1.1.1 Foodborne Pathogenic diseases
Foodborne unwellnesss are defined as diseases, which are caused by agents that entered the organic structure from the consumption of nutrient by World Health Organization ( WHO ) . They are normally infective or toxic in nature: the unwellnesss caused by presence of infective bacteriums or other species of bugs are infective while the unwellnesss caused by the consumption of toxins contained within the nutrient are toxic. In the recent decennary, the foodborne diseases have attracted major concern of public and progressively threaten people ‘s wellness. There are 6million to 81 million individuals were affected by foodborne diseases which resulted in 9,000 deceases in the United States each twelvemonth ( Mead et al. 2000 ) . In Hong Kong, though much manpower have been spent on puting all the foodborne diseases outbreak and advancing nutrient safety, the figure of people infected by foodborne diseases and the incidence of foodborne disease eruptions are continuously increasing from 1996-2005 ( Fig 1.1.1 ) ( Chan and Chan 2008 ) .
Fig 1.1.1 The figure 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 infective micro-organisms, including viruses, infective bacteriums, parasites, Fungis, toxins and prions, which cause foodborne diseases, infective bacteriums are the most foodborne pathogens, accounting for 91 % of all eruptions of foodborne diseases in the United States ( Yang and Bashir 2008 ) . Table 1.1.1 shows the of import foodborne infective bacteriums with the caused disease, secreted toxins, infection beginnings and dosage. The one-year cost of human unwellness caused by six major infective bacteriums, including Salmonella, Campylobacter jejuni, E.coli O157: H7, Listeria monocytogenes, Staphylococcus aureus and Clostridium perfringens, is estimated to account for & A ; 9.3- $ 12.9 billion in USA, among which 30 % -50 % are attributed to foodborne diseases ( Buzby et al. 1996 ) , and the six major infective bacteriums are characterized in Table 1.1.2 with their estimated one-year instances, hospitalizations and deceases ( Leonard et al. 2003 ) .
Table 1.1.1 Important foodborne pathogenic bacteriums
Table 1.1.2 A sum-up of estimated foodborne unwellnesss, hospitalizations and deceases caused six major infective bacteriums in the US yearly as calculated by the USDA ‘s economic research service.
1.1.3 Escherichia ( E.coli ) O157: H7 and Staphylococcus aureus
E. coli was foremost discovered by Escherich in 1885, it is a Gram negative bacillar bacteria that is a typical dweller of the human enteric piece of land and is frequently motile by agencies of scourge ; it is unicellular with about 1 micron in breadth and 2-4 microns in length. Most strains of E.coli is harmless, nevertheless, some strains such as serotype O157: H7 which is an enterohemorrhagic strain of the bacteria E.coli, can do serious foodborne unwellnesss or deceases in the aged, the really immature or the immunocompromised. The “ O ” in the name means the bodily cell wall antigen figure and the “ H ” means the scourge antigen, hence, E.coli O157: H7 expresses the 157th bodily antigen identified and the 7th scourge antigen. It was foremost recognized as a pathogen unwellness 1982 two eruptions of bloody diarrhoea in Oregon and Michigan of United States ( Riley et al. 1983 ) ( Wells et al. 1983 ) , In 1983, Karmali and his squad found an association between E.coli strains ( including O157: H7 ) which produced a cytotoxin lethal ( Shiga toxin ) and enteropathic hemolytic azotemic syndrome ( HUS ) ( Karch et al. 2005 ) which is characterized by thrombopenia, acute nephritic hurt and microangiopathic hemolytic anemia and shortly 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 organic structures from contaminated nutrient such as natural milk and undercooked land meat.
Fig 126.96.36.199. E.coli O157: H7 at 10000x ( POPSIC )
E.coli O157: H7 foremost attracted people ‘s attending after the eruption in 1993 Seattle-Tacoma, when more than 700 individuals infected from hamburger-associated nutrient 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 ) , unpasteurised apple juice ( [ Anon ] 1996 ) ( Hilborn et al. 2000 ) , Jerky made from cervid 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 decennary, the figure of eruptions caused by E.coli O157: H7 increased dramatically and the beginning extended to H2O, In August 1999, several kids were sick with E.coli O157: H7 infection reported swimming in lake ( Bruce et al. 2003 ) ( Bopp et al. 2003 ) . Large eruptions 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 studies about E.coli O157: H7 isolated from cowss and hogs in an butchery ( Leung et al. 2001 ) .
