Validation of the organic electrochemical transistor for in vitro toxicology.

BACKGROUND: The gastrointestinal epithelium provides a physical and biochemical barrier to the passage of ions and small molecules; however this barrier may be breached by pathogens and toxins. The effect of individual pathogens/toxins on the intestinal epithelium has been well characterized: they disrupt barrier tissue in a variety of ways, such as by targeting tight junction proteins, or other elements of the junctions between adjacent cells. A variety of methods have been used to characterize disruption in barrier tissue, such as immunofluorescence, permeability assays and electrical measurements of epithelia resistance, but these methods remain time consuming, costly and ill-suited to diagnostics or high throughput toxicology. METHODS: The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the organic electrochemical transistor (OECT), whose low cost materials and potential for easy fabrication in high throughput formats represent a novel solution for assessing epithelial tissue integrity. RESULTS: In this study, OECTs were integrated with gastro-intestinal cell monolayers to study the integrity of the gastrointestinal epithelium, providing a very sensitive way to detect minute changes in ion flow across the cell layer due to inherent amplification by the transistor. MAJOR CONCLUSIONS: We validate the OECT against traditional methods by monitoring the effect of toxic compounds on epithelial tissue. We show a systematic characterization of this novel method, alongside existing methods used to assess barrier tissue function. GENERAL SIGNIFICANCE: The toxic compounds induce a dramatic disruption of barrier tissue, and the OECT measures this disruption with increased temporal resolution and greater or equal sensitivity when compared with existing methods. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

New biorecognition molecules in biosensors for the detection of toxins.

Biological and synthetic recognition elements are at the heart of the majority of modern bioreceptor assays. Traditionally, enzymes and antibodies have been integrated in the biosensor designs as a popular choice for the detection of toxin molecules. But since 1970s, alternative biological and synthetic binders have been emerged as a promising alternative to conventional biorecognition elements in detection systems for laboratory and field-based applications. Recent research has witnessed immense interest in the use of recombinant enzymatic methodologies and nanozymes to circumvent the drawbacks associated with natural enzymes. In the area of antibody production, technologies based on the modification of in vivo synthesized materials and in vitro approaches with development of "display "systems have been introduced in the recent years. Subsequently, molecularly-imprinted polymers and Peptide nucleic acid (PNAs) were developed as an attractive receptor with applications in the area of sample preparation and detection systems. In this article, we discuss all alternatives to conventional biomolecules employed in the detection of various toxin molecules We review recent developments in modified enzymes, nanozymes, nanobodies, aptamers, peptides, protein scaffolds and DNazymes. With the advent of nanostructures and new interface materials, these recognition elements will be major players in future biosensor development.

Sorption of his-tagged Protein G and Protein G onto chitosan/divalent metal ion sorbent used for detection of microcystin-LR.

A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR) by using strategies for oriented immobilization of functionally intact polyclonal antibodies on chitosan surface. Several physicochemical parameters such as metal ion adsorption, hexahistidine-tagged Protein G sorption, the dilution ratio polyclonal antibody concentration, and peroxidase-labeled MC-LR concentration were studied and optimized. The sorption in batch system of G-histidine and G-proteins was studied on a novel sorbent consisting of chitosan/divalent metal ions. Transition metals as Ni++ and Zn++ were immobilized through interaction with -NH2 groups of chitosan in order to supply a material capable to efficiently remove the proteins from aqueous solutions. The maximum uptake of divalent metals onto the chitosan material was found to be 230 mg g-1 for Zn++ and 62 mg g-1 for Ni++. Experimental data were evaluated using the Langmuir and Freundlich models; the results were well fitted with the Langmuir model; chitosan/Ni++ foam was found to be the best sorbent for G-protein, maximum sorption capacity obtained was 17 mg g-1, and chitosan/Zn++ was found to be the best for G-histidine with a maximum sorption capacity of 44 mg g-1. Kinetic data was evaluated with pseudo-first- and pseudo-second-order models; the sorption kinetics were in all cases better represented by a pseudo-second-order model. Under optimum conditions, the calibration curve obtained for MC-LR gave detection limits of 0.5 ± 0.06 µg L-1, the 50 % inhibition concentration (IC50) was 2.75 ± 0.03 µg L-1, and the quantitative detection range was 0.5-25 µg L-1. The limit of detection (LOD) attained from the calibration curves and the results obtained demonstrate the potential use of CLEIA with chitosan support as a screening tool for the analysis of pollutants in environmental samples.

