Nucleic Acid Biosensors for Environmental Pollution Monitoring

The Royal Society of Chemistry

Contents

Biosensor Techniques for Environmental Monitoring

ILARIA PALCHETTI AND MARCO MASCINI

Dipartimento di Chimica, Universita` degli Studi di Firenze, 50019 Sesto Fiorentino (Fi), Italy

1.1 Introduction: Role of Biosensors in Environmental Analysis

Monitoring of contaminants in the air, water and soil is an instrumental component in understanding and managing risks to human health and ecosystems. The increasing number of potentially harmful pollutants in the environment calls for fast and cost-effective analytical techniques to be used in extensive monitoring programs. Given this requirement, as well as the time and cost involved in traditional chemical analysis of environmental samples (e.g., chromatographic methods, atomic spectroscopy and related hyphenated techniques), there is an expanding need for innovative methods.

Biosensors appear well suited to complement standard analytical methods for a number of environmental monitoring applications. The main advantages offered by biosensor technology over conventional analytical techniques are fast and economical measurements; the possibility of miniaturization and portability; the possibility of continuous monitoring; and, in some cases, the ability to measure pollutants in complex matrices with minimal sample preparation. Although many of the systems that have been developed cannot compete with conventional analytical methods in terms of accuracy and reproducibility, they can be used by regulatory authorities and by industry to provide enough information for routine testing and screening of samples. The peculiar characteristics of biosensors allow these devices to complement current field screening and monitoring methods such as the ELISA test, especially when continuous, real-time, in situ monitoring is required.

Some excellent reviews have summarized the recent progress in use of biosensors for environmental applications. Biosensors have been used in the analysis of chemical compounds, such as pesticides, heavy metals, endocrine disruptors (e.g., phenol derivatives) and persistent organic pollutants (e.g., PCB), as well as for the evaluation of environmental quality parameters (related to chemical pollution) such as biological oxygen demand (BOD). Moreover, biosensors have been also used for the monitoring of microbial pollution and detection of environmentally relevant organisms — microorganisms and plant or animal species. In other words, they can be used to control chemical and microbiological contamination and pollution as well as to check ecosystem biodiversity. Moreover, in recent years, the use of biosensors has been successfully proposed in the areas of environmental toxicity, cytotoxicity and genotoxicity. All these applications lead to the next challenge for environmental biosensor applications — the assessment of environmental pollution exposure and its impact on fundamental biological processes. Obviously, this "will require sophisticated computer models that can handle the immense volume and complexity of data generated for each individual and, also, would allow for integration of data on environmental exposures with genetic factors for the individual and the population".

Recently, biosensor technology has benefited from results obtained in other fields, such as biotechnology, nanoengineering and nanotechnology. "Lab on a chip" technology has greatly increased the possibility of obtaining easy-to-use and self-contained devices, minimizing the need for sample processing and analysis in the laboratory. Developments in micro- and nanotechnology continually help to miniaturize the devices. Biotechnologies help to increase the number of stable, sensitive and selective sensing elements that can be used for obtaining reliable biosensors.

Nevertheless, the commercial exploitation of biosensors for environmental applications is still at an early stage (with some exceptions). From a technical point of view this is still predominately tied to the stability, detection sensitivity and reliability of the biomolecular recognition element. In addition (as clearly stated in ref. 11), before a biosensor gains market acceptance, it must prove capable of being validated by well-established procedures. Research studies, as reported in literature, with few real samples or treated samples, often fail to provide an adequate measure of capability for "real-world" samples, leading to failed technology transfer and further investment. Such activities require appropriate sources of finance for technology development and demonstration, and undoubtedly these economic resources are more concentrated in the clinical and medical fields than in environmental science. Finally, the success of biosensors must prove that they are the inevitable choice as a cost-effective analytical tool.

Bearing these facts in mind, in this chapter we aim to give an overview of the biosensing elements used for environmental applications, focusing on their innovative aspects. Descriptions of the technical attempts to increase stability and sensitivity of the (bio)receptors are reported. Particular emphasis has been given to their use in environmental analysis.

