Hadar Ben-Yoav

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Over the last few years, the physical dimensions of microchip devices have decreased, enhancing the interest in the integration of various devices and complex operations onto a compatible "lab on a chip" system with desirable characteristics and capabilities. This work presents a novel μ-fluidics whole cell biosensor for water toxicity analysis. The biosensor is based on bacterial cells genetically "tailored" to generate an electrochemical bio-signal in the presence of toxic materials. The μ-chip was electrochemically characterized, and demonstrated the potential toxicity analysis with a model toxicant. A novel concept of bacterial biosensors deposition by means of electrophoretic force was examined for the first time. Preliminary results demonstrated the ability to detect electrochemical signal generated by the deposited bacterial cells indicating the potential use for patterning of bacterial cells on solid-state surfaces for the use in bio-sensing.

Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental mon- itoring applications. The advantages of these devices are that they can be produced as a single-chip, low-power, rugged, inexpensive component, and can be deployed in a variety of non-laboratory settings. However, such biosensors encounter inherent problems in overall system light collection efficiency. The light emitted from the bioluminescent microbial cells is isotropic and passes through various media before it reaches the photon detectors. We studied the bioluminescence distribution and propagation in microbial whole cell biochips. Optical emission and detection were modeled and simulated using an optical ray tracing method. Light emission, transfer and detection were simulated and optimized with respect to two fundamental system parameters: system geometry and bacterial concentration. Optimiza- tion elucidated some of the optical aspects of the biochip, e.g. detector radius values between 300 and 750  m, and bacterial fixation radius values between 800 and 1200  m. Understanding theses aspects may establish a basis for future optimization of similar chips.

This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosen- sor presented here is based on bacterial cells that are genetically “tailored” to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemi- cal active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5  m and 300  m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different geno- toxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10  g/ml for NA using 30 min for induction and 0.31  M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alter- native to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl  -d- galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the stan- dard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by micro- system-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such whole- cell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the bio- logical component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the micro- electronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by in- teraction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that con- verts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.

Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotox- icity assays based upon the use of genetically engi- neered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induc- tion of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu-test, has been fully validated and ISO- and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual geno- toxicity testing devices.

A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bio- reporter responded in a dose-dependent manner to three model DNA-damaging agents—hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)—detected 30–60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensi- tivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2- aminoanthracene following metabolic activation with an S9 mammalian liver fraction.

Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.

This paper presents an integrated whole-cell biochip system where functioning cells are deposited on the solid micro-machined surfaces while specially designed indium tin oxide electrodes that can be used to apply controllable electric fields during various stages; for example during cell deposition. The elec- trodes can be used also for sensing currents associated with the sensing mechanisms of electrochemical whole-cell biosensors. In this work a new approach integrating live bacterial cells on a biochip using electrophoretic deposition is presented. The biomaterial deposition technique was characterized under various driving potentials and chamber configurations. An analytical model of the electrophoretic deposi- tion kinetics was developed and presented here. The deposited biomass included genetically engineered bacterial cells that may respond to toxic material exposure by expressing proteins that react with spe- cific analytes generating electrochemically active byproducts. In this study the effect of external electric fields on the whole-cell biochips has been successfully developed and tested. The research hypothesis was that by applying electric fields on bacterial whole-cells, their permeability to the penetration of external analytes can be increased. This effect was tested and the results are shown here. The effect of prolonged and short external electric fields on the bioelectrochemical signal generated by sessile bacte- rial whole-cells in response to the presence of toxins was studied. It was demonstrated that relatively short 10 ms external DC electric pulse improves the performance of bacterial biosensors by 15% relative to un-biased biosensors. The application of prolonged 1 h external alternating electric fields deteriorated the whole-cell performance in the presence of toxins. In this paper we present the electrode apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of this new concept.

Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of hazardous materials. However, their use in the field has been limited by harsh and unstable environmental condi- tions that shorten shelf-life, introduce significant noise, and reduce the signal and signal-to-noise ratio; such conditions may thus decrease the probability of correct decisions, increasing both false positive and false negative outcomes. Therefore, there is a need for a stable cell-on-chip integration that offers long- term storage and resilience to environmental factors. The use of intact microbial biofilms as biological elements in a whole-cell biosensor, and their integration into specialized biochips, holds promise for enhancing sensor stability as well as providing an innovative platform for biofilm research. We report here for the first time on the integration of a bacterial biofilm as the sensing element of a whole-cell biosensor, as a means to stabilize and preserve reproducibility, viability and functionality of the bacte- rial sensor cells. We have employed a genetically engineered Escherichia coli sensor strain, tailored to respond to the presence of genotoxic (DNA damaging) agents by the induction of a reporter enzyme, alkaline phosphatase, and tested its functionality in colorimetric and electrochemical assays. Three dif- ferent bacterial integration forms were examined: planktonic cells, electronically deposited sessile cells, and biofilms. For all integration forms, a clear dose-dependent positive response to the presence of the model toxicant nalidixic acid was observed, with biofilms displaying higher current density and detec- tion sensitivity than planktonic and sessile cells. We present the electrode apparatus and methods and biochip characterization of such chips, e.g. signal vs. time and induction factor, and discuss the advantage and potential problems of the new biofilm-biochip technology.

Integrated polypyrrole, a conductive polymer, interconnects on polymeric substrates were microfabricated for flexible sensors and actuators applications. It allows manufacturing of moving polymeric microcomponents suitable, for example, for micro- optical-electromechanical (MOEMS) systems or implanted sensors. This generic technology allows producing “all polymer” components where the polymers serve as both the structural and the actuating materials. In this paper we present two possible novel architectures that integrate polypyrrole conductors with other structural polymers: (a) polypyrrole embedded into flexible polydimethylsiloxane (PDMS) matrix forming high aspect ratio electrodes and (b) polypyrrole deposited on planar structures. Self-aligned polypyrrole electropolymerization was developed and demonstrated for conducting polymer lines on either gold or copper seed layers. The electropolymerization process, using cyclic voltammetry from an electrolyte containing the monomer, is described, as well as the devices’ characteristics. Finally, we discuss the effect of integrating conducting polymers with metal seed layer, thus enhancing the device durability and reliability.

The electrode geometry and material have a significant effect on the electrochemical biochip transduction of chemical signals into electrical current or voltage. In this work we focus on the working electrode aiming to improve the signal level of live cell sensors integrated on solid-state microchips. We present here a model and measurements describing the effect of the electrode material and dimensions on the biochip performance. The research hypothesis was that the electrode transduction efficiency increases as its effective area increases. Therefore, we investigated two methods to increase the electrode effective area: 3D structures and a polymer modified electrode. An electrochemical microchip was fabricated with a working electrode that was further modified, resulting in two structure types: 3D metallic pillar-based and polypyrrole-coated. The electrochemical performance of both modified electrodes was characterized and their utilization as working electrodes in whole-cell biochips for toxicity sensing was studied. Bio- detection efficiency analysis demonstrated higher biosensing performance for the metallic (e.g. Cu/Au) pillar-based microchip than for the polypyrrole-modified and the non-modified microchips. Therefore, we conclude that the enhanced signal of the modified geometry electrode is most probably due to the increased effective surface area and the improved charge transfer efficiency.

In this article the research for developing whole- cell biochips has been presented using both bioluminescent and electrochemical methods. The research was on integrating an electrode cell with both electrochemical and bioluminescent detection using a single VLSI chip. The authors have investigated the signal conditioning system that can work with any kind of amperometric and bioluminescent sensor. During this research the authors focused on the analog front-end unit. The work includes investigating the electronic model for simulation for an electrochemical cell and conceiving a fully integrated 8X8 electrochemical sensor array. The authors are focusing on signal conditioning system and its functionality. The main concern for the authors was to maintain the complexity and the number of electronic devices as low as possible.

The response modeling of whole-cell biochip represents the link between cellular biology and transducer output, allowing better system engineering. It provides the mathematical background for signal and noise modeling, performance prediction and data analysis. Here we describe an analytical model for whole- cell biosensors with electrochemical detection for single use, test and dispose applications. In this system the electrochemical signal is generated by the oxidation of the by-products of the reaction between an external substrate and the enzyme alkaline phosphatase. The enzyme expression can be either normal or enhanced due to the response of the biological cell to an external excitation. The electrochemical oxidation current is measured as a function of time. The model is based on the electrochemical reaction rate equations; an analytical solution is presented, compared to data and discussed.

