The invention uses induced pluripotent stem cells (iPSCs) for screening anti-neoplastic agents by examining the capability of a single agent, compound or drug, or a combination of multiple agents, compounds or drugs, for inhibiting or suppressing one or more neoplastic activities or processes, including aerobic glycolysis and neoplastic anabolism and, thus, inhibiting rapid growth and excessive reproduction of neoplastic cells. Agents, compounds or drugs may also be screened for their potential in inhibiting an invasion and/or migration of neoplastic cells into healthy or normal tissues and/or cells and a metastasis of neoplastic cells into other sites of the body. Anti-neoplastic agents, compounds and/or drugs found through these methods may represent broad-spectrum anti-neoplastic agents, compounds and/or drugs that preferentially target and damage neoplastic tumor or cancer cells but exert limited or minimal harm to normal or healthy cells.

Mutagenesis, Tumorigenesis and Carcinogenesis
Today's understanding of mutagenesis of human and animal cells that result in the formation of tumor (tumorigenesis) and cancer (carcinogenesis) is still far from complete. Two opposing hypotheses of the origin of tumor/cancer have existed for many decades. Although old and new hypotheses and observations coexist, in vitro and in vivo studies have not always been consistent, and there seems to be no clear resolution of how tumors/cancers really occur and what roles the genetic mutagenesis and the epigenetic alterations play in the origin and formation of tumors/cancers and how environmental factors affect the respective processes.
The tumorigenic/carcinogenic process in humans and animals appears to involve interactive roles of both genetic factors (tumor suppressor genes, proto-oncogenes, other genes) that direct cell behavior, errors in one or more of the various stages of DNA replication and the like and epigenetic factors that alter cell behavior by modifying on DNA "decorations" and change chromatin "structures". On top of these complex interplays between genetic and epigenetic factors in the origin and formation of tumors/cancers environmental factors (different physical or cultural environments, X-rays, ultraviolet radiation, mutagenic pollutants, tumor- or cancer-promoting chemicals and environmental toxicants, gender (hormones), diet and the like) also influence the tumorigenesis/carcinogenesis during different stages of life development (maternal/embryonic, neonatal, adolescent, adult and senescent/geriatric). Thus, there is a huge need for further advance our knowledge on mutagenesis, tumorigenesis and carcinogenesis.
Tumor/Cancer Therapies
Current therapies and drugs for treating tumor/cancer have significant difficulties and are, and have been, relatively ineffective. Many tumors and cancers are not treated successfully. This may be because various drug therapies do not properly target the source of the tumor or the cancer, or that they have multi-drug resistance (due to a presence of one or more drug-resistant genes in the genomes of tumor/cancer cells). Also, various genes present in cancer cells and tumor cells are able to be turned "on" or "off" Moreover, there are many different types of tumor/cancer cell lines. Additionally, cells within a tumor/cancer are heterogeneous, rather than homogeneous, in nature. These cells represent a wide variety of genotypic and phenotypic characteristics. Further, no two tumors/cancers of a given origin in either an animal or a human being would contain tumor/cancer cells that are uniformly genetically and phenotypically identical. Moreover, as a tumor/cancer grows, the microenvironment within the tumor/cancer changes, which is caused by decreasing oxygen tension and blood nutrient availability. The tumor/cancer can also be influenced by the environmental factors that are described above, such as hormones and diet. Further, some tumor/cancer cells express drug-resistant genes. Still further, some anti-tumor/anti-cancer drugs, such as thalidomide, cause serious side effects, such as inducing birth defects that would not otherwise occur, causing a new type of tumor/cancer or a toxicity in a patient and/or causing a "stress response" that functions to protect, rather than destroy, cancer cells. Many of the foregoing explanations also explain discrepant or contrasting results that are often obtained from an experimental testing of a potential anti-tumor/anti-cancer drug in vitro as compared with in vivo. For example, typical cell cultures have a high-level of oxygen content as a result of both the culture media and the atmosphere, whereas tumors/cancers growing in an animal or human are typically not exposed to such oxygen-containing factors.
Medical Needs
There has been, and remains, a very significant long-felt, and unresolved, need in the tumor/cancer field for identifying and developing reliable broad-spectrum anti-tumor/cancer drugs and agents that: (i) are effective in killing a variety of tumor/cancer cells; (ii) do not kill or harm normal cells or tissues, which may be adjacent to tumor/cancer cells, the result of which could lead to the death of a patient; and (iii) do not have harmful, uncomfortable or other disadvantageous side effects. Presently existing anti-tumor/cancer drugs and agents typically do not possess each of these three characteristics, and tumor/cancer researchers have tried, and failed, to identify and develop such drugs and agents, which is very challenging for the reasons that are set forth above. Additionally, improper hypotheses, logic and approaches at the cellular level may have been employed by cancer researchers in an attempt to identify and develop such drugs and agents.
