Background
ELISA (Enzyme-Linked Immunosorbent Assay) is a powerful laboratory technique for screening disease analytes in biological samples. By utilizing the principles of immunoassays and enzymatic reactions, ELISA enables the detection and quantification of specific molecules known as analytes. The procedure begins by coating a microtiter plate or solid support with capture antibodies designed to bind to the target analyte of interest. These antibodies act as "molecular traps" that capture and immobilize the analyte molecules. The coated surface is then blocked to prevent any non-specific binding. The test sample, which may contain the analyte, is then placed on the microplate and incubated. If the analyte is present, it binds to the capture antibodies and forms a "sandwich" complex. To detect the presence of the analyte, a secondary antibody is introduced. This secondary antibody is conjugated to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The secondary antibody binds to either the captured analyte or the captured antibodies, forming a secondary "sandwich" complex. After thorough washing to remove all unbound components, a substrate specific for the enzyme conjugate is added. When the enzyme acts on the substrate, it produces a detectable signal, usually a color change directly proportional to the amount of analyte in the sample. The resulting signal is measured with a microplate reader, which quantifies the amount of analyte in the sample by comparing it to a standard curve generated from known analyte concentrations. Although ELISA is a reliable method that can detect analytes sensitively and selectively even at low concentrations, there are several drawbacks: (i) the need for careful handling and expertise in performing multiple assay steps that are prone to error, (ii) a limited dynamic range, (iii) a time-consuming incubation period that can delay obtaining results, which can be a problem when early detection of disease is critical. (iv) cross-reactivity with similar molecules may affect specificity, leading to false positives or interferences, and (v) the expensive facility to perform the assays.To overcome the above problems, this work aims to develop a simple and cost-effective platform for analyte detection and quantification. To achieve this goal, electrochemical impedance spectroscopy (EIS) analyses are performed to evaluate the changes in impedance/resistance of the solution containing the sample caused by enzymatically initiated hydrogelation upon binding of the analyte. For proof of concept, a sandwich immunoassay platform will be developed to test the biomarker for acute kidney injury (AKI): neutrophil gelatinase-associated lipocalin (NGAL).

Objectives/Work Packages:
The work packages to be performed are as follows.
- Literature study to gain knowledge on immunoassays, hydrogelation and EIS,
- Development of a sandwich immunoassay for colorimetric detection of NGAL and optimization of the ELISA assay.
- Transfer the optimal assay into a magnetic bead-based immunoassay and perform analyte detection with EIS.
- Comparison of analyte detection performance of the developed impedimetric detection method with colorimetric ELISA.
The results will be presented in a final report and presentation. Only the written paper will count for evaluation and grading.