Detection of telomerase activity at the single-cell level is one of the central challenges in cancer diagnostics and therapy. Herein, we describe a facile and reliable point-of-care testing (POCT) strategy for detection of telomerase activity via a portable pressure meter. Telomerase primer (TS) was immobilized onto the surface of magnetic beads (MBs), and then was elongated to a long single-stranded DNA by telomerase. The elongated (TTAGGG)n repeat unit hybridized with several short PtNP-functionalized complementary DNA (PtNPs-cDNA), which specifically enriched PtNPs onto the surfaces of magnetic beads (MBs), which were separated using a magnet. Then, nanoparticle-catalyzed gas-generation reaction converted telomerase activity into significant change in gas pressure. Because of the self-amplification of telomerase and enrichment by magnetic separation, the diluted telomerase equivalent to a single HeLa cell was facilely detected. More importantly, the telomerase in the lysate of 1 HeLa cell can be reliably detected by monitoring change in gas pressure, indicating that it is feasible and possible to study differences between individual cells. The difference in relative activity between different kinds of cancer cells was easily and sensitively studied. Study of inhibition of telomerase activity demonstrated that our method has great potential in screening of telomerase-targeted antitumor drugs as well as in clinical diagnosis.

•A PIET strategy first proposed to study activity and inhibition of EcoRI endonuclease.•The real-time monitoring of enzymatic cleavage was facilely achieved.•G-quenching led to a specific and cost-effective assay.DNA digestion by endonucleases is significant in various biological and medicinal processes. Herein, we describe a facile and sensitive fluorescent assay for real-time studying endonuclease activity and inhibition based on the photo-induced electron transfer (PIET) between deoxyguanosines and a fluorophore. The carboxyfluorescein-labeled DNA (FDNA) hybridizes with its complementary DNA (cDNA) to form substrate of EcoRI endonuclease. Then, the fluorescence of FDNA was quenched by nearby deoxyguanosines due to PIET. In the presence of EcoRI endonuclease, DNA substrate was digested into short fragments. The fluorophore is far away from deoxyguanosines, leading to a recovery of FDNA fluorescence. The fluorescence enhancement efficiency was linear with logarithm of EcoRI concentration over the range of 5 × 10−5–1 × 10−2 U/μL, and the detection limit of was 4 × 10−5 U/μL, which was more sensitive than many previous reports. The influence of inhibitor on the activity of EcoRI endonuclease was facilely and reliably studied. More importantly, the activity of EcoRI endonuclease can be real-time monitored, which is vital for studying functionality and inhibition of endonuclease. The ingenious design of DNA probes ensures high specificity and low cost. Therefore, it offers a fast, sensitive, cost-effective and specific homogeneous analysis protocol for studying the activity and inhibition of EcoRI endonucleases.

The visual detection of a disease biomarker is a promising strategy due to its simplicity and low cost, but the complexity of biological samples limits its application. Herein, a reliable and ultrasensitive colorimetric assay was proposed for detecting telomerase activity in crude cancer cell extracts. A telomerase substrate (TS) primer was immobilized onto magnetic beads (MBs) to form a MB/TS complex. In the presence of telomerase and deoxyribonucleotide triphosphates (dNTPs), the TS primer was elongated by adding multiple telomeric repeats (TTAGGG)n to the 3′ end of the TS. The telomeric repeats of the elongated TS (ETS) hybridized with its short complementary DNA (cDNA) to specifically capture peroxidase onto MBs. After magnetic separation, the activity of telomerase was detected by monitoring the change in the color or absorbance of the peroxidase-catalyzed H2O2/TMB reaction. Magnetic separation greatly eliminated the non-specific interference to ensure reliability and improve the signal-to-noise (S/N) ratio. The mean telomerase activity equivalent to 5 HeLa cells and 1 HeLa cell was reliably detected with the naked eye and UV-vis spectroscopy, respectively. More importantly, the telomerase activity of 5 and 20 HeLa cells was detected via UV-vis spectroscopy and the naked eye, respectively. The differences in the telomerase activity of four carcinoma cell lines and one normal cell line were discriminated visually. Even more strikingly, the telomerase activity of 10 and 50 HeLa cells in human serum was detected by change in the absorbance and color of the TMB/H2O2 solution, respectively. Therefore, it offers an ultrasensitive and reliable colorimetric assay for the visual detection of telomerase activity, which holds promising potential to detect telomerase activity in a complex pathological sample.

