Nanotechnology (sometimes shortened to "nanotech") is the study of manipulating 04/11/2011 SeminarVenkatesh Prathibha Kavia INTRODUCTION TO NANO-BIOSENSORS NANO-BIOSENSORS matter

on an atomic and molecular scale. Generally, nanotechnology deals with de veloping materials, devices, or other structures possessing at least one dimensi on sized from 1 to 100 nanometers. A biosensor is a measurement system for the d etection of an analyte that combines a biological component with a physicochemic al detector. A bio sensor designed using nanotechnology in nano-scale size is ca Recently, nanotechnology has revolutionized research in important areas of molec lled a biosensor. ular biology. Probes has been developed to study interactions at cellular and mo lecular level in real time, and to provide sensing probes that have far higher s ensitivity than conventional methods. The use of this technology is not only lim ited to cutting edge research, but is presently making its way into clinical use where it may represent a fast and cost efficient method for diagnostics and scr eening.the affinity between complementary structures such as enzyme-substrate, a ntibody-antigen and receptor- hormone, is a property in biosensor used for the p roduction of concentrationproportional signals. Biosensors selectivity and specifi city highly depend on biological recognition systems connected to a suitable tra nsducer.In recent years, with the development of nanotechnology, a lot of novel nanomaterials are being fabricated, their novel properties are being gradually d iscovered, and the applications of nanomaterials in biosensors have also advance d greatly. For example, nanomaterials-based biosensors, which represent the inte gration 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 g reat potential in applications such as biomolecular recognition, pathogenic diag One of the earliest modern ways of nosis and environment monitoring. studying biological systems HISTORY is radio-isotope measurements that can be used to detect specificity and affinity of different mo lecular interactions. From the mid 1980:s fluorescence techniques started to evo lve together with powerful techniques for image analysis which resolved the issu es 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 lab eling, 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 elect The integration of nanomaterials into biosensing systemsNANOMATERIALS rical sensors. In all of these areas nanotechnology can be utilized.one of the m represents ost crucial process nanotechnology and nanoscience. It is due to the capacity of nanomaterials that an improved stability, minimization of sensors surface foulin g, an increased sensitivity, multiplexing capacity along with an improved cost-e The immobilization achieved. fficiency are beingof nanomaterials onto sensing devices generates novel interfa ces that enable the sensitive optical or electrochemical detection of molecular and biomolecular analytes. Moreover nanomaterials are being used as effective la bels to amplify the analysis and to design novel biomaterial architectures with To date, modern controlled functions reached a high degree of applications. pre-designed andmaterials science has with interest for severalsophistication. A s a result of continuous progress in synthesizing and controlling materials on t he submicron and nanometer scales, novel advanced functional materials with tail ored properties can be created. When scaled down to a nanoscale, most materials exhibit novel properties that cannot be extrapolated from their bulk behavior. T he interdisciplinary boundary between materials science and biology has become a fertile ground for new scientific and technological development. For the fabric ation of an efficient biosensor, the selection of substrate for dispersing the s ensing material decides the sensor performance. Various kinds of nanomaterials, such as gold nanoparticles, carbon nanotubes (CNTs), magnetic nanoparticles and quantum dots, are being gradually applied to biosensors because of their unique physical, chemical, mechanical, magnetic and optical properties, and markedly en hance the sensitivity and specificity of detection. TYPES OD NANO-MATERIALS Gold nanoparti Gold cles nanoparticles (GNPs) show a strong absorption band in the visible region du e to the collective oscillations of metal conduction band electrons in strong re sonance with visible frequencies of light, which is called surface plasmon reson ance (SPR). There are several parameters that influence the SPR frequency. For e

