International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 76 Nanobiosensors: Diagnostic Tool for Pathogen DetectionSrivastava Priyanka1,2*Pandey Shashank3*, M. K.. Muhammad Aslam, Singh Prashant1,2 and Singh Krishan PalDepartment of Chemistry, D.A.V (P.G.) College, Dehradun-248001, Uttarakhand, INDIA District coordinator, Uttarakhand State council for Science and Technology (UCOST), Dehradun, Uttarakhand, INDIA Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-29, INDIA Theriogenology Lab, National Dairy Research Institute, Karnal - 132001, Haryana, INDIA Biophysics and Nanotechnology, C.B.S.& H.G.B. Pant University of Agriculture and technology, Pantnagar-263145, Uttarakhand, INDIAAvailable online at: www.isca.in, www.isca.me Received 16th June 2013, revised 30th July 2013, accepted 2nd October 2013Abstract Biosensors could be a thrilling alternative to the conventional systems for the detection of toxins and pathogens. Biosensors are reliable which allow the specific detection of target analyte at minimum cost. Recent advancement in the field of nanotechnology has been used to develop highly sensitive bio-sensor’s chips by using multidisciplinary approach. This strategy could be seen as a key for developing bio-sensing devices and demonstrates rapid responses combined with high sensitivity and specificity. Indeed, these traits have nearly become standard attributes of this technological development and come up from the extremely high surface and small size nanostructure areas as nanotubes, nanowires and nanoparticles. In view of above, it is acceptable to state that biosensor technology has the prospective to augment the sensitivity and specificity, hasten the detection and enable high-throughput analysis. Keywords: Nanobiosensors, diagnostic tool, pathogen detection, clinical diagnostics, quartz crystal microbalance (QCM), nanoparticles. Introduction What are Bio-sensors? Biosensor could be defined as a biological sensor i.e. a device which combines a biological component with a physicochemical detector component for the detection of an analyte. Recent advancement in the field of nanotechnology facilitates the material science-based approach to nanoscale synthesis of nanomaterial. Nanoparticles could be used as quantum dots and as chemical catalysts. These particles are a link between bulk materials and atomic or molecular structures. In comparison to bulk nanomaterials, nanoparticles display many unique properties. For example; it can be used for nanobiosensor to recognize a particular type of analyte through a chemical process for qualitative and quantitative analysis of target analyte. According to the behaviour and role of biosensors, there are different types such as photometric biosensor, biological biosensors, electrochemical biosensors etc. Optical biosensors are developed according to surface plasmon resonance and are evanescent wave techniques. This type of sensor are having the properties of gold and can absorb laser light which produces electron waves (surface plasmons) on the surface of gold, while thin layer of gold are also having a high refractive index glass surface. This is highly dependent on the surface of the gold because of the specificity of angle and wavelength of incident light. Target analyte is bound to a receptor located on the surface of gold and generates a signal that can be quantified. The chip consists of a plastic cassette supporting a glass plate and microscopic layer of gold is coated on one side of the chip. This side is used for the contact of optical detection apparatus and opposite side is used for microfluidic flow system. The contact with the micro-fluidic system produces channels across where reagents are passed in solution. The glass sensor chip could be customized by several ways which allow easy and effective attachment of the molecules of interest. Coating of the chip is done by carboxymethyl dextran or similar compound. Binding of the molecule of the interest can effect on the refractive index and through this, biological interactions can be analysed to a high degree of sensitivity with some sort of energy; for example, dual polarisation interferometry. However, optical biosensors are working according to the variations in absorbance of the indicator compound and do not require a total internal reflection geometry. Biosensors are often incorporated with some forms of native proteins or enzymes, which are genetically modified. This receptor protein is attached to specifically detect the analyte. The resulting signal will be analysed by fluorometer or luminometer. Biosensor for determining the concentration of the analyte cAMP (cyclic adenosine monophosphate) in cytosol is an example for this. Moreover, most of the cAMP assays require lysis of the cells before analysing the concentration of cAMP. For solving this problem, a live-cell biosensor for cAMP is developed to use without lysis of cells and having added benefits of multiple reads to understand the kinetics of receptor response. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 77 Electrochemicalbiosensors are mainly based on enzymatic catalysis of a reaction and produce or consume electrons. These enzymes are known as redox enzymes. In electrochemical biosensors three electrodes are present viz. a reference electrode, an active electrode and a sink electrode. There may be an auxiliary electrode (counter electrode) also, which function as the ion source. The target analyte is engaged in the reaction that happen on the active electrode surface, and the ions that arises produce a potential which is deducted from that of the reference electrode to produce a signal; for example, potentiometric biosensor. On the contrary, there are two more type of biosensors such as thermometric biosensors and magnetic biosensors but these sensors are not used frequently and are very rare. Bio-sensor Components: There are three different components of a biosensor. These are described below: i. the sensitive biological element (biological materials such as tissues, microorganisms, cell organelles and receptors, enzymes, antibodies, nucleic acids etc.), a biologically derived material or bio-mimic and these sensitive elements can be developed by biological engineering; ii. the transducer or the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, colorimetric etc.) that modifies the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified; iii. associated electronics or signal processors which are mainly associate with the display of the results in a user-friendly way. This is the mainly costlier component of the sensor device; yet it is probable to produce a user friendly display consisting the transducer and sensitive element. The biochemical interactions are converted into a quantifiable electronic signal by the transducer. Discovery of Bio-sensors and Its History: Biosensors have imperative and significant position in medical science, industries and the environment, offering regular examinations, critical monitoring, and timely diagnosis of problems and crisis points. The history of biosensors started many years back. During the year 1956, a scientist named Leland C Clark Jr., published his classic findings on the oxygen electrode and he is recognized as the father of biocensors. The history of biocensors is summarised in tables-1 and tables-2. Types of Bio-sensors: The detection by biocensors depends upon the principle of alterations in different parameters like electric distribution (conductance, current and potential), light intensity, frequency, pressure etc. However, as described above in brief, details of various types of biosensors are explained below in table-3. Electrochemical Sensor: Ions or electrons are produced or consumed in many chemical reactions. These particles in turn may cause many alterations in the electrical properties of the solution which can be used for quantifying the event. For example; for the detection of hybridized DNA, DNA-binding drugs, glucose concentration, electrochemical biosensors are chiefly employed. Table-1 Major events in the history of development of biocensorsfrom 1916 to 2004 Year Biosensor development 1916 First report on the immobilization of proteins: adsorption of invertase on activated charcoal 1922 First glass pH electrode 1956 Invention of the oxygen electrode 1962 First description of a biosensor: an amperometric enzyme electrode for glucose 1969 First potentiometric biosensor: urease immobilized on an ammonia electrode to detect urea 1970 Invention of the Ion-Selective Field-Effect Transistor (ISFET) 1972 First commercial biosensor: Yellow Springs Instruments glucose biosensor 1976 First bedside artificial pancreas (Miles) 1980 First fibre optic pH sensor for in vivo blood gases 1982 First fibre optic-based biosensor for glucose 1983 First surface Plasmon resonance (SPR) immunosensor 1984 First mediated amperometric biosensor: ferrocene used with glucose oxidize for the detection of glucose 1987 Launch of the MediSense ExacTech™ blood glucose biosensor 1990 Launch of the Pharmacia BIACore SPR-based biosensor system 1992 i-STAT launches hand-held blood analyzer 1996 Glucocard launched 1996 Abbott acquires MediSense for $867 million 1998 Launch of LifeScan Fast Take blood glucose biosensor 1998 Merger of Roche and Boehringer Mannheim to form Roche Diagnostics 2001 LifeScan purchases Inverness Medical's glucose testing business for $1.3billion 