Four assay designs and on-chip calibration: gadgets for a sepsis protein array.

A protein microarray for the early stage diagnosis of sepsis that allows the simultaneous detection of C-reactive protein (CRP) (2-200 µg/mL), procalcitonin (PCT) (0.2-50 ng/mL), and interleukin 6 (IL-6) (2-2000 pg/mL) has been developed. To enable the parallel detection of the differently abundant analytes, the low binding affinity between CRP and phosphocholine is exploited in a "low-sensitive" sandwich assay for CRP. The calibration is integrated directly on the chip resulting in a "one patient-one array" format, to provide a user-friendly and rapid diagnostic tool. Four different assay designs are introduced: (I) the classical assay that works with biotin-streptavidin chemistry, (II) the rapid assay that is performed in a single detection step, and two ultrasensitive assay designs accomplished either by (III) an enzymatic or (IV) an antibody mediated amplification resulting in high density labeling. The assay designs were evaluated by the repetitive measurement of low, medium, and high concentration levels of commercially available certified control sera. The precision was similar across all assay designs (coefficient of variation (CV), CVintra: 8-14%; CVinter: 18-34%), while the sensitivity (limits of detection (LODs)) increased by 1 order of magnitude for the ultrasensitive assays (III, IV) and the accuracy was analyte dependent but best for the classical (I) and the antibody amplified (IV) assays.

Signaling by the extracellular matrix protein Reelin promotes granulosa cell proliferation in the chicken follicle.

Chicken oocytes develop in follicles and reach an enormous size because of a massive uptake of yolk precursors such as very low density lipoprotein and vitellogenin. Oocyte growth is supported by theca cells and granulosa cells, which establish dynamic and highly organized cell layers surrounding the oocyte. The signaling processes orchestrating the development of these layered structures are largely unknown. Here we demonstrate that the Reelin pathway, which determines the development of layered neuronal structures in the brain, is also active in chicken follicles. Reelin, which is expressed in theca cells, triggers a signal in granulosa cells via apolipoprotein E receptor 2 and the very low density lipoprotein receptor, resulting in the phosphorylation of disabled-1 and consecutive activation of the phosphatidylinositol 3-kinase/Akt pathway. This signaling pathway supports the proliferation of differentiated granulosa cells to keep up with the demand of cells to cover the rapidly increasing surface of the giant germ cell.

Thermo-nanoimprinted biomimetic probe for LPS and LTA immunosensing.

A complex prepolymerized film comprising monomers, cross-linkers, and initiator is usually used to create molecularly imprinted polymers. We herein exploit ready-to-use resist materials and link molecular surface imprinting with UV- and thermo-nanoimprinting techniques to create a sensor layer for the specific recognition of the bacterial surface markers lipopolysaccharide (LPS) and lipoteichoic acid (LTA). To account for the highly polar moieties of LPS and LTA, we evaluate different resist and stamp materials of distinct surface properties by AFM and molecularly imprinted sorbent assays. Thermo nanoimprinting of LPS and LTA micelles to Epon 1002F films exhibits excellent sensitivity of up to 13 times increased signals compared to those of the nonimprinted films and negligible cross-reaction with the tested nonspecific analyte. Additionally, the sensitivity and selectivity of the thermo nanoimprints is compared to conventional molecular surface imprints using a cocktail of acrylic monomers in QCM measurements.

Miniaturized protein microarray with internal calibration as point-of-care device for diagnosis of neonatal sepsis.

Neonatal sepsis is still a leading cause of death among newborns. Therefore a protein-microarray for point-of-care testing that simultaneously quantifies the sepsis associated serum proteins IL-6, IL-8, IL-10, TNF alpha, S-100, PCT, E-Selectin, CRP and Neopterin has been developed. The chip works with only a 4 µL patient serum sample and hence minimizes excessive blood withdrawal from newborns. The 4 µL patient samples are diluted with 36 µL assay buffer and distributed to four slides for repetitive measurements. Streptavidin coated magnetic particles that act as distinct stirring detection components are added, not only to stir the sample, but also to detect antibody antigen binding events. We demonstrate that the test is complete within 2.5 h using a single step assay. S-100 conjugated to BSA is spotted in increasing concentrations to create an internal calibration. The presented low volume protein-chip fulfills the requirements of point-of-care testing for accurate and repeatable (CV < 14%) quantification of serum proteins for the diagnosis of neonatal sepsis.