text:
|Publication number
|Publication type
|Application number
|Publication date
|Dec 28, 2010
|Filing date
|Feb 5, 2007
|Priority date
|Jul 7, 1994
|Fee status
|Also published as
|Publication number
|Jing Cheng, Lei Wu, Michael J. Heller, Edward L. Sheldon, Jonathan M. Diver, James P. O'Connell, Dan Smolko, Shila Jalali, David Willoughby
|Original Assignee
|Gamida For Life B.V.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (179), Non-Patent Citations (44), Referenced by (1), Classifications (17), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 10/163,835, filed Jun. 5, 2002, entitled "Integrated Portable Biological Detection System", issued on Feb. 6, 2007 as U.S. Pat. No. 7,172,896, which is a continuation of application Ser. No. 09/470,448, filed Dec. 22, 1999, entitled "Integrated Portable Biological Detection System", now U.S. Pat. No. 6,403,367 which claims benefit of Provisional Patent Application Ser. No. 60/113,730, filed Dec. 23, 1998, entitled "Fluorescent Imaging of Cells and Nucleic Acids in Bioelectronic Chips", and which is a continuation-in-part of application Ser. No. 09/016,596, filed Jan. 30, 1998, now U.S. Pat. No. 6,071,394, which is a continuation-in-part of application Ser. No. 08/709,358, filed Sep. 06, 1996, now U.S. Pat. No. 6,129,828, application Ser. No. 09/016,596 also being a continuation-in-part of application Ser. No. 08/986,065, filed Dec. 5, 1997, now U.S. Pat. 6,051,380, which is a continuation-in-part of application Ser. No. 08/304,657, filed Sep. 09, 1994, now U.S. Pat. No. 5,632,957, which is a continuation-in-part of application Ser. No. 08/271,882, filed Jul. 07, 1994, now U.S. Pat. No. 6,017,696, the specifications of which are hereby expressly and fully incorporated by reference as if fully set forth herein.
The Government has rights in this invention pursuant to Grant No. ATP: 70NANB7H3001 awarded by the Advanced Technology Program.
This invention relates to devices and methods for performing active, multi-step sample preparation and molecular diagnostic analysis of biological materials. More particularly, it relates to integrated, compact, portable devices for self-contained sample to answer systems. Specifically, this invention relates to a device and method for performing multi-step sample preparation and assay on either two or even a single microchip. Examples of applications for this integrated system include food and/or quality monitoring, diagnosis of infectious diseases and cancers, bone marrow plastesis (e.g., stem cell separation and analysis), and genetics-based identification of individuals for forensic purposes.
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
Generally, analysis of biological-derived sample materials cannot occur until the sample is processed through numerous pre-analysis steps. Often, the preparation process is time consuming and laborious. For example, many immuno and molecular-biological diagnostic assays on clinical samples, such as blood or tissue cells, require separation of the molecules of interest from the crude sample by disrupting or lysing the cells to release such molecules including proteins and nucleic acids (i.e., DNA and RNA) of interest, followed by purification of such proteins and/or nucleic acids. Only after performing processing steps can analysis of the molecules of interest begin. Additionally, protocols used for the actual analysis of the samples require numerous more steps before useful data is obtained.
For example, charged and uncharged microparticles in solution (such as cellular material or crude extracts of protein or nucleic acids thereof) may be separated by dielectrophoresis. On a microscale, dielectrophoresis can be performed using a glass slide-based device having exposed, i.e., naked, interdigitated electrodes plated on the surface of the slide and having a flow chamber with a volume of several hundred microliters. With such a device, cells, proteins, and nucleic acids can be separated based on their respective dielectric properties by using separation buffers having appropriate conductivity and an AC signal with a suitable amplitude and frequency. Such devices, however, have several problems including the nonspecific binding of both separated and unseparated cells to exposed portions of the glass surface and the electrodes. Such devices are also problematic in that the flow chamber volume (several hundred microliters) is so large that thermal convection can disturb and push materials such as cells and large molecules initially attracted to and retained by the electrodes off of the electrodes. Additionally, undesired cells and molecules are not easily washed off the surface without disturbing and loosing the desired cells as such cells and molecules can interfere with fluidic flow and, hence, block the flow during wash steps.
Conventional methods to disrupt whole cells for the release of proteins and nucleic acids have employed the use of a series of high voltage DC pulses in a macrodevice, as opposed to a microchip-based device. Such conventional electronic lysis techniques have several problems. For example, some commercial macro-devices use lysis conditions that do not release high molecular weight (larger than 20 Kb) nucleic acids because the high molecular weight molecules can not fit through pores created in the cell membranes using such methods. Additionally, released nucleic acids are often lost due to their non-specific binding to the surface of the lysis chamber. Such loss of material, especially when molecules of interest are in low concentration, is further compounded by the fact that the dielectrophoretic cell separation macro-device systems are stand alone systems allowing for loss of sample in the transfer of material from one device to the other as sample preparation is carried forward.
Processing of the crude lysate often requires chemical reactions to remove undesired cellular components from the specifically desired ones. These reactions typically include subjecting the lysate to enzymatic reactions such as proteinase K and restriction enzymes or nucleases. Processing can also include enhancing the presence of desired molecules, particularly nucleic acids, by performing amplification reactions such as by strand displacement amplification (SDA) or polymerase chain reaction (PCR) methodologies. These reactions are also carried out in stand-alone processes. Only after these sample preparation and processing steps can assaying for data retrieval begin. Because of the numerous steps between sample collection and assay, many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility.
Attempts have been made to use dielectrophoresis to separate and identify whole cells. For example, U.S. Pat. No. 4,326,934 to Pohl discloses a method and apparatus for cell classification by continuous dielectrophoresis. With such method cells are separated by making use of both the positive and negative dielectrophoretic movement of cell particles. Separated cells are allowed to be characterized and/or classified by viewing the characteristic deflection distance of cells moving through the positive and negative electrodes.
In another example, U.S. Pat. No. 5,344,535 to Belts et al. discloses a method and apparatus for the characterization of micro-organisms and other particles by dielectrophoresis. In this system, cells are characterized by matching their signature dielectrophoretic collection rates.
In yet another example, U.S. Pat. No. 5,569,367 to Belts et al. discloses a method and apparatus for separating a mixture of cells using a pair of energized interdigitated electrodes comprised of interweaved grid-like structures arranged to obstruct flow of cells through the apparatus and cause differentiation of cell types into fractions by applying a non-uniform alternating field.
In addition, other attempts have been made to combine certain processing steps or substeps together. For example, various microrobotic systems have been proposed for preparing arrays of DNA probes on a support material. Beattie et al., disclose in "The 1992 San Diego Conference: Genetic Recognition", November, 1992, use of a microrobotic system to deposit micro-droplets containing specific DNA sequences into individual microfabricated sample wells on a glass substrate.
Various other attempts have been made to describe integrated systems formed on a single chip or substrate, wherein multiple steps of an overall sample preparation and diagnostic system would be included. A. Manz et al., in "Miniaturized Total Chemical Analysis System: A Novel Concept For Chemical Sensing", Sensors And Actuators, B1(1990), pp. 244-248, describe a 'total chemical analysis system' (TAS) that comprises a modular construction of a miniaturized TAS. In that system, sample transport, chemical reactions, chromatographic separations and detection were to be automatically carried out.
