Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing

Tuomas Näreoja,Pekka E Hänninen,Jessica M Rosenholm,Urpo Lamminmäki

doi:10.1007/s00216-017-0284-z

Abstract

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L−1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates.Graphical We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

Highlights

Eu-doped nanoparticles can be manufactured to afford high colloidal stability; good protection of Eu-chelates from solvent; very low self-quenching owing to long Stoke’s shift of the Eu-chelates; density that is close to water’s density to reduce sedimentation; and a dense coating of carboxyl groups on their surface for efficient bioconjugation [1]
The colloidal stability of nanoparticle bioconjugates in suspension is determined by their surface potential [28], stability of conjugated antibodies [29], ionic strength and pH of the assay buffer [30], concentration of nanoparticle bioconjugates in the suspension, detergents in the assay buffer, presence of blocking proteins, and stability of their protein corona [12]
Unbound nanoparticle bioconjugates were washed away, and wells were read for time-resolved Eu-fluorescence. c To reduce nonspecific binding in a nanoparticle-based immunoassay we used a nonspecific affinity purification step to remove compounds interfering with the assay

Summary

Introduction

Eu-doped nanoparticles can be manufactured to afford high colloidal stability; good protection of Eu-chelates from solvent; very low self-quenching owing to long Stoke’s shift of the Eu-chelates; density that is close to water’s density to reduce sedimentation; and a dense coating of carboxyl groups on their surface for efficient bioconjugation [1]. Most importantly the Eu-doped nanoparticles facilitate sensitive timeresolved low background detection of Eu-emission and are perfect label candidates for assays that require high sensitivity. While the nanoparticle-based assay concepts have high signal output, their applicability has been limited because of assay matrix-related interference and relatively high level of nonspecific binding observed in the assays [1,2,3]. We have developed a rational basis for development of immunoassays using nanoparticles as labels [5,6,7] and expand this work to describe assay matrix-derived interactions [8]

Results

Discussion

Conclusion