SELEX-Based Direct Enzyme-Linked Aptamer Assay (DELAA) for Diagnosis of Toxoplasmosis by Detection of SAG1 Protein in Mice and Humans

Background: Toxoplasma gondii is a single-celled parasite commonly found in mammals. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma-derived DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent cysts in latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key marker for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology.Methods: Recombinant truncated SAG1（r-SAG1）of Toxoplasma WH3 strain (type Chinese 1) was prokaryotically expressed and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamers which target the r-SAG1, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The specific aptamer-2 was screened out and used in direct enzyme-linked aptamer assay (DELAA) for detection of native SAG1 obtained from tachyzoite lysates (n-SAG1), mouse sera of acute infection, and human sera that had been verified to be positive for Toxoplasma DNAs by PCR amplification. Results: The soluble r-SAG1 protein was obtained from E.coli lysates by purification and identification with immunoblotting, and then labelled with biotin. The selected aptamers were amplified by PCR, followed by DNA sequencing. The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for Toxoplasma circulating antigen test.Conclusions: A new direct enzyme-linked aptamer assay (DELAA), with aptamer-2 as the recognition probe, was developed for detection of native SAG1 protein of Toxoplasma. With increased sensitivity and specificity, stability, easy and cheap preparation, the aptamer-based technology is considered as a efficient method for the diagnosis of active and reactivated toxoplasmosis.

The Role of 8-Amidoquinoline Derivatives as Fluorescent Probes for Zinc Ion Determination

Mass-spectrometry-based and X-ray fluorescence-based techniques have allowed the study of the distribution of Zn2+ ions at extracellular and intracellular levels over the past few years. However, there are some issues during purification steps, sample preparation, suitability for quantification, and the instruments’ availability. Therefore, work on fluorescent sensors based on 8-aminoquinoline as tools to detect Zn2+ ions in environmental and biological applications has been popular. Introducing various carboxamide groups into an 8-aminoquinoline molecule to create 8-amidoquinoline derivatives to improve water solubility and cell membrane permeability is also a recent trend. This review aims to present a general overview of the fluorophore 8-aminoquinoline and its derivatives as Zn2+ receptors for zinc sensor probes. Various fluorescent chemosensor designs based on 8-amidoquinoline and their effectiveness and potential as a recognition probe for zinc analysis were discussed. Based on this review, it can be concluded that derivatives of 8-amidoquinoline have vast potential as functional receptors for zinc ions primarily because of their fast reactivity, good selectivity, and bio-compatibility, especially for biological applications. To better understand the Zn2+ ion fluorophores’ function, diversity of the coordination complex and geometries need further studies. This review provides information in elucidating, designing, and exploring new 8-amidoquinoline derivatives for future studies for the improvement of chemosensors that are selective and sensitive to Zn2+.

A fluorescent and colorimetric dual-recognition probe based on copper(ii)-decorated carbon dots for detection of phosphate

In the present article, we report a novel fluorescent and colorimetric dual-signal sensing probe based on a CD–Cu2+ complex for the detection of the phosphate ion (Pi).

SELEX-Based Direct Enzyme-Linked Aptamer Assay(DELAA) for Diagnosis of Toxoplasmosis by Detection of SAG1 Antigen in Sera of Mice and Humans

Background: Toxoplasma gondii is a single-celled parasite commonly found in mammals. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma-derived DNAs due to the presence of tissue dwelling quiescent cysts and latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key marker for laboratory diagnosis. However, at present, there are no methods available for SAG1 detection using aptamer-based technology.Methods: Recombinant truncated SAG1（r-tSAG1）of Toxoplasma WH3 strain (type Chinese 1) was prokaryotically expressed and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamer which targets the r-tSAG1, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The screened specific aptamer-2 was used in direct enzyme-linked aptamer assay (DELAA) to detect native SAG1 obtained from tachyzoite lysates, mouse sera of acute infection, and human sera that had been verified to be positive for ToxoDNAs by PCR amplification. Results: The soluble r-tSAG1 protein was obtained from E.coli lysates by using 0.01M Tris-Cl in PBS, and was purified and identified by immunoblotting, and then labelled with biotin. The screened aptamers were amplified by PCR, followed by DNA sequencing. The results showed that the aptamer-2, with the highest affinity to nSAG1 among the four aptamer candidates, has a higher specificity and sensitivity when used in detection of nSAG1 in the sera of both animals and humans when compared with the commercial Toxoplasma circulating antigen testing kit.Conclusions: A new direct enzyme-linked aptamer assay (DELAA), with aptamer-2 as the recognition probe, was developed for detection of native SAG1 protein secreted by T.gondii. With increased sensitivity and specificity, stability during storage, easy and cheaper production, the aptamer-based technique is considered as a efficient method for the diagnosis of active and reactivated toxoplasmosis.

Investigating the Potential of 8-Amidoquinoline Derivatives as a Fluorescent Probe for Zinc Detection: A Systematic Literature Review

Abundant of preparatory works have recognized that fluorescent sensors based on 8-aminoquinoline are popular tools to detect Zn2+ ions in environmental and biological applications. Along with these studies, researchers started to introduce a variety of carboxamido group into an 8-aminoquinoline molecule in forming 8-amidoquinoline derivatives. Therefore, this systematic review aims to introduce a general overview of the fluorophore 8-aminoquinoline as Zn2+ receptors and to provide comparisons of collected studies that related to 8-amidoquinoline derivatives as fluorophore probe of the sensor. According to PRISMA systematic searches strategy, 13 articles were analyzed for trends, research designs, results and discussion, subject samples, and remarks or conclusions. We found cross-sectional studies with four aspects in zinc sensing that have been targeted; binding studies via titration, detection's limit, interferences studies, and validation of the study. Hence, this paper also included assessments of those criteria and the trends of development of 8-amidoquinoline derivatives based-zinc fluorescent chemosensor. It also showed that most of the researches conducted in China. In conclusion, this study identified various research designs of fluorescent chemosensors based on 8-amidoquinoline prolong with the effectiveness and potential as a recognition probe to assist the detection of zinc. Hence, elucidation of those derivatives essential to be explored because more studies are needed to improve the sensing criteria of the zinc sensor

Functional Molecular Interfaces for Impedance-Based Diagnostics

In seeking to develop and optimize reagentless electroanalytical assays, a consideration of the transducing interface features lies key to any subsequent sensitivity and selectivity. This review briefly summarizes some of the most commonly used receptive interfaces that have been employed within the development of impedimetric molecular sensors. We discuss the use of high surface area carbon, nanoparticles, and a range of bioreceptors that can subsequently be integrated. The review spans the most commonly utilized biorecognition elements, such as antibodies, antibody fragments, aptamers, and nucleic acids, and touches on some novel emerging alternatives such as nanofragments, molecularly imprinted polymers, and bacteriophages. Reference is made to the immobilization chemistries available along with a consideration of both optimal packing density and recognition probe orientation. We also discuss assay-relevant mechanistic details and applications in real sample analysis.