Staphylococcus aureus ( S. aureus ) is a Gram-positive and non-motile bacteria which appears as grape-like bunchs ( staphylo means grape in Greek ) with a big, unit of ammunition and golden-yellow settlements. S. aureus was discovered by the sawbones Alexander Ogston in Scotland in 1880 in Pus from surgical abscesses. It is a spherical cell with 1 micron in diameter and ever haemolytic on blood agar ; S. aureus can take to different types of suppurative infections and toxinoses in human, such as furuncles and furuncule. Furthermore, with several virulent features, staphylococci aureus caused more serious infective diseases such as endocarditis, pneumonia and bacteriemia. The S. aureus infections are normally blocked by normal host defences at the portal of entry, nevertheless, if the host defences are destroyed even by a infinitesimal needle-stick or a surgical lesion, the organic structures could be easy colonized by S. aureus, which makes it difficult to command the infections. S. aureus could besides colonise in human through respiratory piece of land and cause infections such as Staphylococcal pneumonia. Therefore, S. aureus should be considered a serious and of import pathogen. Methicillin-resistant Staphylococcus aureus ( MRSA ) are strains of Staphylococcus aureus that could defy to beta-lactam antibiotics, including the penicillins and Mefoxins. MRSA ever occurs in infirmary among patients with invasive setup, injury and weak immune systems. It is commonly causes serious infections such as blood toxic condition ( blood poisoning ) and bosom valve infection.
Fig 188.8.131.52 Staphylococcus aureus at 9500x ( Center of Disease Control, CDC stock exposure )
It was reported in the early twenty-first century that 14 patients were involved in the MRSA infection in the surgical sections of the Atrium Medical Center, Netherlands. Tow months subsequently, another MRSA eruption happened affecting 7 patients in the nursing place and five months subsequently, one patient developed an MRSA infected after an abdominal surgery ( Wagenvoort et al. 2000 ) . Subsequent eruption were reported in southwesterly Alaska caused by MRSA tegument 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 of import pathogen in Hong Kong and it was foremost reported in Hong Kong in 2004 ( Ho et al. 2004 ) . Hong Kong is one of the parts which have the highest prevalence rates of MRSA among the whole Asia Pacific Region from old surveies ( Bell et al. 2002 ) ( Ip et al. 2004 ) . The Hong Kong Government shortly established supervising group to surveillance MRSA in research lab of Hong Kong with the aid of Center of Health Protection ( CHP ) and the Center of Infection of University of Hong Kong ( Ho et al. 2007 ) . All the infirmary and university microbiologist of Hong Kong were encouraged to roll up MRSA infection instances for this monitoring group since 2005 and the voluntaries from all industries besides reported instances to CHP. The Hong Kong authorities besides began a plan which could roll up wound swabs from patients with pussy tegument infection for MRSA civilization in 2006, MRSA infection was regarded as a statutory notifiable infection to assist monitoring group for surveillance efficaciously. During the first half twelvemonth of 2007, there were 70 individuals got MRSA infection in Hong Kong and the instances were equally 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 nutrient toxic condition. S. aureus could last in a big scope of temperature from 7a„? to 48a„? ( Schmitt et al. 1990 ) and big Na chloride concentrations ( up to 15 % ) , which makes S. aureus hard to command in nutrient. The symptoms of staphylococcal nutrient toxic condition are sickness, abdominal spasms, purging and sometimes followed by diarrhoea. Outbreaks of community-acquired foodborne unwellness caused by methicillin-resistant staphylococci aureus were besides reported in 2002, in which 3 kids were confirmed to infected by MRSA after eating chopped porc and coleslaw from a convenience-market delicatessen ( Jones et al. 2001 ) . Staphylococcus aureus nutrient toxic condition eruptions besides associated with egg yolk ( Miwa et al. 2001 ) , spaghetti and meat sauce ( Mouallem et al. 2003 ) , and a bite made up of murphy balls fried in vegetable oil ( Nema et al. 2007 ) .
1.2 Traditional Methods for Pathogenic Bacteria Detection
Pathogenic bacteriums sensing is one of the most of import for nutrient safety and public wellness. To avoid diseases caused by infective bacteriums, the procedure of sensing and designation is the first control point. Therefore, it is significantly necessary to command these bacteriums in nutrient and H2O supply by effectual sensing and review attacks. Traditional methods for bacteriums sensing chiefly relies on microbiological and biochemical techniques. For illustration: the methods of civilization and settlement numeration, fluorescent-antibody ( FA ) technique and negatron microscopy are based on numbering bacteriums cells, the method of immunology involves microbiological reaction between antibody and antigen, and the method of polymerase concatenation reaction ( PCR ) is based DNA analysis ( Velusamy et al. 2010 ) .