Shortcomings of Current Anti-Tumor/Anti-Cancer Drugs
Many conventional anti-neoplastic agents or anti-cancer drugs suffer from drawbacks of severe side effects due to their inhibition on some common processes shared by normal cells [1-3]. On the other hand, highly-specific anti-neoplasm drugs that target at the specific "leaf" level genetic mutation unique to different neoplastic cells are not only too narrow spectrum in their anti-neoplastic effect, but also suffer from losing effect as surviving neoplastic cells can develop resistance by using alternative routes [4-5]. More ironically, some anti-cancer agents can kill the targeted original cancer cells but, in the meantime, lead to the formation of new cancer in the patients [6-8]. The sad reality is that most anticancer drugs [9-10] really do not offer much meaningful benefit to patients' quality of life because they add just an extra week or two of suffering time to the patients' lifespan [11-12].
It turns out that the past as well as the current research on tumors and cancers have been focused too much on the distinct types of tumors and cancers in efforts for identifying their unique genetic mutations and thus develop highly specific anti-tumor/anti-cancer therapy. However, these scattered peripheral "leaf"-level hunting efforts for anti-tumor/anti-cancer drugs often meet with disappointments because many tumors/cancers have the capability of by-passing the very specific route targeted by those narrow-spectrum anti-tumor/anti-cancer drugs specifically designed for a "leaf"-level mutation. Therefore, there has been, and remains, a very significant long-felt, and unresolved, need in the tumor/cancer field for methods for reliably identifying broad-spectrum anti-tumor/cancer drugs that will target on the "root"-level process of tumors/cancers and thus cannot be by-passed by any tumor/cancer cell.
Neoplastic Metabolism: The Root-Process for all Neoplasms Including Tumors and Cancers
The "root" of neoplasia or the "Achilles' heel" of neoplasm, which includes tumor and cancer, resides in the unique metabolic properties and the living processes of the neoplastic cells.
A hallmark of all neoplasms is their oncogenic metabolism, such as a high rate of glycolysis, even under a high oxygen concentration. This phenomenon has been known as the "Warburg Effect" since the 1930s, but remains poorly understood even today [14]. During aerobic glycolysis, pyruvate generated from glucose is not transported into mitochondria for total oxidation for yielding more energy but, rather, is converted to lactate in cytosol and then is excreted outside the cell [15]. For a long time, it was unknown why neoplastic cells would "waste" glucose and choose an energy-"inefficient" metabolism. However, the finding of a "neoplastic or pathological Cori cycle" in which the excreted lactate is carried by the blood to the liver and converted to glucose for reuse by the neoplasm may shed some light in understanding Cachexia, a condition exists in neoplastic patients who suffer massive loss of normal body mass as the neoplasm continues its growth [16]. It turns out that, by avoiding a complete "burn" of glucose to CO2, neoplastic cells preserved some key carbon "skeleton" for neoplastic anabolism which represents another aspect of the oncogenic metabolism. The combined result of the "Warburg Effect" (the aerobic glycolysis) and the pathological "Cori Cycle" (the neoplastic anabolism) thus causes a metabolic imbalance: shifting resource toward neoplastic cells and away from normal cells. This metabolic imbalance ultimately results in systematic failure of patients suffering from a neoplastic disease and their death.
In addition, recent studies have shown that some of the molecular mechanisms underlying the neoplastic metabolism also influence invasion and migration of malignant neoplastic cells which are responsible for metastasis and a wider range of neoplastic diseases [17].
Needs for "Root"-Killing Broad-Spectrum Anti-Neoplastic Agents
Despite great progresses in understanding its etiology and pathology of neoplasia, neoplasm especially the malignant neoplasm such as cancer, still remains as a great risk to human life. Thus, an urgent need in combating neoplasia is to obtain some "root killers" for neoplastic cells [13]. In other words, we need to find wide-spectrum anti-neoplastic agents that are harmful for a variety of tumor/cancer cells but not harmful for normal or healthy cells.