To realize facile and reliable analyzing telomerase activity in homogeneous, herein, for the first time, a fluorescent polarization (FP) strategy was developed for polymerase chain reaction (PCR) free monitoring activity of human telomerase at single-cell level ground on gold nanoparticle (GNP) enhancement of FP. First, thiolated telomerase substrate (TS) primer is modified to the surface of GNP via Au–S bond. In the presence of telomerase, TS primer was extended via adding hexamer repeats (GGGTTA), leading to the formation of a long elongation DNA. Several short carboxyfluorescein (FAM)-modified complementary DNA (F-cDNA) can hybridize with the hexamer repeats, resulting in a sharp increase in FP value. Because of the GNP enhancement and self-amplification of telomerase, telomerase activity accounting to one HeLa cell can be rapidly detected in homogeneous solution. Telomerase activities of various cell lines were also favorably estimated. Meanwhile, the inhibition efficiency of telomerase inhibitor was studied, which holds great potential in screening telomerase-targeted anticancer drugs as well. So, a facile method was put forward to reliably and ultrasensitively detect telomerase activity.

Since the level of human telomerase RNA (hTR) in tumor cells is higher than that in normal somatic cells, the quantitative assay of hTR is of significant importance in tumor diagnosis. Herein, graphene oxide (GO) was simultaneously exploited as a fluorescence quencher and a carrier of nucleic acid to successfully deliver two hairpin DNA probes of hybridization chain reaction (HCR) into the cancer cell for detecting telomerase RNA based on DNA nanoassembly of HCR. The sticky end of HCR probes could tightly absorb on the surface of GO, resulting in fluorescence quenching of the dye which was tagged at the sticky end of two hairpin probes. When faced with hTR, the fluorescence of DNA probes is subsequently recovered because hTR could trigger HCR to autonomous assembly of a DNA polymer which released from the GO and led to fluorescence recovery. Taking advantage of nucleic acid nanoassembly of HCR, this intracellular HCR strategy creates enormous signal amplification, and enables ultra-sensitive fluorescence imaging of hTR expression. By monitoring fluorescence change, human telomerase RNA could be specifically studied and this method can also be used for detecting single-base mutation. The GO-aided HCR strategy allowed us to sensitively detect hTR in a living cell, which holds great potential for analyzing other low-abundance biomolecules in living cells via HCR.

G-quadruplex formation at the end of telomeres is one of the effective pathways for inhibiting the growth of cancer cells induced by up-regulation of telomerase. The lack of effective G-quadruplex-binding ligands has strongly limited the discovery of telomerase-targeted cancer drugs. To address these limitations, the development of a high-throughput screening technique is urgent and meaningful for the discovery of potent telomerase inhibitors from the large chemical libraries of candidates. Here, we present a fast and cost-effective method to realize high-throughout screening of G-quadruplex-binding ligands based on G-quadruplex formation induced fluorescence quenching. Carboxyfluorescein-labeled hairpin DNA (F-hpDNA) was designed as a molecular recognition probe. In the presence of G-quadruplex ligands, the conformation of the F-hpDNA switched from hairpin into G-quadruplex, leading to a significant decrease in the fluorescence of F-hpDNA due to photo-induced electron transfer (PIET) between fluorophore and G-quartet. So, ligands were selected by using the G-quadruplex as a natural fluorescence quencher, which means the DNA probe is just single-labeled. A variety of ligands can be simultaneously screened within several minutes. To verify the formation of G-quadruplex, circular dichroism (CD) analysis has been performed to study ligand-induced conformation change. Moreover, the fluorescence microscopy image and MTT cell proliferation assay all demonstrated that the selected ligand has the potential to inhibit the growth of cancer cells. For the first time, a high-throughout method based on PIET was established to quickly, cost-effectively and reliably identify G-quadruplex ligands, which is of great theoretical and practical importance for the discovery of telomerase-targeted anticancer drugs.

•A PCR-free, label-free fluorescence strategy was used for telomerase activity assay.•Combining SDR with formation of two G-quadruplexes ensures high sensitivity.•Hairpin DNAs were used to ensure the high specificity.•The method can measure telomerase activity down to 1 Hela cell.•Telomerase activities of different cell lines can be reliably and facilely evaluated.As a universal biomarker for cancer diagnostics and cancer therapeutics, telomerase has attracted extensive attention concerning its detection and discovery of its inhibitors. Herein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase elongation product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3′-azido-3′-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability.

