Gold nanorods-based FRET assay for sensitive detection of Pb2+ using 8-17DNAzyme

In this paper, we have reported a sensitive assay for fluorescence "turn-on" detection of Pb(2+) in aqueous solutions based on FRET between gold nanorods (GNRs) and the FAM-labeled substrate strand of 8-17DNAzyme. The fluorescence of the FAM-labeled substrate strand is quenched when 8-17DNAzyme is adsorbed on GNRs surface through electrostatic interaction. In the presence of lead ions, the fluorescence is restored due to the decrease of FRET efficiency caused by the specific cleavage of the FAM-labeled substrate strand by the enzyme, which weakens the electrostatic interaction between the GNRs and short FAM-labeled DNA fragment. The interference of eleven common metal ions has been tested, indicating that Pb(2+) can be selectively detected. This method exhibits a high sensitivity for Pb(2+) with a detection limit of 61.8 pM and a linear range from 0.1 nM to 100 nM. It is a simple, sensitive, and selective method for Pb(2+) detection. Moreover, this sensing system obtained satisfying results for Pb(2+) detection in tap water samples.     

Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.     

A SERS DNAzyme biosensor for lead ion detection

SERS biosensor for sensitive and selective detection of lead ions (Pb(2+)) based on DNAzyme was developed by taking advantage of the specific catalytic reaction of DNAzyme upon binding to Pb(2+) ions. Detection was accomplished by SERS nanoprobe labeled with DNA and Raman reporters for signal amplification.     

DNAzyme-based fluorescent microarray for highly selective and sensitive detection of lead(II)

A facile microarray-based fluorescent sensor for the detection of lead (II) was developed based on the catalytic cleavages of the substrates by a DNAzyme upon its binding to Pb(2+). The release of the fluorophore labelled substrates resulted in the decrease of fluorescence intensity. The sensor had a quantifiable detection range from 1 nM to 1 μM and a selectivity of >20 fold for Pb(2+) over other metal ions.     

Zn(2+)-ligation DNAzyme-driven enzymatic and nonenzymatic cascades for the amplified detection of DNA

A generic fluorescence sensing platform for analyzing DNA by the Zn(2+)-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the target DNA induced ligation of two substrate subunits and the subsequent opening of a beacon hairpin probe by the ligated product. The strand displacement of the ligated product by the beacon hairpin is, however, of limited efficiency. Two strategies are implemented to overcome this limitation. By one method, a "helper" nucleic acid sequence is introduced into the system, and this hybridizes with the DNAzyme components and releases the ligated product for opening of the hairpin. By the second method, a nicking enzyme (Nt.BspQI) is added to the system, and this nicks the duplex between the beacon and ligated product while recycling the free ligation product. By combining the two coadded components ("helper" sequence and nicking enzyme), the sensitive detection of the analyte is demonstrated (detection limit, 20 pM). The enzyme-free amplified fluorescence detection of the target DNA is further presented by the Zn(2+)-dependent ligation DNAzyme-driven activation of the Mg(2+)-dependent DNAzyme. According to this method, the Mg(2+)-dependent DNAzyme subunits displace the ligated product, and the resulting assembled DNAzyme cleaves a fluorophore/quencher-modified substrate to yield fluorescence. The method enabled the detection of the target DNA with a detection limit corresponding to 10 pM. The different sensing platforms are implemented to detect the Tay-Sachs genetic disorder mutant.     

Pb(2+)-introduced activation of horseradish peroxidase (HRP)-mimicking DNAzyme

A novel nucleic acid hairpin structure composed of Pb(2+)-dependent DNAzyme and HRP-mimicking DNAzyme was developed. This hairpin structure can be used as a sensor for the detection of Pb(2+) based on colorimetry.     

Accelerated color change of gold nanoparticles assembled by DNAzymes for simple and fast colorimetric Pb2+ detection

The combination of high metal selectivity of DNAzymes with the strong distance-dependent optical properties of metallic nanoparticles has presented considerable opportunities for designing colorimetric sensors for metal ions. We previously communicated a design for a colorimetric lead sensor based on the assembly of gold nanoparticles by a Pb(2+)-dependent DNAzyme. However, heating to 50 degrees C followed by a cooling process of approximately 2 h was required to observe the color change. Herein we report a new improved design that allows fast (<10 min) detection of Pb(2+) at ambient temperature. This improvement of sensor performance is a result of detailed studies of the DNAzyme and nanoparticles, which identified "tail-to-tail" nanoparticle alignment, and large (42 nm diameter) nanoparticle size as the major determining factors in allowing fast color changes. The optimal conditions for other factors such as temperature (35 degrees C) and concentrations of the DNAzyme (2 microM), its substrate (3 nM), and NaCl (300 mM) have also been determined. These results demonstrate that fundamental understanding of the DNAzyme biochemistry and nanoparticle science can lead to dramatically improved colorimetric sensors.     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

Amplified fluorescence detection of mercury(II) ions (Hg2+) using target-induced DNAzyme cascade with catalytic and molecular beacons

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg(2+)) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg(2+)-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg(2+), the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction induced the two fragments to produce the activated Mg(2+)-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg(2+) sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg(2+) to the T-T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg(2+) detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg(2+) detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg(2+) from a wide range of real samples.     

