Real‐Time Fluorescence Turn‐On Detection of Alkaline Phosphatase Activity with a Novel Perylene Probe

A tetracationic perylene probe (probe 2 ) was designed and synthesized. Probe 1 was used for the real‐time fluorescence turn‐on assay of alkaline phosphatase (ALP) activity and inhibitor screening. Probe 1 monomer fluorescence could be very efficiently quenched by ATP through the formation of an ATP/probe 1 complex. ALP triggered the degradation of ATP, the breakdown of the ATP/probe 1 complex, and the recovery of the probe 1 monomer fluorescence. In the presence of an ALP inhibitor, a decrease in fluorescence recovery was observed.     

Label free fluorescence turn-on detection of polynucleotide kinase activity with a perylene probe

A single stranded oligonucleotide could induce aggregation of a perylene probe, the probe's monomer fluorescence was efficiently quenched. However, when the oligonucleotide was 5'-phosphorylated by polynucleotide kinase, it could be very efficiently degraded by lambda exonuclease, probe monomers were released, and a turn on fluorescence signal was detected.     

Ionic Self‐Assembled Platform of Perylenediimide–Sodium Dodecylsulfate for Detection of Spermine in Clinical Samples

The detection and quantification of spermine in clinical practice is important for early diagnosis of many diseases. Chromatographic and immunoassay‐based methods are commonly used. However, a fluorescence‐based assay could provide real‐time detection. Herein, the synthesis and aggregation properties of a dicationic perylene probe (N ¹‐dodecyl‐N ³‐(4‐phenyl)benzimidazolium‐functionalized perylenediimide ( DAB‐PDI )) used to develop a fluorescent “turn‐on” ensemble for the detection of spermine are discussed. The fluorescence of DAB‐PDI (10 μm , Φ =0.55) is efficiently quenched by negatively charged sodium dodecylsulfate (SDS) through the formation of ionic self‐assembled aggregates (charge ratio of negative (N) in SDS to positive (P) in DAB‐PDI (N/P)=9). This negatively charged ionic self‐assembly between DAB‐PDI and SDS has been characterized by using photophysical, microscopic, dynamic light scattering, isothermal titration calorimetry, and HRMS techniques. The addition of spermine to this ensemble solution results in the breakdown of the DAB‐PDI –SDS ensemble owing to strong binding of spermine with SDS and, as a result, the fluorescence of DAB‐PDI is recovered. This ensemble exhibits high sensitivity and selectivity for spermine detection in water, urine, and blood serum. The lowest limit of detection is 27.5 nm , which is at least about 36 times lower than that required for early diagnosis of cancer (1 to 10 μm for urinary spermine).     

5‐(Pyren‐1‐yl)uracil as a Base‐Discriminating Fluorescent Nucleobase in Pyrrolidinyl Peptide Nucleic Acids

A pyrene‐labeled uridine (U^Py) monomer for a pyrrolidinyl peptide nucleic acid with an alternating proline/2‐aminocyclopentanecarboxylic acid backbone (acpcPNA) was synthesized and incorporated into the PNA. The U^Py base in acpcPNA could specifically recognize the base A in its complementary DNA strand as determined by thermal denaturation (T_m) experiments. The fluorescence of the U^Py‐containing single‐stranded acpcPNA was very weak in aqueous buffer. In the presence of a complementary DNA target, the fluorescence was enhanced significantly (2.7–41.9 folds, depending on sequences). The fluorescence enhancement was specific to the pairing between U^Py and dA, making the U^Py‐modified acpcPNA useful as a hybridization‐responsive fluorescence probe for DNA‐sequence determination.     

Superquencher formation via nucleic acid induced noncovalent perylene probe self-assembly

A fluorophore labeled oligonucleotide could induce aggregation of a positively charged perylene probe. The perylene aggregate could very efficiently quench the fluorescence of the labeled fluorophore. Based on this observation, a new method for the highly sensitive and selective detection of a protein has been developed.     

