Label-free Fluorescence Strategy Research Articles

A Fluorescent Biosensor for Streptavidin Detection Based on Double-Hairpin DNA-Templated Copper Nanoparticles

In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.

A label-free fluorescence strategy for analysis of aflatoxin M1 by self-protected DNAzyme and aptamer recognition triggered DNA walker cascade amplification

Monitoring the information of aflatoxin M1 (AFM1) is of great significance for food safety since its widespread presence in milk and other dairy products. In view of this, we developed a label-free fluorescence DNA walker based on self-protected DNAzyme for determination of AFM1. By inserting the AFM1 aptamer into DNAzyme, the complete DNAzyme was split into two separated parts and designed to be an arch-like shield to avoid its degradation in complex matrix. This excellent structure of DNAzyme and the report probe were pre-assembled on the surface of gold nanoparticles (AuNPs). Through the high efficiency affinity between AFM1 and its aptamer, the DNAzyme could be activated and show its high catalytic cleavage towards its substrate of report probe. The releasing G-quardruplex (G4) of this report probe from AuNPs could combine with berberine and exhibit obvious fluorescence values. Under optimized experimental conditions, the AFM1 detection limit was achieved at 6.40 ng/L. This DNA walker exhibited good selectivity, reproductivity and stability. Furthermore, the feasibility for analysis in real sample of our method was performed towards the target spiked in milk, indicating its potential application for real sample determination.

Pre-Folded G-Quadruplex as a Tunable Reporter to Facilitate CRISPR/Cas12a-Based Visual Nucleic Acid Diagnosis.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based detection strategies with a fluorophore quencher-labeled ssDNA reporter or gold nanoparticle ssDNA reporter have been widely used in point-of-care (POC) molecular diagnostics. However, the potential of these CRISPR/Cas12a strategies for POC molecular diagnostics is often compromised due to the complex labeling, high cost, and low signal-to-noise ratio. Herein, we show a pre-folded G-quadruplex (G4) structure with tunable tolerance to CRISPR/Cas12a trans-cleavage and explore its mechanism. Two G4 structures (i.e., Tel22-10 and G16C) sensitive or tolerant to CRISPR/Cas12a trans-cleavage are designed and used as signal elements to fabricate a label-free visible fluorescent strategy or "signal-on" colorimetric strategy, respectively. These two strategies facilitate an ultrasensitive visual nucleic acid determination of Group B Streptococci with a naked-eye limit of detection of 1 aM. The feasibility of the developed G4-assisted CRISPR/Cas12a strategies for real-world applications is demonstrated in clinical vaginal/anal specimens and further verified by a commercial qPCR assay. This work suggests that the proposed G4 structures with tunable tolerance can act as promising signal reporters in the CRISPR/Cas12a system to enable ultrasensitive visible nucleic acid detection.

Label-free fluorescent aptasensor for chloramphenicol based on hybridization chain reaction amplification and G-quadruplex/N-methyl mesoporphyrin IX complexation.

The use of the broad-spectrum antibiotic chloramphenicol (CAP) in food is strictly regulated or banned in many countries. Herein, for the sensitive, rapid, and specific detection of CAP in milk, a label-free fluorescence strategy was established based on guanine (G)-quadruplex/N-methyl mesoporphyrin IX (NMM) complex formation and hybridization chain reaction (HCR) amplification. In this system, CAP can specifically bind to an aptamer (Apt) to release an Apt-C sequence from double-stranded DNA (Apt·Apt-C). Apt-C, can further hybridize with a functional hairpin DNA probe to release a primer sequence. The released primer sequence causes HCR and the formation of a nicked double-helix polymer, which contains G-quadruplex DNA. The recognition of G-quadruplex DNA by the NMM fluorochrome results in fluorescence enhancement. Consequently, CAP can be quantitatively detected by measuring the fluorescence intensity at 612 nm. The reliability of the aptasensor method was confirmed by comparison with an enzyme-linked immunosorbent assay. The proposed aptasensor was found to have a limit of detection of 0.8 pg mL−1 for CAP. Moreover, when the aptasensor was applied to the detection of CAP in milk samples, the average recoveries were 99.8–108.3% with relative standard deviations of 4.5–5.2%. Thus, this CAP detection method, which is rapid with high sensitivity and selectivity, has considerable potential for a wide range of food analysis applications.

