BIOSENSOR STRATEGIES FOR PATHOGEN DETECTION IN WATER SECURITYSECURITY

As described in Chapter 1, there is an increasing demand for testing the quality of public water systems, because of both natural contaminations, and biological terroristic attacks. Being able to perform accurate and fast tests to reveal the presence of hazardous microorganisms can be critical in certain situations.

Unfortunately, currently available tests are not as fast as desirable, and the results are usually available after one or more days, which makes them not very effective. In this scenario, a number of alternative technologies are under development to solve the issue of accurately and timely measuring very small amounts of microbes in large water supply systems.

Among the many technologies that can be used to detect microorganisms, the ones based on biosensors are certainly the most promising, also because such type of sensors are already well established in the market of portable medical devices. There are research projects aiming at the translation of the required knowledge from the medical field to security, and the DINAMICS project was one of them. As an example, the most successful biosensor application in the medical market is the glucometer, which measures glucose in

diabetic patients. The device is portable and the measurement is fast (less than a minute), as required for security specifications. Furthermore, the instrument can be used by untrained personnel, generally by the patient itself, and the active element that is used for the measurement, the biochip, is cheap and disposable, to avoid cross contamination between samples and to guarantee testing quality.

The choice of the specific biosensor technologies to be used depends strongly on the application, and in the following sections, after a short description of conventional laboratory analytical techniques, we will discuss the biosensor solutions that have highest potential for water security, in particular the technologies based on affinity recognition through antibodies and on DNA hybridization.

7.2.1 Conventional laboratory analysis

Most of the currently used microbiological procedures consist of two fundamental steps:

preconcentration/enrichment and detection/quantification. The first step commonly uses filtration or elution to increase the number of microbes to be tested, so that their concentration becomes measurable.

In fact, due to the variability of the infectious dose in the population, even a small number of viruses, bacteria or protozoa can be considered as potentially able to cause a disease if ingested. For example, the median hazardous dose for Salmonella is ten thousand cells, while this number lowers to just a few cells for Cryptosporidium parvum. The detection of such small quantities is very challenging, and a preconcentration step of those microbes is required with all available technologies. However, the amplification yield of microorganisms preconcentration must be proportional to the limit of detection of the measurement technique: if a lower concentration of microbes can be recognized by the instrument, the amplification can be more modest. In the case of conventional laboratory techniques, the limit of detection is often not small enough, so it is required to cultivate the microbes for one or two days in optimal temperature and nutrient conditions before they can reach a detectable concentration.

Following the preconcentration steps, the organisms are usually cultured for further amplification until plaques, colonies, or cysts are formed and visible. Finally a counting procedure is used for the calculation of the initial concentration of pathogens in water. All the cultivation procedures are optimized so that the organisms of interest are selectively stimulated to grow faster than the others, resulting in an improvement of assay selectivity. Also, the cultivation steps exclude dead organisms from the assay response, as they do not replicate.

This schematization of the general architecture of water pathogens recognition is performed through a number of technologies, each optimized for a specific microbial targets, as summarized in Table 7.1.

(Kosteret al., 2003)

Table 7.1 Schematization of the steps involved in the analysis of different microorganisms in conventional laboratory analysis.

A consequence of the described multi-step approach is that the currently used protocols have a very long response time, mainly due to the cultivation step, which can take more than one day. In this way, the results are obtained after one or two days of laboratory analysis since the collection of the sample. This scenario is clearly not optimal for current security requirements, and motivates the search for novel, faster technologies.

7.2.2 Biosensor analysis

Biosensors are analytical devices for the detection of a target analyte. The working principle is based on two main elements: a biological recognition unit, called the receptor, which can interact specifically with the target, and a physicochemical detection unit, the transducer, which converts a change in property (optical, electrochemical, mechanical, etc.) of the sample surface or solution, into an electric output signal. The signal is then analyzed and elaborated by a computing unit to output the response of the measurement.

