Development of reliable and affordable quantitative methods for miRNAs with high specificity and sensitivity is a central challenge to make miRNA testing a routine part of medical care with respect to cancer. Herein, we propose a strategy for glucoamylase-encapsulated liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification (P-ERCA) for portable and accurate quantification of miRNA by using a glucometer readout.

An enzyme-free three-dimensional DNA walker powered by catalytic assembly has been constructed. Automatic movements of the walker were achieved through purely DNA hybridization. This DNA walking device provided a reliable, sensitive and selective strategy for nucleic acid and protein analysis.

•The DNA nanostructure can serve as efficient carrier in anticancer drug delivery.•Different types of DNA nanostructure-based anticancer drug delivery nanosystems are reviewed and summarized.•Active targeting, mutidrug co-delivery and construction of stimuli-responsive/intelligent nanosystems are classified.DNA as a novel biomaterial can be used to fabricate different kinds of DNA nanostructures based on its principle of GC/AT complementary base pairing. Studies have shown that DNA nanostructure is a nice drug carrier to overcome big obstacles existing in cancer therapy such as systemic toxicity and unsatisfied drug efficacy. Thus, different types of DNA nanostructure-based drug delivery nanosystems have been designed in cancer therapy. To improve treating efficacy, they are also developed into more functional drug delivery nanosystems. In recent years, some important progresses have been made. The objective of this review is to make a retrospect and summary about these different kinds of DNA nanostructure-based drug delivery nanosystems and their latest progresses: (1) active targeting; (2) mutidrug co-delivery; (3) construction of stimuli-responsive/intelligent nanosystems.Download high-res image (150KB)Download full-size image

•An autocatalytic cascade amplification strategy for folate receptor was proposed.•Sensitive detection of folate receptor was achieved with the range of 1.0 pM to 2.5 nM.•The maximally amplified signal was achieved with 40 min.•The strategy was applied for folate receptor analysis in spiked human serum samples.Folate receptor (FR) is over-expressed in most human tumors and has been regarded as biomarker and therapeutic target. Specific and sensitive detection of FR is essential for tumor treatment and drug development. Here, a specific, sensitive and rapid FR detection strategy was proposed based on terminal protection-mediated autocatalytic cascade amplification coupled with graphene oxide fluorescence switch. Firstly, the specific binding of FR to the folate terminally-labeled on a primary trigger DNA (PT-DNA) could protect the PT-DNA from exonuclease I degradation, converting FR detection to PT-DNA detection. Subsequently, the PT-DNA hybridized with the overhang of 3′-FAM labeled hairpin probe to initiate exonuclease III-assisted hydrolysis, accompanied with the PT-DNA recycling and autonomous generation of secondary trigger DNA and fluorophore. The secondary trigger DNA could as a PT-DNA analogue for the successive hybridization and hydrolysis process, liberating numerous fluorophores within 40 min. Finally, The fluorophores kept away from the surface of graphene oxide, achieving significantly amplified fluorescence signal. The specific interaction between FR and folate guaranteed the FR could be distinguished from other proteins with high selectivity. The high fluorescence quenching efficiency of graphene oxide guaranteed a low background. Due to the highly autocatalytic cascade amplification efficiency and low background, sensitive detection of FR had been achieved with the detection limit of 0.44 pM. The recoveries from 92% to 107% were achieved by detecting FR in spiked human serum. These results indicate this strategy holds a great potential for reliable quantification of FR in clinical diagnosis and disease treatment.Download high-res image (104KB)Download full-size image

Adenosine is a potent physiological and pharmacological regulator, and its abnormal level is closely related to disease development. The sensitive and specific detection of adenosine is crucial for health evaluation and disease diagnosis. In this work, a target triggered proximity combination-based fluorescence sensing strategy is developed for the sensitive and specific detection of adenosine. A difunctional probe showing target recognition and signal amplification is designed, by integration of DNA linker-connected split aptamer fragments with a fragment-elongated polymerase/nicking template. The presence of adenosine would glue the split aptamers, which triggers the two distal aptamer fragments to combine with each other into proximity. The approaching aptamer fragment ends then initiate the strand displacement amplification (SDA) reaction, generating numerous DNA primers. The DNA primers further hybridize with a padlock probe and initiate the rolling circle amplification (RCA) reaction, producing numerous G-quadruplex sequences. The G-quadruplex sequences finally bind with Thioflavin T to obtain enhanced fluorescence signals. The method exhibits a linear correlation within the adenosine concentration range from 5.0 × 10−7 M to 2.0 × 10−5 M (R = 0.999) with a detection limit of 8.4 × 10−8 M, and a good selectivity to distinguish adenosine from its analogues. The recoveries of adenosine in human serum are from 91% to 94%, demonstrating that the system works well in biological fluids. The proposed sensing strategy is anticipated to hold promise in biochemical research, clinical diagnosis and disease treatment.

Sensitive and specific detection of uracil-DNA glycosylase (UDG) activity is crucial in biomedical study and disease diagnosis. Here, we developed a uracil removal-inhibited ligase reaction in combination with catalytic hairpin assembly (CHA) for the sensitive and specific detection of UDG activity. A hairpin probe is specially designed, which contains two uracil bases in the loop and is extended with toehold and branch-migration domains at the ends of the stem. Two short oligonucleotides are separately hybridized to one-half of the loop of the hairpin probe to form a DNA complex with a nick. Under the action of UDG, two uracil bases in the hairpin-loop are removed to generate apurinic/apyrimidinic (AP) sites. The AP sites locating at the 3′-side of the nick inhibit the ligase reaction, leaving the toehold and branch-migration domains at the ends of the hairpin probe still adjacent. The adjacent toehold and branch-migration domains initiate CHA, producing numerous G-quadruplex (G4) structures, which interact with N-methyl-mesoporphyrin IX (NMM) to generate an enhanced fluorescence signal. The excessive probes would be masked by the ligase reaction that closes the nick and forms a long DNA strand fully complementary to the hairpin domain. The probes then get opened and the toehold/branch-migration domains are not associated, prohibiting the CHA reaction and minimizing false-positive interferences. The detection limit is as low as 0.00028 U mL−1, and UDG can be well distinguished from other DNA glycosylases. Furthermore, this method is successfully applied for detecting UDG activity from HeLa cell lysates. Additionally, the inhibition of UDG activity is analyzed, which shows inhibitor dose-dependent activity suppression. This strategy will provide a promising tool for assaying UDG activity in biomedical study and disease diagnosis.

