Recent developments in bio-molecular electronics techniques for food pathogens

Analytica Chimica Acta 568 (2006) 259–274

Recent developments in bio-molecular electronics techniques for food pathogens Kavita Arora a,b,∗ , Subhash Chand b , B.D. Malhotra a a

Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, K.S. Krishnan Road, New Delhi 110012, India b Department of Biochemical Engineering and Biotechnology, Indian Institute Technology Delhi, Hauz Khas, New Delhi 110016, India Received 2 November 2005; received in revised form 20 March 2006; accepted 23 March 2006 Available online 30 March 2006

Abstract Food borne illnesses contribute to the majority of infections caused by pathogenic microorganisms. Detection of these pathogens originating from different sources has led to increased interest of researchers. New bio-molecular techniques for food pathogen detection are being developed to improve the sensor characteristics such as sensitivity, reusability, simplicity and economic viability. Present article deals with the various methods of food pathogen detection with special emphasis on bio-molecular electronics techniques such as biosensors, microarrays, electronic nose, and nano-materials based methods. © 2006 Elsevier B.V. All rights reserved. Keywords: Food pathogens; Bio-molecular electronics; Biosensors; Microarrays; Nano-devices; Electronic nose

1. Introduction There is increased demand for the availability of simple and reliable protocols for the analysis of molecules of biological importance. The requirement of skilled manpower, excessive external reagents, complicated sample preparation methods and time-consuming procedures, etc., has limited the applications of most of the known classical techniques for food pathogen detection. In this context, bio-molecular electronics techniques have emerged as one of the important alternatives as these methods are likely to lead to the development of assay methods with desired sensitivity, selectivity, reusability, portability, reversibility, simplicity, the choice of transducers (optical/mass/electrochemical/thermal) and the possibility to monitor simultaneously a wide range of parameters. Besides this, use of a biological element in close vicinity of a transducer has led to the evolution of smart and intelligent bio-molecular electronic devices such as molecular switches; molecular logic gates, biosensors, microarrays, molecular motors, ion-sensitive-field-effecttransistors (ISFETs), chemical-field-effect-transistors (CHEM-

∗

Corresponding author. Tel.: +91 11 25734273; fax: +91 11 25726938. E-mail address: [email protected] (K. Arora).

0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.03.078

FETs), micro-fluidics and nano-materials based bio-molecular electronic devices, etc. Many pathogenic bacteria have been considered as biological warfare agents since humans are susceptible to diseases and most of these pathogens have been found to be resistant to environmental conditions [1,2]. Increasing incidences of food poisoning outbreaks in countries like USA, UK, India and other countries have led to increased demand of monitoring pathogens in food materials [3–5]. Despite the fact that America’s food supply is one of the safest in the world, the Center for Disease Control, USA, reports millions of food borne illnesses and food poisoning outbreaks every year indicating an upward trend [6–8]. Besides this, there are many reports for the recall of products due to contamination of pathogens. One of the largest recalls in the world history is by Wampler Foods Inc. (Franconia, PA) in October 2002 when 28 million pounds of fresh and frozen readyto-eat chicken and turkey products were spoiled due to Listeria monocytogenes [8]. The on-site tests require shorter assay times resulting in considerable reduction in the storage cost of food products. Additionally, if a recall is issued, this often results in reducing the corporate liability costs. Food borne illnesses are known to share a major part of infections caused among human beings. Infectious microbes either viable or non-viable could become harmful if they produce toxins. Usually factors like inadequate refrigeration, preparation

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of food in advance, inadequate cooking, infected food handling, poor hygiene, inadequate holding temperature, inadequate reheating, contaminated raw ingredients, cross-contamination, and poorly maintained equipments, etc., are some of the major causes of food contamination. The emergence and dissemination of microbial food borne pathogens is affected by factors related to the nature of pathogens, type of host, process of food production, food storage, while transportation, and during consumption. Besides this, there has been increased concern over the use of genetically modified organisms (GMOs) in food products. Therefore, there is considerable need to develop detection, control and prevention strategies as well as further research to ensure the quality of food in both domestic and international trade [9,10]. Various food borne pathogens have been identified for causing food borne diseases. However, Campylobacter, Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7 have been generally found to be responsible for majority of food borne outbreaks [11–13]. Food producers have been using simple and empirical scheme for assessing and managing risks for food contamination. This detection process usually includes detection of the causatives, collection of information about the causatives, determination of ways to control the causatives, selection of a method to control the presence of causative, control of the causative and rejection of a lot containing infection causing agents [13]. It may be noted that the present-day need to monitor the presence, of food-pathogens at each step of production, storage and transport cannot be solved by existing locations such as government laboratories or reference laboratories. Many companies are, now employing the Hazard Analysis and Critical Control Point (HACCP) concept in their quality assurance and control programs where strong emphasis is given to the rapid screening of raw material quality before processing and to the rapid “in-line” testing of process efficacy [14,15]. The advances in biotechnology, bio-molecular electronics, nanotechnology and other sciences have led to the spawning of new and rapid methods of food pathogen detection that can perhaps be used for on-site testing. This interesting devolvement has motivated the industries to develop newer methods of food pathogen detection. Table 1 contains the list of various food pathogens that are responsible for various food borne illnesses. The present article is an attempt to review the literature relating to the application of bio-molecular electronic techniques for detection of food pathogens. 2. Contemporary methods for detection of food pathogens Some of the conventional methods being used for pathogens detection rely on specific microbiological, biochemical immunological identification techniques. Some of these detection methods are: microbiological methods (culturing onto desired medium) for detection of Listeria monocytogenes [16], S. aureus, Salmonella, Coliforms, E. coli, etc. [17] (e.g., ALOA® method [16] and VIT® technology [18] for Listeria; BIO DISC B, E and O for Streptococci, S. pneumonia, P. aeruginosa and Nesseria sp.; TNT kit for S. aureus in food and cultures [19]);

