presence of cocaine, the conformational cence lifetime offers an excellent sequence in the target strand to be change of the aptamer makes the 30 end alternative approach that is independent recognized and cleaved by the nicking of the stem shorter than the 50 end, of the fluorescence intensity and the endonuclease. By incubating the reac- resulting in digestion by Exo III. initial excitation conditions such as tion solution at 4 8C for 24 h, a PCR- Incorporation of the signal amplification wavelength, thus largely eliminating level sensitivity (20 aM) was reported system into the aptamer-based assay for the interference of background substanc- for the Exo III amplification system. cocaine enhanced the sensitivity by two es and fluctuations in excitation.65 Willner and co-workers demonstrated orders of magnitude. The work demon- When an ensemble of fluorophores is a DNAzyme-based signal amplification strated the great potential of DNA-based 10 excited with an ultrashort pulsed light, method. In the method, one target signal amplification systems to improve the fluorescence emission of excited DNA strand triggers two well-designed the detection sensitivity of a wide fluorophores will decay exponentially oligonucleotides to form a DNAzyme, variety of target molecules beyond with time via energy loss. The decay which subsequently cleaves multiple nucleic acids themselves. process may be quantitatively described dually-labeled probes. More interesting- Very recently, our group developed by the following equation: ly, when the cleaved fragment of the an apurinic/apyrimidinic probe-based -t=s probe is designed to be capable of endonuclease IV (Endo IV) signal FðtÞ¼F0e ð1Þ triggering the formation of the same amplification system that extended the kind of DNAzyme, the method achieves application of nucleic-acid-based signal where F0 is the initial fluorescence exponential autocatalytic amplification amplification systems to the area of intensity at t = 0, t is the time after with a detection limit of 1 pM target ultrasensitive and ultraselective detec- absorption, and s is the fluorescence strand. The method shows the promise tion of low-abundance DNA muta- lifetime. s equals the time for the of in vivo application by virtue of its tions.11 By artificially producing an emission to decay to 1/e of the initial independence of extra enzymes. The abasic site in the dually labeled single- value (F0). same group also designed a special MB stranded probe, a universal strategy is The two predominantly used tech- that contains the sequence of cocaine proposed for rapid cleavage of the niques for the fluorescence lifetime aptamer and several additional nucleo- fluorescent probe once it binds to the measurement are time-domain and fre- tides.13 Without addition of cocaine, the target DNA strand by using Endo IV, quency-domain methods. In the time 30 end of the stem is longer than the 50 which enables sensitive detection of 1.0 domain, the intensity decay of a fluo- end, so Exo III won’t degrade the stem- fmol of target strands. Additionally, the rescent sample is directly measured as a loop structure of the MBs. In the high activity of Endo IV at relatively function of time, following absorption of

1254 Volume 66, Number 11, 2012 close proximity to the donor results in a reduction in the donor’s lifetime. FRET efficiency is sensitive to the local environments of donors and acceptors, allowing the method to be applicable in many areas such as genotyping,74–76 nucleic acids detection,77 and probing DNAs with complicated structures.78,79 McGuinness and co-workers used a DNA Holliday junction nanoswitch labeled with FAM and TAMRA to discriminate SNPs.74 A perfectly matched target would reduce the fluorescence lifetime of FAM while single- base mismatched target shows no difference. Magennis and co-workers in- FIG.5. Schematic depiction of base flipping. The base in red is 2-aminopurine [adapted vestigated the global structure of a three- from Ref. 71]. way DNA junction by labeling it with three FRET pairs at specific nucleo- tides.78 The distances between donors a short excitation pulse. In the frequency in a cell sample without any sample and acceptors were calculated from the domain, a sample is excited by an pretreatment. donor’s lifetime by fitting the lifetime intensity-modulated light source, and Fluorescent base analogs have little histogram of the FRET subpopulation in thus the fluorescence emission is mod- influence on the structure of host nucleic multi-parameter fluorescence detection ulated at the same frequency but with a acids, but they show sensitive response plots to a single Gaussian. phase shift due to the intensity decay to the change of the structures, which is Overall, fluorescence-lifetime-based law of the sample and a reduction in the well suited for investigating the structure measurements have unique advantages 66 modulation depth. Detailed informa- changes of nucleic acids, especially in terms of eliminating background tion on the instrumentation and data when the change involves protein rec- 71,72 signal and probing DNA structures for analysis of these two approaches has ognition. Jones and co-workers used applications involving complicated been extensively reviewed previously.65 2-aminopurine to investigate base-flip- background substances or structures of In recent years, fluorescence lifetime ping in M.Hha1–DNA complexes (Fig. 71 nucleic acids. has been utilized to detect various 5). From time-resolved fluorescence targets, particularly in probing compli- measurements of the crystalline com- FLUORESCENCE cated DNA structures. Pyrene has been plexes of DNA methyltransferase, POLARIZATION/ widely used in fluorescence lifetime M.Hha1, and its cognate DNAs, the ANISOTROPY-BASED authors found a characteristic response measurements as the excimer formed BIOSENSING of 2-aminopurine to base flipping: the by the excited monomer and ground- loss of the short decay component (100 Fluorescence polarization (FP) and state monomer has a concomitant in- ps) and a large increase in the amplitude fluorescence anisotropy (FA) have been crease of fluorescence lifetime (tens of of the long decay component (10 