xample, the size and shape of nanoparicles, surface charges, dielectric constant of surrounding medium etc. By changing the shape of gold nanoparticles from sph erical to rod, the new SPR spectrum will present two absorption bands: a weaker short-wavelength in the visible region due to the transverse electronic oscillat ion and a stronger long-wavelength band in NIR due to the longitudinal oscillati on of electrons. The change of aspect ratio can greatly affect the absorption sp ectrum of gold nanorods (GNRs). In the same vein, increasing the aspect ratio ca n lead to longitudinal SPR absorption band redshifts. Different GNP structures s hows different properties. In comparison with a gold nanoparticle-conjugating pr obe, the gold nanowire-functionalized probe could Sensors avoid the leakage of b iomolecules from the composite film, and enhanced the stability of the sensor. T his interesting phenomenon will be enormously beneficial in practical applicatio It is well known that ns such as biosensors.well-dispersed solutions of GNPs display a red color, whil e aggregated GNPs appear a blue color. Based on this phenomenon, Jena et al. est ablished a GNPs-based biosensor to quantitatively detect the polyionic drugs suc h as protamine and heparin. GNPs in biosensors can also provide a biocompatible microenvironment for biomolecules, greatly increasing the amount of immobilized biomolecules on the electrode surface, and thus improving the sensitivity of the 1)The glassy carbon electrode (GCE) was widely used in biosensor, and GNP modifi biosensor. 2)GNPs showed much blue electrochemical stability a sensitivity. ed GCEsand methylenebetter(MB) could be assembled viaandlayer-by-layer (LBL) tec hnique into films on the GCE modified for detection of human chorionic gonadotro Due (HCG). phinto the high surface area of the nanoparticles for loading anti-HCG, this imm unosensor can be used to detect the HCG concentration in human urine or blood sa 3)For mples.the detection of reduction of H2O2, GNP-modified electrodes also showed mu 4)Due to the large specific surface area and good biocompatibility of GNPs, hors ch wider pH adaptive range and larger response currents. eradish peroxidase (HRP) can be adsorbed onto a GNP layer for the detection of H 2O2 without loss of biological activity. Shi et al. confirmed that this kind of 5)GNPs/CNTs multilayers can also provide a suitable microenvironment to retain e HRP-GNP biosensor exhibited long-term stability and good reproducibility. nzyme activity and amplify the electrochemical signal of the product of the enzy 6)The gold nanorods (GNR) modified electrode layer shows a better analytical res matic reaction. 7)GNR based immunosensors have advantages such as simplicity, being label free, ponse than GNPs. low sample volume, reusability and being more suitable for lab-on-chip devices o 8)GNRs are sensitive to ver gold nanoparticles. the dielectric constant of the surrounding medium due to surface plasmon resonance, therefore a slight change of the local refractive in It around examined the quantification of the plasmonic binding events shift. dexwas alsoGNRs will result in an observable plasmon resonance frequencyand esti mation of ligand binding kinetics tethered to GNRs via a mathematical method. Th e GNRs sensors were found to be highly specific and sensitive with a dynamic res ponse in the range between 10-9 M and 10-6 M. For higher-target affinity pair, o ne can expect to reach femtomolar levels limit of detection. This is promising f or developing sensitive and precise sensors for biological molecule interactions . Since Iijima discovered carbon nanotubes (CNTs) in 1991, CNTs have attracted eno CNTs in Biosensors rmous interest due to their many novel properties such as unique mechanical, phy sical, chemical properties. CNTs have great potential in applications such as na noelectronics, biomedical engineering, and biosensing and bioanalysis. For examp le, polymer-CNTs composites can achieve high electrical conductivity and good me chanical properties, which offer the exciting possibility of developing ultrasen 1)Amperometric biosensors was constructed by incorporation of single-walled carb sitive, electrochemical biosensors. on nanotubes modified with enzyme into redox polymer hydrogels. First, an enzyme was incubated in a single-walled carbon nanotube (SWNT) solution, then cross-li nked within a poly[(vinylpyridine)Os(bipyridyl)(2)Cl2+/3+] polymer film, and fin ally formed into composite films. The redox polymer films incorporated with gluc ose oxidase modified SWNTs resulted in a 2 to 10-fold increase in the oxidation and reduction peak currents during cyclic voltammetry, while the glucose electro oxidation 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 p 2)The bionanocomposite layer of into redox carbon nanotubes (MWNT) in chitosan eroxidase prior to incorporationmultiwalled hydrogels. (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 the surface of GCE modified with MWNTs/ZnO/CHIT composite film. The sensor can effec