molecular biological methods (using techniques such as southern blotting, northern blotting, nucleic acid-based assays, restriction fragment length polymorphism (RFLP), rapid amplified polymorphic DNA (RAPD), etc.) [20]; immunological techniques (like immuno-precipitation reactions, agglutination tests, ELISA, RIA, flow injection immuno-assay for E. coli and Salmonella [21], western blotting, etc.) [22]; and gas chromatography (GC), high performance liquid chromatography (HPLC) for detection of gases that are volatile marker for food spoilage [23]. These methods, however, yield both qualitative and quantitative information on the nature and number of pathogens being tested. Besides this, these techniques are greatly restricted by the long assay time, initial enrichment of desired pathogen, maintenance of strict sterile environment, essential pre-requisite of skilled manpower and sometimes costly preparation. Some of the other contemporary methods for food pathogen detection being used are: flow cytometry (e.g., fluorescent activation cell sorter or FACS) [23]; immunodiagnostic kits (based on ELISA, agglutination, FIA, etc.) for detection of E. coli, Campylobacter jejuni, Listeria monocytogenes, Salmonella, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, Fungi, Mycotoxins, etc. [8,24,25]; immuno-magnetic separation bacteriophage assay (IMSBA) for Salmonella entreitids [23,26], E. coli O157:H7, Salmonella typhi, Listeria monocytogenes, Compylobacter jejuni [27]; polymerase chain reaction (PCR) for detection of Campylobacter jejuni, Salmonell spp., E. coli O157:H7 and Listeria [8,28–33]; nucleic acid sequence-based amplification (NASABA) for detection of Campylobacter sp., Cryptosporidium parvum, E. coli, Hepatitis A virus, Listeria monocytogenes, Rotavirus, Salmonella enterica, etc. [34,35]; conductance microbiology for detection of Salmonella, Listeria and Campylobacter [36]; optical detection via FTIR Raman spectroscopy for detection of Legionella pneumophila, Aeromonades, E. coli, Pseudomonas aeruginosa, and Salmonella typhimurium [8,37]; and DNA probe assay Mycobacterium kansasii, Mycobacterium tuberculosis, M. avium, Listeria monocytogenes, Ligeonell pneumophila, plant pathogenic bacteria, B. forsythus, liver fluke or Fasciola hepatica [38], etc. These methods have been reported to be used for detection of the microbial presence in a wide variety of samples. However, their application to real time monitoring of food pathogen has not yet succeeded. Some of the disadvantages of these methods relate to higher assay time, lower sensitivity, the reduced specificity, the size of the equipment used, complicated procedures and wide acceptance [39]. Table 2 describes the details relating to various contemporary methods that are used for food pathogen detection. 3. Bio-molecular electronics based techniques for food pathogen detection Application of new advanced materials such as nanomaterials (carbon, Au, Zn), quantum dots, nano-composites, core-shell structures, conducting polymers, Langmuir–Blodgett (LB) films, self-assembled monolayers (SAMs), microfluidics, molecular switches, and molecular gates have recently led to the evolution of bio-molecular electronics-based techniques for

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Table 1 Various pathogens found responsible for generation of food-borne illnesses Organism

Type

Food source

Disorder/disease symptoms

Reference

Bacillus cereus

Bacteria

Diarrhea

[210]

Campylobacter jejuni

Bacteria

Diarrhea, abdominal cramps, bloody stool

[7,12,195,211,212]

Clostridium botulinum

Bacteria

Milk products, sprouted cereals, cakes, cucumbers Raw milk, meat, poultry, unchlorinated water, under cooked food In low acid food i.e. canned food foil wrapped and meat products which are kept at room temperature

[213,214]

Clostridium perfringens

Bacteria

Cooked meat, chafing dishes

Cryptosporidium parvum Entamoeba histolytica Escherichia coli

Protozoa Protozoa Bacteria

Hepatitis A

Virus

Fecal contaminated water Poor water, and food quality Partially cooked meat, milk, fruits, vegetables, etc. Canned food, fruits, meat, sea food

Food poisoning, nervous system disturbances such as double vision, droopy eyelids, trouble speaking, swallowing, breathing. Untreated botulism can be fatal Enterotoxin causes diarrhea, gas pains, nausea, and sometimes vomiting lasting only a day. Usually mild, but can be serious in ulcer patients, the elderly, ill, or immune-compromised Gastrointestinal diseases/symptom less Amoebiosis Gastroenteritis

[217]

Klebsiella aerogenes Klebsiella pniumaniae Listeria monocytogenes

Bacteria Bacteria Bacteria

Enteric diseases, fatigue, fever, nausea, vomiting, abdominal cramps, appetite loss, followed by liver enlargement, jaundice, and darkened urine. May cause liver damage and death Diarrhea Enteric disease Flue, abortion (in pregnant women), still births

Moulds

Moulds

Liver and/or kidney disease depending on the type of mycotoxin

[212]

Noro virus

Virus

[212]

Norwalk virus Penicillium digitatum Penicillium italicum Phytopthora cactorum Rhizoctonia sp. Rota virus Salmonella Shigella

Virus Fungi Fungi Fungi Fungi Virus Bacteria Bacteria

Diarrhea, vomiting, nausea, abdominal cramps, fever, chills, and body aches – – – – – Acute gastroenteritis Bacterial diarrhea, salmonellosis Shigellosis i.e. diarrhea, abdominal pain, fever, sometimes vomiting, and blood, pus or mucus in stool; lasts 5–6 days

Staphylococcus aureus

Bacteria

[214]

Vibrio parahaemolyticus Vibrio vulnificus

Bacteria Bacteria

Gastroenteritis i.e. vomiting, diarrhea, nausea, abdominal cramps but rarely fatal Diarrhea –

Yersinia enterocolitica

Bacteria

–

[213]

Fruits Fast food Canned foods, fruits, vegetables, stored food Beans, peanuts, corn, and other grains that have been stored in warm moist places Contaminated shellfish raw or partially cooked Water, food Fruits, walnuts Fruits Fruits and their products Fruits and their products Sewage contaminated water Meat, milk, raw eggs Spreads from poor sanitary habits handles liquid or moist food that is not thoroughly cooked afterwards, e.g., celery, fruits, vegetables Milk, fruit, vegetables, diary products, eggs Sea food, contaminated fish Coconut milk, vegetables like lettuce, cabbage Milk

food pathogen detection. This has been attributed to smaller size, quicker response time, reusability, portability and multi-analyte detection, etc. Some of the bio-molecular electronics-based techniques that are gradually gaining importance include electronic nose, biosensors, microarrays and nano-materials based methods (Table 3). 3.1. Electronic nose Electronic nose measures the change in resistance of a patterned conducting polymer surface upon interaction with microbes/volatile compounds that can be amplified and ana-

[213,215]

[34,158] [213] [216]

[218] [218] [18,24,219]

[213] [214] [214] [214] [214] [220] [13,21,212,221–223] [214]

[213] [214]

lyzed through a database capture software system [40–42]. Electronic noses based on a variety of materials such as conducting polymers, ionic compounds, electronic conductors (SnO2 , TiO2 , Ta2 O3 , IrOx , etc.), mixed conductors (Ga2 O3 , SrTiO3 , Niy O3 , etc.), ionic conductors (Na2 Co3 , ZrO2 , CaF3 , CeO2 , CaF2 , ␤-alumina, etc.), molecular crystals (pthalocyanines, porphyrins), self-assembled monolayers (alkane thiols, dialkylsulfides), Langmuir–Blodgett films (pthalocyanines, porphyrins), polymer films (polyethenes, polypyrroles, polythiophenes), super molecular structures (zeolites, cyclophanes, cyclopeptides) and components of bio-molecular functional system (synthetic lipids), etc., have recently been reviewed [41]. It

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Table 2 Comparative chart containing various contemporary methods of food pathogen detection S. no.