ns). effective tools for the study of molecular nanoseconds). DNA probes covalently Results in the solution phase further interactions.80 By using nucleic acid linked with pyrene for the detection of confirmed this response, which cannot probes, FA has been applied for assay nucleic acids and proteins using time- be discerned from the present X-ray of large molecules. The designed oligo- resolved spectroscopy have been dem- structures. In a more recent study, QDs nucleotide probes usually show signifi- 67–70 onstrated. Tan and co-workers la- have been used for time-resolved detec- cant increases of fluorescent signals beled the aptamer of platelet-derived tion of nucleic acids.73 The fluorescence upon binding with large molecules such growth factor (PDGF) with two pyrene lifetime of QDs labeled to the probe will as proteins. Thus, the signal change 70 molecules at each end. In the presence decrease about 20 to 50% when the could indicate that the binding event of PDGF, the conformational change of probe hybridizes with the complemen- occurred between nucleic acid and other aptamer would bring the two pyrene tary strand. The sensitivity of this molecules. Protein assays by FP have molecules together, forming pyrene lifetime-based detection is comparable been reported by using fluorophore- excimers that have a much longer to that of fluorescence-intensity-based labeled aptamer probes.81,82 The rota- fluorescence lifetime (~40 ns) than that detection. tional motion of the fluorophore at- of the background (~5ns).Time- In a FRET system, energy transfer is a tached to the aptamer becomes slower resolved measurements were used to quenching process that reduces the due to the increase of molecular weight eliminate the biological background, donor’s intensity and lifetime. Thus, a caused by the bound protein. Thus the enabling quantitative detection of PDGF binding event that brings an acceptor in concentration of the target protein can be

APPLIED SPECTROSCOPY 1255 focal point review quantitatively measured according to the cence-labeled bound and unbound li- FRET-FCS based on dual-labeled nu- increased anisotropy value.80 gands. Nucleic acid fluorescent probe- cleic acid probes was also developed, in In addition to the assays of large based FCS measurements usually use a which the fluorescence can be detected molecules by FA or FP, the interactions singly labeled strand to monitor the only when the two dyes are in close between protein and target DNA could interactions with other molecules, in- enough proximity. Although the FRET be further used to screen small mole- cluding the complementary strand,89 signal is weaker than the fluorescence cules that specifically bind the proteins, proteins, or other biomolecules.90 With signal obtained by direct excitation, the including inhibitors,83 antagonists,84 and a highly photostable fluorophore At- specificity of interaction is notably promoters.85 For qualitative analysis, the to647N, RNA dimerization was moni- improved. Using a FRET nucleic acid FP and FA assay could be well applied tored by FCS. The fractions of single- probe, the dynamics of nucleic acid because the small molecules indirectly and double-stranded RNA were quanti- itself, including hybridization/dehybrid- affect the binding between protein and fied by applying multicomponent model ization and self-assembly, can be stud- the nucleic acid probe. Due to its analysis of autocorrelation functions and ied.94–97 Majima and co-workers used a simplicity and rapidity, FA and FP globally fitting several autocorrelation dual-labeled DNA probe to investigate technology is broadly applicable to functions. The study demonstrated that the detailed conformational dynamics of high-throughput drug screening. the resolution limit of FCS is signifi- i-motif shapes.94 A donor and an Although aptamer-based nucleic acid cantly lower than previously assumed acceptor are labeled at the terminal of probes have many small molecular (1.6-fold difference in translational dif- the cytosine (C)-rich ssDNA to monitor targets, it is difficult to use FA or FP fusion coefficients) and extended the the change of diffusion rates under to detect their binding process due to the application of FCS to the study of different pH conditions. The quantitative small increase of molecular weight molecular interactions of equally sized analysis of the FCS signal demonstrated before and after the binding. Several molecules based on their diffusional that the gradual decrease of the diffusion groups have proposed alternative ways behavior. coefficient of the i-motif with increasing to address the issue.86–88 A complemen- Recently, FCS was also used to pH was caused not only by the interac- tary oligonucleotide probe is used to investigate the intramolecular properties tions between DNA and the solvent but compete with the target small molecule of nucleic acids.91,92 By using an also by the change of the shape of the for the aptamer, resulting in sensitive ATTO665 labeled DNA probe, Kawai DNA. Furthermore, FCS analysis response to different concentrations of et al. successfully investigated the showed that the intrachain contact 86 small molecule targets. The strategy charge-recombination dynamics in formation and dissociation for i-motifs can be readily extended to a large DNA at the single-molecule level.91 are 5 to 10 times faster than that for the variety of small molecules by using Because the photo-induced charge-trans- open form, which is helpful for further their corresponding aptamers. Enzymat- fer can quench the fluorescence of the understanding of i-motif-shaped DNA ic cleavage protection ensures the FP labeled dye and subsequent charge and their functions. signal has a notable change before and recombination leads to reversible de- As an extension of FCS, fluorescence after binding with the small molecule quenching, monitoring the microenvi- cross-correlation spectroscopy (FCCS) target as the cleavage occurs only on the ronment of the labeled dye by FCS can measures the cross-correlation between 87 unbinding aptamer probe. Direct small allow the charge-transfer dynamics to be two spectrally distinct fluorophores, molecule FP assay is even