tively discriminate different DNA sequences related to PAT gene in the transgeni 3)Carbon nanofibers are limit of 2.8 effective strategy for building a biosens c corn, with a detectionfound to be anmol/L of target molecues. or platform. Bai et al. found that the synergistic effects of MWNTs and ZnO impr oved the performance of the biosensors formed. They reported an amperometric bio sensor for hydrogen peroxide, which was developed based on adsorption of horsera dish peroxidase at the GCE modified with ZnO nanoflowers produced by electrodepo 4)Zhang et MWNTs film. sition ontoal. described a controllable layer-by-layer self-assembly modificatio n technique of GCE with MWNTs and introduce a controllable direct immobilization of acetylcholinesterase (AChE) on the modified electrode. By the activity decre asing of immobilized AChE caused by pesticides, the composition of pesticides ca Magnetic nanoparticles (MNP), because of their special magnetic properties, have n be determined. Magnetic Nanoparticales been widely explored in applications such as hyperthermia, magnetic resonance i maging (MRI) contrast agent, tissue repair, immunoassay, drug/gene delivery, cel 1)Zhang et al. prepared new l separation, GMR-sensoraetc. kind of magnetic dextran microsphere (MDMS) by sus pension crosslinking using iron nanoparticles and dextran. HRP was then immobili zed on a MDMS-modified GCE. On the basis of the immobilized HRP-modified electro de with hydroquinone (HQ) as mediator, an amperometric H2O2 biosensor was fabric 2)Lai ated. et al. prepared a magnetic chitosan microsphere (MCMS) using carbon-coated MNPs and chitosan. Hemoglobin (Hb) was successfully immobilized on the surface 3)Janssen et al.demonstrated cross-linking magnetic field can of MCMS modified GCE with thethat a rotatingof glutaraldehyde. be used to apply a controlled torque on superparamagnetic beads which leads to a tunable bead ro tation frequency in fluid and develop a quantitative model, based on results fro m a comprehensive set of experiments. This control of torque and rotation will e 4)The amperometric biosensor in based on the reaction nable novel functional assayswas bead-based biosensors.of alkaline phosphatase ( ALP) with the substrate ascorbic acid 2-phosphate (AA2P), where the Fe3O4 nanopa rticles have led to the enhancement of the biosensor response with an improved l inear response range. This biosensor was applied to the determination of the her 5)In fact, a wide variety of methods have 4-D). bicide 2, 4-dichlorophenoxyacetic acid (2,been developed for sensing and enumera ting individual micron-scale magnetic particles. Direct detection of magnetic pa rticle labels includes Maxwell bridge, Frequency-dependent magnetometer, Superco nducting quantum interference device (SQUID) and methods of magnetoresistance. I ndirect detection includes Micro-cantilever-based Force Amplified Biological Sen 6)Recently, a Magnetic Relaxation Switches (MRS). Two examples follow. sor (FABS) andhighly sensitive, giant magnetoresistance-spin valve (GMR-SV) bios ensing device with high linearity and very low hysteresis was fabricated by phot olithography. The signal from even one drop of human blood and nanoparticles in Quantum dots have been subject to their detectionQDs in Biosensors distilled water was sufficient forintensive investigations because of their uniq and analysis. ue photoluminescent properties and potential applications. So far, several metho ds have been developed to synthesize water-soluble quantum dots for use in biolo gically relevant studies. For example, quantum dots have been used successfully in cellular imaging, immunoassays, DNA hybridization, biosensor, and optical bar coding. Quantum dots also have been used to study the interaction between protei n molecules or detect the dynamic course of signal transduction in live cells by Fluorescence Resonance Energy Transfer (FRET). These synthesized quantum dots h ave significant advantages over traditional fluorescent dyes, including better s tability, stronger fluorescent intensity, and different colors, which are adjust ed by controlling the size of the dots. Therefore, quantum dots provide a new fu 1)CdTe quantum dots led to an increased effective surface area for immobilizatio nctional platform for bioanalytical sciences and biomedical engineering. n of enzyme and their electrocatalytic activity promoted electron transfer react ions and catalyzed the electro-oxidation of thiocholine, thus amplifying the det 2)Deng et al. reported that green and orange CdTe QDs can be used as pH-sensitiv ection sensitivity. Sensors probes, e fluorescent which could monitor the proton (H+) flux driven by ATP synthesis for dual simultaneous and independent detection of viruses on the basis of anti Aside from GNPs, CNTs, bodyantigen reactions.magnetic nanoparticles and quantum dots, there are still many other nanomaterials such as metals, metal oxides, polymers and other compou 4)Hollow nanospheres CdS (HS CdS) were nds, which could be used in biosensors.first used to study the direct electroche mical behavior of Hb and the construction of nitrite biosensors. The HS CdS nano structure provides a microenvironment around the protein to retain the enzymatic 5)Metal nanoparticles, nano Cu, with great surface area and high surface energy, bioactivity. are used as electron conductors and show good catalytic ability to the reductio 6)Nanoscale metal oxides have also been widely used in used in biosensors. n of H2O2. Platinum nanoparticles have also been widelyimmobilization of protein