Microfabricated biosensor for the simultaneous amperometric and luminescence detection and monitoring of Ochratoxin A.

The low molecular weight hapten, Ochratoxin A (OTA), is a natural carcinogenic mycotoxin produced by Aspergillus and Penicillium fungi and so it commonly appears in wines, other foods, and in the environment. An amperometric biosensor has been developed that uses the immobilized synthetic peptide, NFO4; which possesses a high binding affinity and thus provides for molecular recognition of OTA; simulating the mycotoxin-specific antibody. Biotransducers were produced from a microlithographically fabricated electrochemical cell-on-a-chip that uses the microdisc electrode array working electrode format augmented with microporous graphitized carbon (MGC) that was electrodeposited within a poly(aniline-co-meta-aminoaniline) electroconductive polymer layer. A redox mediator, iron-nickel hexacyanoferrate (Fe|NiHCF) was amperometrically deposited onto the MGC. The device was then dip-coated with monomer cocktail that yielded poly(HEMA-co-AEMA) foam that was prepared in-situ by UV crosslinking and by sequentially freezing followed by freeze drying of the chip to yield a 3-D support for the chelation of Zn(2+) ions (ZnCl2) and the subsequent immobilization of N-terminus his-tagged peptide, NFO4. To conduct the biosensors assay, HRP conjugated OTA was added to the free OTA solutions and together competitively incubated on the biospecific MDEA ECC 5037-Pt|MGC|HCF|Hydrogel-NFO4 biotransducer. The amperometric response to peroxide was determined after 5 min of enzymatic reaction following addition of standard substrate H2O2/luminol. Simultaneous analysis of light emission signals (λmax=425 nm) allowed direct comparison of amperometric and luminescence performance. Using chitosan foam and a luminescence bioassay we obtained maximum inhibition at 10 µg L(-1) and half inhibition occurred at 2.1 µg L(-1). Using poly(HEMA-co-AEMA) hydrogel and an amperometric bioassay (50s) we obtained maximum inhibition at 10 µg L(-1) and half inhibition occurred at 2.8 µg L(-1).

Sensing of barrier tissue disruption with an organic electrochemical transistor.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

Dynamic monitoring of Salmonella typhimurium infection of polarized epithelia using organic transistors.

Ion flow across polarized epithelia is a tightly regulated process. Measurement of the transepithelial resistance is a highly relevant parameter for assessing the function or health of the tissue. Dynamic, electrical measurements of transepithelial ion flow are preferred as they provide the most accurate snapshot of effects of external stimuli. Enteric pathogens such as Salmonella typhimurium are known to disrupt ion flow in gastrointestinal epithelia. Here, for the first time, the use of organic transistors as a powerful potential alternative for front-line, disposable, high-throughput diagnostics of enteric pathogens is demonstrated. The transistors' ability to detect early and subtle changes in transepithelial ion flow is capitalized upon to develop a highly sensitive detector of epithelial integrity. Stable operation of the organic devices under physiological conditions is shown, followed by dynamic, pathogen-specific diagnosis of infection of epithelia. Further, operation of the device is possible in complex matrices, showing particular promise for food and safety applications.

Measurement of barrier tissue integrity with an organic electrochemical transistor.

The integration of an organic electrochemical transistor with human barrier tissue cells provides a novel method for assessing toxicology of compounds in vitro. Minute variations in paracellular ionic flux induced by toxic compounds are measured in real time, with unprecedented temporal resolution and extreme sensitivity.