possible via examined. The blinking of the FCS which are particularly useful for moni- elaborate design of the tertiary structure 88 signal proves the principle of charge toring molecular interactions or simulta- of aptamer probes. transfer and enables the read-out of neously detecting multiple targets. DNA sequence information including FCCS shows advantages over conven- FLUORESCENCE data on SNPs at the single-molecule tional FCS in monitoring of the reac- CORRELATION level. A new approach was developed tions in which the molecular weight or SPECTROSCOPY-BASED by Delon and co-workers to estimate the diffusion rate changes are very small. By BIOSENSING number of fluorescent labels by com- using a dually labeled dsDNA probe, Fluorescence correlation spectroscopy bining FCS and photobleaching.93 FCS FCCS has been applied to monitor the (FCS) offers a useful tool to investigate was used to measure the effective mean denaturing,98 unwinding,99 and degra- molecular interactions under native con- number of molecules in the observation dation100 of DNA molecules. Compared ditions. It measures the fluctuation of the volume together with their brightness, with FRET, FCCS requires much small- fluorescence intensity arising from the while photobleaching of the molecules er sample volume and works better with diffusion of biomolecules into and out of confined in the small volume offered an large chromophore distances. It is capa- an excitation volume or by the confor- additional degree of freedom to provide ble of detecting sandwich formations mational fluctuation of biomolecules more information on the system. The without strict distance limitations. By due to interactions with other molecules method is very helpful for interpretation using two spectrally distinct fluoro- at extremely low concentrations. FCS of the data of single-molecule detection phores to label aptamer probes to form usually requires a large difference be- systems. sandwich complexes with thrombin, tween the molecular weights or the In addition to the singly labeled FCCS detection was directly used to diffusion coefficients of the fluores- probe-based FCS described above, detect thrombin in serum.101

1256 Volume 66, Number 11, 2012 FLUORESCENCE IMAGING Seitz and co-workers demonstrated a the presence of the target nucleic acids. WITH NUCLEIC ACID PROBES novel type of probe that responds to Moreover, these probes also share other changes in the local structure in the benefits such as fast reaction kinetics Fluorescence imaging is an attractive vicinity of the dye rather than to the more and improved signal amplification. For technique that can create quantitative global changes in conformation.112 These example, Winssinger and co-workers maps of the concentration of desired analytes in living samples. Although the FIT-PNA probes contain a single thiazole designed quenched bis-azidorhodamin- sensitivity and selectivity of many orange (TO) intercalator serving as an PNA probes for miRNA imaging. In the nucleic acid probes have proven suffi- artificial fluorescent nucleobase, provid- presence of the target, the bis-aziderho- ing superior signal-to-background ratios damin PNA and phosphine PNA were cient for in vitro detection of various 112–114 targets, the in situ and in vivo fluores- in comparison to MB probes. The held together and the reduction of azide cence imaging of these targest remains a authors successfully applied the probes to led to fluorescence enhancement. This great challenge. the detection of influenza H1N1 mRNA strategy was successfully applied for the in living infected cells, which represents quantification of miRNA in different cell Due to the important functions of 119 RNA for living organisms, considerable the first example of an artificial nucleo- lines (Fig. 6). tidic probe that can be used for imaging Specific visualization of carcinogene- effort has been devoted to the develop- 115 ment of nucleic acid fluorescent probes of mRNA in living cells. The probes sis or established tumor cells at the early that are capable of imaging RNA achieved an 11-fold increase in TO stage may greatly increase the chances expression in living cells. Conventional emission upon hybridization with the of positive prognosis and reduce treat- 120 methods for mapping the intracellular complementary RNA target. ment costs. As a newly emerging distribution of mRNAs are represented Another novel and versatile tool for in technology with a high spatio-temporal by fluorescence in situ hybridization situ fluorescence imaging is nucleic acid resolution, fluorescence imaging has with labeled nucleic acid probes.102 A templated reactions, which have proven distinct advantages in cancer diagnosis. notable drawback of this method is the to be viable for the detection of highly By using the specific aptamers with requirement of removing excess probes expressed mRNA in living cells or strong affinity to the cancer cell surface 116,117 to reduce the background for clear bacteria. A commonly used nu- markers, a number of aptamer-based observation of the targets, leading to cleic acid templated reaction is Stau- methods for in vivo fluorescence imag- contrast compromise and long diagnos- dinger ligation, a fast reaction between ing of tumor have been reported.121–125 tic time. An alternative approach to azide and phosphine.115,118 The remark- Tan and his co-workers designed cell- visualize the DNA or RNA in cells is able advantage of this type of probe is SELEX strategies for generating multi- to use MBs with a stem-loop structure that the reaction only occurs when the ple cancer cell specific aptamers and that generates a fluorescent signal by two reactive groups are anchored with obtained six aptamers from two types of hybridizing with specific sequences.103 close proximity, and thus fluorescence leukemia cells, Toledo cells and CCRF- However, MBs are subject to unintend- enhancement can only be observed in CEM cells.126 By labeling the aptamers ed protein binding or degradation by nonspecific nucleases when applied to in situ fluorescence imaging or detection of real biological samples.104,105 To cir- cumvent the problem, many alternative approaches such as locked nucleic acids (LNA, Fig. 1C),106 peptide nucleic acids (PNA, Fig. 1D),107,108 and the 20-O- methyl nucleic acids109 (Fig. 1E) have been developed to avoid false positive signals. Hrdlicka and co-workers developed a kind of ‘‘glowing LNA’’ probe that contained one or more 20-N-(pyren- 1-yl) carbonyl-20-amino LNA mono- mers, a highly versatile building block for construction of efficient hybridization probes and quencher-free MBs.110 Due to the change of arrangement state of the labeled pyrene molecule upon hybridization, the probe generates bright fluorescence.111 This probe is demonstrated to be applicable for in vitro transcription assays and direct micro- scopic observation of probes bound to FIG.6. Schematic representation of nucleic acid-templated reduction of bis-azidorhod- mRNA in its native form in living cells. amine for miRNA imaging [adapted from Ref. 119].

