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Kavia VenkateshPrathibhaSeminar04/11/2011 NANO-BIOSENSORSINTRODUCTION TO NANO-BIOSENSORSNanotechnology (sometimes shortened to "nanotech") is the study of manipulatingmatter on an atomic and molecular scale. Generally, nanotechnology deals with developing materials, devices, or other structures possessing at least one dimension sized from 1 to 100 nanometers. A biosensor is a measurement system for the detection of an analyte that combines a biological component with a physicochemical detector. A bio sensor designed using nanotechnology in nano-scale size is called a biosensor. Recently, nanotechnology has revolutionized research in important areas of molec

ular biology. Probes has been developed to study interactions at cellular and molecular level in real time, and to provide sensing probes that have far higher sensitivity than conventional methods. The use of this technology is not only limited to cutting edge research, but is presently making its way into clinical usewhere it may represent a fast and cost efficient method for diagnostics and screening.the affinity between complementary structures such as enzyme-substrate, antibody-antigen and receptor- hormone, is a property in biosensor used for the production of concentrationproportional signals. Biosensors selectivity and specificity highly depend on biological recognition systems connected to a suitable transducer.In recent years, with the development of nanotechnology, a lot of novelnanomaterials are being fabricated, their novel properties are being gradually discovered, and the applications of nanomaterials in biosensors have also advance

d greatly. For example, nanomaterials-based biosensors, which represent the integration of material science, molecular engineering, chemistry and biotechnology,can markedly improve the sensitivity and specificity of biomolecule detection,hold the capability of detecting or manipulating atoms and molecules, and have great potential in applications such as biomolecular recognition, pathogenic diagnosis and environment monitoring. HISTORYOne of the earliest modern ways of studying biological systems is radio-isotopemeasurements that can be used to detect specificity and affinity of different molecular interactions. From the mid 1980:s fluorescence techniques started to evolve together with powerful techniques for image analysis which resolved the issues with safety and signal clarity often found with the previous method. However,both these techniques use tagged molecules to visualize the molecular processes. In all areas involving tagged reporter molecules problems like non-uniform labeling, noise and signal quenching are readily observed1. To overcome the problem

s many other non-labeling techniques for sensing molecules have been developed.These new types of sensors can be categorized into optical, mechanical and electrical sensors. In all of these areas nanotechnology can be utilized.NANOMATERIALSThe integration of nanomaterials into biosensing systems represents one of the most crucial process nanotechnology and nanoscience. It is due to the capacity ofnanomaterials that an improved stability, minimization of sensors surface fouling, an increased sensitivity, multiplexing capacity along with an improved cost-efficiency are being achieved.The immobilization of nanomaterials onto sensing devices generates novel interfaces that enable the sensitive optical or electrochemical detection of molecularand biomolecular analytes. Moreover nanomaterials are being used as effective labels to amplify the analysis and to design novel biomaterial architectures withpre-designed and controlled functions with interest for several applications.To date, modern materials science has reached a high degree of sophistication. As a result of continuous progress in synthesizing and controlling materials on t

he submicron and nanometer scales, novel advanced functional materials with tailored properties can be created. When scaled down to a nanoscale, most materialsexhibit novel properties that cannot be extrapolated from their bulk behavior. The interdisciplinary boundary between materials science and biology has become afertile ground for new scientific and technological development. For the fabrication of an efficient biosensor, the selection of substrate for dispersing the sensing material decides the sensor performance. Various kinds of nanomaterials,such as gold nanoparticles, carbon nanotubes (CNTs), magnetic nanoparticles andquantum dots, are being gradually applied to biosensors because of their uniquephysical, chemical, mechanical, magnetic and optical properties, and markedly enhance the sensitivity and specificity of detection. TYPES OD NANO-MATERIALS

Gold nanoparticlesGold nanoparticles (GNPs) show a strong absorption band in the visible region du

e to the collective oscillations of metal conduction band electrons in strong resonance with visible frequencies of light, which is called surface plasmon resonance (SPR). There are several parameters that influence the SPR frequency. For e


