Toon Hendrik Evers

Rapid integrated biosensor for multiplexed immunoassays based on actuated magnetic nanoparticles

The realization of biomolecular detection assays for diagnostic purposes is technologically very challenging because such tests demand full integration for ease of use and need to deliver a high analytical performance with cost-effective use of materials. In this article an optomagnetic immunoassay technology is described based on nanoparticles that are magnetically actuated and optically detected in a stationary sample fluid. The dynamic control of nanoparticles by magnetic fields impacts the key immunoassay process steps, giving unprecedented speed, assay control and seamless integration of the total test. The optical detection yields sensitive and multiplexed assays in a low-cost disposable cartridge. We demonstrate that the optomagnetic technology enables high-sensitivity one-step assays in blood serum/plasma and whole saliva. Drugs of abuse are detected at sub-nanogram per millilitre levels in a total assay time of 1 min, and the cardiac marker troponin I is detected at sub-picomole per litre concentrations in a few minutes. The optomagnetic technology is fundamentally suited for high-performance integrated testing and is expected to open a new paradigm in biosensing.

Rapid, high sensitivity, point-of-care test for cardiac troponin based on optomagnetic biosensor

BACKGROUND We present a prototype handheld device based on a newly developed optomagnetic technology for the sensitive detection of cardiac troponin I (cTnI) in a finger-prick blood sample with a turnaround time of 5 min. METHODS The test was completed in a compact plastic disposable with on-board dry reagents and superparamagnetic nanoparticles. In our one-step assay, all reaction processes were precisely controlled using electromagnets positioned above and below the disposable. Nanoparticle labels (500 nm) bound to the sensor surface via a sandwich immunoassay were detected using the optical technique of frustrated total internal reflection. RESULTS A calibration function measured in plasma demonstrates a limit of detection (mean of blank plus 3-fold the standard deviation) of 0.03 ng/mL cTnI. A linear regression analysis of the region 0.03-6.5 ng/mL yields a slope of 37+/-4, and a linear correlation coefficient of R2=0.98. The measuring range could be extended substantially to 100 ng/mL by simultaneously imaging a second spot with a lower antibody concentration. CONCLUSIONS The combination of magnetic particles and their fine actuation with electromagnets permits the rapid and sensitive detection of cTnI. Because of the potential high analytical performance and ease-of-use of the test, it is well suited for demanding point-of-care diagnostic applications.

How antibody surface coverage on nanoparticles determines the activity and kinetics of antigen capturing for biosensing

The antigen-capturing activity of antibody-coated nanoparticles is very important for affinity-based bioanalytical tools. In this paper, a comprehensive study is reported of the antigen-capturing activity of antibodies that are nondirectionally immobilized on a nanoparticle surface. Superparamagnetic nanoparticles (500 nm) were covalently functionalized with different quantities of monoclonal antibodies against cardiac troponin I (cTnI). At a low antibody surface coverage, up to 4% of the immobilized antibodies could capture antigen molecules from solution. At high antibody coverage (≥50 × 10(2) antibodies per nanoparticle, i.e., ≥ 64 × 10(2) antibodies per μm(2)), the fraction of antigen-capturing antibodies drops well below 4% and the number of active antibodies saturates at about 120 per nanoparticle. The fraction of active antibodies is small, yet surprisingly their dissociation constants (Kd) are low, between 10 and 200 pM. In addition, the surface-binding activity of the antibody-coated nanoparticles was analyzed in an optomagnetic sandwich immunoassay biosensor, measuring cTnI in undiluted blood plasma. The data show that the immunoassay response scales with the number of active antibodies, increasing initially and saturating at higher antibody densities. The observations are summarized in a molecular sketch of the attachment, ordering, and functionality of antibodies on the nanoparticle surface.

Protein biomarker enrichment by biomarker antibody complex elution for immunoassay biosensing

It is very challenging to perform sample enrichment for protein biomarkers because proteins can easily change conformation and denature. In this paper we demonstrate protein enrichment suited for high-sensitivity integrated immuno-biosensing. The method enhances the concentration of the biomarkers and simultaneously removes matrix components that could interfere with the immunoassay. Biomarkers are captured using antibody coated magnetic particles and the biomarker antibody complexes are released by enzymatic elution. The eluted complexes are subsequently detected in a sandwich immunoassay biosensor. A scaling study of the enrichment process demonstrates an enrichment factor of 15 in buffer and plasma. We analyze the enrichment factor in terms of the three basic steps of the assay (capture, concentration, elution) and we quantify their respective efficiencies. The process is suited for integration into bio-analytical tools.