Developing a sensitive and selective sensing platform for the p53 gene and its mutation analysis is essential and may aid in early cancer screening and assessment of prognosis. Here, we developed a highly sensitive and selective p53 gene assay based on the coupling of a triple-helix magnetic probe (THMP) to a fluorescent liposome hybridization assembly, a process initiated by rolling circle amplification (RCA). In the presence of p53, the THMP unfolds and activates an enzymatic cleavage reaction, thus releasing the RCA primer and initiating the RCA product-assisted fluorescent liposome hybridization assembly. The resultant double-stranded DNA structures bind the intercalating SG dye from the fluorescent liposomes, thus dramatically enhancing the fluorescence signal. In the absence of p53, the THMP remains intact and blocks the trigger release and fluorescent liposome assembly, thus resulting in a low background signal. The THMPs were designed with integrated target recognition by Watson–Crick base-pairing, site-specific cleavage by an endonuclease and background signal elimination by magnetic isolation, thus avoiding the need to design multiple probes. Moreover, the use of fluorescent liposome assembly and magnetic isolation helps in avoiding sample matrix interference and nonspecific staining. Through cooperative amplification coupling with enzyme cleavage recycling, the RCA-assisted fluorescent liposome assembly and magnetic isolation improved the sensitivity, with a detection limit of 0.07 fM. The excellent capacity of the THMP to specifically detect the involved targets and the precise site-specific endonuclease cleavage ensured remarkable selectivity for p53 against single-base mismatches. This proposed approach worked well in biological samples, thus demonstrating great potential for biomedical and clinical diagnosis applications.

Here we developed a simple, sensitive and accurate PLD detection method based on a target-controlled gating liposome (TCGL) “off–on” cascade amplified strategy and personal glucose meters (PGMs). It showed excellent sensitivity with a detection limit of 0.005 U L−1 and well performed PLD activity analysis in breast cancer cells and inhibitor drug screening.

An ultrasensitive and highly selective electrochemical assay was first attempted by combining the rolling circle amplification (RCA) reaction with poly(thymine)-templated copper nanoparticles (CuNPs) for cascade signal amplification. As proof of concept, prostate specific antigen (PSA) was selected as a model target. Using a gold nanoparticle (AuNP) as a carrier, we synthesized the primer–AuNP–aptamer bioconjugate for signal amplification by increasing the primer/aptamer ratio. The specific construction of primer–AuNP–aptamer/PSA/anti-PSA sandwich structure triggered the effective RCA reaction, in which thousands of tandem poly(thymine) repeats were generated and directly served as the specific templates for the subsequent CuNP formation. The signal readout was easily achieved by dissolving the RCA product-templated CuNPs and detecting the released copper ions with differential pulse stripping voltammetry. Because of the designed cascade signal amplification strategy, the newly developed method achieved a linear range of 0.05–500 fg/mL, with a remarkable detection limit of 0.020 ± 0.001 fg/mL PSA. Finally, the feasibility of the developed method for practical application was investigated by analyzing PSA in the real clinical human serum samples. The ultrasensitivity, specificity, convenience, and capability for analyzing the clinical samples demonstrate that this method has great potential for practical disease diagnosis applications.Keywords: electrochemical detection; poly(thymine)-templated copper nanoparticle; prostate specific antigen; rolling circle amplification; signal amplification

A novel fluorescence detection system was developed for DNA methyltransferase (MTase) activity assay based on a target-protected dumbbell molecular probe mediated cascade rolling circle amplification. It showed excellent specificity and sensitivity with a detection limit of 0.0024 U mL−1, and potential application in quantitatively monitoring MTase activity and screening of anticancer drugs.

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a “plug-and-play” fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil–DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil–DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

A wide range of analytical techniques in bioanalysis relies on surface-based biomolecular detection, which requires the confinement of probes onto a heterogeneous surface to react with targets. Probe arrangement on the interface is critical for target recognition and determines assay performance. Much effort has been devoted to screen the optimized probe arrangement according to experimental tests. Such a data-driven posteriori pattern faces low efficiency, ambiguous orientation, and possible deviated tested ranges from the best case. Herein, we demonstrate that a model can effectively guide probe arrangement onto the interface to facilitate probe–target recognition, embodied by the assay of human telomerase activity with DNA-conjugated gold nanoparticles (AuNPs). Both theoretical calculation and experimental results indicate that telomerase activity is maximized on the AuNP surface under guidance of the model. The detection limit is at least 1 order of magnitude lower than that of AuNP bearing densely packed DNA, comparable to that of the telomeric repeat amplification protocol (TRAP). The model-guided interface probe arrangement is proved to be highly useful in regulating interface recognition and may offer a new paradigm to promote surface-based biomolecular detection.

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL−1 for UDG and 0.11 U mL−1 for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

The sensitive detection of clinically significant DNA is of critical importance in early clinical diagnostics and medical research. Herein, we developed a sensitive fluorescent method for the detection of DNA fragments from the breast cancer 1 gene based on Au nanoparticles (AuNPs) fluorescence switch-mediated target recycling amplification. First, the designed FAM-labeled single-stranded DNA recognition probes at the 5′-terminus (donated as F-DNA) were adsorbed on the surface of AuNPs, followed by the substantial fluorescence quenching of the FAM. Then, the F-DNA specifically hybridized with the complementary region of the target DNA (T-DNA) and desorbed from the AuNPs surface, leading to the recovery of the partially quenched fluorescence. Finally, under the action of Exo III, T-DNA was released and hybridized with F-DNA for the target recycling; thereby large amounts of fluorescent probe fragments were obtained, resulting in significant fluorescent amplification for T-DNA detection. The proposed strategy exhibited a detection limit of 1.0 × 10−11 mol L−1 and good selectivity towards the mismatched T-DNA, which was better than or comparable to the existing nanomaterial-based fluorescent methods. The method possessed perfect recoveries in the human serum and cell lysate. Therefore, the proposed strategy would offer a new potential for quantification of specific DNA sequences in early clinical diagnostics and medical research.