APPLIED SPECTROSCOPY 1257 focal point review with fluorescent dyes or fluorescent bridization-based probes, higher sensi- Molecular Beacons’’. Angew. Chem. Int. Ed. nanoparticles such as QDs or upconver- tivity for in vivo imaging and higher 2009. 48(5): 856-870. 3. A.D. Ellington, J.W. Szostak. ‘‘In vitro sion nanoparticles, it is possible to selectivity for identification of ultra-low selection of RNA molecules that bind visualize the cancer cells and tumor abundance mutations remain great chal- specific ligands’’. Nature. 1990. 346(6287): position in vivo. Jon and his co-workers lenges. Efforts to improve the perfor- 818-822. reported an in vitro targeted cancer mance of aptamer-based sensors will 4. C. Tuerk, L. Gold. ‘‘Systematic evolution of ligands by exponential enrichment: RNA imaging, therapy, and sensing system continue to focus on new probe designs ligands to bacteriophage T4 DNA polymer- based on a bi-FRET system containing a with more chemical functionalities and ase’’. Science. 1990. 249(4968): 505-510. QD-aptamer-doxorubicin (Dox) conju- less nonspecific interactions for more 5. J. Liu, Z. Cao, Y. Lu. ‘‘Functional nucleic gate.127 In the ‘‘OFF’’ state, the fluores- specific molecular recognition and novel acid sensors’’. Chem. Rev. 2009. 109(5): 1948-1998. cence of QDs was quenched by signaling generation strategies for more 6. C. Song, M. Zhao. ‘‘Real-time monitoring of doxorubicin, while the fluorescence of accurate quantification of target mole- the activity and kinetics of T4 polynucleotide doxorubicin was quenched by intercala- cules. Due to the high background and kinase by a singly labeled DNA-hairpin tion within the aptamer. Upon entering complexity of the intracellular environ- smart probe coupled with lambda exonucle- the cancer cell, the doxorubicin released ase cleavage’’. Anal. Chem. 2009. 81(4): ment, in vivo imaging of enzymatic 1383-1388. from the conjugates and the bi-FRET catalytic processes is also one of the 7. N. Dai, E.T. Kool. ‘‘Fluorescent DNA-based system was disrupted, resulting in the remaining scientific and technical chal- enzyme sensors’’. Chem. Soc. Rev. 2011. recovery of fluorescence from both QD lenges. More principles and demonstra- 40(12): 5756-5770. and Dox (‘‘ON’’ state). The fluorescence 8. J.J. Li, Y. Chu, B.Y. Lee, X.S. Xie. tions are needed to track the enzymatic ‘‘Enzymatic signal amplification of molecu- imaging and cell viability experiments processes with greater temporal and lar beacons for sensitive DNA detection’’. demonstrated that the multifunctional spatial resolution. Novel fluorophores Nucleic Acids Res. 2008. 36(6): e36. nanoparticles can detect cancer cells at 9. X. Zuo, F. Xia, Y. Xiao, K.W. Plaxco. with high quantum yield in the near- ‘‘Sensitive and selective amplified fluores- the single-cell level while intracellularly infrared and low perturbation to the releasing a cytotoxic dose of a thera- cence DNA detection based on exonuclease normal biological process need to be III-aided target recycling’’. J. Am. Chem. peutic agent in a reportable manner. A further explored. In addition to intensity- Soc. 2010. 132(6): 1816-1818. notable advance in cancer imaging in 10. F. Wang, J. Elbaz, C. Teller, I. Willner. based measurement, other fluorescence vivo is represented by recent work by ‘‘Amplified detection of DNA through an signaling techniques, such as lifetime, Wang and his co-workers who designed autocatalytic and catabolic DNAzyme-medi- polarization, and localization, showed ated process’’. Angew. Chem. Int. Ed. 2011. an activatable aptamer probe targeting various advantages in addressing specif- 50(1): 295-299. membrane proteins of living cancer ic issues. Further combination of these 11. X. Xiao, C. Song, C. Zhang, X. Su, M. Zhao. cells. The probe could be specifically ‘‘Ultra-selective and sensitive DNA detection techniques with different probe designs activated by target cancer cells with a by a universal apurinic/apyrimidinic probe- dramatic fluorescence enhancement, will achieve significant advancement in based endonuclease IV signal amplification the future. Finally, integration of nucleic system’’. Chem. Commun. 2012. 48(14): which achieved contrast-enhanced can- 1964-1966. cer visualization inside mice.128 acid fluorescent probes with high- 12. X.J. Xiao, C. Zhang, X. Su, C. Song, M.P. DNA-scaffolded oligodeoxyriboside throughput platforms such as micro- Zhao. ‘‘A universal mismatch-directed signal fluorophores (ODFs) have been devel- fluidic chips or microarray chips will amplification platform for ultra-selective and be a rapidly expanding field. These new sensitive DNA detection under mild isother- oped by Kool and co-workers for mal conditions’’. Chem. Sci. 2012. 3(7): cellular enzyme imaging.7 In the ODF methodologies will make significant 2257-2261. system, the DNA bases are replaced by new contributions to real-time monitor- 13. X. Liu, R. Freeman, I. Willner. ‘‘Amplified several fluorophores that are directly ing of biochemical events, rapid identi- fluorescence aptamer-based sensors using fication, and characterization of a large exonuclease III for the regeneration of the attached to the backbone in close analyte’’. Chem. Eur. J. 2012. 