Sequence Specific Electrochemical DNA Detection Based on Solution‐Phase Competitive Hybridization

We report a novel electrochemical method for detecting sequence‐specific DNA based on competitive hybridization that occurs in a homogeneous solution phase instead of on a solution‐electrode interface as in previously reported competition‐based electrochemical DNA detection schemes. The method utilizes the competition between the target DNA (t‐DNA) and a ferrocene‐labeled peptide nucleic acid probe (Fc‐PNA) to hybridize with a probe DNA (p‐DNA) in solution. The neutral PNA backbone and the electrostatic repulsion between the negatively‐charged DNA backbone and the negatively‐charged electrode surface are then exploited to determine the result of the competition through measurement of the electrochemical signal of Fc. Upon the introduction of the t‐DNA, the stronger hybridization affinity between the t‐DNA and p‐DNA releases the Fc‐PNA from the Fc‐PNA/p‐DNA hybrid, allowing it to freely diffuse to the negatively charged electrode to produce a significantly enhanced electrochemical signal of Fc. Therefore, the presence of the t‐DNA is indicated by the appearance or enhancement of the electrochemical signal, rendering a signal‐on DNA detection, which is less susceptible to false positive and can produce more reliable results than signal‐off detection methods. All the competitive hybridizations occur in a homogeneous solution phase, resulting in very high hybridization efficiency and therefore extremely short assay time. This simple and fast signal‐on solution‐competition‐based electrochemical DNA detection strategy has promising potential to find application in fields such as nucleic acid‐based point‐of‐care testing.     

Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

We present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.     

A Fluorescent Probe with Aggregation‐Induced Emission for Detecting Alkaline Phosphatase and Cell Imaging

Alkaline phosphatase (ALP) is associated with many diseases, and its accurate detection is of great significance. Fluorescent compounds with aggregation‐induced emission (AIE) feature show beneficial advantages for serving as fluorescent probes. Herein, an AIE‐active “turn on” probe for ALP detection was synthesized through incorporating a strong electron‐withdrawing group (cyano) in the middle and the recognition moiety phosphate group at the end, thereby rendering a D–A–D structure with a relatively high conjugation degree and good water solubility. It was found that the probe TPE‐CN‐pho is highly sensitive to ALP in aqueous solution. In the presence of ALP, the hydrophilic phosphate group on the probe is rapidly removed, resulting in a decrease in water solubility and subsequent formation of aggregates, thereby achieving aggregation‐induced emission. Moreover, the probe TPE‐CN‐pho has also been successfully applied to imaging ALP in living cells.     

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation‐sensitive endonuclease DpnⅡ

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED‐induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation‐sensitive endonuclease DpnⅡ which could recognize the same specific site 5′‐GATC‐3′ with Dam MTase in double‐stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME‐LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5–20 U/mL with a detection limit of 0.12 U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME‐LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs     

A Peptide Nucleic Acid (PNA) Heteroduplex Probe Containing an Inosine–Cytosine Base Pair Discriminates a Single‐Nucleotide Difference in RNA

Selective discrimination of a single‐nucleotide difference in single‐stranded DNA or RNA remains a challenge with conventional DNA or RNA probes. A peptide nucleic acid (PNA)‐derived probe, in which PNA forms a pseudocomplementary heteroduplex with inosine‐containing DNA or RNA, effectively discriminates a single‐nucleotide difference in a closely related group of sequences of single‐stranded DNA and/or RNA. The pseudocomplementary PNA heteroduplex is easily converted to a fluorescent probe that distinctively detects a member of highly homologous let‐7 microRNAs.     

A Competitive Strategy Coupled with Endonuclease‐Assisted Target Recycling for DNA Detection Using Silver‐Nanoparticle‐Tagged Carbon Nanospheres as Labels

A simple competitive strategy was designed for the sensitive detection of sequence‐specific DNA by combining endonuclease‐assisted target recycling and electrochemical stripping analysis of silver nanoparticles (AgNPs). The AgNP‐tagged carbon nanospheres were synthesized by means of in situ reduction of Ag⁺ adsorbed onto a negatively charged polyelectrolyte layer and functionalized with streptavidin for binding biotin‐labeled DNA strands. The labeled strand was captured on the DNA sensor surface by competitive hybridization of biotinated primer 1 and its cleaved product. The cleaved product could be amplified in homogeneous solution by endonuclease‐assisted target recycling with a Y‐shaped junction DNA structure, thus leading to the correlation of the stripping signal to the target concentration. The functionalized nanosphere was characterized with X‐ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The proposed method showed a linear range from 0.1 to 1000 fM with a limit of detection of 0.066 fM (3σ) and good selectivity for base discrimination. The designed strategy provided a sensitive tool for DNA analysis and could be widely applied in bioanalysis and biomedicine.     

