Magnetite-Quantum Dot Immunoarray for Plasmon-Coupled-Fluorescence Imaging of Blood Insulin and Glycated Hemoglobin

Abstract

New microarray chip strategies that are sensitive and selective and that can measure low levels of important biomarkers directly in a blood sample are significant for improving human health by allowing timely diagnosis of an abnormal condition. Herein, we designed an antibody–aptamer immunoarray chip to demonstrate simultaneous measurement of blood insulin and glycated hemoglobin (HbA1c) levels relevant to diabetic and prediabetic disorders using a surface plasmon microarray with validation by fluorescence imaging. To accomplish both surface plasmon and fluorescence imaging on the same sample, we decorated magnetite nanoparticles with quantum dots for covalent immobilization of aptamers for subsequent capture and isolation of the aptamers specific for insulin and HbA1c markers from 20-times diluted whole blood samples. Direct clinically relevant analysis, along with fluorescent imaging of the two markers, was achieved by this new immunoarray platform. The limit of detection was 4 pM for insulin and 1% for HbA1c. Examination of cross-talk using thrombin and platelet-derived growth factor confirmed that the designed immunoarray was highly selective for insulin and HbA1c. Surface plasmon kinetic analysis provided apparent binding constants of 0.24 (±0.08) nM and 37 (±3) μM, respectively, for the binding of insulin and HbA1c onto their surface immobilized monoclonal antibodies. Thus, quantitative imaging of ultralow levels of blood biomarker levels with binding kinetics is uniquely obtained in the designed immunoarray chip. In conclusion, this report demonstrates considerable significance of the developed magnetite-quantum dot-bioconjugate strategy for clinical diagnostics of whole blood biomarkers with characterization of molecular binding interactions.

Electrochemical and surface plasmon insulin assays on clinical samples

“…Our subsequent study focused on a much more complex matrix such as whole blood to detect two biomarkers, insulin and HbA1c, in one assay by an aptamer-surface antibody sandwich array format. 76 Sensitive detection was achieved by decorating magnetic NPs with dual quantum dots (QDs; one for insulin and the second for HbA1c) to simultaneously accomplish both the surface plasmon and fluorescence imaging of the two biomarkers with binding kinetics analysis (Fig. 8a, 8b).…”

Epitope‐Resolved Detection of Peanut‐Specific IgE Antibodies by Surface Plasmon Resonance Imaging

“…Secondary antibodies (Ab 2 ) specific for the IgE ϵ ‐chain were immobilized onto the MB to bind IgE antibodies from serum. This tended to lower the nonspecific binding of other serum proteins to the array surface . Differential epitope responses to allergen‐specific IgE antibodies were achieved by providing a binding environment designed to detect low IgE concentrations with minimal cross‐reactivity.…”

Building Biomimetic Potency Tests for Islet Transplantation

“…Some use synthetic strands of RNA or DNA, known as "aptamers," to bind to the insulin. These approaches often use an electrochemical (121)(122)(123)(124)(125)(126)(127)(128) or surface plasmon resonance (129,130) sensing principle, wherein insulin-targeting aptamers conjugated to various active elements are adhered to a metal surface and can alter the electromagnetic properties of the system as a result of aptamer conformation change during insulin binding (131). In some cases, the aptamers adhered to a surface do not release their target compounds once bound, which necessitates cycles of wash steps to regenerate the sensors for repeated measurements (122,132,133).…”