The mercuric ion is a highly toxic contaminant and causes severe harm to the environment and human health. Herein, a T–Hg2+–T metallo-base pair-mediated dual amplification fluorescent strategy was proposed for the selective and sensitive detection of Hg2+ based on a target cycle and DNAzyme cycle. First, Hg2+ selectively bound with T–T mismatches in H-DNA and A-DNA to form stable T–Hg2+–T metallo-base pairs. This initiated the strand displacement between H-DNA and A-DNA to obtain the Hg2+-mediated partial double-stranded structure, with a blunt 3′-terminus of the opened H-DNA (donated as the Hg-complex). Next, under the action of Exo III, the Hg-complex was digested to release DNAzyme, A-DNA and Hg2+. The released Hg2+ could bind with another A-DNA and H-DNA, and the target cycle started anew, eventually generating numerous DNAzymes. DNAzymes then catalyzed the cleavage of a molecular beacon (MB) to generate free fluorophores. Upon cleavage, DNAzymes were released and continuously hybridized with another MB to trigger a second DNAzyme cycle. Finally, numerous fluorophores were liberated, resulting in a significantly amplified signal. The strategy showed a good linear relationship in the range from 2.0 × 10−10 mol L−1 to 1.0 × 10−8 mol L−1, with a detection limit of 7.2 × 10−11 mol L−1. The proposed strategy exhibited remarkable selectivity towards Hg2+ against other metal ions. Furthermore, this strategy was successfully applied to detect Hg2+ in real water samples. The proposed strategy provided a reliably quantitative candidate for potential application in environmental monitoring and biotoxicity analysis.

Transcription factors (TFs) are DNA-binding proteins that regulate gene transcription and their expression levels are closely associated with disease development. Sensitive and specific detection of TFs is significant to clinical diagnostics and drug development. Herein, a label-free fluorescent strategy for the sensitive and specific detection of TFs was developed based on protein binding-protected DNA three-way junction (TWJ)-mediated rolling circle amplification (RCA). A trifunctional TWJ was designed including a target binding site, AlwI recognition site and sutured primer of RCA. Firstly, TFs bound with a target binding site, protecting the four and five bases downstream from the AlwI recognition site against cleavage by AlwI. Next, the sutured primer in the protected TWJ hybridized with the padlock probe, initiating RCA. Finally, the sutured primer was extended with multiple G-quadruplex sequences, binding with N-methyl-mesoporphyrin IX (NMM) to yield an enhanced fluorescence response. Residual TWJs were digested by AlwI, effectively blocking the RCA reaction, thus suppressing nonspecific amplification. Taking NF-κB p50 as a model target of TFs, high sensitivity was achieved with a low detection limit of 6.8 pM and a broad linear range from 8 pM to 15 nM. We successfully measured NF-κB p50 in HeLa cell nuclear extracts with a low detection limit of 0.34 ng μL−1. The strategy was also effectively used to assay the inhibition effect of a model inhibitor of NF-κB, oridonin. The results indicated the proposed strategy holds great promise for studying TFs in disease diagnostics and drug developments.

•A dual amplification strategy for detection of M.SssI MTase activity was proposed.•A trifunctional double-stranded DNA probe was designed.•The strategy could detect M.SssI MTase activity as low as 0.0082 U/mL.•It showed a good selectivity and could detect target in complex biological matrix.•The strategy provided a potential tool for early cancer diagnosis and therapeutics.DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8–17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8–17 DNAzymes. Finally, the released 8–17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082 U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2′-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

Transcription factors (TFs) play pivotal roles in the regulation of a variety of essential cellular processes and some of them have been recognized as potential diagnostic markers and therapeutic targets of some diseases. Sensitive and accurate detection of TFs is of great importance to better understanding their roles in gene regulation and evaluation of disease state. Here, we developed a simple, label-free and enzyme-free new fluorescent strategy for the detection of TFs by graphene oxide (GO) fluorescence switch-based multifunctional G-quadruplex-hairpin probe (MGHP). The MGHP possessed of three functions simultaneously, adsorbing onto GO with the loop part, binding to target with the stem part and serving as signal carrier with the terminal G-quadruplex. First, the MGHP was adsorbed quickly to GO. Next, the TF bound to the stem part of MGHP to form a huge target-MGHP complex, which led to desorption of the complex from GO. Finally, NMM was inserted into G-quadruplex in the complex to yield an enhanced fluorescence response. The GO used here, as a fluorescence switch, could quickly and efficiently quench the fluorescence of NMM inserted into the MGHP absorbed on the GO, guaranteeing a high signal-to-noise ratio. Sensitive detection of purified NF-κB p50 and HeLa cell nuclear extracts were achieved with detection limits of 0.2 nM and 7.8 ng/µL, respectively. Moreover, this proposed strategy could be used to screen inhibitors of NF-κB p50 activity. The strategy proposed here might offer a new potential approach for reliable quantification of TFs in clinical diagnostics and treatment research of some diseases.

We developed a one-pot label-free molecular beacon-mediated quadratic isothermal exponential amplification strategy (LFMB-QIEA) for simple, rapid and sensitive DNA methyltransferase (MTase) activity detection.

A novel toehold activation strategy was developed based on hairpin-reconfiguration which was initiated by the interaction between the hairpin and different environmental stimuli (Hg2+ or ATP). Through variation of the concentration of environmental stimuli, it allowed the fine control of the DNA strand displacement rates almost 2 orders of magnitude.

Effective detection of nicotinamide adenine dinucleotide (NAD+) is crucial for better understanding its roles in biological process and further validating its function in clinical diagnosis. Herein, we developed a ligation-triggered exonuclease III (Exo III)-assisted signal amplification strategy for label-free and sensitive detection of NAD+. We ingeniously designed an oligo1–oligo2–cDNA double-strand DNA (dsDNA) probe and a G-quadruplex (G4)-template dsDNA probe. In the presence of NAD+, oligo1–oligo2–cDNA dsDNA probe containing a ligatable nick was ligated by E. coli DNA ligase and formed a complete trigger DNA (tDNA). Then, Exo III digested the ligated oligo1–oligo2–cDNA dsDNA probe and released the formed tDNA. Successively, tDNA hybridized with the G4-template dsDNA probe and initiated the Exo III-assisted cycling cleavage, ultimately releasing tDNA and G4 DNA. Finally, the released large amounts of G4 DNA interacted with N-methylmesoporphyrin IX (NMM) to form G4–NMM complex, generating a label-free and enhanced fluorescence signal. The proposed assay provided a detection limit as low as 3.0 pM and exhibited high selectivity against NAD+ analogues. Thus, the strategy provided a facile and convenient tool for sensitive quantification of NAD+ in NAD+ related biological processes and clinical diagnosis.

