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

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.

Keywords: internal calibration; miniaturization; neonatal sepsis; point-of-care testing; protein microarray; streptavidin coated magnetic particles.

Figure 1.

On-chip sandwich immunoassay steps for (a) the classical chip (b) the miniaturized chip and (c) the optimized miniaturized chip. Capture antibodies are immobilized equally providing same array format for the classical chip and the miniaturized chip. a(I) incubation of analytes (2.5 h), a(II) incubation of biotinylated detection antibodies (45 min), a(III) detection of bound antigens/antibodies via Dy647 Streptavidin incubation (45 min); b(I) Incubation of analytes, biotinylated antibodies and streptavidin conjugated magnetic particles in one step (2.5), b(II) detection via biotinylated antibody against Cy3/Cy5 and Cy3 labeled streptavidin; c(I) Incubation of analytes, biotinylated antibodies and Dy647 Streptavidin conjugated magnetic particles in one step (2.5 h).

Figure 2.

Calibration curves for (a) IL-6 (b) S-100 (c) E-Selectin and (d) CRP. The black curve ( ) displays the 10 μL assay and the red curve ( ) shows the 50 μL assay when adding strep.MPs; the blue curve ( ) displays the classical 50 μL assay and the green curve ( ) displays the 10 μL assay without addition of strep.MPs.

Figure 3.

Calibration curves for (a) IL-6 (b) S-100 (c) E-Selectin and (d) CRP in human serum 1:10 diluted with assay buffer after 2.5 h ( ), 2 ( ) and 1.5 h ( ) processing time.

Figure 4.

Calibration curves (a) and Scatter plot (b) for the internal ( ) and external chip calibration ( ). Internal and external calibration curves cover a very similar measurement range and most fluorescence intensity values cluster within the 95% confidence band (grey dotted lines) of the fit line. The coefficient of determination (R²) was 0.968.

Figure 5.

Processed chip showing spots obtained for the external (a) and internal (b) calibration at a concentration of 8.1 ng/mL S-100.