The ability of such tests to be highly accurate depends on two factors: on one side, their use of a biological receptor element enables the biochip to bind molecules of the desired target with low interference from other organisms and molecules present in the sample. On the other side, the transducer unit transforms the biochemical recognition of the target into an electric signal through technologies that can sense small differences in the presence of microbes or microbe markers close to the surface of the biochip.

In the medical field, the principal advantage of biosensors on conventional assays is that the detection of molecular interactions happens as they take place, at the point of care, and without requiring auxiliary procedures thanks to their high sensitivity and low limit of detection. As explained above, in the case of water security systems, a preconcentration step is anyways required, as there is the need to recognize tiny quantities of highly toxic pathogens. However, the limit of detection of biosensors allows to be less stringent on the amplification steps, as described herein.

Of the many existent types of biosensors, developed primarily for medical applications, we herein describe in detail the two that can be translated towards the detection of pathogens in drinking waters.

Immunosensors

Immunosensors use antibodies as receptors for the identification or quantification of the target material in a sample. Antibodies are an efficient recognition tool because, if properly developed, they rely on their unique ability to bind their respective antigen with much higher affinity than other molecules. Furthermore, immunogenic diversity allows to monitor potentially any compound that can produce a response of an immune system. In fact, antibodies are produced by B-cells of the immune system to identify and neutralize non-self antigens such as bacteria and viruses.

Antibodies recognize a specific part of a foreign molecule, called the antigen, and bind it. Through this molecular interaction, an antibody can fasten to the surface of a microbe, hence providing a tag for the target selection and target recognition. A further advantage of this technology is that the general structure of all antibodies is very similar, with just an extremely variable small region at the tip of the protein, known as the hypervariable region (Figure 7.1), which is responsible for the recognition of the antigens. This allows the development of generally functioning transduction techniques, relatively independently on the specific target specie out of the wide variety of pathogenic organisms.

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A wide range of immunological methods that take advantage of the interaction antibody-antigen is available. As an example, the target organism can be caught by capture antibodies that are stably deposited on the surface of the biochip transducer. The persistence of the target organism on the surface can be directly detected by the transducer, or the detection can be aided by the addition of a label, normally carried by a secondary reporter antibody in a so called sandwich assay, as pictured in Figure 7.2. While direct label-free detection uses an easier biochemical protocol, its limit of detection is generally not sufficient for most applications, therefore the sandwich assay is a more frequent configuration.

In another type of immunosensor, magnetic beads coated with capture antibodies specific for a target organism are used (Gijs, 2004). After incubation and efficient mixing of the particles in the cell suspension, the target and the magnetic beads are bound through affinity recognition, so they can be separated from the rest of the suspension with the help of a magnetic field. As in the previous example, a set of reporter antibodies can be used for the signal transduction.

To operate an immunosensor, a basic lab infrastructure could be an advantage, but it is not necessary. The assays are developed to be easy to perform and fast, with a typical response time inferior to one hour.

Immunosensors have a potential for applications in water security, but the limit of detection needs to be lowered for direct application of such devices in the field, in order to decrease the requirements on the preconcentration procedure, and hence the response time.

A further use of immunosensors stems from them being also sensitive to non-viable microorganisms.

Poorly perpetrated deliberate contamination attempts might result in unusual but dead microbial agents Figure 7.1 Structure of an antibody. The general structure of all antibodies is very similar, but different antibodies can specifically bind a certain antigen thanks to a short part of the sequence which is extremely variable, called the hypervariable region (indicated with the arrow).

Figure 7.2 Schematization of an immunosensor sandwich assay. Generally a layer of probe antibodies deposited on the biosensor surface is used to capture the target analyte and separate it from the whole sample. Secondary reporter antibodies then bind to the captured target antigens and are used for the signal transduction and detection.

flowing in the water system: it might still be desirable to detect the breach in the microbiological security system even if it does not directly lead to an increased health risk.

DNA sensors

The use of nucleic acid recognition layers is a relatively new and exciting area in sensors technology.

