NANO SENSORS AND DETECTORS-THEIR APPLICATIONS (NEMS)
Nanotechnology is an extremely powerful emerging technology, which is expected to have a substantial impact on medical technology now and in the future. The potential impact ofnovel nanomedical applications on disease diagnosis, therapy, and prevention is foreseen to change health care in a fundamental way.
Biomedical nanotechnology presents revolutionary opportunities in the fight against many diseases . An area with near-term potential is detecting molecules associated with diseases such as cancer, diabetes mellitus, neurodegenerative diseases, as well as detectingmicroorganisms and viruses associated with infections, such as pathogenic bacteria, fungi, and HIV viruses. Macroscale devices constructed from exquisitely sensitive nanoscale components, such as micro-/nanocantilevers, nanotubes, and nanowires, can detect even the rarest biomolecular signals at a very early stage of the disease. Development of these devices is in the proof-of-concept phase, though entering the market may be sooner than expected. However, a different approach of molecular sensing in vivo involves the use of implantable sensors which is still hampered by unwanted biofouling impairing long-term stability of continuous sensors caused by blood components and factors of the immune system. Nanotechnology might yield nano-structured surfaces preventing this non-specific protein adsorption.
RANJITH KONETI (astute_ranjith[at]yahoo.co.in)
IV Yr. B.Tech-BME
Department of Biomedical Engineering, J.B Institute of Engineering and Technology,
A biosensor is generally defined as a measurement system that consists of a probe with a sensitive biological recognition element, or bioreceptor, a physicochemical detector component, and a transducer in between.
A biosensor consists of usually three components 1) BIORECEPTORS 2) TRANSDUCERS
Bio receptorsâ€œ It is a sensitive biological element . The interaction of an analyte, e.g. a particular chemical component, virus or micro-organism, with the bioreceptor is designed to generate an effect picked up by a transducer, which converts the information into a measurable effect by the detector, for instance an electric signal. Bioreceptors are used because of their specificity. They enable measurement with minimum interference from other components in complex mixtures. The bioreceptor is a biological molecule (e.g., an antibody/antigen, DNA, protein, or enzyme), or a living biological system (e.g., cells, tissues, or whole organisms) that utilises a biochemical mechanism of recognition. The sampling component of a biosensor contains a bio-sensitive layer that can either contain bioreceptors or be made of bioreceptors covalently attached to the transducer. Transducer -Transduction can be accomplished by optical, electrochemical, and mass detection methods. A nanobiosensor or nanosensor is a biosensor that has dimensions on the nanometre .size scale. Nanosensors could provide thetools to investigate important biological processes at the cellular level in vivo.
The three types of Nano sensors with medical application possibilities are a) Cantilever array sensors and b) Nanotube sensors c) Nanowire sensors.
a) Cantilever array sensors
Microfabricated cantilever array sensors are used as ultra-sensitive mechanical sensors converting (biochemical or physical processes into a recordable signal in microelectromechanical systems (MEMS) or nanoelectromechanical systems (NEMS).
Cantilevers are typically rectangular-shaped silicon bars. The unique feature of microcantilevers is their ability to undergo bending due to molecular adsorption or bindinginduced changes in surface tension.
Applications of Cantilever array sensors
i) Cantilever sensors for diagnosis of diabetes mellitus
Medical applications of cantilever-based sensors have been proposed for early diagnosis of diabetes mellitus and can improve blood glucose monitoring using small and ultra-sensitive analytical platforms .In patients with diabetes mellitus, ketones are produced due to the deterioration of blood insulin concentrations. Acetone is one of these ketones which is excreted in urine or expired as vapour in exhaled air. Disposable test kits are used to detect acetone in urine. Acetone in exhaled air can only be detected by the physician as a putrid smell without any quantification. Small amounts of acetone in a patientâ„¢s breath can be detected by cantilever array sensor technique which may attribute to early diagnosis of diabetes mellitus.
ii) Cantilever sensors for bacteria, fungi, viruses
The Devices have also been developed to detect bacteria, fungal spores and viruses.The interaction between specific antibodies, for instance antibodies to Escherichia coli, immobilised on the surface of the cantilever, and antigens on cell membrane surface results in additional mass loading detected by the device. The detection sensitivity is in the order of a single bacterium corresponding to a mass of ~1 pg , single fungal spore, and single vaccinia virus particles
corresponding to a mass of ~10 fg . Cantilever arrays allow detection of vital functionalised fungal spores in situ within ~4 hours, which is more than ten times faster than current applied procedures for fungal detection.