Yet another proposed integrated system by Stapleton, U.S. Pat. No. 5,451,500, a system for automated detection of target nucleic acid sequences is described. In this system multiple biological samples are individually incorporated into matrices containing carriers in a two-dimensional format.
Various multiple electrode systems are also disclosed which purport to perform multiple aspects of biological sample preparation or analysis. Pace, U.S. Pat. No. 4,908,112, entitled "Silicon Semiconductor Wafer for Analyzing Micronic Biological Samples" describes an analytical separation device in which a capillary-sized conduit is formed by a channel in a semiconductor device, wherein electrodes are positioned in the channel to activate motion of liquids through the conduit. Additionally, Soane et al., in U.S. Pat. No. 5,126,022, entitled "Method and Device for Moving Molecules by the Application of a Plurality of Electrical Fields", describes a system by which materials are moved through trenches by application of electric potentials to electrodes in which selected components may be guided to various trenches filled with antigen-antibodies reactive with given charged particles being moved in the medium or moved into contact with complementary components, dyes, fluorescent tags, radiolabels, enzyme-specific tags or other types of chemicals for any number of purposes such as various transformations which are either physical or chemical in nature. Further, Clark, et al. in U.S. Pat. No. 5,194,133, entitled "Sensor Devices", discloses a sensor device for the analysis of a sample fluid which includes a substrate having a surface in which is formed an elongate micro-machined channel containing a material, such as starch, agarose, alginate, carrageenan or polyacrylamide polymer gel, for causing separation of the sample fluid as the fluid passes along the channel. The biological material may comprise, for example, a binding protein, an antibody, a lectin, an enzyme, a sequence of enzymes, or a lipid.
Various devices for eluting DNA from various surfaces are known. For example, Shukla U.S. Pat. No. 5,340,449, entitled "Apparatus for Electroelution" describes a system and method for the elution of macromolecules such as proteins and nucleic acids from solid phase matrix materials such as polyacrylamide, agarose and membranes such as PVDF in an electric field. Materials are eluted from the solid phase into a volume defined in part by molecular weight cut-off membranes. Also, Okano, et al. in U.S. Pat. No. 5,434,049, entitled "Separation of Polynucleotides Using Supports Having a Plurality of Electrode-Containing Cells" discloses a method for detecting a plurality of target polynucleotides in a sample, the method including the step of applying a potential to individual chambers so as to serve as electrodes to elute captured target polynucleotides, the eluted material is then available for collection.
Other devices for performing nucleic acid diagnosis have been designed wherein at least two reaction chambers are necessary for carryout the sample preparation and analysis such as R. Lipshutz, et al., entitled "Integrated Nucleic Acid Diagnostic Device" (U.S. Pat. No. 5,856,174) and R. Anderson, et al., entitled "Integrated. Nucleic Acid Diagnostic Device", (U.S. Pat. No. 5,922,591).
Still other achievements have been made toward partial integration of a complete sample handling system such as P. Wilding, et al., "Integrated cell isolation and PCR analysis using silicon microfilter-chambers," Anal. Biochem. 257, pp. 95-100, 1998; and P. C. H. Li and D. J. Harrison, "Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects," Anal. Chem., 69, pp. 1564-1568, 1997.
Still others have attempted to integrate chemical reactions with detection such as M. A. Burns, et al., "An integrated nanoliter DNA analysis device," Science, 282, pp. 484-487, 1998; S. C. Jacobson and J. M. Ramsey, "Integrated microdevice for DNA restriction fragment analysis," Anal. Chem., 68, pp. 720-723, 1996; L. C. Waters, et al., "Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing," Anal. Chem., 70, pp. 158-162, 1998; and A. T. Woolley, et al., "Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device," Anal. Chem., 68, pp. 4081-4086, 1996.
Generally, as is understandable from the forgoing examples, systems and methods have been described that do not fully provide for a completely integrated self-contained sample to answer system that uses electronically active microchips. Moreover, numerous of the described systems are extremely labor and time intensive requiring multiple steps and human intervention either during the process or between processes which together are suboptimal allowing for loss of sample, contamination, and operator error. Further, the use of multiple processing steps using multiple machines or complicated robotic systems for performing the individual processes is often prohibitive except for the largest laboratories, both in terms of the expense and physical space requirements. For the reasons stated above, these techniques are limited and lacking. They are not easily combined to form a system that can carry out a complete self-contained integrated diagnostic assay, particularly assays for data retrieval for nucleic acids and protein-derived information, on a single electronically addressable microchip. Despite the long-recognized need for such an integrated system without a complicated fluidics and inadequate valve systems, no satisfactory solution has previously been proposed. There is therefore a continuing need for methods and devices which lead to improved dielectrophoretic separation of biological cells as well as improved biological stability of the separated cells and further a continuing need for methods and devices which improve cell preparation and analysis, and which are capable of integrating cell separation, preparation, purification, and analysis in a single self-contained system without complicated fluidics.
Accordingly, provided herein are integrated, portable systems, devices, and methods for performing active, integrated multi-step sample preparation and molecular diagnostic analysis of biological samples using electronically addressable microchips.
In one aspect of the present invention, an integrated system for the analysis of eukaroytic and/or prokaryotic cells in a biological sample is provided. The system comprises an electronic system for cell separation, cell lysis, sample preparation, and sample analysis further comprising a flow cell, an input port coupled to the flow cell, an output port coupled to the flow cell, a plurality of individually addressable electrodes positioned within the flow cell and coupled to a power source configured to electronically disrupt cell membranes within the sample, and an array of probes coupled to the electrodes and adapted to bind to predetermined components within the sample for analysis of the components, and a heating element coupled to the flow cell, wherein the flow cell is adapted to receive the sample via the input. The system further includes a detector operatively positioned to detect the molecules bound to the probes by a detectable signal, a power source coupled to the flow cell, and a portable housing configured to contain the electronic system, the detector, and the power source.
The system may include an illumination source operatively positioned to direct radiation to the probes. In one embodiment, the illumination source is a laser, and the system further comprises a beam splitter. This system may also comprise a desalting column coupled to the flow cell. The desalting column may be configured to introduce a buffer into the flow cell.
The electronic system may include a permeation layer overlaying the electrodes, and the probes may be coupled to the permeation layer. Additionally, in one embodiment, the electrodes are addressable to form a square-wall dielectric force pattern. Alternatively, in another embodiment, the electrodes are addressable to form a checkerboard dielectric force pattern. The system may also include a wave form generator coupled to the electrodes. The electronic system may also include first and second flow cells, wherein the first flow cell is configured for sample preparation, and the second flow cell is configured for sample analysis. Furthermore, a computer may be coupled to the portable housing and located outside of the portable housing.