Highly sensitive and selective colorimetric sensors for uranyl (UO2(2+)): development and comparison of labeled and label-free DNAzyme-gold nanoparticle systems

Colorimetric uranium sensors based on uranyl (UO2(2+)) specific DNAzyme and gold nanoparticles (AuNP) have been developed and demonstrated using both labeled and label-free methods. In the labeled method, a uranyl-specific DNAzyme was attached to AuNP, forming purple aggregates. The presence of uranyl induced disassembly of the DNAzyme functionalized AuNP aggregates, resulting in red individual AuNPs. Once assembled, such a "turn-on" sensor is highly stable, works in a single step at room temperature, and has a detection limit of 50 nM after 30 min of reaction time. The label-free method, on the other hand, utilizes the different adsorption properties of single-stranded and double-stranded DNA on AuNPs, which affects the stability of AuNPs in the presence of NaCl. The presence of uranyl resulted in cleavage of substrate by DNAzyme, releasing a single stranded DNA that can be adsorbed on AuNPs and protect them from aggregation. Taking advantage of this phenomenon, a "turn-off" sensor was developed, which is easy to control through reaction quenching and has 1 nM detection limit after 6 min of reaction at room temperature. Both sensors have excellent selectivity over other metal ions and have detection limits below the maximum contamination level of 130 nM for UO2(2+) in drinking water defined by the U.S. Environmental Protection Agency (EPA). This study represents the first direct systematic comparison of these two types of sensor methods using the same DNAzyme and AuNPs, making it possible to reveal advantages, disadvantages, versatility, limitations, and potential applications of each method. The results obtained not only allow practical sensing application for uranyl but also serve as a guide for choosing different methods for designing colorimetric sensors for other targets.     

Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer

The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ > Zn2+ >Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.     

Core-shell silica nanoparticles synthesized for quantitative study of DNA cleavage by laser-induced fluorescence microscopy

Dye-doped silica nanoparticles (C dots) were synthesized in reverse microemulsions and used to quantitatively examine DNA cleavage in the presence of transition metal ions. The cores were synthesized as fluorescein isothiocyanate (FITC)-doped silica nanoparticles and the shells' surfaces were modified with single-stranded DNA oligomers tagged with Cy5 fluorophores. DNA cleavage induced by heavy metal ions was estimated by comparing the fluorescence of Cy5 before and after reaction with metal ions. For this, a lab-built laser-induced fluorescence microscope equipped with a charge coupled device (CCD) camera, for imaging, and photomultiplier tube, for photon counting, was used. FITC fluorescence from the core was measured as an internal standard to compensate for possible loss of the beads during the treatment. The cleavage of DNA in air in the presence of Pb(2+), Cd(2+), and Hg(2+) at 1 ng/mL was found to be 14%, 6%, and 20%, respectively, and was significantly reduced to below 9% under N(2) gas, indicating that the main cleavage source was oxygen in air. The most significant DNA cleavage was observed with the addition of hydrogen peroxide. This analytical method using dye-doped C dots provided convenient handling and quantification of the estimation of metal-DNA interaction with a detection limit of 34.9 pmol/mL.     

A thin-layer chromatography plate prepared from BODIPY-based receptor immobilized SiO2 nanoparticles as a portable chemosensor for Pb2+

A new fluorescence receptor based on BODIPY-immobilized silica nanoparticles (BODIPY-SiO(2)) exhibits a high affinity and selectivity for Pb(2+) over competing metal ions in water. An overall emission change of ca. 100-fold at the emission maximum was observed for Pb(2+). The fluorescence receptor BODIPY-SiO(2) can remove 97% and 95% of the initial 100 ppb Pb(2+) from human blood and waste solution, respectively. Experiments show the fluorescence receptor BODIPY-SiO(2) can be a potentially useful and effective agent for the selective separation and rapid removal of Pb(2+)in vivo. We also prepared a portable chemosensor kit by coating a 4 μm thick film of BODIPY-SiO(2) onto a glass substrate. We found that this BODIPY-SiO(2) film detects Pb(2+) ions at pH 7.4 with a sensitivity of 3.2 nM. Finally, we tested the effect of pH on BODIPY-SiO(2) with Pb(2+) ions between pH 3.0 and 11.0. The fluorescence changes of BODIPY-SiO(2) were almost constant between pH 3 and 11. The results imply that the BODIPY-SiO(2) film is applicable as a portable chemosensor for detection of Pb(2+) ions in the environmental field.     

