MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 5. Techniques for microbiological analyses

Lesson 31
BIOSENSORS FOR MONITORING CONTAMINANTS

31.1 Introduction

In order to meet strict regulations on food safety issues and owing to greater public awareness of environmental contaminants there is a huge need to monitor wider range of food contaminants linked with supply chain so that quality and safe foods can be ensured to consumers. Analysts currently have a range of portable analytical techniques at their disposal for monitoring across a variety of contaminant namely pesticides, aflatoxin M1, drug residues, heavy metals and microbial pathogens in food matrixes. More recently, biosensors have emerged as another promising technology in the analyst's armoury, especially for applications requiring persistent monitoring. Biosensors are defined as analytical devices integrating biological elements and signal transducers. The biological elements such as enzymes, antibodies, receptors proteins, nucleic acids, cells, or tissue sections or bacterial spores interact specifically with an analyte, producing a signal that the transducer recognizes and converts into measurable parameters (Fig. 31.1). The amount of signal generated is proportional to the concentration of the analyte, allowing for both quantitative and qualitative measurements in time. Although biosensors are of the essence for detection of contaminants but still operation of biosensors is a challenging task for their utility owing to the cost and shelf life of bio-recognition molecule. The resolution to above challenges is spore based biosensor which has evolved as robust, easy to use, simple, and inexpensive method for long term preservation, storage and transport of biosensing element.

Fig. 31.1 Schematic diagram showing the main components of a biosensor

The spore based biosensing systems are much superior in terms their activity, viability and analytical performance can be retained up to a period of 8 months when kept as dried spores at room temperature. The biosensors based on spore germination are real time sensing systems as germination process completes within minutes of sensing germinants in the environment. The spore production is a low priced process and its immobilization effortless process which curtails the cost of bio-recognition molecule employed in a biosensor.

31.2 Biosensor Technology

The two main elements in a biosensor are a biological recognition element or bio-receptor and a signal transducer. The bio-receptor is a bio-molecule that recognizes the target analyte and can be divided into three distinct groups: bio-catalytic, bio-affinity, and microbe-based systems. Biocatalysis-based biosensors depend on the use of pure or crude enzymes to moderate a biochemical reaction. For environmental applications, enzyme-based reactions involve enzymatic transformation of a pollutant or inhibition of enzyme activity by the pollutant. Enzyme inhibition approaches tend to cater for a larger number of environmental pollutants, usually of a particular chemical class such as antibiotic/ drug residues, aflatoxin M1, pesticides and heavy metals in food system. However, such methods requires the use of chromogen/ or fluorogens for measuring the presence of target contaminants in food matrix. A spore inhibition based enzyme substrate assay (SIB-ESA) for detection of aflatoxins M1 milk has been developed. Spores of Bacillus spp. have been lyophilized/ immobilized in micro centrifuge tube /sensor disk to which milk and substrate is added. In case where analyte is absent in milk system, specific indicator enzyme(s) are produced by active bio-sensing molecules which will act specifically on chromogenic/or fluorogenic substrate resulting in colored reaction (Patent Reg # 3064/DEL/2010)/ or fluorescence as end product which is measured semi-quantitatively by either visually/ or using optical system at specific excitation/emission spectra.

31.3 Inhibition Principle

Another system based on enzyme inhibition principle has been invented for monitoring of β-lactam antibiotics in milk. It is based on the principle of resistance mechanism of some β-lactamase generating Bacillus spp. Some spore forming bacteria such as B. cereus and B. licheniformis produce β-lactamase enzyme due to induction by β-lactam antibiotics and the enzyme production is proportional to the concentration of inducer present in milk. A real time microbial assay based on β-lactamase enzyme using starch iodine as colour indicator has been developed. The microbial assay is working on principle of non competitive enzyme action on inducer (β-lactam) resulting in indirect reduction of starch iodine mixture through penicilloic acid. A comparison of the intensity of the test reaction with that of a control was taken as criteria to determine whether the sample is positive or negative. The assay can detect specifically β- lactam groups in spiked milk within 15-20 min at regulatory codex limits with negligible sensitivity towards non β- lactam groups. The presence of Inhibitors other than antibiotic residues in milk did not interfere with the working principle of microbial assays (Patent Reg No. 115/DEL/2009).