In another aspect of the present invention, a method for performing an analysis of a biological sample having desired cells and undesired cells using an integrated portable system comprising a flow cell, a plurality of individually addressable electrodes positioned within the flow cell, and a portable housing containing the flow cell is provided. The sample may be introduced into the flow cell via an input port coupled to the flow cell. A dielectric force pattern may be created by individually biasing the electrodes positioned within the flow cell. The sample may then be subjected to the dielectric force pattern in order to separate the undesired cells from the desired cells in the sample. Then, the desired cells may be isolated by maintaining an attractive bias for the desired cells and introducing a flow of wash buffer through the flow cell via the input port to eliminate the undesired cells. Here, the undesired cells may be removed from the flow cell via an output port coupled to the flow cell. The desired cells may be lysed by applying an electric force within the flow cell to electronically disrupt the desired cells into a plurality of components. The components of the desired cells may then be analyzed by hybridizing the components with probes to form probe-target hybrids within the flow cell, and detecting the probe-target hybrids while the hybrids are within the flow cell, wherein the probes are coupled to the electrodes.
In one embodiment, the attractive bias maintained during the isolating the desired cells step comprises a square-wall dielectric force pattern. In another embodiment, the attractive bias maintained during the isolating the desired cells step comprises a checkerboard dielectric force pattern.
The isolation of the desired cells may be accomplished by generating a dielectric force pattern having a plurality of field maxima and a plurality of field minima, wherein the attractive bias comprises the field maxima, and the undesired cells collect at the field minima.
Creating a dielectric force pattern may be performed by biasing all of the individual electrodes as a single array. Alternatively, creating a dielectric force pattern may be performed by biasing the individual electrodes as a plurality of subarrays, wherein each subarray may be used for a function such as, e.g., cell separation, cell lysis, or cell analysis.
After lysing the desired cells, the desired cells may be purified by introducing a protease into the flow cell via the input port, maintaining a temperature of substantially 60� C. in the flow cell to treat the desired cells with the protease, and maintaining a temperature of substantially 95� C. in the flow cell to inactivate the protease after treating the desired cells with the protease. Maintaining a temperature may be accomplished using a heat element coupled to the flow cell and contained within the portable housing. Additionally, the purified desired cells may be treated with an enzyme to release a specific protein of interest from the desired cells. Here, the enzyme may be introduced into the flow cell via the input port. The specific protein of interest may then be amplified and labeled with a marker. Amplifying the specific protein of interest may be accomplished by suitable methods such as, e.g., PCR, or SDA.
The method may also include the detection of the probe-target hybrids by using fluorophore-labeled reporter probes and an optical imaging system configured to detect the reporter probes.
In another aspect of the present invention, a method for performing an analysis of a biological sample having desired cells and undesired cells using an integrated portable system is provided. An integrated portable system comprising an electronic system having a flow cell, a plurality of individually addressable electrodes positioned within the flow cell, a plurality of probes coupled to the electrodes, a detector configured to detect the probes, a power source coupled to the flow cell, and a portable housing containing the electronic system, the detector, and the power source may be provided. The sample may be introduced into the integrated portable system by injecting the sample into the flow cell via an input port coupled to the flow cell. The input port may be accessed through the portable housing. A dielectric force pattern may be created by individually biasing the electrodes positioned within the flow cell. The sample may be subject to the dielectric force pattern in order to separate the undesired cells from the desired cells in the sample. The desired cells may be isolated by maintaining an attractive bias for the desired cells and introducing a flow of wash buffer through the flow cell via the input port to eliminate the undesired cells. The undesired cells may be removed from the flow cell via an output port coupled to the flow cell. The desired cells may be lysed by applying an electric force within the flow cell to electronically disrupt the desired cells into a plurality of components. The components of the desired cells may then be analyzed by hybridizing the components with probes to form probe-target hybrids within the flow cell, and then detecting the probe-target hybrids while the hybrids are within the flow cell.
Regardless of the embodiment, the systems and methods of the invention generally provide the ability to (1) separate eukaryotic cell types from one another as well as eukaryotic cell types from prokarotic cell types, (2) directly process the sample materials from a crude state to a more refined state, and (3) directly analyze the sample materials on the microchip grid. Such an ability is possible by the novel use of electronic biasing at one level of voltage in the form of a dielectric current to cause dielectrophoresis of cells, followed by an increase in voltage to lyse captured cells, followed in turn by changing the manner of biasing from an alternating current mode to direct current mode for the addressing of specific electrodes on the arrays of the flow cell(s) to cause the transport of molecules of interest for capture/hybridization to probes previously bound to the electrode array. The present invention further contemplates that other appropriate sample preparation reagents may be transported to and away from the flow cell(s) by simplified arrangement of tubing and solenoid operated valves and pump. Additionally, in embodiments having a first flow chamber that is without a microchip, the system contemplates the ability to directly lyse the cells in the sample and analyze materials of interest without a need for separating the cell types. In such embodiment, the flow cell has a heating element that can be used to raise the temperature for direct lysis of the cells in the sample. Following such lysis, sample preparation steps such as protease treatment or nucleic acid amplification may be performed followed by transporting the amplified species to the second flow cell containing the electronically addressable microchip.
Also, numerous techniques can be performed in the preparation of molecules of interest including, but not limited to, enzymatic treatment using protease K to remove proteinaceous materials from nucleic acids of interest, enzymatic treatment using nucleases to remove nucleic acids from proteins, digestive residue adsorption, nucleic acid amplification (e.g., by PCR and SDA), in situ buffer exchange and binding of antibodies or other protein-protein binding reactions such as receptor-ligand or enzyme-substrate for binding to proteins of interest.
Analysis of prepared sample materials can also comprise any number of preselected hybridization formats. For example, nucleic acids of interest may be hybridized selectively through an electronically directed process as is known to those skilled in the art of electronically addressable microchips. Such hybridization formats comprise binding of nucleic acids (RNA, DNA, pNA) to probes anchored to the microarray. Other formats contemplated for use with the systems and methods of the present invention include selective capture of proteins of interest such as by antibody or other protein binding probes attached to the electronic grid. These can include other protein-protein interactions such as receptor-ligand and enzyme-substrate binding.
The present invention also contemplates the use of elements (e.g. buffer vials, tubing, miniature solenoid valves, and at least one pump) for carrying and transporting samples, buffers, enzymes and reagents to and from said flow cell(s). Additionally, other elements that may be used with the present invention include a battery operated diode laser (preferably having a wavelength of 635 nm), LEDs, and a CCD camera coupled with filters and zoom lens for astronomy of the individual electrodes of the microchip grid(s) of the first and/or second flow cell chamber(s). Still other methods of detection are also contemplated not requiring illumination with a light emitting device such as direct electrochemical detection as is well known by those in the art of such detection as described in P. Ropp and H. Holden Thorp, Chemistry & Biology, 1999, Vol. 6, No. 9, pp. 599-605. Each of these electronic components are further contemplated to be coordinated through a computer and appropriate programming software as is well understood by those skilled in the electronic arts.
The invention will be further described with reference to the accompanying drawings in which:
Referring now to specific embodiments of the invention, a portable lab-on-a-chip system is provided wherein a sample may be processed and analyzed in at least a single flow cell comprising an electronically addressable microchip. The elements of the device are housed in a portable casing or housing which contains the sample preparation and analysis flow chamber, fluid handling systems, and illumination source (such as a battery operated 635 nm diode laser) and detection electronics (such as a CCD camera coupled with a set of filters and a zoom lens for astronomy of the microchip). External to the portable casing is a computer, such as a personal computer connected to the portable housing by a cable.