Schizophrenia is a lifelong mental disorder with few recent advances in treatment. Clozapine is the most effective antipsychotic for schizophrenia treatment. However, it remains underutilized since frequent blood draws are required to monitor adverse side effects, and maintain clozapine concentrations in a therapeutic range. Micro-system technology utilized towards real-time monitoring of efficacy and safety will enable personalized medicine and better use of this medication. Although work has been reported on clozapine detection using its electrochemical oxidation, no in situ monitoring of clozapine has been described. In this work, we present a new concept for clozapine in situ sensing based on amplifying its oxidation current. Specifically, we use a biofabricated catechol-modified chitosan redox cycling system to provide a significant amplification of the generated oxidizing current of clozapine through a continuous cycle of clozapine reduction followed by re-oxidation. The amplified signal has improved the signal-to- noise ratio and provided the required limit-of-detection and dynamic range for clinical applications with minimal pre-treatment procedures. The sensor reports on the functionality and sensitivity of clozapine detection between 0.1 and 10  g/mL. The signal generated by clozapine using the catechol-modified chitosan amplifier has shown to be 3 times greater than the unmodified system. The sensor has the ability to differentiate between clozapine and its metabolite norclozapine, as well as the feasibility to detect clozapine in human serum in situ within the required dynamic range for clinically related applications. This new biosensing approach can be further developed towards its integration in miniaturized devices for improved personalized mental health care.

This work presents a thorough electrochemical
and reliability analysis of a sensing scheme for the
antipsychotic clozapine. We have previously demonstrated a
novel detection approach for this redox-active drug, highly
effective in schizophrenia treatment, based on a catechol-
modified chitosan film. The biomaterial film enables
amplification of the oxidative current generated by clozapine
through redox cycling. Here, we study critical electrochemical
and material aspects of the redox cycling system to overcome
barriers in point-of-care monitoring in complex biological
samples. Specifically, we explore the electrochemical parameter
space, showing that enhanced sensing performance depends
on the presence of a reducing mediator as well as the
electrochemical technique applied. These factors account for up to 1.75-fold and 2.47-fold signal enhancement, respectively. Looking at potential interferents, we illustrate that the redox cycling system allows for differentiation between selected redox- active species, clozapine’s structurally largely analogous metabolite norclozapine as well as the representative catecholamine dopamine. Furthermore, we investigate material stability and fouling with reuse as well as storage. We find no evidence of film fouling due to clozapine; slow overall biomaterial degradation with successive use accounts for a 2.2% absolute signal loss and can be controlled for. Storage of the redox cycling system appears feasible over weeks when kept in solution with only 0.26%/day clozapine signal degradation, while ambient air exposure of three or more days reduces performance by 58%. This study not only advances our understanding of the catechol-modified chitosan system, but also further establishes the viability of applying it toward sensing clozapine in a clinical setting. Such point-of-care monitoring will allow for broader use of clozapine by increasing convenience to patients as well as medical professionals, thus improving the lives of people affected by schizophrenia through personalized medicine.

Microelectronic devices that contain biological components are typically used to interrogate biology1,2 rather than control biological function. Patterned assemblies of proteins and cells have, however, been used for in vitro metabolic engineering3–7, where coordinated biochemical pathways allow cell metabolism to be characterized and potentially controlled8 on a chip. Such devices form part of technologies that attempt to recreate animal and human physiological functions on a chip9 and could be used to revolutionize drug development10. These ambitious goals will, however, require new biofabrication methodologies that help connect microelectronics and biological systems11,12 and yield new approaches to device assembly and communi- cation. Here, we report the electrically mediated assembly, interrogation and control of a multi-domain fusion protein that produces a bacterial signalling molecule. The biological system can be electrically tuned using a natural redox molecule, and its biochemical response is shown to provide the signalling cues to drive bacterial population behaviour. We show that the biochemical output of the system correlates with the electrical input charge, which suggests that electrical inputs could be used to control complex on-chip biological processes.