31.4 Affinity Based Biosensors

Bioaffinity-based biosensors rely on the use of proteins, DNA or microbial receptor to recognize and bind a particular target. For environmental applications such systems depend primarily on the use of antibodies. This is due to the ready availability of monoclonal and polyclonal antibodies directed toward a wide range of environmental pollutants, as well as the relative affinity and selectivity of these recognition proteins for a specific compound or closely related groups of compounds. Nucleic acid-based affinity and electrochemical biosensors for potential environmental applications have recently been reported. Application areas for these include the detection of chemically induced DNA damage and the detection of microorganisms through the hybridization of species-specific sequences of DNA. Charm assay (Charm Sciences Inc., USA) is an example of bio-affinity biosensor which employs an immune reaction to bind the antibiotic to a microbial receptor and detects this complex using a low-level ³H or ¹⁴C radio-label. The Charm assay can detect a family of antibiotics, notably ß-lactams, sulphonamides, tetracyclines, novobiocin, aminoglycosides and macrolides, as well as various other substances such as chloramphenicol. The Charm II assay is an immune receptor test but is suitable for large laboratories only, requiring a range of laboratory equipment, including a centrifuge and sample mixers to prepare samples as well as a scintillation counter to detect the radio-label.

31.5 Microbial Biosensors

Microbial biosensors involves application of microorganisms as such/ or their spores as biological recognition element. These generally involve the measurement of microbial respiration, or its inhibition, by the analyte of interest. Compared to enzyme-based approaches, microorganism-based biosensors are relatively inexpensive to construct and can operate over a wide range of pH and temperature. The broad specificity of microbial biosensors to environmental toxins make them particularly applicable for general toxicity screening like biological oxygen demand (BOD) or in situations where the toxic compounds are well defined, or where there is a desire of measure total toxicity through a common mode of action.

A signal transducer is the second essential component of a biosensor. It converts the recognition event into a measurable signal. The transducer can take many forms depending upon the parameters being measured. The most well developed classes of transducers are potentiometric, amperometric, conductometric, optical, acoustic or piezoelectric etc. These utilize various electrochemical responses to measure changes in the electrical properties of the biological recognition element. Most of the reported potentiometric biosensors for detection of environmental pollutants have used enzymes that catalyze the consumption or production of protons. Phosphoric and carbamate pesticides can be evaluated through the use of a pH electrode that measures the activity of acetyl cholinesterase. The activity of the enzyme is affected by the presence of pesticides.

Further application of spore as signal transducer application targets real time detection of bacterial contamination using the inhibition of enzyme acetyl esterase coupled to spore germination using optical device for measurement .The use of spore as signal transducer is feasible if an illustrative knowledge of spore germination process and germinants are required. It involves selective enrichment of target bacteria in a selective media. The enriched bacterial cells will produce specific marker enzymes which act on germinogenic substrate and produce specific germinant (sugars and amino acids).The germinants induce spore germination and germination mediated concomitant de novo acetyl esterase enzymatic activity. As a consequence germination derived product can be easily detected by quantification of fluorescent signal produced as result of DAF hydrolysis by acetyl esterase. Based on above principle of germinogenic substrate detection of enterococci detection system has been developed which targets specific marker enzyme β-D glucosidase of enterococci will and aesculin as germinogenic substrate which releases germinant β-D glucose. The sensitivity of spore based bioassay was 5.66 log counts of cells in 5-6 hrs in spiked milk.

31.6 Optical Biosensors

In the field of biosensors transducers based on optical detection techniques are also emerging. These may employ linear optical phenomenon, including fluorescence, phosphorescence, polarization, rotation, interference, surface plasmon resonance (SPR), total internal reflection fluorescence (TIRF), etc. or non-linear phenomena, such as second harmonic generation. Advantages of optical techniques involve the speed and reproducibility of the measurement. Microbial spore germination based optical biosensor for the detection of enterococci in milk is being developed in our laboratory (Fig. 31.2). The detection technique being used is electron multiple charged couple device (EMCCD), as optical transducer which improvises the sensitivity as it equipped to detect germination of single spore (Patent file no. Ref. # IPR 119/DEL/2011).