Referring now to
Sample handling in stage (1) processing generally comprises processing of crude biological samples (e.g., cells from blood, urine, stool, mixed cell populations, etc.) for the purpose of isolating molecules of interest such as nucleic acids and proteins. Sample handling at the chemical reaction stage (2) involves potentially many types of molecular biological reactions for clean-up and further isolation, purification, or amplification of molecules of interest including, but not limited to, enzymatic-based reactions such a treatment with proteases, nucleases and restriction enzyme digestion, PCR and SDA-based nucleic acid amplification, in situ buffer exchanges, chemical labeling such as by radioisotope and fluorescence markers, and immuno-based and protein-protein reactions such as antibody-antigen, ligand-receptor, and enzyme-substrate reactions. Sample handling at the analyte detection stage (3) may be accomplished through numerous formats including optical detection of fluorescent emissions, electrochemical detection, and radioisotope detection. In a preferred embodiment, this detection comprises hybridization of nucleic acids or captured proteins to the electronic grid and detection using fluorescent imaging.
As will be understood by one of skill in the art, the flow cell, as well as the other electrically operated components, such as solenoid operated valves and pumps, laser and detection camera, are interconnected electronically with the computer 20 by cabling 19 to the housing 18 for all programming and control purposes. By way of example, the power source and computer are connected to the electrodes of the flow cell 11 for programming and manipulation. In the preferred embodiment, the electronic signals that may be generated by the computer software used with the system include an AC component, such as where the AC component is sinusoidal and used in dielectrophoresis, and such as where a DC component is square and used in hybridization. In yet a further aspect, it is preferred that a time varying electronic signal also used in the system includes an offset signal, such as where the offset signal is a DC signal.
As an example of a flow cell design,
The microchip can be then attached to a molded or machined medical grade plastic flow cell 27 so that the electronic microarray 23 makes up the inner bottom surface of a well within the flow chamber. The flow cell 27 provides a compartment 29 for containing biological sample materials and buffers to be layered on top of the microchip. The flow cell may be further designed to have at least 2 to 4 ports 30 for sample delivery and extraction. The flow cell 27 may further be constructed so as to accept tubing for interfacing with exterior fluidic delivery and removal systems as well as desalting. Additionally, the flow cell compartment is covered and sealed by a fused quartz window or lid 28 for visual access to the microarray analysis sites or capture pads. This window 28 can comprise any thickness of quartz but generally is about 0.015 inches thick.
The window 28 may be attached to the flow cell and the flow cell to the chip by any number of methods of which a preferred method uses an ultraviolet (UV) curing adhesive 26 developed for optics assembly. Attachment of the flow cell to the chip may be either directly to a bare surface of the chip or to the permeation layer overlaying the microarray also using adhesive 26. Tubing is attached to the inlet and outlet ports using a variety of methods depending on the tubing size, fittings, and the tubing base material.
In an alternative embodiment, the integrated system may use two flow cells, one for sample preparation and the second for analysis.
The second flow cell 32 is used for sample analysis. In a preferred embodiment both flow cells 31 and 32 are disposed on the same side of the support 10. However, these flow cells may be disposed in alternative arrangements, including a back-to-back or a stacked configuration. Additionally, these two flow cells may be connected via a hollow fiber in order to purify, desalt and introduce different buffers into the flow stream. Moreover, each flow cell may be designed to have independent temperature control elements as desired. As will be understood by one of skill in the art, each of the electronically addressable microchips within the flow cells, as well as the other electrically operated components, such as solenoid operated valves and pumps, are interconnected electronically with the computer 20 by cabling for all programming and control purposes as in the single flow cell arrangement.
Whether using a single or multiple flow cell integrated system of the invention, cell separation is achieved by a dielectrophoresis technique. In such method, polarizeable particles (e.g., eukaryotic and prokaryotic cells) including those with no net charge are subject to a "dielectrophoretic" force of a non-uniform electrical field. As long as the effective polarizability of the particles is different from that of the surrounding medium, the cells will remain subject to the dielectric force. The direction of migration of different cell types is determined by: (1) surface charges of the cell walls or membranes of the cell bilipid membranes, (2) the conductivity and permitivity of such cell membranes and walls, and (3) the morphologies and structural architectures of the cells. Dielectrophoresis as practiced in the current invention has been used to selectively separate from mixed cell populations (e.g., blood cells) several types of bacteria including Escherichia coli, Salmonella typhimurium, Micrococcus lysodeikticus and Staphylococcus epidermidis as well as cancer cells such as cultured cervical carcinoma cells.
In a preferred embodiment, a flow cell useful for separating cells as practiced in this invention may be designed to include an electronically addressable microchip having at least 100 individually addressable microelectrodes capable of being biased individually so that a dielectric force pattern may be generated across the microchip electronic grid. For example,
Additionally, the electrodes of the microarray in the flow cell(s) may be biased either all together as a single array for carrying out cell separation, lysis, and analysis, or may be programmed to form subarrays in any number of patterns for carrying out the separation, lysis, and analysis steps. In other words, one subset may be biased to separate cells, another set may be biased to cause cell lysis, and yet another set may be used for analysis.
In an example of cell separation,
Undesired cells are washed from the flow chamber while retaining the cells of interest. This is accomplished by maintaining an attractive bias for the cells of interest and creating a flow of wash buffer through the flow chamber. Once the cells of interest are isolated in the flow chamber they may be treated in any number of ways for further processing. In one embodiment, the cells are lysed by applying high voltage pulses of up to 450 volts with a pulse width between 10 μs and 50 μs as described in J. Cheng, et al., "Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips," Nature Biotechnol., 16, pp. 541-546, 1998. In another example, E. coli cells were separated from blood cells as shown in
In another example, mammalian cells are separated on the electronic grid of the flow chamber.
Following lysis, both cellular proteins and nucleic acids may be retained for analysis. An example showing that nucleic acid may be retained from lysed cells is provided in
Where nucleic acids are desired for analysis, further clean-up and purification may include treatment with proteases such as with Proteinase K. After such treatment, the lysate may either be analysed immediately, or amplified then analysed. If the alternate embodiment is used, the sample may be further treated in the flow chamber or may be exported to the second chamber for such protease treatment, amplification and analysis. Generally, a protease treatment may be carried out in an appropriate buffer at 60� C. for 15 min. Temperature control may be achieved by use of the ceramic heater element attached to the back of the microchip. Inactivation of proteinase K may be achieved by heating the sample at 95� C. for 2 min. Overall, the cell separation, lysis, and protease treatment process can take between 15-25 minutes.
Where proteins are desired, nucleic acids may be removed by treating the sample with restriction enzymes and nucleases. The proteins may further be treated with various enzymes and partial protease treatments to release specific proteins of interest from cell membranes and other cellular components. Following the above sample processing steps additional processing may be carried out including amplification (e.g., by PCR and SDA) and labeling with radioisotopic or fluorescent markers.
