A weak organic acid is used to effect the release of CaDPA from Bacillus or Clostridium endospores, rapidly and at room temperature, to enable detection and measurement of DPA and thereby the assessment of risk associated with exposure to Bacillus anthracis, Clostridium botulinum, and like spores. The method can be applied to airborne, food-borne, and water-borne spores, as well as to spores collected from surfaces or contained in body fluids, and analysis is advantageously carried out using surface-enhanced Raman spectroscopy.

Calcium dipicolinate (CaDPA, calcium 2,6-pyridinedicarboxilate) represents 5 to 15% by weight of Bacillus anthracis spores (Murrell, W G, in The Bacterial Spores, Gould, G W, A Hurst, Eds. Academic Press, London, 1969, p. 215), and consequently a number of methods are being developed and used to detect CaDPA or its derivatives as an indicator or biochemical signature for the presence of B. anthracis, a bacterium that has been used in bioterrorism due to its ability to cause anthrax. The appropriateness of measuring CaDPA or its derivatives as a signature for B. anthracis is supported by the fact that only spore-forming bacteria contain CaDPA, of which there are 13 genera, two of which, Bacillus and Clostridium, are common and of interest; while the most widespread, potentially interfering spores, such as pollen and mold spores, do not contain CaDPA. This ability to discriminately detect anthrax-causing spores is one of the important measurement parameters that must be satisfied if a method is to have value in minimizing terror. Three additional germane measurement parameters include sensitivity, speed and ease-of-use. The Center for Disease Control estimates that inhalation of 10,000 spores, or 100 nanograms, will be lethal to 50 percent of an exposed population (Ingelsby, T V et al., "Anthrax as a biological weapon, 2002: Updated recommendations for management" J Amer Med Ass, 287, 2236-52, 2002). The ideal measurement device would have as a minimum sensitivity the ability to detect the presence of 10,000 spores within minutes of detection, such that precautions could be executed to prevent infection. Such a device could be used to detect spores in suspicious mail or on contaminated surfaces, and prevent distribution or exposure.
Methods that have successfully been used to detect CaDPA or its derivatives include mass spectrometry and fluorescence, luminescence, and Raman spectroscopies. Mass spectrometry has been used to identify dipicolinic acid (DPA), the acid form of CaDPA, that was separated from spores by pyrolysis (Beverly M B, F Basile, K J Voorhees, T L Hadfield, "A rapid approach for the detection of dipicolinic acid in bacterial spores using pyrolysis/mass spectrometry", Rapid Commun. Mass Spectrom, 10, 455-458, 1996). Although this method provides a relatively high degree of discrimination and sensitivity, it requires significant time due to sample handling and data analysis.
Fluorescence involves of course the absorption of electromagnetic radiation into an electronic transition of a molecule and the re-emission of radiation at longer wavelengths. It has been shown that fluorescence can be obtained from CaDPA (Nudelman, R, B V Bronk, S Efrima, "Fluorescence Emission Derived from Dipicolinate Acid, its Sodium, and its Calcium Salts" Appl Spectrosc 54, 445-449, 2000), however the emission spectrum is not sufficiently unique to differentiate it from common biological materials that also fluoresce. In an effort to overcome this limitation, others have explored the formation and the use of a terbium-DPA complex to generate a distinctive, highly luminescence spectrum (here defined as fluorescence that employs a chemical modification, Rosen D L, C Sharpless, L B McBrown, "Bacterial spore detection and determination by use of terbium dipicolinate photoluminescence", Anal Chem, 69, 1082-1085, 1997; or Rosen D L et al. U.S. Pat. No. 5,876,960). In a similar study, hot dodecylamine (DDA, a cationic surfactant) was used to rapidly break apart the spore to release CaDPA, as the acid, to form the terbium complex (Pellegrino P M, N F Fell Jr., J B Gillespie, "Enhanced spore detection using dipicolinate extraction techniques" Analyt Chim Acta, 455, 167-177, 2002). Unfortunately, it has been found that as many as three concentration-dependent complexes can form, each with different lifetimes (Rosen D L, S Niles, "Chelation Number of Terbium Dipicolinate: Effects on Photoluminescence Lifetime and Intensity", Appl Spectrosc 55, 208-216, 2001). This, coupled with the fact that the Tb3+ cation produces the same luminescence spectrum, makes determinations of low spore concentrations problematic. Furthermore, the combination of heat and the DDA surfactant severely degrade the spore, generating cell debris (here defined as biochemical fragments of the spore). This requires sample cleanup and in this particular case, AlCl3 had to be added to remove phosphates that would interfere with the photoluminescent measurement.
It has long been known that Raman spectra of bacilli spores are dominated by peaks associated with CaDPA, and that these spectra may provide a suitable anthrax signature at the genus level (Woodruff W H, T G Spiro, C Gilvarg, "Raman Spectroscopy In Vivo: Evidence on the Structure of Dipicolinate in Intact Spores of Bacillus Megaterium", Biochem Biophys Res Commun, 58, 197-203, 1974). Since that time, considerable improvements in Raman instrumentation have led to field measurements of spores captured from a mail-sorting system (Farquharson S, L Grigely, V Khitrov, W W Smith, J F Sperry, G Fenerty, "Detecting Bacillus cereus spores on a mail sorting system using Raman Spectroscopy", J Raman Spectrosc, 35, 82-86, 2004). However, Raman spectroscopy is inherently an insensitive technique, and these measurements were of milligram, and not the required nanogram, sample amount.
Two approaches are widely used to improve the sensitivity of Raman spectroscopy, i.e., resonance Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS). The former method involves laser excitation at or near the wavelength of an electronic absorption to substantially increase the interactions between the radiation and molecular states, and was used more than a decade ago to analyze Bacillus spores (Ghiamati E, R S Manoharan, W H Nelson, J F Sperry, "UV Resonance Raman spectra of Bacillus spores", Appl Spectrosc, 46, 357-364, 1992). The value of this technique is limited by the extremely low energy conversion of ultraviolet lasers, which require substantial power supplies and thus confine measurements to laboratory settings, and which also require spectral acquisition times as long as one hour.
SERS involves the absorption of incident laser photons within nanoscale metal structures, generating surface plasmons, which couple with nearby molecules (the analyte) and thereby enhance the efficiency of Raman scattering by six orders of magnitude or more (Jeanmaire D L, R P Van Duyne, "Surface Raman Spectroelectrochemistry", J Electroanal Chem, 84, 1-20, 1977; or Weaver M J, S Farquharson, M A Tadayyoni, "Surface-enhancement factors for Raman scattering at silver electrodes: Role of adsorbate-surface interactions and electrode structure", J Chem Phys, 82, 4867-4874, 1985). In addition to affording high levels of sensitivity, the rich molecular vibrational information provided by Raman scattering yields exceptional selectivity and allows the identification of virtually any chemical as well as the ability to distinguish multiple chemicals in mixtures (see Garrel R L, "Surface-Enhanced Raman Spectroscopy", Anal Chem, 61, 401A-411A, 1989; or Storey J M E, T E Barber, R D Shelton, E A Wachter, K T Carron, Y Jiang, "Applications of Surface-Enhanced Raman Scattering (SERS) to Chemical Detection", Spectroscopy, 10, 20-25, 1995).
Four methods have become common in the practice of generating SERS. They are: (1) activated electrodes in electrolytic cells (see for example Jeanmaire or Weaver above); (2) activated silver and gold colloids (Kerker M, O Siiman, L A Bumm, D-S Wang, "Surface-enhanced Raman Scattering of citrate ion adsorbed on colloidal silver", Appl Opt, 19, 3253-3255, 1980, or Angel S M, L F Katz, D D Archibold, L T Lin, D E Honigs, "Near Infrared Surface-enhanced Raman Spectroscopy. Part II: Copper and gold colloids", Appl Spectrosc, 43, 367-372, 1989); (3) activated silver and gold substrates (Seki H, "Surface-enhanced Raman Scattering of pyridine on different silver surfaces", J Chem Phys, 76, 4412-4418, 1982, or Li Y-S, T Vo-Dinh, D L Stokes, Y Wang, "Surface-Enhanced Raman Analysis of p-Nitroaniline on Vacuum Evaporation and Chemical Deposited Silver-Coated Alumina Substrates", Appl Spectrosc, 46, 1354-1357, 1992); and (4) sol-gels doped with silver or gold particles (Farquharson et al. U.S. Pat. Nos. 6,623,977, 6,943,031, and 6,943,032 and corresponding International Application Publication No. WO 01/33189 A2, which are commonly owned herewith and the entire specification of which the United States patents are hereby incorporated by reference thereto).
The first measurement of dipicolinic acid by SERS was reported in 1999 followed by a more in-depth analysis in 2004 (Farquharson S, W W Smith, S Elliott, J F Sperry, "Rapid biological agent identification by surface-enhanced Raman spectroscopy", SPIE, 3855, 110-116, 1999; Farquharson S, A Gift, P Maksymiuk, F Inscore, and W Smith, "pH dependence of methyl phosphonic acid, dipicolinic acid, and cyanide by surface-enhanced Raman spectroscopy", SPIE, 5269, 117-125, 2004). In these two reports, the measurement of DPA by SERS was performed to demonstrate the possible use of such a measurement to identify spores, such as Bacillus anthracis. However, both measurements were of pure DPA in water and no method to extract the DPA from spores was mentioned. In any event, no prior art describes the use of chemicals to cause the release of CaDPA so that it or DPA is available for measurement by SERS; that concept is disclosed in recent papers authored by the present inventors (see Farquharson S, A Gift, P Maksymiuk, F E Inscore, "Rapid dipicolinic acid extraction from Bacillus spores detected by surface-enhanced Raman spectroscopy", Appl Spectrosc, 58, 351-54, 2004; and Inscore F E, A D Gift, S Farquharson, "Detect-to-treat: development of analysis of Bacilli spores in nasal mucus by surfaced-enhanced Raman spectroscopy", SPIE, 5585, 53-57, 2005).
Since the Farquharson, et al. publication (Appl Spectrosc, 2004) one research group has attempted to improve upon these measurements (Zhang X, M A Young, O Lyandres, R P Van Duyne, "Rapid Detection of an Anthrax Biomarker by Surface-Enhanced Raman Spectroscopy", J Am Chem Soc, 127, 4484-4489, 2005). In this paper, published on Mar. 30, 2005, 0.02M nitric acid was used in conjunction with 10 minutes of ultrasonification to obtain a SER spectrum of DPA. Although a 200 fold improvement in sensitivity was claimed, the lowest reported amount measured, 187,000 spores per microliter, was considerably less sensitive than the 10,000 spores per microliter reported by Farquharson et al. Xhang et al. also reported that no signal was obtained unless sonication was used.
Even so, in these measurements, the use of DDA or nitric acid alone was insufficient to separate a significant amount of DPA (defined, for present purposes, to be at least 10% by weight, on average, of that which is available) in a timely manner (defined, for present purposes, as a period that is less than 30 minutes, and preferably less than 10 minutes), without the addition of heat or ultrasound. The use of heat or ultrasound had two deleterious consequences, 1) it produced spore debris that likely reduced the sensitivity of the SERS measurement, and 2) it unduly complicated the device by requiring a heat or ultrasound source.
Significant research to understand the sporulation and corresponding germination of spores has been performed for more than 50 years. Bacterial spores are considered marvels of nature, in that when conditions for life become unfavorable, they become dormant through a process known as sporulation (forming an endospore). These spores are resistant to severe environmental conditions, such as heat, ultraviolet radiation, vacuum, and many chemicals including oxidizing agents and strong acids (e.g. nitric, hydrochloric, sulfuric, etc., see Russell, A D, The destruction of bacterial spores, Acad Press, New York, N.Y., 1982 or Nicholson, et al. "Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments", MicroBio Molec Bio Rev, 64, 584-572, 2000). However, when conditions are again favorable they re-initiate cell growth through a process known as germination (see Dricks, A, "From Rings to Layers: Surprising patterns of protein deposition during bacterial spore assembly", J Bacteriol, 186, 4423-4426, 2004). Numerous methods have been examined to initiate germination to elucidate this process. Germinants studied naturally included amino and nucleic acids, sugars, carbohydrates, and also dodecylamine (Setlow B, A E Cowan, P Setlow, "Germination of spores of Bacillus subtilis with dodecylamine", J Appl Microbiol, 95, 637-648, 2003). It is now known that certain amino acids and inosine, a nucleic acid variant, initiate germination, which includes an early step involving the release of CaDPA, which becomes DPA as it enters the surrounding solution. It is also now known that DDA initiates germination through a different mechanism. The details of these mechanisms whereby CaDPA is released using natural nutrients or DDA, through one or multiple spore wall channels, are still unknown. In fact, endospores may contain a small percent of DPA along with the CaDPA. In either case, however, germination takes several hours at room temperature, and consequently none of these chemicals provide sufficient speed for the purpose to satisfy the objects of this invention.
In another area of research, methods have been investigated for the purpose of killing spores, such as in the case of sterilizing medical equipment and disinfecting food. A comprehensive review is given by Russel A D, "Bacterial Spores and Chemical Sporicidal Agents", Clin Microbiol Revs, 3, 99-119, 1990. In summary, only few chemicals kill spores effectively, glutaraldehyde, formaldehyde, chlorine releasing agents, peroxygens (e.g. peroxide), and ethylene oxide, of which only the first two have practical value. It is worth noting that according to this review organic acids, specifically, benzoic acid and sorbic acid, can be used to kill bacterial cells, but not spores. In recent years the mechanisms by which these chemicals, and others, kill spores have been examined. However, only limited studies have involved the analysis of DPA. These reports measure the release of DPA as a method of improving the understanding of germination, as well as spore death. These reports include the use of inorganic acids, alkali, peroxides, and haloginated chemicals (Young S B, P Setlow, "Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid", J Appl Microbiol, 89, 330-338, 2000, Setlow B, C A Loshen, P C Genest, A E Cowan, C Setlow, P Setlow, "Mechanisms of killing spores of Bacillus subtilis by acid, alkali, and ethanol", J Appl Microbiol, 92, 362-375, 2002, and Young S B, P Setlow, "Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide", J Appl Microbiol, 95, 54-67, 2003). In all of these cases, hours of treatment are required to generate significant quantities of DPA. Again, however, the inclusion of a physical means, such as heat, pressure, sonication, or their combination (e.g. autoclaving), are required if the DPA release times are to be substantially reduced, and in such cases significant cell debris is generated, thus compromising the sensitivity of DPA detection.
It is therefore surprising that treatment of spores by a weak acid compound, having a pKa value of 0.1 to 11, at room temperature, in accordance with the present invention and as is described hereinafter, quickly (e.g., within 10 minutes) releases most, if not all, of the CaDPA contained therein.