Mental health disorders are complex and poorly understood but would benefit from real-time chemical analysis capable of assessing a patient’s current status, personalizing a therapeutic action, and monitoring compliance. Here, an elec- trochemical sensor is reported for detecting the antipsychotic drug clozapine which is one of the most effective but under-utilized drugs for managing schizo- phrenia. This sensor employs a composite film of multiwalled carbon nano- tubes (CNTs) embedded within a matrix of the aminopolysaccharide chitosan. Chitosan allows programmable assembly of the composite film at an electrode address while the CNTs confer electrocatalytic activities that displace interfering serum peaks from the voltage region where clozapine oxidation occurs. Using differential pulse voltammetry, high sensitivities (limit of detection 0.05 × 10–6 M) are demonstrated for clozapine analysis in buffer. In serum, clozapine sensi- tivity is reduced by an order of magnitude but still sufficient for clinical analysis. Finally, the detection of clozapine from the serum of a schizophrenia patient is demonstrated without the need for serum pretreatment. In the long term, it is envisioned that the CNT-chitosan coated electrode could be integrated within
a small array of other sensor types to enhance information-extraction to allow mental health disorders to be better managed and better understood.

Background: Clozapine is the most effective antipsychotic drug for schizophrenia treatment, however it is currently underused. In order to understand the barriers of frequent blood draws for white blood cell counts (WBCs) and clozapine levels, we developed a psychiatrist survey and began and integrative approach of designing a point-of-care device that could eventually have real-time monitoring with immediate results. Methods: We ascertained barriers related to clozapine management and the acceptance of possible solutions by sending an anonymous survey to physicians in psychiatric practice (N=860). In parallel we tested clozapine sensing using a prototype point-of-care monitoring device. Results: 255 responses were included in the survey results. The two barriers receiving mean scores with the highest agreement as being a significant barrier were patient nonadherence to blood work and blood work’s burden on the patient (out of 28). Among nine solutions, the ability to obtain lab results in the physician’s office or pharmacy was top-ranked (mean±sd Likert scale (4.0±1.0)). Physicians responded that a point-of-care device to measure blood levels and WBCs would improve care and increase clozapine use. Residents ranked point-of-care devices higher than older physicians (4.07±0.87 vs. 3.47±1.08, p<0.0001). Also, the prototype device was able to detect CLZ reliably in 1.6, 8.2, and 16.3g/mL buffered solutions. Discussion: Survey results demonstrate the physician’s desire for point-of-care monitoring technology, particularly among younger prescribers. Prototype sensor results identify that clozapine can be detected and integrated for future device development. Future development will also include integration of WBCs for a complete detection device.

BACKGROUND/OBJECTIVES: The use of electric fields in combination with small doses of antibiotics for enhanced treatment of biofilms is termed the ‘bioelectric effect’ (BE). Different mechanisms of action for the AC and DC fields have been reported in the literature over the last two decades. In this work, we conduct the first study on the correlation between the electrical energy and the treatment efficacy of the bioelectric effect on Escherichia coli K-12 W3110 biofilms.
METHODS: A thorough study was performed through the application of alternating (AC), direct (DC) and superimposed (SP) potentials of different amplitudes on mature E. coli biofilms. The electric fields were applied in combination with the antibiotic gentamicin (10 μg/ml) over a course of 24 h, after the biofilms had matured for 24 h. The biofilms were analysed using the crystal violet assay, the colony-forming unit method and fluorescence microscopy.
RESULTS: Results show that there is no statistical difference in treatment efficacy between the DC-, AC- and SP-based BE treatment of equivalent energies (analysis of variance (ANOVA) P40.05) for voltages o 1 V. We also demonstrate that the efficacy of the BE treatment as measured by the crystal violet staining method and colony-forming unit assay is proportional to the electrical energy applied (ANOVA Po0.05). We further verify that the treatment efficacy varies linearly with the energy of the BE treatment
(r2 = 0.984). Our results thus suggest that the energy of the electrical signal is the primary factor in determining the efficacy of the BE treatment, at potentials less than the media electrolysis voltage.
CONCLUSIONS: Our results demonstrate that the energy of the electrical signal, and not the type of electrical signal (AC or DC or SP), is the key to determine the efficacy of the BE treatment. We anticipate that this observation will pave the way for further understanding of the mechanism of action of the BE treatment method and may open new doors to the use of electric fields in the treatment of bacterial biofilms.

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 in- vestigating cross-reactions in complex fluids. First, a systematic screening of matrix compo- nents 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 informa- tion 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 com- plex 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 infor- mation regarding therapeutic efficacy and psychiatric symptoms. This work proposes an in- vestigational tool that can guide multi-analyte sensor design, chemometric modeling and biomarker discovery.