Using an In Vivo Mouse Model to Determine the Exclusion Criteria of Preexisting Anti-AAV9 Neutralizing Antibody Titer of Pompe Disease Patients in Clinical Trials.

The efficacy of adeno-associated virus (AAV)-based gene therapy is dependent on effective viral transduction, which might be inhibited by preexisting immunity to AAV acquired from infection or maternal delivery. Anti-AAV neutralizing Abs (NAbs) titer is usually measured by in vitro assay and used for patient enroll; however, this assay could not evaluate NAbs' impacts on AAV pharmacology and potential harm in vivo. Here, we infused a mouse anti-AAV9 monoclonal antibody into Balb/C mice 2 h before receiving 1.2 × 1014 or 3 × 1013 vg/kg of rAAV9-coGAA by tail vein, a drug for our ongoing clinical trials for Pompe disease. The pharmacokinetics, pharmacodynamics, and cellular responses combined with in vitro NAb assay validated the different impacts of preexisting NAbs at different levels in vivo. Sustained GAA expression in the heart, liver, diaphragm, and quadriceps were observed. The presence of high-level NAb, a titer about 1:1000, accelerated vector clearance in blood and completely blocked transduction. The AAV-specific T cell responses tended to increase when the titer of NAb exceeded 1:200. A low-level NAbs, near 1:100, had no effect on transduction in the heart and liver as well as cellular responses, but decreased transduction in muscles slightly. Therefore, we propose to preclude patients with NAb titers > 1:100 from rAAV9-coGAA clinical trials.

Separation and Enrichment of Sudan III Using Surface Modified Hollow Glass Microspheres and Colorimetric Detection.

BACKGROUND: Sudan III has been shown to be carcinogenic to human beings due to the azo chemical structure. A simple, highly selective, and environmentally friendly pretreatment method is usually required before the analysis of Sudan III in complex practical samples due to low concentration and matrix interference. OBJECTIVE: The aim of this research was to prepare buoyant adsorbents, octyl trimethoxysilane caped hollow glass microspheres (HGMs), and establish a new pretreatment method for the detection of Sudan III in real samples. METHOD: HGMs were activated and transferred to a flask containing 80 mL ethanol solution (9:1, v/v) and 0.9 mL ammonia. The octyl trimethoxysilane was added to the slurry and covalently coupled on the surface of the HGMs. The modified HGMs were used as adsorbents for the enrichment of Sudan III. After adsorption and desorption, the UV-Vis absorption spectrum was recorded under excitation at 506 nm. RESULTS: Under the optimum conditions, the linear range and detection limit were 0.10-4.0 mg/L and 0.048 mg/L, respectively. The proposed method was successfully employed to detect Sudan III in chili products with acceptable recoveries of spikes (90.7-102%). CONCLUSIONS: The adsorbent, which could be separated by flotation, provided a new solid phase extraction method for the pretreatment of complex samples. HIGHLIGHTS: A new solid phase extraction method was provided for the pretreatment of complex samples. In addition, the adsorbents with high enrichment efficiency can be easily separated by flotation and repeatedly used for separation and enrichment of Sudan III.

Selective determination of DNA based on the fluorescence recovery of carbon dots quenched by Ru(bpy)2(dppz)2.

A quick, highly selective and sensitive method for the determination of DNA was constructed based on the recovery of red fluorescence of carbon dots quenched by a ruthenium complex (Ru (bpy)2 (dppz)2+, bpy = 2,2'-bipyridine; dppz = dipyrido [3.2-a:2',3'-c]phenazine). The carbon dots showed two emission peaks at 450 and 683 nm under excitation wavelength of 420 nm. After addition of Ru (bpy)2 (dppz)2+, the fluorescence of carbon dots at 683 nm was markedly quenched. Due to the stronger interaction between DNA and Ru (bpy)2 (dppz)2+, the quenched fluorescence of carbon dots was recovered with DNA added. Under the optimum conditions, there was a good linear relationship between the degree of fluorescence recovery of carbon dots and DNA concentration in range of 0.0150-9.60 µg mL-1. The detection limit was 0.00536 µg mL-1. The carbon dots were successfully applied to detect DNA in the simulated sample and the spiked recoveries were between 97.4% and 106%.

ZNF597 is a maternally expressed imprinted gene in the Holstein breed.