18(8): proximity.129,130 By conjugation with a number of enzymes in a complex 2207-2211. specific substrate structure, these ODF- system, drug screening, and disease 14. C.A.M. Seidel, A. Schulz, M.H.M. Sauer. based fluorescence sensors can be used diagnosis at the early stages. ‘‘Nucleobase-specific quenching of fluores- as the reporters in turn-on sensing of cent dyes .1. Nucleobase one-electron redox ACKNOWLEDGMENTS potentials and their correlation with static and cleaving activities of the corresponding dynamic quenching efficiencies’’. J. Phys. enzymes. A tetrapyrene ODF (Fig. 1F) This work was supported by National Natural Chem. 1996. 100(13): 5541-5553. Science Foundation of China (91132717, has been successfully applied for the 15. J.P. Knemeyer, N. Marme, M. Sauer. 21175007) and The Research Fund for the ‘‘Probes for detection of specific DNA detection of esterases and lipases in vitro Doctoral Program of Higher Education of China 7,131 sequences at the single-molecule level’’. and in cells. (20110001110083). Anal. Chem. 2000. 72(16): 3717-3724. 16. K. Stohr, B. Hafner, O. Nolte, J. Wolfrum, FUTURE DIRECTIONS M. Sauer, D.P. Herten. ‘‘Species-specific 1. S. Tyagi, F.R. Kramer. ‘‘Molecular beacons: identification of mycobacterial 16S rRNA By virtue of their versatile functions probes that fluoresce upon hybridization’’. PCR amplicons using smart probes’’. Anal. and low cost, nucleic acid fluorescent Nat. Biotechnol. 1996. 14(3): 303-308. Chem. 2005. 77(22): 7195-7203. 2. K.M. Wang, Z.W. Tang, C.Y.J. Yang, Y.M. 17. M.J. Snare, P.J. Thistlethwaite, K.P. Ghiggi- probes represent a class of broadly Kim, X.H. Fang, W. Li, Y.R. Wu, C.D. no. ‘‘Kinetic-Studies of Intramolecular Exci- applicable fluorescent biosensors in Medley, Z.H. Cao, J. Li, P. Colon, H. Lin, mer Formation in Dipyrenylalkanes’’. J. Am. biological studies. Regarding the hy- W.H. Tan. ‘‘Molecular Engineering of DNA: Chem. Soc. 1983. 105(10): 3328-3332.

1258 Volume 66, Number 11, 2012 18. H. Nohta, H. Satozono, K. Koiso, H. aptamer sensor of small molecules’’. Biosen. and Biological Sensing’’. Chem. Rev. 2012. Yoshida, J. Ishida, M. Yamaguchi. ‘‘Highly Bioelectron. 2011. 28(1): 33-37. 112(5): 2739-2779. selective fluorometric determination of poly- 32. M. Zhang, B.C. Ye. ‘‘Label-free fluorescent 46. B. Dubertret, M. Calame, A.J. Libchaber. amines based on intramolecular excimer- detection of copper(II) using DNA-templated ‘‘Single-mismatch detection using gold- forming derivatization with a pyrene-labeling highly luminescent silver nanoclusters’’. An- quenched fluorescent oligonucleotides’’. reagent’’. Anal. Chem. 2000. 72(17): alyst. 2011. 136(24): 5139-5142. Nat. Biotechnol. 2001. 19(4): 365-370. 4199-4204. 33. F. Auzel. ‘‘Upconversion and anti-Stokes 47. D.J. Maxwell, J.R. Taylor, S. Nie. ‘‘Self- 19. C. Eggeling, J. Widengren, R. Rigler, processes with f and d ions in solids’’. Chem. assembled nanoparticle probes for recogni- C.A.M. Seidel. ‘‘Photobleaching of fluores- Rev. 2004. 104(1): 139-173. tion and detection of biomolecules’’. J. Am. cent dyes under conditions used for single- 34. P. Zhang, S. Rogelj, K. Nguyen, D. Wheeler. Chem. Soc. 2002. 124(32): 9606-9612. molecule detection: Evidence of two-step ‘‘Design of a highly sensitive and specific 48. L. Dyadyusha, H. Yin, S. Jaiswal, T. Brown, photolysis’’. Anal. Chem. 1998. 70(13): nucleotide sensor based on photon upcon- J.J. Baumberg, F.P. Booy, T. Melvin. 2651-2659. verting particles’’. J. Am. Chem. Soc. 2006. ‘‘Quenching of CdSe quantum dot emission, 20. H.P. Lu, L. Xun, X.S. Xie. ‘‘Single-molecule 128(38): 12410-12411. a new approach for biosensing’’. Chem. enzymatic dynamics’’. Science. 1998. 35. I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Commun. 2005. (25): 3201-3203. 282(5395): 1877-1882. Mattoussi. ‘‘Quantum dot bioconjugates for 49. J. Zhang, L. Wang, H. Zhang, F. Boey, S. 21. W.C. Chan, S. Nie. ‘‘Quantum dot bioconju- imaging, labelling and sensing’’. Nat. Mater. Song, C. Fan. ‘‘Aptamer-based multicolor gates for ultrasensitive nonisotopic detec- 2005. 4(6): 435-446. fluorescent gold nanoprobes for multiplex tion’’. Science. 1998. 281(5385): 2016-2018. 36. Z. Chen, H. Chen, H. Hu, M. Yu, F. Li, Q. detection in homogeneous solution’’. Small. 22. W.R. Algar, U.J. Krull. ‘‘Developing mixed Zhang, Z. Zhou, T. Yi, C. Huang. ‘‘Versatile 2010. 6(2): 201-204. films of immobilized oligonucleotides and synthesis strategy for carboxylic acid-func- 50. D.A. Giljohann, D.S. Seferos, A.E. Prigo- quantum dots for the multiplexed detection tionalized upconverting nanophosphors as dich, P.C. Patel, C.A. Mirkin. ‘‘Gene regu- of nucleic acid hybridization using a combi- biological labels’’. J. Am. Chem. Soc. 2008. lation with polyvalent siRNA-nanoparticle nation of fluorescence resonance energy 130(10): 3023-3029. conjugates’’. J. Am. Chem. Soc. 2009. transfer and direct excitation of fluores- 37. Y. Wang, L. Bao, Z. Liu, D.W. Pang. 131(6): 2072-2073. cence’’. Langmuir. 2010. 26(8): 6041-6047. ‘‘Aptamer biosensor based on fluorescence 51. J.I. Cutler, K. Zhang, D. Zheng, E. Auyeung, 23. E.R. Goldman, G.P. Anderson, P.T. Tran, H. resonance energy transfer from upconverting A.E. Prigodich, C.A. Mirkin. ‘‘Polyvalent Mattoussi, P.T. Charles, J.M. Mauro. ‘‘Con- phosphors to carbon nanoparticles for throm- nucleic acid nanostructures’’. J. Am. Chem. jugation of luminescent quantum dots with bindetectioninhumanplasma’’.Anal. Soc. 2011. 133(24): 9254-9257. antibodies using an engineered adaptor Chem. 2011. 83(21): 8130-8137. 52. A.E. Prigodich, A.H. Alhasan, C.A. Mirkin. protein to provide new reagents for fluo- 38. F. Zhang, Q.H. Shi, Y.C. Zhang, Y.F. Shi, ‘‘Selective enhancement of nucleases by roimmunoassays’’. Anal. Chem. 2002. 74(4): K.L. Ding, D.Y. Zhao, G.D. Stucky. ‘‘Fluo- polyvalent DNA-functionalized gold nanoparticles’’. J. Am. Chem. Soc. 2011. 133(7): 841-847. rescence Upconversion Microbarcodes for 2120-2123. 24. X. Michalet, F.F. Pinaud, L.A. Bentolila, Multiplexed Biological Detection: Nucleic 53. A.E. Prigodich, D.S. Seferos, M.D. Massich, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, Acid Encoding’’. Adv. Mater. 2011. 23(33): D.A. Giljohann, B.C. Lane, C.A. Mirkin. A.M. Wu, S.S. Gambhir, S. Weiss. ‘‘Quan- 3775-3779. ‘‘Nano-flares for mRNA regulation and tum dots for live cells, in vivo imaging, and 39. X. Feng, L. Liu, S. Wang, D. Zhu. ‘‘Water- detection’’. ACS Nano. 2009. 