diseases caused by foodborne pathogenic bacteria

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1.1.1 Foodborne Pathogenic diseases

Foodborne illnesses are defined as diseases, which are caused by agents that entered the body from the ingestion of food by World Health Organization (WHO). They are usually infectious or toxic in nature: the illnesses caused by presence of pathogenic bacteria or other species of microbes are infectious while the illnesses caused by the ingestion of toxins contained within the food are toxic. In the recent decade, the foodborne diseases have attracted major concern of public and increasingly threaten people's health. There are 6million to 81 million persons were affected by foodborne diseases which resulted in 9,000 deaths in the United States each year (Mead et al. 2000). In Hong Kong, though much manpower have been spent on investing all the foodborne diseases outbreak and promoting food safety, the number of people infected by foodborne diseases and the incidence of foodborne disease outbreaks are continuously increasing from 1996-2005 (Fig 1.1.1) (Chan and Chan 2008).

Fig 1.1.1 The number of foodborne diseases outbreaks in the Hong Kong Special Administrative Region from 1996 to 2005 (Department of Health 2006)

1.1.2 Foodborne Pathogenic Bacteria

Among the pathogenic microorganisms, including viruses, pathogenic bacteria, parasites, fungi, toxins and prions, which cause foodborne diseases, pathogenic bacteria are the most foodborne pathogens, accounting for 91% of all outbreaks of foodborne diseases in the United States (Yang and Bashir 2008). Table 1.1.1 shows the important foodborne pathogenic bacteria with the caused disease, secreted toxins, infection sources and dose. The annual cost of human illness caused by six major pathogenic bacteria, including Salmonella, Campylobacter jejuni, E.coli O157:H7, Listeria monocytogenes, Staphylococcus aureus and Clostridium perfringens, is estimated to account for &9.3-$12.9 billion in USA, among which 30%-50% are attributed to foodborne diseases (Buzby et al. 1996), and the six major pathogenic bacteria are characterized in Table 1.1.2 with their estimated annual cases, hospitalizations and deaths (Leonard et al. 2003).

Table 1.1.1 Important foodborne pathogenic bacteria

Table 1.1.2 A summary of estimated foodborne illnesses, hospitalisations and deaths caused six major pathogenic bacteria in the US annually as calculated by the USDA's economic research service.

1.1.3 Escherichia (E.coli) O157:H7 and Staphylococcus aureus

E. coli was first discovered by Escherich in 1885, it is a Gram negative rod-shaped bacterium that is a typical inhabitant of the human intestinal tract and is often motile by means of flagella; it is unicellular with about 1 micrometer in width and 2-4 micrometers in length. Most strains of E.coli is harmless, however, some strains such as serotype O157:H7 which is an enterohemorrhagic strain of the bacterium E.coli, can cause serious foodborne illnesses or deaths in the elderly, the very young or the immunocompromised. The "O" in the name means the somatic cell wall antigen number and the "H" means the flagella antigen, therefore, E.coli O157:H7 expresses the 157th somatic antigen identified and the 7th flagella antigen. It was first recognized as a pathogen illness 1982 two outbreaks of bloody diarrhea in Oregon and Michigan of United States (Riley et al. 1983) (Wells et al. 1983), In 1983, Karmali and his team found an association between E.coli strains (including O157:H7) which produced a cytotoxin lethal (Shiga toxin) and enteropathic haemolytic uraemic syndrome (HUS) (Karch et al. 2005) which is characterized by thrombocytopenia, acute renal injury and microangiopathic haemolytic anaemia and soon after this, the strain of O157:H7 was recognized the first of several strains referred to as enterohaemorrhagic E.coli (EHEC) (Levine et al. 1987), which is transmitted to bodies from contaminated food such as raw milk and undercooked ground meat.