Method for effecting the rapid release of a signature chemical from bacterial endospores, and for detection thereof


A fuel injector needle has a separate pin element fixed in an axial bore in a tip end of the needle, the bore surrounded by an annular partially spherical region sealing to a valve seat. The pin element is separately manufactured and has a conical spray pattern shaping feature at one end protruding from the needle tip. The annular region can be accurately formed and finished prior to installation of the pin element.

This invention concerns fuel injectors for internal combustion engines. Fuel injection has become the preferred mode of delivering fuel charges to the cylinders of engines used in automobiles and trucks.
Fuel injectors typically include a thin elongated valve element commonly referred to as a valve "needle", which has a partially spherical tip, the tip engaging a conical seat when the injector is closed to prevent fuel from being injected into the engine from the injector until the injector is opened. Energizing an injector solenoid operator causes the needle to be lifted from the seat when fuel is to be injected, allowing fuel under pressure to be discharged through an aligned opening in the seat, and through an orifice in a plate below the valve seat. The orifice influences the shape of a spray pattern formed when the pressurized fuel discharges from the injector tip.
The shape and surface finish of the needle tip is critical for proper operation of the injector, primarily because the tip must very reliably establish a sealing engagement with the valve seat whenever the solenoid is deenergized.
The tip end initially is ground to a partially spherical shape. A "superfinishing" procedure is then conducted to reliably achieve proper sealing of each fuel injector tip on its seat. The superfinishing is carried out using a very fine grit stone and a compliant mounting of the needle to achieve a submicron surface finish.
Current multivalve engines employ injectors which divide the discharge into two distinct spray patterns, each directed at a respective intake valve associated with a given engine cylinder. This dual spray pattern is achieved by an orifice disc having two orifice openings and by a modification of the partially spherical needle tip, comprised of a conical spray-shaping feature extending from the center of the tip. Other spray pattern affecting features are also possible.
The spray-shaping feature has been difficult to form while carrying out shaping and superfinishing of the annular region of the spherical needle tip which engages the conical valve seat.
It is the object of the present invention to provides a fuel injector needle tip which enables a central spray pattern shaping feature to be provided on the tip while allowing a properly finished annular valve seat engaging region on the needle tip.


The present invention relates to polymers having the formula: ##STR1## wherein: M, Q, X, Y, Z, R.sup.3 and R.sup.4 are as defined in the text.