1.2.1 Culture and Colony Counting Methods
The civilization and settlement numeration methods is the oldest and standard method for bacteriums sensing. It includes the processs of microbiological culturing, isolation of pathogen, enrichment and plating, and so infective bacteriums can be detected by mensurating physicochemical alterations caused by their metabolic activities or growing ( Swaminathan and Feng 1994 ) . Although dependable, the process of this conventional method is labour intensive and overly time-consuming, taking up to several yearss to give confirmed consequences ( de Boer and Beumer 1999 ) . Therefore, it is non suited for doing timely appraisals on nutrient quality.
1.2.2 Fluorescent-Antibody Technique
The fluorescent-antibody ( FA ) technique could be applied to observe specific bacteriums in situ. In general, antibody conjugated with a fluorochrome binds with specific bacteriums or unlabelled antibody binds with specific bacteriums, so the antibody-antigen composite is labeled with a fluorescent antibody. If the bacterium cells present, the combined specific antibodies would do them to fluoresce and the figure 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 bacteriums sensing ; nevertheless, most fluorochromes are prone to photobleaching which makes the sensing procedure non stable.
1.2.3 Electron Microscopy Methods
Some traditional methods used for infective bacteriums designation focused on the morphology characteristic alterations by bacteriums metamorphosis with the aid of microscope techniques. Bacterial cells were counted and sized by scanning negatron microscope ( SEM ) on membrane filters ( Krambeck et al. 1981 ) and analyzed to mensurate the cell volume and dry weight by transmittal negatron microscope ( TEM ) ( Borsheim et al. 1990 ) , ( Loferer-Krossbacher et al. 1998 ) .
Bacterias have besides been detected by scanning investigation microscopy ( SPM ) . Howell et Al. developed patterned antibody microarrays to analyze their ability for adhering targeted bacteriums. Infective bacteriums such as E.coli O157: H7 and Renibacterium salmoninarum were detected by the microarrays with the aid of high-resolution SPM imagination ( Howell et al. 2003 ) . The antibody microarrays were fabricated by the method of microcontact printing ( I?CP ) , the high specificity of bacteriums adhering 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 investigation is a sensitive and specific manner for bacteriums sensing.
Huff et Al. utilised surface alterations of the high-resolution topographical imagination provided by AFM to observe and qualify the viral atoms and other pathogens ( Huff et al. 2004 ) . The AFM is capable of 2 nanometres sidelong declaration and 1 nm perpendicular declaration which allow it monitor the minute alterations in topography. This pathogen sensing system coupled with AFM could supply detail three-dimensional surface information, real-time informations acquisition and necessitate no signal elaboration.
These microscope methods could obtain the morphology information in a direct, label-free mode and supply real-time informations acquisition. Compared with fluorescent-based techniques, these label-free read-out methods have the advantages of cheap and easy-operation ( without multiple rinsing stairss ) . Furthermore, these methods could forestall the procedure of labeling which may alter the morphological information of antibodies or other proteins and mostly affect sensing consequences. Meanwhile, jobs such as exposure bleaching and label stableness could besides be avoided ( Huff et al. 2004 ) . However, these microscopic techniques for bacteriums sensing require the high-cost instrumentalities and ever necessitate skilled operators.
1.2.4 Polymerase Chain Reaction
Polymerase concatenation reaction ( PCR ) is a nucleic acerb elaboration technique which is widely used for bacteriums sensing by magnifying a few measures of DNA including the targeted bacterium ‘s familial stuff. It is based on the thermal cycling which consists of rhythms of denaturation by heating, polymerisation and extension by chilling ( Lazcka et al. 2007 ) .
Ke et Al. described a conversional PCR and Real-time PCR assays for sensing Group B Streptococci. The conversional PCR check could accomplish sensitive sensing with a high specificity and the real-time PCR was comparable with conversional PCR in sensitiveness and specificity, in add-on, it achieved the rapid thermic cycling for elaboration clip and real-time fluorescence monitoring ( Ke et al. 2000 ) .