Fig E.coli O157:H7 at 10000x (POPSIC)

E.coli O157:H7 first attracted people's attention after the outbreak in 1993 Seattle-Tacoma, when more than 700 persons infected from hamburger-associated food and 4 of them died (Obrien et al. 1993). After that, Outbreaks of E.coli O157:H7 infections were reported which associated with Roast Beef (Rodrigue et al. 1995), unpasteurized apple juice ([Anon] 1996) (Hilborn et al. 2000), Jerky made from deer meat (Keene et al. 1997), Mesclun Lettuce (Hilborn et al. 1999), white radish sprouts (Michino et al. 1999) and Genoa salami (Williams et al. 2000). In recent decade, the number of outbreaks caused by E.coli O157:H7 increased dramatically and the source extended to water, In August 1999, several children were ill with E.coli O157:H7 infection reported swimming in lake (Bruce et al. 2003) (Bopp et al. 2003). Large outbreaks have been reported from Europe (Dundas et al. 2001) (Sartz et al. 2008), Japan (Ahmed et al. 2005), Canada (Ali 2004) (MacDonald et al. 2004), United States (Kotewicz et al. 2008) (Goode et al. 2009). In Hong Kong, there were still many reports about E.coli O157:H7 isolated from cattle and pigs in an abattoir (Leung et al. 2001).

Staphylococcus aureus (S. aureus) is a Gram-positive and non-motile bacterium which appears as grape-like clusters (staphylo means grape in greek) with a large, round and golden-yellow colonies. S. aureus was discovered by the surgeon Alexander Ogston in Scotland in 1880 in pus from surgical abscesses. It is a spherical cell with 1 micrometer in diameter and always hemolytic on blood agar; S. aureus can lead to different types of suppurative infections and toxinoses in human, such as boils and furuncule. Furthermore, with several virulent characteristics, staphylococcus aureus caused more serious infectious diseases such as endocarditis, pneumonia and bacteremia. The S. aureus infections are usually blocked by normal host defenses at the portal of entry, however, if the host defenses are destroyed even by a minute needle-stick or a surgical wound, the bodies could be easily colonized by S. aureus, which makes it hard to control the infections. S. aureus could also colonize in human through respiratory tract and cause infections such as Staphylococcal pneumonia. Therefore, S. aureus should be considered a serious and important pathogen. Methicillin-resistant Staphylococcus aureus (MRSA) are strains of Staphylococcus aureus that could resist to beta-lactam antibiotics, including the penicillins and cephalosporins. MRSA always occurs in hospital among patients with invasive apparatus, trauma and weak immune systems. It is commonly causes serious infections such as blood poisoning (septicemia) and heart valve infection.

Fig Staphylococcus aureus at 9500x (Center of Disease Control, CDC stock photo)

It was reported in the early 21st century that 14 patients were involved in the MRSA infection in the surgical departments of the Atrium Medical Center, Netherlands. Tow months later, another MRSA outbreak happened involving 7 patients in the nursing home and five months later, one patient developed an MRSA infected after an abdominal surgery (Wagenvoort et al. 2000). Subsequent outbreaks were reported in southwestern Alaska caused by MRSA skin infections (Baggett et al. 2001), San Diego, California, (Campbell et al. 2004), France (Guerin et al. 2000), Australia (O'Brien et al. 2004), Norway (Larssen et al. 2005), Brazil (d'Azevedo et al. 2008) and in Singapore (Chan et al. 2009).

MRSA is a epidemic and important pathogen in Hong Kong and it was first reported in Hong Kong in 2004 (Ho et al. 2004). Hong Kong is one of the regions which have the highest prevalence rates of MRSA among the whole Asia Pacific Region from previous studies (Bell et al. 2002) (Ip et al. 2004). The Hong Kong Government soon established monitoring group to surveillance MRSA in laboratory of Hong Kong with the help of Center of Health Protection (CHP) and the Center of Infection of University of Hong Kong (Ho et al. 2007). All the hospital and university microbiologist of Hong Kong were encouraged to collect MRSA infection cases for this monitoring group since 2005 and the volunteers from all industries also reported cases to CHP. The Hong Kong government also began a program which could collect wound swabs from patients with purulent skin infection for MRSA culture in 2006, MRSA infection was regarded as a statutory notifiable infection to help monitoring group for surveillance effectively. During the first half year of 2007, there were 70 persons got MRSA infection in Hong Kong and the cases were evenly distributed geographically with 30% in Kowloon, 26% in New Territories West, 19% in New Territories East and 26% in Hong Kong Island (Tsang and Tsui 2007).