s and enzymes for bioanalytical applications.Metal oxide based semiconducting na nowires or nanotubes play an important role on electric, optical, electrochemica 7)Cheng et al. reported a nano TiO2 based biosensor for the detection of lactate l and magnetic transducers. dehydrogenase (LDH). Waxberry like nanoscale ZnO balls, as shown in Figure 8, c an act as excellent materials for immobilization of enzymes and the rapid electr on transfer agent for the fabrication of efficient biosensors due to the wide di rect band gap . The porous structure can greatly enhances the active surface are a available for protein binding, provide a protective microenvironment for the e nzymes to retain their enzymatic stabilityNANO activity. Electrochemicaland BIOSENSORS Viral Nanosensors TYPES OF Optical Biosensors Nanoshell Biosensor Electrical Biosensors Nanotube Based Biosensors s Central to detection is the signal transductionBiosensors Introduction EXAMPLES OF NANO BIOSENSORS selective recogn Nanowire associated with Nanowire Biosensors ition of a biological or chemical species of interest. Nanostructures, such as n anowires and nanocrystals, offer new and sometimes unique opportunities in this rich and interdisciplinary area of science and technology. The diameters of thes e nanostructures are comparable to the sizes of biological and chemical species being sensed, and thus intuitively represent excellent primary transducers for p roducing signals that ultimately interface with macroscopic instruments. Inorgan ic nanowires and nanocrystals exhibit unique electrical and optical properties t hat can be exploited for sensing. The size tunable colors of semiconductor nanoc rystals, together with their highly robust emission properties, are opening up o pportunities for labeling and optical based detection of biological species that offer advantages compared with conventional organic molecular dyes widely used today8 12. The electronically switchable properties of semiconducting nanowires provide a sensing modality direct and label free electrical readout that is exce ptionally attractive for many applications29 37. The signals from electrically b ased devices can be directly routed to the outside world, electronic nanodevices are readily integrated into miniaturized systems, and, moreover, direct electri cal detection dispenses with time consuming labeling chemistry. These characteri together stics, with ultrahigh sensitivity, suggest that nanowire devices could revolut The underlying mechanism for nanowire sensorsin Constructionmedicine.is transduc ionize many aspects of sensing and detection isbiologyfield andthatsensors Nanowire and effect a field effect Working ed using field effect transistors (FETs)29, the ubiquitous switches of the micro electronics industry. In a standard FET illustrated in Fig. 1A, a semiconductor such as p type silicon (p Si) is connected to metal source and drain electrodes through which a current is injected and collected, respectively. The conductance of the semiconductor between source and drain is switched on and off by a third gate electrode capacitively coupled through a thin dielectric layer38. In the c ase of p Si or another p type semiconductor, applying a positive gate voltage de pletes carriers and reduces the conductance, while applying a negative gate volt age leads to an accumulation of carriers and an increase in conductance. The dep endence of the conductance on gate voltage makes FETs natural candidates for ele resulting from sensing since the electric to the ctrically basedbinding of a charged speciesfield gate dielectric is analogous to applying a voltage using a gate electrode. This idea for sensing with FETs wa s introduced several decades ago39 41, although the limited sensitivity of these Semiconductor nanowires composed offromand other large impact. also function as planar devices has precluded them Si having a materials can FET 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 nm 1 4,42,43 and have been shown, for both p type and n type materials, to exhibit performance characteristics comparable