In an example of chemical reaction stage processing, specific nucleic acid sequences, (i.e., the invA and spa Q genes of Salmonella enterica) were amplified using SDA. SDA is preferred in this portable system as the amplification may be performed under isothermal conditions at between 50� C. and 60� C. thereby eliminating high temperature denaturation cycles associated with PCR. However, in one embodiment, the portable system is capable of performing PCR amplification as the flow chamber is equipped with the aforementioned heating element that has the capability of achieving repetitive high temperature cycling.
As shown in
The total nucleic acid from the bacteria cells was isolated by first lysing the cells by energizing the electrodes with pulsed direct current at 200 V having a square wave form af 10 ms duration and a total of 40 separate pulses. (Alternatively, the cells can be lysed by heating the chamber to 95� C. for five minutes). A concentrated SDA reagent and buffer stock mix was introduced into the flow cell and mixed with the denatured target nucleic acid to give the following final concentrations of SDA reaction components: 500 nM amplification primers, 50 nM 'bumper' primers, 9.5 mM magnesium acetate, 35 mM potassium phosphate buffer pH 7.6, 80 μg/ml bovine serum albumin, and 1.4 mM each of dATP, dGTP, TTP, and alpha-thiolated dCTP. The amplification primers were designed to amplify 81 base pairs of the invA or spa Q gene and comprised the following nucleic acid sequences:
spa Q Seq. Id. No. 1 5′-accgcatcgaatgcatgtctcgggtcctggtagggttattc-3′ spa Q Seq. Id. No. 2 5′-cgattccgctccagacttctcgggaacacacgccaagta-3′ inv A Seq. Id. No. 3 5′-accgcatcgaatgcatgtctcgggtttcaacgtttcctgcg-3′ inv A Seq. Id. No. 4 5′-cgattccgctccagacttctcgggatcgataatgccagacg-3′
Bumper primers comprised the following sequences:
spa Q
Seq. Id. No. 5
spa Q
Seq. Id. No. 6
inv A
Seq. Id. No. 7
inv A
Seq. Id. No. 8
The released nucleic acid was then denatured by heating the chamber to 90� C. for five minutes. The chamber was then brought to 60� C. and 10 μl of SDA reagent buffer containing enzymes was introduced into the chamber to initiate amplification (i.e., 40 units of BsoB1 restriction endonuclease and 16 units of exo-Bst DNA polymerase).
In the system of the invention, amplification using SDA may be carried out for between 25 and 35 minutes. In the present example, after 30 minutes of reaction a 5 μl aliquot of the reaction volume was removed for PAGE analysis (
The third stage of sample handling comprising detection of the molecules of interest is preferably carried out for proteins and nucleic acids of interest. Such detection can use various forms of hybridization to probes previously attached to the microarray. For example, nucleic acids (e.g., RNA, DNA, and pNA) may be used for binding sample-derived nucleic acid analyte (e.g., amplified or unamplified target nucleic acids) by hybridization. Proteins may also be made to bind to capture molecules (i.e., protein-ligand binding interactions) attached to the array. Such capture molecules may comprise proteins or other molecules and the binding interaction can comprise such interactions as antigen-antibody, enzyme-substrate, and receptor-ligand binding.
With respect to nucleic acids, target species, whether amplified or not, are electronically addressed to specified capture pads of the microarray of either the single (or the secondary) flow chamber for capture by oligonucleotide probes that are anchored thereto. Preferably, the electrode array of the flow cell, (i.e., flow cell 11, 31 a or 32 depending upon the embodiment and protocol used) has at least 25 individually-addressable electrodes coated with a permeation layer (e.g. for example, an acrylamide-based hydro-gel). The target nucleic acid is biased using a positive sinusoidal signal generated using a function generator/arbitrary wave form generator (33120A, Hewlett Packard, Santa Clara, Calif.). The capture probe-target hybrids are then detected using fluorophore-labeled reporter probes and the CCD-based optical imaging system employed for the portable instrument shown in
Continuing with the above example, the amplification products of the spa Q and inv A genes were available for capture and analysis. Prior to capture, the SDA reaction solution and amplification products were passed through a desalting column in a volume of about 75 ul followed by exchanging the buffer with a 50 mM Histidine buffer. The amplification products were then addressed to specific pads of the electrodes which contained gene specific probes attached to the permeation layer overlaying the electrodes. This was followed by washing the chamber with 200 mM NaCl, 10 mM Tris pH 8.0, 1 mMEDTA, followed in turn by introduction of a bodipy 630-labeled probe oligonucleotide specific for the amplification product at a concentration of 0.5 μM in 200 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, and 100 ug/ml Calf Thymus DNA. The reporter probe solution was left in the assay cell for 10 minutes then washed with 7-800 μl of 50 mM Histidine buffer. The chamber was then visualized using a 630 nM helium-neon laser and a computer controlled CCD camera.
Visual detection of amplification reaction products by probe hybridization may be performed by directing a battery operated diode laser having at least the capability of generating approximately 2 m Watts of laser power with an emission wavelength of 635 nm. The laser is used to excite fluorescent dye-label reporter probe (such as BODIPY-630). The wavelength of the emission filter is 670 nm. The dichromatic mirror has a wavelength cutoff at 645 nm. Alternatively, a direct electrochemical voltammetric detection system may also be used instead of light based detection as is understood by those skilled in the art.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
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|Jacobsen et al., Integrated Microdevice For DNA Restriction Fragment Analysis, Analytical Chemistry, 68, 5, Mar. 1, 1996, 720-723.
|Kakerow et al., A Monolithic Sensor Array Of Individually Addressable Microelectrodes, Sensors & Actuators, A43, 1994, 296-301.
|Li et al., Transport, Manipulation & Reaction Of Biological Cells Using Electrokinetic Effects , Analytical Chemistry, 69, 1997, 1564-1568.
|Manz et al., Miniaturized Total Chemical Analysis System: A Novel Concept For Chemical Sensing, Sensors & Actuators B1, 1990, 244-248.
|Mathews et al., Analytical Strategies For The Use Of DNA Probes, Analytical Biochemistry, 169, Feb. 1, 1988, 1-25.
|Palecek, New Trends In Electrochemical Analysis Of Nucleic Acids, Bioelectrochemistry & Bioenergetics, 20, 1988, 179-194.
|Ranki et al., Sandwich Hybridization As A Means For The Detection Of Nucleic Acids In Crude Samples, Gene, 21, 1983, 77-85.
|Saiki et al., Amplification Of Genomic DNA, PCR Protocols: A Guide To Methods & Amplifications , (Academic Press), 1990, 13-20.
|Sato et al., Individual & Mass Operation Of Biological Cells Using Micromechanical Silicon Devices, Sensors & Actuators A21, A23, 1990, 948-953.
|Scanning Laser Microscopy Lab Home page, website printout, http://sciborg.uwaterloo.ca/physics/research/confocal/main html, retreived Aug. 23, 2004, created 1996, redesigned 1999.
|Sosnowski, et al., "Rapid Determination Of Single Base Mismatch Mutations In DNA Hybrids By Direct Electric Field Control", Proc. Natl. Acad. Sci. USA, vol. 94, Feb. 1997, 1119-1123.
|Southern et al., Analyzing & Comparing Nucleic Acid Sequences By Hybridization To Arrays Of Oligonucleotides Evaluation Using Experimental Modes, Genomics, 13, 1992, 1008-1017.