Detection technique

Organism detected

Assay time

Detection limit

Limitations of technique

Reference

1

Microbiological analysis

∼10−5 dilutions – – – 103 –106 cfu/ml

[91] [17] [16]

Biochemical methods

∼7 days – – – 1–3 days – – – – – – – 1–3 days

Longer assay time, detection of only live cells, no detection of toxins

2

L. monocytogenes, S. aureus, Salmonella, E. coli, Coliforms, etc. L. monocytogenes, Pseudomonas, Nesseria S, pneumonia Bacillus, Campylobacter, Citrobacter, Escherichia, Lactobacillus, Pediococcus, Salmonella, Streptococcus, Yersinia E. coli, all microorganisms

Longer assay time, detection of only live cells, no detection of toxins

[16,18,19]

[16,19,160] [52]

3

Molecular biological methods

4

Serological tests or immunologicaldetection methods or assays Western blotting

E. coli, Salmonella

1–2 days

–

E. coli, Salmonella

1–2 days

–

Gas sensing methods like GC, HPLC, etc. Flow cytometry

Non specific but senses only the gases like from contaminated food Non specific

2–3 h

–

–

–

Immuno diagnostic kits

Salmonella, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, E coli, Fungi Mycotoxins Listeria spp., Campylobacter jejuni Salmonella entretids, S. typhi, E. coli O157: H7, L. monocytogenes, Campylobacter jejuni Salmonella entretids, E. coli O157: H7, L. monocytogenes, Campylobacter jejuni E. coli, Campylobacter, Cryptosporidium parvum, Hepatitis A virus, L. monocytogenes, Salmonella enterica, P. falciparam

Within minutes to hours

–

5 6

7

8

9

10

11

12 13 14

15

Immuno magnetic separation bacteriophage assay Polymerase chain reaction

Nucleic acid sequence-based amplification (NASABA)

Conductance microbiology Optical detection Fourior transform infrared and Raman spectroscopy

DNA probe assay

Salmonella, Listeria, Campylobacter, E. coli O 157 Salmonella Legionella pneumophila, Aeromonades E. coli Pseudomonas aeruginosa, Salmonella typhimurium Samonella, Listeria spp., E. coli, Staphylococcus aureus, Campylobacter spp. Mycobacterium kansasii, M. tuberculosis, M. avium, Listeria monocytogenes, Ligionella pnuemophila, Fasciola hepatica

–

Longer assay time, skilled manpower, high cost, no on-site measurement Costly, requirement of monoclonal antibodies, longer assay time, etc. Costly, longer assay time, skilled manpower Costly, no on-site measurement

Applicable mostly in fermentation processes, incapable of distinguishing dead and live cells Costly, in some cases it may require enrichment

[54] [20,224]

[21]

[225,226] [42]

[23]

[224]

[23] ∼20 h – – – ∼2–4 h – –

–

Cannot be applied to on-site testing, long assay time

[23,26] [27]

∼1–10 cells/ml, 1–10 cells/ml, 1000 cells/ml

Can not detect live or dead cells, no on-site detection, need of enrichment, etc.

[227] [30] [227]

<1 h

1 cfu/ml – – –

Onsite testing can not be performed

[34,228]

On-site testing can not be performed Long detection time Costly and complicated instrumentation

[228]

– – –

∼24 h

10–108 cells/ml blood –

16 h to 2 days ∼5–10 min

– –

– –

– –

– – – – – – – – –

1 cell/25 g food

–

Longer assay time, pre-enrichment, various pre-experimental preparations, etc.

[37] [229]

[230] [38,36]

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263

Table 3 Comparative chart enlisting various recent biomolecular electronics-based methods of food pathogen detection S. no.

Detection technique

Organism detected

Assay time

Detection limit

Limitations of technique

Reference

1

Electronic nose

Psuedomonas aureofaciens

1–2 min

ppm and sometimes ppb

[42–44]

2

Immunosensors

Salmonella (after enrichment), Salmonella (QCM based), after enrichment Salmonella typhimunrium, S. aureours

∼100 cfu 5 × 103 cells/ml 5.3 × 105 –107 cfu/ml

15 min <2 h ∼25 min

Less stability, sensitivity, reproducibility and life time of sensor Some times reusability is not possible

E. coli O157:H7,

BioflashTM based on CANARYTM Neurotoxic amino acid in shellfish, S. aureous, Yersinia meningitis Yesinia pestis L. monocytogenes

4

Phage display peptide biosensor

DNA biosensor

[102] – –

– ∼20 min – <2 h

Listeria monocytogenes, E. coli, Yersinia pestis, Hepatitis C, Herpes simplex virus, Human cytomegalovirus, Rabies virus, Vaccinia virus, Ebola virus, Baccillus spores Staphyloccoccal enterotoxin B

– – – – – – – – – – –

– – – – – – – – – – – – – –

E. coli

10 cells/0.075 ml volume ∼40 cfu/ml – –

Listeria monocytogenes Bacillus anthracis

– – – –

PNA biosensor

No specific microorganism

Comparable to DNA sensing

–

6

LNA sensing

Not yet explored for microbial detection

–

–

7

Microarrays

DNA micro array for: Listeria, Campylobacter, C. perfringes Plasmodium falciparum Staphylococcus E.coli Protein microarrays Tissue microarrays

–

–

– – – – –

– – – – –

2.67 × 10−2 cfu/ml and 10−12 M oligos 10−14 M 10−9 –10−11 mol/l

∼30 min

Nano-materialsbased sensing

QCM based E.coli O157:H7

[97] [97]

150 s

5

8

[103,104] [95]

∼7 min 2h – <5 min –

104 –105 cfu/ml 103 cells/ml 4.7 × 106 –2.4 × 109 cfu/ml 107 cells/ml – –

Francixoli tularensis, Coxiella burnetti, Brucella militensis, Campylobacter 3

20 cfu/ml 350 ± 150 cells/ml 104 –105 cfu/ml 6–102 cells/ml 103 –104 cells ∼10 pg analyte/mm2 103 –104 cells/ml 5 ng/ml –

[96,98,99,101,102] [96] [104]