•A PCR-free fluorescence strategy was proposed for telomerase activity.•Arch-structure DNA and MB ensure the reliability and specificity of signal transfer.•Telomerase activity equivalent to 5 HeLa cells and 10 CCRF-CEM cells were determined.•The strategy holds great promise for discovering potential telomerase inhibitors.As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its application has been significantly limited by amplification related errors and time-consuming procedure. To address the limitations of PCR-based protocol, a dual amplification fluorescence assay was developed for PCR-free detecting telomerase activity. Briefly, we designed an arch-structure DNA probe to specifically control strand displacement reaction and subsequent enzyme-aided amplification. Telomerase substrate (TS) primer was extended by telomerase to form long elongation products which contain several TTAGGG repeat units. So, one elongation product can release more than one trigger DNA (t-DNA) via strand displacement reaction to realize first amplification. Subsequently, t-DNA specifically opened molecular beacon (MB) to restore the fluorescence of MB. Meanwhile, t-DNA was recycled by the aid of nicking endonuclease to continuously open more and more MBs, leading to a second amplification. Owing to the double amplification strategy, the proposed method allowed the measurement of telomerase activity in crude cell extracts equivalent to 5 HeLa cells and 10 CCRF-CEM cells without PCR amplification. Besides, the influence of telomere-binding ligands on the telomerase activity demonstrated that the proposed method holds the potential to evaluate the inhibition efficiency of telomerase inhibitors.

Several fluorescence methods have been developed for sensitive detection of PNK activity based on signal amplification techniques, but they need fluorescently labeled DNA probes and superabundant assistant enzymes. We have addressed these limitations and report here a label-free and enzyme-free amplification strategy for sensitively and specifically studying PNK activity and inhibition via hybridization chain reaction (HCR). First, the phosphorylation of hairpin DNA H1 by T4 PNK makes it be specifically digested by lambda exonuclease (λ exo) from 5′ to 3′ direction to generate a single-stranded initiator which can successively open hairpins H2 and H3 to trigger an autonomous assembly of long DNA nanowires. Meanwhile, an intermolecular G-quadruplex is formed between H2 and H3, thereby providing fluorescence enhancement of N-methyl mesoporphyrin IX (NMM) which is a highly quadruplex-selective fluorophore. So, the PNK activity can be facilely and sensitively detected by using NMM as a signal probe which provides a low background signal to improve the overall sensitivity, resulting in the detection limit of 3.37 × 10−4 U mL−1. More importantly, its successful application for detecting PNK activity in a complex biological matrix and studying the inhibition effects of PNK inhibitors demonstrated that it provides a promising platform for screening PNK inhibitors as well as detecting PNK activity. Therefore, it is a highly sensitive, specific, reliable and cost-effective strategy which shows great potential for biological process research, drug discovery, and clinical diagnostics.

A label-free and enzyme-free amplification protocol has been proposed for studying endonuclease activity and inhibition on the basis of the enzyme-digested product triggered hybridization chain reaction (HCR). Three hairpin oligonucleotides were designed as probes which could not open or hybridize with each other at room temperature until the initiator DNA was released by specific enzymatic cleavage in the presence of endonuclease to trigger the hybridization chain reaction. SYBR Green I was chosen as a signal probe which intercalated into the grooves of the nicked double DNA polymer, generating a substantially apparent increase in fluorescence intensity. Once the activity of endonuclease is inhibited by enzyme inhibitors, the efficiency of HCR will be greatly decreased. Therefore, screening of endonuclease inhibitors can be achieved effectively as well as the assay of endonuclease activity. Meanwhile, the assay of endonuclease activity and inhibition achieves a better performance as compared to the previous reports. Importantly, it is a more universal method that can be simply used to study activity and inhibition of other endonucleases by changing the specific recognition site. So, the protocol was proved to be a sensitive and cost-effective approach for studying endonuclease activity and inhibition, and as such, it is promising for broad potential application in various biological reactions.

