Abstract
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.
Highlights
Sepsis is a life-threatening systemic body infection manifested by symptoms ranging from elevated heartbeat, breathing difficulties and fever to severe necrosis of the internal organs
While sensitivity expressed by limit of detection (LOD), limit of quantification (LOQ) and IC50 is similar for interleukin 6 (IL-6) and E- Selectin after 2 h and 2.5 h incubation, there are significant differences observed for S-100 and C-reactive protein (CRP)
We have presented a protein chip that fulfills the requirements for point-of-care diagnostics in terms of miniaturization, assay time and internal calibration
Summary
Sepsis is a life-threatening systemic body infection manifested by symptoms ranging from elevated heartbeat, breathing difficulties and fever to severe necrosis of the internal organs. Sepsis is a condition caused by pathogenic microorganisms but induced by the immune response through excessive release of growth factors, cytokines and inflammation- and coagulation factors into the systemic circulation Those factors can be exploited as specific biomarkers enabling early and reliable diagnosis. We aim at the further development of a sepsis chip for point-of-care testing of neonatal sepsis This is achieved by: (a) the reduction of required patient sample volume, (b) the reduction of assay steps and incubation times and (c) the integration of an internal calibration. The chip for biomarker detection of sepsis comprises multi-analyte on-chip immunoassays which include both sandwich (cytokines, PCT, S-100, E-Selectin) and binding inhibition formats (CRP, neopterin). The calibration was integrated on the chip (internal calibration) presenting a point-of-care device suitable for use in neonates
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