10–15 min – –

Comparatively higher time of detection

[119] [120] [120]

[231] [121] [118] Reusability is not possible except hybridization process; extraction of target DNA High cost and extraction of target DNA High cost, initial stage of development, extraction of target DNA, etc. Problems while fabrication at such a miniaturized level

[43,44] [97,149] [160] [148,149] [174–179] [199–202]

[189]

[190] [192] [199] [198,199] [197] Simplicity in detection better sensitivity and quick response

[232]

[204] [206]

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has recently been reported that different levels of milk spoilage caused by single bacterial species such as Psuedomonas aureofaciens can be differentiated [42]. This portable technique provides advantage over gas chromatographic procedures like GC-MS (gas chromatography-mass spectrophotometer) that use complicated procedures using sophisticated instruments [43,44]. Recently, Research Centre Karlsruhe, Germany, has developed the Karlsruhe Micro Nose (KAMINA) based on gradient microarray of 38 Pt doped SnO2 sensor strips for water pollution screening and discrimination of volatile pollutants such as ammonia and chloroform (Table 3). The KAMINA has high sensitivity, gas discrimination power, small size, reduced power and is low-priced [45]. Microorganisms such as Bacillus subtilis CRA 14160, Penicillium verrucosum Vmmope 20-07 and Pichia anomala NCPF present on the bread prior to visible spoilage have been shown to be detected using Bloodhound BH-114 electronic nose via cluster analysis of volatile compounds that are caused by lipoxygenase of contaminants [46]. 3.2. Biosensors A biosensor is an analytical device that converts a biological response into an electrical signal including sensor devices that determine concentration of substrates and other parameters of biological interest even where they do not utilize a biological system directly. Biological recognition element produces a signal (electrochemical/optical/piezoelectric (mass)/thermal) [5,47–90] that is proportional to the concentration of an analyte or a group of analytes. The bioactive element can be an enzyme, an antibody, a whole cell (bacterial or fungal), a tissue slice, a receptor molecule [52–63], nucleic acid, an organelle, or bio-mimetic components such as engineered proteins, peptide nucleic acids [53–56], aptamers (synthetic/semi-synthetic) [57,58], ribozymes [59], synzymes [60] leading to either a catalytic or affinity biosensor [61,62]. Various characteristics of a biosensor include accuracy, response time, sensitivity, specificity, easy handling and reproducibility. Above all, it must be robust to changes in the pH, temperature and ionic strength [42]. These biosensing devices can be utilized to measure biological effects such as genotoxicity, immunotoxicity, biotoxins, endocrine effects and various parameters such as glucose [72,73], cholesterol [74,75], lactate [76–78], urea [79,80] and creatinine in blood/urine samples [81,82]. Besides this, these can be used for the detection of specific analytes that are difficult to detect and are important contaminants of drinking water, waste water, soil or air (e.g., surfactants, chlorinated hydrocarbons, pesticides, drugs, carcinogens, pollutants or other toxicants) [83,84]. Recent developments in biosensors have recently been reported in literature [63]. Biosensors for pathogen detection offer advantages over existing large number of highly automated machines, from major manufacturers, placed in centralized laboratories for routine analysis. This area requires serious considerations from several angles to successfully channel sound research ideas towards finished products to fulfill the drifting demand towards measurement of a range of parameters using a single micro-fabricated portable analyzer with either disposable or reusable cartridge.

There have been reports about the use of enzymes and whole cell-based biosensors for biomedical, agro-food application and for the interrogation of general metabolic status [69,70]. Discovery of surface receptors, development of artificial receptors using computer (molecular) modeling and molecularly imprinted polymer (MIP) or combinatorial synthesis has increased the range of receptors that can be used for the fabrication of biosensors. The automated manufacturing technologies have demonstrated the commercial production of devices where large numbers of inexpensive, reproducible electrochemical devices are used. The ability to print materials at high precision and speed has been developed for the mass production of biosensors [66–68]. Several techniques like screen-printing, inkjet printing, air-brush and Cavro deposition have been developed and adapted, under micro-processor control especially thick film biosensors. However, advances in antibody production, emergence of phage-displayed peptide biosensors, and discovery of conducting properties of DNA have increased the possibilities for rapid detection of pathogens using biosensors [71,85–90]. The combinations of various biological receptors with desired transducers allow detection of a broad spectrum of analytes in the area of food analysis, clinical diagnostics, bioprocess and environmental monitoring [42]. 3.2.1. Immunosensors Immunological methods rely on specific binding of an antibody (monoclonal, polyclonal or engineered) to an antigen. Detection of specific microorganisms and microbial toxins requires immobilization of specific antibodies onto a given transducer that can produce signal upon attachment of typical microbe/microbial toxin [91]. Inherent features of immunosensors such as specificity, sensitivity, speed, ease and on-site analysis have attracted much interest of the scientific community. Various immunosensors based on fluorescent antibodies, acoustic gravimetric wave transduction, surface plasmon resonance, electrochemical, etc., have been reported [92] (Table 3). Light-addressable potentiometric sensor (LAPS)-based immunosensing system has been reported to be used to detect Salmonella [65,94]. These LAPS are electrolyte-insulatorsemiconductor (EIS) structures that measure the change in capacitance across the depletion layer formed at insulatorsemiconductor junction as a function of ion-concentration, providing advantages of flat surface, no wirings or passivation and capability of measuring pH and concentration [22,93]. The immuno-complex thus formed is captured on biotin-coated nitrocellulose membrane and is detected via an anti-fluorescein urease conjugate. These immuno-sensors have been reported to detect the presence of pathogenic bacteria like Nesseria meningitides, Y. pestis [95], Brucella militensis, Francisella tularensis, Coxiella burnetti, and E. coli [96,97], etc. These types of biosensors have several difficulties associated with the light sensitive material, reproducibility and selectivity, etc. Optical immunosensors include optical fiber or surface plasmon resonance (SPR)-based sensing systems that measure luminescence, fluorescence, reflectance and absorbance, etc. Specific antibodies immobilized onto different fiber probes have been reported for the simultaneous detection of Salmonella, E. coli