Facile and label-free fluorescence strategy for evaluating the influence of bioactive ingredients on FMO3 activity via supramolecular host-guest reporter pair

Screening inhibitors of flavin monooxygenase 3 (FMO3) is very important for treating trimethylamine N-oxide (TMAO) derived thrombotic diseases. Herein, focusing on Xuefu Zhuyu decoction (XFZYD) as a Chinese traditional medicine with antithrombotic efficacy, a facile and label-free fluorescence strategy was developed for evaluating the influence of the bioactive ingredients in XFZYD on FMO3 activity through indicator displacement assay. To this end, the optimized supramolecular host-guest (p-sulfonatocalix[4]arene-oxazine 1) reporter pair and FMO3 catalytic system were exploited to determine the influence of the bioactive compounds in XFZYD on the conversion from TMA to TMAO. From the nine compounds tested, naringin, paeoniflorin, β-ecdysterone, 18β-glycyrrhizic acid, amygdalin, albiflorin, and saikosaponin A downregulated FMO3 activity and reduced TMAO biosynthesis. Moreover, molecular docking was successfully applied to simulate the optimal conformation of a receptor-ligand complex between FMO3 and all tested compounds except for β-ecdysterone. Therefore, this approach provides a novel and promising strategy for screening FMO3 inhibitors from Chinese traditional medicine by supramolecular sensing.

Label-free fluorescence strategy for methyltransferase activity assay based on poly-thymine copper nanoclusters engineered by terminal deoxynucleotidyl transferase

The assay of detecting DNA methyltransferase activity has been identified as one of the central challenges in cancer diagnostics as DNA methylation is closely related to the diagnosis and treatment of tumors. In this study, a label-free fluorescence probe based on poly-thymine copper nanoclusters engineered by terminal deoxynucleotidyl transferase is proposed for methyltransferase activity assay. Taking advantage of the efficient polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase and the copper nanoclusters with large Stokes shift instead of labeling fluorescent dyes, the strategy exhibits a broader linear scope from 1 to 300 U mL−1 with a detection limit of 0.176 U mL−1. The economical method is specificity for M.SssI and also provides a convenient and high-throughput platform for screening the DNA methylation inhibitors, which displays a great potential for the practical applications of the drug development and clinical cancer diagnosis in the future.

Probing Transient DNA Conformation Changes with an Intercalative Fluorescent Excimer.

Variation of DNA conformation is important in regulating gene expression and mediating drug-DNA interactions. However, directly probing transient DNA conformation changes is challenging owing to the dynamic nature of this process. We show a label-free fluorescence method to monitor transient DNA conformation changes in DNA structures with various lengths and shapes using a DNA intercalator, K21. K21 can form transient excimers on the surface of DNA; the ratiometric emission of monomer and excimer correlate to DNA transient conformation stability in numerous DNA structures, including i-motifs, G-quadruplex structures, and single nucleotide mutation at random position. We analyzed the conformation dynamics of a single plasmid before and after enzyme digestion with confocal fluorescence microscopy. This method provides a label-free fluorescence strategy to probe transient conformation changes of DNA structures and has potential in uncovering transient genomic processes in living cells.

Label-free and enzyme-free fluorescence detection of microRNA based on sulfydryl-functionalized carbon dots via target-initiated hemin/G-quadruplex-catalyzed oxidation

