Self-corrected dual-optical immunosensors using carbon dots@SiO2@MnO2 improving diethyl phthalate detection accuracy.

The traditional immunoassay is widely used for pollutant detection and bioanalysis, but there are still some challenges in ensuring its sensitivity and reliable accuracy. Dual-optical measurement can prove mutual evidence to effectively improve the accuracy of the method by self-correction, which will overcome this problem. In this study, we developed a "visualization and sensing" dual-modal immunoassay based on blue carbon dots@SiO2@MnO2 (B-CDs@SiO2@MnO2) as "color and fluorescence" immunosensors. Here, MnO2 nanosheets have the activity of simulating oxidase. 3,3', 5,5'-Tetramethylbenzidine (TMB) can be oxidized to TMB2+ under acidic conditions and the color of the solution from colorless to yellow. On the other hand, the MnO2 nanosheets can quench the fluorescence of B-CDs@SiO2. After adding ascorbic acid (AA), MnO2 nanosheets were reduced to Mn2+, thereby the fluorescence of B-CDs@SiO2 was restored. Under the optimum conditions, as the concentration of target substance (diethyl phthalate) increased from 0.05 to 100 ng/mL, the method showed a good linear relationship. The fluorescence measurement signal and the color change signal of the solution visualization support each other and give the information of the corresponding material content. The results of the dual-optical immunoassay maintain good consistency, which proves the accuracy of the developed dual-optical immunoassay for detection of diethyl phthalate is reliable. Additionally, it is demonstrated that the dual-modal method exhibits high accuracy and stability in the assays, pointing to a broad range of application prospects in pollutant analysis.

Fluorescence Signal Amplification: Red Carbon Dots@SiO2-Induced Ultra-sensitive Immunoassay for Diethyl Phthalate.

Carbon dots as new nanomaterials, have been widely used in rapid detection because of their nondestructive, real-time detection characteristics. Improving the sensitivity and selectivity of the method in complex real samples is new challenge and requirement for sensing technology. Here, we report an ultrasensitive fluorescence immunoassay (FIA) for trace diethyl phthalate (DEP) using red carbon dots@SiO2 (R-CDs@SiO2) as tags. SiO2 as a nanocarrier can effectively improve the bio-functionalization and utilization rate of carbon dots. Moreover, several R-CDs embedded in SiO2 nanospheres can magnify the fluorescence signal and improve sensitivity. R-CDs@SiO2 conjugate anti-DEP antibody (Ab) as fluorescent immunosensor, which can specifically recognize DEP. Under optimization conditions, the detection limit (LOD) of this FIA was calculated as 0.0011 ng/mL. In addition, the recoveries of this established FIA ranged from 96.8 to 108.5%, showing satisfactory accuracy. Compared with GC-MS/MS (LOD µg/mL), the sensitivity of the FIA was significantly improved. As a result, the FIA developed using R-CDs@SiO2 as tags has a high potential for determining trace DEP.

Nano-Bio Interface-Guided Nanoparticle Protein Corona Antigen for Immunoassays and Immunoimaging in a Complex Matrix.

Engineered nanoparticles are widely used in biological imaging and drug delivery because of their excellent physical and chemical properties, but almost all the original functions of engineered nanoparticles suffer from a complex matrix. Herein, we proposed a strategy of preparing nanoparticle protein corona antigens (NPCAgs) through exposing a magnetic core silicon shell (Fe3O4@SiO2) fluorescent probe to an antigen protein solution, which could reduce the adsorption of nanoparticles (NPs) with other proteins in serum. In the presence of target anti-BSA IgG, a competitive-type displacement reaction was implemented between NPs@BSA and other proteins by target anti-BSA IgG through the specific antigen-antibody reaction. In addition, secondary structure analysis showed that almost all of the NPCAgs retained their natural conformation, which ensured the function of the NPCAgs, specifically capturing an antibody. Therefore, the NPCAgs showed good performance in immunoassays and immunoimaging, which should shed light on the application in imaging and identification of other nanomaterials.

Nanoparticle/Nanocarrier Formulation as an Antigen: The Immunogenicity and Antigenicity of Itself.