Mesoporous Silica Nanoparticles Decorated with Carbosilane Dendrons as New Non‐viral Oligonucleotide Delivery Carriers

A novel nanosystem based on mesoporous silica nanoparticles covered with carbosilane dendrons grafted on the external surface of the nanoparticles is reported. This system is able to transport single‐stranded oligonucleotide into cells, avoiding an electrostatic repulsion between the cell membrane and the negatively charged nucleic acids thanks to the cationic charge provided by the dendron coating under physiological conditions. Moreover, the presence of the highly ordered pore network inside the silica matrix would make possible to allocate other therapeutic agents within the mesopores with the aim of achieving a double delivery. First, carbosilane dendrons of second and third generation possessing ammonium or tertiary amine groups as peripheral functional groups were prepared. Hence, different strategies were tested in order to obtain their suitable grafting on the outer surface of the nanoparticles. As nucleic acid model, a single‐stranded DNA oligonucleotide tagged with a fluorescent Cy3 moiety was used to evaluate the DNA adsorption capacity. The hybrid material functionalised with the third generation of a neutral dendron showed excellent DNA binding properties. Finally, the cytotoxicity as well as the capability to deliver DNA into cells, was tested in vitro by using a human osteoblast‐like cell line, achieving good levels of internalisation of the vector DNA/carbosilane dendron‐functionalised material without affecting the cellular viability.     

An Enzyme‐Responsive “Turn‐on” Fluorescence Polymeric Superamphiphile as a Potential Visualizable Phosphate Prodrug Delivery Vehicle

The development of inexpensive and highly efficient enzyme‐responsive polymers has significantly contributed to targeted drug delivery systems. Here, a superamphiphile with a capability of fluorescent dissociation sensing is designed. It is constructed with negatively charged adenosine 5′‐triphosphate (ATP) and negatively charged fluorescein diphosphate (FDP), which are used as fluorescence detection, and a cationic diblock copolymer methoxy‐poly(ethylene glycol)₁₁₃‐b‐poly(2‐dimethyl‐aminoethyl methacrylate)₇₀. Upon addition of calf intestinal alkaline phosphatase, the superamphiphile disintegrates, presumably due to the enzymatic hydrolysis of ATP. This process is accompanied by an increase in the fluorescence emission intensity of fluorescein owing to the hydrolysis of FDP. The in vitro application of the superamphiphile is also proven. Thus, the “turn‐on” fluorescence of the superamphiphile serves as a real‐time module for detection of the disintegration of superamphiphile.     

Aggregation‐Induced Emission Active Probe for Light‐Up Detection of Anionic Surfactants and Wash‐Free Bacterial Imaging

Anionic surfactants are widely used in daily life and industries, but their residues can cause serious damage to the environment. The current detection methods for anionic surfactants suffer from various limitations and a new detection strategy is highly desirable. Based on 2‐(2‐hydroxyphenyl)benzothiazole fluorogen with aggregation‐induced emission characteristics, we have developed a fluorescent probe HBT‐C₁₈ for selective and sensitive detection of anionic surfactants. By in situ formation of catanionic aggregates or micelles with anionic surfactants, the emission intensity of the HBT‐C₁₈ probe can increase with increasing keto/enol emission ratio through restriction of intramolecular motion and excited‐state intramolecular proton‐transfer mechanisms. The probe can also be used for wash‐free imaging of bacteria enveloped by a negatively charged outer membrane. The results of this study provide a new strategy for sensitive detection of anionic surfactants and wash‐free bacterial imaging.     