•A sensitive fluorescence protein assay was proposed based on cooperative DNA machine.•The DNA machine achieved cooperatively mediation of two amplification process.•The strategy could detect folate receptor as low as 0.23 pM.•The method was label-free, and it was a potential tool for clinical diagnosis.Sensitive detection of protein is essential for both molecular diagnostics and biomedical research. Here, taking folate receptor as the model analyte, we developed a label-free and dual-amplified strategy via small molecular-ligand linked DNA and a cooperative DNA machine which could perform primary amplification and mediate secondary amplification simultaneously. Firstly, the specific binding of folate receptor to the small-molecule folate which linked to a trigger DNA could protect the trigger DNA from exonuclease I digestion, translating folate receptor detection into trigger DNA detection. Subsequently, trigger DNA initiated the DNA machine through hybridizing with the hairpin of the DNA machine, resulting in hairpin conformational change and stem open. The open stem further hybridized with a primer which initiated circular strand-displacement polymerization reaction; meanwhile the rolling circle amplification templates which were initially blocked in the DNA machine were liberated to mediate rolling circle amplification. In such a working model, the DNA machine achieved cooperatively controlling circular strand-displacement polymerization reaction and rolling circle amplification, realizing dual-amplification. Finally, the rolling circle amplification process synthesized a long repeated G-quadruplex sequence, which strongly interacted with N-methyl mesoporphyrin IX, bringing label-free fluorescence signal. This strategy could detect folate receptor as low as 0.23 pM. A recovery over 90% was obtained when folate receptor was detected in spiked human serum, demonstrating the feasibility of this detection strategy in biological samples.

•A fluorescence amplification strategy for detection of UDG activity was proposed.•A unique dsDNA probe and a label-free and enzyme-free DNA machine were designed.•The background reduction and signal amplification were achieved.•The strategy could detect UDG activity as low as 0.00044 U/mL.•A potential tool for UDG and UGI functional study and clinical diagnosis was provided.Sensitive detection of uracil-DNA glycosylase (UDG) activity is critical for function study of UDG and clinical diagnosis. Here, we developed a novel fluorescent strategy for sensitive detection of UDG activity based on the signal amplification by a label-free and enzyme-free DNA machine. A double-strand DNA (dsDNA) probe P1–P2 with uracil bases and trigger sequence was designed for UDG recognition and signal transduction. Two hairpin probes H1 and H2 which were partially complementary were employed to construct the label-free and enzyme-free DNA machine. Under the action of UDG, uracil bases were removed from the P1-P2 dsDNA probe, and then a strand P2ʹ with abasic sites was released. Subsequently, the liberated P2ʹ activated the DNA machine and generated numerous H1–H2 complexes containing G-quadruplex (G4) structures in the end. Finally, the G4 structures could bind with N-methylmesoporphyrin IX (NMM) to form G4-NMM complexes with the enhanced fluorescence responses. This strategy could detect UDG activity as low as 0.00044 U/mL. In addition, the strategy was also applied for the analysis of UDG activity in HeLa cells lysate with low effect of cellular components. Moreover, this strategy was successfully applied for assaying the inhibition of UDG using uracil glycosylase inhibitor (UGI). This strategy provided a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis.

•A Fok I cleavage–inhibition strategy was proposed for transcription factor detection.•Cleavage activity of Fok I can be inhibited by transcription factor–DNA complex.•Fok I–DNA interaction converted the protein signal to the DNA signal.•The specificity of Fok I–DNA interaction ensured the detection accuracy.Specific and accurate detection of transcription factors is critical for disease diagnosis and drug development. Here, we developed a novel and versatile fluorescent sensing strategy for specific and accurate detection of transcription factors based on the inhibition of endonuclease Fok I-catalyzed DNA cleavage reaction. A FAM-labeled double-stranded DNA probe (dsDNA probe) with transcription factor binding site and Fok I recognition site was designed for target recognition and signal transduction. With the binding of transcription factors, the dsDNA probes are protected from cleavage by Fok I. These protected dsDNA probes then cannot hybridize with the added BHQ-DNA, keeping the fluorescence of FAM in an on state. However, in the absence of targets, the dsDNA probes were cleaved to release the FAM-DNA, which subsequently hybridized with BHQ-DNA, resulting in the fluorescence of the FAM being quenched. With the Fok I–DNA interaction that can specifically recognize the transcription factor-DNA binding and accurately convert the detection of transcription factors to the detection of DNA, the proposed strategy realized the reliable detection of model target NF-κB p50 with a nanomolar detection limit. This strategy was also employed to detect the inhibition effect of oridonin, a known inhibitor of NF-κB. Furthermore, perfect recoveries were obtained when detecting the targets in HeLa cells lysate, demonstrating the feasibility of this strategy for transcription factor detection in biological samples.

•A fluorescent method was developed for human immunodeficiency virus type 1 DNA assay.•The method was based on a label-free DNA templated Ag NCs fluorescent probe.•The DNA templated Ag NCs fluorescent probe was separating from recognition process.Based on DNA templated Ag NCs (DNA/Ag NCs) fluorescent probe, a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1 (HIV-1) DNA. Firstly, a hairpin probe, containing target DNA recognition sequence and guanine-rich sequence, was designed to hybridize with the target DNA and form a blunt 3′-terminus DNA duplex. Then, exonuclease III (Exo III) was employed to stepwise hydrolyze the mononucleotides from formed blunt 3′-terminus DNA duplex, releasing the target DNA and guanine-rich sequence. Finally, DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence, leading to an enhanced fluorescence signal for detection. The proposed method could detect as low as 2.9×10−10 mol L−1 HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA. Furthermore, the method possessed perfect recoveries in cells lysate and human serum, showing potential to be used in biological samples.

Based on a Ag+-stabilized self-assembly triplex DNA molecular switch (Ag+-STDMS), a simple, enzyme-free and sensitive new fluorescent strategy for detection of transcription factors was developed, achieving high sensitivity towards purified targets and real biological samples.