A facile, sensitive and rapid method has been developed for detection of disease-related DNA based on lambda exonuclease-aided signal amplification by utilizing graphene oxide (GO) as a fluorescence quencher. The fluorescence of the carboxyfluorescein-labeled DNA probe (F-DNA) was sharply quenched due to the electron or energy transfer between the fluorescence dye and GO. While in the presence of target DNA, the formation of a DNA hybrid released F-DNA from the surface of GO, leading to a fluorescence recovery. Then, the fluorescence enhancement was further amplified by using lambda exonuclease (λexo) to liberate target DNA for cyclic hybridization. Fluorescence polarization and gel electrophoresis further verified the reliability of the principle. Disease-related DNA can be sensitively detected based on the enzyme-aided amplification strategy. More importantly, single-base mismatched DNA can be effectively discriminated from complementary target DNA and random DNA. Therefore, it offered a universal, simple, sensitive and specific method for detection of disease-related genes.

Sensitive and specific detection of disease-related gene and single nucleotide polymorphism (SNP) is of great importance in cancer diagnosis. Here, a colorimetric and fluorescent approach is described for detection of the p53 gene and SNP in homogeneous solution by using gold nanorods (GNRs) as both colorimetric probe and fluorescence quencher. Hairpin oligonucleotide was utilized as DNA probe to ensure highly sequence-specific detection of target DNA. In the presence of target DNA, the formation of DNA duplex greatly changed the electrostatic interaction between GNR and DNAs, leading to an obvious change in fluorescence and colorimetric response. The detection limit of fluorescent and colorimetric assay is 0.26 pM and 0.3 nM, respectively. Both fluorescence and colorimetric strategies were able to effectively discriminate complementary DNA from single-base mismatched DNA, which is meaningful for cancer diagnosis. More important, target DNA can be detected as low as 10 nM by the naked eye. Furthermore, transmission electron microscopy and fluorescence anisotropy measurements demonstrated that the color change as well as fluorescence quenching is ascribed to the DNA hybridization-induced aggregation of GNRs. Therefore, the assay provided a fast, sensitive, cost-effective, and specific sensing platform for detecting disease-related gene and SNP.Keywords: colorimetric assay; fluorescence; gene mutation; gold nanorods; hairpin DNA;

A label-free fluorescent AND logic gate has been developed utilizing ion-tuned configuration conversion of DNA probe with K+ and Pb2+ as two inputs. A well-designed hairpin DNA which is composed of a poly-G loop and a GR-5 DNAzyme stem serves as a recognition probe, and an derivative of aloe-emodin (AED) was designed and synthesized as signal probe. In the presence of Pb2+, the substrate strand of DNAzyme is irreversibly and specifically cleaved at the cleavage site, which made the poly-G loop form G-quadruplex in the presence of a constant concentration of K+. Such a structural change significantly affects the spectral behaviors of AED, which can be explored to ultra-sensitively detect Pb2+ with a limit of detection of 22.8 pM. By combing the high specificity of hairpin DNA and GR-5 DNAzyme, Pb2+ can be highly selectively detected even when coexisted with other metal ions. Circular dichroism (CD), UV–vis absorption spectrometry and fluorescence polarization (FP) measurements further verified the reliability and reasonability of the sensing mechanism. Therefore, it provides a simple and label-free approach to detect ions with high sensitivity and specificity, and promises to provide a solid sensing platform for the detection of targets by altering the specific sequence of nucleic acid probe.Highlights► This method has high selectivity due to the high specificity of hairpin DNA and DNAzyme. ► DNA probe is label-free, resulting in cost-effective analysis and high activity of DNA. ► A new derivative of aloe emodin was designed and synthesized by us acts as signal probe. ► It has the advantages of quick response and high sensitivity compared with other strategies. ► It can be explored to detection of various analytes by change the stem sequence of hairpin DNA probe.

Specific and homogeneous detection of heavy metal ion is of great importance for both human health care and environmental protection. We reported a highly specific and sensitive assay for fluorescent detection of Pb2+ based on the difference in quenching ability between deoxyguanosines and G-quartet by using carboxyfluorescein-labeled hairpin DNA (F-hpDNA) as a recognition probe. In the absence of target, the fluorescence of F-hpDNA can be quenched through photoinduced electron transfer from the dye to deoxyguanosines because the formation of hairpin brings deoxyguanosines close to the FAM. In the presence of Pb2+, the formation of G-quadruplex DNA leads to a significant decrease in fluorescence due to the effective stack of dye on the G-quartet, which obviously intensified the quenching of fluorophore. In comparison with linear DNA probe, hairpin DNA probe greatly improved the specificity, and Pb2+ can be highly selective detected even when coexisted with other metal ions. The quenching efficiency is linear with the concentration of lead(II) over the range of 0.5–500 nM, with a limit of detection of 0.4 nM. Conformational switch from hairpin to G-quadruplex was verified by CD measurements. Moreover, the application for detection of real samples further demonstrated its reliability. Therefore, it is a selective, simple and sensitive approach for detection of lead ion, as such, it promises to provide a solid foundation for developing universal analytical method for heavy metal ions.