Homogeneous DNA Detection Based on Fluorescence Quenching by Nanoparticles in Single-step Format :Target-Induced Configuration Transform

A new strategy for homogeneous detection of DNA hybridization in single-step format was developed based on fluorescence quenching by gold nanoparticles. The gold nanoparticle is functionalized with 5’-thiolated 48-base oligonucleotide (probe sequence), whose 3’-terminus is labeled with fluorescein (FAM), a negatively charged fluorescence dye. The oligonucleotide adopts an extended configuration due to the electrostatic repulsion between negatively charged gold nanoparticle and the FAM-attached probe sequence. After addition of the complementary target sequence, specific DNA hybridization induces a conformation change of the probe from an extended structure to an arch-like configuration, which brings the fluorophore and the gold nanoparticle in close proximity. The fluorescence is efficiently quenched by gold nanoparticles. The fluorescence quenching efficiency is related to the target concentration, which allows the quantitative detection for target sequence in a sample. A linear detection range from 1.6 to 209.4 nmol/L was obtained under the optimized experimental conditions with a detection limit of 0.1 nmol/L. In the assay system, the gold nanoparticles act as both nanoscaffolds and nanoquenchers. Furthermore, the proposed strategy, in which only two DNA sequences are involved, is not only different from the traditional molecular beacons or reverse molecular beacons but also different from the commonly used sandwich hybridization methods. In addition, the DNA hybridization detection was achieved in homogenous solution in a single-step format, which allows real-time detection and quantification with other advantages such as easy operation and elimination of washing steps.     

Label-free fluorescence turn-on detection of microRNA based on duplex-specific nuclease and a perylene probe

A novel fluorescence turn-on microRNA (miRNA) detection method based on duplex-specific nuclease (DSN) and a perylene probe is presented in this study. A positively charged perylene derivative (compound 1) was used as the fluorescent probe. Compound 1 exhibits strong monomer fluorescence in an aqueous buffer solution. It is well known that single-stranded DNA is a polyanion in nature. Thus, it can induce the aggregation of compound 1 through strong electrostatic, hydrophobic and π−π stacking interactions. As a result, the fluorescence of compound 1 was efficiently quenched. When the target miRNA was added, the formation of DNA-RNA hybridized duplex initiated the cleavage of the DNA strand by DSN cycle reaction, which resulted in disaggregation of compound 1. A fluorescence turn-on signal was detected, and a novel miRNA sensing method was therefore established. The presented method is label-free, simple, cost effective, sensitive and selective.     

Efficient Preparation of Large‐Sized Rings of Single‐Stranded DNA through One‐Pot Ligation of Multiple Fragments

Circular single‐stranded DNA (c‐ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c‐ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one‐pot ligation method to efficiently prepare large c‐ssDNA. By ligating several short fragments of linear single‐stranded DNA (l‐ssDNA) in one‐pot by using T4 DNA ligase, longer l‐ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large‐sized single‐stranded c‐ssDNAs (e.g., 200‐nt) were successfully synthesized in high selectivity and yield.     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.     

Near‐Infrared Fluorescent Molecular Probe for Sensitive Imaging of Keloid

Early detection of skin diseases is imperative for their effective treatment. However, fluorescence molecular probes that allow this are rare. The first activatable near‐infrared (NIR) fluorescent molecular probe is reported for sensitive imaging of keloid cells, skin cells from abnormal scar fibrous lesions. As keloid cells have high expression levels of fibroblast activation protein‐alpha (FAPα), the probe (FNP1) is designed to have a caged NIR dye and a FAPα‐cleavable peptide substrate linked by a self‐immolative segment. FNP1 can quickly and specifically turn on its fluorescence at 710 nm by 45‐fold in the presence of FAPα, allowing it to effectively recognize keloid cells from normal skin cells. Integration of FNP1 with a simple microneedle‐assisted topical application enables sensitive detection of keloid cells in metabolically‐active human skin tissue with a theoretical limit of detection down to 20 000 cells.     

Enzyme‐Regulated Activation of DNAzyme: A Novel Strategy for a Label‐Free Colorimetric DNA Ligase Assay and Ligase‐Based Biosensing

The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label‐free and DNAzyme‐based strategy to detect DNA ligase activity. This novel strategy relies on the ligation‐trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin‐DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL^?1 and a detection limit of 0.2 U mL^?1. Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a “split DNA machine” to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL^?1.