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xample, the size and shape of nanoparicles, surface charges, dielectric constantof surrounding medium etc. By changing the shape of gold nanoparticles from spherical to rod, the new SPR spectrum will present two absorption bands: a weakershort-wavelength in the visible region due to the transverse electronic oscillation and a stronger long-wavelength band in NIR due to the longitudinal oscillation of electrons. The change of aspect ratio can greatly affect the absorption spectrum of gold nanorods (GNRs). In the same vein, increasing the aspect ratio ca

n lead to longitudinal SPR absorption band redshifts. Different GNP structures shows different properties. In comparison with a gold nanoparticle-conjugating probe, the gold nanowire-functionalized probe could Sensors avoid the leakage of biomolecules from the composite film, and enhanced the stability of the sensor. This interesting phenomenon will be enormously beneficial in practical applications such as biosensors.It is well known that well-dispersed solutions of GNPs display a red color, while aggregated GNPs appear a blue color. Based on this phenomenon, Jena et al. established a GNPs-based biosensor to quantitatively detect the polyionic drugs such as protamine and heparin. GNPs in biosensors can also provide a biocompatiblemicroenvironment for biomolecules, greatly increasing the amount of immobilizedbiomolecules on the electrode surface, and thus improving the sensitivity of thebiosensor.1)The glassy carbon electrode (GCE) was widely used in biosensor, and GNP modifi

ed GCEs showed much better electrochemical stability and sensitivity.2)GNPs and methylene blue (MB) could be assembled via a layer-by-layer (LBL) technique into films on the GCE modified for detection of human chorionic gonadotrophin (HCG).Due to the high surface area of the nanoparticles for loading anti-HCG, this immunosensor can be used to detect the HCG concentration in human urine or blood samples.3)For the detection of reduction of H2O2, GNP-modified electrodes also showed much wider pH adaptive range and larger response currents.4)Due to the large specific surface area and good biocompatibility of GNPs, horseradish peroxidase (HRP) can be adsorbed onto a GNP layer for the detection of H2O2 without loss of biological activity. Shi et al. confirmed that this kind ofHRP-GNP biosensor exhibited long-term stability and good reproducibility.5)GNPs/CNTs multilayers can also provide a suitable microenvironment to retain enzyme activity and amplify the electrochemical signal of the product of the enzymatic reaction.6)The gold nanorods (GNR) modified electrode layer shows a better analytical response than GNPs.7)GNR based immunosensors have advantages such as simplicity, being label free,low sample volume, reusability and being more suitable for lab-on-chip devices o

ver gold nanoparticles.8)GNRs are sensitive to the dielectric constant of the surrounding medium due tosurface plasmon resonance, therefore a slight change of the local refractive index around GNRs will result in an observable plasmon resonance frequency shift.It was also examined the quantification of the plasmonic binding events and estimation of ligand binding kinetics tethered to GNRs via a mathematical method. The GNRs sensors were found to be highly specific and sensitive with a dynamic response in the range between 10-9 M and 10-6 M. For higher-target affinity pair, one can expect to reach femtomolar levels limit of detection. This is promising for developing sensitive and precise sensors for biological molecule interactions. CNTs in BiosensorsSince Iijima discovered carbon nanotubes (CNTs) in 1991, CNTs have attracted enormous interest due to their many novel properties such as unique mechanical, physical, chemical properties. CNTs have great potential in applications such as nanoelectronics, biomedical engineering, and biosensing and bioanalysis. For example, polymer-CNTs composites can achieve high electrical conductivity and good mechanical properties, which offer the exciting possibility of developing ultrasensitive, electrochemical biosensors. 1)Amperometric biosensors was constructed by incorporation of single-walled carbon nanotubes modified with enzyme into redox polymer hydrogels. First, an enzymewas incubated in a single-walled carbon nanotube (SWNT) solution, then cross-linked within a poly[(vinylpyridine)Os(bipyridyl)(2)Cl2+/3+] polymer film, and finally formed into composite films. The redox polymer films incorporated with glucose oxidase modified SWNTs resulted in a 2 to 10-fold increase in the oxidationand reduction peak currents during cyclic voltammetry, while the glucose electrooxidation current was increased 3- fold to close to 1 mA/cm2 for glucose sensors. Similar effects were also observed when SWNTs were modified with horseradish peroxidase prior to incorporation into redox hydrogels.2)The bionanocomposite layer of multiwalled carbon nanotubes (MWNT) in chitosan(CHIT) can be used in the detection of DNA. The biocomponent, represented by dou

ble-stranded herring sperm DNA, was immobilized on this composite using layer-by-layer coverage to form a robust film. SsDNA probes could be immobilized on thesurface of GCE modified with MWNTs/ZnO/CHIT composite film. The sensor can effec