A sensitive and label-free fluorescence assay for DNA detection has been developed based on cascade signal amplification combining exonuclease III (Exo III)-catalyzed recycling with rolling circle amplification. In this assay, probe DNA hybridized with template DNA was coupled onto magnetic nanoparticles to prepare a magnetic bead-probe (MNB-probe)–template complex. The complex could hybridize with the target DNA, which transformed the protruding 3′ terminus of template DNA into a blunt end. Exo III could then digest template DNA, liberating the MNB-probe and target DNA. The intact target DNA then hybridized with other templates and released more MNB-probes. The liberated MNB-probe captured the primer, circular DNA and then initiated the rolling circle amplification (RCA) reaction, realizing a cascade signal amplification. Using this cascade amplification strategy, a sensitive DNA detection method was developed which was superior to many existing Exo III-based signal amplification methods. Moreover, N-methyl mesoporphyrin IX, which had a pronounced structural selectivity for the G-quadruplex, was used to combine with the G-quadruplex RCA products and generate a fluorescence signal, avoiding the need for any fluorophore-label probes. The spike and recovery experiments in a human serum sample indicated that our assay also had great potential for DNA detection in real biological samples.

•TSDR triggered DNA amplification for single-base mutations detection was developed.•The selective and sensitive method has potential for clinical application.•The method could be performed in homogeneous format without fluorescent labeling.Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples.

•A high sensitive fluoroimmunoassay based on a dual amplification was developed for detection of protein.•The strategy exhibited ultrahigh sensitivity and potential for clinical application.•The method could be extended to detect a wide range of trace proteins.An ultrasensitive fluorescence method for determination of protein is developed based on hybridization chain reaction (HCR). In this assay, the streptavidin-magnetic nanobeads were conjugated to biotinylated initiators and biotinylated anti-IgG. In the presence of human IgG, the magnetic nanobeads were fixed on the substrate and the carried initiators propagated the chain reaction of hybridization to form the nicked polymers. Because the nanobead probe carries with a large number of oligonucleotides per protein binding event, there is obvious amplification in the nicked polymers. Then, numerous SYBR Green I molecules were intercalated into the grooves of the long dsDNA polymers, generating a substantially apparent increase in the corresponding fluorescence intensity. With HCR amplification and magenetic nanobead to preamplify the fluorescence signal and reduce the background signal, the detection limit of this assay was 14 aM. Compared with the reported protein detection methods, our method exhibited ultrahigh sensitivity. In addition, the proposed method possessed excellent selectivity and low matrix effect. What is more, the assay was also studied for clinical application in human serum with a satisfactory and reliable result.

•We assembled nanolabels by inserting SYBR Green I into DNA tetrahedron nanostructure.•The nanolabel shows good photostability and biocompatiblity, and exhibits no blinking.•The nanolabel was used as a detectable signal in single-molecule counting to detect the human IgG.•This method has an excellent specificity and a low matrix effect.A highly sensitive method for single-molecule quantitative detection of human IgG is presented by the employment of a new fluorescent nanolabel. In this method, fluorescent nanolabels were assembled by inserting SYBR Green I into DNA tetrahedron nanostructure. The bio-nanolabels were attached to the streptavidin-antihuman antibody by a specific reaction between biotin and streptavidin. The antibody was combined with the target antigen, human IgG, which was immobilized on the silanized glass subtrate surface. Finally, epi-fluorescence microscopy (EFM) coupled with an electron multiplying charge-coupled device was employed for fluorescence imaging. The fluorescent spots corresponding to single protein molecule on images were counted and further used for the quantitative detection. It was found that the new nanolabel shows good photostability, biocompatiblity and exhibits no blinking compared to traditional labels like fluorescence dyes and quantum dot (QDs). In addition, the number of fluorescence spots on the images has a linear relationship with the concentration of human IgG in the range of 3.0×10−14 to 1.0×10−12 mol L−1. What is more, this method showed an excellent specificity and a low matrix effect.

A novel label-free fluorescent sensing scheme for sensitive and selective detection of NAD+ and ATP has been developed based on dumbbell probe-mediated rolling circle amplification (D-RCA)-responsive G-quadruplex formation. This approach can detect 0.5 pM for ATP and 100 fM for NAD+, much lower than those of previously reported biosensors, and exhibits high discrimination ability.

Porous CuO–Ag nanofibers have been synthesized via a combination of the electrospinning technique and the PVP-assisted sol–gel technique in the absence of any templates. The thermal behavior of the as-spun fibers was characterized by TG-DSC while FT-IR was employed to prove the removal of the polymer, which resulted in a porous structure. The structure and morphology of the resultant products were characterized by XRD, TEM and SEM. Porous CuO–Ag nanofibers fabricated on a Si substrate exhibited large Raman enhancement ability, which was also investigated. The SERS signals on the obtained substrate had perfect temporal stability under continuous laser radiation and a good uniform response, enabling quantitative detection of analyte azobenzene. The log–log plot of the SERS intensity to the azobenzene concentration exhibited a good linear relationship.

This work reports a novel signal amplification method for Hg2+ detection based on quantum dots (QDs) and nicking endonuclease (NEase). In this assay, streptavidin-coated QDs were conjugated to biotinylated hairpin-shaped probe A which was modified with a quencher BHQ-2. The fluorescence of the QDs was quenched by BHQ-2 through FRET quenching. In the presence of Hg2+, probe B hybridized with the loop of probe A due to specific binding between thymine–thymine mismatches and Hg2+, thus probe A was opened. Then NEase recognized specific nucleotide sequences and cleaved probe A. After the dissociation of probe A fragments, the distance between QDs and BHQ-2 increased, which led to increase of QDs fluorescence. The released Hg2+ and probe B could hybridize with another probe A to start a new cycle, therefore a remarkable signal amplification was achieved. Under optimal conditions, the detection limit of this assay was 8.0×10−10 mol L−1.Highlights► A signal amplification method based on QDs and NEase was developed to detect Hg2+. ► The assay exhibited good sensitivity with a detection limit of 8.0×10−10 mol L−1. ► The assay exhibited good selectivity due to Hg2+ and T–T mismatch specific binding.