Carbon dots (CDs)-based biosensors have attracted considerable interest in reliable and sensitive detection of microRNA (miRNA) because of their merits of ultra-small size, excellent biosafety and tunable emission, whereas complicated labeling procedure and expensive bioenzyme associated with current strategies significantly limit their practical application. Herein, we developed a label-free and enzyme-free fluorescence strategy based on strand displaced amplification (SDA) for highly sensitive detection of miRNA using sulfydryl-functionalized CDs (CDs-SH) as probe. CDs-SH displayed excellent response to G-quadruplex DNA against other DNAs based on based on the catalytic oxidation of –SH into –S–S– by hemin/G-quadruplex. Further, CDs-SH were employed to detect miRNA, using miRNA-21 as target model, which triggered the SDA reaction of P1 and P2 to generate hemin/G-quadruplex, subsequently making CDs-SH transform from dot to aggresome along with the quenched fluorescence. Therefore, label-free, enzyme-free, and highly sensitive analysis of miRNA-21 was readily acquired with a limit of detection at 0.03 pM. This proposed biosensor couples the advantages of CDs and label-free/enzyme-free strategy, and thus has a significant potential to be used in early and accurate diagnosis of cancer.

A Light-Up Strategy with Aggregation-Induced Emission for Identification of HIV-I RNA-Binding Small Molecules.

Fluorescence methods are important tools to identify RNA-binding small molecules and further employed to study RNA-protein interactions. Most reported fluorescence strategies are based on covalent labeling of ligand or RNA, which can impede the binding between them to some extent, or light-off fluorescent indicator displacement methods, which ask for particular indicators. Herein, a label-free fluorescence strategy based on the light-on aggregation-induced emission (AIE) feature of tetraphenylethene (TPE) derivative to screen RNA-binding small molecules is presented. As a result of electrostatic interaction, the selected peptides can induce self-assembly of the TPE derivative to produce strong fluorescent emission; when the peptides are bound to RNA molecules, the TPE derivative is in the deaggregated form and shows no or minimum fluorescence. Based on the phenomenon, a competitive displacement assay combined with the TPE reporter was employed to characterize selected small molecules for their binding abilities to HIV-I RNAs. This AIE feature enables the fluorescence-off state of the TPE derivative in the presence of RNA-peptide complex to be "lightened up" quickly as the RNA-binding molecule is introduced and the peptide is competitively released. This strategy was carried out to test several small molecule binders, and the results are consistent with previous reports. This report gives an inspiring example of AIE-based fluorescent assay for HIV-I RNA-binding molecule screening, which may further be explored to build a drug screening platform for RNA-protein interference.

Terminal Deoxynucleotidyl Transferase and Rolling Circle Amplification Induced G-triplex Formation: A Label-free Fluorescent Strategy for DNA Methyltransferase Activity Assay

It is of great significance to sensitively detect DNA methyltransferase (MTase) activity and screen its inhibitory drugs in the field of human cancer diagnosis and disease therapeutics. In this work, based on terminal deoxynucleotidyl transferase (TdT)-induced rolling circle amplification (RCA), we construct a label-free and highly sensitive fluorescence biosensing strategy for DNA MTase activity analysis. The hairpin substrate is methylated by Dam MTase and cleaved by Dpnl to generate a DNA fragment with a free 3′-hydroxy (3′−OH) terminus that is extended by TdT to obtain a long adenine (poly-A) tail. Subsequently, the resulting poly-A work as primer triggers RCA reaction and generate lots of G-triplexes which can combine with Thioflavin T (ThT) to achieve the apparent fluorescence signal. Integrate TdT-induced RCA with G-triplex@ThT, Dam MTase can be detected down to 0.058 U/mL with a wide linear range from 0.1 U/mL to 40 U/mL. In addition, the method exhibits high selectivity to other MTases. We also successfully demonstrate this work can be used in complex matrixes assay and screen MTase inhibitors. Thus, in the area of DNA MTase-related cancer diagnosis and disease therapeutics, the developed method may be a promising tool for the detection of Dam MTase.

An exonuclease-assisted fluorescence sensor for assaying alkaline phosphatase based on SYBR Green I

In this report, we propose a fast, reliable and convenient approach to determine the alkaline phosphatase (ALP) activity based on a label-free fluorescence strategy. Upon catalysis of ALP, dephosphorylated dsDNA hampers the λ exonuclease (λexo) cleavage, shows high affinity to SYBR Green I (SG I), resulting in a strong fluorescence emission peak at 520 nm. In the absence of ALP, the dsDNA with 5′-phosphoryl-termini could be employed as a substrate of λexo. After cleavage, a weak fluorescence emission peaks at 520 nm could be observed. The assay was both selective and sensitive, and the detection limit was found to be as low as 3 U/L. This method was utilized to evaluate Na3VO4 as ALP inhibitor. The method was successfully applied to the determination of the activity of ALP in spiked human serum samples.