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tively discriminate different DNA sequences related to PAT gene in the transgenic corn, with a detection limit of 2.8 mol/L of target molecues.3)Carbon nanofibers are found to be an effective strategy for building a biosensor platform. Bai et al. found that the synergistic effects of MWNTs and ZnO improved the performance of the biosensors formed. They reported an amperometric biosensor for hydrogen peroxide, which was developed based on adsorption of horseradish peroxidase at the GCE modified with ZnO nanoflowers produced by electrodeposition onto MWNTs film.4)Zhang et al. described a controllable layer-by-layer self-assembly modificatio

n technique of GCE with MWNTs and introduce a controllable direct immobilizationof acetylcholinesterase (AChE) on the modified electrode. By the activity decreasing of immobilized AChE caused by pesticides, the composition of pesticides can be determined. Magnetic Nanoparticales Magnetic nanoparticles (MNP), because of their special magnetic properties, havebeen widely explored in applications such as hyperthermia, magnetic resonance imaging (MRI) contrast agent, tissue repair, immunoassay, drug/gene delivery, cell separation, GMR-sensor etc.1)Zhang et al. prepared a new kind of magnetic dextran microsphere (MDMS) by suspension crosslinking using iron nanoparticles and dextran. HRP was then immobilized on a MDMS-modified GCE. On the basis of the immobilized HRP-modified electrode with hydroquinone (HQ) as mediator, an amperometric H2O2 biosensor was fabricated.2)Lai et al. prepared a magnetic chitosan microsphere (MCMS) using carbon-coatedMNPs and chitosan. Hemoglobin (Hb) was successfully immobilized on the surface

of MCMS modified GCE with the cross-linking of glutaraldehyde.3)Janssen et al.demonstrated that a rotating magnetic field can be used to applya controlled torque on superparamagnetic beads which leads to a tunable bead rotation frequency in fluid and develop a quantitative model, based on results from a comprehensive set of experiments. This control of torque and rotation will enable novel functional assays in bead-based biosensors.4)The amperometric biosensor was based on the reaction of alkaline phosphatase (ALP) with the substrate ascorbic acid 2-phosphate (AA2P), where the Fe3O4 nanoparticles have led to the enhancement of the biosensor response with an improved linear response range. This biosensor was applied to the determination of the herbicide 2, 4-dichlorophenoxyacetic acid (2, 4-D).5)In fact, a wide variety of methods have been developed for sensing and enumerating individual micron-scale magnetic particles. Direct detection of magnetic particle labels includes Maxwell bridge, Frequency-dependent magnetometer, Superconducting quantum interference device (SQUID) and methods of magnetoresistance. Indirect detection includes Micro-cantilever-based Force Amplified Biological Sen

sor (FABS) and Magnetic Relaxation Switches (MRS). Two examples follow.6)Recently, a highly sensitive, giant magnetoresistance-spin valve (GMR-SV) biosensing device with high linearity and very low hysteresis was fabricated by photolithography. The signal from even one drop of human blood and nanoparticles indistilled water was sufficient for their detection and analysis.QDs in Biosensors Quantum dots have been subject to intensive investigations because of their unique photoluminescent properties and potential applications. So far, several methods have been developed to synthesize water-soluble quantum dots for use in biologically relevant studies. For example, quantum dots have been used successfullyin cellular imaging, immunoassays, DNA hybridization, biosensor, and optical barcoding. Quantum dots also have been used to study the interaction between protein molecules or detect the dynamic course of signal transduction in live cells byFluorescence Resonance Energy Transfer (FRET). These synthesized quantum dots have significant advantages over traditional fluorescent dyes, including better stability, stronger fluorescent intensity, and different colors, which are adjusted by controlling the size of the dots. Therefore, quantum dots provide a new functional platform for bioanalytical sciences and biomedical engineering.1)CdTe quantum dots led to an increased effective surface area for immobilization of enzyme and their electrocatalytic activity promoted electron transfer reactions and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity.2)Deng et al. reported that green and orange CdTe QDs can be used as pH-sensitive fluorescentSensors probes, which could monitor the proton (H+) flux driven by ATP synthesisfor dual simultaneous and independent detection of viruses on the basis of antibodyantigen reactions.Aside from GNPs, CNTs, magnetic nanoparticles and quantum dots, there are stillmany other nanomaterials such as metals, metal oxides, polymers and other compounds, which could be used in biosensors.4)Hollow nanospheres CdS (HS CdS) were first used to study the direct electrochemical behavior of Hb and the construction of nitrite biosensors. The HS CdS nanostructure provides a microenvironment around the protein to retain the enzymatic