A novel enzyme-free and label-free fluorescence aptasensor based on target-catalyzed hairpin self-assembly is developed for amplified detection of adenosine. This aptasensor contains four DNA strands termed as aptamer-catalysis strand, inhibit strand, hairpin structures H1 and H2 which are partially complementary. Meanwhile, a sequence that can form DNA G-quadruplex is partly hidden in the stem of H2. In the absence of adenosine, aptamer-catalysis strand is inhibited, and cannot trigger the self-assembly between H1 and H2. Upon the addition of adenosine, the binding event of aptamer and adenosine triggers the self-assembly between H1 and H2, resulting in the formation of G-quadruplex at the end of H1–H2 complex. The addition of N-methyl mesoporphyrin IX, which has a pronounced structural selectivity for G-quadruplex, generates label-free fluorescence signal. In the optimum conditions, we could detect adenosine as low as 6 μM by monitoring the change in fluorescence intensity. Furthermore, this amplified aptasensor shows high selectivity toward adenosine against its analogs due to the specific recognition ability of the aptamer for the target. Thus, the proposed aptasensor could be used as a simple and selective platform for target detection.Highlights► A novel fluorescence amplified aptasensor was developed. ► The aptasensor was based on target-catalyzed self-hairpin assembly. ► The aptasensor was enzyme-free and label-free, making it simple and inexpensive. ► The limit of detection of the aptasensor for adenosine was 6 μM. ► This strategy provided a simple, rapid and high selective versatile platform.

We report a label-free fluorescent strategy to study the distribution and stability of DNA origami nanostructures in live, cellular systems, using carbazole-based biscyanine as a probe molecule which has the characteristic property of restriction of intramolecular rotation (RIR) induced emission.

A single DNA molecule detection method on DNA tetrahedron decorated substrates has been developed. DNA tetrahedra were introduced onto substrates for both preventing nonspecific adsorption and sensitive recognition of single DNA molecules.

A novel rolling circle amplification (RCA) immunoassay based on DNA enriching magnetic nanoparticles and assembled fluorescent DNA nanotags, magnetic nanoparticles-RCA immunoassay, is developed as a versatile fluorescence assay platform for highly sensitive proteins detection.

This study effectively demonstrates a strategy to enable the ferricyanide-based second-generation biosensors for selective in vivo measurements of neurochemicals, with glucose as an example. The strategy is based on regulation of redox potential of ferricyanide mediator by carefully controlling the different adsorption ability of ferricyanide (Fe(CN)63-) and ferrocyanide (Fe(CN)64-) onto electrode surface. To realize the negative shift of the redox potential of Fe(CN)63-/4-, imidazolium-based polymer (Pim) is synthesized and used as a matrix for surface adsorption of Fe(CN)63-/4- due to its stronger interaction with Fe(CN)63- than with Fe(CN)64-. The different adsorption ability of Fe(CN)63- and Fe(CN)64- onto electrodes modified with a composite of Pim and multiwalled carbon nanotubes (MWNTs) eventually enables the stable surface adsorption of both species to generate integrated biosensors and, more importantly, leads to a negative shift of the redox potential of the surface-confined redox mediator. Using glucose oxidase (GOD) as the model biorecognition units, we demonstrate the validity of the ferricyanide-based second-generation biosensors for selective in vivo neurochemical measurements. We find that the biosensors developed with the strategy demonstrated in this study can be used well as the selective detector for continuous online detection of striatum glucose of guinea pigs, by integration with in vivo microdialysis. This study essentially paves a new avenue to developing electrochemical biosensors effectively for in vivo neurochemical measurements, which is envisaged to be of great importance in understanding the molecular basis of physiological and pathological events.

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin–streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.

The reduction of graphene oxide (GO) can be induced by the photolysis of silver or gold ions under UV light, resulting in the formation of G/M (M=Ag/Au) hybrid nanomaterials. This one-pot reduction method was performed using GO aqueous solution and silver ammonia solution or chloroauric acid solution under UV light. No additional chemical reductant was involved during the synthetic process. The existence of Ag nanoparticles on the surface of graphene nanosheets and the reduction of GO were characterized by XRD, TEM, SAED, FTIR and XPS. The obtained hybrid nanomaterial exhibits SERS activity and thus has a potential application as SERS substrate.Highlights► G/M (M=Ag/Au) were obtained under ultraviolet light without any additional reductant. ► Photolysis of M ions under ultraviolet light induced reduction of GO simultaneously. ► The existence of Ag nanoparticles on graphene was characterized by XRD, TEM and SAED. ► The reduction of GO was characterized by FTIR and XPS. ► G/Ag film exhibits SERS activity and has a potential application as SERS substrate.

A novel and simple method was presented for micrococcal nuclease (MNase) detection based on fluorescence resonance energy transfer (FRET) realized by electrostatic interaction. In this study, mercaptoacetic acid capped quantum dots (MAA-QDs) and ROX-modified single-stranded DNA (ROX-ssDNA) were chosen as energy donor and acceptor, respectively. At slightly basic pH, the positively charged peptide served as a bridge to bring negatively charged QDs and negatively charged ROX-ssDNA into close contact to energy transfer. When the ROX-ssDNA was cleaved into small fragments by MNase, the relatively weak electrostatic interaction between the fragmented ssDNA chains and the QDs/peptide complex should make the ROX away from the QDs/peptide complex, and thus the FRET efficiency decreased. Consequently, the fluorescence intensity of acceptor decreased and a quantification of the MNase was enabled. Under the optimal conditions, experimental results showed that the fluorescence intensity of acceptor was proportional to the logarithm of MNase concentration in a range of 4.0×10−3–8.0×10−2 U mL−1. The proposed approach offered adequate sensitivity for the detection of the MNase at 2.9×10−3 U mL−1.Highlights► A fluorescence resonance energy transfer system based on electrostatic interaction was developed. ► The system was applied for the detection of micrococcal nuclease. ► This method did not require covalent modification to the quantum dots and doubly labeled DNA strand.

A sensitive and selective method for the paraoxon detection based on enzyme inhibition and fluorescence quenching was presented in this study. Under the catalytic effect of acetylcholinesterase (AChE), acetylthiocholine (ATCh) hydrolysis released thiocholine (TCh) which could react with N-(7-dimethylamino-4-methylcoumarin-3-yl) maleimide (DACM) to produce a blue fluorescence compound. Subsequently, AChE catalytic activity was inhibited with the addition of paraoxon, which caused TCh decreased, leading to a significant decrease of the blue fluorescent compound. Meanwhile, p-nitrophenol, the hydrolysis product of paraoxon, would lead to a quenching of the fluorescence. Therefore, fluorescence intensity of the system would decrease dramatically by a combined effect of enzyme inhibition and fluorescence quenching. Under optimal experimental conditions, an excellent linear relationship between the decrease of fluorescence intensity and paraoxon concentration over the range from 5.5 × 10−12 to 1.8 × 10−10 mol L−1 was obtained. Fluorescence background caused by nonenzymatic hydrolysis of ATCh or other matters was relatively low, the proposed approach offered adequate sensitivity for the detection of paraoxon at 3.5 × 10−12 mol L−1.

