In situ biosensor for detection miRNA in living cells based on carbon nitride nanosheets with catalytic hairpin assembly amplification

In this study, an ultrasensitive fluorescence turn‐on assay for in situ sensing of intracellular microRNA (miRNA) was developed utilizing a carbon nitride nanosheet (CNNS) and a catalytic hairpin assembly (CHA). The CHA showed favourable signal amplification for low‐level biomarkers, and CNNS was an excellent candidate as a fluorescence quencher and gene vector. Moreover, the hairpin DNA of CHA could be adsorbed onto the surface of CNNS. An enzyme‐free fluorescence biosensor for ultrasensitive sensing of intracellular miRNA in cells based on CHA and CNNS was designed. When faced with target miRNA, the fluorescence was recovered due to the miRNA, which could trigger cycling of CHA circuits, leading to the production of a marked enhanced fluorescence signal. Compared with traditional methods, the proposed method is convenient, with low cytotoxicity, and high specificity and ultrasensitivity. It has promising potential for detection low‐level biomarkers.     

Large-Stokes-shift-based folded DNA probing systems targeting DNA and miRNA 21 with signal amplification

Large-Stokes-shift based simple folded DNA probing system (LSFP) had a simple folded DNA structure and exhibited a large Stokes-shifted (194?nm) fluorescence signal upon excitation at a single wavelength (386?nm). This Stokes shift was achieved through a simple combination of donor and acceptor fluorophores and employing multi-FRET systematically. This unique large Stokes-shifted fluorescence signal was used to detect target DNA with large increases in the fluorescence signal (9.7–14.2 fold). This LSFP exhibited enough selectivity, distinguishing a perfectly matched sequence from the probe itself and mismatched sequences. Surprisingly, when DSN was used for signal amplification with miR21P probing system whose target is miRNA 21, it showed high sensitivity (13.7?aM) and selectivity (one base mismatch discrimination). This system has several advantages over other molecular beacons (MBs): (i) it is easy to design and synthesize the probing system that does not require the construction of a finely designed stem and loop, as in most MBs (this can prevent the degradation of miR21P itself by DSN enzyme without special backbone modification); (ii) it can control unique fluorescence, such as large Stokes-shifted fluorescence through a simple combination of donor and acceptor fluorophores; and (iii) through signal amplification using DSN, it can efficiently detect extremely small amounts of target miRNA with high sensitivity (13.7?aM).     

A T7exonuclease‐assisted target recycling amplification with graphene oxide acting as the signal amplifier for fluorescence polarization detection of human immunodeficiency virus (HIV) DNA

We report a fluorescence polarization (FP) platform for human immunodeficiency virus (HIV) DNA detection based on T7exonuclease‐assisted target recycling amplification with graphene oxide (GO) acting as a FP signal amplifier. In the sensing method, the presence of the target DNA leads to target recycling with the assistance of T7exonuclease, furthermore, the amplification products are absorbed onto the surface of GO, so the all FP values are enhanced by GO. More importantly, this FP sensor exhibits high detection sensitivity; under optimal conditions, the change in FP is linear with the concentration of the target DNA within a concentration range of 50–2000 pmol/L, and the detection limit of this method is as low as 38.6 pmol/L. This FP sensor also exhibits high selectivity, even single‐base mismatched DNA can be effectively discriminated from complementary target DNA. Above all, the proposed FP sensor may serve as a general platform for the sensitive assay of disease‐related genes. Copyright © 2015 John Wiley & Sons, Ltd.     

A highly sensitive fluorescence sensor based on lucigenin/chitosan/SiO2 composite nanoparticles for microRNA detection using magnetic separation

In this paper, a convenient reverse‐phase microemulsion method for the synthesis of SiO₂ nanoparticles (NPs) by simply introducing the chitosan and fluorescent dye of lucigenin during the formation reaction of SiO₂ NPs was proposed. Addition of chitosan can make the SiO₂ NPs porous, and increases lucigenin molecule incorporation into chitosan/SiO₂ NPs nanopores based on electrostatic interaction and supermolecular forces. Therefore, fluorescence quantum yield of the lucigenin/chitosan/SiO₂ composite nanoparticles was increased by introduction of chitosan and compared with lucigenin/SiO₂ NPs without chitosan. Because the number of negative charges carried when using single‐stranded DNA (ssDNA) was different from that of double‐stranded DNA (dsDNA), the numbers of lucigenin/chitosan/SiO₂ composite nanoparticles with positive charge adsorbed using ssDNA or dsDNA were different. Consequently, fluorescence intensity caused using ssDNA or dsDNA/miRNA was clearly discriminative. With increase in target DNA/miRNA concentration, the difference in fluorescence intensity also increased, resulting in a good linear relationship between fluorescence intensity sensitizing value and target miRNA concentrations. Therefore, a new fluorescence analysis method for direct detection of let‐7a in human gastric cancer cell samples without enzyme, label free and no immobilization was established using lucigenin/chitosan/SiO₂ composite nanoparticles as a DNA hybrid indicator. The proposed method had high sensitivity and selectivity, low cost and the detection limit was 10 fM (S/N = 3).     