bioactivity.5)Metal nanoparticles, nano Cu, with great surface area and high surface energy,are used as electron conductors and show good catalytic ability to the reduction of H2O2. Platinum nanoparticles have also been widely used in biosensors.6)Nanoscale metal oxides have also been widely used in immobilization of protein


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s and enzymes for bioanalytical applications.Metal oxide based semiconducting nanowires or nanotubes play an important role on electric, optical, electrochemical and magnetic transducers.7)Cheng et al. reported a nano TiO2 based biosensor for the detection of lactatedehydrogenase (LDH). Waxberry like nanoscale ZnO balls, as shown in Figure 8, can act as excellent materials for immobilization of enzymes and the rapid electron transfer agent for the fabrication of efficient biosensors due to the wide direct band gap . The porous structure can greatly enhances the active surface are

a available for protein binding, provide a protective microenvironment for the enzymes to retain their enzymatic stability and activity.TYPES OF NANO BIOSENSORSOptical BiosensorsNanotube Based BiosensorsElectrical BiosensorsViral NanosensorsElectrochemical Biosensors Nanoshell Biosensors Nanowire BiosensorsEXAMPLES OF NANO BIOSENSORSNanowire BiosensorsIntroductionCentral to detection is the signal transduction associated with selective recognition of a biological or chemical species of interest. Nanostructures, such as nanowires and nanocrystals, offer new and sometimes unique opportunities in thisrich and interdisciplinary area of science and technology. The diameters of these nanostructures are comparable to the sizes of biological and chemical speciesbeing sensed, and thus intuitively represent excellent primary transducers for producing signals that ultimately interface with macroscopic instruments. Inorganic nanowires and nanocrystals exhibit unique electrical and optical properties that can be exploited for sensing. The size tunable colors of semiconductor nanocrystals, together with their highly robust emission properties, are opening up o

pportunities for labeling and optical based detection of biological species thatoffer advantages compared with conventional organic molecular dyes widely usedtoday8 12. The electronically switchable properties of semiconducting nanowiresprovide a sensing modality direct and label free electrical readout that is exceptionally attractive for many applications29 37. The signals from electrically based devices can be directly routed to the outside world, electronic nanodevicesare readily integrated into miniaturized systems, and, moreover, direct electrical detection dispenses with time consuming labeling chemistry. These characteristics,together with ultrahigh sensitivity, suggest that nanowire devices could revolutionize many aspects of sensing and detection in biology and medicine.Nanowire field effect sensorsConstruction and WorkingThe underlying mechanism for nanowire sensors is a field effect that is transduced using field effect transistors (FETs)29, the ubiquitous switches of the microelectronics industry. In a standard FET illustrated in Fig. 1A, a semiconductorsuch as p type silicon (p Si) is connected to metal source and drain electrodes

