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The development of biochips is a major thrust of the rapidly growing biotechnology industry, which encompasses a very diverse range of research efforts including genomics, proteomics, and pharmaceuticals, among other activities.
Advances in these areas are giving scientists new methods for unravelling the complex biochemical processes occurring inside cells, with the larger goal of understanding and treating human diseases. At the same time, the semiconductor industry has been steadily perfecting the science of micro-miniaturization. The merging of these two fields in recent years has enabled biotechnologists to begin packing their traditionally bulky sensing tools into smaller and smaller spaces, onto so-called biochips. These chips are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions. Biochips enable researchers to quickly screen large numbers of biological analytes for a variety of purposes, from disease diagnosis to detection of bioterrorism agents.
Biosensors are powerful tools aimed at providing selective identification of toxic chemical compounds at ultratrace levels in industrial products, chemical substances, environmental samples (e.g., air, soil, and water) or biological systems (e.g., bacteria, virus, or tissue components) for biomedical diagnosis. Combining the exquisite specificity of biological recognition probes and the excellent sensitivity of laser-based optical detection, biosensors are capable of detecting and differentiating big/chemical constituents of complex systems in order to provide unambiguous identification and accurate quantification. A new generation of biosensors discussed in this presentation uses antibody and DNA probes.
Antibody-based fluoroimmunosensors (FISs)
Antibody-based fluoroimmunosensors (FISs) have been developed for the carcinogen benzo[a]pyrene (BaP) and related adducts such as benzopyrene tetrol (BPT). Polyclonal or monoclonal antibodies produced are immobilized at the terminus of a fiber optics probe or contained in a micro-sensing cavity within the FIS for use both in in-vitro and in-vivo fluorescence assays. High sensitivity is provided by laser excitation and optical detection. The FIS device utilizes the back-scattering of light emitted at the remote sensor probe. A single fiber is used to transmit the excitation radiation into the sample and collect the fluorescence emission from the antigen. The laser radiation reaches the sensor probe and excites the BaP bound to the antibodies immobilized at the fiberoptics probe. The excellent sensitivity of this device illustrates that it has considerable potential to perform trace analyses of chemical and biological samples in complex matrices. Measurements are simple and rapid (~ 5 min), and the technique is applicable to other compounds provided appropriate antibodies are used.
Antibody-based fluoroimmunosensors (FISs) In the recent investigations a new generation of biosensors using DNA probes (DNA Biochip). Probe recognition is based on the molecular hybridization process, which involves the joining of a strand of nucleic acid with a complementary sequence. Biologically active DNA probes are directly immobilized on optical transducers which allow detection of Raman, SERS, or fluorescent probe labels. DNA biosensors could have useful applications in areas where nucleic acid identification is involved. The DNA probes could be used to diagnose genetic susceptibility and diseases. The Biochip using antibody probes has recently been developed to detect the p53 protein system.
Cancer Diagnostics Using LIF
Cancer Diagnostics Using LIF: A minimally invasive method using laser-induced fluorescence (LIF) for in vivo cancer diagnosis has been developed by scientists at the Oak Ridge National Laboratory (ORNL) and the Thompson Cancer Survival Center (TCSC). Autofluorescence of normal and malignant tissues was measured directly using a fiberoptic probe inserted through an endoscope. The measurements were performed in vivo during routine endoscopy and did not require biopsies. The results of this LIF approach were compared with histopathology results of conventional biopsy samples and indicated excellent agreement in the classification of normal and malignant tumors for the samples investigated. The LIF procedure could lead to the development of a rapid and cost-effective technique for cancer diagnosis.
Exploring the Sanctuary of Individual Living Cell: The combination of nanotechnology, biology, advanced materials and photonics opens the possibility of detecting and manipulating atoms and molecules using nano-devices, which have the potential for a wide variety of medical uses at the cellular level. We have recently reported the development of nano-biosensors and in situ intracellular measurements of single cells using antibody-based nanoprobes. The nano-scale size of this new class of sensors also allows for measurements in the smallest of environments. One such environment that has evoked a great deal of interest is that of individual cells. Using these nanosensors, it is possible to probe individual chemical species and molecular signalling processes in specific locations within a cell. We have shown that insertion of a nano-biosensor into a mammalian somatic cell not only appears to have no effect on the cell membrane, but also does not affect the cell's normal function. The possibilities to monitor in vivo processes within living cells could dramatically improve our understanding.
Acoustic and Ultrasound Techniques for Monitoring Brain Injury: Providing windows into the body, ultrasound-based imaging has become an essential tool in the diagnostic arsenal of the physician. Ultrasonic technology can be made portable, easy to use, and safe. However, the use of ultrasound for diagnosing pathologies within the adult cranium have largely been limited to assessing blood flow in the major vessels. The skull has proven a challenging obstacle to the coherent propagation of ultrasonic waves, making imaging difficult. A largely unexplored method for the assessment of brain injury is ultrasonic tissue characterization. Ultrasonic tissue characterization has proven to provide sensitive and selective indicators of the state of vital tissues in more sonically accessible organs such as the heart. The backscattered signals were acquired using various probes that permitted us to cover a decade in the ultrasonic spectrum (500 kHz to 5 MHz). We investigate the utility of various parameters in detecting induced asymmetries in the brains of the subjects. The goal of this work is develop a rapid screening
Plasmonics refers to the research area of enhanced electromagnetic properties of metallic nanostructures. The term plasmonics is derived from “plasmons”, which are the quanta associated with longitudinal waves propagating in matter through the collective motion of large numbers of electrons. Incident light irradiating these surfaces excites conduction electrons in the metal, and induces excitation of surface plasmons leading to enormous electromagnetic enhancement of spectral signature, such as surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) for ultrasensitive detection of chemical and biological species. Biological process such as cell differentiation, cell division, apoptosis, phagocytosis and necrosis are associated with spatial reorganization of cellular components. Therefore new techniques are being developed in our laboratory to monitor the behavior of the molecules in key processes of the cell’s existence. External labeling of molecules of interest by chemical or recombinant techniques has enabled tracking of individual molecules using fluorescence microscopy with great sensitivity. In our laboratory, novel alternative microscopic techniques such as Surface-enhanced Raman scattering (SERS) that are chemically specific and allow the interrogation of molecules are being investigated.