3(8): diagnostics’’. Science. 2005. 307(5709): soluble fluorescent conjugated polymers and 2147-2152. 538-544. their interactions with biomacromolecules for 54. M. Zheng, A. Jagota, E.D. Semke, B.A. 25. C.Y. Zhang, L.W. Johnson. ‘‘Single quan- sensitive biosensors’’. Chem. Soc. Rev. 2010. Diner,R.S.McLean,S.R.Lustig,R.E. tum-dot-based aptameric nanosensor for 39(7): 2411-2419. Richardson, N.G. Tassi. ‘‘DNA-assisted dis- cocaine’’. Anal. Chem. 2009. 81(8): 40. X. Duan, L. Liu, F. Feng, S. Wang. ‘‘Cationic persion and separation of carbon nanotubes’’. 3051-3055. conjugated polymers for optical detection of Nat. Mater. 2003. 2(5): 338-342. 26. A.J. Tavares, M.O. Noor, C.H. Vannoy, DNA methylation, lesions, and single nucle- 55. R. Yang, J. Jin, Y. Chen, N. Shao, H. Kang, W.R. Algar, U.J. Krull. ‘‘On-chip transduc- otide polymorphisms’’. Acc. Chem. Res. Z. Xiao, Z. Tang, Y. Wu, Z. Zhu, W. Tan. tion of nucleic acid hybridization using 2010. 43(2): 260-270. ‘‘Carbon nanotube-quenched fluorescent oli- spatial profiles of immobilized quantum dots 41. B.S. Gaylord, A.J. Heeger, G.C. Bazan. gonucleotides: probes that fluoresce upon and fluorescence resonance energy transfer’’. ‘‘DNA detection using water-soluble conju- hybridization’’. J. Am. Chem. Soc. 2008. Anal. Chem. 2012. 84(1): 312-319. gated polymers and peptide nucleic acid 130(26): 8351-8358. 27. R. Freeman, X. Liu, I. Willner. ‘‘Chemilu- probes’’.Proc.Natl.Acad.Sci.U.S.A. 56. Z. Zhu, R. Yang, M. You, X. Zhang, Y. Wu, minescent and chemiluminescence resonance 2002. 99(17): 10954-10957. W. Tan. ‘‘Single-walled carbon nanotube as energy transfer (CRET) detection of DNA, 42. H.A. Ho, M. Boissinot, M.G. Bergeron, G. an effective quencher’’. Anal. Bioanal. metal ions, and aptamer-substrate complexes Corbeil, K. Dore, D. Boudreau, M. Leclerc. Chem. 2010. 396(1): 73-83. using hemin/G-quadruplexes and CdSe/ZnS ‘‘Colorimetric and fluorometric detection of 57. R. Yang, Z. Tang, J. Yan, H. Kang, Y. Kim, quantum dots’’. J. Am. Chem. Soc. 2011. nucleic acids using cationic polythiophene Z. Zhu, W. Tan. ‘‘Noncovalent assembly of 133(30): 11597-11604. derivatives’’. Angew. Chem. Int. Ed. Engl. carbon nanotubes and single-stranded DNA: 28. J.T. Petty, J. Zheng, N.V. Hud, R.M. Dick- 2002. 41(9): 1548-1551. an effective sensing platform for probing son. ‘‘DNA-templated Ag nanocluster forma- 43. B. Kim, I.H. Jung, M. Kang, H.K. Shim, biomolecular interactions’’.Anal.Chem. tion’’. J. Am. Chem. Soc. 2004. 126(16): H.Y. Woo. ‘‘Cationic conjugated polyelec- 2008. 80(19): 7408-7413. 5207-5212. trolytes-triggered conformational change of 58. Z. Wu, Z. Zhen, J.H. Jiang, G.L. Shen, R.Q. 29. C.I. Richards, S. Choi, J.C. Hsiang, Y. molecular beacon aptamer for highly sensi- Yu. ‘‘Terminal protection of small-molecule- Antoku, T. Vosch, A. Bongiorno, Y.L. tive and selective potassium ion detection’’. linked DNA for sensitive electrochemical Tzeng, R.M. Dickson. ‘‘Oligonucleotide- J. Am. Chem. Soc. 2012. 134(6): 3133-3138. detection of protein binding via selective stabilized Ag nanocluster fluorophores’’.J. 44. C. Zhang, X. Su, Y. Liang, X.C. Zhu, C. carbon nanotube assembly’’. J. Am. Chem. Am. Chem. Soc. 2008. 130(15): 5038-5039. Song, M.P. Zhao. ‘‘A transformer of molec- Soc. 2009. 131(34): 12325-12332. 30. H.C. Yeh, J. Sharma, J.J. Han, J.S. Martinez, ular beacon for sensitive and real-time 59. Y. Liu, X. Dong, P. Chen. ‘‘Biological and J.H. Werner. ‘‘A DNA–silver nanocluster detection of phosphatases with effective chemical sensors based on graphene materi- probe that fluoresces upon hybridization’’. inhibition of the false positive signals’’. als’’. Chem. Soc. Rev. 2012. 41(6): Nano Lett. 2010. 10(8): 3106-3110. Biosen. Bioelectron. 2011. 28(1): 13-16. 2283-2307. 31. Z. Zhou, Y. Du, S. Dong. ‘‘DNA-Ag nano- 45. K. Saha, S.S. Agasti, C. Kim, X. Li, V.M. 60. Y. Shi, W.T. Huang, H.Q. Luo, N.B. Li. ‘‘A clusters as fluorescence probe for turn-on Rotello. ‘‘Gold Nanoparticles in Chemical label-free DNA reduced graphene oxide-

APPLIED SPECTROSCOPY 1259 focal point review

based fluorescent sensor for highly sensitive sibility of single nucleotide polymorphism Peyrin. ‘‘Fluorescence polarization biosensor and selective detection of hemin’’. Chem. genotyping with a single-probe by time- based on an aptamer enzymatic cleavage Commun. 2011. 47(16): 4676-4678. resolved Forster resonance energy transfer’’. protection strategy’’. Anal. Bioanal. Chem. 61. X. Su, X. Zhu, C. Zhang, X. Xiao, M. Zhao. Mol. Cell Probes. 2009. 23(2): 119-121. 2011. 401(10): 3229-3234. ‘‘In Situ, Real-Time Monitoring of the 30 to 75. A. Andreoni, M. Bondani, L. Nardo. ‘‘Time- 88. J. Ruta, S. Perrier, C. Ravelet, J. Fize, E. 50 Exonucleases Secreted by Living Cells’’. resolved FRET method for typing polymor- Peyrin. ‘‘Noncompetitive fluorescence polar- Anal. Chem. 2012. 84(11): 5059-5065. phic alleles of the human leukocyte antigen ization aptamer-based assay for small mole- 62. S.L. Allinson. ‘‘DNA end-processing enzyme system by using a single DNA probe’’. cule detection’’. Anal. Chem. 2009. 81(17): polynucleotide kinase as a potential target in Photochem. Photobiol. Sci. 2009. 8(8): 7468-7473. the treatment of cancer’’. Future Oncol. 2010. 1202-1206. 89. A. Werner, V.V. Skakun, C. Meyer, U. 6(6): 1031-1042. 76. C.D. McGuinness, M.K. Nishimura, D. Hahn. ‘‘RNA dimerization monitored by 63. C. Song, C. Zhang, M. Zhao. ‘‘Singly labeled Keszenman-Pereyra, P. Dickinson, C.J. fluorescence correlation spectroscopy’’. Eur. smart probes for real-time monitoring of the Campbell, T.T. Bachmann, P. Ghazal, J. Biophys. J. 2011. 40(8): 907-921. kinetics of dNTP misincorporation and single Crain. ‘‘Detection of single nucleotide poly- 90. T. Kral, J. Leblond, M. Hof, D. Scherman, J. nucleotide extension in DNA intra-molecular morphisms using a DNA Holliday junction Herscovici, N. Mignet. ‘‘Lipopolythiourea/ polymerization’’. Biosens. Bioelectron. 2009. nanoswitch—a high-throughput fluorescence DNA interaction: a biophysical study’’. 25(2): 301-305. lifetime assay’’. Mol. Biosyst. 2010. 6(2): Biophys. Chem. 2010. 