This invention relates to polymers formed by the radical-initiated polymerization of unsaturated species of low molecular weight. Polymers of low molecular weight, or oligomers, are important as precursors in producing other polymeric materials and such polymers have been found to be useful in a variety of products, for example, in the production of high solids (low VOC) surface coatings, in adhesives and as plasticizers in polymeric composites.
In conventional polymerization practice, the manufacture of oligomers requires the use of an initiator which acts as a free radical source, and of a chain transfer agent. The chain transfer agent controls the molecular weight of the polymer by reacting with the propagating polymer radical to terminate its growth. It then initiates a new polymer chain thus transferring the growth process from one discrete polymer molecule to another discrete polymer molecule. The most commonly used chain transfer agents are alkanethiols, which normally are associated with an objectionable odor and lead to a wide distribution of molecular weight with certain monomers. Also, the residual thiols and the end thio-ether linkage of the polymers may have an adverse effect on the properties of the ultimate product from the polymer.
The polymers of the present invention are prepared without the disadvantages of polymerizations regulated with thiols by using alternative polymerization regulators. These regulators have good stability and shelf life while maintaining many of the advantages over thiols. In the majority of cases, the materials that are part of the present process present a different range of chain transfer activities, allowing more opportunity for an optimal process to be selected for a given polymerization system of monomers and polymerization conditions. The chain transfer constant that a given regulator possesses is an important consideration in selecting the optimum process for producing low molecular weight polymers.

Polymers formed from allylic chain transfer agents

Commonwealth Scientific and Industrial Research Organisation


A multiple diffusion channel filter assembly is disclosed. The filter assembly provides filtration of air entering and exiting an electronic enclosure through a breather hole. In one implementation, the filter assembly contains a housing with an adsorbent component and a first diffusion channel and a second diffusion channel. In another implementation, the filter assembly contains a first layer and a second layer which surround an adsorbent component and a first and a second diffusion channel that are in fluid communication with the interior space defined by the first and second layers. In yet another implementation, the invention, includes a filter accessory containing a diffusion channel configured to work in conjunction with a separate filter assembly thereby adding an additional diffusion channel to the existing design.

Filters are useful in a variety of devices. For example, filters are often used in electrical or optical equipment. An air pressure differential between the interior and exterior of a housing containing the equipment can be produced as the electrical or optical equipment heats and cools. Often the housing includes a vent or breather hole to allow airflow that equalizes the pressure. A filter is typically provided over the vent to reduce the flow of contaminants into and/or out of the housing.
Computer disk drives, and in particular, hard disk drives, are one example of a device that uses filters in this manner. Disk drives are sensitive to moisture, chemical contamination, and particulate contamination, particularly, as the drive heads become smaller and aerial densities increase. Chemical contaminants, such as hydrocarbons and acid gases, can condense onto a disk and degrade the head/disk interface and/or corrode the heads. Particulate contaminants can lead to stiction and can cause read/write errors and head crashes.
Therefore, a need exists for filters that provide beneficial removal of particulate and chemical contaminants.


An automatic change-over ball lock punch retainer including a punch retainer having a punch, a retainer block, and an arm member in a sliding engagement retainer with the retainer housing block. The arm member having a camming means which interengages with an inclined surface of the punch retainer to move a punch into an active or inactive punch position.

1. Field of the invention
The present invention relates generally to a punch retainer for use in a punch and die assembly, and more particularly to an automatic or programmable change-over ball lock punch retainer for use in a punch and die assembly.
2. Description of the Related Art
Punch retainers have been used in punch and die assemblies for many years. The punch retainers retain a punch in the die shoe while the punch is moved to form an aperture in a sheet of metal. The punch retainers are used in combination with an upper die shoe and a punch press which can hold a plurality of punches for various jobs that place apertures in a sheet of metal. A typical die shoe for multiple sheet metal parts has a plurality of punches that are not needed for each particular metal stamping process. In that case gagging retainers as known in the art have been used to place a punch in an active position for stamping of the material or to recess the punch so that punch will not form its aperture in the metal stamping process. A typical example of a die shoe having a plurality of punches includes fifteen punches that are fixed in position and form an aperture every time the stamping process is completed along with five gagging retainer punches which are liftable and capable of being deactivated into a position such that an aperture is not formed in the stamping process.
Prior art gagging retainer systems have used spring biased means to physically lift a punch away from its proper punching position or used a toggle mechanism to lower the tool holder thus making the lower punching tool inactive with respect to the work piece positioning means. However, the prior art techniques springs were not very reliable and caused down time of the stamping process and the servicing of the prior art punches required basic disassembly of the press to remove the punch and service the punch. The prior art also did not actively confirm that a punch had been placed in its retracted position.
Therefore, there is a need in the art for a punch retainer to overcome the above shortcomings.

Automatic or programmable change-over ball lock punch retainer apparatus


Low sulfur content fuel compositions containing additive compounds are described which exhibit improved lubricity. The additive compounds include a carboxylic acid substituted by at least one hydroxyl group, derivatives of the carboxylic acid substituted by at least one hydroxyl group, and an ester which is the reaction product of a carboxylic acid which does not contain any hydroxy-substitution in the acid backbone and an alkanolamine.

The present invention relates to the use of certain compounds to improve the lubricating properties of low sulfur-content fuels and to fuels and additive concentrates comprising the compounds.
Sulfur contained in fuel, for example middle distillate fuels such as diesel fuel and jet fuel, is said to constitute a serious environmental hazard. Hence strict regulations limiting the amount of sulfur which may be present in such fuels have been introduced. Unfortunately, fuels having a suitably low sulfur content exhibit very poor inherent lubricity and this can lead to problems when the fuel is used. For example, the use of low sulfur fuel in diesel engines frequently results in damage to the fuel injector pump which relies on the natural lubricating properties of the fuel to prevent component failure. There is therefore a need to improve the lubricating properties of low sulfur fuels.
EP-A-0608149 discloses the use of an ester as an additive in a liquid hydrocarbon compression-ignition fuel oil for reducing consumption of the fuel oil.
WO 92/09673 discloses additives which are the reaction products of (1) anhydrides and/or poly-acids and (2) aminoalcohols or amino/alcohol/amides with long chain hydrocarbyl groups attached used to improve the low-temperature properties of distillate fuels.
U.S. Pat. No. 4,617,026 (Shaub et al.) discloses the use of hydroxyl-containing esters of a monocarboxylic acid and a glycol or trihydric alcohol to reduce fuel consumption in automobiles.
U.S. Pat. No. 3,681,038 (Gaydasch) discloses middle distillate fuel compositions containing N,N-dialkylricinoleamide pour point depressants.
U.S. Pat. No. 5,194,068 (Mohr et al.) discloses fuel compositions containing small amounts of an ester of a mono- and/or poly-carboxylic acid with an alkyl alkanolamine or alkyl aminopolyalkylene glycol.
U.S. Pat. No. 4,683,069 (Brewster et al.) discloses lubricating oil compositions containing a glycerol partial ester of a fatty acid.
U.S. Pat. No. 4,491,455 (Ishizaki et al.) teaches adding esters of nitrogen containing compounds having polyhydroxyl groups with linear saturated fatty acids to fuel oils in order to improve cold flow.
U.S. Pat. No. 4,253,876 (Godar et al.) discloses corrosion inhibitors comprising triesters of an alkenyl or alkyl succinic acid or anhydride and a trialkanolamine.


An electric power steering apparatus includes a steering assist torque control system in which a steering torque signal is input and a motor speed signal is output. The control system includes a main control value setting section having a gain KTT and setting a target assist torque value corresponding to the steering torque, a differential-value correction value setting section having a gain KTD and setting a differential-value correction value corresponding to a differential steering torque value, a motor having an inertial efficiency JM and a counter electromotive force (induced voltage) constant KE, a motor drive section incorporated in a controlled object and having a proportional gain KP, a steering speed correction value setting section having a gain KTR and setting a steering speed correction value corresponding to the motor speed signal. The ratio of the gain KTD to the gain KTT is set to satisfy the condition: KTD/KTT>JM/{.alpha.(KP.multidot.KTR+KE)} where .alpha.=KT/(R+KP), R is the resistance (winding resistance) of the electric motor, and KT is the torque constant of the electric motor. With this arrangement, the phase of a gain of the steering assist torque control system is advanced, thereby improving the response characteristic to a level at least comparable to the manual steering mechanism.

