Highly Sensitive Detection of miRNA-155 Using Molecular Beacon-Functionalized Monolayer MoS2 Nanosheet Probes with Duplex-Specific Nuclease-Mediated Signal Amplification

MicroRNA-155 (miRNA-155) as a characteristic myeloma-associated biomarker exhibits significant potential application in the diagnosis of multiple myeloma (MM). In this paper, a novel type of molecular beacon (MB)-functionalized monolayer MoS2 nanosheet probe was proposed as fluorescent probe for high-sensitive assays of miRNA-155that uses a duplexspecificnuclease (DSN) enzyme to amplify the fluorescence signal. The preparation and detection conditions of the fluorescent probes were optimized in some aspects, such as the concentration of MoS2 (0.80 μM) and DSN (0.2 U), and the incubation time of DSN (30 min). The probesexhibited a sensitive fluorescence response to miRNA-155 and the fluorescence signal of the assay was significantly amplified by the cleavage of DSN. The relationship between F/F0 and logC miRNA follows a linear calibration curve, and the limit of detection (LOD) of miRNA-155 in 10% human serum is calculated to be 10.96 fM based on this relationship. The good performance and fluorescence amplification effect of the fluorescent probe were confirmed by studying the recovery of miRNA-155 in 10% human serum, which was ranged from 98.32% to 106.3% with a relative standard deviation of less than 4.14%. Besides, the high expression of miRNA-155 in clinic blood of MM patients was sensitively distinguished from healthy peoples by using the proposed probes. The proposed novel fluorescent probe based on the DSN can be used to detect miRNA-155 in human serum and provide a potential, convenient and reliable tool for diagnosis of MM.

A Facile Aptasensor for Instantaneous Determination of Cadmium Ions Based on Fluorescence Amplification Effect of MOPS on FAM-Labeled Aptamer

Analytical performance and efficiency are two pivotal issues for developing an on-site and real-time aptasensor for cadmium (Cd2+) determination. However, suffering from redundant preparations, fabrications, and incubation, most of them fail to well satisfy the requirements. In this work, we found that fluorescence intensity of 6-carboxyfluorescein(FAM)-labeled aptamer (FAM-aptamer) could be remarkably amplified by 3-(N-morpholino)propane sulfonic acid (MOPS), then fell proportionally as Cd2+ concentration introduced. Importantly, the fluorescence variation occurred immediately after addition of Cd2+, and would keep stable for at least 60 min. Based on the discovery, a facile and ultra-efficient aptasensor for Cd2+ determination was successfully developed. The sensing mechanism was confirmed by fluorescence pattern, circular dichroism (CD) and intermolecular interaction related to pKa. Under the optimal conditions, Cd2+ could be determined rapidly from 5 to 4000 ng mL−1. The detection limit (1.92 ng mL−1) was also lower than the concentration limit for drinking water set by WHO and EPA (3 and 5 ng mL−1, respectively). More than a widely used buffer, MOPS was firstly revealed to have fluorescence amplification effect on FAM-aptamer upon a given context. Despite being sensitive to pH, this simple, high-performance and ultra-efficient aptasensor would be practical for on-site and real-time monitoring of Cd2+.

Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA)

This report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.

Randomly positioned gold nanoparticles as fluorescence enhancers in apta-immunosensor for malaria test

A plasmon-enhanced fluorescence-based antibody-aptamer biosensor — consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly — is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies — covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) — and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10–15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays. Graphical abstract

Detection of meat from horse, donkey and their hybrids (mule/hinny) by duplex real-time fluorescent PCR

Meat adulteration is currently a common practice worldwide. In China, adulteration of donkey meat products with the similar species (horse and mule/hinny) meat and mislabeling are becoming widespread concerns. In this study, a sensitive and species-specific duplex real-time PCR assay based on the simultaneous amplification of fragments of the creatine kinase muscle gene family, was developed and optimized for the identification of horse, donkey and mule /hinny species in raw and heat-processed meat products. Duplex real-time PCR results showed different fluorescence amplification curves for horse and donkey. Both kinds of fluorescence amplification curves appeared simultaneously for mule/hinny. The limit of detection (LOD) of the method was up to 0.01 ng /μL. The method and strategy developed in this study could be applied to detect the presence of adulterants from horse and mule /hinny meat in raw donkey meat and meat products.