Fig. 31.2 Principle of microbial spore germination based optical biosensor for the detection of bacterial contaminants

The basic requirement of a biosensor is that the biological material should bring the physico-chemical changes in close proximity of a transducer. In this direction immobilization technology has played a major role. Immobilization not only helps in forming the required close proximity between the biomaterial and the transducer, but also helps in stabilizing it for reuse. The biological material is immobilized directly on the transducer or in most cases, in membranes, which can subsequently be mounted on the transducer. Selection of a technique and/or support would depend on the nature of the bio-material and the substrate and configuration of the transducer used.

31.7 Immobilization Techniques

Some of the widely used immobilization techniques include adsorption, entrapment, covalent binding and cross-linking. Immobilization of enzymes and whole cells through adsorption perhaps is the simplest of all the techniques and was achieved successfully in monitoring of aflatoxin M1 and enterococci on sensor disc/ or bio-chip using EMCCD system and plate reader. Most of these techniques have the drawbacks of weak adhesion as well as complexity of the process. Novel techniques have been developed for immobilizing viable or non-viable cells through adhesion on a variety of polymeric surfaces including glass, cotton fabric and synthetic polymeric membranes using polyethylene-imine (PEI). This technique is gaining importance in the introduction of enzymes and microbes on transducer surfaces.

31.8 Commercial Biosensors

Although most biosensors systems have been tested only on non-real samples (such as in distilled water or buffer solutions), a few biosensors applied to real samples have appeared in recent years. Some representative examples of their application to the determination of different classes of key pollutants and environmental quality parameters, such as biological oxygen demand (BOD), toxicity or endocrine effects, in a variety of matrices are listed in Table 31.1. The application of biosensors to real samples must be a necessary step before their commercialization, which is, in general, the aim of the device development. Results must also be validated by comparison with those obtained with standard protocols in order to get the acceptance of end users. Most commercial biosensors developed are focused in clinical applications, such as for glucose and lactate. Prospective biosensor market for food, pharmaceutical, agriculture, military, veterinary and environment are still to be explored.

Table. 31.1 Biosensors applied to the determination of pollutants in real samples

31.9 Future Prospects

The hurdles to application of biosensors include:

• Diversity and complexity of samples.
• Relatively high development costs for single analyte systems,
• Limited shelf and operational life

Nevertheless, there are a number of areas where the unique capabilities of biosensors might be exploited to meet the requirements of environmental monitoring. Advances in areas such as multi-pollutant-screening could allow these techniques to be more competitive. The present scenario demands for increased range of detectable analytes with portable device structure. Solving the resulting integration issues will require further convergence with associated technologies such as biochemistry, polymer chemistry, electronics, micro-fluidics and separation technology. Micro-electro-mechanical systems or MEMS technology is one of the promising areas that may be going to fulfill these demands in future. The technology is an integration of mechanical elements, sensors, actuators, and electronics on a common silicon substrate through micro fabrication technology. Bio-chips and sensor arrays for detection of a wide range of hazardous chemical and biological agents can be made out of these MEMS based devices, making it feasible for simultaneous detection of multiple analytes. This also brings the lab-on-chip concept. However, Immobilization and stabilization of bio-molecules on these nano-devices may be a greater challenge. Some of the works in these areas have already been initiated. Utilization of molecular recognition ability of bio-molecules like avidin-biotin or streptavidin-biotin in conjunction with a lithographic technique is being investigated for the micro immobilization of enzymes on silicon wafers for biosensor applications. Immobilization of enzymes on silicon supports has attracted attention in biosensor chip technology and a variety of classical techniques have been proposed.

There are interesting possibilities within the field of biosensors. Given the existing advances in biological sciences, coupled with advances in various other scientific and engineering disciplines, it is imminent that many analytical applications will be replaced by biosensors. A fruitful fusion between biological sciences and other disciplines will help to realize the full potential of this technology in the future.