to or better than the best achieved in th e microelectronics industry20,21,24 27. These attractive performance characteris tics are also achieved with high reproducibility24; that is, the electronic char acteristics of nanowires are well controlled during growth in contrast to carbon nanotubes. The high performance switching characteristics of Si nanowires are i mportant since it is one factor that affects sensitivity. More important to over coming the sensitivity limitations of previous planar FET sensors is the one dim nanoscale structures since binding to the surface of a nanowire leads to depleti ensional morphology of these on or accumulation of carriers in the bulk of the nanometer diameter structure (ve rsus only the surface region of a planar device)29 and increases sensitivity to A general sensing device can bedetection might be possible. the point that single molecule configured from the high performance, field eff ect nanowire transistors, as illustrated in Fig. 1C, where specific sensing is a chieved by linking a recognition group to the surface of the nanowire. Si nanowi res with their natural oxide coating make this receptor linkage straightforward

since extensive data exists for the chemical modification of silicon oxide or gl ass surfaces from planar chemical and biological sensors44,45. When the sensor d evice with surface receptor is exposed to a solution containing a macromolecule like a protein that has a net positive charge in aqueous solution, specific bind ing will lead to an increase in the surface positive charge and a decrease in co nductance for a p type nanowire device. Practically, we have developed a very re that incorporates a Si nanowire with well defined p or n shown in Fig. 1D, liable and flexible integrated nanowire sensor device, as type doping, source dr ain electrodes that are insulated from the environment (so that only processes o ccurring at the Si nanowire surface contribute to electrical signals), and a mic VG > 0 A) rofluidic device forcarriers conductance decreases depletion of delivery of solutions being examined. VG < 0 accumulation of carriers conductance increases Nanowire FET sensor. (A) Schematic of a regular planar FET device, where S, D, a C) B) nd G correspond to source, drain, and gate, respectively. (B) Schematic of a Si nanowire based FET device configured as a sensor with antibody receptors (green) , where binding of a protein with net positive charge (red) yields a decrease in the conductance. (C) Cross sectional diagram and scanning electron microscopy i mage of a single Si nanowire sensor device, and a photograph of a prototype nano wire sensor biochip withApplication microfluidic sample delivery. integrated Detection of sing The studies le viruses reviewed above demonstrate some of the exciting capabilities of nano wire sensors for the detection of both biological and chemical species in soluti on. While these studies implicitly show exquisite sensitivity unmatched by exist ing label free sensor devices, they do not define the ultimate sensitivity of na nowire FET devices. To address this critical issue, our group recently carried o ut studies of the detection of viruses35, which are among the most important cau ses of human disease57 and an increasing concern as agents for biological warfar e and terrorism58,59, with the goal of determining whether the ultimate limit of The underlying concept ofbe detected reliably. one single entity could our experiments is illustrated schematically in Fig. 5 A. When a virus particle binds to an antibody receptor on a nanowire device, the virus unbindsof that device will change from the baseline value, and when the conductance again, the conductance will return to the baseline value. Signific antly, delivery of highly dilute influenza A virus solutions, on the order of 80 aM (10 18 M) or 50 viruses/l, to p type Si nanowire devices modified with monocl onal antibody for influenza A produces well defined, discrete conductance change s (Fig. 5B) that are characteristic of binding and unbinding of single negativel y charged influenza viruses35. Definitive proof that the discrete conductance ch anges observed in these studies are the result of the detection of single virus binding/unbinding was obtained from simultaneous optical and electrical measurem ents using fluorescently labeled influenza viruses. The optical and electrical d ata in Fig. 5B show that, as a virus diffuses near a nanowire device, the conduc tance remains at the baseline value, and only after binding at the nanowire surf ace does the conductance drop in a quantized manner similar to that observed wit h unlabeled viruses; as the virus unbinds and diffuses from the nanowire surface the conductance returns rapidly to the baseline value. These parallel measureme nts also show that a virus