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and Listeria [22]. Among optical immunosensors, surface plasmon resonance-based biosensors that measure changes in refractive index have been reported for the detection of Salmonella enteridis and Salmonella typhimurium using antigens immobilized onto a metal surface [98]. Detection of IGF-1 in cow’s milk [99] and E. coli O157:H7 using BIAcore system [100] has been reported. Advantages of very low amount of reagents and elimination of antibody labeling and secondary reagents have provided this method a lead. Enzyme-linked chemiluminescence (ELIMCL) has been reported to detect E. coli O157:H7 incorporating the selectivity of antibodies with magnetic particle isolation [96]. A fluorescent fiber-optic biosensor has been developed to detect Salmonella typhimurium using specific antibodies immobilized on the surface of polystyrene micro-spheres [101]. A disposable electrochemical (differential pulse voltammetry) immunosensor based on polyclonal antibodies has been developed for the detection of neuro-toxic amino acid (domoic acid) in muscle tissue of shell-fish [102]. An enzyme-linked amperometric immunosensor has been utilized for the detection of S. aureus and Salmonella in pure cultures and in foods [103]. Recently, immuno-magnetic beads have been applied in immuno-electrochemical assays for the detection of S. typhimurium [105]. E. coli was detected amperometrically in chicken carcass, wash water, ground beef and fresh cut broccoli after immunomagnetic separation, flow injection and bi-enzyme electrode [104]. Detection of Salmonella [106] and E. coli [107] has been reported too. Novel liposome-based amperometric biosensors for haemolytic microorganisms, L. monocytogenes, Listeria welshimeri and E. coli [97] have been demonstrated. Another report relates the ion-channel electrochemical biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species at stainless steel electrode [97]. Detection kits for Listeria monocytogenses and Bacillus cereus have recently been demonstrated [108]. Micro-gravimetric or acoustic sensors measure mass changes occurring at a bio-molecular surface [109,112]. Quartz is the most frequently used piezoelectric crystal as mass-to-frequency transducer by immobilizing specific polyclonal/monoclonal antibodies to detect the presence of microbial pathogens. A quartz crystal micro-balance (QCM)-based immuno-sensor has been shown to detect Salmonella [110–112,114]. Another QCMbased immuno-sensor using SAM of thiosalicyclic acid has been reported for the detection of Listeria monocytogenes with reusability of about 10 times [113]. An Automated Water Analyzer Computer Supported System (AWACSS) based on immuno-multi-sensor chip to sense various organic pollutants in drinking water and waste water samples via inhibition assay within few minutes has been reported [68]. Innovative Biosensors Inc. (IBI) Maryland has recently launched BioFlashTM system based on CANARYTM (Cellular Analysis and Notification of Antigen Risks and Yields) for E. coli O157:H7 detection [235]. This was developed at Massachusetts Institute of Technology, USA. The exploitation of surface plasmon resonance concept has been successfully implemented by BIAcore® system too [64].

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Immunosensors offer great advantages of detecting various microorganisms with high degree of sensitivity, specificity and reusability. The recent advances in new materials and methods are likely to help towards the present problems of obtaining monoclonal antibodies, cross-reactivities and high costs. 3.2.2. Phage display peptide biosensor Phage display peptides are short peptides displayed on the minor coat protein (pIII) or major coat protein (pVIII) of bacteriophage M13 [115,116]. These have been used to isolate peptide ligands for a variety of targets including antibodies, enzymes, and cell surface receptors [116]. Peptide phage display may be utilized to select peptides that selectively bind microbial cells, their toxins or even environmental pollutants in a range of non-physiological solvents [117]. These fusion proteins upon combination with suitable transducer systems can reveal the presence of a target analyte in a given sample. It has been reported that SPR-based sensing can be used for the detection of analytes [118] such as Listeria monocytogenes using antibody fragment (100 kDa) [119], staphylococcal enterotoxin B (SEB) [120], E. coli, Yersinia pestis [120]; hepatitis C, herpes simplex virus, human cytomegalovirus, rabies virus, Vaccinia virus, Ebola virus [121]. 3.2.3. DNA biosensor Gene-based research has paved the way for simpler, lessexpensive tactics for distinguishing similar species or strains of various microbes that are infectious. Therefore various pathogens in food poisoning outbreaks caused by undistinguished strains can be identified. Various genes are in the process of being decoded and the specific sequences are being identified for detection of pathogenic strains. In this context, DNA (deoxyribonucleic acid) is known to have interesting chemical and physical properties that can be used to fabricate biosensors such as DNA-based electronic devices, biochips and microarrays. The DNA structure is ideal for electron transfer since some of the electron orbitals belonging to the stacked base pairs overlap quite well with each other along the long axis that could be used in DNA-based electronics [122]. A DNA biosensor is a diagnostic device having immobilized single stranded DNA onto a suitable matrix to detect hybridization signal (mass, electrical charge, or optical property) upon exposure to complementary DNA molecule [7,123]. Therefore, target organism can be detected by specific target sequence hybridization at the surface of a transducer [124]. Unlike enzymes or antibodies, nucleic acid recognition layers are known to be stable and can be readily synthesized or regenerated for repeated use that have high degree of specificity [128]. Although, there has been debate about the origin of conducting properties of DNA, it has been found that DNA can serve as an elegant model for one dimensional charge transport [122–125]. The variability in conductivity of DNA can be attributed to various experimental conditions, guanine content, immobilization matrices and the difficulties of attaining non-resistive contact [86,125–127]. A wide variety of new matrices can be used to immobilize DNA for application to sensors with varied specifications. Some of these matrices include graphite [129], glassy carbon electrode

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[130], conducting polymers [131–136] and glass substrate [137], Langmuir–Blodgett films [138,139], self-assembled monolayers (SAMs) [140], gold [141,142], silver [143], platinum [144], etc. Immobilization methods such as physical adsorption [145], electrochemical adsorption [83,84,146], electrochemical entrapment [236], biotin–avidin coupling [147], covalent immobilization, etc., have been used for this purpose. Recent discoveries and advances in the bio-molecular techniques have led to the generation of various new intelligent and smart materials with interesting properties that can be used to fabricate new generation miniaturized bio-molecular electronics-based devices. Nucleic acids specifically DNA have been utilized to investigate the affinity interactions using electrochemical and optical techniques Many efforts have recently been directed to the development of optical DNA sensing arrays [147]. It is possible to monitor weak or reversible DNA–DNA complementary binding interactions. Detection of DNA of Bacillus anthracis and other Bacillus species has been reported using fluorescent molecule [7,148,149]. It has been shown that alkane-thiol modified oligonucleotide can be immobilized onto gold surface to detect DNA hybridization using SPR-based detection in agrofood industry [150]. Electrochemical sensing offers several advantages such as easy immobilization of DNA (either physical or electrochemical), reduced response time, compatibility with the immobilization matrix, improved stability, sensitivity and portability, etc. A chrono-potentiommetric DNA hybridization biosensor based on 38 base DNA probe immobilized onto carbon paste electrode for the detection of pathogenic Cryptosporidium [151] and Micobacterium tuberculosis [152] has been reported. Soon after the discovery of Bacillus anthracis in the mailed letters during 2001, a DNA biosensor for the specific identification of the bacterium was developed [153]. The intercalation of electro-active components into doublestranded DNA or the applications of oligonucleotide–redoxenzyme conjugates both have been used for the amperometric transduction of DNA detection [155]. An HRPO coupled antibody against digoxygenine has been demonstrated to detect electrochemically the DNA-hybridization for toxic dinoflagellate A. ostenfeldii using 18s RNA target sequences [154]. While voltammetric DNA biosensor based on 21 base probes immobilized onto carbon paste electrode has been reported for detection of hepatitis B virus using methylene blue as the hybridization indicator [128]. Similarly, di(2,2 -bipyridine)osmium (III) has been reported as hybridization indicator to detect hepatitis B virus using cyclic voltammetry [156]. Besides this, a portable and reusable RNA biosensor, for the detection of E. coli in drinking water has been reported targeting mRNA sequences amplified using the isothermal NASBA technique [97,157,158]. The Langmuir–Blodgett (LB) film technique coupled with self-assembled monolayer containing immobilized single stranded oligo-nucleotide has been used to detect bacterial pathogens [159]. 16S rDNA probes generated by PCR amplification have been reported for the detection of bacterial pathogens like Listeria monocytogenes [160]. Microfluidic biochips have been used for the monitoring of eukaryotic and prokaryotic cell growth and DNA hybridization through