through which a current is injected and collected, respectively. The conductanceof the semiconductor between source and drain is switched on and off by a thirdgate electrode capacitively coupled through a thin dielectric layer38. In the case of p Si or another p type semiconductor, applying a positive gate voltage depletes carriers and reduces the conductance, while applying a negative gate voltage leads to an accumulation of carriers and an increase in conductance. The dependence of the conductance on gate voltage makes FETs natural candidates for electrically based sensing since the electric fieldresulting from binding of a charged species to the gate dielectric is analogousto applying a voltage using a gate electrode. This idea for sensing with FETs was introduced several decades ago39 41, although the limited sensitivity of theseplanar devices has precluded them from having a large impact.Semiconductor nanowires composed of Si and other materials can also function asFET devices13 15,19 27. One of the best studied examples, Si nanowires (Fig. 1B), can be prepared as single crystal structures with diameters as small as 2 3 nm1 4,42,43 and have been shown, for both p type and n type materials, to exhibitperformance characteristics comparable to or better than the best achieved in the microelectronics industry20,21,24 27. These attractive performance characteristics are also achieved with high reproducibility24; that is, the electronic characteristics of nanowires are well controlled during growth in contrast to carbonnanotubes. The high performance switching characteristics of Si nanowires are important since it is one factor that affects sensitivity. More important to overcoming the sensitivity limitations of previous planar FET sensors is the one dimensional morphology of thesenanoscale structures since binding to the surface of a nanowire leads to depletion or accumulation of carriers in the bulk of the nanometer diameter structure (versus only the surface region of a planar device)29 and increases sensitivity tothe point that single molecule detection might be possible.A general sensing device can be configured from the high performance, field eff

ect nanowire transistors, as illustrated in Fig. 1C, where specific sensing is achieved by linking a recognition group to the surface of the nanowire. Si nanowires with their natural oxide coating make this receptor linkage straightforward


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since extensive data exists for the chemical modification of silicon oxide or glass surfaces from planar chemical and biological sensors44,45. When the sensor device with surface receptor is exposed to a solution containing a macromoleculelike a protein that has a net positive charge in aqueous solution, specific binding will lead to an increase in the surface positive charge and a decrease in conductance for a p type nanowire device. Practically, we have developed a very reliable and flexible integrated nanowire sensor device, as shown in Fig. 1D,that incorporates a Si nanowire with well defined p or n type doping, source dr

ain electrodes that are insulated from the environment (so that only processes occurring at the Si nanowire surface contribute to electrical signals), and a microfluidic device for delivery of solutions being examined.A)VG > 0 depletion of carriers conductance decreases

VG < 0 accumulation of carriers conductance increasesB) C)Nanowire FET sensor. (A) Schematic of a regular planar FET device, where S, D, and G correspond to source, drain, and gate, respectively. (B) Schematic of a Sinanowire based FET device configured as a sensor with antibody receptors (green), where binding of a protein with net positive charge (red) yields a decrease inthe conductance. (C) Cross sectional diagram and scanning electron microscopy image of a single Si nanowire sensor device, and a photograph of a prototype nanowire sensor biochip with integrated microfluidic sample delivery.Application Detection of single virusesThe studies reviewed above demonstrate some of the exciting capabilities of nano

wire sensors for the detection of both biological and chemical species in solution. While these studies implicitly show exquisite sensitivity unmatched by existing label free sensor devices, they do not define the ultimate sensitivity of nanowire FET devices. To address this critical issue, our group recently carried out studies of the detection of viruses35, which are among the most important causes of human disease57 and an increasing concern as agents for biological warfare and terrorism58,59, with the goal of determining whether the ultimate limit ofone single entity could be detected reliably.The underlying concept of our experiments is illustrated schematically in Fig. 5A. When a virus particle binds to an antibody receptor on a nanowire device, theconductance of that device will change from the baseline value, and when thevirus unbinds again, the conductance will return to the baseline value. Significantly, delivery of highly dilute influenza A virus solutions, on the order of 80aM (10 18 M) or 50 viruses/l, to p type Si nanowire devices modified with monoclonal antibody for influenza A produces well defined, discrete conductance change