A NEMS device with molecular recognition for virus particle detection has been developed, allowing improvement of the detection sensitivity up to 6 bound baculovirus particles. Once these devices with on-chip antibody-based recognition are integrated with sample concentrators, nanomechanical oscillators may prove to present a viable strategy for ultra-sensitive detection of airborne bacteria, fungi, and virus particles.
iii) Cantilever sensors for cancer diagnosis
Cantilever arrays can aid cancer diagnosis and can be engineered to bind to molecules associated with cancer, such as DNA sequences , single nucleotide polymorphisms ,and proteins. When the cancer-associated molecules bind to the cantilevers, changes in surface tension cause the cantilever to bend. By monitoring whether or not the cantilevers are bending, the presence of cancer-associated molecules can be demonstrated. Significant bending should be evident when the molecules are present in very low DNA concentrations. The mass detection limitation of NEMS cantilevers is improved to the enumeration of a single DNA molecule consisting of ~1600 base pairs and weighing ~1000 kD, which is ~1 ag (atto (a) = 10-18).
The cantilever technology could be useful in high-throughput nanomechanical genomic analysis and proteomics detecting early molecular events in the development of cancer. The specificity and sensitivity of these arrays do not yet offer substantial advantages over conventional detection methods, although the use of nanoparticle probes might allow for individual single-pair mismatch discrimination. Rather, the breakthrough potential of micro- and nanomechanical cantilevers resides in their extraordinary multiplexing capabilities. It is realistic to envision arrays of thousands of cantilevers constructed on individual centimetre-sized chips, enabling the simultaneous reading of proteomic profiles or, ultimately, the entire proteome.
Cantilever array sensor. The biomarker are affinity-bound to the cantilevers and
cause them to bend. The deflections of the cantilever beams can be directly observed with
lasers. Alternatively, the shift in resonant frequencies caused by the binding can be
electronically detected. The breakthrough potential in cantilever technology is the
multiplexing modality, i.e. the ability to sense a large number of different proteins at the
same time, in real time.
b) Nano tube based sensors
Nano-sized carbon tubes coated with strands of DNA can create tiny sensors with abilities to detect odors and tastes, According to the researchers, arrays of these nanosensors could detect molecules on the order of one part per million, akin to finding a one-second play amid 278 hours of baseball footage or a single person. The researchers tested the nanosensors on five different chemical odorants, including methanol and dinitrotoluene, or DNT, a common chemical that is also frequently a component of military-grade explosives. The nanosensors could sniff molecules out of the air or taste them in a liquid, suggesting applications ranging from domestic security to medical detectors.
Applications of Nano tube based sensors
i) Nanotube-based sensors for blood glucose monitoring
Carbon nanotubes are promising sensing candidates to monitor glucose in blood and urine. MWCNTs as well as SWCNTs have been used to develop enzymatic amperometric biosensors or fluorimetric biosensors . The enzyme glucose oxidase is either immobilised inside MWCNTs or non-covalently attached to the surface of SWCNTs enabling the catalysis of glucose with hydrogen peroxide as co-product. For the amperometric biosensor the enzyme immobilisation allows for the direct electron transfer from the enzyme to a gold or platinum transducer producing the response current. The fluorescence biosensor could be used in a new type of implantable biological sensor such as near-infrared nanoscale.