Hybridization biosensors are in fact considerably promising for obtaining sequence-specific information in a simple, fast and cheap manner compared to conventional assays. Their advantages are very similar to those offered by immunosensors, with an additional opportunity coming from the existence of established and fast DNA amplification procedures, such as the polymerase chain reaction (PCR) or other newer strategies (see Chapter 8 of this book), which are significantly faster than the time consuming amplification of whole organisms through selective cultivation.

The working principle of these devices is the Watson-Crick DNA base pairing. The biosensors rely on the immobilization of a single-stranded DNA sequence, defined as probe, on the transducer surface. In turn the transducer gives an electrical signal upon hybridization with the desired complementary region of the target nucleic acid (Figure 7.3) (Wang, 2000).

The required DNA sample extraction and preparation steps, including amplification processes such as PCR, have been demonstrated at the microscale (Miret al., 2004), with examples reported also in this book, in Chapters 5 and 6. This makes such devices very suitable for complete on-chip DNA analysis.

PCR exponentially multiplies the number of target DNA strands by using the DNA target as a template, and replicating it through the DNA polymerase enzyme, which is natively used by eukaryotic and prokaryotic cells, and by some viruses for replication and repair of DNA. The oligonucleotide sequence to be amplified is selected by the primers, which are short oligonucleotides (usually less than 30 bases) that bind to complementary strands flanking the sequence of interest of the target DNA. A thermocycler allows iteration of the three necessary steps for the reaction: denaturation, annealing and extension of the primers. On each cycle, the amount of the DNA sequence specifically selected by the primers is roughly doubled, allowing for a selective amplification of only the nucleic acid of organisms of interest. The Figure 7.3 Concept of the formation of a DNA double helix through hybridization.

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DNA amplification is a particularly desirable step when very small amounts of microorganisms are available, since it can specifically amplify the desired DNA sequence by many orders of magnitude in a timescale of about one-two hours.

The PCR output can then be injected on the biochip, where probe oligonucleotides complementary to a part of the amplified target sequence capture the target molecules by base-sequence recognition. The double helix can be directly recognized, or it can be labelled for example with a reporter DNA sequence that binds to a part of the target that is still free (in a sandwich assay configuration). A readily detectable signal can be produced (Figure 7.4).

The DNA biosensor technology has been developed at the end of the 20th century for biomedical applications, and it led to the well known products called DNA microarrays, where the probe can be produced by conventional or in situ synthesis (20 to 30 bases probe) (Lemieux et al., 1998; Lipshutz et al., 1999), or it can be synthesized through the enzymes reverse transcriptase and DNA polymerase from a biological source (500 to 5000 bases cDNA probe) (Ekinset al., 1999).

As in the case of immunosensors, DNA devices do not require a lab infrastructure for the measurement, and the assays are sensitive, easy and fast to perform, with a typical time of 2–4 hours including the target amplification step. DNA biosensors are very promising for applications in water security, with the only downside that they need to be integrated with a sample preconcentration module and a DNA extraction module. Of particular relevance is the great improvement in response time with respect to conventional laboratory assays. Furthermore, like immunosensors, DNA biosensors maintain the ability to recognize both viable and non-viable microbes.

In Table 7.2, the main advantages and disadvantages of the classes of biosensors so far mentioned are summarized.

Figure 7.4 Schematization of a DNA sandwich assay. Generally a layer of probe oligonucleotides deposited on the biosensor surface is used to capture the target DNA sequence only. A secondary reporter oligonucleotide then forms a double-helix to an unbound segment of the target sequence and is used for the signal transduction and detection.

7.2.3 Conclusions

In this section, we have analyzed the characteristics of the most promising technologies that can be translated from the biomedical field for water security, with a clear competitive advantage for the fast, sensitive and selective DNA biosensors. In the following section, the most common methods for the electrochemical conversion of the hybridization event into an electrical signal are presented.

7.3 COMMON ELECTROCHEMICAL DETECTION SYSTEMS FOR DNA