2003 i-STAT acquired by Abbott for $392 million 2004 Abbott acquires TheraSense for $1.2 billion Table-2 Biosensor development from 2005 to present 2005 A portable automated multianalyte biosensor 2006 Electrochemical deoxyribonucleic acid (DNA) biosensor was developed using an electroactive label based on human interleukine-2 gene 2007 Development of thermal immune biosensor for detecting nonylphenol in the environment 2008 Development of amperometric glucose biosensor 2009 Piezoelectric sensor was developed by Romero and co-workers for detecting methamphetamine 2010 SERS nanosensors 2011 Optical biosensors in the field of nano bioelectronics 2012 For determining organophosphate pesticides, novel automated flow based- biosensor was developed International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 78 Table-3 Principle of detection Types of Biosensors Determine the variations in Electrochemical Electric distribution Optical Intensity of light Resonant Frequency/mass deposit/resistance Piezoelectric Pressure, acceleration, strain or force Depending upon the measuring of electrical parameters, electrochemical biosensors are classified as i. Conductimetric, ii. Amperometric and iii. Potentiometric. The consequence of the immobilization and interaction of biomolecules between a pair of metal electrodes in a bulk solution can be measured using conductimetric sensors because of the effect caused by chemical and biological changes upon the conductance. The overall conductivity or resistivity of the solution can be changed due to ions or electrons produced by electrochemical reactions. These changes can be quantified and standardized to an appropriate degree. Conductance measurements have comparatively lower sensitivity. Amperometric biosensors measure current fluctuations. The biosensors depend on linear concentration and quantify alterations in the current on the working electrode resulting from the direct oxidation/reduction of the products of a biochemical reaction in direct or indirect measuring systems. Amperometric transducers have advantages of good sensitivity, speedy response, low expensive, and disposable in comparison to other analytical devices7, 8 and are preferentially used for the design of biosensors. The amperometric transducers with best sensitivity are glassy carbon10, 11, carbon paste12 and diamond paste electrodes13,14. The diamond paste are used as a transducer in the biosensor technology owing to their qualities like minimum background current, broadened potential range, little adsorption, high signal-to-noise and signal-to-background ratios. The oxidation or reduction potential of an electrochemical reaction is measured in potentiometric type of sensor. This is based on the principle that by applying ramp voltage through an electrode in a solution, a current flow can be elicited due to electrochemical reactions. There may be differences among the voltage which elicit these reactions in particular reaction and particular species, which can be used as an indicator. These biosensors detect the accumulation of charge created by selective binding at the electrode surface15. Optical Biosensor: Optical biosensors have been invented for quick and accurate diagnosis of contaminants, poisonous substances or medicines and even pathogenic bacteria. These sensors utilize light waves to detect the presence or alterations due to a chemical on certain biological system. It is based upon optical diffraction or electrochemiluminescence. In case of optical diffraction based devices, protein is coated on a silicon wafer through covalent bonds. The exposure of wafer to ultra violet rays using a photo-mask may cause inactivation of the antibodies in certain areas which are exposed to the rays. After this, when the wafer chip is exposed to an analyte, Ag-Ab (antigen-antibody) binding takes place at the active regions and there forms a diffraction grating. This leads to the production of diffraction signal if it get excited through some source of light like laser. Then the signals produced through this can be quantified. Optical biosensor includes vibration (IR, Raman) and chemiluminescence phenomenon. The three important optical transducers employed in biosensors are optical fibres16, chemiluminescence and surface plasmon resonance17. The most common types of optical transducers are fluorescence and chemiluminescence transducers. Bioluminescence comes under the broad classification of chemiluminescence and these take place in living creatures such as fireflies, glow-worms, bacteria and others18. Resonant Sensor: In resonant sensor, the change in frequency is measured which appears due to the attachment of analyte molecule with a biological