Imaging electrochemiluminescence layer to dissect concentration-dependent light intensity for accurate quantitative analysis

This study is led by Dr. Bin Su (Key Laboratory of Excited-State Materials of Zhejiang Province, Institute of Analytical Chemistry, Department of Chemistry, Zhejiang University). Electrochemiluminescence (ECL) is light emission produced by radiative decay of excited states formed by exergonic electron–transfer reactions involving electrochemically generated species. One of the most frequently studied ECL systems is that composed of tris(2,2′-bipyridyl)ruthenium (Ru(bpy)₃²⁺) as luminophore and tri-n-propylamine (TPrA) as coreactant, showing a high analytical sensitivity because of its near-zero background, continuous regeneration and emission of (Ru(bpy)₃²⁺) near the electrode surface. With this system, various strategies have been elaborated to match diverse applications where the intensity of ECL is often measured as the signal, e.g., in the immunoassay of disease biomarkers. In addition, a variety of coreactant-type compounds, such as oxalate , biomolecules, amides and others, can be also detected in solutions using freely diffusing or immobilized (Ru(bpy)₃²⁺) as luminophore.

However, ECL generation from (Ru(bpy)₃²⁺) and TPrA follows a rather complex scheme involving several parallel competitive reaction routes (Scheme S1), depending on the analytical strategy and concentration of (Ru(bpy)₃²⁺) and TPrA. For example, in microbead based immunoassays, (Ru(bpy)₃²⁺) bound to the bead surface yields ECL by reacting with electrogenerated TPrA⁺ and TPrA^• via the so-called “low oxidation potential route”, in which TPrA acts as the sacrificial reagent and the measured intensity of ECL is directly proportional to the quantity of immunologically captured protein biomarkers. When both (Ru(bpy)₃²⁺) and TPrA are freely diffusing in solution, the reaction process is dominated by the so-called “oxidative-reduction route” at a low concentration of (Ru(bpy)₃²⁺), whereas the contribution of “catalytic route” will emerge if the concentration of (Ru(bpy)₃²⁺) is beyond 100 μM. Moreover, some other routes also exist and all of them are always involved together and the deconvolution according to the concentration of (Ru(bpy)₃²⁺) is ambiguous.

With a primary objective of unveiling the contribution of different reaction routes to overall ECL process, herein they report a new approach to monitor the variation of thickness of ECL layer (TEL) by combined use of microwire electrode and ECL microscopy. As illustrated in Fig. 1, a piece of carbon fiber is horizontally positioned in the electrochemical cell made by polydimethylsiloxane (PDMS) to act as working electrode, around which TEL can be visualized by microscopic imaging. In comparison with the microtube imaging approach they reported recently, the current microwire-based platform is easy to build without complicated chemical preparation. Moreover, the cylindrical wire structure of carbon fiber allows a three-dimensional free diffusion of reactants and intermediates in solution, thus avoiding spatial overlap of ECL layer observed with microtube electrode and being capable of revealing the extension of ECL layer more accurately. Further in conjunction with finite element simulations, they intend to explore the variation of TEL with the concentration ratio of (Ru(bpy)₃²⁺) to TPrA (c_Ru/c_TPrA) and to understand the mechanistic origin behind. Finally, the specific effect of this concentration-dependent mechanism on accurate quantitative analysis is deciphered for the detection of coreactant-type analytes.

In summary, by combined use of carbon fiber electrode and microscopic imaging, they observed an apparent extension of ECL layer at the electrode surface, with a thickness varied from a few micrometers to several tens of micrometers upon increasing the concentration ratio of (Ru(bpy)₃²⁺) to TPrA. The thickness is remarkably larger than that estimated in terms of the “oxidative-reduction route”, even if the concentration of (Ru(bpy)₃²⁺) is as low as 50 μM. This phenomenon indicates the significant contribution of “catalytic route” as long as the concentration ratio of (Ru(bpy)₃²⁺) to TPrA is sufficiently large, regardless of the concentration of (Ru(bpy)₃²⁺). Moreover, in the quantitative analysis of coreactant-type analytes, the “catalytic route” helps achieve a high sensitivity in the low concentration range yet results in a nonlinear calibration plot in the full concentration range. For coreactant-type analytes that are hard to oxidize, the contribution of “catalytic route” is insignificant or absent, the calibration curve is thus monotonically linear. They believe fundamental understanding of concentration-dependent reaction mechanisms help conduct precise quantitative analysis. This microwire-based imaging approach also applies to other ECL reaction systems.

See the article:

Imaging electrochemiluminescence layer to dissect concentration-dependent light intensity for accurate quantitative analysis

https://doi.org/10.1016/j.asems.2022.100028