|Strezoska et al., DNA Sequence Determination By Hybridization: 100 Bases Ready By A Non-Gel Based Method, Proc. Natl. Sci. USA, 88, Nov. 1991, 10089-10093.
|Wallace et al., Hybridization Of Synthetic Oligodcoxyribonucleotides To �174 DNA: The Effect Of Single Base Pair Mismatch, Nucleic Acids Research, 6, 11, 1979, 3543-3557.
|Washizu et al., Electrostatic Manipulative Of DNA In Microfabricated Structures, IEEE Transactions On Industry Applications, 26, 6, Nov.-Dec. 1990, 1165-1172.
|Washizu, Electrostatic Manipulation Of Biological Objects, Journal Of Electrostatics, 25, 1990, 109-123.
|Waters et al., Microchip Device For Cell Lysis, Multiplex PCR Amplification, & Electrophoretic Sizing, Analytical Chemistry, 70, 1998, 158-162.
|Wilding et al., Integrated Cell Isolation & PCR Analysis Using Silicon Microfilter-Chambers, Analytical Biochem, 257, 1998, 95-100.
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|Citing Patent
|Filing date
|Publication date
|Mar 17, 2014
|Aug 1, 2017
|Syracuse University
|Microfluidics polymerase chain reaction and high resolution melt detection
|U.S. Classification
|204/600, 204/643, 435/306.1, 435/259
|International Classification
|Cooperative Classification
|European Classification
|Jul 25, 2008
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Dissertation Paul Val Ry
There is a dissertation merely a crucial looking title for a lengthy essay – according to your own personal research. Publishing a dissertation can be an exciting, challenging, worthwhile and frequently amazing experience. In the time it may be stressful, time-consuming, exasperating and frustrating.Precisely the same standard construction is followed by many dissertations and therefore are comprised of an abstract five pieces approaches and talk, ideas and sources. Even though specific depth of each element can differ (such as the numbers of terms permitted while in the subjective), the inclusion of every element is standard and mounted. I prefer to assess the dissertation's design with all the structure of an orchestral work – generally your dissertation having a prelude, principal moves as well as an end. A great musician gets an encore also – and thus does a great dissertation.If you've completed in your study topic – welldone! Now you need to turn detective – accumulating every one of the information you can to support and verify your concepts and trying to find. Now is the time for analyzing and collecting together the literature within your discipline of research; data from wide and far – together with on your doorstep.As there are different varieties of data available addressing grips together with the massive array of methods is quite a task alone. By hotfooting it for your university catalogue to planning online and surfing the Planet Wide Web, you could possibly well uncover all of the info you need.Then publishing up a low -empirical dissertation is not unlikely to be described as a gratifying experience. A low-empirical, or theoretical, dissertation doesn't have any knowledge you've produced whatsoever. You won't have interviews, questionnaires or observations presenting but you do must control the framework and circulation of disagreement properly so your realization is sensible and also the part that is accomplished is coherent and clear.Investigators desire to be able to check out any reference in your work up by viewing most of the data you've used in a recognisable and clear format and simply flipping to the bibliography.Students generally get into a spin about referencing and bibliographies. But making a superior bibliography can be a quite straightforward method to obtain items. A properly-designed listing of resources is definitely a requirement and prerequisite so you can get a significant level. Not joining for references and the bibliography is a definitely unnecessary way be irrational to not spending some time on this task and you'd to work.List Of Dissertation Topic
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subset: commoncrawl


text:
I woke up on November 26, 2011 with hopes that a win over Michigan would redeem a season that had just followed up a three-game winning streak with back to back losses. The difference between the high of Braxton Miller's 40-yard touchdown pass to Devin Smith in the final minute against Wisconsin, and the low of the pathetic ending in West Lafayette was colossal.
Actually, scratch that. The low got even lower, when Ohio State would give Penn State their first Joe Paterno-less victory since 1965.
Either way, a win over that team up north heals all wounds. Especially in a season that had already made its way down the toilet, a win over the Wolverines would rescue the season, and continue what had become a November tradition. Never mind that Ohio State was 6-5 going into the game, Michigan was 9-2, and the Buckeyes hadn't lost to Michigan since 2003. Society hadn't yet seen a single Facebook status update about a Michigan win over Ohio State, because Facebook didn't exist the last time the Wolverines won The Game1.
Much like several other games in 2011, Ohio State had that one within reach. Just one overthrown pass from a true freshman QB is what kept the Buckeyes from keeping that streak alive, against what was supposed to be a Michigan team that was back. As that ball dropped, so did my heart. I wasn't quite sure what to do with myself for the rest of the day, so I hibernated and wasted away in my desolate room.
Later that night, when I decided to rejoin the real world, my brother and I went out for a drink and a bite to eat. We sat in front of TVs and watched as USC beat UCLA.
Actually, scratch that. We watched USC pummel UCLA in that kind of game that makes you abandon your basic human instinct to root for the underdog, because now you're just mad at the underdog for showing absolutely no resolve to at least make this assault semi-respectable.
This was now funny. The only thing that could redeem UCLA now was to lose so poorly they'd have no choice but to fire Rick Neuheisel. Even my UCLA alumnus brother (whose second UCLA degree was in progress at the time) was cheering on the Trojans in their quest to hang half a hundred on the apathetic Bruins.
Following the game, the 6-6 Bruins would be on their way to represent the Pac-12 South in the first-ever Pac-12 Championship, while the Trojans would be headed to the locker room for the final time that year. As they poured into that locker room, USC had Pac-12 South Division Champions t-shirts waiting for them. Unofficial, obviously, since they were at the tail end of their two year postseason ban, yet still... awesome.
The reminder to themselves, and to the college football world, that they were the rightful owners of that title was fitting. Not only did they certainly earn that recognition by what they did on the field, but USC was exactly the kind of program that would spin NCAA sanctions and a postseason ban into something you couldn't poke fun at them for.
How could you poke fun at a team that just put up 50 against their crosstown rival, made a point to remind everyone they were clearly the best team in their division, and laughed at the NCAA in the process?
As much as that moment impressed me, it impressed me even more so weeks later, when Ohio State's new head coach Urban Meyer was officially in place, and the NCAA handed Ohio State their own postseason ban, effective in a year that instantly came with high expectations. As a spectator, how do you keep a season exciting when what the team "plays for" each year has been removed?
USC fans started by reminding themselves that "you can't sanction the end zone." The traditional goals for the season are no longer available? Make a new one. Root for your team to destroy every team they face that year, and then just tell everyone where you deserved to be post-season.
As an outsider, it actually looked kind of fun! And frankly, it appears way less stressful than actually having to win a bowl game. Your team never loses a hypothetical gloat-fest! They are literally undefeated when you get to completely make up a bowl game that never happens.
Others may roll their eyes at first, but when the team caps off an impressive season with total domination of their rival, and they hear that conviction in your voice, how can they argue? Oh my gosh, your team IS awesome! And they DEFINITELY would have crushed in the conference championship game! And in their BCS bowl game? Forget it!