In antibody preparation, the immunogenicity of small molecules is limited due to the instability of adjuvant/hapten emulsions. Nanoparticle-based adjuvants overcome instability and effectively improve immune responses. Immunogenicity and antigenicity are fundamentally important, yet understudied, facets of nanoparticle formulations themselves. Herein, we studied the immunogenicity and antigenicity of nanoparticle formulations. In experiments in a rabbit model, simple inorganic nanoparticle (e.g., gold nanoparticle (AuNP) and silver nanoparticle (AgNP)) immunogens induced higher titers of antiserum. Moreover, several promising nanoparticle drug carrier immunogens (e.g., SiO2, oleylamine graft polysuccinimide (PSIOAm), oleylamine and N-(3-aminopropyl)imidazole cograft polysuccinimide (PSIOAm-NAPI), Fe3O4@O-dextran, etc.) showed excellent immunogenicity. Cross-reactivity calculations revealed that the antigenicity properties of AgNP and AuNP antigens are highly size-dependent. Meanwhile, four nanoparticle drug carriers generate antibody-specific immune responses to their antigens. The reactivity of the anti-NP antibodies with nanoparticle antigens was confirmed using immunoassays. This study systematically identified the immunogenicity and antigenicity of the nanoparticle formulation itself. These findings provide insights into the immunological properties of the nanoparticle formulation itself in an organism.

An evaporation induced self-assembly approach to prepare polymorphic carbon dot fluorescent nanoprobes for protein labelling.

Herein, we report a novel route to prepare polymorphic carbon dot fluorescent probes via the evaporation-induced self-assembly of glutaraldehyde and carbon dots, which first usually form carbon nanoclusters which then could self-assemble to form carbon nanocrystals, nanospheres or nanofibers in different ionic strength solutions at room temperature. The aldehyde functionalized polymorphic C-dot fluorescent probe can easily covalently bond with amino groups on proteins.

Peptide and peptide nucleic acid syntheses using a DNA/RNA synthesizer.

The use of an ABI 394 DNA/RNA synthesizer for peptide and peptide nucleic acid (PNA) syntheses is described. No additional physical part or software is needed for the application. A commercially available large DNA synthesis column was used, and only about half of its volume was filled with resin when the resin was fully swollen. With additional space in the top portion of the column, agitation of reaction mixture was achieved by bubbling argon from the bottom without losing solution. Removing solutions from column was achieved by flushing argon from top to bottom. Two peptide and two PNA sequences were synthesized. Good yields were obtained in all the cases. The method is easy to follow by researchers who are familiar with DNA/RNA synthesizer.

Purification of synthetic peptides using a catching full-length sequence by polymerization approach.

During automated solid-phase peptide synthesis, failure sequences were capped with acetic anhydride. After synthesis, a polymerizable methacrylamide tag was attached to the full-length sequences. Peptide purification was then achieved by polymerizing the full-length sequences, washing away impurities, and cleaving the peptide product from the polymer.

Determination of dimethyl phthalate in environment water samples by a highly sensitive indirect competitive ELISA.

Recent controversy over the discovery of clouding agents containing the banned chemical di(2-ethylhexyl) phthalate in beverages in 2011 in Taiwan has caused public concerns. For the detection of dimethyl phthalate (DMP) in environment water samples, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed in this paper. Dimethyl 4-aminophthalate (4-DMAP) was covalently attached to bovine serum albumin as immunogen by a diazotization method. The conjugation of DMAP and ovalbumin as coating antigen was obtained in the same way. Polyclonal antibody was obtained from New Zealand white rabbits. Under the optimized conditions, DMP was detected in the concentration range of 0.02-419 ng/mL with a detection limit of 0.01 ng/mL. The proposed method has been applied to the analysis of river water, lake water, and rain water samples. Satisfactory recoveries were obtained ranging from 90.6% to 105.5%. The cross-reactivities of the anti-DMP antibody to seven structurally related phthalate esters were below 10%. The data demonstrated that the ic-ELISA method described in our study is a simple, sensitive, and specific method and showed that this assay is a reliable tool to detect DMP in water samples.

A direct competitive enzyme-linked immunosorbent assay by antibody coated for diethyl phthalate analysis.

A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detection of diethyl phthalate (DEP). Protein-hapten conjugate was synthesized to produce polyclonal antibodies against DEP. Experimental parameters were optimized, including immunoreaction conditions, the dilution ratio of horseradish peroxidase (HRP)-antigen conjugate, time of the antibody coated, effect of pH, and ionic strength. The limit of detection was 0.096ng/ml, and the linear range was 0.1-3500ng/ml with a regression coefficient (R(2)) of 0.9957. Recoveries were between 96.4 and 106.2%. The cross-reactivities of the anti-DEP antibody to six structurally related phthalate esters were less than 9%. The method was successfully applied to the determination of DEP in tap water, river water (Yangtze River), and leachate from plastic drinking bottles. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DEP monitoring. The results obtained were compared with those obtained using the high-performance liquid chromatography method.