148(1-3): 68-73. 64. C. Song, C. Zhang, M. Zhao. ‘‘Rapid and 386-390. 91. K. Kawai, E. Matsutani, A. Maruyama, T. sensitive detection of DNA polymerase 77. V. Laitala, A. Ylikoski, H.M. Raussi, P. Majima. ‘‘Probing the charge-transfer dy- fidelity by singly labeled smart fluorescent Ollikka, I. Hemmila. ‘‘Time-resolved detec- namics in DNA at the single-molecule level’’. probes’’. Biosens. Bioelectron. 2011. 26(5): tion probe for homogeneous nucleic acid J. Am. Chem. Soc. 2011. 133(39): 2699-2702. analyses in one-step format’’. Anal. Biochem. 15568-15577. 65. J.R. Lakowicz. Principles of fluorescence 2007. 361(1): 126-131. 92. P. Qu, X. Yang, X. Li, X. Zhou, X.S. Zhao. spectroscopy. New York: Springer, 2006. 3rd 78. T. Sabir, A. Toulmin, L. Ma, A.C. Jones, P. ‘‘Direct measurement of the rates and barriers ed. McGlynn, G.F. Schroder, S.W. Magennis. on forward and reverse diffusions of intra- 66. R. Richards-Kortum, E. Sevick-Muraca. ‘‘Branchpoint expansion in a fully comple- molecular collision in overhang oligonucle- ‘‘Quantitative optical spectroscopy for tissue mentary three-way DNA junction’’. J. Am. otides’’. J. Phys. Chem. B. 2010. 114(24): diagnosis’’. Annu. Rev. Phys. Chem. 1996. Chem. Soc. 2012. 134(14): 6280-6285. 8235-8243. 47: 555-606. 79. S. Chatterjee, J.B. Lee, N.V. Valappil, D. 93. A. Delon, I. Wang, E. Lambert, S. Mache, R. 67. P. Conlon, C.J. Yang, Y. Wu, Y. Chen, K. Luo, V.M. Menon. ‘‘Probing Y-shaped DNA Mache, J. Derouard, V. Motto-Ros, R. Gal- Martinez, Y. Kim, N. Stevens, A.A. Marti, S. structure with time-resolved FRET’’. Nano- land. ‘‘Measuring, in solution, multiple- Jockusch, N.J. Turro, W. Tan. ‘‘Pyrene scale. 2012. 4(5): 1568-1571. fluorophore labeling by combining fluores- excimer signaling molecular beacons for 80. X. Fang, Z. Cao, T. Beck, W. Tan. cence correlation spectroscopy and photo- probing nucleic acids’’. J. Am. Chem. Soc. ‘‘Molecular aptamer for real-time oncopro- bleaching’’. J. Phys. Chem. B. 2010. 114(8): 2008. 130(1): 336-342. tein platelet-derived growth factor monitor- 2988-2996. 68. A.A. Marti, X. Li, S. Jockusch, Z. Li, B. ing by fluorescence anisotropy’’.Anal. 94. J. Choi, S. Kim, T. Tachikawa, M. Fujitsuka, Raveendra, S. Kalachikov, J.J. Russo, I. Chem. 2001. 73(23): 5752-5757. T. Majima. ‘‘pH-induced intramolecular fold- Morozova, S.V. Puthanveettil, J. Ju, N.J. 81. M. Zou, Y. Chen, X. Xu, H. Huang, F. Liu, ing dynamics of i-motif DNA’’. J. Am. Turro. ‘‘Pyrene binary probes for unambigu- N. Li. ‘‘The homogeneous fluorescence Chem. Soc. 2011. 133(40): 16146-16153. ous detection of mRNA using time-resolved anisotropic sensing of salivary lysozyme 95. S. Kim, J. Choi, T. Majima. ‘‘Self-assembly fluorescence spectroscopy’’. Nucleic Acids using the 6-carboxyfluorescein-labeled Res. 2006. 34(10): 3161-3168. DNA aptamer’’. Biosens. Bioelectron. 2012. of polydeoxyadenylic acid studied at the 69. Y.J. Seo, H. Rhee, T. Joo, B.H. Kim. ‘‘Self- 32(1): 148-154. single-molecule level’’. J. Phys. Chem. B. duplex formation of an A(py)-substituted 82. W. Li, K. Wang, W. Tan, C. Ma, X. Yang. 2011. 115(51): 15399-15405. oligodeoxyadenylate and its unique fluores- ‘‘Aptamer-based analysis of angiogenin by 96. M. Levitus. ‘‘Relaxation Kinetics by Fluo- cence’’. J. Am. Chem. Soc. 2007. 129(16): fluorescence anisotropy’’. Analyst. 2007. rescence Correlation Spectroscopy: Determi- 5244-5247. 132(2): 107-113. nation of Kinetic Parameters in the Presence 70. C.J. Yang, S. Jockusch, M. Vicens, N.J. 83. E.J. Cho, S. Xia, L.C. Ma, J. Robertus, R.M. of Fluorescent Impurities’’. J. Phys. Chem. Turro, W. Tan. ‘‘Light-switching excimer Krug, E.V. Anslyn, G.T. Montelione, A.D. Lett. 2010. 1: 1346-1350. probes for rapid protein monitoring in Ellington. ‘‘Identification of influenza virus 97. X. Chen, Y. Zhou, P. Qu, X.S. Zhao. ‘‘Base- complex biological fluids’’. Proc. Natl. Acad. inhibitors targeting NS1A utilizing fluores- by-base dynamics in DNA hybridization Sci. U.S.A. 2005. 102(48): 17278-17283. cence polarization-based high-throughput as- probed by fluorescence correlation spectros- 71. R.K. Neely, D. Daujotyte, S. Grazulis, S.W. say’’. J. Biomol. Screen. 2012. 17(4): copy’’. J. Am. Chem. Soc. 2008. 130(50): Magennis, D.T. Dryden, S. Klimasauskas, 448-459. 16947-16952. A.C. Jones. ‘‘Time-resolved fluorescence of 84. M. Hafner, E. Vianini, B. Albertoni, L. 98. A. Chen, M.M. Eberle, E.J. Lunt, S. Liu, K. 2-aminopurine as a probe of base flipping in Marchetti, I. Grune, C. Gloeckner, M. Leake, M.I. Rudenko, A.R. Hawkins, H. M.HhaI-DNA complexes’’. Nucleic Acids Famulok. ‘‘Displacement of protein-bound Schmidt. ‘‘Dual-color fluorescence cross- Res. 2005. 33(22): 6953-6960. aptamers with small molecules screened by correlation spectroscopy on a planar opto- 72. C.J. Campbell, C.P. Mountford, H.C. Sto- fluorescence polarization’’.Nat.Protoc. fluidic chip’’. Lab Chip. 2011. 11(8): quert, A.H. Buck, P. Dickinson, E. Ferapon- 2008. 3(4): 579-587. 1502-1506. tova, J.G. Terry, J.S. Beattie, A.J. Walton, J. 85. C. Joce, R. White, P.G. Stockley, S. 99. X.G. Xi, E. Deprez. ‘‘Monitoring helicase- Crain, P. Ghazal, A.R. Mount. ‘‘A DNA Warriner, W.B. Turnbull, A. Nelson. ‘‘De- catalyzed DNA unwinding by fluorescence nanoswitch incorporating the fluorescent sign, synthesis and in vitro evaluation of anisotropy and fluorescence cross-correlation base analogue 2-aminopurine detects single novel bivalent S-adenosylmethionine ana- spectroscopy’’. Methods. 2010. 51(3): nucleotide mismatches in unlabelled targets’’. logues’’. Bioorg. Med. Chem. Lett. 2012. 289-294. Analyst. 2009. 134(9): 1873-1879. 22(1): 278-284. 100. A. Sasaki, M. Kinjo. ‘‘Monitoring intracellu- 73. S.H. Lim, P. Buchy, S. Mardy, M.S. Kang, 86. J.A. Cruz-Aguado, G. Penner. ‘‘Fluorescence lar degradation of exogenous DNA using A.D. Yu. ‘‘Specific nucleic acid detection polarization based displacement assay for the diffusion properties’’. J. Control Release. using photophysical properties of quantum determination of small molecules with ap- 2010. 143(1): 104-111. dot probes’’. Anal. Chem. 2010. 82(3): tamers’’. Anal. Chem. 2008. 80(22): 101. X. Zhou, Y. Tang, D. Xing. ‘‘One-step 886-891. 8853-8855. homogeneous protein detection based on 74. A. Andreoni, M. Bondani, L. Nardo. ‘‘Fea- 87. A. Kidd, V. Guieu, S. Perrier, C. Ravelet, E. aptamer probe and fluorescence cross-corre-