1. Field of the Invention
The present invention relates to improvements in an electric power steering apparatus which provides power assist of an electric motor directly to a steering system so as to reduce manual steering effort to be applied by the driver.
2. Description of the Related Art
FIG. 1 of the accompanying drawings diagrammatically shows the general construction of an electric power steering apparatus of the type concerned. The electric power steering apparatus 1 includes an electric motor 10 incorporated in a steering system, and a control unit 20 for controlling power assist supplied from the electric motor 10, so as to reduce the manual steering effort or force required by the driver.
The steering system includes a steering wheel 2 attached to an end of a steering shaft 3. The opposite end of the steering shaft 3 is connected to one end of a connecting shaft 4 via a first universal joint 4a, the other end of the connecting shaft 4 being connected via a second universal joint 4b to a pinion 6 of a rack-and-pinion mechanism 5. The pinion 6 is in mesh with a rack 7 which is a long bar with gear teeth 7a cut into one side. The rack-and-pinion mechanism 5 translates a rotary motion of the pinion 6 into an axial reciprocating motion of the rack 7. Opposite ends of the rack 7 are connected via tie rods 8 to steerable left and right front wheels 9, 9. When the steering wheel 2 is manually turned or rotated in a desired direction, the rack-and-pinion mechanism 5 and the tie rods 8 cause the front wheels 9 to pivot in the same direction to thereby change the direction of movement of a motor vehicle.
In order to reduce the manual steering effort or force required by the driver, the electric motor 10 is disposed in concentric relation to the rack 7 and supplies an assist torque (steering assist torque) to the rack 7 via a ball screw mechanism 11. The ball screw mechanism 11 converts rotational power of the electric motor 10 into an axial thrusting force acting on the rack 7. The ball screw mechanism 11 is generally comprised of a nut 12 connected to a rotor of the electric motor 10, and a threaded screw portion 7b formed along a longitudinal portion of the rack 7. By virtue of the threaded engagement between the nut 12 and the threaded screw portion 7b, a rotational force of the nut 12 is converted into an axial thrusting force of the rack 7. Thus, the assist torque generated by the electric motor 10 is translated into the axial thrusting force of the rack 7 by which manual steering effort required by the driver to turn the steering wheel 2 is reduced.
A steering torque detecting section (steering torque sensor) 18 detects a manual steering torque Ts acting on the pinion 6 and supplies a torque signal Tp indicative of the detected steering torque Ts to the control unit 20. The control unit 20 outputs, on the basis of the torque signal Tp, a motor control signal 20a to control output power (steering assist torque) of the electric motor 10.
FIG. 2 of the accompanying drawings shows in block diagram the general arrangement of a conventional control unit. The control unit 20A includes a target assist torque determining section 201 and a motor drive section 202. The target assist torque determining section 201 determines a target assist torque on the basis of the torque signal Tp and outputs the determined target assist torque in the form of a target assist torque signal 201a. More specifically, the target assist torque determining section 201 sets the target assist torque to be zero when an absolute value of the steering torque is less than a predetermined dead zone threshold. Conversely, when the absolute value of steering torque is greater than the predetermined dead zone threshold, a target assist torque which is proportional to the steering torque is output from the target assist torque determining section 201. The target assist torque output from the target assist torque determining section 201 is limited below an upper limit even when the steering torque increases excessively.
The motor drive section 202 determines an offset between the target assist torque signal 201a supplied from the target assist torque determining section 201 and a motor current signal IM supplied from a current detector (not shown) provided to detect a current actually flowing in the electric motor 10, and generates a motor drive signal 20a in such a manner as to render the offset zero. The motor drive signal 20a is supplied to the electric motor 10 with the result that the target assist torque is supplied from the electric motor 10.
FIG. 3 shows in block diagram the general arrangement of another conventional control unit. The control unit 20B includes a first target assist torque determining section 211, a steering torque differentiating section 212, a second target assist torque determining section 213, an adding section or adder 214, and a motor drive section 202.
The first target assist torque determining section 211 determines a first target assist torque on the basis of the torque signal Tp and outputs the determined first target assist torque in the form of a first target assist torque signal 211a. More specifically, the first target assist torque determining section 211 sets the first target assist torque to be zero when an absolute value of the steering torque is less than a predetermined dead zone threshold. Conversely, when the absolute value of steering torque is greater than the predetermined dead zone threshold and less than a predetermined threshold, a first target assist torque which is proportional, with low gain, to the steering torque is output from the first target assist torque determining section 211. A steering torque greater than the predetermined threshold causes the first target assist torque determining section 211 to output a first target assist torque which is proportional, with high gain, to the steering torque. The first target assist torque output from the first target assist torque determining section 211 is limited below an upper limit even when the steering torque increases excessively.
The steering torque differentiating section 212 determines a variation per unit time of the torque signal Tp and outputs the determined variation in the form of a differential torque signal 212a (Tp.multidot.s in a Laplace transform range).
The second target assist torque determining section 213 determines a second target assist torque on the basis of the differential torque signal 212a and outputs the determined second target assist torque in the form of a second target assist torque signal 213a. The second target assist torque output from the second target assist torque determining section 213 is limited below an upper limit even when the differential torque value becomes excessively large.
The adder 214 adds together a signal 211a corresponding to the first target assist torque and a signal 213a corresponding to the second target assist torque and outputs the result of arithmetic operation (addition) in the form of a target assist torque signal 214a.
The motor drive section 202 determines an offset between the target assist torque signal 201a supplied from the target assist torque determining section 201 and a motor current signal IM supplied from a current detector (not shown) provided to detect a current actually flowing in the electric motor 10, and generates a motor drive signal 20a in such a manner as to render the offset zero. The motor drive signal 20a is supplied to the electric motor 10 with the result that the target assist torque is supplied from the electric motor 10.
The conventional control unit 20A shown in FIG. 2 seeks to improve the response characteristic by adjusting gain of the gain of a steering power assist system in which the input is the steering torque signal Tp and the output is the motor drive signal 20a or the steering assist torque supplied from the electric motor 10. The conventional control unit shown in FIG. 3 seeks to enhance the response characteristic by achieving adjustment of the gain of the steering power assist system in response to variations of the steering torque. The conventional control units, however, have a drawback that an excessively high gain of the steering power assist system would harm the operational stability of the steering power assist system. Another drawback with the conventional control units is that due to the influence of an inertial efficiency of the electric motor and a friction produced in a speed reducing mechanism, a sufficiently high level of response cannot be achieved. The conventional electric power steering apparatus as a whole cannot perform steering operation with steering feeling (response characteristic) at least comparable to that of the manual steering apparatus.


A pipe splitting assembly includes a pipe splitter, for instance, a cutter with one or more blades. An expander is flexibly coupled with the pipe splitter and allows the pipe splitter to rotate out of alignment with the longitudinal axis of the expander. In one example, the pipe splitter includes a cylindrical base portion and a frusto-conical base portion proximal to the cyclindrical base portion, and an exterior surface of the frusto-conical base portion is flush with an exterior surface of the expander. In another example, the expander has a larger outer perimeter than the cutter. Optionally, the pipe splitting assembly further includes a puller shoulder sized and shaped to couple with the expander, and a puller sleeve adapted to receive and hold a replacement pipe.

Pipe, such as plastic, copper, lead pipe and the like, has been used for connecting homes and creating networks for utilities, for instance, water, sewage and natural gas. As pipes become older, they break down, crack, develop scaling on interior surfaces that can clog the pipe, etc. and thereby require replacement.
A technique know as pipe bursting is currently used as a convenient method to replace underground pipe without the need to completely dig up the pipe to be replaced. A pipe breaking device, such as an expander or a mole, is pulled by a cable through the existing pipe while it is still underground. The expander is designed to break, split or burst the pipe, and at the same time to push the old pipe into the surrounding soil. The expansion of the old pipe allows the expander to pull a replacement pipe into place.
Pipe splitters typically use one or more cutting surfaces to split pipe material. Typically, the cutting surfaces engage against the end of the pipe and are pulled longitudinally to split the pipe. Large pulling forces are required to pull cutting surfaces with this configuration through the pipe material and thereby split the pipe. In some examples, where the existing pipe is located in soft soil, the pulling force transmitted from the cutting surfaces to the pipe will move the pipe through the soil instead of splitting the pipe material.
In other examples, pipe splitters are used to burst small diameter pipes and thereby must navigate tight bends in the pipe. An elongate pipe splitter experiences stress as it attempts to navigate tight bends and sometimes fractures within the pipe. This requires extraction and replacement of the pipe splitter to continue the operation. Replacement of the pipe splitter increases labor. Additionally, the pipe splitter is constructed with hardened steel in some examples, and is expensive. Replacing the pipe splitter thereby increases the cost of splitting small diameter pipes.
What is needed is a pipe splitter that provides consistent cutting without requiring excessive pulling forces. What is further needed is a pipe splitter capable of navigating bends in small diameter pipes without fracturing.