Nanoparticles are increasingly finding a wide application in the biological studies due to their unique physical and chemical properties. However, biological and medical applications would require nanoparticles to be conjugated to biomolecules. A universal approach for conjugation of silver colloidal nanoparticles to biomolecules has been developed in our group. Surface functionalized silver colloids were labeled with a Raman active dye and bioreceptor molecule and used as labels for cellular imaging. The silver colloidal nanoparticles are efficient substrates that exhibit SERS phenomenon by enhancing the scattering cross sections of conjugated Raman active molecules thus enabling highly sensitive biological probes. In addition SERS nanotechnology and confocal surface-enhanced Raman imaging (SERI) using a acousto-optic tunable filter (AOTF)-based hyperspectral surface-enhanced Raman imaging (HSERI) system equipped with an intensified charged-coupled device has been developed to monitor the intracellular distribution of molecular species associated with biological abnormalities and localization of drugs and other cellular components within cells and thus offering a promising application for molecular signaling monitoring for nanomedicine applications.
Near-field scanning optical microscopy (NSOM)
Near-field scanning optical microscopy (NSOM) investigated in the laboratories is an emerging technique with its excellent resolving power less than 100 nanometer domains, and nondestructive nature compared to other scanning probe microscopic techniques. Non-destructive imaging of biomolecules in nanoscale domains is of considerable interest. At the single-molecule level resolution, it is possible to use the NSOM as a critical tool for visualization of cellular components labeled with fluorescent molecules. NSOM technology obtains more fundamental information about the cellular component’s orientation and locality without disturbing their original orientation and position, and level of interaction with surface. Several areas of science and medicine can benefit from this type of studies especially for molecular imaging, biomedical and biochip applications.
Currently we have investigated confocal microscopy and NSOM to study the localization of multidrug resistance (MDR) transport protein and their effect on chemotherapeutic drugs. Multidrug resistance (MDR) is the generic term used to indicate the variety of strategies that tumor cells are able to develop in order to evade cytotoxic effects of anticancer drugs. Previous studies have demonstrated that there are three major changes in cells that develop MDR: 1) decreased accumulation of cytotoxic drugs; 2) changes in activity or expression of certain cellular proteins, including the P-glycoprotein (Pgp, P for permeability), MDR-associated protein (MRP1), glutathione S-transferase p , protein kinase C and DNA topoisomerase II; and 3) changes in cellular physiology affecting the structure of the plasma membrane, the cytosolic pH, and the rates and extent of intracellular transport of membranes, as well as lysosomal structure and function. Current studies in our laboratory are focused to the investigation of these processes at the molecular and single-cell level.
Real-time monitoring of molecular signaling processes in a live cell
Real-time monitoring of molecular signaling processes in a live cell is a significant challenge for the next phase of genomics and proteomics studies. Conventional methods for determining molecule interactions often employ in vitro approaches using fixed cells or cell lysates. These methods are often time-consuming or less sensitive and may not represent true physiological conditions of cells in vivo. Furthermore, they cannot provide us any dynamic information about molecular interactions during cell division or environmental changes. To bridge the gap created by the inability of these conventional techniques to produce in vivo measurements in living cells, we have recently developed a class of unique optical nanobiosensors that can be inserted into single living cells to monitor and measure biomolecules and biochemicals of biomedical interest without disrupting normal cellular processes. Optical nanobiosensors are integrated nanoscale devices consisting of a biological recognition molecule coupled to the optical transducing element such as an optical nanofiber interfaced to a photometric detection system. They are capable of providing specific quantitative, semi-quantitative or qualitative analytical information using biological recognition elements (e.g., DNA, protein) in direct spatial contact with a solid-state optical transducer element. This nanobiotechnology-based devices are being developed in our laboratory and could provide unprecedented insights into intact cell function, allowing, for the first time, studies of molecular functions (such as apoptosis, DNA-protein interactions, protein-protein interaction, functioning of nanomachines, etc.) in the context of the functional cell architecture in a systems biology approach. These devices will lead to novel and powerful tools for fundamental biological research, ultra-high throughput drug screening and medical diagnostics applications.
The development of biochips is a major thrust of the rapidly growing biotechnology industry, which encompasses a very diverse range of research efforts including genomics, proteomics, and pharmaceuticals, among other activities. Advances in these areas are giving scientists new methods for unravelling the complex biochemical processes occurring inside cells, with the larger goal of understanding and treating human diseases. At the same time, the semiconductor industry has been steadily perfecting the science of micro-miniaturization. The merging of these two fields in recent years has enabled biotechnologists to begin packing their traditionally bulky sensing tools into smaller and smaller spaces, onto so-called biochips. These chips are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions. Biochips enable researchers to quickly screen large numbers of biological analytes for a variety of purposes, from disease diagnosis to detection of bioterrorism agents.