Multiplex PCR was used to observe different types of bacteriums at the same time by utilizing multiple sets of primers and investigation that were specific for bacterium. Hu et Al. described a coincident designation of serotype O157: H7 of E.coli and its virulency factors in a individual reaction by manifold PCR check ( Hu et al. 1999 ) . Salmonella spp. and Listeria monocytogenes were besides been detected in nutrient samples at the same time following the processs of civilization enrichment and manifold real-time PCR. Two intentional sets of primers specific to L. monocytogenes and Salmonella spp. were used to compose the manifold check. Primers used for L. monocytogenes sensing were complementary to a part of the hly cistron while the primers to observe Salmonella were complementary to a part of the bipA cistron. This manifold real-time PCR achieved sensitive and specific sensing of two sorts of bacteriums at the same time and shortened the assay clip from 5-7 yearss to less than 2 yearss ( Jofre et al. 2005 ) .
The attack of PCR has a high sensitiveness with a good specificity ; nevertheless, it is mostly restricted by assay clip. The processs of PCR need require enrichment of nutrients before elaboration in order to observe bacterium which by and large present with low concentration in nutrient samples. In add-on, the sensing of bacteriums with PCR is expensive and complex which requires skilled operators.
Since the traditional methods mentioned above for bacteriums sensing are ever time-consuming and labour-intensive, the immunological sensing has been successfully employed with the advantage of less assay clip. The immunology-based methods are widely used to observe different sorts of foodborne infective bacteriums, 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 check ( ELISA ) ( Johnson et al. 1995 ) , enzyme-linked fluorescent check ( ELFA ) , enzyme-linked immunomagnetic chemiluminescence ( ELIMCL ) ( Gehring et al. 2004 ) , immunomagnetic separation ( Pyle et al. 1999 ) and so on.
Compared with traditional PCR and civilization and settlement numeration method, the immunology-based methods are rapid ; nevertheless, the sensitiveness is low and could non observe in real-time. Therefore, there is an pressing demand for a technique that could quickly, merely, faithfully detect infective bacteriums in real-time with high sensitiveness and specificity. In add-on, in position of future market, this technique should non be expensive.
1.3 Biosensors for Pathogenic Bacteria Detection
Biosensor engineering is pulling a batch of research workers ‘ involvements for its advantages of easy-operation, time-saving, portable, with low cost and the same dependable consequences. It has overcome the restrictions of traditional engineering for bacteriums sensing. Biosensor is an analytical device which incorporates a biological constituent and a physicochemical transducer or transducing micro-system. The biological constituent could be micro-organism, 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 important function in process of bacteriums sensing. The transduction methods such as optical, piezoelectric and electrochemical are the most common methods used in today ‘s research for bacteriums sensing ( Velusamy et al. 2010 ) .
1.3.1 Optical Biosensor
Optical biosensor is a rapid, sensitive and direct method in sensing of bacteriums. It detects the alterations in optical belongingss of reagents, such as light surface assimilation, contemplation, refraction, scattering, chemiluminescence, fluorescence and light energy. Due to the high sensitiveness, surface plasmon resonance ( SPR ) becomes the most popular technique in all optical biosensor techniques to observe bacteriums. For case, Subramanian et Al. used SPR biosensor to observe E.coli O157: H7 with high sensitiveness and specificity. With aid of polythene ethanediol terminated alkanethiol assorted self-assemble monolayer ( SAM ) , antibodies against E.coli O157: H7 were immobilized on a detector bit and direct and sandwich checks were carried out to observe E.coli O157: H7. The surface of biosensor was monitored during processs of sensing by optical microscope. The sensing bound was every bit low as 103 CFU/ml of E.coli O157: H7, with high specificity against Salmonella. Meanwhile, the sensitiveness was enhanced by 1000 times by utilizing sandwich check when compared with direct check ( Subramanian et Al. 2006 ) .
Waswa et Al. used the SpreetaTM, SPR-based biosensor to observe E.coli O157: H7 in different nutrient samples with specific antibody. Milk, apple juice and land beef cakes spiked with assorted concentration E.coli O157: H7 were injected on to sensor surface, and the visible radiation from an LED was reflect off a gold surface, the lower limit mensurable alterations in refractile index ( RI ) caused by the antibody-antigen reaction was recorded as the sensing bound. This check demonstrated rapid and real-time sensing with the sensitiveness every bit high as 102-103 CFU/ml ( Waswa et al. 2007 ) .
1.3.2 Piezoelectric Biosensors
Piezoelectric biosensors which depend on the usage of piezoelectric crystals are highly appropriate for sensitive bacteriums sensing. Crystals such as vitreous silica is made to hover at a specific frequence under the influence of an electric field. This frequence depends on the applied electrical frequence, hence, when bacterial cells bond to the surface of crystal due to the antibody-antigen reaction, the thickness of crystal alterations, ensuing in the frequence alteration of oscillation which can be detected electrically ( Velusamy et al. 2010 ) .