s (Fig. 5B) that are characteristic of binding and unbinding of single negatively charged influenza viruses35. Definitive proof that the discrete conductance changes observed in these studies are the result of the detection of single virusbinding/unbinding was obtained from simultaneous optical and electrical measurements using fluorescently labeled influenza viruses. The optical and electrical data in Fig. 5B show that, as a virus diffuses near a nanowire device, the conductance remains at the baseline value, and only after binding at the nanowire surface does the conductance drop in a quantized manner similar to that observed with unlabeled viruses; as the virus unbinds and diffuses from the nanowire surfacethe conductance returns rapidly to the baseline value. These parallel measurements also show that a virus must be in contact with the nanowire device to yieldan electrical response, suggesting that it will be possible to develop ultradense nanowire device arrays without crosstalk in future, where the minimum size scale is set by that of the virus. In addition to meeting the ultimate sensitivitychallenge of single particle electrical detection with nanowire FET devices, this achievement of single particle or stochastic sensing offers scientific advantages and opens up opportunities60,61: the sensor detection limit is not set by the receptor affinity for the target of interest as in equilibrium measurements; the analysis of single particle on/off times provides direct information about binding kinetics crucial to understanding virus receptor interactions; and singleparticle sensitivity enables simple charge based detection of macromolecules.(A) Schematic of a single virus binding and unbinding to the surface of a Si nanowire device modified with antibody receptors and the corresponding time dependent change in conductance. (B) Simultaneous conductance and optical data recorded for a Si nanowire device after the introduction of influenza A solution. The images correspond to the two binding/unbinding events highlighted by time points

1 3 and 4 6 in the conductance data, with the virus appearing as a red dot in the images. (Reprinted with permission from35. 2004 National Academy of Sciences,USA.)OPTICAL NANOSENSORSFiberoptical bioprobesNSOM (Near field Scanning Optical Microscopy) is a technique that has been aroun


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d for some years now that gives a very good spatial resolution. The technique isbased on optical fibers where one end is very thin (20 500 nm). When light is led in the fiber, it will create an evanescent field in the thin end since the diameter here is smaller than the wavelength of the light. The spatial resolutionis very good because of the fact that the evanescent field only excites a very small volume since the intensity falls off exponentially from the tip of the fiber. Since the evanescent field mostly is used to excite fluorescent particles thi

s method inherits the problems of fluorescent dyes. The fluorescent proteins ordyes can damage the functionality of the molecules they attach to and deliveringthe dyes inside a living cell involves penetrating the cell wall. On the otherhand, probes based on this technology are not sensitive to static electricity, strong magnetic fields or surface potentials. The most common method of manufacturing NSOM probes is the so called heat and pull method. A strand of glass is heated locally, and then pulled apart. The shape of the tip depends strongly on the temperature used and the timing of the pulling6.A few years ago, a new method has been developed that uses the basics of NSOM but where the tip is covered with antibodies6. This makes it possible to sense molecules not labeled with GFP or similar fluorescent agents within a cell to determine the local distribution of the analyte within the cell (fig. 2). The methodinvolves puncturing the cell membrane with a probe, but because of the small tip

diameter of the probe (~40 nm) the damage caused to the cell is non fatal, andthe cells are able to undergo further cell division7. The cells in this experiment were treated with BPT (benzo pyrene tetrol) and the tip of the probe coveredwith antibodies against BPT. The minimum amount of BPT that could be detected was 300 zeptomoles (10 21 moles). The fact that no fluorescent marker is left within the cell also improves cell vitality. This also helps to make a more realistic measurement of the analyte distribution since the fluorescent dye may be transported to locations within the cell where it is more likely to stay, and not necessarily to the sites that one may want to investigate.Fig. 2. Image of a fiberoptical bioprobe probing a single cell (reproduced fromreference [7]).4In the future, nanosized optical probes may be useful in studying the cell structure/ function and intracellular transport. The mode of entry of viruses, molecules and signaling substances are areas of great interest for life sciences where these probes may play an important roll.Biology has entered a new era with the