A camshaft drive system is described for a selectable two-stroke/four-stroke internal combustion engine with exhaust and intake poppet valves opened and closed by at least one camshaft. In a preferred embodiment of the system, the camshaft is connected permanently to a planetary gear set which is driven continuously by the engine crankshaft, and means are provided for setting the planetary gear set in one of two selectable stable modes to function either as a gear box or as a rigid coupling.

The present invention relates to a camshaft drive system for operating an internal combustion engine according to the two-stroke cycle or the four-stroke cycle.
Conventional internal combustion engines operate according to thermodynamic principles following either the two-stroke cycle or the four-stroke cycle and are commonly classified as two-stroke engines or four-stroke engines respectively. Both type of engines can operate using a range of fuels including gasoline, diesel, alcohol and gaseous fuels; the fuel can be introduced into the engine by a range of devices including carburettors and fuel injectors into the air intake system and fuel injectors aimed directly into the engine cylinder; and the fuel-air mixture can be ignited by a range of methods including spark ignition and compression ignition. Each engine cycle type has different merits and shortcomings making it a subject of continuous debate as to which is more suitable for automotive application taking into account various parameters such as power density, fuel consumption, exhaust emissions, noise, vibration, engine size, weight and cost etc. Currently, the automotive manufacturer must first decide on the engine cycle type, then design the engine accordingly.
With a view of combining the merits of both engine cycle types, there is provided a camshaft drive system for a selectable two-stroke/four-stroke internal combustion engine with exhaust and intake poppet valves opened and closed by at least one camshaft, wherein the camshaft is connected permanently to a planetary gear set which is driven continuously by the engine crankshaft, and wherein means are provided for setting the planetary gear set in one of two selectable stable modes to function either as a gear box or as a rigid coupling.
In a preferred embodiment of the invention, the camshaft is driven by the planetary gear set according to the selectable mode at either the same speed as the engine crankshaft or half the speed of the engine crankshaft, and the phasing of the camshaft in relation with the crank angle position of the piston is adjustable using the planetary gear set to position the exhaust and intake gas exchange events appropriately for either the two-stroke or the four-stroke engine cycle respectively.
Preferably, the planetary gear set is mounted concentrically to the camshaft. Alternatively, it may be mounted concentrically to the engine crankshaft or it may be mounted separately next to the camshaft and the crankshaft.
Various configurations are possible in using the planetary gear set to drive the camshaft according to the present invention. The input from the engine may be connected to any one of the three members of the planetary gear set which may be the ring gear, the sun gear or the planetary gear carrier, the output to the camshaft may be connected to any one of the remaining two members, and the mode setting means may be provided in the remaining last member which is the reaction member between the input and output members.
In the case where the planetary gear carrier is the reaction member, idler gears coupled with the planetary gears may be additionally provided on the planetary gear carrier such that the ring gear and the sun gear rotate in the same direction.
In the invention, the means for setting the planetary gear set to function as a gear box is a device for holding the reaction member of the gear set stationary relative to a fixed datum so that the gears rotate relative to one another within the gear set, whereas the means for setting the planetary gear set to function as a rigid coupling is a device for holding the reaction member of the gear set stationary relative to either the rotating input member or the rotating output member so that the gears are locked relative to one another within the gear set and the whole assembly rotate together.
In a preferred example, the ring gear is the input member driven by the engine at half crankshaft speed, the sun gear is the output member connected to the camshaft, and the planetary gear carrier is the reaction member of the gear set. The speed ratio between the ring gear and the sun gear is 1:2.
In this example, means are provided for setting the planetary gear carrier in one of two selectable stable modes such that in the two-stroke mode the carrier is held stationary relative to the engine frame so that the gear set acts as a gear box, and in the four-stroke mode the carrier is held stationary relative to the rotating ring gear so that the entire gear set acts as a rigid coupling. Additionally, means are provided for allowing the carrier to slip and rotate relative to the engine frame or relative to the rotating ring gear in order to adjust the phasing and the speed of the camshaft during a mode change.
In operation, when the two-stroke mode is selected, the carrier, which has been held stationary relative to the rotating ring gear in the four-stroke mode, is first allowed to slip and rotate relative to the rotating ring gear in order to adjust the phasing and the speed of the camshaft progressively towards the two-stroke mode, at which point the carrier is then held stationary relative to the engine frame thereafter. In this case, the planetary gear set acts as a gear box in which the camshaft is driven at twice the speed of the ring gear and each engine valve along the camshaft is opened and closed at a predetermined two-stroke valve timing once during every engine revolution consistent with the two-stroke cycle.
In addition to the above, a combination of various operating parameters in the engine necessary for executing the two-stroke cycle is implemented, and this would be obvious to a person familiar with the state-of-the-art. For example, an air blower would be necessary to supply pressurised air for the two-stroke scavenging process and the frequency and timing of the fuel injection and/or spark ignition would be scheduled appropriately.
When the four-stroke mode is selected, the carrier, which has been held stationary relative to the engine frame in the two-stroke mode, is first allowed to slip and rotate relative to the engine frame in order to adjust the phasing and the speed of the camshaft progressively towards the four-stroke mode, at which point the carrier is then held stationary relative to the rotating ring gear thereafter. In this case, the planetary gear set acts as a rigid coupling in which the camshaft is driven at the same speed as the ring gear and each engine valve along the camshaft is opened and closed at a predetermined four-stroke valve timing once during every two engine revolutions consistent with the four-stroke cycle.
In addition to the above, a combination of various operating parameters in the engine necessary for executing the four-stroke cycle is implemented, and this would be obvious to a person familiar with the state-of-the-art. The air blower for two-stroke operation may in this case be used to supply boosted air for four-stroke operation.
The above example is only one of many possible configurations for the camshaft to be driven by a mode selectable planetary gear set of the present invention. In another example, the planetary gear carrier is the input member driven at half crankshaft speed, the sun gear is the output member connected to the camshaft, and the ring gear is the reaction member of the gear set. The speed ratio between the carrier and the sun gear is 1:2.
In this case, the ring gear may be set in one of two selectable modes such that in the two-stroke mode the ring gear is held stationary relative to the engine frame so that the planetary gear set acts as a gear box and the camshaft is driven at crankshaft speed, and in the four-stroke mode the ring gear is held stationary relative to the carrier so that the entire gear set acts as a rigid coupling and the camshaft is driven at half crankshaft speed.
In a further example, the sun gear is the input member driven at crankshaft speed, the planetary gear carrier is the output member connected to the camshaft, and the ring gear is the reaction member of the gear set. The speed ratio between the sun gear and the carrier is 2:1.
In this case, the ring gear may be set in one of two selectable modes such that in the two-stroke mode the ring gear is held stationary relative to the carrier so that the entire gear set acts as a rigid coupling and the camshaft is driven at crankshaft speed, and in the four-stroke mode the ring gear is held stationary relative to the engine frame so that the epicycle gear set acts as a gear box and the camshaft is driven at half crankshaft speed.
The principle of operating a planetary gear set in several selectable modes is well known and is commonly used in automotive automatic transmission systems for driving a vehicle. The present invention proposes to apply the same principle for driving a camshaft in order to enable the engine to be operated in either the two-stroke or the four-stroke mode with smooth and rapid transition and with the minimum risk of misfire during a mode change even at high speed. This provides a camshaft drive which is permanently in mesh and avoids the abruptness and uncertainty caused by temporarily decoupling and then re-coupling the camshaft and the crankshaft as proposed in the previously known systems such as U.S. Pat. No. 4,907,544 and U.S. Pat. No. 2,178,152 which are unacceptable for automotive application.
The technology for controlling the planetary gear set to operate in the selectable modes and to change mode smoothly and imperceptibly using brakes, clutches, and sensors is well advanced in the automatic transmission field and may be transferred readily to the camshaft drive system of the present invention.

Selectable 2-stroke/4-stroke camshaft drive system


In multi-phase driving systems of electric motors in a switching mode employing distinct integrated half-bridge output stages, the use of multiple external components for each integrated half-bridge stage to implement functions of adjustment of the switching dead-time and of disabling/enabling of the half-bridge stage can be avoided and an improved matching obtained by using single external components connected in common to the dedicated pins of all the distinct integrated half-bridge output stages. This is achieved by duplicating the disabling comparator, respectively for the power transistor of the pull-up branch and for the power transistor of the pull-down branch in each integrated half-bridge stage.