Staphylococcal enterotoxins (SEs) can be generated in some strains of S. aureus, which makes S. aureus the major pathogen for food poisoning. S. aureus could survive in a large range of temperature from 7℃ to 48℃ (Schmitt et al. 1990) and large sodium chloride concentrations (up to 15%), which makes S. aureus difficult to control in food. The symptoms of staphylococcal food poisoning are nausea, abdominal cramps, vomiting and sometimes followed by diarrhea. Outbreaks of community-acquired foodborne illness caused by methicillin-resistant staphylococcus aureus were also reported in 2002, in which 3 children were confirmed to infected by MRSA after eating shredded pork and coleslaw from a convenience-market delicatessen (Jones et al. 2001). Staphylococcus aureus food poisoning outbreaks also associated with egg yolk (Miwa et al. 2001), spaghetti and meat sauce (Mouallem et al. 2003), and a snack made up of potato balls fried in vegetable oil (Nema et al. 2007).

1.2 Traditional Methods for Pathogenic Bacteria Detection

Pathogenic bacteria detection is one of the most important for food safety and public health. To avoid diseases caused by pathogenic bacteria, the process of detection and identification is the first control point. Therefore, it is significantly necessary to control these bacteria in food and water supply by effective detection and inspection approaches. Traditional methods for bacteria detection mainly relies on microbiological and biochemical techniques. For example: the methods of culture and colony counting, fluorescent-antibody (FA) technique and electron microscopy are based on counting bacteria cells, the method of immunology involves microbiological reaction between antibody and antigen, and the method of polymerase chain reaction (PCR) is based DNA analysis (Velusamy et al. 2010).

1.2.1 Culture and Colony Counting Methods

The culture and colony counting methods is the oldest and standard method for bacteria detection. It includes the procedures of microbiological culturing, isolation of pathogen, enrichment and plating, and then pathogenic bacteria can be detected by measuring physicochemical changes caused by their metabolic activities or growth (Swaminathan and Feng 1994). Although reliable, the procedure of this conventional method is labor intensive and excessively time-consuming, taking up to several days to yield confirmed results (de Boer and Beumer 1999). Therefore, it is not suitable for making timely assessments on food quality.

1.2.2 Fluorescent-Antibody Technique

The fluorescent-antibody (FA) technique could be applied to detect specific bacteria in situ. In general, antibody conjugated with a fluorochrome binds with specific bacteria or unlabeled antibody binds with specific bacteria, then the antibody-antigen complex is labeled with a fluorescent antibody. If the bacteria cells present, the combined specific antibodies would cause them to fluoresce and the number of fluorescing cells is counted by an epifluorescence microscope (Hobson et al. 1996). The most widely used fluorochrome is fluorescein, such as fluorescein isothiocyanate (FITC). The fluorescent-antibody technique is a simple and direct method for bacteria detection; however, most fluorochromes are prone to photobleaching which makes the detection process not stable.

1.2.3 Electron Microscopy Methods

Some traditional methods used for pathogenic bacteria identification focused on the morphology feature changes by bacteria metabolism with the help of microscope techniques. Bacterial cells were counted and sized by scanning electron microscope (SEM) on membrane filters (Krambeck et al. 1981) and analyzed to measure the cell volume and dry weight by transmission electron microscope (TEM) (Borsheim et al. 1990), (Loferer-Krossbacher et al. 1998).

