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.
Novel hot electron-emitting working electrodes and conventional counter electrodes were created by screen printing. Thus, low-cost disposable electrode chips for bioaffinity assays were produced to replace our older expensive electrode chips manufactured by manufacturing techniques of electronics from silicon or on glass chips. The present chips were created by printing as follows: (i) silver lines provided the electronic contacts, counter electrode and the bottom of the working electrode and counter electrode, (ii) the composite layer was printed on appropriate parts of the silver layer, and (iii) finally a hydrophobic ring was added to produce the electrochemical cell boundaries. The applicability of these electrode chips in bioaffinity assays was demonstrated by an immunoassay of human C-reactive protein (i) using Tb(III) chelate label displaying long-lived hot electron-induced electrochemiluminescence (HECL) and (ii) now for the first time fluorescein isothiocyanate (FITC) was utilized as an a low-cost organic label displaying a short-lived HECL in a real-world bioaffinity assay.
This paper presents a simple and inexpensive method to fabricate chemically and mechanically resistant hot electron-emitting composite electrodes on reusable substrates. In this study, the hot electron emitting composite electrodes were manufactured by doping a polymer, nylon 6,6, with few different brands of carbon particles (graphite, carbon black) and by coating metal substrates with the aforementioned composite ink layers with different carbon-polymer mass fractions. The optimal mass fractions in these composite layers allowed to fabricate composite electrodes that can inject hot electrons into aqueous electrolyte solutions and clearly generate hot electron-induced electrochemiluminescence (HECL). An aromatic terbium (III) chelate was used as a probe that is known not to be excited on the basis of traditional electrochemistry but to be efficiently electrically excited in the presence of hydrated electrons and during injection of hot electrons into aqueous solution. Thus, the presence of hot, pre-hydrated or hydrated electrons at the close vicinity of the composite electrode surface were monitored by HECL. The study shows that the extreme pH conditions could not damage the present composite electrodes. These low-cost, simplified and robust composite electrodes thus demonstrate that they can be used in HECL bioaffinity assays and other applications of hot electron electrochemistry.
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