Integrated circuits for driving systems through half-bridge power stages are widely used in industrial applications, such as for driving three-phase motors, stepper motors, brushless motors or a generic resistance-inductance (R-L) actuator. When these integrated devices possess only one logic command input, it becomes necessary to implement a circuit that regulates the switching dead-time to avoid a simultaneous state of conduction of the two branches, that is, of the two power devices (HIGH SIDE and LOW SIDE) of the half-bridge stage. In the majority of cases, it is necessary to program or adjust the value of the switching dead-time by taking into account the electrical characteristics of the driven R-L actuator.
FIG. 1 shows a classical way of realizing such a dead-time programming function, using a dedicated PIN of the integrated device IC1. This dedicated pin serves to implement both the programming function of the switching dead-time by suitably dimensioning the external resistor R.sub.ex, and the disabling/enabling logic function of the half-bridge power stage of the integrated device through a dedicated external switch S1, controlled by a specific logic command SHUTDOWN.
By referring to the conventional scheme of FIG. 1, the amplifier (buffer) A1 restores a certain reference voltage Vbg (typically a band-gap voltage) on the dedicated PIN. Through the external resistor R.sub.ex it is then possible to regulate the switching dead-time (Tdt) generated by the DEAD-TIME block, by considering that: ##EQU1## where C is the delay capacitance within the DEAD-TIME circuit block, V is the voltage existing on such capacitance C at the switching instant, and I.sub.R is the current that charges the capacitance C.
The comparator COMP1 compares the voltage on the dedicated pin with ceratin reference voltage REF1, and produces a shutdown or enabling signal of the output power stage HALF-BRIDGE. In practice, the logic function of "shutdown" is implemented by forcing a low logic stage on the pin PIN as referred to the threshold REF1 established on the inverting input node of COMP1.
According to this typical circuit implementation of a programming function of the switching dead-time and an enabling/disabling function of the half-bridge power stage, the output impedance of the operational amplifier (buffer) A1 is necessarily low to allow a good degree of precision for the circuit, as well as immunity to noise. These characteristics of known circuits do not allow connecting in common all the respective dedicated pins (PIN) and the use of a unique set of external components to implement the programming function of the switching dead-time and of enabling/disabling of all the integrated power devices of the multi-phase driving system. This is so especially when they include several output stages, as in the case for driving multi-phase motors. In practice, according to the prior art, it is necessary to duplicate the external components and the respective lines for each integrated device (IC1, IC2, IC3), as depicted in FIG. 2.

Sharing of external components for the control of the switching dead-time of a plurality of integrated devices cooperating in driving an R-L multi-phase actuator


Prepared first are a mounting substrate having, on its principal surface, a plurality of recesses which correspond in plan configuration to a plurality of functional chips, and a flat plate disposed on the mounting substrate and having a plurality of confinement regions formed of openings, each of which exposes one of the recesses and a peripheral portion of the recess and is designed to confine one of the functional chips. Next, the functional chips are put into the respective confinement regions on the mounting substrate. The mounting substrate having the functional chips thereon is then immersed in a fluid together with the flat plate. Subsequently, the flat plate immersed in the fluid is displaced (vibrated) with the mounting substrate fixed, thereby fitting the functional chips into the recesses in the mounting substrate.

The present invention relates to a semiconductor-device assembly method and a semiconductor-device assembly apparatus, in which an assembly method for simultaneously assembling a plurality of micro functional chips, i.e., semiconductor devices or the like, onto a mounting substrate is used.
Techniques for assembling micro functional chips, such as electronic components, onto a mounting substrate, such as a wafer or substrate, simultaneously in a single operation to fabricate semiconductor devices have been attracting considerable attention recently. At present, assembly process for mass production is normally being performed by pick and place technique in which assembly robots pick functional chips and place them in predetermined locations. However, since the cost and speed of the assembly robots are limited, it is necessary to find an alternative means which enables the assembly of a large number of functional chips with higher speed and lower cost.
As one of the technologies considered for this purpose, fluidic self assembly (FSA) has been proposed recently. The FSA technology is a technique for assembling in a fluid a plurality of functional chips onto a mounting substrate in a self-aligned manner.
Hereinafter, a conventional FSA process will be described with reference to FIGS. 7 and 8.
FIG. 7 schematically illustrates a semiconductor-device assembly apparatus 300A in which a FSA process according to a first conventional example is used. As shown in FIG. 7, a mounting substrate 410 is a wafer or a printed circuit board, has on its surface a plurality of recesses 410a for specifying assembling positions, and is held in a fluid with its surface inclined with respect to the surface of the fluid. Then, a fluid 311 in the form of slurry, in which a large number of functional chips 400 has been spread, is poured over the surface of the mounting substrate 410 through a pipette 310. The functional chips 400 are typically in the shape of a square, disc, or sphere. The numerous functional chips 400 contained in the fluid 311 poured over the surface of the mounting substrate 410 fit, by chance and naturally, into the empty recesses 410a having the matching shape, while sliding on the surface of the mounting substrate 410.
FIG. 8 schematically illustrates a semiconductor-device assembly apparatus 300B with a bubble pump according to a second conventional example. As shown in FIG. 8, the bubble-pump-equipped semiconductor-device assembly apparatus 300B is composed of a container 312, in which a fluid 311 and a mounting substrate 410 are placed, and a bubble pump system 313 for circulating functional chips 400.
The bubble pump system 313 carries the functional chips 400 that have not yet been disposed into recesses in the mounting substrate 410, to a position higher than the mounting substrate 410 by the buoyancy of bubbles 311a. The carried functional chips 400 are then poured over the surface of the mounting substrate 410 again. The bubbles 311a are created by a bubble-velocity controller 313a provided in the lower portion of the bubble pump system 313 from nitrogen gas 314 supplied to the bubble pump system 313.
However, in the conventional semiconductor-device assembly apparatus, the probability that the functional chips fit into the recesses in the mounting substrate depends upon the density of the recesses. Therefore, in order to achieve a high assembling efficiency, an enormous number of functional chips have to be prepared as compared with the density of the recesses. On the other hand, in the bubble pump shown in FIG. 8, the functional chips that have not been placed into the recesses can be reused, which allows the number of necessary functional chips to be reduced. Nevertheless, the assembling efficiency achievable by these types of semiconductor-device assembly apparatuses is limited and depends upon the shape of the semiconductor devices, for example. For instance, it has been reported that when disc-like functional chips having a given thickness and a given size were replaced with square-shaped functional chips, the assembling efficiency dropped from over 90% to less than 40%. This indicates that functional chips having a smaller number of symmetry axes have lower assembling efficiency. Therefore, it can be concluded that if square-shaped functional chips are replaced with rectangular functional chips, the assembling efficiency may be reduced to about half, as the square has four symmetric edges, while the rectangle has only two symmetric edges. If these rectangular functional chips are further made directional as required for semiconductor lasers, the assembling efficiency may be further reduced to half, and the assembling efficiency finally obtained may not exceed 10%. Since the assembling efficiency of functional chips is usually required to exceed 99% (at least 90%), there is a demand for a method which enables functional chips to be assembled with high assembling efficiency and accuracy, irrespective of the size and shape of the functional chips.

Semiconductor device assembly method and semiconductor device assembly apparatus

Matsushita Electric Industrial Co., Ltd.


Methods are provided for high-throughput screening for the presence of genetic alterations and disease-causing microorganisms in a biological sample. These methods are particularly useful for identifying individuals with gene mutations indicative of early colorectal cancer.