Label‐free,zeptomole cancer biomarker detection by surface‐enhanced fluorescence on nanoporous gold disk plasmonic nanoparticles

We experimentally demonstrate a label‐free biosensor for the ERBB2 cancer gene DNA target based on the distance‐dependent detection of surface‐enhanced fluorescence (SEF) on nanoporous gold disk (NPGD) plasmonic nanoparticles. We achieve detection of 2.4 zeptomole of DNA target on the NPGD substrate with an upper concentration detection limit of 1 nM. Without the use of molecular spacers, the NPGD substrate as an SEF platform was shown to provide higher net fluorescence for visible and NIR fluorophores compared to glass and non‐porous gold substrates. The enhanced fluorescence signals in patterned nanoporous gold nanoparticles make NPGD a viable material for further reducing detection limits for biomolecular targets used in clinical assays.

With patterned nanoporous gold disk (NPGD) plasmonic nanoparticles, a label‐free biosensor that makes use of distance‐dependent detection of surface‐enhanced fluorescence (SEF) is constructed and tested for zeptomole detection of ERBB2 cancer gene DNA targets.     

A dual discrimination mode for improved specificity towards let‐7a detection via a single‐base mutated padlock probe‐based exponential rolling circle amplification

MicroRNA (miRNA) family members are usually highly homologous sequences, and it is a challenging task to selectively detect one miRNA member from other family members in medical diagnosis. Here, we describe the design of a dual discrimination mode for improved specificity towards let‐7a detection over the other members of the let‐7 family, in which an intentional base mutation was introduced into the padlock probe of an exponential rolling circle amplification. The inherent discrimination power of the padlock probe and the introduced base mutation constituted a dual discrimination mode, which provided enhanced specificity for let‐7a, even over single‐base mismatched family sequences. Furthermore, the assay enabled the quantitative detection of let‐7a in a dynamic range from 200 amol to 100 fmol. This technique has also been successfully applied to real small RNA samples extracted from human lung cancers. For the first time, through intentionally mutating one base on the padlock probe of the exponential rolling circle amplification (RCA), we improved the discrimination capability for let‐7 family members, while maintaining adequate sensitivity. Overall, this dual discrimination mode and the high amplification strategy have the potential to be extended to other short, but highly homologous, miRNA sequences.     

Electrochemical DNA biosensor for screening of chlorinated benzene pollutants

In this work, an electrochemical DNA biosensor based on double-stranded DNA modified Au electrode (dsDNA/Au) was proposed for the rapid screening and detection of chlorinated benzenes pollutants, in which redox-active methylene blue (MB) was used to amplify the interaction between dsDNA and the target analyte. Using hexachlorobenzene (HCB) as a model analyte of chlorinated benzenes, the biosensor demonstrated a linear response with the logarithm of HCB concentrations from 100 pmol L(-1) to 100 nmol L(-1). The obtained detection limit was 30 pmol L(-1), which was remarkably superior to other biosensors. The interaction mechanism of the biosensor with HCB was proposed based on systematical characterization by cyclic voltammetry (CV), differential pulse voltammetry (DPV), UV-vis spectrometry and electrochemical quartz crystal microbalance (EQCM). Further studies revealed that the biosensor could screen chlorinated benzenes in the presence of 100 fold amount of other co-existing chemicals (ethyl acetate and sodium oxalate, etc.), and the response signal of the biosensors for different chlorinated benzenes was correlative to their respective toxicity. The proposed biosensor proved to be a promising "alarm" tool for rapid screening of chlorinated benzenes in real water samples.     

Enzyme-amplified electrochemical biosensor for detection of PML-RARα fusion gene based on hairpin LNA probe

In this study, an enzyme-amplified electrochemical biosensor was developed for detection of the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia (APL). This new sensor employs a hairpin locked nucleic acids (LNAs) probe dually labeled with biotin and carboxyfluorescein molecule (FAM). The probe is immobilized at a streptavidin-modified electrode surface via the biotin-streptavidin bridge, and FAM serves as an affinity tag for the peroxidase conjugate binding. Initially, the immobilized hairpin probe was in the "closed" state in the absence of the target, which shielded FAM from being approached by the bulky anti-FAM-HRP conjugate due to the steric effect. Target binding opens the hairpin structure of the probe, the probe undergoes a significant conformational change, forcing FAM away from the electrode. As a result, the FAM label becomes accessible by the anti-FAM-HRP, and the target hybridization event can be sensitively transduced via the enzymatically amplified electrochemical current signal. This new biosensor demonstrates its excellent specificity for single-base mismatch and able to detect as little as 83 fM target DNA even in the presence of human serum. We also employed this sensor to directly detect PCR real sample with satisfactory results.     