Bacteria have also been detected by scanning probe microscopy (SPM). Howell et al. developed patterned antibody microarrays to study their ability for binding targeted bacteria. Pathogenic bacteria such as E.coli O157:H7 and Renibacterium salmoninarum were detected by the microarrays with the help of high-resolution SPM imaging (Howell et al. 2003). The antibody microarrays were fabricated by the method of microcontact printing (μCP), the high specificity of bacteria binding to their specific antibody was observed by SPM compared with the low-binding selectivity to non-specific antibody. It demonstrated that the method of microarray coupled with high-resolution scanning probe is a sensitive and specific way for bacteria detection.

Huff et al. utilized surface changes of the high-resolution topographical imaging provided by AFM to detect and characterize the viral particles and other pathogens (Huff et al. 2004). The AFM is capable of 2 nanometers lateral resolution and 1 nm vertical resolution which allow it monitor the minute changes in topography. This pathogen detection system coupled with AFM could provide detail 3-dimensional surface information, real-time data acquisition and require no signal amplification.

These microscope methods could obtain the morphology information in a direct, label-free manner and provide real-time data acquisition. Compared with fluorescent-based techniques, these label-free readout methods have the advantages of inexpensive and easy-operation (without multiple washing steps). Moreover, these methods could prevent the process of labeling which may change the morphological information of antibodies or other proteins and largely affect detection results. Meanwhile, problems such as photo bleaching and label stability could also be avoided (Huff et al. 2004). However, these microscopic techniques for bacteria detection require the high-cost instrumentations and always need skilled operators.

1.2.4 Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a nucleic acid amplification technique which is widely used for bacteria detection by amplifying a few quantities of DNA including the targeted bacteria's genetic material. It is based on the thermal cycling which consists of cycles of denaturation by heating, polymerization and extension by cooling (Lazcka et al. 2007).

Ke et al. described a conversional PCR and Real-time PCR assays for detection Group B Streptococci. The conversional PCR assay could achieve sensitive detection with a high specificity and the real-time PCR was comparable with conversional PCR in sensitivity and specificity, in addition, it achieved the rapid thermal cycling for amplification time and real-time fluorescence monitoring (Ke et al. 2000).

Multiplex PCR was used to detect different types of bacteria simultaneously by using multiple sets of primers and probe that were specific for bacteria. Hu et al. described a simultaneous identification of serotype O157:H7 of E.coli and its virulence factors in a single reaction by multiplex PCR assay (Hu et al. 1999). Salmonella spp. and Listeria monocytogenes were also been detected in food samples simultaneously following the procedures of culture enrichment and multiplex real-time PCR. Two designed sets of primers specific to L. monocytogenes and Salmonella spp. were used to compose the multiplex assay. Primers used for L. monocytogenes detection were complementary to a region of the hly gene while the primers to detect Salmonella were complementary to a region of the bipA gene. This multiplex real-time PCR achieved sensitive and specific detection of two kinds of bacteria simultaneously and shortened the assay time from 5-7 days to less than 2 days (Jofre et al. 2005).

The approach of PCR has a high sensitivity with a good specificity; however, it is largely restricted by assay time. The procedures of PCR need require enrichment of foods before amplification in order to detect bacteria which generally present with low concentration in food samples. In addition, the detection of bacteria with PCR is expensive and complex which requires skilled operators.

Since the traditional methods mentioned above for bacteria detection are always time-consuming and labor-intensive, the immunological detection has been successfully employed with the advantage of less assay time. The immunology-based methods are widely used to detect different kinds of foodborne pathogenic bacteria, such as E.coli O157:H7 (Gehring et al. 2004), Salmonella typhimurium, Listeria monocytogenes (Chen and Durst 2006) (Magliulo et al. 2007) and staphylococcal enterotoxin (Schlosser et al. 2007). The immunology-based methods include enzyme linked immunosorbent assay (ELISA) (Johnson et al. 1995), enzyme-linked fluorescent assay (ELFA), enzyme-linked immunomagnetic chemiluminescence (ELIMCL) (Gehring et al. 2004), immunomagnetic separation (Pyle et al. 1999) and so on.