Visualization of the intracellular location and stability of DNA flowers with a label-free fluorescent probe.

Rolling circle amplification (RCA) and rolling circle transcription (RCT) can be used to fabricate various structures and organize functional materials for biological applications. The full understanding of the interactions between RCA/RCT-derived structures and live cells is urgently demanded. Here, we present a label-free fluorescent strategy to study the intracellular location and stability of RCA-based DNA flowers in live cells. The DNA flower structures are co-assembled with carbazole-based biscyanine fluorophores, which are DNA detecting molecules and characterized by restriction of intramolecular rotation (RIR) induced strong fluorescent emission. When biscyanine molecules are encapsulated in the DNA flowers via electrostatic attraction, these confined RIR dyes can produce strong luminescent emission. Using this advantage, we use the RIR enhanced technique for direct visualization of the distribution and degradation of DNA flowers in live cellular systems. Our current research could be adapted to other advanced DNA-based materials, providing a new strategy to fabricate fluorescent DNA materials and realize controllable release of payloads.

A label-free ratiometric fluorescence strategy for 3′–5′ exonuclease detection

A label-free ratiometric fluorescent biosensor for detection of exonuclease was proposed through utilizing two individual DNA conformation-specific dyes (DAPI and NMM).

Ultrasensitive microRNA-21 detection based on DNA hybridization chain reaction and SYBR Green dye

It is extremely important for quantifying trace microRNAs in the biomedical applications. In this study, an ultrasensitive, rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction (HCR). DNA H1 and DNA H2 were designed and used as hairpin probes, the HCR was proceeded in the presence of target microRNAs. Amounts of SYBR Green І dyes were used as signal molecules to intercalate long DNA concatemers from HCR, which guaranteed the model of label-free fluorescence and strong fluorescence density. The detection method showed a wide linear region from 1 fM to 105 fM, and the limit of detection was 0.2554 fM (at S/N = 3) for microRNAs. The results showed that this method had an excellent specificity and reproducibility. Furthermore, the label-free fluorescence strategy exhibited a sensitive response to microRNA-21 in real serum samples of gastric cancer patients and the results obtained were in accordance with reference method (R2 = 0.994). Overall, the proposed strategy could be satisfactory for rapid, ultrasensitive and efficient detection of microRNA-21, and held great potentials in clinic diagnosis of gastric cancer.

Label-free fluorescent strategy for sensitive detection of tetracycline based on triple-helix molecular switch and G-quadruplex

In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch (THMS) and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T (ThT) as a signal transduction probe (STP). In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to ThT which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0nmol/L. The detection limit of tetracycline was determined to be 970.0pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.

A Label-free and Turn-on Fluorescence Strategy for DNA Detection with a Wide Detection Range Based on Exonuclease III-aided Target Recycling Amplification.

In the present work, a simple and sensitive turn-on fluorescence method for DNA detection was developed. It was explored based on the N-methyl mesoporphyrin IX (NMM)/G-quadruplex DNA system as a reporter and exonuclease III (Exo III)-aided target recycling amplification to ensure sensitivity. Our method showed an ultra-wide detection range from 10 fM to 100 nM with a low linear detection limit of 0.76 nM. It also had excellent selectivity in a selectivity experiment.