Antibody Tumour biomarker proteins Bent cantilever sensor. This sensor could be inserted into tissue, excited with a laser pointer, and provide real-time, continuous monitoring of blood glucose levels. It consists of protein-encapsulated SWCNTs functionalised with potassium ferrocyanide, a substance that is sensitive to hydrogen peroxide. The ferrocyanide ion adsorbs on the surface through the porous monolayer. When present, hydrogen peroxide will form a complex with the ion, which changes the electron density of the carbon nanotube and consequently its optical properties. The more glucose that is present, the brighter the carbon nanotube will fluoresce. The sensor can be loaded into a porous capillary and inserted into tissue. As carbon nanotubes do not degrade like organic molecules that fluoresce, these nanoparticle optical sensors would be suitable for long-term monitoring applications. Proof-of-concept studies to detect glucose levels have been performed in vitro, i.e. in blood samples. Practical use is five to ten years ahead, according to the researchers .Self-assembled peptide nanotubes can be used in an electrochemical biosensor. The presence of the peptide nanotubes improves the sensitivity of the device severalfold. Peptide nanotubes offer several advantages over carbon nanotubes, since they arebiocompatible, water-soluble, inexpensive, easy to manufacture, and can be chemically
modified by targeting their amino or carboxyl groups. The sensing technique can be used as a
platform for ultra-sensitive detection of biological and chemical agents.
i) Nanotube-based sensors for DNA detection
MWCNT-based nanoelectrode arrays embedded in SiO2 matrix have been integrated into a electrochemical system for ultra-sensitive and rapid DNA detection. A bottom-up approach is used for the fabrication of individually addressed nanoelectrode arrays, that results in precisely positioned and well aligned MWCNT arrays on a silicon wafer. Subsequently, the open ends of MWCNTs are functionalised with oligonucleotide probes. Combining the nanoelectrode arrays with redoxactive molecule-mediated guanine oxidation, the hybridisation of less than a few attomoles of oligonucleotide targets (~3.5Ãƒâ€”106 DNA molecules) can be easily detected by voltametric measurement. The proof-of-concept has been demonstrated for clinical relevant DNA molecules related to wild-type alleles associated with cancer genes. Furher optimisation of the system could yield detections below one attomole.
ii) Nanotube-based sensors for capnography
Carbon nanotube-based chemical gas sensors have great potential in medical applications. Capnography is the measurement of carbon dioxide concentration in human respiration and is a indicator of patient status during administration of anaesthesia. The tiny, low-power sensor will be the first disposable electronic capnography sensor and has the potential to extend the reach of quantitative respiratory monitoring beyond the operating room and into ambulatory and emergency settings as well as doctorsâ„¢ offices.
c) Nanowire-based sensors
IT is a hybrid of two molecules that are extremely sensitive to outside signals: single stranded DNA, which serves as the 'detector,' and a carbon nanotube, which functions as 'transmitterâ„¢. By putting the two together and they become an extremely versatile type of sensor, capable of finding tiny amounts of a specific molecule." The size of such sensors each carbon nanotube is about a billionth of a meter wide
Applications of Nanowire based sensors
i) Nanowire-based electrical detection of single viruses
Semiconducting silicon nanowires can be configured as field-effect transistors for the electrical detection of viruses in solutions. When a single charged virus binds to receptors (e.g., antibodies) linked to the nanodevice the conductance of a semiconducting nanowire changes from the baseline value, and when the virus unbinds, the conductance returns to the baseline value. The conductance of a second nanowire device without receptors should show no change during the same time period and can serve as an internal control. Nanowires are confined to a central region that is coupled to a microfluidic channel for sample delivery and the conductance response can be recorded while solutions with viruses flow at a constant rate. Modification of different nanowires within an array with receptors specific for different viruses provides a means for simultaneous detection of multiple viruses at the single particle level. The potential of nanowire-based electrical detection of viruses exceeds the capabilities of other methods such as polymerase chain reaction-based assays and micromechanical devices .
ii) Nanowire-based electrical detection of biomolecules
Silicon nanowire field-effect transistor devices have been used for detection of small molecule inhibitors of ATP binding to AbI, which is a protein kinase whose activity is responsible for chronic myelogenous leukemia. Silicon nanowire sensors functionalised with peptide nucleic acid receptors can distinguish wild-type from the mutation type in the cystic fibrosis transmembrane receptor. Cystic fibrosis is one of the most common fatal genetic diseases among populations of European origin.