Compared with traditional PCR and culture and colony counting method, the immunology-based methods are rapid; however, the sensitivity is low and could not detect in real-time. Therefore, there is an urgent need for a technique that could rapidly, simply, reliably detect pathogenic bacteria in real-time with high sensitivity and specificity. In addition, in view of future market, this technique should not be expensive.

1.3 Biosensors for Pathogenic Bacteria Detection

Biosensor technology is drawing a lot of researchers' interests for its advantages of easy-operation, time-saving, portable, with low cost and the same reliable results. It has overcome the limitations of traditional technology for bacteria detection. Biosensor is an analytical device which incorporates a biological component and a physicochemical transducer or transducing micro-system. The biological component could be microorganism, enzyme, cell, antibody, DNA or a biomimic, while the transducers may be optical, piezoelectric or electrochemical (Lazcka et al. 2007). The biosensor could be classified based on the employed transducer which plays a significant role in procedure of bacteria detection. The transduction methods such as optical, piezoelectric and electrochemical are the most common methods used in today's research for bacteria detection (Velusamy et al. 2010).

1.3.1 Optical Biosensor

Optical biosensor is a rapid, sensitive and direct method in detection of bacteria. It detects the changes in optical properties of reagents, such as light adsorption, reflection, refraction, dispersion, chemiluminescence, fluorescence and light energy. Due to the high sensitivity, surface plasmon resonance (SPR) becomes the most popular technique in all optical biosensor techniques to detect bacteria. For instance, Subramanian et al. used SPR biosensor to detect E.coli O157:H7 with high sensitivity and specificity. With help of polyethylene glycol terminated alkanethiol mixed self-assemble monolayer (SAM), antibodies against E.coli O157:H7 were immobilized on a sensor chip and direct and sandwich assays were carried out to detect E.coli O157:H7. The surface of biosensor was monitored during procedures of detection by optical microscope. The detection limit was as low as 103 CFU/ml of E.coli O157:H7, with high specificity against Salmonella. Meanwhile, the sensitivity was enhanced by 1000 times by using sandwich assay when compared with direct assay (Subramanian et al. 2006).

Waswa et al. used the SpreetaTM, SPR-based biosensor to detect E.coli O157:H7 in different food samples with specific antibody. Milk, apple juice and ground beef patties spiked with various concentration E.coli O157:H7 were injected on to sensor surface, and the light from an LED was reflect off a gold surface, the minimum measurable changes in refractive index (RI) caused by the antibody-antigen reaction was recorded as the detection limit. This assay demonstrated rapid and real-time detection with the sensitivity as high as 102-103 CFU/ml (Waswa et al. 2007).

1.3.2 Piezoelectric Biosensors

Piezoelectric biosensors which depend on the use of piezoelectric crystals are extremely appropriate for sensitive bacteria detection. Crystals such as quartz is made to oscillate at a specific frequency under the influence of an electric field. This frequency depends on the applied electrical frequency, therefore, when bacterial cells bond to the surface of crystal due to the antibody-antigen reaction, the thickness of crystal changes, resulting in the frequency change of oscillation which can be detected electrically (Velusamy et al. 2010).

The piezoelectric biosensors were widely applied for rapid detection of pathogenic bacteria. Su et al. developed a piezoelectric immunosensor to monitor E.coli O157:H7 in a short assay time based on the SAM modified surface of a quartz crystal's Au electrode. The biosensor's resonant frequency was decreased by the binding assay of antibody-bacteria during the detection procedures and the frequency shift was closely related to the concentration of E.coli O157:H7. By using the method, the bacteria concentration ranging from 103-108 could be easily detected in 30-50 minutes (Su and Li 2004).