1.2 Biosensors: Definition, Classification and a Brief History

The history of biosensors starts around 60 years ago. In 1956 Professor Leland C. Clark published his paper on the development of an oxygen probe; from this initial research activity he expanded the range of analytes that could be measured, and in 1962, in a conference at the New York Academy of Sciences, he described how "to make electrochemical sensors (pH, polarographic, potentiometric or conductometric) more intelligent" by adding "enzyme transducers as membrane enclosed sandwiches". The first example was illustrated by entrapping the enzyme glucose oxidase in a dialysis membrane over an oxygen probe. The addition of glucose proportionally determined the decrease of oxygen concentration. This first biosensor was described as an enzyme electrode. Then subsequently, in 1967, Updike and Hicks used the same term to describe a similar device where again glucose oxidase was immobilized in a polyacrylamide gel onto the surface of an oxygen electrode for the rapid and quantitative determination of glucose. In addition to amperometry, Guilbault and Montalvo in 1969 used glass electrodes coupled with urease to measure urea concentration by potentiometric measurement.

Since then there has been a rapid proliferation of biosensors with different biological elements and different transducers. This intensive research effort has led to the commercial exploitation of some devices. The first biosensor that appeared on the market was the glucose biosensor for diabete care. In 1984 a paper by Cass et al. described the use of ferrocene and its derivatives as mediators for amperometric biosensors. A few years later the Medisense Exatech (now Abbott) Glucose Meter was launched in the market and become the world's best-selling biosensor product. The initial product was a pen-shaped meter with disposable screen-printed electrodes; nowadays many formats are commercially available from different companies worldwide. During the same years (mid 1980s), researchers from Pharmacia started to work jointly with physics and biochemistry faculty at Linkoping University in Sweden, in order to develop a new bioanalytical instrument that could monitor the interactions between biomolecules. In 1984 a new company, Pharmacia Biosensor, was created. This company introduced a new instrument, the BIAcore, in 1990 (it is nowadays distributed by General Electric).

The concept of a biosensor has evolved enormously during the years. In the early days of this research area, many authors considered a biosensor to be a self-contained analytical device that responded to the concentration of chemical species in biological samples, without mentioning the role of the biologically active material involved in the device. This obviously caused many misunderstandings, since any physical or chemical sensor operating in biological samples could be considered a biosensor. Thus, many authors started to reserve the use of the term "biosensor" for a chemical sensor in which the recognition system utilizes a biochemical mechanism.

In early 2000, two Divisions of the International Union of Pure and Applied Chemistry (IUPAC), namely Physical Chemistry (Commission I.7 on Biophysical Chemistry, formerly Steering Committee on Biophysical Chemistry) and Analytical Chemistry (Commission V.5 on Electroanalytical Chemistry) prepared recommendations on the definition, classification and nomenclature relating to electrochemical biosensors; these recommendations have been then extended to other types of biosensors. Following these IUPAC recommendations,

a biosensor is defined as a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element. Because of their ability to be repeatedly calibrated, a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. A device that is both disposable after one measurement, i.e., single use, and unable to monitor the analyte concentration continuously or after rapid and reproducible regeneration, should be designated a single use biosensor.

Another important concept was introduced, some years later, by Turner and Newman; they referred to a biosensor as "a compact analytical device incorporating a biological or biologically-derived sensing element either integrated within or intimately associated with a physicochemical transducer", thus including synthetic chemical compounds that mimic the biological material in the development of biosensors. Nowadays, the concept of "biologically derived element" is fully accepted in the scientific community.

Biosensors are classified according to the biological specificity-conferring mechanism (Figure 1.1) or, alternatively, to the mode of physicochemical signal transduction. They may be further classified according to the analytes or reactions that they monitor; for example, direct monitoring of analyte concentration or of reactions producing or consuming such analytes. Alternatively, the indirect monitoring of inhibitor or activator of the biological recognition element (biochemical receptor) may be used.

In terms of transduction principles, biosensors can be classified as optical, electrochemical, mass, magnetic, calorimetric or micromechanical biosensors.

• Optical detection by fluorescence spectroscopy is a popular method, largely because of the ease with which biomolecules (especially nucleic acids) can be fluorescently labeled, the availability of many different fluorophores and quenchers, and the inherent capability for real-time multiplex detection. Chemiluminescence is another widely used optical technique. A different type of optical transduction, based on an evanescent wave device, can offer real-time label-free optical detection. These biosensors rely on monitoring changes in surface optical properties (shift in resonance angle due to change in the interfacial refractive index) resulting from the surface binding reaction.