The piezoelectric biosensors were widely applied for rapid sensing of infective bacteriums. Su et Al. developed a piezoelectric immunosensor to supervise E.coli O157: H7 in a short check clip based on the SAM modified surface of a quartz crystal ‘s Au electrode. The biosensor ‘s resonating frequence was decreased by the adhering check of antibody-bacteria during the sensing processs and the frequence displacement was closely related to the concentration of E.coli O157: H7. By utilizing the method, the bacteriums concentration runing from 103-108 could be easy detected in 30-50 proceedingss ( Su and Li 2004 ) .
Quartz crystal microbalance ( QCM ) is the chief type of piezoelectric biosensor, due to the simpleness and cost effectivity, it has been greatly applied for sensing DNA immobilisation and hybridisation ( Caruso et al. 1997 ) and infective bacteriums ( Mo et al. 2002 ) . Mao et Al. demonstrated a QCM-based Deoxyribonucleic acid detector for E.coli O157: H7 sensing based on the nanoparticle elaboration. The sensor surface of QCM was modified by a thiolated single-strand DNA ( ssDNA ) which was specific to eaeA cistron of E.coli O157: H7. The DNA hybridisation between the ssDNA and the complementary Deoxyribonucleic acid from E.coli O157: H7 caused the frequence displacement. Nanoparticles coated with streptavidin were used for frequence displacement elaboration. As a consequence, this QCM-based DNA detector gave a sensing bound of E.coli O157: H7 every bit low as 2.67A-102 CFU/ml ( Mao et al. 2006 ) .
1.3.3 Electrochemical Biosensors
Electrochemical biosensors are highly of import attacks for infective bacteriums designation and quantification. The chief rule of electrochemical biosensors is that biochemical reactions produce ions and negatrons or barricade the flow of ions and negatrons, ensuing in some mensurable alterations of the electrical belongings, which are detected by electrochemical instruments. Compared to optical and piezoelectric biosensor, electrochemical biosensor is portable which is conformable to miniaturisation and can be used to observe bacteriums in situ ; in add-on, it allows the analyst to work in cloudy media. Electrochemical biosensors can be classified by the mensural transduction parametric quantities, such as amperometric biosensor and potentiometric biosensor ( Lazcka et al. 2007 ) .
The amperometric biosensor is a sensitive method for bacteriums sensing compared with potentiometric biosensor. apmerometric biosensors map by the production of current by analyte when the potency which is served as a driving force of the negatron transportation reaction, is set at a value. Therefore, the amperometric signal related with bacterial concentration is detected.
A flow through immunochemical assay system based on amperometric technique coupled with high-dispersed C atoms was developed to observe bacteriums such as E.coli Listeria monocytogenes and Campylobacter jejuni ( Chemburu et al. 2005 ) . In this instance, infective bacteriums cells were captured by specific antibodies immobilized with C atoms served as solid stage, and so labeled by horseradish peroxidase ( HRP ) conjugated secondary antibodies to organize a sandwich construction. When the peroxidase flowed through the biosensor, the amperometric signal was produced. This method provided bacteriums sensing bound every bit low as 50 cells/ml.
Amperometric technique combined with DNA hybridisation and enzyme elaboration is besides used for E.coli sensing. A micro-electromechanical system ( MEMS ) based amperometric sensor for E.coli was designed. With the aid of DNA hybridisation and enzyme elaboration, this check was able to observe 1000 cells without PCR. In add-on, a little sample volume on order of a few micro litres was another advantage of this system ( Gau et al. 2001 ) .
A potentiometric biosensor is based on ion selective electrodes and ion selective field consequence transistors ( FET ) . It consists of ion selective membrane bioactive stuffs such as enzyme. The species caused by enzyme-catalyst reaction are detected by the ion selective electrodes. The light addressable potentiometric detector ( LAPS ) based on FET is reported to successfully observe infective bacteriums ( Gehring et al. 1998 ) ( Ercole et al. 2003 ) . The LAPS consists of an electrolyte-insulator-semiconductor construction, the possible alterations caused by biochemical reaction are detected by the difference in charge distribution. A LAPS measures the jumping photocurrent produced by light beginning, so that possible alterations are converted to voltage ( Leonard et al. 2003 ) .