element like Abs which in turn is coupled with an acoustic wave transducer. This attachment of analyte results in the mass change of the acoustic wave transducer. A binding incident can be transformed into a quantifiable signal with the help of a piezoelectric transducer, for example resonance frequency changes, mass deposit and resistance. This type of biosensor can detect two or more species simultaneously depending upon the way of development of piezoelectric transducer with a simple and multifunctional recognition element19. Piezoelectric Biosensor: Piezoelectric sensors are based on piezoelectric effect discovered by Curie brothers. Pierre Curie and Jacques Curie recognized that piezoelectric objects like tourmaline and quartz have the capacity to convert energy of a mechanical input into an electrical output. There is a mechanical deformation and displacement of charges, when pressure is exerted over a piezoelectric object. The inner dipoles are re-orientated and a crystalline mechanical strain is observed. These charges are directly proportional to the exerted pressure (Piezoelectricity). QCM (Quartz Crystal Microbalance) is a device which can be modified by using different schemes in order to develop different immunosensors. Immunosensors are those biosensors in which the recognition element is Ab. It is a device comprising of an Ag or Ab species coupled to a single transducer which detects the binding of the complementary species. QCM is made up of quartz and belongs to the trigonal crystal system. Quartz is a continuous framework of SiO (silicon–oxygen tetrahedral) in which two tetrahedral are being shared by each oxygen molecules and gives an overall formula of SiO2 (figure-2). QCM is an easy, less expensive, high-resolution mass sensing practice. It is mainly based upon the alterations in the frequency of a quartz crystal resonator by mass per unit area and measures International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 79 piezoelectric effect. QCM has high sensitive solution-surface interface measurement capability and therefore in largely analytical chemistry and electrochemistry applications, evolved solution quantification potential. In this system large variety of molecules are detected such as low mass is detected by monolayer surface coverage by using tiny molecules or polymer films while at the greater end, it is competent to detect heavy masses attached to its surface. It could be considered as a multifaceted array of biopolymers and biomacromolecules, even the entire cells. Moreover, details regarding the energy dispersing qualities of the bound surface mass can also be provided by the QCM. The progress of piezoelectric tools is mainly associated with a set of intrinsic benefits such as i. high modulus of elasticity as compared to many metallic substances ii. piezoelectric sensors are electromechanical systems that react on compression, iii. The deflection showed by this sensing elements are almost nil, iv. this technology is insensitive to electromagnetic fields and radiation and v. some piezoelectric materials (especially gallium phosphate or tourmaline) remain highly stable even above 1000°C temperature, which facilitate the sensors to perform under unfavorable conditions also. At the same time, piezoelectric sensors have the disadvantage that they cannot be employed for true static measurements. A static force may lead to some fixed quantity of charges on the piezoelectric material. Only those piezoelectric materials which can maintain high internal resistance can be used at elevated temperatures as, the increased temperature may lead to an added reduction in internal resistance. Besides this piezoelectric devices can be classified into TSM (Thickness Shear Mode) Sensor, SAW (Surface Acoustic Wave) Sensor and APM (Acoustic Plate Mode) Sensor on the basis of mode of acoustic wave propagation. Theory of QCM Device Sauerbrey Equation: Sauerbrey investigated the association between the variation in resonance frequency and the added mass20. This equation is applicable only for thin, even and purely elastic layers. (1) Where is frequency change in Hz, = Sensitivity factor of the crystal in Hz/ng/cm, and = Change in mass per unit area in g/cm. According to Sauerbrey equations, the additional mass/film deposited over the crystal has the same acousto-elastic properties as quartz and this assumption resulted in a sensitivity factor, C which is a fundamental property of the QCM crystal as shown in equation 2. (2) In this formula, n stands for number of the harmonic at which the crystal is driven, is resonant frequency of the fundamental mode in Hz, is the density of quartz (2.648 g.cm-3) and is the effective piezoelectrically