Bioconjugated fluorescent zeolite L nanocrystals as labels in protein microarrays.

Zeolite L nanocrystals, as inorganic host material containing hydrophobic fluorophore N,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide in the unidirectional channels, are developed as new labels for biosensor systems. The external surface of the particles is modified with carboxylic acid groups for conjugation to primary amines of biomolecules such as antibodies. Anti-digoxigenin (anti-DIG) is selected to be immobilized on zeolite L via N-hydroxysulfosuccinimide ester linker. Together with DIG, it serves as a good universal binding pair for diverse analyte detection owing to the high binding affinity and low background noise. The conjugates are characterized by the dynamic light scattering technique for their hydrodynamic diameters and by enzyme-linked immunosorbent assay for antigen-antibody binding behavior. The characterizations prove that anti-DIG antibodies are successfully immobilized on zeolite L with their binding activities maintained. The microarray fluorescent sandwich immunoassay based on such nanocrystalline labels shows high sensitivity in a thyroid-stimulating hormone assay with the lower detection limit down to the femtomolar range. These new fluorescent labels possess great potential for in vitro diagnostics applications.

Performance evaluation of three platforms with ultrasensitive ligand-binding assay potential.

AIM: We evaluated three immunoassay-based technologies and their biomarker kits, by determining precision, parallelism and detectability of analytes of interest. MATERIALS & METHODS: We compared ultrasensitive assays for three biomarkers: interleukins IL-6, IL-13 and IL-17A using kits obtained from Roche (IMPACT platform - proprietary platform), Singulex (Erenna®) and Quanterix (Simoa™). We defined the true LLOD as the LLOQs, and provided disease-specific parallelism results and detectability levels for endogenous analyte, which were good across platforms, though they varied from analyte to analyte. CONCLUSION: We highlight a simplified approach employed for evaluating ultrasensitive kits and provide an overview of the methodologies used to compare available assays. All three platforms are able to detect very low-level analytes. We recommend all three platforms for detection of very low-level analytes.

Preparation of plasma samples for chromatographic analyses using functionalized ferromagnetic micro-particles manipulated in a high pressure liquid system.

OBJECTIVES: To study if functionalized ferromagnetic micro-particles used for extraction procedures prior to chromatographic analyses can be handled within an HPLC system. METHOD: We used C18-functionalized ferromagnetic micro-particles, manipulated within a high-performance liquid chromatography system for extraction of plasma samples, with a novel extractor device enabling reversible immobilization of the particles within HPLC tubing. A pilot analytical system for the quantification of itraconazole in plasma--as a representative small molecule analyte--by ultraviolet detection was configured and tested. RESULTS: The system allowed linear and reproducible quantification of the representative target analyte. CONCLUSIONS: Sample preparation of biological samples can be achieved by handling of functionalized ferromagnetic micro-particles which are handled within a high pressure liquid chromatography configuration; this offers interesting perspectives for the development of automated sample preparation devices.

Sample preparation for liquid chromatography-tandem mass spectrometry using functionalized ferromagnetic micro-particles.

OBJECTIVES: To study the applicability of functionalized ferromagnetic micro-particles in the sample preparation for quantification of small molecules by LC-MS/MS. DESIGN AND METHODS: A representative analyte (itraconazole) was extracted from plasma samples using small quantities of C18-modified ferromagnetic micro-particles. RESULTS: 125 microg of functionalized micro-particles was sufficient to extract the target analyte from 10 microL of plasma with a recovery rate of approximately 100%; linearity (r>0.99) and reproducibility (inter-assay CV< or =7%) of quantitative results was found. CONCLUSIONS: C18-functionalized ferromagnetic particles can be used to efficiently extract small molecule analytes from complex biological matrices for quantitative analyses using LC-MS/MS.