Genomic imprinting is an epigenetic phenomenon that leads to the preferential expression of genes from either the paternal or maternal allele. Imprinted genes play important roles in mammalian growth and development and a central role in placental function. ZNF597 and NAA60 are two paternally imprinted genes in the human ZNF597-NAA60 imprinted locus, both of which show biallelic expression in the mouse, but their imprinting status in cattle is still unknown. In this study, we examined the allelic expression of ZNF597 and NAA60 in adult bovine placental and somatic tissues. By comparing the mRNA-based genotypes with the genomic DNA-based genotypes, we identified monoallelic expression of ZNF597 in the placenta and in seven other tissues, including the cerebrum, heart, liver, spleen, lung, kidney, and muscle. Nevertheless, analysis revealed biallelic expression of the NAA60 gene in these tissues. Moreover, we tested the imprinting status of ZNF597 and confirmed that the maternal allele is expressed in the bovine placenta. To determine the role of DNA methylation in regulating monoallelic/imprinted expression of bovine ZNF597, the methylation status of two CpG-enriched regions in the bovine ZNF597-NAA60 locus was analyzed using the bisulfite sequencing method. Differentially methylated regions were detected on ten CpG loci in the bovine ZNF597 promoter region. In summary, the bovine ZNF597 gene is a maternally expressed gene, and its expression is regulated by DNA methylation, whereas the NAA60 gene is not imprinted in cattle.

Determination of thiourea based on the reversion of fluorescence quenching of nitrogen doped carbon dots by Hg2.

Herein, a facile and quick strategy to detect thiourea was conducted based on the reversion of fluorescence quenching of nitrogen doped carbon dots (NCDs) by Hg2+. The NCDs with good water solubility and 17% of quantum yield was synthesized by one-step hydrothermal method, using ammonium citrate and dextrin as carbon source and nitrogen source, respectively. The fluorescence of NCDs was obviously quenched by Hg2+ and can be recovered, due to stronger interaction between thiourea and Hg2+. There was a good linear relationship between the recovered fluorescence and the concentration of thiourea within range of 0.90-10.0 µM and the detection limit for thiourea detection was 0.15 µM. The as-prepared NCDs can be used for determination of thiourea in tap water, lake water and rice flour products, and the spike recoveries were between 91.6 and 108%.

Ratio fluorometric determination of ATP base on the reversion of fluorescence of calcein quenched by Eu(III) ion using carbon dots as reference.

A kind of nitrogen doped carbon dots (NCDs) with excellent stable luminescence performance was prepared by pyrolysis using ethanolamine as precursor. By simply mixing solution of NCDs and calcein-Eu3+, a ratio fluorometric probe with carbon dots as "internal reference" and calcein-Eu3+ as recognition group was constructed for ATP detection. The fluorescence of the calcein can be selectively quenched by Eu3+, and can be restored when ATP was added because Eu3+ ions exhibit higher affinity to the oxygen-donor atoms originated from phosphates than that from carboxylate groups. Meanwhile, fluorescence of NCDs was not affected by Eu3+, calcein or ATP. By adding NCDs as "internal reference" in the above system, a new ratiometric strategy for detecting ATP was conducted. The dynamic linear range for ATP detection was 5.0 × 10-8 mol L-1~ 2.0 × 10-6 mol L-1, and the detection limit was 2.0 × 10-8 mol L-1.The method was successfully applied to detecting ATP in adenosine disodium triphosphate injection. Compared with calcein- Eu3+ probe without NCDs as reference, the ratio fluorometric probe effectively reduced interference and improved the accuracy and sensitivity.

Determination of DNA based on fluorescence quenching of terbium doped carbon dots.

This work describes the preparation of carbon dots doped with terbium(III) (Tb-CDs) via a hydrothermal method, starting from terbium ion and ethylenediamine. The size, composition and spectral properties of the Tb-CDs were characterized by transmission electron microscopy, infrared spectra, and fluorescence spectra. The results show that doping of the CDs with Tb(III) reduces the particle size and results in more uniform particles, while fluorescence (at excitation/emission peaks of 380/475 nm) is strongly enhanced. The interaction between Tb-CDs and ct-DNA results in fluorescence quenching of Tb-CDs. The findings were exploited to design a quenchometric method for the determination of ct-DNA. The signal drops linearly in the 80 ng·mL-1 to 50 µg·mL-1 ct-DNA concentration range, and the detection limit is 53 ng·mL-1. The method was applied to the determination of ct-DNA in spiked samples and gave satisfactory results. The possible fluorescence quenching mechanism (which is mainly static) was investigated using the Stern-Volmer equation and thermodynamic equations. Graphical abstract A kind of carbon dots doped with terbium(III) (Tb-CDs) were prepared via a hydrothermal method, using terbium ion and ethylenediamine as precursor. Doping with Tb(III) reduced the particle size of CDs and results in uniform particle size and stronger fluorescence. The interaction between the Tb-CDs and dsDNA results in quenching of the fluorescence of Tb-CDs and can be applied to determination of dsDNA.