recentTip ~ 40 nmFigure 2 Photograph of an Antibody based Nanoprobe. (The small size of the probe(200 nm diameter) allows manipulation of the nanoprobe at specific locations within single cells).advances in nanotechnology, which have recently led the development of biosensordevices having nanoscale dimensions that are capable to probe the innerspace ofsingle living cells. Nanosensors provide new and powerful tools for monitoringin vivo processes within living cells, leading to new information on the inner workings of the entire cell [5, 6]. Such a systems biology approach could greatlyimprove our understanding of cellular function, thereby revolutionizing cell biology. Fiberoptic sensors provide useful tools for remote in situ monitoring. Fiberoptic sensors could be fabricated to have extremely small sizes, which make them suitable for sensing intracellular/intercellular physiological and biological parameters in microenvironments. A wide variety of fiberoptic chemical sensorsand biosensors have been developed in our laboratory for environmental and biochemical monitoring [1].We have developed nanosensors for in situ intracellular measurements of single cells using antibody based nanoprobes. In this work, we describe the developmentof nano biosensors for in situ monitoring of single cells using biosensors having antibody based nanoprobes having 40 nm diameter (Fig. 2).Figure 3 Photograph of Single Cell Sensing Using the Nano Biosensor.Fig. 3 shows a photograph of an antibody based nanoprobe used to measure the presence of BPT inside a single cell. The small size of the nanoprobe allowed it tobe manipulated to specific locations within the Clone 9 cells.The 1st International Symposium on Micro & Nano Technology, 14 17 March, 2004, Honolulu, Hawaii, USA2The small size of the nanoprobe allowed it to be manipulated to specific locations within the Clone 9 cells. This study illustrates the use of antibody based nanoprobes for measurements of chemicals inside a single cell. These nanodevices could also be used to develop advanced biosensing systems in order to study in situ intracellular signaling processes and to investigate gene expression inside individual living cells. Programmed cell death called apoptosis is an importa

nt process for multi cellular organisms, from which they benefit during development and homeostasis. The nanosensor was used to detect caspase 9 activition following apoptosis.Dynamic information of signaling processes inside living cells is important to f


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undamental biological understanding of cellular processes. Many traditional microscopy techniques involve incubation of cells with fluorescent dyes or nanoparticles and examining the interaction of these dyes with compounds of interest. However, when a dye or nanoparticle is incubated into a cell, it is transported tocertain intracellular sites that may or may not be where it is most likely to stay and not to areas where the investigator would like to monitor. The fluorescence signals which are supposed to reflect the interaction of the dyes with chemic

als of interest, is generally directly related to the dye concentration as opposed to the analyte concentration. Only with optical nanosensors can excitation light be delivered to specific locations inside cells. An important feature of nanosensors is the minimal invasiveness of the monitoring process. A cell survivalstudy was previously performed whereby an investigation was performed to determine whether penetration of the cell by the nanosensor resulted in intracellular or membrane damage of such a nature as to compromise cellular viability. It was determined that the process of mitosis continued normally and that nanosensor insertion and withdrawal did not affect the life cycle of the cell. Nanosensors arean important technology that can be used to measure biotargets in a living celland that does not significantly affect cell viability. Combined with the exquisite molecular recognition of antibody probes, nanosensors could serve as powerfu

l tools capable for exploring biomolecular processes in sub compartments of living cells. They have a great potential to provide the necessary tools to investigate multi protein molecular machines ofcomplex living systems and the complex network that control the assembly and operation of these machines in a living cell. Future developments would lead to thedevelopment of nanosensors equipped with nanotool sets that enable tracking, assembly and disassembly of multi protein molecular machines and their individualcomponents. These nanosensors would have multifunctional probes (antibody as well as DNA probes) that could measure structure of biological components in singlecells. Until now, scientists have been limited to investigate the workings of individual genes and proteins by breaking the cell apart and study its individualcomponents in vitro. The advent of nanosensors will hopefully permit research on entire networks of genes and proteins in an entire living cell in vivo [5 7]APPLICATIONS OF NANO BIOSENSORBiological ApplicationsDNA Sensors; Genetic monitoring, diseaseImmunosensors; HIV, Hepatitis,other viral diseas, drug testing, environmental monitoringCell based Sensors; functional sensors, drug testingPoint of care sensors; blood, urine, electrolytes, gases, steroids,drugs, hormones, proteins, otherBacteria Sensors; (E coli, streptococcus, other): food industry,medicine, environmental, other.Enzyme sensors; diabetics, drug testing, other.Environmental ApplicationsDetection of environmental pollution and toxicityAgricultural monitoringGround water screeningOcean monitoringReport Name Venkatesh 3Report Name Venkatesh 3Report Name Venkatesh 3

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