must be in contact with the nanowire device to yield an electrical response, suggesting that it will be possible to develop ultradens e nanowire device arrays without crosstalk in future, where the minimum size sca le is set by that of the virus. In addition to meeting the ultimate sensitivity challenge of single particle electrical detection with nanowire FET devices, thi s achievement of single particle or stochastic sensing offers scientific advanta ges and opens up opportunities60,61: the sensor detection limit is not set by th e receptor affinity for the target of interest as in equilibrium measurements; t he analysis of single particle on/off times provides direct information about bi nding kinetics crucial to understanding virus receptor interactions; and single particle sensitivitysingle virus binding and unbinding to the surface of a Si na (A) Schematic of a enables simple charge based detection of macromolecules. nowire device modified with antibody receptors and the corresponding time depend ent change in conductance. (B) Simultaneous conductance and optical data recorde d for a Si nanowire device after the introduction of influenza A solution. The i mages 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 th e images. (Reprinted with permission from35. 2004 National Academy of Sciences, NSOM Fiberoptical bioprobes OPTICAL NANOSENSORS USA.)(Near field Scanning Optical Microscopy) is a technique that has been aroun

d for some years now that gives a very good spatial resolution. The technique is based on optical fibers where one end is very thin (20 500 nm). When light is l ed in the fiber, it will create an evanescent field in the thin end since the di ameter here is smaller than the wavelength of the light. The spatial resolution is very good because of the fact that the evanescent field only excites a very s mall volume since the intensity falls off exponentially from the tip of the fibe r. Since the evanescent field mostly is used to excite fluorescent particles thi s method inherits the problems of fluorescent dyes. The fluorescent proteins or dyes can damage the functionality of the molecules they attach to and delivering the dyes inside a living cell involves penetrating the cell wall. On the other hand, probes based on this technology are not sensitive to static electricity, s trong magnetic fields or surface potentials. The most common method of manufactu ring NSOM probes is the so called heat and pull method. A strand of glass is heate d locally, and then pulled apart. The shape of the tip depends strongly on the t A few years ago, a the timing of been developed emperature used andnew method has the pulling6. that uses the basics of NSOM bu t where the tip is covered with antibodies6. This makes it possible to sense mol ecules not labeled with GFP or similar fluorescent agents within a cell to deter mine the local distribution of the analyte within the cell (fig. 2). The method involves 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, and the cells are able to undergo further cell division7. The cells in this experime nt were treated with BPT (benzo pyrene tetrol) and the tip of the probe covered with antibodies against BPT. The minimum amount of BPT that could be detected wa s 300 zeptomoles (10 21 moles). The fact that no fluorescent marker is left with in the cell also improves cell vitality. This also helps to make a more realisti c measurement of the analyte distribution since the fluorescent dye may be trans ported to locations within the cell where it is more likely to stay, and not nec Fig. 2. to the a fiberoptical bioprobe probing a single essarilyImage ofsites that one may want to investigate. cell (reproduced from 4In the future, reference [7]). nanosized optical probes may be useful in studying the cell stru cture/ function and intracellular transport. The mode of entry of viruses, molec ules and signaling substances are areas of great interest for life sciences wher e these probes may play an important roll.Biology has entered a new era with the Figure 2 Photograph of an Antibody based Nanoprobe. (The small size of the probe Tip ~ 40 nm recent (200 nm diameter) allows manipulation of the nanoprobe at specific locations w advances in cells). ithin singlenanotechnology, which have recently led the development of biosensor devices having nanoscale dimensions that are capable to probe the innerspace of single living cells. Nanosensors provide new and powerful tools for monitoring in vivo processes within living cells, leading to new information on the inner w orkings of the entire cell [5, 6]. Such a systems biology approach could greatly improve our understanding of cellular function, thereby revolutionizing cell bi ology. Fiberoptic sensors provide useful tools for remote in situ monitoring. Fi beroptic