Nanowire-based sensors deployed within a microfluidic system. Different colours
indicate that different molecules/viruses adsorb or affinity-bind to different nanowire sensors. The
binding causes a change in conductance of the wires, which can be electronically and
quantitatively detected in real time. The working principle is that of a (biologically) gated
transistor. The nanosize of the wire is required to attain high signal-to-noise ratios.
a) Nanoarray-based biodetection
These nanoarrays occupy a surface area thousands of times smaller than a standard microarray and therefore present many novel applications. Three of the applications we will demonstrate are cytokine expression profiling nanoarrays for small animal model systems, reverse-phase nanoarrays for profiling malignant progression from laser capture microdissected tissue samples, and a nanoarray-based pathogen detection platform with biodefense applications called the ViriChip.
Application of nanoarray-based detectors
Ultra-sensitive virus detection
Viruses in human blood samples, such as HIV-1, can be detected using nanoscale antibody array-based devices. Dip-pen nanolithography was used to pattern 16-mercaptohexadecanoic acid into an array of 60 nm dots on a gold thin film. Monoclonal antibodies to the HIV-1 p24 antigen were immobilised on the dots. The analysis consists of immersing the array for one hour in a blood plasma sample. Subsequently, the signal from the antigen-array binding was amplified using gold nanoparticles probes functionalised with polyclonal antibodies in a solution for one more hour. A measurable amount of HIV-1 p24 antigen in blood plasma from humans with less than 50 copies of RNA/ml is feasible demonstrating that nano-based assays can far exceed the 5 pg/ml (pico (p) = 10-12) detection limit of conventional enzyme-linked immunosorbent assays and provide sensitivity comparable to a polymerase chain reaction-based assay, without target amplification. Nanobased array biodetection could enable HIV-1 diagnosis in mother-to-child transmission.
b) Nanoparticle-based biodetection
Applications of nanoparticle-based detectors
i) Ultra-sensitive detection of pathogenic biomarkers
One of the major drawbacks of conventional protein or antigen detection methods (e.g., enzyme-linked immunoassays, blotting assays) is the relative insensitivity for the target. Ultra-sensitive tests are needed for patient screening and diagnosis in the early stage of diseases enabling detection of very low concentrations of pathogenic biomarkers and conclusive confirmation of the disease in living patients. Recently, an ultra-sensitive bio-bar code assay has been developed for the detection of protein/antigen analytes at clinically relevant attomolar (atto = 10-18) concentrations which is five to six orders of magnitude less compared to conventional clinical assays.
The bio-bar code assay uses two types of probes
a) gold nanoparticle (13-30 nm indiameter) probes heavily functionalised with hundreds of identical hybridized oligonucleotides (DNA strands or bar-code DNA acting as an identification label) and polyclonal antibodies, and magnetic microparticle (1-Ã‚Âµm diameter polyamine particle with magnetic iron oxide core) probes functionalised with monoclonal antibodies. The polyclonal and monoclonal antibodies recognize and bind to the same target protein, sandwiching the protein between the nano- and microparticle. After the sandwich is removed magnetically from the solution, the bar-code DNA strands are released and read using standard DNA detection methodologies. The increased sensitivity of the assay derives mainly from the very effective sequestration of the protein/antigen and the amplification process that occurs as a result of the large number of barcode DNA strands (for 13 nm nanoparticles, each nanoparticle can support up to 100 strands of DNA) released for each recognition and binding event.
The bio-bar code assay technology has been tested to detect very low concentrations free of prostate-specific antigens. Prostate-specific antigens are associated with prostate and breast cancer. In women with breast cancer, free prostate-specific antigen is found in serum at much lower concentration than in men and it is being explored as a breast cancer screening target. The bio-bar code assay technology has successfully been applied for the first time to detect amyloid-ÃƒÅ¸-derived diffusible ligands in cerebrospinal fluid of living patients with Alzheimerâ„¢s disease. Amyloid-ÃƒÅ¸-derived diffusible ligands are found in brain tissue of individuals with Alzheimerâ„¢s disease where they cause neurological damage but ligand concentrations in blood were too low to be detected until now. The bio-bar code assay technology can be used to identify these markers before symptoms develop and the disease may be treated in its nascent form when treatments may be most effective. In fact, the assay could be extended to potential applications such as blood screening concerning HIV, prions, many forms of cancer, and certain cardiac and pulmonary markers.