DNA cleavage reaction catalyzed by nucleases is essential in many important biological processes and medicinal chemistry. Therefore, it is important to develop reliable and facile methods to assay nuclease activity. With this goal in mind, we report a fluorescent assay for label-free, facile, and real-time monitoring of DNA cleavage by EcoRI endonuclease using SYBR Green I (SGI) as a signal probe. The fluorescence of SGI dramatically increased when the free SGI was mixed with double-stranded DNA (dsDNA) substrate. Upon interacting with EcoRI, which cleaves the dsDNA into small fragments, the weakened interaction between SGI and the shortened DNA fragments caused a decrease in fluorescence of SGI. EcoRI–DNA interaction was real-time studied by monitoring fluorescence change with the prolonging of interaction time. The important kinetic parameters, including Michaelis–Menten constant (KM) and maximum initial velocity (Vmax), were accurately calculated, which is consistent with previously reported studies. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis analysis, which indicated that this method is a simple and effective approach to assay DNA cleavage reaction. Specificity investigation demonstrated that EcoRI–DNA interactions can be studied with high selectivity. Compared with previously reported methods, this approach is selective, simple, convenient and cost-efficient without any labeling of the probe or of the target.

The G-rich overhang of human telomere tends to form a G-quadruplex structure, and G-quadruplex formation can effectively inhibit telomerase activity in most cancer cells. Therefore, it is important to identify the formation and properties of the G-quadruplex, with the particular aim of selecting G-quadruplex-binding ligands that could potentially lead to the development of anticancer therapeutic agents. With this goal in mind, we report a fluorescence resonance energy transfer (FRET) assay system for the identification of G-quadruplex ligands using DNA-functionalized gold nanoparticles (DNA-GNPs) as the fluorescence quencher and a carboxyfluorescein (FAM)-tagged human telomeric sequence (F-GDNA) as the recognition probe. A thiolated complementary strand of human telomeric DNA (cDNA), which first adheres to the surface of the GNPs and then hybridizes with F-GDNA, results in the fluorescence quenching of F-GDNA by the GNPs. However, fluorescence is restored when single-stranded F-GDNA folds into a G-quadruplex structure upon the binding of quadruplex ligands, leading to the release of F-GDNA from the surface of the GNPs. Combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to form a G-quadruplex. Therefore, this FRET G-quadruplex assay is a simple and effective approach to identify quadruplex-binding ligands, and, as such, it promises to provide a solid foundation for the development of novel anticancer therapeutic agents.

It is important to develop reliable and sensitive methods for assay of nuclease activity. With this goal in mind, we report a new strategy for nuclease assay by taking advantage of efficient fluorescence resonance energy transfer (FRET) between gold nanorods (GNRs) and fluorescein-tagged single-stranded DNA (FDNA). Upon mixing with GNRs, the FRET between positively charged GNRs and negatively charged FDNA caused a decrease in fluorescence of FDNA. The formation of FDNA/cDNA duplex further improved the FRET efficiency, leading to a significant decrease in fluorescence intensity. However, fluorescence is restored when FDNA1/cDNA1 hybrid was cleaved into small fragments by EcoRI endonucleases, resulting in a decrease in FRET efficiency because of weakened electrostatic interaction between GNRs and the shortened DNA fragments. Activity of EcoRI endonuclease has been real-time studied by monitoring fluorescence change with the prolonging of interaction time. Under optimized conditions, the cleaved fraction is linear with EcoRI concentration over the range of 1.0 × 10−3 to 1.0 × 10−1 U μL−1, with a limit of detection of 6.5 × 10−4 U μL−1 which is much better or at least comparable to previous reports. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis, fluorescence anisotropy and TEM analysis, which indicated that this method is a feasible and reasonable approach to study sequence-specific protein–DNA interactions. Assay of BamHI activity demonstrated that it is a more universally applied method for studying the activity of endonuclease. Furthermore, this fluorescence assay has been also used for studying the inhibition of EcoRI endonuclease activity. Importantly, experimental results suggested that endonuclease inhibitors can be screened by monitoring the change of fluorescence change. Therefore, this FRET assay is a simple, sensitive and effective approach to study endonuclease activity and inhibition, and as such, it promises to provide a feasible method to screen nuclease inhibitors.Highlights► We have developed a reliable and sensitive method to assay nuclease activity by taking advantage of efficient FRET between gold nanorods and carboxyfluorescein-tagged single-stranded DNA. ► The protocol also afforded a convenient approach for assay of nucleases inhibition in homogeneous solution without separation, precipitation, or washing. ► This new methodology can be easily extended to analyze other endonucleases and to screen their inhibitors. ► This assay only requires one dye labeled on the oligonucleotide probe, leading to less laborious and more cost-effective synthesis.