Reduced expression of let‐7f activates TGF‐β/ALK5 pathway and leads to impaired ischaemia‐induced neovascularization after cigarette smoke exposure

This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large‐scale miRNA profiling and qRT‐PCR analyses, we identified let‐7f as a pro‐angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let‐7f mimic restored ischaemia‐induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let‐7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let‐7f mimic could also rescue pro‐angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF‐βR1), an important modulator of angiogenesis, is a target of let‐7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti‐angiogenic factors SMAD2/3 and PAI‐1. Importantly, treatment with let‐7f mimic reduces the expression of ALK5, SMAD2/3 and PAI‐1 both in vitro and in vivo. Moreover, let‐7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let‐7f and activation of the anti‐angiogenic TGF‐β/ALK5 pathway. Overexpression of let‐7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia‐induced neovascularization in pathological conditions.     

A fluorescent aptasensor for analysis of adenosine triphosphate based on aptamer–magnetic nanoparticles and its single‐stranded complementary DNA labeled carbon dots

A new fluorimetric aptasensor was designed for the determination of adenosine triphosphate (ATP) based on magnetic nanoparticles (MNPs) and carbon dots (CDs). In this analytical strategy, an ATP aptamer was conjugated on MNPs and a complementary strand of the aptamer (CS) was labeled with CDs. The aptamer and its CS were hybridized to form a double helical structure. The hybridized aptamers could be used for the specific recognition of ATP in a biological complex matrix using a strong magnetic field to remove the interfering effect. In the absence of ATP, no CDs–CS could be released into the solution and this resulted in a weak fluorescence signal. In the presence of ATP, the target binds to its aptamer and causes the dissociation of the double helical structure and liberation of the CS, such that a strong fluorescence signal was generated. The increased fluorescence signal was proportional to ATP concentration. The limit of detection was estimated to be 1.0 pmol L^–1 with a dynamic range of 3.0 pmol L^–1 to 5.0 nmol L^–1. The specific aptasensor was applied to detect ATP in human serum samples with satisfactory results. Moreover, molecular dynamic simulation (MDS) studies were used to analyze interactions of the ATP molecule with the aptamer.     

Folate deficiency disturbs hsa‐let‐7 g level through methylation regulation in neural tube defects

Folic acid deficiency during pregnancy is believed to be a high‐risk factor for neural tube defects (NTDs). Disturbed epigenetic modifications, including miRNA regulation, have been linked to the pathogenesis of NTDs in those with folate deficiency. However, the mechanism by which folic acid‐regulated miRNA influences this pathogenesis remains unclear. It is believed that DNA methylation is associated with dysregulated miRNA expression. To clarify this issue, here we measured the methylation changes of 22 miRNAs in 57 human NTD cases to explore whether such changes are involved in miRNA regulation in NTD cases through folate metabolism. In total, eight of the 22 miRNAs tested reduced their methylation modifications in NTD cases, which provide direct evidence of the roles of interactions between DNA methylation and miRNA level in these defects. Among the findings, there was a significant association between folic acid concentration and hsa‐let‐7 g methylation level in NTD cases. Hypomethylation of hsa‐let‐7 g increased its own expression level in both NTD cases and cell models, which indicated that hsa‐let‐7 g methylation directly regulates its own expression. Overexpression of hsa‐let‐7 g, along with its target genes, disturbed the migration and proliferation of SK‐N‐SH cells, implying that hsa‐let‐7 g plays important roles in the prevention of NTDs by folic acid. In summary, our data suggest a relationship between aberrant methylation of hsa‐let‐7 g and disturbed folate metabolism in NTDs, implying that improvements in nutrition during early pregnancy may prevent such defects, possibly via the donation of methyl groups for miRNAs.     

Assembly of a miRNA‐modified QCM sensor for miRNA recognition through response patterns

In this paper, a miRNA‐based quartz crystal microbalance (QCM) biosensor was fabricated and used to the rapid and effective sensing of miRNA. The specific hybridization between probe miRNA and different selected miRNAs (miR‐27a, miR‐27b, and Let‐7a) cause a different interaction mode, thus display different frequency change and response patterns in the QCM sensor, which were used to detect miR‐27a and miR‐27b. The selective sensing of miR‐27a in mixed miRNA solution was also achieved. This miRNA‐based QCM biosensor has the advantages of real‐time, label‐free, and short cycle detection.     