Quartz crystal microbalance (QCM) is the main type of piezoelectric biosensor, due to the simplicity and cost effectiveness, it has been greatly applied for detection DNA immobilization and hybridization (Caruso et al. 1997) and pathogenic bacteria (Mo et al. 2002). Mao et al. demonstrated a QCM-based DNA sensor for E.coli O157:H7 detection based on the nanoparticle amplification. The sensor surface of QCM was modified by a thiolated single-strand DNA (ssDNA) which was specific to eaeA gene of E.coli O157:H7. The DNA hybridization between the ssDNA and the complementary DNA from E.coli O157:H7 caused the frequency shift. Nanoparticles coated with streptavidin were used for frequency shift amplification. As a result, this QCM-based DNA sensor gave a detection limit of E.coli O157:H7 as low as 2.67Ã-102 CFU/ml (Mao et al. 2006).

1.3.3 Electrochemical Biosensors

Electrochemical biosensors are extremely important approaches for pathogenic bacteria identification and quantification. The main principle of electrochemical biosensors is that biochemical reactions produce ions and electrons or block the flow of ions and electrons, resulting in some measurable changes of the electrical property, which are detected by electrochemical instruments. Compared to optical and piezoelectric biosensor, electrochemical biosensor is portable which is amenable to miniaturization and can be used to detect bacteria in situ; in addition, it allows the analyst to work in turbid media. Electrochemical biosensors can be classified by the measured transduction parameters, such as amperometric biosensor and potentiometric biosensor (Lazcka et al. 2007).

Amperometric Biosensor

The amperometric biosensor is a sensitive method for bacteria detection compared with potentiometric biosensor. apmerometric biosensors function by the production of current by analyte when the potential which is served as a driving force of the electron transfer reaction, is set at a value. Therefore, the amperometric signal related with bacterial concentration is detected.

A flow through immunoassay system based on amperometric technique coupled with high-dispersed carbon particles was developed to detect bacteria such as E.coli Listeria monocytogenes and Campylobacter jejuni (Chemburu et al. 2005). In this case, pathogenic bacteria cells were captured by specific antibodies immobilized with carbon particles served as solid phase, and then labeled by horseradish peroxidase (HRP) conjugated secondary antibodies to form a sandwich structure. When the peroxidase flowed through the biosensor, the amperometric signal was produced. This method provided bacteria detection limit as low as 50 cells/ml.

Amperometric technique combined with DNA hybridization and enzyme amplification is also used for E.coli detection. A micro-electromechanical system (MEMS) based amperometric detector for E.coli was designed. With the help of DNA hybridization and enzyme amplification, this assay was able to detect 1000 cells without PCR. In addition, a small sample volume on order of a few micro liters was another advantage of this system (Gau et al. 2001).

Potentiometric Biosensor

A potentiometric biosensor is based on ion selective electrodes and ion selective field effect transistors (FET). It consists of ion selective membrane bioactive materials such as enzyme. The species caused by enzyme-catalyst reaction are detected by the ion selective electrodes. The light addressable potentiometric sensor (LAPS) based on FET is reported to successfully detect pathogenic bacteria (Gehring et al. 1998) (Ercole et al. 2003). The LAPS consists of an electrolyte-insulator-semiconductor structure, the potential changes caused by biochemical reaction are detected by the difference in charge distribution. A LAPS measures the alternating photocurrent produced by light source, so that potential changes are converted to voltage (Leonard et al. 2003).