sensors could be fabricated to have extremely small sizes, which make t hem suitable for sensing intracellular/intercellular physiological and biologica l parameters in microenvironments. A wide variety of fiberoptic chemical sensors and biosensors have been developed in our laboratory for environmental and bioc We have developed [1]. hemical monitoringnanosensors for in situ intracellular measurements of single c ells using antibody based nanoprobes. In this work, we describe the development of nano biosensors for in situ monitoring of single cells using biosensors havin Figure 3 Photograph of Singlean antibody diameterthe Nanoused to measure the pre g antibody based nanoprobes having 40 nm based nanoprobe2). Fig. 3 shows a photograph of Cell Sensing Using (Fig. Biosensor. sence of BPT inside a single cell. The small size of the nanoprobe allowed it to The manipulated to specific locations within theTechnology, 14 17 March, 2004, H be 1st International Symposium on Micro & Nano Clone 9 cells. 2The small size USA onolulu, Hawaii,of the nanoprobe allowed it to be manipulated to specific locati ons within the Clone 9 cells. This study illustrates the use of antibody base d nanoprobes for measurements of chemicals inside a single cell. These nanodevic es could also be used to develop advanced biosensing systems in order to study i n situ intracellular signaling processes and to investigate gene expression insi de individual living cells. Programmed cell death called apoptosis is an importa nt process for multi cellular organisms, from which they benefit during developm ent and homeostasis. The nanosensor was used to detect caspase 9 activition foll Dynamic information of signaling processes inside living cells is important to f owing apoptosis.

undamental biological understanding of cellular processes. Many traditional micr oscopy techniques involve incubation of cells with fluorescent dyes or nanoparti cles and examining the interaction of these dyes with compounds of interest. How ever, when a dye or nanoparticle is incubated into a cell, it is transported to certain intracellular sites that may or may not be where it is most likely to st ay and not to areas where the investigator would like to monitor. The fluorescen ce 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 oppos ed to the analyte concentration. Only with optical nanosensors can excitation li ght be delivered to specific locations inside cells. An important feature of nan osensors is the minimal invasiveness of the monitoring process. A cell survival study was previously performed whereby an investigation was performed to determi ne whether penetration of the cell by the nanosensor resulted in intracellular o r membrane damage of such a nature as to compromise cellular viability. It was d etermined that the process of mitosis continued normally and that nanosensor ins ertion and withdrawal did not affect the life cycle of the cell. Nanosensors are an important technology that can be used to measure biotargets in a living cell and that does not significantly affect cell viability. Combined with the exquis ite molecular recognition of antibody probes, nanosensors could serve as powerfu l tools capable for exploring biomolecular processes in sub compartments of liv ing cells. They have a great potential to provide the necessary tools to investi complex living systems and the complexof gate multi protein molecular machines network that control the assembly and ope ration of these machines in a living cell. Future developments would lead to the development of nanosensors equipped with nanotool sets that enable tracking, as sembly and disassembly of multi protein molecular machines and their individual components. These nanosensors would have multifunctional probes (antibody as wel l as DNA probes) that could measure structure of biological components in single cells. Until now, scientists have been limited to investigate the workings of i ndividual genes and proteins by breaking the cell apart and study its individual components in vitro. The advent of nanosensors will hopefully permit research o n entire networks ofHepatitis,other viral diseas, drug testing, environmental mo Immunosensors; HIV, monitoring,APPLICATIONS entire BIOSENSOR DNA Sensors; Genetic genes and proteins in anOF NANO living cell in vivo [5 7] Biological Applications disease Report sensors;hormones, proteins, other other): Oceanbasedof sensors; Groundmonitoringdiabetics, drugsensors, toxicity Agricultural screening Detectioncaremonitoringblood, urine, electrolytes, gases, steroids, Environmentalenvironmental3streptococcus,other. food Enzymeofmedicine, environmental,testing,drug testing industry, Point water Applications pollution and Cell NameSensors; coli, nitoringSensors; (Efunctional Bacteriadrugs, Venkatesh other.