impedance measurements while cells grow at micro-scale level [237]. Piezoelectric sensors are well known for linking molecular interactions like antibody–antigen, hybridization (DNA–DNA, DNA–RNA, DNA–peptide nucleic acids (PNA)) and binding of proteins to nucleic acids. A DNA biosensor for the detection of Hepatitis B virus has been reported using quartz crystal microbalance technique. A specific probe immobilized onto gold surface with polyethyleneimine adhesion, glutaraldehyde crosslinking method and physical adsorption were used to detect the target DNA. In addition, the specific binding of antibodies or proteins to double stranded DNA was detected by micro-gravimetric quartz-crystal microbalance analyses [161,162]. The fluorescence resonance energy transfer (FRET) is characterized by the excitation of a donor fluorophore with an emission spectrum that overlaps the excitation spectrum of an acceptor fluorophore lying in close proximity [163]. FRET is limited to distances <10 nm where DNA oligo-nucleotides of 30–90 bases length suit the requirement effectively [165]. A signal is generated only when both probes are bound to a template DNA. Specific intercalating dye SYBR Green I as FRET donor with FRET acceptor affixed to one of the DNA molecules has been reported to detect DNA hybridization [164]. FRETbased sensing can be used for the detection of food pathogens in various food samples. Recent reports indicate application of DNA biosensors for toxicity detection [83,84,166–169]. Molecules such as rifampicine [170], 2-chlorophenol, 2-aminoanthracene [84,85,236], drugs screening [171], mutagens or probable human carcinogens like aromatic amines, endocrine disruptors, surfactants, pesticides, etc. [83,84] in drinking water, waste water and food beverages that alter the redox properties of DNA, etc., have been shown to interact and alter the electrochemical properties of DNA. Use of various electro-chemical indicators like daunomycene, methylene blue [128], ferrocenes, di(2,2 bipyridine)osmium(III), etc., has been reported to differentiate single stranded and double stranded DNA [61,172,173]. New bio-molecular electronics-based approaches on direct analysis of DNA (RNA) in its environment have been demonstrated to monitor the types of bacterial species present in milk before and after the refrigeration [238]. It was found that the presence of L. moncytogenes drastically increased in refrigerated milk as compared to Lactobacillus that was present in fresh milk [238]. Molecular devices with single molecule resolution are likely to be the next generation of DNA sequence biosensors for detection of food pathogens. Efforts are being made to commercialize the DNA-based products for food pathogen detection and to combat bio-terrorism. 3.2.4. PNA biosensor Peptide nucleic acid (PNA) is a synthetic nucleic acid reported in early 1990s that has achiral neutral polyamide backbone formed by repetitive units of N-(2-aminoethyl) glycine linked to N bases [174]. Unique properties of PNA have been found to have applications in research and diagnostic applications, such as human pathology, virology, mycology, bacteriology and chromosome analysis [175]. PNA molecule that

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mimics DNA is advantageous as a probe molecule, owing to superior hybridization characteristics and improved chemical and enzymatic stability relative to nucleic acids. Furthermore, its different molecular structure enables new modes of label-less detection contributing significantly towards the establishment of faster, stable and more reliable analytical processes. PNAs have been reported for various applications including hybridization biosensors and microarrays [176]. PNA hybridization biosensors fabricated using self-assembled monolayers (SAMs) of PNA molecules onto gold surfaces have been used to recognize complementary DNA [177,178]. Carbon paste electrode has been reported for the immobilization of PNA as well as DNA for hybridization detection of the target sequence using methylene blue as hybridization indicator [179]. PNA hybridization probebased FRET principle has been reported, which allows for the direct, rapid detection and quantification of activating GNAS mutations with a sensitivity of ∼2.5% of mutant alleles (corresponding to 5% of mutant cells) from different tissues [180]. PNA molecule being resistive to the nucleases attack provides an extra edge over the use of conventional or naturally existing nucleic acids. PNA sensing can be utilized to detect desired contaminating organisms within various food and water samples providing high stability and increased affinity for target sequences. Efforts are being made to lower the cost of PNA synthesis. 3.3. Microarrays Biosensors can be used for the fabrication of arrays. An array is an orderly arrangement of samples. In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots. Microarrays require specialized robotics and imaging equipments for spotting. Immobilization of biological capture agents (e.g., DNA, RNA, PNA, anti-bodies, streptavidin, proteins) on silicon oxide IOWs (integrated optical waveguides) is widely reported [233]. DNA or protein microarray chips are fabricated by high-speed robotics, generally on glass (ITO coated or conducting polymer based), silicon surfaces or nylon substrates. Probes (either protein or DNA) with known identity have been used to determine binding that allows massively parallel gene expression, gene discovery studies, genome typing, community profiling, disease screening, diagnosis and simultaneous detection of various microorganisms or toxicants (e.g., pesticides, herbicides, pollutants, etc). Use of both a target molecule and reporter molecule tagged with a fluorescent label has been utilized to detect the presence of hybridization or interaction using laser scanners [181,182]. Microarrays utilize different platforms, devices, approaches and electric fields on a microelectronic device to regulate nucleic acid transport, hybridization, and stringency such that speed, convenience and full automation can be manipulated [183,184]. The 16S rRNA gene being the conserved region is commonly used for the study of microbial population in complex systems [185,188]. DNA electronic microarray has been used to quickly and reliably detect pathogenic bacteria (Edwardsiella tarda, Photobacterium damselae, Flexibacter maritimus, Nocardia seiolae, Lactococcus garvieae, Vibrio ichthyoenteri, Streptococ-