Implementation of the bio-barcode assay within a microfluidic device. First, magnetic particles functionalized with monoclonal PSA antibodies are introduced into the separation area of the chip. The particles are then immobilized by placing a permanent magnet under the chip, followed by introduction of the sample and gold nanoparticles that are decorated with both polyclonal antibodies and barcode DNA.
Sandwiched target protein for bio-bar code assay. DNA-coated (oligonucleotides) gold
nanoparticles form the basis of the bio-bar code assay using larger magnetic macroparticles to
detect attomolar concentrations of serum proteins. In this case a monoclonal antibody to prostate
specific antigen (PSA) is attached to the magnetic macroparticle capturing free PSA. A second
polyclonal antibody to PSA, attached to the nanoparticle, creates a sandwich of the captured
protein and the two particles that is easily separated using a magnetic field.
These scientific breakthroughs could have profound clinical implications for research, therapeutic cerebrospinal fluid screening as well as wide scale blood screening. The molecular detection method has the potential for massive multiplexing and simultaneous detection of many analytes in one solution.
ii) Ultra-sensitive detection of single bacteria
Recently, dye-doped silica nanoparticles have been used to develop an assay tool for in situ pathogen quantification in water samples enabling the detection of one bacterium cell . This ultra-sensitive detection method uses fluorescent-bioconjugated silica nanoparticles (~60 nm in diameter). Within each silica nanoparticle thousands of fluorescent dye molecules are trapped. The silica matrix not only provides high photostability of the dye molecules inside the nanoparticle, but it also enables easy modification of the surface by conjugation of various biomolecules to the nanoparticles. Monoclonal antibodies against antigens of bacteria are covalently immobilised onto the nanoparticles, which are then used in an immunoassay. High fluorescent signal amplification is achieved when the antibodybioconjugated nanoparticles bind to antigens on the surface of the bacteria enabling detection of bacteria using a spectrofluorometer. The single-bacterium assay can be adapted for multiple-sample determination (>300 samples at one time) and is rapid, taking <20 minutes to complete sample preparation, instrumentation preparation, and sample determination. In addition, the bioassay can be used for multiple-pathogen quantification in situ with high specificity.
Current developments in cantilever array sensors are towards improvement of medical diagnostics tools, e.g. new ways to characterise complex solutions such as small amounts of blood or body-fluid samples. On the other hand, from a scientific point of view, the challenge lies in optimising cantilever sensors to improve the sensitivity until the ultimate limit is reached, which may be the nanomechanical detection of individual molecules. Further refinement of in vitro nanotechnology systems (cantilevers, nanowires) for rapid, sensitive analysis of disease biomarkers might take place within the next five years. Such systems could be easily expanded as new biomarkers are identified. Current implantable biosensors, equipped with technology to relay sensed information extracorporeally, are facing serious problems such as unwanted biofouling, i.e. non-specific adsorption of blood components and factors of the immune system on the sensing surfaces resulting in rapid loss of the ability of the sensor to detect the particular protein
over the background signal . Developing surface nanostrucures for implantable molecular sensors might tackle this still unsolved problem of biofouling. More realistically, nanotechnology might be expected to yield novel, biofouling-indifferent sensing strategies, based for instance on the measurement of physical properties.
Nanotechnology offers important new tools expected to have a great impact on many areas in medical technology. It provides extraordinary opportunities not only to improve materials and medical devices but also to create new smart devices and technologies where existing and more conventional technologies may be reaching their limits. It is expected to accelerate scientific as well as economic activities in medical research and development.
Nanotechnology has the potential to make significant contributions to disease detection, diagnosis, therapy, and prevention. Tools are important and integral parts for early detection. Novel tools and tools complementing existing ones are envisaged. It offers opportunities in multiple platforms for parallel applications, miniaturisation, integration, and automation. Nanotechnology could have a profound influence on disease prevention efforts because it offers innovative tools for understanding the cell as well as the differences between normal and abnormal cells. It could provide insights into the mechanism of transformation, which is fundamental in designing preventive strategies. Further, it provides novel non-invasive observation modalities into the cellular machinery. It allows for the analysis of such parameters as cellular mechanics, morphology, and cytoskeleton, which have been difficult to achieve using conventional technologies.
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