Abstract
The mechanism of action (MOA) of the first line type-2 diabetes drug metformin remains unclear despite its widespread usage. However, recent evidence suggests that the mitochondrial copper (Cu)-binding action of metformin may contribute toward the drug’s MOA. Here, we present a novel biosensing platform for investigating the MOA of metformin using a magnetic microbead-based agglutination assay which has allowed us to demonstrate for the first time the interaction between Cu and metformin at clinically relevant low micromolar concentrations of the drug, thus suggesting a potential pathway of metformin’s blood-glucose lowering action. In this assay, cysteine-functionalized magnetic beadswere agglutinated in the presence of Cu due to cysteine’s Cu-chelation property. Addition of clinically relevant doses of metformin resulted in disaggregation of Cu-bridged bead-clusters, whereas the effect of adding a closely related but blood-glucose neutral drug propanediimidamide (PDI) showed completely different responses to the clusters. The entire assay was integrated in an automated microfluidics platform with an advanced optical imaging unit by which we investigated these aggregation–disaggregation phenomena in a reliable, automated, and user-friendly fashion with total assay time of 17 min requiring a sample (metformin/PDI) volume of 30 μL. The marked difference of Cu-binding action between the blood-glucose lowering drug metformin and its inactive analogue PDI thus suggests that metformin’s distinctive Cu-binding properties may be required for its effect on glucose homeostasis. The novel automated platform demonstrating this novel investigation thus holds the potential to be utilized for investigating significant and sensitive molecular interactions via magnetic bead-based agglutination assay.
Highlights
Disaggregation phenomena in a reliable, automated, and user-friendly fashion with total assay time of 17 min requiring a sample volume of 30 μL
The MBs functionalized with biotinylated L-cysteine forms clusters through L-cysteine-Cu bond after the addition of Cu2+ followed by magnetic incubation, which are again disaggregated after adding metformin followed by the second magnetic incubation
We investigated the effect of higher doses (0.5−50 mM) of metformin on Cu-MB disaggregation
Summary
Disaggregation phenomena in a reliable, automated, and user-friendly fashion with total assay time of 17 min requiring a sample (metformin/PDI) volume of 30 μL. The novel automated platform demonstrating this novel investigation holds the potential to be utilized for investigating significant and sensitive molecular interactions via magnetic bead-based agglutination assay This drug has been proven to interact with different metals[10−14] and the most stable of these interactions is with the metal copper (Cu).[15,16] Recently the relationship of Cubinding to the clinical action of metformin has been under investigation.[16−18] The studies found that metformin binds with mitochondrial Cu under conditions where it inhibits mitochondrial respiration. We present an integrated and automated biosensing platform with a substantial improvement in sensitivity compared to our previous work while using microsized magnetic beads (hereafter represented by “MB”) These improvements have allowed us to quantify, for the first time, the effects of micromolar concentrations of metformin on copper-bridged cysteine MB aggregates. The experimental setup has been improved compared to our previous study[18] by integrating an automated optical imaging unit (oCelloScope, Philips Biocell) for automated operation of the entire study, allowing rapid optimization of the Cu-MB clusters
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