Amplified impedimetric immunosensor based on instant catalyst for sensitive determination of ochratoxin A.

A new impedimetric immunosensor for the fast determination of ochratoxin A (OTA) in food samples was developed based on the instant catalyst as enhancer. Initially, the signal tags were prepared via co-immobilization of anti-OTA antibody and amine-terminated dendrimer (PAMAM) on the graphene oxide nanosheets through the covalent interaction, which were utilized as a good platform for combining manganese ion (anti-OTA-GO-PAMAM-Mn(2+)). Upon target OTA introduction, a competitive-type immunoreaction was implemented between the analyte and the immobilized OTA-BSA on the electrode for the anti-OTA antibody on the graphene oxide nanosheets labels. After a competitive immunoassay format, the anti-OTA-GO-PAMAM-Mn(2+) were captured onto the electrode surface, which could induce the in situ formation of MnO2via classical redox reaction between Mn(2+) and KMnO4 on the immunesensing platform. Moreover, the generated MnO2 nanoparticles act as efficient catalyst could catalyze the 4-chloro-1-naphthol (4-CN) oxidation without H2O2 to generate an insoluble precipitation on the platform. Under the optimal conditions, the instant catalyst based impedimetric immunosensor displayed a wide dynamic working range between 0.1pgmL(-1) and 30ngmL(-1). The detection limit (LOD) of the assay was 0.055pgmL(-1). The developed method exhibited high selectivity and can be used for the determination of OTA in real red wine samples.

Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification.

A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B1 (AFB1) in foodstuff. The system consisted of anti-AFB1 antibody labeled DNA1 (Ab-DNA1), AFB1-bovine serum albumin (BSA)-conjugated DNA2 (AFB1-DNA2), and methylene blue functionalized hairpin DNA. Owing to a specific antigen-antibody reaction between anti-AFB1 and AFB1-BSA, the immunocomplex formed assisted the proximity hybridization of DNA1 with DNA2, thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB1, the analyte competed with AFB1-DNA2 for the conjugated anti-AFB1 on the Ab-DNA1, subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB1 in a dynamic working range of 0.01-30 ng mL-1 with a detection limit of 4.8 pg mL-1. In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB1-spiked peanut samples, giving results that matched well with those obtained with a commercial AFB1 ELISA kit.

Novel glucometer-based immunosensing strategy suitable for complex systems with signal amplification using surfactant-responsive cargo release from glucose-encapsulated liposome nanocarriers.

Methods based on surfactant-responsive controlled release systems of cargoes from nanocontainers have been developed for bioanalytical applications, but most were utilized for drug delivery and a few reports were focused on immunoassays. Herein we design an in situ amplified immunoassay protocol for high-efficient detection of aflatoxins (aflatoxin B1, AFB1 used in this case) based on surfactant-responsive cargo release from glucose-encapsulated liposome nanocarriers with sensitivity enhancement. Initially, biotinylated liposome nanocarrier encapsulated with glucose was synthesized using a reverse-phase evaporation method. Thereafter, the nanocarrier was utilized as the signal-generation tag on capture antibody-coating microplate through classical biotin-avidin linkage after reaction with biotinylated detection antibody. Upon addition of buffered surfactant (1X PBS-Tween 20 buffer) into the medium, the surfactant immediately hydrolyzed the conjugated liposome, and released the encapsulated glucose from the nanocarriers, which could be quantitatively determined by using a low-cost personal glucometer (PGM). The detectable signal increased with the increment of target analyte. Under the optimal conditions, the assay could allow PGM detection toward target AFB1 as low as 0.6 pg mL(-1) (0.6 ppt). Moreover, the methodology also showed good reproducibility and high specificity toward target AFB1 against other mycotoxins and proteins, and was applicable for quantitatively monitoring target AFB1 in the complex systems, e.g., naturally contaminated/spiked peanut samples and serum specimens, with the acceptable results. Taking these advantages of simplification, low cost, universality and sensitivity, our design provides a new horizon for development of advanced immunoassays in future point-of-care testing.