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cus iniae, and Vibrio anguillarum) in fish [181] and nitrifying bacteria Nitrosomonas eutropha and Nitrosospira briensis [188]. Microarray technology has been applied to detect bacterial genes in environmental samples [186,187]. Single-chip multipathogen oligonulceotide microarray (FDA-1) for the simultaneous analysis of Listeria sp., Campylobacter sp., and C. perfringerns toxin genes has been reported [189]. Microarray-based comparative genomic hybridization has been done for Plasmodium falciparum for the rapid detection of genomic variation for its virulence and disease [190]. These findings demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefence application to detect microbial pathogens. Complex bacterial population has been assessed directly using rRNA detection from bacterial communities [191]. In a similar manner flow through microarrays (PamChip® ) has been reported to detect and characterize bacterial rRNA that showed sufficient sensitivity to detect directly the Staphylococcus aureus rRNA. This method can be used to detect highly expressed genes such as mecA, katA and ileS [192]. Generally, microarrays are prepared by the deposition of probes onto membranes or plastic using radioactive and fluorescent dyes for detection [193]. Micro-electromechanical systems (MEMS) or microarray technology provides transducers for both sensing and actuation in various engineering applications. E. coli and a range of bacteria have been detected utilizing MEMS and SAMs (self assembled monolayers) in solution having volume of the order of a few microliters. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction [194], Campylobacter sp. [195], genotyping for Streptococcus, Saphylococcus, Pseudomonas, Listeria, Coliforms, etc. Pathogen detection can be done using DNA microarray chip both for direct sample of nucleic acid from given sample or after amplification of conserver sequences using PCR. Genotypic application provides alternative to various techniques like pulse field gel electrophoreseis (PFGE), amplified fragment length polymorphism (AFLP), rapid amplified polymerized DNA (RAPD), REP-PCR (repetitive extragenic palindromic) and ERIC-PCR (enterobacterial repetitive intergenic consensus), etc. [196]. DNA chip technology is an enabling technology, which is now used in various fields of research in biology. The tissue microarrays (TMAs) are emerging as complete evaluation strategy for drug targets allowing simultaneous and rapid analysis of thousands of human tissues to determine frequency of target gene expression, e.g., tumors, disease progression, normal tissues, etc. [197]. Tissue microarrays have enabled simultaneous analysis, thereby combining increased statistical accuracy and high throughput. Microarrays based on proteins or antibodies have been reported to detect various microorganisms. Various methods and equipment for spotting, processing, and analyses of cDNA hybridization microarrays have recently been used to fabricate microarrays for specific detection of proteins in complex biological fluids [198]. The specificity provided via antibody is very high; some of the challenges include problems of cross-reactivity, production of monoclonal antibodies, and the cost of antibodies production, etc. There have been few reports

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for microarrays, efforts are continuously being made towards the application of microarrays for development of lab-on-chip. 3.4. Locked nucleic acid-based sensing A novel series of nucleotide analogs called Locked Nucleic Acids (LNA) have recently been reported [199]. A locked nucleic acid (LNATM ) is a modified RNA nucleotide. LNA is a bicyclic nucleic acid where a ribonucleoside is linked between the 2 -oxygen and the 4 -carbon atoms with a methylene unit. LNA can be used in any hybridization assay that requires high specificity and/or reproducibility, e.g., dual labeled probes, in situ hybridization probes, molecular beacons and PCR primers [202]. Furthermore, LNA offers the possibility to adjust Tm values of primers in multiplex assays to increase the sensitivity and specificity. General properties of LNA oligonucleotides include highly stable base pairing with DNA and RNA [200]; exceptionally high thermal stability; improved discrimination; compatibility with most enzymes and predictable melting behavior. In fact the high binding affinity of LNA oligomers allows the use of short probes in, e.g., small nucleotide polymorphism (SNP), genotyping, etc. [201]. Application of LNA has yielded good results in allele specific PCR and mRNA sample preparation. The LNA molecules can be utilized for diagnostics and drug development [234]. There exists enormous possibility of developing LNA devices that can be used to detect various food pathogens by using the specific oligo-nucleotide probes. Although, real time applications of LNA appear promising, the cost incurred for LNA synthesis is presently too high. 3.5. Nano-material-based devices for food pathogen detection Manipulation of matter at the level of single atom or small groups of atoms at nano-meter level and characterization of the properties of these materials has been treated as nanoscience/nano-technology. It has been found that clusters of small numbers of atoms or molecules (nano-materials) have properties such as strength, electrical conductivity, and optical properties significantly different from the properties of the same matter at either of the single molecule or bulk state. These unique properties can be further manipulated for the same material to develop new capabilities with potential applications across all fields of science, engineering, technology, and medicine. Titanium, zinc, gold, carbon, silver, ruthenium, etc., have been routinely used to prepare various nano-materials for a wide range of applications. Present-day applications vary from plant pigments, cutting tools, resistant coatings, pharmaceuticals, drugs, paints, cosmetics, thin film electronic devices and jewellery, etc., to biosensors, transducers, detectors, drug delivery vehicles, biomagnetic separations, wound healing, propellants, nozzles, valves and functional designer fluids, etc., that can be used to develop nanooptical devices, nano-electronics, nano-power sources, high end flexible displays, artificial organs, NEMS-based devices, faster switches, ultra sensitive sensors, etc., in very near future. New detection technologies have been developed using derivatized fluorescent nano-spheres and nano-particles. Mag-