基于脱氧核酶的新型铅离子荧光探针

将荧光猝灭基团修饰的17E脱氧核酶(17E DNAzyme)与荧光基团修饰的底物链通过6个脱氧核苷酸相连, 得到了一种新型的对Pb2+敏感的荧光探针. 由于DNAzyme与底物链发生分子内杂交, 荧光基团与猝灭基团相互靠近, 导致荧光猝灭. 当Pb2+存在时, DNAzyme被激活, 底物链被切断后释放出荧光基团标记的DNA片段, 从而产生明显的荧光信号. 据此可在常温下快速检测Pb2+, 检测下限为10 nmol/L. 在Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Mg2+和Ni2+等多种二价金属离子中, 除Zn2+, Mn2+和Cd2+略有干扰外, 其它几种金属离子均无响应, 表明该荧光探针对Pb2+具有良好的选择性.     

A highly sensitive sensor for Cu2+ with unmodified gold nanoparticles and DNAzyme by using the dynamic light scattering technique

Copper ion (Cu(2+)) plays an important role in many biological reactions, and a suitable level of Cu(2+) is necessary for the regular metabolism of life. Thus developing a sensitive and simple method for determination of Cu(2+) is essential. Here, a novel and sensitive Cu(2+) sensor was developed based on detecting the average hydrodynamic diameter of AuNPs by using dynamic light scattering (DLS). Cu(2+)-specific DNAzyme was double-strand and could not adsorb on the surface of AuNPs, accordingly AuNPs aggregation would occur with the addition of NaCl. However, Cu(2+) could cleave DNAzyme and release single-stranded DNA (ssDNA) fragments, which could adsorb on the surface of AuNPs and prevent them from aggregation. Such differences in DNA adsorption ability on AuNPs before and after the addition of Cu(2+) affected the disperse state of AuNPs directly, and then affected their average hydrodynamic diameter, which could be detected with the DLS technique. Based upon the above mentioned principle, detection of Cu(2+) could be realized over the range from 100 pM to 2.0 nM, with a linear regression equation of D = 306.73 - 89.66C (C: nM, R = 0.9953) and a detection limit of 60 pM (3δ/slope). Moreover, satisfactory results were obtained when the assay was applied in the detection of Cu(2+) in water samples.     

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol?L^?1 and detect ATP specifically with a detection limit as low as 13 nmol?L^?1. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.     

Surface modified microarray chip and laser induced fluorescence microscopy to detect DNA cleavage

This work describes a quantitative method to detect DNA damage in the presence of Pb and Cd ions using a surface modified microarray chip and a laser induced fluorescence microscopy (LIFM). The detection was carried out by the immobilization of a single-stranded DNA oligomer, tagged with a Cy5 fluorophore on a polydimethylsiloxane (PDMS) microarray chip followed by LIFM. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) was attached as a cross-linker via the formation of covalent amide bonds. Then, the single-stranded DNA oligomer containing Cy5 as a fluorophore and thiol functional groups at both terminals, was bonded to the linker by reaction with sulfhydryl group. As the DNA oligomers were reacted with metal ions of Pb and Cd, the un-cleaved DNA oligomers were quantitatively identified by monitoring Cy5 fluorescence. Cadmium showed a quenching constant of 0.84 in the Stern–Volmer plot, whereas lead gave 0.22, indicating that cadmium ions suppress fluorescence more than lead ions. When optimized, fluorescence reductions of 23% (± 2.1) for Pb and 25% (± 1.4) for Cd were observed in air and decreased to almost < 5.0% in a radical scavenger of 5 mM. The cleaved DNA was also confirmed by MALDI-TOF-MS. In result, this experimental method using a microarray chip with surface modification provided quantitative determination of DNA oligomer damage with reproducible results, significantly reduced sample volumes and analysis times.     

DNAzyme crosslinked hydrogel: a new platform for visual detection of metal ions

We propose the use of DNAzyme as a crosslinker of hydrogel to develop a catalytic platform for the sensing of metal ions. The DNAzyme crosslinked hydrogel can undergo gel-sol transition in response to Cu(2+) ions, which enables sensitive visual detection of Cu(2+) by observing the release of pre-trapped AuNPs.     

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.     

Hetero-oligophenylene-based AIEE material as a multiple probe for biomolecules and metal ions to construct logic circuits: application in bioelectronics and chemionics

New hetero-oligophenylene derivative (2) was synthesized which exhibits aggregation-induced emission enhancement (AIEE) in H(2)O/THF (80:20). The aggregates serve as a biological probe for three different proteins, that is bovine serum albumin (BSA), cytochrome?c, and lysozyme, and DNA in contrasting modes. Further, among 29 metal ions tested, the contrasting fluorescence behavior of aggregates of 2 is observed with only Pb(2+) and Pd(2+) ions. Multiple output logic circuits based upon the fluorescence behavior between BSA and cytochrome?c and between Pb(2+) and Pd(2+) ions are constructed.