The knowledge of molecular defects causative of diseases, such as inherited disorders and cancer, is increasing rapidly. Inherited diseases thought to be caused by genetic mutations include sickle cell anemia, .alpha.- and .beta.-thalassemias, phenylketonuria, hemophilia, .alpha..sub.i -anti-trypsin deficiency, and cystic fibrosis. Sickle cell anemia, for example, is reported to result from homozygosity resulting from a single base pair substitution in the sixth codon of the .beta.-globin gene. Antonarakis, New England J. Med., 320: 153-163 (1989). Mutations in the insulin receptor gene and in the insulin-responsive glucose transporter gene have been detected in insulin-resistant diabetes. Krook et al., Human Molecular Genetics, 1: 391-396 (1992).
Cancer has been associated with genetic mutations in a number of oncogenes and tumor suppressor genes. Duffy, Clin. Chem., 41: 1410-1413 (1993). For example, point mutations in the ras genes have been shown to convert those genes into transforming oncogenes. Bos et al., Nature, 315: 726-730. Mutations and the loss of heterozygosity at the p53 tumor suppressor locus have been correlated with various types of cancer. Ridanpaa et al., Path. Res. Pract., 191: 399-402 (1995); Hollstein et al., Science, 253: 49-53 (1991). In addition, the loss or other mutation of the apc and dcc tumor suppressor genes has also been associated with tumor development. Blum, Europ. J. Cancer, 31A: 1369-1372 (1995). Those mutations can serve as markers for early stages of disease and for predisposition thereto. Early diagnosis is not only important for successful treatment, but can also lead to prevention or treatment before chronic symptoms occur.
Colorectal cancer is an example of a disease that is highly curable if detected early. With early detection, colon cancer may be effectively treated by, for example, surgical removal of the cancerous tissue. Surgical removal of early-stage colon cancer is usually successful because colon cancer begins in cells of the colonic epithelium and is isolated from the general circulation during its early stages. Thus, detection of early mutations in colorectal cells would greatly increase survival rate. Current methods for detection of colorectal cancer focus on extracellular indicia of the presence of cancer, such as the presence of fecal occult blood or carcinoembryonic antigen circulating in serum. Such extracellular indicia typically occurs only after the cancer has become invasive. At that point, colorectal cancer is very difficult to treat.
Methods have been devised to detect the presence of mutations within disease-associated genes. One such method is to compare the complete nucleotide sequence of a sample genomic region with the corresponding wild-type region. See, e.g., Engelke et al., Proc. Natl. Acad. Sci, U.S.A., 85: 544-548 (1988); Wong et al., Nature, 330: 384-386 (1988). However, such methods are costly, time consuming, and require the analysis of multiple clones of the targeted gene for unambiguous detection of low-frequency mutations. As such, it is not practical to use extensive sequencing for large-scale screening of genetic mutations.
A variety of detection methods have been developed which exploit sequence variation in DNA using enzymatic and chemical cleavage techniques. A commonly-used screen for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of southern blots, as reported by Botstein et al., Am. J. Hum. Genet., 32: 314-331 (1980) and White et al., Sci. Am., 258: 40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of the DNA. Sequences are compared by looking for differences in restriction fragment lengths. A problem with this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect mutations that do not affect cleavage with a restriction endonuclease. One study reported that only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA were detected using RFLP analysis. Jeffreys, Cell, 18:1-18 (1979).
Single base mutations have been detected by differential hybridization techniques using allele-specific oligonucleotide (ASO) probes. Saiki et al., Proc. Natl. Acad. Sci. USA, 86: 6230-6234 (1989). Mutations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Disadvantages of this approach for mutation analysis include: (1) the requirement for optimization of hybridization for each probe, and (2) the nature of the mismatch and the local sequence impose limitations on the degree of discrimination of the probes. In practice, tests based only on parameters of nucleic acid hybridization function poorly when the sequence complexity of the test sample is high (e.g., in a heterogeneous biological sample). This is partly due to the small thermodynamic differences in hybrid stability generated by single nucleotide changes. Therefore, nucleic acid hybridization is generally combined with some other selection or enrichment procedure for analytical and diagnostic purposes.
In enzyme-mediated ligation methods, a mutation is interrogated by two oligonucleotides capable of annealing immediately adjacent to each other on a target DNA or RNA molecule, one of the oligonucleotides having its 3' end complementary to the point mutation. Adjacent oligonucleotide sequences are only covalently attached when both oligonucleotides are correctly base-paired. Thus, the presence of a point mutation is indicated by the ligation of the two adjacent oligonucleotides. Grossman et al., Nucleic Acid Research, 22: 4527-4534 (1994). However, the usefulness of this method for detection is compromised by high backgrounds which arise from tolerance of certain nucleotide mismatches or from non-template directed ligation reactions. Barringer et. al., Gene, 89: 117-122 (1990).
A number of detection methods have been developed which are based on a template-dependent, primer extension reaction. These methods fall essentially into two categories: (1) methods using primers which span the region to be interrogated for the mutation, and (2) methods using primers which hybridizes proximally and upstream of the region to be interrogated for the mutation.
In the first category, Caskey and Gibbs U.S. Pat. No. 5,578,458! report a method wherein single base mutations in target nucleic acids are detected by competitive oligonucleotide priming under hybridization conditions that favor the binding of the perfectly-matched primer as compared to one with a mismatch. Vary and Diamond U.S. Pat. No. 4,851,331! described a similar method wherein the 3' terminal nucleotide of the primer corresponds to the variant nucleotide of interest. Since mismatching of the primer and the template at the 3' terminal nucleotide of the primer inhibits elongation, significant differences in the amount of incorporation of a tracer nucleotide result under normal primer extension conditions.
It has long been known that primer-dependent DNA polymerases have, in general, a low replication error rate. This feature is essential for the prevention of genetic mistakes which would have detrimental effects on progeny. Methods in a second category exploit the high fidelity inherent in this enzymological reaction. Detection of mutations is based on primer extension and incorporation of detectable, chain-terminating nucleoside triphosphates. The high fidelity of DNA polymerases ensures specific incorporation of the correct base labelled with a reporter molecule. Such single nucleotide primer-guided extension assays have been used to detect aspartylglucosaminuria, hemophilia B, and cystic fibrosis; and for quantifying point mutations associated with Leber Hereditary Optic Neuropathy (LHON). See. e.g., Kuppuswamy et al., Proc. Natl. Acad. Sci. USA, 88:1143-1147 (1991); Syvanen et al., Genomics, 8: 684-692 (1990); Juvonen et al., Human Genetics, 93: 16-20 (1994); Ikonen et al., PCR Meth. Applications, 1: 234-240 (1992); Ikonen et al., Proc. Natl. Acad. Sci. USA, 88: 11222-11226 (1991); Nikiforov et al., Nucleic Acids Research, 22: 4167-4175 (1994).
Strategies based on primer extension require considerable optimization to ensure that only the perfectly annealed oligonucleotide functions as a primer for the extension reaction. The advantage conferred by the high fidelity of the polymerases can be compromised by the tolerance of nucleotide mismatches in the hybridization of the primer to the template. Any "false" priming will be difficult to distinguish from a true positive signal.
The selectivity and stability of the oligonucleotide primer extension assay is determined by the length of the oligonucleotide primer. Under typical reaction conditions, short primers (i.e., less than about a 15-mer) exhibit transient, unstable hybridization and, consequently, do not readily prime the extension reaction. Moreover, in a complex heterogeneous biological sample, short primers exhibit non-specific binding to a wide variety of perfectly-matched complementary sequences. Thus, because of their low stability and high non-specific binding, short primers are not very useful for reliable identification of a mutation at a known location. Therefore, detection methods based on primer extension assays use oligonucleotide primers ranging in length from 15-mer to 25-mer. See e.g., PCT Patent Publications WO 91/13075; WO 92/15712; and WO 96/30545. Lengthening the probe to increase stability, however, has the effect of diminishing selectivity. Due to the small thermodynamic differences in hybrid stability generated by single nucleotide changes, a single base mismatch usually does not affect binding efficiency of longer oligonucleotide primers. This tolerance of nucleotide mismatches in the hybridization of the primer to the template can result in significant levels of non-specific "false" priming in complex heterogeneous biological samples.
Methods in the art reduce the possibility of false priming by decreasing the sequence complexity of the test sample. Thus, genomic DNA is isolated from the biological sample and/or amplified with PCR using primers which flank the region to be interrogated. The primer extension analysis is then conducted on the purified PCR products. See PCT Patent Publications WO 91/13075; WO 92/15712; and WO 96/30545. Moreover, since considerable optimization is required to ensure that only the perfectly annealed oligonucleotide functions as a primer for the extension reaction, only limited multiplexing of the primer extension assays is possible. Krook et al., supra report that multiplexing can be achieved by using primers of different lengths and by monitoring the wild-type and mutant nucleotide at each mutation site in two separate single nucleotide incorporation reactions. Given that the selectivity and stability of the oligonucleotide primer extension assay is determined by the length of the oligonucleotide primer, the number of primers that can be tested simultaneously in a given reaction mixture is very limited. Accordingly, these methods are limited in scope due to low throughput.
Therefore, there is a need in the art for simple and efficient detection methods for reliable large-scale screening for a large number of genomic mutations in heterogeneous biological samples. Such methods are provided herein.

High-throughput screening method for identification of genetic mutations or disease-causing microorganisms using segmented primers


A center bond flip chip device carrier and a method for making and using it are described. The carrier includes a flexible substrate supporting a plurality of conductive traces. A cut out portion is formed in each trace at a position within a gap of a layer of elastomeric material provided over the traces. Each cut out portion is sized and configured to receive a solder ball for electrically connecting the carrier with a semiconductor die.