netic nano-particles have been reported to be used in the analysis of blood, urine, and other body fluids to speed up separation and improved selectivity. Producers of optical materials and electronics substrates use silicon and gallium arsenide nanosize particles for chemo-mechanical polishing of various substrates. Functionalized nano-sized semiconductors called ‘quantum dots’ have been used as a tool for the analysis of biological systems that emit with specific colors of light upon irradiation. In fact, quantum dots of different size can be attached to different molecules in a biological reaction, allowing researchers to follow all molecules simultaneously during biological processes with only one screening tool. These can be used as a quick and easy screening tool for laborious DNA and antibody screenings [203]. These quantum dots, as a marker for DNA diagnostics, are possible component to be exploited. A QCM-based biosensor has been reported using 50 nm gold nano-particles as amplification probes for DNA detection [204]. 1-Dodecanethiol-encapsulated colloidal gold array has been used to establish DNA-based nano-electronic devices [205]. A recent report suggests that complementary DNA sequences could be sensed by immobilizing thiol-modified DNA probe onto gold nano-particle coated electrode [206,209]. A gold nano-particle coated quartz crystal microbalance-based DNA sensor has been reported for the detection of E. coli O157:H7 synthesized oligonucleotides [232]. The use of nano-particles effectively amplifies the signals and results in improved detection limit paving the way to develop quantitative sensor for pathogenic bacteria detection [232]. There exists enormous possibility of fabricating nano-biosensing devices that could detect various contaminating food pathogens with very high degree of sensitivity and specificity. Micro-fluidics coupled with microarrays, micro-motors and micro-heaters could generate low power consumption devices that could be applied to simultaneous in situ detection of various pathogenic microorganisms present in different samples. Nano-sensors for chemical detection using semiconductor nano-wires are being explored for various applications. Sensors for food pathogen detection in industrial food process control, chemical and biological hazard detection, environmental monitoring and a wide variety of scientific instruments may be the market niches in which nano-devices will become established in the next few years. This has led to the possibility of fabrication of the devices that were thought to be impossible earlier. Time is not far when these nano-devices would serve the common people needs instantly. Table 3 gives the characteristics of various bio-molecular electronics-based methods for food pathogen detection. Fig. 1 shows a schematic of conventional as well as biomolecular electronics-based techniques. Compared to conventional techniques, it can be seen that the bio-molecular electronics techniques can be directly used to detect food pathogens without initial enrichment or PCR amplification in desired test specimens. Figs. 2 and 3 show the response times and detection limits observed for various contemporary and bio-molecular electronics-based techniques, respectively. It can be noted that there is a considerable decrease in both the response time and

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Fig. 1. Schematic of various conventional and bio-molecular electronics-based techniques used to detect food borne pathogens. A bold arrows show the conventional methods while dotted arrows show the bio-molecular electronics based methods.

the detection limit in the bio-molecular electronics-based techniques. 4. Commercialization of bio-molecular electronics-based devices for food pathogen detection Rapid technological advances in the field of bio-molecular electronics allow various materials to be manipulated at ‘nano’ level providing tremendous opportunities to develop sensors that can cater to a wide range of applications such as home diagnostics, medical, bio-defense and food pathogen detection. In 1999, the food industry performed 144 million microbial tests for biological agents. Besides this, 24 million pathogen-specific tests were performed by the food industry representing a market of 122 million US$ [11]. Trend is moving beyond detection of biological threats such as anthrax and there is increase in the number of non-biological applications. Emerging technolo-

gies that can sense toxic agents and avert related food borne illnesses in a timely manner, especially during wars hold huge growth potential. According to experts, the army can network thousands of sensors to detect these agents. With the move toward miniaturization and developments in the lab-on-a-chip technologies, researchers can offer refined products to meet the expanding market requirements. Definitely, these bio-molecular electronics-based techniques are set to gain a competitive edge. The global market for bio-molecular electronic devices including biosensors is projected to grow from $6.1 billion in 2004 to $8.2 billion in 2009, at an average annual growth rate of about 6.3% [207]. Literature trend portrays that majority of researchers have been working on immunosensors [240,242] the trend is now drifting towards nucleic acid based sensing, multianalyte sensing and nano-materials based sensing. Patents pertaining to DNA hybridization detection have started appearing that require initial amplification using PCR for enteric bacteria

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Fig. 2. Bar chart showing response time (minutes) for various food pathogens detection methods with dark color indicating various response time reported in literature [23,30,34,42–45,92,94,97,102–104,110,151,152,224, 227–229,232,235]. PCR = polymerase chain reaction; NASABA = nucleic acid sequence-based amplification; FTIR = Fourier transform infrared spectroscopy; NM = nano-material based.

present in food [239] as well as the optical DNA hybridization sensor that detect color changes occurring in some biopolymeric materials [241]. It is known that, biomedical and life sciences applications dominate the market, accounting for 99%, with environmental monitoring and remediation applications a distant second. Continued success in commercializing nanomaterials based bio-electronic technologies largely has been proposed to depend on the extent to which major companies have focused their attentions on bio-electronic technologies. In the wake of the 9/11 terrorist attacks new markets for bioelectronics devices are being developed as heightened security concerns to detect dangerous chemical and biological agents. The first protein-based optical memory media has begun appearing on the market. Innovative Biosensors Inc., have launched

immunosensor for detection of E. coli [235]. Although, the applications of nano-technology to the life sciences are relatively new, the academic projects undertaken at present far outnumber the marketed products in the field. Nano-materials such as carbon buckyballs, dendrimers, and metal nano-particles have begun to reach the market for pharmaceutical and diagnostic use both in vitro and in vivo. Currently available products fall into three major segments of nano-biotechnology: drug delivery, imaging agents, and biosensors. The market for nano-materials based bio-molecular electronics devices is expected to grow rapidly to reach over $3B in 2008, reflecting growth at an annual rate of 28% worldwide. The current estimated worldwide market breakdown is: USA at 65%, Europe at 20%, Japan at 10%, and the rest of the world at 5% [208]. 5. Conclusions Attempts have been made to bring out the characteristics such as detection limit and response time, etc., of the biomolecular electronics based techniques and contemporary methods for food pathogen detection. It has been shown that the biomolecular electronics-based devices provide interesting option for the detection of various organisms that may cause fatal infections within hours (e.g., Neisseria meningitidis) [50]. Among the various bio-molecular electronic techniques, nucleic acids and antibody-based sensors appear promising for the detection of a wide variety of food borne microorganisms, diseases, pollutants, dyes, toxicants. However, the commercialization of the bio-molecular electronic devices for food pathogen detection is closely linked with the problems associated with the interferents and the stability of the bio-molecules. Acknowledgements We are grateful to Dr. Vikram Kumar, Director, NPL, for his interest in this work. KA is grateful to Council of Scientific and Industrial Research (CSIR), India for award of Senior Research Fellowship (SRF). We thank Dr. S.P. Singh for interesting discussions during the preparation of manuscript. Financial support received under the DST sponsored project DST/TSG/ME/2002/19 and the CSIR sponsored project CMM011 are gratefully acknowledged. References

Fig. 3. Bar chart showing detection limit (cell/ml) for various food pathogen detection methods. Dark color shows the range of detection limits reported in literature [16–19,22,30,34,43,44,91,94,96–98,102–104,106,107,110,149, 227–229,232,235]. MB = micro-biological methods; BC = biochemical methods; PCR = polymerase chain reaction; NASABA = nucleic acid sequence-based amplification; NM = nano-material based.

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