Perhaps the Buckeyes weren't meant to redeem 2011 with a win over the Wolverines after all. Perhaps 2011 needed to be nothing more than a reminder that the Michigan game isn't a sure thing every year, so that it could serve as The Game, the conference championship, and the bowl game for the Buckeyes in 2012.
But even before Michigan heads to Columbus on November 24th, there are other things to play for as well. The chance to beat two other teams with postseason bans in 2012. The chance for victory over a team with a fire-breathing dragon as its mascot... The 2012 Ohio State Buckeyes, FIRE-BREATHING DRAGON SLAYERS! The chance to beat a Pac-12 team, and call it our Rose Bowl. The chance to see Bielema's face when the Buckeyes beat his Badgers, again2.
The chance to see John Simon eat people alive. The chance to remind Purdue of their proper place, as they seem to forget from time to time. The chance to avenge the 2011 Michigan State and Nebraska losses as if they're Albanians that kidnapped our daughter, because WOW those days sucked. The chance to show the college football world that Ohio State can hire an offensive coordinator that isn't completely obsessed with Dave. The chance to watch this team develop into what is hopefully a contender the following year. The chance to climb the AP poll, just for fun. The chance to witness the effects of Urban's man-crush on Braxton Miller, on the field.
So, what do you look forward to as a fan? What do you not look forward to? College football is in a league of its own because every game means so much. That's true this year for Ohio State, just as much as it's always been. And while it won't serve to determine the conference champion this year, The Game has an even greater sense of purpose than it typically does. Did you even think that was possible?
What could possibly set up 2013 better than a dominant year in 2012, and the added motivation of coming off a postseason ban? The results are still yet to be seen, but it appears it may work for USC this year.
Even without a postseason, Ohio State should have a pretty memorable year, with something new to root for each week. And if not, there's always basketball...
- 1 Yes, "The Game." You're cute, Harvard and Yale.
- 2 Let's be honest, there's really no other time when seeing his face is pleasant.


text:
Has SLI Improved Yet?
Subject: Graphics Cards | March 23, 2005 - 12:17 PM | Ryan Shrout
HardOCP has come back to the table with an updated look at the status of SLI gaming. Since we last heard from them on this topic, a new driver has been released that promised additional SLI support for everyone! Did it happen?
We are still very concerned with the frequency of driver releases and bug fixes, however. As you can see in our gameplay testing, this driver has quite a few problems. Right now, there is a temperature issue bug, and a bug with Coolbits not allowing overclocking in SLI. Then there are gameplay bugs that affect performance and gameplay, such as not being able to run in SLI in The Chronicles of Riddick and having stuttering in EverQuest II, which is hampering the performance benefit of SLI in that game. It worries us that it has been four months since the last official driver release. It makes us wonder if it will be another four months before those issues are fixed.
Source: [H]ard|OCP
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Written on: 04/02/2005 by i love horses 4eva 2005 (1 review written)
Barnston riding school is the best stables ever, they look after the horses. They only do 3 lessons so they don't get tired. All the staff are really nice, I worked there for a year but had to leave due to getting my own, I didn't want to leave my new friends and the staff I got on with. All the horses have energy when they are ridden. They do teaching stages so if you wanted a teaching career its great.


text:
One of the American missionaries infected with the Ebola virus while working in Liberia who was brought back to the U.S. for further treatment was discharged this week from the hospital where he had been kept in isolation, the mission said, according to Reuters.
Dr. Kent Brantly and Nancy Writebol were brought back from West Africa earlier this month and transported directly to Emory University Hospital in Atlanta. Franklin Graham, president of the mission Samaritan's Purse, said Brantly has recovered.
"Today I join all of our Samaritan's Purse team around the world in giving thanks to God as we celebrate Dr. Kent Brantly's recovery from Ebola and release from the hospital," Graham said in a statement, according to CBS News.
The hospital, however, would not say whether it would be discharging the Ebola patients in its care, both of whom received an experimental drug to treat the virus, or if it already had discharged either of them. Hospital officials said they would provide more details at a news conference later Thursday morning.
Last week, Brantly released a statement from isolation saying that he was "recovering in every way."
"I thank God for the health care team here who is giving me compassionate, world-class care. I am more grateful every day to the Lord for sparing my life and continuing to heal my body. There are still a few hurdles to clear before I can be discharged, but I hold on to the hope of a sweet reunion with my wife, children and family in the near future," the doctor said on August 15.
The World Health Organization reported that as of August 18, that the Ebola virus has spread to nearly 2,500 patients in several West African countries, resulting in about 1,350 deaths.
The current outbreak of the virus, which is contagious through contact with infected bodily fluids and causes hemorrhagic fever among other symptoms, has a fatality rate of 47 percent, according to WHO.
Watch WSB-TV's report about the discharge plans for Ebola patients:
Update: A source told WSB-TV that Nancy Writebol was already discharged from the hospital this week.
The Associated Press contributed to this report.


text:
|low graphics version | feedback | help
|You are in: Health
Thursday, 8 February, 2001, 11:55 GMT
Complaints culture 'threatens medicine'
Nine out of ten doctors fear that the growing complaints culture threatens the future of the NHS, a survey has found.
Many say they have already adopted a take-no-chances style of "defensive" medicine where patients may undergo a battery of tests for even the most trivial conditions simply to minimise the risk of ligitation.
Doctors do not think the rise in complaints reflects a drop in clinical standards.
More than two-thirds said clinical practice in the NHS was good or excellent, and one in three said standards had risen in the last five years.
Defensive medicine
The poll, of more than 700 doctors, also found that the "compensation culture" is contributing to low morale and the recruitment difficulties within the health service.
The overwhelming majority of doctors say that patients have become more demanding in recent years.
More than a third believe the best way to reduce litigation levels is to increase the number of doctors and nurses in the health service.
But there is also widespread support for the establishment of a special authority to deal with compensation matters.
The Medical Defence Union, which commissioned the survey, paid out more than Ł77 million in compensation to patients during 1999.
At the moment, medical insurance experts such as the MDU deal with complaints and pay out compensation to patients.
Dr Frances Szekely, senior medical claims handler at the MDU, said: "The strength of feeling among the profession about rising litigation is clearly evident in the number of doctors who have taken the time to reply to this survey.
"There is no doubt that this climate is having an impact on the profession both in terms of clinical practice and morale."
See also:
13 Dec 00 | Background Briefings
Protecting patients
30 Nov 00 | Health
Elderly 'failed by NHS complaints system'
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text:
Time Schedule:
Susan A Glenn
Seattle Campus
Introduction to gender as category of historical analysis, examining the impact of feminist theory within the discipline of history. Course traces historiographical debates in women's and gender history and explores, through cross-cultural comparisons, how scholars have conceived the relationship between gender and categories such as class, race, ethnicity, and sexuality.
Class description
Please note there are several articles to read IN ADVANCE OF THE FIRST meeting. These are posted as pdf's on the Catalyst Site for this course.