1.4 Electrochemical Impedance Spectroscopy (EIS) for Bacteria Detection

In the last few years, researchers have done some improvements to this method, which made the impedance technique more valuable. These improvements involve different electrodes and equivalent circuit analysis. The electrode-based impedance method is based on the electrical properties change of these electrodes when the bacteria cells adhered to the electrodes. Compared with traditional methods, this technique do has shortened the procedure time to some extent; however, the sensitivity has been limited, because when the size of metal electrodes decreases, the electrode impedance caused by the electrode polarization is increased at the low frequency range. If the impedance related with bacteria is much smaller than the high electrode impedance, it is hard to extract the impedance of bacteria from total measured spectrum. Moreover, it only can be used for detect one type of bacteria, and the specificity is poor as well. So it is interesting and urgent to develop a method that could detect multiple bacteria simultaneously with a good sensitivity.

1.5 Nanoporous Anodic Aluminum Oxide (AAO) Membrane

Nanoporous Anodic Aluminum Oxide (AAO) membranes with their fairly well-defined nanopores provide a convenient substrate for biosensor applications. The properties of small pore diameter between 20nm to 200nm combine with the high density of nanopores (1Ã-109/cm2) result in a high surface area which is appropriate and easily for surface modification, the critical procedure in biosensing.

1.5.1 Nanoporous AAO membrane based Cell sensor

Nanoporous AAO membrane in various forms has been widely applied as a scaffold for tissue engineering research due to its biocompatibility with bone cells. It is also reported to be biocompatible with neuronal cells (Wolfrum et al. 2006). In addition, the AAO membrane is served as an appropriate template for study the cell behaviors due to the surface morphology, for example: cell proliferation and cell adhesion.

Swan et al. fabricated nanoporous alumina with uniform pore size and distribution based on the two-step anodization process. Osteoblast adhesion and morphology on different diameters of nanopores (30-80 nm) were studied. Images captured by scanning electron microscopy showed the cell extending into the nanopores (Swan et al. 2005).

The nanoporous alumina membrane is also used as a substrate for cancer cell studies. Yu et al. cultured human esophageal squamous epithelial KYSE30 cancer cells on the nanoporous AAO membrane with PEG hydrogel microwells. The impedance spectroscopy measurement was used to study of anti-cancer drug effect of retinoic acid (RA) on KYSE 30 cancer cells. The impedance magnitude was reduced with time from the value with cell layer after adding RA, and returned to the initial base line after 12 hours (Yu et al. 2009). This electrochemical based system was successfully testified morphology-sensitive for cell growth and drug induced change by impedance spectroscopy.

1.5.2 Nanoporous AAO membrane based DNA sensor

Recent science report has proven that the nanoporous AAO membrane has beaten other materials in the application of DNA analysis. Due to the advantages of low auto-fluorescence, high porosity which allows for high flow rates through the membrane, good transparency as well as the small pore diameter which is comparable to the DNA length, the nanoporous AAO membrane is widely used in for the application of DNA detection and sensing.

A capacitance sensor based on a nanoporous AAO structure was fabricated for DNA hybridization sensing (Kang et al. 2010). The AAO membrane served as a template and the gold nanowires which was made by depositing gold film on surface of membrane were used as the working and counter electrode respectively. The capacitance of the sensor decreased greatly when the complementary DNA molecules were provided. The selectivity was proved good by integrating three sensors into the capacitance sensor array. One of these sensors was unmodified and the other two were modified with complementary DNA and non-complementary DNA respectively.

Kim et al. described a microfluidic system made of polydimethylsiloxane (PDMS) with AAO embedded with it for DNA extraction from blood sample. The permeation rate was used to measure the extraction efficiency. A low permeation rate indicates that the DNA was captured on the membrane and not allowed to pass through the membrane. The eluted DNA from blood sample was further amplified by PCR (Kim and Gale 2008).

In addition, DNA molecules were also detected by method of fluorescence (Kim et al. 2006), surface charge effect (Wang and Smirnov 2009), electrochemical (Vlassiouk et al. 2005), Optical and IR adsorption (Vlassiouk et al. 2004) because of the low auto-fluorescence, high negative charge, small pore diameter and optical transparency of the nanoporous AAO membrane.

1.5.3 Nanoporous AAO membrane based bacteria sensor