stiffened shear modulus of quartz (2.947 x 1011 g cm-1sec-2). The sensitivity factor for different crystal can be summarized as 0.0566 Hz/ng/cm2 for a 5 MHz crystal, 0.0815 Hz/ng/cm for a 6 MHz crystal and 0.1834 Hz/ng/cm for a 9 MHz crystal at 20 C. Solving these equations for yields (3) Where, is the resonant frequency of unloaded crystal and is resonant frequency of loaded crystal in Hz. It should be noted that in these hypothesis, the variation in frequency is a function of mass per unit area. As a result, theoretically calibration is not required for the QCM mass sensor. But, Sauerbrey equation is appropriate for uniform, rigid, thin-film deposits only. More complicated frequency-mass correlations may be shown by vaccum and gas phase thin film depositions which could not satisfy these conditions; therefore they require some calibration for providing perfect results. Nanoparticles Since nanomaterials have more surface to volume ratio and can provide more space for binding of antigen and antibody, the efficacy and sensitivity of sensors can be increased by immobilization of functional nanomaterials like silver and gold nanoparticles on the surface of quartz crystal. The nanoparticles possess specific optical, thermal, catalytic and electric properties drawing major attention with the advancement of Nanotechnology21. Nowadays, metal nanoparticles like silver and gold nanoparticles, micelles, dendrimers, polymeric nanoparticles, quantum dots play significant role in biomedical applications. A polymeric nanoparticle is the one which is derived from a polymer. It has its application in drug delivery. Abraxane is the first polymeric nanoparticle made. A metal nanoparticle is made up of metal like Gold and Silver. These gold and silver nanoparticles have their uses in biosensors, optics and catalysts. Recent research is focused on the synthesis of nanoparticles based on gold chemistry. They are point of interest for many researchers as these are multipurpose agents with very much biomedical relevance. For example: antibody modified gold nanoparticles detect prostate specific antigen which is having million times more sensitivity than the routinely using ELISA technique22 On the other hand, silver nanoparticles also hold great application as catalysts23. Due to the unique ability to terminate International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 80 bacterial division, silver nanopartcles can also be used as bactericidal agents24. Another application of silver NPs (nanoparticles) is a real time optical sensor. Among these three metallic substances (Ag, Au, and Cu) which show plasmon resonances in visible spectrum, silver (Ag) possess the highest competence of plasmon excitation. Advancement in Bio-sensors for Bacterial Detection Basic Immunosensor development: The initial step in the development of QCM based immunosensor is to achieve a thiolated crystal surface. Thiolization not only leads to surface activation but also modification in favour of antibody immobilization. Attachment of ATP (-Aminothiophenol) molecules decreases the average frequency of bare quartz crystal. The modification can be done by using ATP because thiol group shows great affinity towards the gold and second, the developing SAM (self-assembled monolayer) film activates the electrode (gold) by providing partial positive charge to crystal surface because –NH lies at right angles to the surface. This partial positive charged surface is the ideal location for antibody immobilization. The ATP modified crystal can then be exposed to the Ab so as to prepare the surface of biosensor specific to the Ag (figure-1). Immunsensor development using AgNP (silver nanoparticles) @ATP: The process of biosensor development starts with quartz crystal surface modification with DTPA (3,3’-dithiopropionic acid). As a result, a SAM will be there on the golden electrode. This SAM film develops partial negative charge on its surface as the –COOH remains at right angles to the surface and prompt the gold electrode. The modification was performed using DTPA because thiol have increased affinity towards the gold and at the same time, the surface which is partially negative charged, due to –COOH groups, also offer better site for AgNPs@ATP which are partially positive charged. When AgNPs@ATP are exerted over the modified gold electrode with DTPA, there is an attraction force building from increased affinity of the partially negative charged –COOH group towards the partially positively charged –NH group. Due to this attraction, the silver nanoparticles will be bound firmly on the gold surface. Immobilisation of silver nanoparticles provides a larger surface area along with a partial positive