1.4 Electrochemical Impedance Spectroscopy ( EIS ) for Bacteria Detection
In the last few old ages, research workers have done some betterments to this method, which made the electric resistance technique more valuable. These betterments involve different electrodes and tantamount circuit analysis. The electrode-based electric resistance method is based on the electrical belongingss alteration of these electrodes when the bacterium cells adhered to the electrodes. Compared with traditional methods, this technique do has shortened the process clip to some extent ; nevertheless, the sensitiveness has been limited, because when the size of metal electrodes lessenings, the electrode electric resistance caused by the electrode polarisation is increased at the low frequence scope. If the electric resistance related with bacteriums is much smaller than the high electrode electric resistance, it is difficult to pull out the electric resistance of bacteriums from entire measured spectrum. Furthermore, it merely can be used for detect one type of bacteriums, and the specificity is hapless every bit good. So it is interesting and pressing to develop a method that could observe multiple bacteriums at the same time with a good sensitiveness.
1.5 Nanoporous Anodic Aluminum Oxide ( AAO ) Membrane
Nanoporous Anodic Aluminum Oxide ( AAO ) membranes with their reasonably chiseled nanopores provide a convenient substrate for biosensor applications. The belongingss of little pore diameter between 20nm to 200nm combine with the high denseness of nanopores ( 1A-109/cm2 ) consequence in a high surface country which is appropriate and easy for surface alteration, the critical process in biosensing.
1.5.1 Nanoporous AAO membrane based Cell detector
Nanoporous AAO membrane in assorted signifiers has been widely applied as a scaffold for tissue technology research due to its biocompatibility with bone cells. It is besides reported to be biocompatible with neural cells ( Wolfrum et al. 2006 ) . In add-on, the AAO membrane is served as an appropriate templet for survey the cell behaviours due to the surface morphology, for illustration: cell proliferation and cell adhesion.
Swan et Al. fabricated nanoporous aluminum oxides with unvarying pore size and distribution based on the two-step anodization procedure. Osteoblast adhesion and morphology on different diameters of nanopores ( 30-80 nanometer ) were studied. Images captured by scanning negatron microscopy showed the cell widening into the nanopores ( Swan et al. 2005 ) .
The nanoporous aluminum oxide membrane is besides used as a substrate for malignant neoplastic disease cell surveies. Yu et Al. cultured human esophageal squamous epithelial KYSE30 malignant neoplastic disease cells on the nanoporous AAO membrane with PEG hydrogel microwells. The electric resistance spectrometry measuring was used to analyze of anti-cancer drug consequence of retinoic acid ( RA ) on KYSE 30 malignant neoplastic disease cells. The electric resistance magnitude was reduced with clip from the value with cell bed 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 growing and drug induced alteration by electric resistance spectrometry.
1.5.2 Nanoporous AAO membrane based DNA detector
Recent scientific discipline study has proven that the nanoporous AAO membrane has beaten other stuffs in the application of DNA analysis. Due to the advantages of low auto-fluorescence, high porousness which allows for high flow rates through the membrane, good transparence every bit good as the little pore diameter which is comparable to the DNA length, the nanoporous AAO membrane is widely used in for the application of DNA sensing and detection.
A electrical capacity detector based on a nanoporous AAO construction was fabricated for DNA hybridisation detection ( Kang et al. 2010 ) . The AAO membrane served as a templet and the gold nanowires which was made by lodging gold movie on surface of membrane were used as the working and counter electrode severally. The electrical capacity of the detector decreased greatly when the complementary Deoxyribonucleic acid molecules were provided. The selectivity was proved good by incorporating three detectors into the electrical capacity detector array. One of these detectors was unmodified and the other two were modified with complementary Deoxyribonucleic acid and non-complementary DNA severally.
Kim et Al. described a microfluidic system made of polydimethylsiloxane ( PDMS ) with AAO embedded with it for DNA extraction from blood sample. The pervasion rate was used to mensurate the extraction efficiency. A low pervasion rate indicates that the Deoxyribonucleic acid was captured on the membrane and non allowed to go through through the membrane. The eluted Deoxyribonucleic acid from blood sample was farther amplified by PCR ( Kim and Gale 2008 ) .
In add-on, DNA molecules were besides detected by method of fluorescence ( Kim et al. 2006 ) , surface charge consequence ( Wang and Smirnov 2009 ) , electrochemical ( Vlassiouk et al. 2005 ) , Optical and IR surface assimilation ( Vlassiouk et al. 2004 ) because of the low auto-fluorescence, high negative charge, little pore diameter and optical transparence of the nanoporous AAO membrane.