An indirect competitive fluorescence immunoassay for determination of dicyclohexyl phthalate in water samples.

A sensitive and specific indirect competitive fluorescence immunoassays (FIA) has been developed for the quantitative determination of dicyclohexyl phthalate (DCHP) using an antigen-coated plate format. The polyclonal antibodies raised against dicyclohexyl 4-amino phthalate conjugated to bovine serum albumin (BSA) by the amino diazotization linkage method. Antiserum with a sufficiently high titer was generated in rabbits and fluorescein isothiocyanate (FITC) was used as sensitive labels to construct the fluorescence immunoassay (FIA) for measurement of targeted compounds. Under optimized FIA condition, the quantitative working range was from 0.1 to 200 µg L−1 with a limit of detection of 0.05 µg L−1. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. Four kinds of water samples (tap water, lake water, river water and leachate) had been detected in this assay, the recovery was 91.3­107.8%. The proposed fluorescence immunoassay turned out to be a powerful tool for monitoring of dicyclohexyl phthalate in water samples at trace level.

[Study of dicyclohexyl phthalate on preparation and characterization of artificial antigen].

OBJECTIVE: To study the preparation and characterization of hapten and artificial antigen of dicyclohexyl phthalate. METHODS: A dicyclohexyl phthalate hapten (4-amino dicyclohexyl phthalate) was synthesized by introducing amino as a substituent on the aromatic ring and retaining the ester group, and characterized by 1HNMR, IR and UV. The hapten was conjugated to BSA via amino diazotization linkage. RESULTS: Lambda1 = 214nm, lambda2 = 256nm for the UV of dicyclohexyl 4-nitrophthalate and lambda1 = 226nm, lambda2 = 288nm for the UV of dicyclohexyl 4-aminophthalate. Artificial antigen was prepared and tested by fluorescence, and lambda(ex) = 307nm, lambda(em) = 468nm, and the approximate molar ratio of dicyclohexyl 4-aminophthalate to BSA was 19. The product was used as an immunogen, demonstrating that it is suitable for polyclonal antibody production. CONCLUSION: It is a good method for preparation of artificial antigen of dicyclohexyl phthalate by introducing amino as a substituent on the aromatic ring and retaining the ester group. It was suggested that could supply excellent immune antigen for further preparation of antibody and immunoassay to dicyclohexyl phthalate.

A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate.

A novel, sensitive, and specific competitive fluorescence immunoassay has been developed for the quantitative determination of dibutyl phthalate (DBP) using an antibody-coated plate format. Hapten was synthesized in order to produce polyclonal antibodies against dibutyl phthalate. Polyclonal antisera to dibutyl phthalate were generated in rabbits and used to construct the fluorescence immunoassay for measurement of dibutylphthalate. The assay had a detection limit of about 0.02 microg L(-1), a dynamic range of approximately 0.1-300 microg L(-1). Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. The study demonstrated that the developed antiserum and fluorescence immunoassay procedure can be used to detect dibutyl phthalate in environmental samples such as tap water, river water, drinking water, and leachate from plastic drinking water bottles.

Fluorescence for the determination of protein with functionalized nano-ZnS.

ZnS nanoparticles have been prepared and modified with sodium thioglycolate. The functionalized nanoparticles are water-soluble. They were used as fluorescence probes in the determination of proteins, which was proved to be a simple, rapid and specific method. In comparison with single organic fluorophores, these nanoparticle probes are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimum conditions, linear relationships were found between the enhanced intensity of fluorescence at 441 nm and the concentration of protein in the range 0.1-4.0 microg mL(-1) for human serum albumin (HSA), 0.2-3.0 microg mL(-1) for bovine serum albumin (BSA) and 0.1-4.5 microg mL(-1) for gamma-globulin (gamma-G). The limits of detection were 0.015 microg mL(-1) for HSA, 0.024 microg mL(-1) and 0.017 microg mL(-1) for BSA and gamma-G, respectively. The method has been applied to the analysis of human serum samples collected from the hospital and the results were in good agreement with those reported by a hospital, indicating that the method presented here is not only sensitive and simple, but also reliable and suitable for practical application.