1260 Volume 66, Number 11, 2012 lation spectroscopy’’. Anal. Chem. 2011. homogeneous SNP detection’’. Bioorg. Med. transfer nanoparticles for multiplexed cancer 83(8): 2906-2912. Chem. 2008. 16(1): 114-125. cell monitoring’’. Anal. Chem. 2009. 81(16): 102. J.M. Levsky. ‘‘Fluorescence in situ hybrid- 113. D.V. Jarikote, N. Krebs, S. Tannert, B. 7009-7014. ization: past, present and future’’. J. Cell Sci. Roder, O. Seitz. ‘‘Exploring base-pair-spe- 123. J.K. Kim, K.J. Choi, M. Lee, M.H. Jo, S. 2003. 116(14): 2833-2838. cific optical properties of the DNA stain Kim. ‘‘Molecular imaging of a cancer- 103. D.P. Bratu. ‘‘Visualizing the distribution and thiazole orange’’. Chem. Eur. J. 2007. 13(1): targeting theragnostics probe using a nucle- transport of mRNAs in living cells’’. Proc. 300-310. olin aptamer- and microRNA-221 molecular Natl. Acad. Sci. U.S.A. 2003. 100(23): 114. E. Socher, D.V. Jarikote, A. Knoll, L. beacon-conjugated nanoparticle’’. Biomateri- 13308-13313. Roglin, J. Burmeister, O. Seitz. ‘‘FIT probes: als. 2012. 33(1): 207-217. 104. A.K. Chen, M.A. Behlke, A. Tsourkas. peptide nucleic acid probes with a fluores- 124. S.E. Lupold, B.J. Hicke, Y. Lin, D.S. Coffey. ‘‘Avoiding false-positive signals with nucle- cent base surrogate enable real-time DNA ‘‘Identification and characterization of nuclease-vulnerable molecular beacons in single quantification and single nucleotide poly- ase-stabilized RNA molecules that bind living cells’’. Nucleic Acids Res. 2007. morphism discovery’’. Anal. Biochem. 2008. human prostate cancer cells via the pros- 35(16): e105-e105. 375(2): 318-330. tate-specific membrane antigen’’. Cancer 105. X. Su, C. Zhang, M. Zhao. ‘‘Discrimination 115. S. Kummer, A. Knoll, E. Socher, L. Bethge, Res. 2002. 62(14): 4029-4033. of the false-positive signals of molecular A. Herrmann, O. Seitz. ‘‘Fluorescence Imag- 125. J.A. Phillips, Y. Xu, Z. Xia, Z.H. Fan, W. beacons by combination of heat inactivation ing of Influenza H1N1 mRNA in Living Tan. ‘‘Enrichment of cancer cells using and using single walled carbon nanotubes’’. Infected Cells Using Single-Chromophore aptamers immobilized on a microfluidic Biosens. Bioelectron. 2011. 26(8): FIT-PNA’’. Angew. Chem. Int. Ed. 2011. channel’’.Anal.Chem.2009.81(3): 3596-3601. 50(8): 1931-1934. 1033-1039. 106. L. Wang, C.J. Yang, C.D. Medley, S.A. 116. H. Abe, E.T. Kool. ‘‘Flow cytometric 126. X. Fang, W. Tan. ‘‘Aptamers generated from Benner,W.Tan.‘‘Locked nucleic acid detection of specific RNAs in native human cell-SELEX for molecular medicine: a chem- molecular beacons’’. J. Am. Chem. Soc. cells with quenched autoligating FRET ical biology approach’’. Acc. Chem. Res. 2005. 127(45): 15664-15665. probes’’.Proc.Natl.Acad.Sci.U.S.A. 2010. 43(1): 48-57. 107. P.E. Nielsen, M. Egholm. ‘‘An introduction 2006. 103(2): 263-268. 127. V. Bagalkot, L. Zhang, E. Levy-Nissenbaum, to peptide nucleic acid’’. Curr. Issues Mol. 117. R.M. Franzini, E.T. Kool. ‘‘Efficient nucleic S. Jon, P.W. Kantoff, R. Langer, O.C. Biol. 1999. 1(1-2): 89-104. acid detection by templated reductive Farokhzad. ‘‘Quantum dot-aptamer conju- 108. Z. Pianowski, K. Gorska, L. Oswald, C.A. quencher release’’.J.Am.Chem.Soc. gates for synchronous cancer imaging, ther- Merten, N. Winssinger. ‘‘Imaging of mRNA 2009. 131(44): 16021-16023. apy, and sensing of drug delivery based on in live cells using nucleic acid-templated 118. K. Gorska, A. Manicardi, S. Barluenga, N. bi-fluorescence resonance energy transfer’’. reduction of azidorhodamine probes’’. J. Am. Winssinger. ‘‘DNA-templated release of Nano Lett. 2007. 7(10): 3065-3070. Chem. Soc. 2009. 131(18): 6492-6497. functional molecules with an azide-reduc- 128. H. Shi, X. He, K. Wang, X. Wu, X. Ye, Q. 109. A.K. Chen, M.A. Behlke, A. Tsourkas. tion-triggered immolative linker’’.Chem. Guo, W. Tan, Z. Qing, X. Yang, B. Zhou. ‘‘Sub-cellular trafficking and functionality Commun. 2011. 47(15): 4364-4366. ‘‘Activatable aptamer probe for contrast- of 20-O-methyl and 20-O-methyl-phosphor- 119. K. Gorska, I. Keklikoglou, U. Tschulena, N. enhanced in vivo cancer imaging based on othioate molecular beacons’’. Nucleic Acids Winssinger. ‘‘Rapid fluorescence imaging of cell membrane protein-triggered conforma- Res. 2009. 37(22): e149-e149. miRNAs in human cells using templated tion alteration’’. Proc. Natl. Acad. Sci. U.S.A. 110. M.E. Ostergaard, P. Cheguru, M.R. Papasani, Staudinger reaction’’. Chem. Sci. 2011. 2011. 108(10): 3900-3905. R.A. Hill, P.J. Hrdlicka. ‘‘Glowing locked 2(10): 1969. 129. J. Gao, S. Watanabe, E.T. Kool. ‘‘Modified nucleic acids: brightly fluorescent probes for 120. J. Teissie´, M. Golzio, V. Ecochard, L. DNA analogues that sense light exposure detection of nucleic acids in cells’’. J. Am. Paquereau, E. Bellard, A. Paganin-Gioanni. with color changes’’. J. Am. Chem. Soc. Chem. Soc. 2010. 132(40): 14221-14228. ‘‘Fluorescence imaging agents in cancerolo- 2004. 126(40): 12748-12749. 111. P.J. Hrdlicka, B.R. Babu, M.D. Sorensen, N. gy’’. Radiol. Oncol. 2010. 44(3): 142-148. 130. Y.N. Teo, J.N. Wilson, E.T. Kool. ‘‘Poly- Harrit, J. Wengel. ‘‘Multilabeled pyrene- 121. C.S. Ferreira, C.S. Matthews, S. Missailidis. fluorophores on a DNA backbone: a multi- functionalized 20-amino-LNA probes for ‘‘DNA aptamers that bind to MUC1 tumour color set of labels excited at one nucleic acid detection in homogeneous marker: design and characterization of wavelength’’.J.Am.Chem.Soc.2009. fluorescence assays’’. J. Am. Chem. Soc. MUC1-binding single-stranded DNA ap- 131(11): 3923-3933. 2005. 127(38): 13293-13299. tamers’’. Tumour Biol. 2006. 27(6): 289-301. 131. N. Dai, Y.N. Teo, E.T. Kool. ‘‘DNA- 112. L. Bethge, D.V. Jarikote, O. Seitz. ‘‘New 122. X. Chen, M.C. Estevez, Z. Zhu, Y.F. Huang, polyfluorophore excimers as sensitive report- cyanine dyes as base surrogates in PNA: Y. Chen, L. Wang, W. Tan. ‘‘Using aptamer- ers for esterases and lipases’’. Chem Com- forced intercalation probes (FIT-probes) for conjugated fluorescence resonance energy mun. 2010. 46(8): 1221-1223.

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