In this reported work, we achieved high-throughput, highly sensitive fluorescent analysis using an enzyme-linked immunosorbent assay (ELISA) that employed a metallic nanoparticle (NP)-immobilized 96-well plate. The immobilization of metallic NPs on a 96-well plate effectively amplified fluorescent signals of the assay. The silver (Ag) NP-immobilized plate showed the best fluorescent enhancement effect of all plates immobilized by metal NPs. Our results demonstrate the potential of applying Ag NPs to enhance the efficiency of direct and indirect ELISA by the labeling of targets.

Extracellular vesicles (EVs) play an important role in cell-to-cell communication by carrying molecular messages that reflect physiological and pathological conditions of the parent cells. EVs have been identified in all body fluids; and among them, urine stands out as a sample that is easy and inexpensive to obtain and can be collected over time to monitor changes. Various protocols have been established to study urinary extracellular vesicles (UEVs) and they have shown great potential as a biomarker source for clinical applications, not only for urological, but also non-urological diseases. Due to the high variability and low reproducibility of pre-analytical and analytical methods for UEVs, establishing a standardized protocol remains a challenge in the field of diagnosis. Here, we review UEV studies and present the techniques that are most commonly used, those that have been applied as new developments, and those that have the most potential for future applications. The workflow procedures from the sampling step to the qualitative and quantitative analysis steps are summarized along with advantages and disadvantages of the methodologies, in order to give consideration for choosing the most promising and suitable method to analyze human UEVs.

Sensitive and specific detection of nucleic acids or proteins, which act as biomarkers, is of great importance in disease diagnosis. By combing the concept and operation of an endonuclease-assisted target-responsive amplification method and peroxidase-mimic DNAzyme generated by terminal deoxynucleotidyl transferase (TdT), a novel and facile colorimetric biosensor was developed for DNA and protein. Target DNA and thrombin were chosen as representative biomolecules. The production of cleavage fragments can only be triggered by specific target binding and the following nicking process, which do not occur spontaneously. In the signal collection part, numerous guanine-rich DNA were produced through the prolongation of cleavage fragments by TdT and formed highly effective DNAzyme with hemin. In this novel amplification method, we succeeded in realizing sensitive and specific detection of target DNA and thrombin. Under optimal conditions, target DNA can be detected as low as 1 pM, and thrombin with a detection limit of 100 pM. The method also proves the potential versatility and feasibility of TdT-generated DNAzyme in various bio-analyses.

Two novel malonyl-based chemosensors, N,N′-bis(ethyl-4′-benzoate)-1,3-propanediamide (1) and N,N′-bis(ethyl-3′-benzoate)-1,3-propanediamide (2), have been synthesized and screened towards various biologically important metal ions such as Na⁺, Mg²⁺, K⁺, Ca²⁺, Al³⁺, Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺, Ti³⁺, and Pb²⁺. The emission spectral studies of both 1 and 2 displayed 84 – 91% turn-off emission responses selectively with Fe³⁺ ion in aqueous buffer (MeCN/H₂O, 1:4, v/v, pH = 7.4) solution. Chemosensors 1 and 2 exhibited remarkable sensing ability towards Fe³⁺ ion over other metal ions with limit of detection (LOD) of 4.28 and 4.33 μM, respectively. The binding stoichiometry of 1 and 2 with Fe³⁺ ion was studied by Benesi–Hildebrand fitting, Stern–Volmer plot and Job’s plots, revealing that both chemosensors (1 – 2) bind with Fe³⁺ metal ion in 1:1 stoichiometric ratio with the apparent association constant (K_a) 8.90 × 10³ and 11.16 × 10³ M⁻¹, respectively. Furthermore, the interactions of chemosensors (1 – 2) with metal ion were also investigated by using density functional theory (DFT) at B3LYP hybrid functional using 6-31G and LanL2DZ basis sets.

We describe here a magnetic molecular imprinted polymeric ionic liquid (MMIPIL) film by using a functionalized ionic liquid (3-vinyl-4-amino-5-imidazole carboxamide chloride, IL) and Fe₃O₄@Polyrutin–COOH as a functional monomer and supporting materials. The change in the direction of the charge density in the structure of MMIPIL polymer resulted in a red shift of about 100 nm for the characteristic group of –C=O. Polyrutin containing an electron-rich benzene ring and multiple hydroxyl groups not only prevented the aggregation of Fe₃O₄, but also benefitted to immobilize template molecules. More symmetric amino groups in the template molecules generated more hydrogen bonds and other synergistic effects between MEL and the functional monomers, which resulted in a highly-matched and highly stable MMIPIL sensor. The proposed magnetic sensor lowered the matching potential, and enhanced the signal for the detection of melamine (MEL) in milk powder. Under the optimum conditions, the MEL template molecule showed a significant linear relationship between 5.0 × 10⁻³ and 0.8 μg/L with a detection limit (S/N = 3) of 1.5 × 10⁻³ μg/L. The MMIPIL sensor showed wonderful selectivity and exhibited facile, fast and efficient results in the monitoring MEL with recoveries of between 96.5 and 108.3%.