Lead poisoning in adults can affect the peripheral and central nervous systems, the kidneys, and blood pressure. Thus, the development of environment-friendly and simple methods for Pb2+ detection is of great importance. Herein, a label-free colorimetric method has been developed for the detection of Pb2+ based on the conformational switch from single-stranded DNA to G-quadruplex. The electrostatic interactions between DNA probe and gold nanorods (GNRs) induce GNRs to space closely. However, the electrostatic interaction is not strong enough to change the suspension state of GNRs. In the presence of Pb2+, the formation of G-quadruplexes increases the surface charge density around DNA, which is expected to strengthen the electrostatic interaction between the GNRs and the DNA. Therefore, the longitudinal absorption of GNRs decreased because the stronger interaction induced aggregation of GNRs. Importantly, the decrease in longitudinal absorption is proportional to concentration of Pb2+. By monitoring the change of absorbance, Pb2+ can be detected at a level of 3 nM with a linear range from 5 nM to 1 μM. The overall test only takes a few minutes and very little interference is observed from other metal ions. The major advantages of this method are its low cost, convenience, simplicity, sensitivity, and specificity.

A fluorescence method for detecting mercury ion in a homogeneous medium is proposed with gold nanorods (GNRs) as a fluorescence quencher on the basis of the fluorescence resonance energy transfer (FRET). Under the optimum conditions, the method exhibits a dynamic response range from 10 pM to 5 nM with a detection limit of 2.4 pM.

In this paper, we have reported a sensitive assay for fluorescence “turn-on” detection of Pb2+ 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 Pb2+ can be selectively detected. This method exhibits a high sensitivity for Pb2+ 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 Pb2+ detection. Moreover, this sensing system obtained satisfying results for Pb2+ detection in tap water samples.

Ligands that bind and stabilize G-quadruplex DNA structures are of significant interest because of their potential to inhibit telomerase and halt tumor cell proliferation. An electrochemical method was proposed to select natural G-quadruplex-binding ligands using ferrocene as the electrochemical indicator.

Single-stranded telomeric DNA tends to fold into G-quadruplexes made of a stack of G-quartets. Ligands that target G-quadruplex DNA have received considerable attention because of their potential anticancer activity. In this paper, we designed a fluorescence resonance energy transfer system to identify drugs that form G-quadruplexes with human telomeric DNA in a homogeneous medium using gold nanorods (GNRs) as fluorescence quencher. Fluorescein-tagged human telomeric DNA (F-GDNA) adsorbed on the surface of GNRs, which can quench the fluorescence of F-GDNA. In the presence of G-quadruplex-binding drugs, the formation of G-quadruplex further quenched the fluorescence of F-GDNA, which can be utilized to select G-quadruplex-binding ligands. Thirteen natural drugs were studied by this proposed method. G-quadruplex formation induced by drugs was investigated by circular dichroism measurements. Results of research indicated that it offers a simple, sensitive and effective method to identify ligands with potential anticancer activity.