Student learning goals
General method of instruction
Recommended preparation
Class assignments and grading


text:
Q&A: ALU Talks Customer Experience as the Final Differentiator
Pricing and device choice just aren't the differentiators they used to be for mobile operators; consumers are still shopping around for the provider that will treat them like a human being. Alcatel Lucent has rolled out a suite of new tools that aim to help call centers reduce the number of calls they take, while improving the quality of service they can provide when they are on the line with a customer. Greg Owens, director of marketing for customer experience at Alcatel-Lucent, sat down with Wireless Week to talk about the increasing importance operators place on the quality of care they offer their customers. In the end, it can have implications on everything from churn rate to cost-savings.
Wireless Week: What consumer behaviors are driving mobile operators to look more and more at Customer Experience Management solutions?
Greg Owens: This is something that we've actually been looking at for a while. What we're seeing, particularly as operators deploy LTE, is that they're looking at their offers and finding that they don't have as much competitive differentiation. I think we're getting to a place where consumers get the point; everybody has fast networks. Even differentiation around devices has pretty much gone away, as almost every carrier offers the iPhone. So, you don't want to get to a point where the operators are just differentiating on price. So, we really feel strongly that a focus on customer experience allows the operators to differentiate in a meaningful way, and we have a lot of customers that are embracing that. Look at AT&T in the United States. We have companies like Telefonica that offer service all over Europe. And these are companies that are really focused on offering a better customer experience.
Customer experience can drive down costs. Many customers want to do everything for themselves. AT&T has launched a service, leveraging our products, where consumers can troubleshoot all of their different services from any device. So if you're having an issue with your home voice or your IPTV service, you can actually fire up an application on your iPad or iPhone and actually figure it out yourself. And we're finding that a large majority of people, especially younger people, would rather do anything than talk to an agent. In fact, around 75 percent of customers prefer self-service over calling the help desk. In this case, you're cutting costs, while actually improving the customer experience at the same time.
WW: What are the unique challenges that mobile operators face in addressing customer experience?
Owens: On a smartphone, we just don't have the same kind of ability to remotely connect to a user's device and help them solve their issues the way we can with a router or a modem. Whereas TR-069 is the one predominate management protocol for connecting with wireline devices, in mobile there are multiple protocols such as OMA-DM, OMA-CP, and other proprietary mechanisms based on device vendor. So here at Alcatel Lucent, we have a number of different solutions to help us deal with mobile devices. We can actually manage an Android device, but it is harder to do with Apple because iOS is a closed operating system. Instead, you actually have to walk them through all the different settings, which obviously takes a lot more time. That's a big challenge. Again, there are standards in the mobile space such as OMA-DM, but they're just not widely accepted the way TR-069standard is in the fixed space.
The other big challenge in mobile is that we're seeing so many different types of phones. With Verizon, we're managing all of their LTE smartphones. Fragmentation is a big challenge. Verizon would go to launch a new service and notice there's a problem with a particular version of the firmware, and it might impact, say, a million of their users, but it doesn't impact all of their users universally. So they have to have a strategy for dealing with that. They found that at any one time there could be dozens and dozens of different versions of Android running on their network.
WW: You recently announced the launch of Motive ServiceView for Mobile – what exactly is it?
Owens: Motive ServiceView for Mobile is the next generation in customer care for handling the growth and complexity of mobile broadband. It's a single integrated solution that handles agent-assisted care, self-care, and preemptive care with visibility and control across networks, devices, and applications. Basically what we're trying to do with ServiceView for Mobile is bring together all of the different touch points that customers have with the service provider and offer the provider a single view of that customer. I was recently visiting a call center and listening in on calls to Tier II agents. They have to access about 30 different systems, depending on what the customer's calling about. So if they're calling about billing, they have to log into the billing system. If they're calling to upgrade their phone, there's another system that they have to log into. They don't have a unified view of the customer, and that's what we're trying to do with ServiceView for Mobile. We have a single console that's integrated with all of the different back end systems, which is something that we're already doing for dozens of operators on the fixed side.
For consumers, there's nothing more frustrating than getting through to an agent and having them say that they're unable to find your account or the relevant information to solve your problem. We want ServiceView to put that information at the agent's fingertips. So when you're at the hockey game, and you can't get a connection, the agent can say, 'Yes, it's our issue, the network is slow.' Or if it's a technical issue, they can actually resolve it. Being able to resolve the customer's issues efficiently and the first time is a very powerful mechanism for enhancing the overall customer experience.
WW: What are Alcatel-Lucent's biggest differentiators – and how do these factor in to value for the service provider?
Owens: As mentioned before, offering a single integrated solution that takes a multi-channel approach to customer care, that also handles agent-assisted, self-assisted, and pre-emptive care with end-to-end visibility across devices, networks, and applications is an extremely powerful starting point for improving the mobile customer experience. It's something the industry is now demanding and something we can offer off-the-shelf.
In addition, our toolset is built on a flexible workflow engine that allows our operators to standardize their processes across multiple customer care channels, automate a large number of tasks/operations, and optimize their business processes easily through an agile methodology. This reduces the time it takes to implement new operations, it increases the consistency of the processes, and lowers the overall cost of customer care.
And finally, we provide standardized templates out of the box that leverage best practices we have discovered over time for handling some of the most frequent support workflows and operations. This basically allows our customers to deploy quickly and optimize after in order to meet their specific needs. These templates and workflows really are a big differentiator for us, because they allow us to offer our customers insight into some of the biggest problems they're going to see. If they're launching a service that's new to them, there's a pretty good chance that one of our customers has launched that service, and we have experience with that service., Through our workflows we can give them a pretty good idea of what kinds of issues they're going to run into, what is root cause for a specific issue, and what are most efficient steps for issue resolution. That's a really big differentiator for us.
WW: What kind of impact do you think CEM systems like Motive ServiceView for Mobile will have on a mobile operator's overall profitability?
Owens: This is almost always the starting point for most of our customers. They almost always say, "Listen, we're interested in being more efficient." Some of them are putting just a generic focus on customer care. AT&T is a good example of that; U.S. Cellular is another good example. But realistically, most of them are doing it from a cost efficiency perspective. Another way to view this is how will enhancing the customer experience improve my profitability. If they don't find better ways to serve customers, customer experience will degrade and their costs are going to go up. Smartphones customers are twice as likely to call the help desk. And the calls last around 30 percent longer. So when we're visiting those call centers, we say to them, 'As you see more smartphone being adopted, do you want to hire twice as many agents?' No, of course they don't want to do that. Some are looking at the off-shore option, but often that doesn't result in a better customer experience. What you want to do is enhance that customer care experience while driving down your costs – both of these go hand in hand.
Specifically, one of our European customers launched a self-care initiative with us, and they saw calls to the help desk reduced by 75 percent. Other customers have seen their first-call resolution rate go up by about 20 percent. Those numbers have a big impact on customer experience, and a big impact on their costs. If you're calling the help desk, and you're spending 20 minutes on the phone with an agent, and then that problem isn't actually resolved, that's a big issue.
Our solutions help reduce average handle time (AHT) and increase that first-call resolution (FCR) rate. You're going to resolve issues more quickly with our tools – and reduce operational expenses. You're going to make sure they get resolved the first time, and you're going to eliminate the need for some calls to be made at all. This has a huge positive impact on customer satisfaction. Customers with our solutions will see stronger brand affiliation from their customers and overall will have reduced churn rates. And all of this together drives improved profitability for the service provider.