Ultra‐sensitive fluorescent sensor for intracellular miRNA based on enzyme‐free signal amplification with carbon nitride nanosheet as a carrier

A novel ultra‐sensitive fluorescent sensor for monitoring microRNA (miRNA) in living cells was constructed by utilizing a hybridization chain reaction (HCR) as the signal amplification with a carbon nitride nanosheet (CNNS) as a carrier. The Cy5‐labeled hairpin DNA could be adsorbed onto the surface of CNNS, resulting in fluorescence quenching of Cy5. When treated with complementary miRNA, the fluorescence was recovered because miRNA could efficiently trigger an HCR, which led to the release of the HCR products from the CNNS. This intracellular HCR strategy can be used for ultra‐sensitive monitoring of intracellular miRNA. The main advantages of the proposed method are its simplicity, high sensitivity, high specificity and low toxicity for monitoring low‐level biomarkers.     

Chip-based visual detection of microRNA using DNA-functionalized gold nanoparticles

In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle(Au NP) probes and a gene chip to detect micro RNAs(mi RNAs). Specific oligonucleotides were attached to the glass surface as capture probes for the target mi RNAs, which were then detected via hybridization to the Au NP probes. The signal was amplified via the reduction of HAu Cl4 by H2O2. The use of a single Au NP probe detected 10 pmol L?1 of target mi RNA. The recovery rate for mi R-126 from fetal bovine serum was 81.5%–109.1%. The biosensor detection of mi R-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR(q PCR) results. The use of two Au NP probes further improved the detection sensitivity such that even 1 fmol L?1 of target mi R-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye. The platform simultaneously detected lung cancer related mi R-126 and mi R-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual mi RNA detection.     

A facile fluorescence turn-on biosensor customized for monitoring of protein kinase activity based on carboxylic carbon nanoparticle–peptide complexes

In this study, we present a facile and low-cost approach for detecting protein kinase A (PKA) by assembling a purpose-designed carboxyfluorescein (FAM)-labelled peptide with carboxylic carbon nanoparticles (CNPs). Fluorescence of the FAM-labelled peptide gradually decreases to a low background signal as a result of the electron transfer from CNPs to FAM-labelled peptide via the peptide, which acts as a bridge. The reaction in the sensor in the presence of adenosine 5′-triphosphate and PKA phosphorylates the substrate peptide and disrupts the electrostatic repulsive force between the CNPs and the peptide, therefore altering the spectroscopic signal of the system. The change in fluorescence signal was directly proportional to the PKA concentration in the range 0–1.8 U/ml with a detection limit of 0.04 U/ml. These results suggest that PKA activity can be effectively measured using the developed PKA biosensor. Moreover, the fluorescence biosensor was successfully used in the investigation of PKA in spiked human embryonic kidney (HEK) 293 cells lysates, indicating its potential applications in protein kinase-related biochemical fundamental research.     

let‐7 regulates radial migration of new‐born neurons through positive regulation of autophagy

During adult neurogenesis, newly formed olfactory bulb (OB) interneurons migrate radially to integrate into specific layers of the OB. Despite the importance of this process, the intracellular mechanisms that regulate radial migration remain poorly understood. Here, we find that microRNA (miRNA) let‐7 regulates radial migration by modulating autophagy in new‐born neurons. Using Argonaute2 immunoprecipitation, we performed global profiling of miRNAs in adult‐born OB neurons and identified let‐7 as a highly abundant miRNA family. Knockdown of let‐7 in migrating neuroblasts prevented radial migration and led to an immature morphology of newly formed interneurons. This phenotype was accompanied by a decrease in autophagic activity. Overexpression of Beclin‐1 or TFEB in new‐born neurons lacking let‐7 resulted in re‐activation of autophagy and restored radial migration. Thus, these results reveal a miRNA‐dependent link between autophagy and adult neurogenesis with implications for neurodegenerative diseases where these processes are impaired.     

Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

Development of surface‐engineered yeast cells displaying phytochelatin synthase and their application to cadmium biosensors by the combined use of pyrene‐excimer fluorescence

The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface‐engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level‐dependent synthesis of PC₂ from glutathiones by AtPCS1‐displaying yeast cells, followed by simple discriminative detection of PC₂ via sensing of excimer fluorescence of thiol‐labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose‐dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross‐reaction, the proposed simple biosensor holds promise as a method useful for cost‐effective screening of Cd contamination in environmental and food samples. The AtPCS1‐displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1197–1202, 2013