A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 × 10−14–3.0 × 10−12 mol L−1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

An ultrasensitive fluorescence immunoassay method for quantitative detection of single molecules is developed on the basis of counting single magnetic nanobeads (MNBs) with combined amplification of DNA and dye/DNA conjugate. Highly amplified fluorescence signal and low background signal are achieved by using mutilabel bioconjugates made by linking multiple dye/DNA conjugates to streptavidin-coated magnetic nanobeads (SA-MNBs) and magnetic separation. In this method, human IgG (Ag) is captured on the silanized glass substrate surface, followed by immunoreaction with biotinylated mouse antihuman antibody (BT-Ab). Then, SA-MNBs are attached to the BT-Ab through the biotin/streptavidin interaction at a ratio of 1:1. Subsequently, a 30 base pair double-stranded oligonucleotide terminated with biotin (BT-dsDNA) is conjugated to the SA-MNBs. The resultant Ag-BT-Ab-SA-MNBs/BT-dsDNA/SYBR Green I is achieved after a fluorescent DNA probe, SYBR Green I, is added to the substrate and bound to the oligonucleotide at high ratios. Finally, epifluorescence microscopy coupled with a high-sensitivity electron multiplying charge-coupled device is employed for human IgG fluorescence imaging and detection. The number of fluorescent spots corresponding to single protein molecules on the images is counted. It is found that the number of fluorescent spots resulting from the SA-MNBs/BT-dsDNA/SYBR Green I immuotargeted on the glass slides is correlated with the concentration of human IgG target antigen in the range 3.0−50 fM.

A novel co-luminescence system based on the formation of a complex between europium (III) (Eu3+) and gatifloxacin (GFLX) in sodium dodecylbenzene sulfonate (SDBS) micelle solution containing lanthanum (III) (La3+) has been developed for the determination of Eu3+. The experimental results show that the complex formed by Eu3+ and GFLX here can emit the characteristic luminescence of Eu3+. With the addition of La3+, the luminescence intensity of the system was enhanced about 7-fold compared with that without La3+. Under the optimal conditions, the luminescence intensity exhibits an excellent linear relationship with Eu3+ concentration in the range of 1.0×10−10–5.0×10−8 mol L−1. The correlation coefficient (r) is 0.9998, and the detection limit (3σ) is 7.0×10−14 mol L−1. A test method with satisfactory accuracy based on this system was applied to determine trace amounts of Eu3+ in rare earth samples. In addition, the detailed luminescence mechanism of this system was investigated by analyzing the ultraviolet absorption spectra, surface tension, fluorescence polarization, quantum yield, and the number of water molecules in the first coordination sphere of the Eu3+ complex.

In this work, we developed a novel DNA quantitative analysis based on fluorescence resonance energy transfer (FRET) realized by combination with a surfactant CPB. The approach was capable of detecting long-stranded DNA in a separation-free format. A sandwich-type FAM-c-DNA-t-DNA-r-DNA-TAMRA conjugate was first formed by the capture probe tagged with FAM, the reporter probe tagged with TAMRA and the target DNA through hybridization. The donor (FAM) and the acceptor (TAMRA) were bridged to afford a FRET system. Subsequently, an addition of the cationic surfactant CPB to the system resulted in a substantial change of the microenvironment and an effective condensation of DNA strands. Consequently, without altering the component of the double strands, an enhanced acceptor fluorescence signal from FRET was achieved and a quantification of the target DNA containing 30 bases was enabled. Under the optimal experimental conditions, an excellent linear relationship between the increase of acceptor fluorescent peak area and the target DNA concentration was obtained over the range from 1.0 × 10−7 to 3.0 × 10−9 mol L−1. The proposed approach offered adequate sensitivity for the detection of the target DNA at 1.0 × 10−9 mol L−1.

On the basis of the supported protein layers (SPLs) substrate, the study presented an ultrasensitive and highly specific platform for single-molecule fluorescence detection of antibody using quantum dots (QDs) as probes. To construct the SPLs surface platform for antibody immobilization, bovine serum albumin (BSA), anti-BSA, and protein G were firstly attached to carboxyl-terminated substrate surfaces by turns. Then nonspecific adsorption of single antibody molecules on SPLs surfaces was investigated. Through the irreversible interaction between streptavidin and biotin, streptavidin-QD conjugates were employed to conjugate with biotinylated antibody, producing QD-antibody conjugates for generating fluorescent signals in fluorescent imaging. Epi-fluorescence microscopy equipped with an electron multiplying charge-coupled device was chosen as the tool for single-molecule fluorescence detection here. The concentration of antibody is quantified based on the direct counting of individual fluorescent spots, one by one. The generated fluorescent signals increased with the increasing concentration of immobilized antibody and were found to be proportional to antibody concentrations. The better brightness and photostability of QDs, and slower increase in the number of counted molecules make a large linear dynamic range of 1.0 × 10−14 to 3.0 × 10−12 mol L−1 between the number of single molecules and antibody concentrations, which is comparable to the previously reported surface-based SMD analysis.

We presented a sensitive method to quantify antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling. In this method, the biotinylated monoclonal anti-human IgG molecules were immobilized on the silanized glass substrate surface. By the strong biotin–streptavidin affinity, streptavidin-coated quantum dots were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots in the evanescent wave field were obtained by a high-sensitivity electron multiplying charge-coupled device. Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted, one by one. The linear range of 8.0 × 10−14 to 5.0 × 10−12 mol L−1 was obtained between the number of single molecules and the sample concentration.