Ligands of G-quadruplex have received great attention because of their potential anticancer activity. Here, we designed a label-free electrochemical method to identify electrochemically active molecules that stabilize G-quadruplexes in homogeneous medium. Daunorubicin (DNR) was used as model drug. In the presence of GDNA, the voltammetry of DNR changed. The binding constant (β) and binding-site number (m) of GDNA – mDNR are simultaneously obtained from dependence of the current on the amount of added GDNA in voltammetry, concluding that β = (2.37 ± 2.25) × 107 M− 1and m was near 1, respectively. Control experiment was performed, indicating that this method can specific identify G-quadruplex-binding ligand. The interaction mechanism of DNR with GDNA was also investigated by circular dichroism (CD). The result demonstrated that it is a simple and selective method for identification electroactive ligand with potential anticancer activity.

Monitoring proteins in real time and in homogeneous solution without using external labels has always been a difficult task. In this paper, we have developed a label-free method for the ultrasensitive detection of thrombin in homogeneous solutions. High-affinity thrombin-binding aptamer (TBA) used as molecular recognition probe, and fluorophore, crystal violet (CV), was chose as fluorescence signal probe. The fluorescence of CV enhanced significantly when the free CV solution was mixed with single-stranded TBA. In the presence of human thrombin, the fluorescence of CV decreased after the specific interaction between TBA and thrombin. Using the fluorescence change, we are able to selectively detect the thrombin in homogeneous solutions. The conformation transformation was investigated by circular dichroism (CD) spectra measurements. Our method has been shown to be simple and effective without any labelling of the probe or of the target, and this procedure poses minimum effects on the binding properties of the proteins. This assay is highly selective and ultrasensitive. Under the optimum conditions, the method exhibits a dynamic response range from 2 × 10−11 to 2 × 10−9 M with a detection limit of 8 × 10−12 M.

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences. Ligands that bind and stabilize G-quadruplex DNA structures are of significant interest because of their potential to inhibit telomerase and halt tumor cell proliferation. This paper demonstrated a label-free electrochemical method for the selection of quadruplex-binding ligands using the electroactive complex [Ru(NH3)6]3+ (RuHex) as signal transducer. The assay exploits the electrostatic interactions between RuHex and anionic phosphate backbones of DNA strands. In the presence of quadruplex-binding ligand, the oligonucleotide of the human telomeric sequence folded into a G-quadruplex structure, as a result, the adsorption amount of RuHex reduced and the peak current decreased. Six traditional Chinese medicine monomers were investigated as potential ligands using proposed method. To further study the interaction of GDNA with drugs, the competition between daidzein and complementary DNA of GDNA has been studied. The combined data from CD spectroscopy, melting curve and electrochemical measurements indicated that ligands selected by this electrochemical method could induce the GDNA folding into G-quadruplex. So this electrochemistry method offers a simple and effective approach to identify ligands with potential anticancer activity.

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. There is considerable interest in the design of ligands that target G-quadruplex DNA because of their potential anticancer activity. We designed a fluorescence resonance energy transfer (FRET) system to identify molecules that stabilize G-quadruplexes in a homogeneous medium using unmodified gold nanoparticles (GNPs) as a fluorescence quencher. The assay exploits the different adsorption abilities of GNPs for single-stranded DNA and double-stranded DNA. Fluorescein-tagged probe DNA adsorbed onto the surface of GNPs can quench the fluorescence of a DNA probe. Intramolecular folding of an oligonucleotide of the human telomeric sequence into a G-quadruplex structure led to fluorescence enhancement in the presence of quadruplex-binding ligands. G-quadruplex formation, induced by specific binding of GDNA ligands, was investigated by CD measurements. Melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the absorbance at 295 nm. Two series of natural drugs were studied, and flavonoids were shown to increase the melting temperature of the G-quadruplex. This increase in the Tm value was well-correlated with an increase in FRET efficiency. The combined data from fluorescence measurements and melting experiments indicate that the FRET approach offers a simple, sensitive, and effective method to identify ligands with potential anticancer activity.

A fluorescence method for detecting mercury ion in a homogeneous medium is proposed with gold nanorods (GNRs) as a fluorescence quencher on the basis of the fluorescence resonance energy transfer (FRET). Under the optimum conditions, the method exhibits a dynamic response range from 10 pM to 5 nM with a detection limit of 2.4 pM.

Ligands that bind and stabilize G-quadruplex DNA structures are of significant interest because of their potential to inhibit telomerase and halt tumor cell proliferation. An electrochemical method was proposed to select natural G-quadruplex-binding ligands using ferrocene as the electrochemical indicator.