surface. This partially positively charged surface also offer an ideal site for partially negative charged antibody for binding to the surface thus favouring greater Ab-Ag binding (figure-3). Immunosensor development using AuNP (gold nanoparticles) @ATP: Quartz cystal is used to wash with Piranha solution (mixture of 70% HSO and 30% H (3:1). And modified with TDPA (3,3’-thiodipropionic acid). Excess of TDPA is removed by using methanol solution. AuNP@ATPs is attached to the crystal surface using DCC (N,N’-dicyclohexyle carbodimide) / NHS (N-Hydrosuccinimide) chemistry. Then, AuNP@ATPs is immobilized onto the modified crystal surface followed by Abs immobilization. The Ab-Ag interactions can then be studied (figure-4). Figure-1 Schematic Diagram of Modified QCM Device Coated with Nanoparticles (Au/Ag) for Bacterial Detection with Increased Sensitivity International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 81 -aminothiophenol Blocking with BSA QuartzFigure-2 Schematic Diagram for Immunosensor Development for Bacterial Detection Figure-3 Schematic Diagram for Immunosensor Development using Agnp@ATP for Bacterial Detection International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 82 QuartzFigure-4 Schematic Diagram for Immunosensor Development using Aunp@ATP for Bacterial Detection ConclusionIn this review we made an effort to discuss the recent developments, industrial applications of pathogen sensing systems and realized that biosensor technology is a young and growing field. It is observed that the efficacy and sensitivity of sensors can be increased by immobilization of functional nanoparticles (Gold, Silver etc) on quartz crystal surfaces. The detection limit will be increased remarkably and will form the basis of presence of minor contamination and presence of pathogens/microbes in various food products, biological fluids etc. Bashir and co workers were the first one to report bacterial detection at Microsystems in 200125. The study involves a microsystem which uses impedance spectroscopy to detect Listeria. Woo and co-workers also described the selective amperometric detection of Escherichia coli in 2001 within 40 minutes which is a very short time span26, while Mura et al., 2012, demonstrated that contamination of E. coli in food and water could be detected in by using FITR spectrophotometry by using titania thin-film substrates as sensors27. However, lab on a chip biosensors are having ample prospects to be used in the area of pathogen detection because of the scope for miniature and automated features, similarly they are rapid and highly sensitive. The biocensors are able to detect microorganisms in various farms, post harvest and process units, logistics channels and even at the end user level. However, there are number of difficulties to be addressed before extending these labs on chip sensors for field applications28. A model experiment is carried out by Wang et al., 2012 in which E. coliwas detected at very low concentrations of 50 cfu/mL, 4 times greater than the sensitivity of detection through regular grating- coupled SPR with direct detection limit29. Cheng et al., 2012 has demonstrated that nanoporous alumina-modified platinum electrode can be used for developing membrane-based electrochemical nanobiosensor which can be used for the diagnosis of dengue type 2 virus (DENV-2)30. The value of nanobiosensors as a diagnostic tool has long been in focus and when the advantages of nanobiosensor based approach in diagnostics were recognized during the last two, International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(10), 76-84, October (2013) Int. Res. J. Biological Sci. International Science Congress Association 83 three decades, it led to an evolution. This sensitive approach towards the diagnostic could be a breakthrough which is filling the gap and advancing the technology for the pathogen detection. This approach is certainly have several advantages over traditional methods of pathogen detection such as culture techniques, PCR (polymerase chain reaction), ELISA (enzyme linked immunosorbant assay), Flow cytometry etc. because the technology is smaller, faster, and more sensitive. Nanobiosensors present as a detection option that can be effectively employed to specific pathogen related requirements. The prospects are low detection limit, less time consuming, economical, biocompatible, precise and accurate like features. In coming years through the application of biosensors, the speed with which water treatment plants, nursing stations, and society can respond to biosecurity threats, unsafe food and water, and medical problem will be changed considerabley. 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