A novel method of luminescence enhancement effect for the determination of balofloxacin (BLFX) was proposed. A new system of the BLFX–Eu3+–SDBS (sodium dodecylbenzene sulfonate) was investigated. It was found that SDBS significantly enhanced the luminescence intensity of the BLFX–Eu3+ complex (about 20-fold). Under the optimized experimental conditions, the system exhibits an excellent linear relationship between the enhanced luminescence intensity and the concentration of BLFX over the range of 1.0×10−8–8.0×10−7 mol L−1 with a correlation coefficient (R) of 0.9994, and the detection limit (3σ) of the method was determined as 2.0×10−9 mol L−1. This method has been successfully applied for the determination of BLFX in pharmaceuticals and human urine/serum samples. Compared with most of the other methods reported, the rapid and simple procedure proposed in the text offers higher sensitivity, wider linear range, and better stability.

A terbium-sensitized spectrofluorimetric method using an anionic surfactant, sodium dodecyl benzene sulfonate (SDBS), was developed for the determination of gatifloxacin (GFLX). A coordination complex system of GFLX–Tb3+–SDBS was studied. It was found that SDBS significantly enhanced the fluorescence intensity of the complex (about 11-fold). Optimal experimental conditions were determined as follows: excitation and emission wavelengths of 331 and 547 nm, pH 7.0, 2.0 × 10−4 mol l−1 terbium (III), and 2.0 × 10−4 mol l−1 SDBS. The enhanced fluorescence intensity of the system (ΔIf) showed a good linear relationship with the concentration of GFLX over the range of 5.0 × 10−10 to 5.0 × 10−8 mol l−1 with a correlation coefficient of 0.9996. The detection limit (3σ) was determined as 6.0 × 10−11 mol l−1. This method has been successfully applied to the determination of GFLX in pharmaceuticals and human urine/serum samples. Compared with most of other methods reported, the rapid and simple procedure proposed in the text offers higher sensitivity, wider linear range, and better stability. The interaction mechanism of the system is also studied by the research of ultraviolet absorption spectra, surface tension, solution polarity and fluorescence polarization.

•A background-eliminated fluorescence assay for T4 PNK was developed.•The magnetic nanoprobes were served as the multi-function recognition.•The “polymerization-nicking” mediated HRCA were used for signal amplify.•The proposed method can detect T4 PNK activity in cell extracts.T4 polynucleotide kinase (PNK) plays critical roles in regulating DNA phosphorylation modes during the repair of DNA lesions. The aberrant activity of T4 PNK has been proven to be associated with a variety of human pathologies. Sensitive detection of T4 PNK activity is critical to both clinical diagnosis and therapeutics. Herein, a background-eliminated fluorescence assay for sensitive detection of T4 PNK activity has been developed by multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification (HRCA). First, the streptavidin-magnetic nanobeads (MBs) were functionalized with the biotin modified hairpin probe (HP) with 3′-phosphoryl, forming multifunctional magnetic probes (HP-MBs). Then, in the presence of T4 PNK, the 3′-phosphoryl of HP-MBs was hydrolyzed to 3′-hydroxyl, thus serving as primers to initiate the polymerization extension and nicking endonuclease cleavage reaction. Next, the primers released from above “polymerization-nicking” cycles were separated out to trigger the subsequently HRCA process, producing plenty of dsDNA. Finally, the intercalating dye SYBR Green I (SG) was inserted into the dsDNA, generating enhanced fluorescence signals. In our design, the HP-MBs here serve together as the T4 PNK, DNA polymerase, and endonuclease recognition probe, and thus avoid the demands of utilizing multiple probes design. Moreover, it performed primary “polymerization-nicking” amplification and mediate secondary HRCA. In addition to, performing the separation function, the binding of HP-MBs and SG could be avoided while a low background was acquired. This method showed excellent sensitivity with a detection limit of 0.0436 mU/mL, and accomplished exceptional characterization T4 PNK activity in cell extracts, offering a powerful tool for biomedical research and clinical diagnosis.

An enzyme-free three-dimensional DNA walker powered by catalytic assembly has been constructed. Automatic movements of the walker were achieved through purely DNA hybridization. This DNA walking device provided a reliable, sensitive and selective strategy for nucleic acid and protein analysis.

A novel fluorescence detection system was developed for DNA methyltransferase (MTase) activity assay based on a target-protected dumbbell molecular probe mediated cascade rolling circle amplification. It showed excellent specificity and sensitivity with a detection limit of 0.0024 U mL−1, and potential application in quantitatively monitoring MTase activity and screening of anticancer drugs.

Here we developed a simple, sensitive and accurate PLD detection method based on a target-controlled gating liposome (TCGL) “off–on” cascade amplified strategy and personal glucose meters (PGMs). It showed excellent sensitivity with a detection limit of 0.005 U L−1 and well performed PLD activity analysis in breast cancer cells and inhibitor drug screening.

A novel toehold activation strategy was developed based on hairpin-reconfiguration which was initiated by the interaction between the hairpin and different environmental stimuli (Hg2+ or ATP). Through variation of the concentration of environmental stimuli, it allowed the fine control of the DNA strand displacement rates almost 2 orders of magnitude.

Based on a Ag+-stabilized self-assembly triplex DNA molecular switch (Ag+-STDMS), a simple, enzyme-free and sensitive new fluorescent strategy for detection of transcription factors was developed, achieving high sensitivity towards purified targets and real biological samples.

A novel label-free fluorescent sensing scheme for sensitive and selective detection of NAD+ and ATP has been developed based on dumbbell probe-mediated rolling circle amplification (D-RCA)-responsive G-quadruplex formation. This approach can detect 0.5 pM for ATP and 100 fM for NAD+, much lower than those of previously reported biosensors, and exhibits high discrimination ability.

A novel rolling circle amplification (RCA) immunoassay based on DNA enriching magnetic nanoparticles and assembled fluorescent DNA nanotags, magnetic nanoparticles-RCA immunoassay, is developed as a versatile fluorescence assay platform for highly sensitive proteins detection.

A single DNA molecule detection method on DNA tetrahedron decorated substrates has been developed. DNA tetrahedra were introduced onto substrates for both preventing nonspecific adsorption and sensitive recognition of single DNA molecules.

We report a label-free fluorescent strategy to study the distribution and stability of DNA origami nanostructures in live, cellular systems, using carbazole-based biscyanine as a probe molecule which has the characteristic property of restriction of intramolecular rotation (RIR) induced emission.

We developed a one-pot label-free molecular beacon-mediated quadratic isothermal exponential amplification strategy (LFMB-QIEA) for simple, rapid and sensitive DNA methyltransferase (MTase) activity detection.