Semiconductor device packaging techniques are well known. In some conventional packaged devices, a die is attached to a carrier, and contacts of each are electrically connected. In one such packaged device called a flip-chip device, a semiconductor chip is flipped and bonded with a carrier such that contacts of the die face and bond to contacts of the carrier.
With reference to FIGS. 1–3, a conventional center bond flip chip device 10 is shown as including a flipped die 30 and a carrier 11. The carrier 11 has a flexible substrate 12 and an elastomeric cover material 14. The elastomeric material 14 may be formed of a silicone or a silicone-modified epoxy. The elastomeric material 14 includes a first portion 15 and a second portion 17 of generally equal size. The flexible substrate 12 is formed of a material exhibiting high temperature stability as well as high mechanical rigidity. The substrate 12 may be a flexible tape, such as, for example, a polyimide tape. Two commercially available polyimide tapes, KAPTON® from E.I. DuPont Nemours and Company and UPILEX® from Ube Industries, Ltd., can be used to form the substrate 12.
Conductive traces 16a, 16b, 16c are formed on the flexible substrate 12 and positioned below the elastomeric material 14. The traces 16a, 16b, 16c may be deposited on the flexible substrate 12 in a variety of ways, the most preferred method being electrolytic deposition. Other suitable methods include sputter coating and laminating a sheet of conductive material and etching away excess material to form the traces.
A gap 20 separates the two portions 15, 17 of the elastomeric material 14. Conductive lands 18a, 18b, 18c are positioned on, respectively, the conductive traces 16a, 16b, 16c within the gap 20. The die 30 has been removed from the FIG. 1 for clarity of illustration of the lands 18a, 18b, 18c. As illustrated, the gap 20 is rectangularly shaped, although any configured gap will suffice as long as the conductive pads 18a, 18b, 18c are not covered by the elastomeric material 14.
A die 30 is positioned on the elastomeric material 14 of the carrier 11. The carrier 11 is electrically connected with the die 30 by way of suitable conductive connecting structures, such as, for example, inner lead solder balls or bumps 19a, 19b, 19c positioned on, respectively, the conductive pads or lands 18a, 18b, 18c. Conductive vias 22a, 22b, 22c respectively extend from each of the underside surfaces of the traces 16a, 16b, 16c. Outer lead solder balls or bumps 24a, 24b, 24c, or other conductive connecting structures, are located in electrical connection with each respective via 22a, 22b, 22c and serve to connect the traces 16a, 16b, 16c to a structure or common base for mounting components, such as, for example, a printed circuit board 35. Preferably, the outer lead balls 24a, 24b, 24c are about 16 mils in diameter.
Conventional center bond flip chip semiconductor devices have several disadvantages, particularly as die 30 sizes decrease and the contacts thereof are positioned closer together. One disadvantage is that adjacent traces 16a, 16b, 16c of the carrier 11 and their associated conductive lands 18a, 18b, 18c must likewise be positioned closer together to such an extent that the inner lead balls 19a, 19b, 19c will occasionally contact one another, thereby shorting out the semiconductor device. Another disadvantage is that in positioning the inner lead balls 19a, 19b, 19c on the conductive lands 18a, 18b, 18c, wicking of the solder balls onto the conductive traces may sometimes occur during the solder process, providing less of a solder ball surface to make good electrical contact between the die 30 bond pad and a conductive land 18 of the carrier 11.
There is, therefore, a need for a center bond flip chip semiconductor device design which alleviates to some extent these disadvantages.

Method of making a center bond flip chip semiconductor carrier


A method for rapid processing of large sets of hyperspectral data. A hyperspectral image, with hundreds of thousands to millions of pixels measured at hundreds of wavelengths, can contain over a gigabyte of data. Even modern computers can be quite slow when performing involved calculations on data sets of this size. An algorithm requiring a minimal amount of floating point calculations that still yielded useful results is disclosed.

The present invention relates generally to spectroscopy, and more specifically the invention pertains to an algorithm for rapidly estimating basis spectra ('endmembers') for use in analysis of hyperspectral data.
Hyperspectral data consists of hundreds of digital images, each spatially coincident image measured at a different wavelength. Each pixel in the image, then, has measured value at hundreds of wavelengths, and a spectrum of measured values vs. wavelength can be plotted for each pixel. This spectrum can also be thought of as a vector with magnitude and direction in come multi-dimensional space, with the perpendicular coordinate axes spanning this space being the wavelengths at which measurements were made. The measured spectra are then lists of coordinates for a point in this space.
When one makes a scatterplot in multiple dimensions with every pixel in the image plotted as such a point, the entire data set can be viewed as a 'data cloud'—the scatterplot with thousands of points plotted from the data resembles a cloud. Points inside the data cloud can often be usefully modeled as a linear combination of points near the 'hull' of the data cloud. The physical interpretation of this model is that points on the hull of the data cloud may correspond to pixels that have uniform and unique spectral composition (uniform surface properties), and points inside the data cloud correspond to pixels with inhomogeneous composition describable as mixtures of the supposedly pure pixels of the hull.
An example of a system that acquires hyperspectral data is in U.S. Pat. No. 5,379,065, Jan. 3, 1995, Programmable hyperspectral image mapper with on-array processing, the disclosure of which is incorporated herein by reference.
An ideal case for hyperspectral data analysis is using the 'convex hull' approach would be data that, when scatterplotted, fell within (and even outlined) an obvious simplex. For two wavelength measurements, a two-dimensional simplex is a triangle. For three wavelength measurements, the corresponding simplex is a tetrahedron. Imagine, for the moment, the two-dimensional case. If the data cloud is of a triangle shape, then one may imagine trying to find a 'best fit' triangle to the data cloud. If this is done, the vertices of the data cloud could then be taken as points (representing spectra) that describe the rest of the data. That is, every point inside the triangle can be described as a linear combination of the triangle vertices, where the multipliers on the vertice vectors sum to one and are all greater or equal to zero. Imagine that the three vertices represent the spectra of a tree, a road, and soil. One would interpret the triangle shape of the data cloud, then, to mean that the measured scene was composed uniquely of there three things. The points near the triangle vertice can be taken to be purely tree, purely road, or purely grass. Points inside the triangle vertice are assumed to be a mixture of these three 'endmembers'. Based on the position of data points inside the triangle one can calculate the exact nature of this mixture for each data point.
This calculation of multipliers for the endmembers is called 'spectral unmixing'. For example, a point dead center in the triangle would be described as 1.3 tree, ⅓ road, and ⅓ grass (note the sum of the multipliers is one). A point along the 'hull' (the edge of the triangle) between the road and grass endmember could be ½ A grass, ½ A road, and 0 tree. The results of this unmixing are useful for an analyst trying to assign physical characteristics to each pixel in a hyperspectral image. Fir higher dimensions (often there are hundreds of wavelengths in a hyperspectral data set), one must imagine a data cloud inside a multi-dimensional simplex.
Often, the measured data may intrinsically be of lower dimensionality that the number of wavelengths. Imagine data being measured in the dimensions that falls along a plane when scatterplotted in three dimensions. The physical interpretation of this is that the measured scene contained only three unique spectral signatures and mixtures of these endmembers. It would then be possible to transform the data into a two-dimensional space and so the endmember identification and spectral unmixing in two dimensions (a simpler problem). In this manner, data measured in 200 wavelengths often is only intrinsically 10-15 dimensional
Data sets may exist where, physically, no pixels in the image were of a uniform composition. In this case, one may try to fit a simplex around the data cloud and use the extrapolated vertices guarantees a neat mathematical solution to the unmixing problem, but leaves room for error in having found physically relevant endmembers.
Fitting a simplex around a multi-dimensional data cloud in a physically meaningful way is a very difficult mathematical and computational problem. I am unaware of any existing software tools for doing this. Instead, a classic approach to finding endmembers is as follows. Firstly, one performs a principal component transformation on the data set to reduce the data to its intrinsic dimensionality. One then scatterplots the data along various axes, looking for obvious outliers that might be useful as endmembers. Having located several endmembers, one then does the linear spectral unmixing. This process is far from automated and can require hours to complete.
One attempt at automating part of the interactive hunt for endmembers is called "Pixel Purity Index'. This is included in the commercial software package called 'ENVI'. This method iteratively creates random direction vectors, projects the data cloud onto these vectors, and flags pixels that lie at the extremes of the resulting distribution. After doing this thousands of time, it then selects the pixels that were most often flagged in this manner as possible endmembers. One then can interactively select pixels from this reduced number of pixels. This method is quite effective, but can require hours or even days of computing.

Algorithm for rapid endmembers determination (ALRED) in hyperspectral data

The United States of America as represented by the Secretary of the Air Force