Electrochemical Biosensors

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 electrodes 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 deposition 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 specific 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 bacterial 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.

A ferrocenyl intercalator was investigated to
develop an electrochemical DNA biosensor employing
a peptide nucleic acid (PNA) sequence as capture probe.
After hybridization with single strand DNA sequence,
a naphthalene diimide intercalator bearing ferrocene moieties
(FND) was introduced to bind with the PNA-DNA
duplex and the electrochemical signal of the ferrocene
molecules was used to monitor the DNA recognition.

Present work displays the preparation of an electrochemical biosensor using a conjugated polymer and laccase enzyme for catechol quantification in samples. The biosensing system is based on an enzyme immobilization on polymer modified graphite transducer surface. For that purpose, a random conjugated polymer, thienothiophene-benzoxadiazole-alt-benzodithiophene (BOTT), was coated onto a graphite electrode surface via drop casting method followed by immobilization of a biomolecule (laccase) for sensing experiments. Herein, for the first time, we proposed a BOTT polymer as an inexpensive and effective way to fabricate highly sensitive and fast response biosensors. The proposed sensing system possessed superior properties with 0.38 μM limit of detection and 110.81 μA mM −1 sensitivity. Furthermore, cyclic voltammetry and scanning electron microscopy techniques were used to examine the surface modifications. The proposed system could be useful for many future studies for catechol quantification in environmental samples.

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.

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.

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.

The application of solar UV radiation as sample digestion method is reported. The method is employed in adsorptive stripping voltammetric determination of nickel and cobalt in river water samples. The river water samples were collected from downstream of Warnow River (Germany) and acidified to pH of 2 ± 0.2 by addition of ultrapure 65% HNO 3. Furthermore, 3.4 mgL −1 ultrapure hydrogen peroxide solution was added to the samples as photochemical reaction initiator. The samples were transferred to UV-A transparent polyethylene terephthalate bottles and put in the sunshine for UV irradiation for six and 12 hours at a UV-A intensity of 3.90 mW/m 2. The comparison of the concentration values showed that, 6 hours of solar UV irradiation at 3.90 mW/m 2 UV-A intensity is not sufficient to complete the digestion process though it yields much better results than the undigested original sample. However, 12 hours of solar UV-A irradiation under similar conditions is almost as effective as a 30 W artificial UV lamp (254 nm) and can be applied to the digestion of dissolved organic carbon in trace nickel (II) and cobalt (II) analysis in natural waters such as river water, lake waters, and well waters.

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.

ARTICLE INFO ABSTRACT A novel step electropolymerization technique, using the natural dye safranine nanoparticles (AgNPs). The dsDNA with silver nanoparticles potensiodynamically on the surface of carbon paste electrode (CPE), using cyclic voltammetry (CV The morphology and performance of bioimprinted polymer were characterized by electron scanning microscopy (SEM. The analytical performance of the bioimprinted sensor was also studied. The modified electrode presented very good reproducibility, satisfact sensitivity and selectivity compared to the conventional DNA and electrochemical sensors. Furthermore, the square wave voltammetric peak current was linear to butyl paraben mass concentration in the range 0.362 to 100 bioimprinted sensor was successfully applied to the determination of butyl paraben in real cosmetic samples with satisfactory recovery ranging from 97.3 to 100.2 %, demonstrating its feasibility for practical ap

This work demonstrates the first utilization of virus molecules as nano-scale biotemplates assembled on an electrochemical biosensor, allowing for an 8-times increased signal and an improved biosensing performance of 9.5-fold. The versatile and inexpensive biological Tobacco mosaic virus was integrated as a high aspect ratio, low footprint, low-cost, easy to genetically functionalize, nanostructured three-dimensional scaffold for the synthesis of novel multifunctional electrodes. The biotemplated scaffold allows for an increased surface area resulting in higher electrochemical currents, better signal-to-noise ratio and improved sensitivity when incorporated into miniaturized biosensors.