Glycoprotein assay based on the optimized immittance signal of a redox tagged and lectin-based receptive interface

Abstract

Glycoproteins play important roles in biological systems such as in process related to cell binding, signaling and disease. Consequently, novel, potentially quantitative, and rapid electroanalytical approaches capable of detecting protein binding are welcome. Herein, we introduce a methodology that is both fast and sensitive, and capable of quantification of the binding affinity in glycoprotein-lectin molecular models. The proposed methodology is based on the electrochemical impedance spectroscopy technique focused on the immittance function approach, wherein a library of analytical parameters can be computed from the raw impedance data obtained, and automatically processed in a label-free, quantifiable and very sensitive assay platform. This approach also avoids redox probe pre-doping of the analytical sample. Avoiding redox pre-doping of the analytical sample is achievable designing an appropriate redox-tagging monolayer containing lectin interface (a carbohydrate binding protein, herein ArtinM) as the bio-receptor, endowing high sensitivity of electrochemical signal when specifically detecting glycoproteins of interest (presently horseradish peroxidase, HRP, a mannose glycoprotein) as the biochemical target for ArtinM. The electroanalytical curves demonstrated that the binding affinity constant could be evaluated as equivalent for all library (immittance function) parameters, allowing optimized single frequency (or a range of frequencies) assessment with high sensitivity. In other words, binding affinity constants between ArtinM and HRP for each of the parameters in the immittance function library at given optimized frequencies were similar, independently of the parameter. Thus, the feasibility of using this immittance function approach for electroanalytical glycoarrays by accessing bio-recognition processes on a rapid (optimized) single frequency and highly multiplexable platform was demonstrated.

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“…(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) inverse of imaginary capacitance as an effective transducer signal of molecular recognition at higher analytical frequencies of a redox tagged interface (~7-100 Hz) when compared with purely capacitance analysis as described in the previous section (Santos and Bueno, 2016;Bedatty Fernandes et al, 2015). Accordingly, the optimized frequency found is of 100 Hz (Fig.…”

“…The benefit of using this approach is that it is, in essence, a "plug and play" concept wherein a sensor "chip" is coupled to the read out device, where an algorithm can be set up to search for the most sensitive function, simultaneously mapping its frequency response. In previous work we have demonstrated this application to a number of markers (Santos and Bueno, 2016;Bedatty Fernandes et al, 2015;Patil et al, 2015).…”

“…In the dominant faradaic approach, it is additionally necessary to pre-dope with an amplifying excess of redox probe (Lisdat and Schäfer, 2008) prior using the resolved charge transfer resistance (R ct ) as signal transduction. As an alternative, we have introduced the use of redox tagged receptor interfaces and redox capacitance (C r ) (from electrochemical impedance-derived capacitance spectroscopy) as underpinning a fully label free sensory approach (Fernandes et al, 2014(Fernandes et al, , 2013Santos and Bueno, 2016). Although this approach brings significant practical advantages, by default, it also requires a full spectrum analysis.…”

“…Although this approach brings significant practical advantages, by default, it also requires a full spectrum analysis. We have also introduced an immittance function methodology that enables a more facile analysis without any data fitting (Santos and Bueno, 2016;Bedatty Fernandes et al, 2015). This concept is based on the relationship between potential perturbation and measured current being described by mathematically exchangeable complex immittance functions (e.g.…”

“…impedance Z*, capacitance C*, admittance Y* and modulus M*). It is possible, within this, to obtain different transduction signals using only one data set without any reference to equivalent circuits (Santos and Bueno, 2016;Bedatty Fernandes et al, 2015;Patil et al, 2015). The benefit of using this approach is that it is, in essence, a "plug and play" concept wherein a sensor "chip" is coupled to the read out device, where an algorithm can be set up to search for the most sensitive function, simultaneously mapping its frequency response.…”