• Electrochemical devices have also proved very useful, because of their inherent miniaturization and their compatibility with advanced microfabrication technology. Electrochemical detection usually involves monitoring a current response under controlled potential conditions. However, other changes in electrochemical parameters such as capacitance, impedance and conductivity have been used.

• Another useful label-free, mass detection scheme relies on the use of quartz crystal microbalance (QCM) transducers. A QCM biosensor consists of an oscillating crystal with a bioreceptor immobilized on its surface. The increased mass, associated with the biorecognition reaction, results in a decrease of the oscillating frequency. Acoustic wave sensors used in thickness-shear mode with a liquid sample detect changes in a number of physical properties including mass, viscosity and charge density.

• Micromechanical transduction based on cantilevers and label-free biosensors capable of detecting biomolecular interactions via the bending of microfabricated cantilevers coated with bioreceptors have been reported in the literature.

• Finally, in magneto-biosensors, magnetic labels are used to detect magnetoresistance, giant magnetoresistive effect (GMR), spin-value GMR and other parameters.

1.3 Innovative Biorecognition Elements for Environmental Analysis

"The biological recognition element may be based on a chemical reaction catalysed by, or on an equilibrium reaction with macromolecules that have been chemically synthesized, naturally isolated, or engineered, or are present in their original biological environment. In the latter cases, equilibrium is generally reached and there is little or no further net consumption of analyte(s) by the immobilized biocomplexing agent incorporated into the sensor".

Enzymes (and all biological elements, such as tissues, cells, microorganisms, which contain enzymes) represent the class of what are now called catalytic elements. Enzymes were historically the first molecular recognition elements included in biosensors, and continue to be the basis for a significant number of publications reported for biosensors in general as well as for environmental applications. Enzyme biosensors have several advantages. These include a stable source of material (primarily through biorenewable sources); the possi- bility of modifying the catalytic properties or substrate specificity by means of genetic engineering; and catalytic amplification of the biosensor response by modulation of the enzyme activity with respect to the target analyte. However, enzyme-based biosensors show also some limitations for use in environmental applications. These include the limited number of substrates for which enzymes have been evolved, the limited interaction between environmental pollutants and specific enzymes, and in the case of inhibitor formats, the lack of specificity in differentiating among compounds of similar classes such as nerve agents or organophosphate (OP) and carbamate pesticides.

Typical examples of enzymes involved in environmental applications are cholinesterase for OP and carbammate pesticide analysis, and tyrosinase for analysis of phenols and related compounds with endocrine disruptor characteristics. As already mentioned, genetic engineering helps in the careful selection of the location and type of mutations giving rise to enzymes with enhanced or particular properties, such as higher affinity towards specific analytes, higher stability, higher electron transfer rates, and residues able to provide an oriented or more stable immobilization. These improvements have already resulted in biosensors with enhanced performance. Recent progress with respect to genetically modified enzyme biosensors for environmental applications has been well reviewed.

An emerging field of biorecognition elements is so-called whole-cell systems. Whole cells have long been used for environmental applications, in particular for BOD monitoring. However, recently they have benefited enormously from the recent improvements in recombinant DNA technology and there is renewed interest in their use in monitoring environmental pollution and toxicity. Whole-cell systems are based on complex cellular functions, among which enzyme-catalysed reactions play an important role. The biosensors are constructed by the fusion of promoters (responsive to the relevant environmental conditions) to easily monitored reporter genes. Depending on the choice of reporter gene, expression can be monitored by the production of colour, light, fluorescence or electrochemical reactions. Although there are numerous examples of genetic modification involving bacteria, yeast, algae and tissue culture cells, genetically engineered bacteria (GEMs) are most often reported in cell-based biosensors.

Marco Mascini is a professor of analytical science in the Department of Chemistry at the University of Florence. He sits on the editorial board of several international journals and has been the coordinator and leader of several participant units in various European Projects.

Dr. Ilaria Palchetti received her degree in Pharmaceutical Chemistry and Technology from the University of Firenze in 1994 and a PhD in Biotechnology at Cranfield University in 1999. She is assistant professor of analytical chemistry in the Faculty of Sciences at the University of Florence. She has considerable expertise in biosensor development and in the environmental application of biosensors.