High-throughput identification of telomere-binding ligands based on the fluorescence regulation of DNA-copper nanoparticles

Formation of the G-quadruplex in the human telomeric DNA is an effective way to inhibit telomerase activity. Therefore, screening ligands of G-quadruplex has potential applications in the treatment of cancer by inhibit telomerase activity. Although several techniques have been explored for screening of telomeric G-quadruplexes ligands, high-throughput screening method for fast screening telomere-binding ligands from the large compound library is still urgently needed. Herein, a label-free fluorescence strategy has been proposed for high-throughput screening telomere-binding ligands by using DNA-copper nanoparticles (DNA-CuNPs) as a signal probe. In the absence of ligands, human telomeric DNA (GDNA) hybridized with its complementary DNA (cDNA) to form double stranded DNA (dsDNA) which can act as an efficient template for the formation of DNA-CuNPs, leading to the high fluorescence of DNA-CuNPs. In the presence of ligands, GDNA folded into G-quadruplex. Single-strdanded cDNA does not support the formation of DNA-CuNP, resulting in low fluorescence of DNA-CuNPs. Therefore, telomere-binding ligands can be high-throughput screened by monitoring the change in the fluorescence of DNA-CuNPs. Thirteen traditional chinese medicines were screened. Circular dichroism (CD) measurements demonstrated that the selected ligands could induce single-stranded telomeric DNA to form G-quadruplex. The telomere repeat amplification protocol (TRAP) assay demonstrated that the selected ligands can effectively inhibit telomerase activity. Therefore, it offers a cost-effective, label-free and reliable high-throughput way to identify G-quadruplex ligands, which holds great potential in discovering telomerase-targeted anticancer drugs.

Synthesis and sensing integration: A novel enzymatic reaction modulated Nanoclusters Beacon (NCB) “Illumination” strategy for label-free biosensing and logic gate operation

A novel fluorescent label-free “turn-on” NAD+ and adenosine triphosphate (ATP) biosensing strategy is proposed by fully exploiting ligation triggered Nanocluster Beacon (NCB). In the presence of the target, the split NCB was brought to intact, which brought the C-rich sequence and enhancer sequence in close proximity resulting in the lightening of dark DNA/AgNCs (“On” mode). Further application was presented for logic gate operation and aptasensor construction. The feasibility was investigated by Ultraviolet–visible spectroscopy (UV–vis), Fluorescence, lifetime and High Resolution Transmission Electron Microscopy (HRTEM) etc. The strategy displayed good performance in the detection of NAD+ and ATP, with the detection limit of 0.002nM and 0.001mM, the linear range of 10–1000nM and 0.003–0.01mM, respectively. Due to the DNA/AgNCs as fluorescence reporter, the completely label-free fluorescent strategy boasts the features of simplicity and low cost, and showing little reliance on the sensing environment. Meanwhile, the regulation by overhang G-rich sequence not relying on Förster energy transfer quenching manifests the high signal-to-background ratios (S/B ratios). This method not only provided a simple, economical and reliable fluorescent NAD+ assay but also explored a flexible G-rich sequence regulated NCB probe for the fluorescent biosensors. Furthermore, this sensing mode was expanded to the application of a logic gate design, which exhibited a high performance for not only versatile biosensors construction but also for molecular computing application.

PCR-free and label-free fluorescent detection of telomerase activity at single-cell level based on triple amplification

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.

Multifunctional Dumbbell-Shaped DNA-Templated Selective Formation of Fluorescent Silver Nanoclusters or Copper Nanoparticles for Sensitive Detection of Biomolecules.

In this work, a multifunctional template for selective formation of fluorescent silver nanoclusters (AgNCs) or copper nanoparticles (CuNPs) is put forward. This dumbbell-shaped (DS) DNA template is made up of two cytosine hairpin loops and an adenine-thymine-rich double-helical stem which is closed by the loops. The cytosine loops act as specific regions for the growth of AgNCs, and the double-helical stem serves as template for the CuNPs formation. By carefully investigating the sequence and length of DS DNA, we present the optimal design of the template. Benefiting from the smart design and facile synthesis, a simple, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection is proposed. Through the systematic comparison, it is found that the strategy based on CuNPs formation is more sensitive for ATP assay than that based on AgNCs synthesis, and the detection limitation was found to be 81 pM. What's more, the CuNPs formation-based method is successfully applied in the detection of ATP in human serum as well as the determination of cellular ATP. In addition to small target molecule, the sensing strategy was also extended to the detection of biomacromolecule (DNA), which illustrates the generality of this biosensor.