Hydrogel based protein biochip for parallel detection of biomarkers for diagnosis of a Systemic Inflammatory Response Syndrome (SIRS) in human serum

Abstract

The Systemic Inflammatory Response Syndrome (SIRS), a sepsis related inflammatory state, is a self-defense mechanism against specific and nonspecific stimuli. The six most extensively studied inflammatory biomarkers for the clinical diagnosis of SIRS are interleukin 4 (hIL-4), interleukin 6 (hIL-6), interleukin 10 (hIL-10), tumor necrosis factor alpha (hTNF-α), interferon gamma (hIFN-γ) and procalcitonin (hPCT). These biomarkers are naturally present (but usually only at low concentration) in SIRS infected patients [1, 2] and thus the development of a highly sensitive detection method is of major clinical interest. However, the existing analytical techniques are lacking in required analytical sensitivity and parallel determination of these biomarkers. We developed a fast, easy and cost-efficient protein microarray biochip where the capture molecules are attached on hydrogel spots, enabling SIRS diagnosis by parallel detection of these six clinically relevant biomarkers with a sample volume of 25 μl. With our hydrogel based protein microarray biochip we achieved a limit of detection for hIL-4 of 75.2 pg/ml, for hIL-6 of 45.1 pg/ml, for hIL-10 of 71.5 pg/ml, for hTNF-α of 56.7 pg/ml, for IFN-γ of 46.4 pg/ml and for hPCT of 1.1 ng/ml in spiked human serum demonstrating sufficient sensitivity for clinical usage. Additionally, we demonstrated successful detection of two relevant SIRS biomarkers in clinical patient samples with a turnaround time of the complete analysis from sample-to-answer in less than 200 minutes.

Macroporous Polymer Monoliths in Thin Layer Format

“…Thus, the most optimal supports for protein analysis are the porous hydrophilic matrices providing the aqueous microenvironment for both immobilized and analyzed proteins. Currently, the supports based on hydrophilic gels as well as nitrocellulose membranes are widely used as base for protein microarrays [108][109][110]. In comparison with swelling hydrogel layers, the washing steps for nitrocellulose membranes and macroporous monoliths, having non-swelling open porous space, takes less time.…”

Biomedical applications of magnetic hydrogels

“…97 The magnetic hydrogel as a diagnostic element constitutes a versatile and profitable platform and can be used for different applications in the field of diagnosis. 98 The use of magnetic hydrogels to create biosensors that respond to external stimuli is increasingly used. This hydrogel must have great stability and hardness and be able to adequately detect the component of interest and translate this interaction into a quantifiable signal.…”

Composite Peptide–Agarose Hydrogels for Robust and High-Sensitivity 3D Immunoassays

“…A growing niche of hydrogel applications is the field of three-dimensional (3D) immunoassays, where they are used as soft matrices to locally confine biomolecules onto analytical surfaces in solution-mimetic conditions. This advanced technology has been recently applied to diverse purposes, including drug screening, enzyme activity assays, miRNA detection, and high-throughput assessment of biomaterials in regenerative medicine applications. − In a broader sense, to be effective in clinical diagnostics, immunoassays must achieve low limits of detection and stringent specificity for the target biomarkers even in complex biological samples. As such, one of the biggest challenges is to set the sensitivity of analytical platforms, including enzyme-linked immunosorbent assay (ELISA) and microarrays, up to a level where high sample dilution can be used, to minimize nonspecific interference of the sample matrix and maximize assay specificity. − …”