Interference Studies Using Multidimensional Mapping of Cross-Reactive Sensors: Applications in Blood Monitoring of Clozapine

Point-of-care sensors are used in clinical applications for diagnosing and monitoring health conditions. For example, a point-of-care sensor for therapeutic drug monitoring of the clozapine antipsychotic can reduce burdens from guidelines suggesting routine monitoring of this medication. However, when measuring chemical markers in complex fluids, there are challenges related to decreased sensor performance due to chemical interference. This work presents a methodology for identifying individual interfering species. A set of cross-reactive electrochemical sensors were developed, whose diversified responses provide a fingerprint-type pattern capable of differentiating various species. By mapping the multidimensional responses, patterns from complex solutions were discerned and matched to those of individual species. Applying this methodology to clozapine sensing in blood, a major source of chemical interference was identified. The understanding matrix components that cause interference can guide the design of reliable sensing systems and can be integrated with pattern recognition tools that can account for it

The Binding Effect of Proteins on Medications and Its Impact on Electrochemical Sensing: Antipsychotic Clozapine as a Case Study

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Clozapine (CLZ), a dibenzodiazepine, is demonstrated as the optimal antipsychotic for patients suffering from treatment-resistant schizophrenia. Like many other drugs, understanding the concentration of CLZ in a patient’s blood is critical for managing the patients’ symptoms, side effects, and overall treatment efficacy. To that end, various electrochemical techniques have been adapted due to their capabilities in concentration-dependent sensing. An open question associated with electrochemical CLZ monitoring is whether drug–protein complexes (i.e., CLZ bound to native blood proteins, such as serum albumin (SA) or alpha-1 acid-glycoprotein (AAG)) contribute to electrochemical redox signals. Here, we investigate CLZ-sensing performance using fundamental electrochemical methods with respect to the impact of protein binding. Specifically, we test the activity of bound and free fractions of a mixture of CLZ and either bovine SA or human AAG. Results suggest that bound complexes do not significantly contribute to the electrochemical signal for mixtures of CLZ with AAG or SA. Moreover, the fraction of CLZ bound to protein is relatively constant at 31% (AAG) and 73% (SA) in isolation with varying concentrations of CLZ. Thus, electrochemical sensing can enable direct monitoring of only the unbound CLZ, previously only accessible via equilibrium dialysis. The methods utilized in this work offer potential as a blueprint in developing electrochemical sensors for application to other redox-active medications with high protein binding more generally. This demonstrates that electrochemical sensing can be a new tool in accessing information not easily available previously, useful toward optimizing treatment regimens

Multidimensional Mapping Method Using an Arrayed Sensing System for Cross-Reactivity Screening

<div><p>When measuring chemical information in biological fluids, challenges of cross-reactivity arise, especially in sensing applications where no biological recognition elements exist. An understanding of the cross-reactions involved in these complex matrices is necessary to guide the design of appropriate sensing systems. This work presents a methodology for investigating cross-reactions in complex fluids. First, a systematic screening of matrix components is demonstrated in buffer-based solutions. Second, to account for the effect of the simultaneous presence of these species in complex samples, the responses of buffer-based simulated mixtures of these species were characterized using an arrayed sensing system. We demonstrate that the sensor array, consisting of electrochemical sensors with varying input parameters, generated differential responses that provide synergistic information of sample. By mapping the sensing array response onto multidimensional heat maps, characteristic signatures were compared across sensors in the array and across different matrices. Lastly, the arrayed sensing system was applied to complex biological samples to discern and match characteristic signatures between the simulated mixtures and the complex sample responses. As an example, this methodology was applied to screen interfering species relevant to the application of schizophrenia management. Specifically, blood serum measurement of antipsychotic clozapine and antioxidant species can provide useful information regarding therapeutic efficacy and psychiatric symptoms. This work proposes an investigational tool that can guide multi-analyte sensor design, chemometric modeling and biomarker discovery.</p></div

Electrochemical Serum Response.

<p>Differential pulse voltammetry (DPV) of serum with and without 5.6 μM clozapine using sensing element A, and heat map signature representation. All solutions were tested using GCE, and represent an average of duplicate measurements.</p

SMA Response of Serum.

<p>(A) Heat map representation of electrochemical responses of the SMA for serum, with signatures highlighted in black outlines. (B) Simplified heat map of the electrochemical responses of the SMA showing only the outlined signatures of serum spiked with 5.6 μM CLZ, overlaid with outlined signatures of un-spiked serum (orange lines) and well as CLZ and UA in buffer (shaded). The simplified heat maps illustrate the overlapping signatures such that they can be matched across the samples. The A-F annotations refer to the various elements in the SMA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116310#pone.0116310.t001" target="_blank">Table 1</a>).</p

Methodology for Bottom-up and Top-Down Investigation.

<p>Schematic representing the systematic methodology for studying the effect of cross-reactive species (CRS) presence on the CLZ measurement by correlating bottom-up and top-down approaches through a sensing methods array (SMA).</p

SMA Response of Simulated Mixtures in PBS.

<p>Simplified heat map representation of electrochemical responses of the sensing methods array for 5.6 μM CLZ in a mixture with (A) 410 μM UA, and (B) 60 μM CySH in PBS buffer (black outline), compared to their individual counterparts (shaded). The rectangular shapes represent the signatures derived from the heat maps of each of the species such that they can be overlaid. The A-F annotations refer to the various elements in the SMA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116310#pone.0116310.t001" target="_blank">Table 1</a>).</p

Electrochemical CLZ Response.

<p>Differential pulse voltammetry (DPV) and heat map representation of 5.6 μM CLZ in PBS (pH 7.4) using GCE. Signal response represents an average of triplicate measurements.</p

SMA Response of Individual Species in PBS.

<p>Heat map representation of electrochemical responses of the sensing methods array for (A) 5.6 μM CLZ, (B) 410 μM UA, and (C) 60 μM CySH tested individually in PBS buffer. The A–F annotations refer to the various elements in the SMA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116310#pone.0116310.t001" target="_blank">Table 1</a>), and the peak signatures are outlined in black rectangles. Each response represents the average of triplicate measurements. Note that two scales are used in each heat map to enhance visualization.</p