Targeting Neoplastic Metabolism as the Achilles' Heel of Neoplasm
Neoplastic metabolism or tumorigenic/oncogenic metabolism is a common process that all tumor/cancer cells must rely on for their abnormal growth and excessive reproduction (multiplication or proliferation). The neoplastic metabolism or tumorigenic/oncogenic metabolism includes elevated aerobic glycolysis which is known as the Warburg Effect and enhanced neoplastic anabolism which is recognized as a pathological Cori Cycle. Thus, a very effective and much needed approach for treating neoplasia should be based on inhibiting aerobic glycolysis (Warburg Effect), neoplastic anabolism (pathological Cori Cycle), or both at the same time. This approach should yield therapeutic schemes that present much less (fewer) side effects than those associated with conventional anti-cell cycle (reproduction)-based therapy, such as "chemotherapy" or "radiotherapy." This approach should also yield much broader spectrum anti-neoplastic drugs than those approaches targeting at some rare mutations specific for only a certain type of neoplastic cells. Understanding unique metabolism common to all, or at least most, neoplastic cells and finding agents inhibiting such neoplastic metabolism may hit the Achilles' heel of neoplasm and eradicate neoplasm from its root.
The Shortage and Shortcomings of Primary Neoplastic Cells and their Cell Lines
In addition to the still lacking full translation of the existing insights into the causes of tumor/cancer from bench to bed, a major obstacle in searching for anti-neoplastic agents is ironically the shortage of neoplastic cells suitable for laboratory drug screening [18]. Despite the high incidence of neoplasia, neoplastic cells preserved for research use are relatively few. Collecting primary neoplastic cells is a complex procedure impeded with many red tapes, not to say the great investments and efforts required for characterizing them for research use. Consequently, only a limited number of primary cancer cells have been established as useful cell lines. Thus, there is a need to find more model neoplastic cells for utilization in screening anti-neoplastic agents.
In addition to this limitation in quantity, some neoplastic cell lines passed many times in the laboratories might have accumulated additional mutations that are atypical for natural neoplasm [18]. Thus, it is not uncommon that drugs effective against laboratory lines of cancer cells actually failed dramatically in clinical trials [19]. Some of the anti-cancer drugs actually lead to opposite effects [20-21]. It is also known that some conventional "chemotherapy" sometimes induces tumor regression while simultaneously elicits stress responses that protect subsets of tumor cells [22]. Thus, there is a need for finding more appropriate model neoplastic cells that are suitable for a reliable screening of anti-neoplastic agents.
In view of the above, it would be extremely beneficial to provide cost-effective and reliable methods for successfully discovering, identifying and/or screening for broad-spectrum (or other) anti-neoplastic agents, compounds and/or drugs that are safe for use by human beings and/or animals and may potentially have a therapeutic activity therein, such as partially or fully inhibiting a neoplastic activity or capability of neoplastic cells that are present in such human beings and/or animals, with minimal or no side effects.
Induced Pluripotent Stem Cells
Recently, cells known as induced pluripotent stem (iPS) cells (iPSCs) have been generated in large quantity and diversity [23-25]. The prototype iPSCs were first reported by Yamanaka's team in 2006 for mice [23] and in 2007 for human [24]. These iPSCs were claimed to be induced from differentiated cells by ectopic expressing exogenous transcription factors Oct4, Sox2, Klf4 and c-Myc [23-24] (abbreviated as OSKM) [123] and later known as Yamanaka factors [124]. Another group of iPSCs were reported in 2007 using Oct4, Sox2, Nanog, and LIN28 as the reprogramming factors [25] (abbreviated as OSNL) [123] and later known as Thomson factors [124].
These iPSCs have been described by others as "indistinguishable" from "embryonic stem cells" (ESCs) [26-28] and, thus, have been perceived as "ethical" and "safe" "replacements" of ESCs for cell therapy [29-31], and even for regenerative medicine [32-35].
Discovery: iPSCs are Man-Made Cancer Cells Due to Metabolic Mutation by iPS Reprogramming
It is a discovery of the present inventor that iPSCs are incorrectly programmed stem cells (still abbreviated as iPSCs) or, in other words, man-made cancer stem cells (mmCSCs) [36-38]. Thus, iPSCs can be used as "replacements" for "naturally-occurring cancer cells" in screening agents against neoplastic cells.
It is a further discovery of the present inventor that iPS reprogramming can be linked with neoplastic aerobic glycolysis and anabolism [39]. Thus, iPSCs can be used as model cells for screening agents that specifically inhibit some aerobic glycolysis and anabolism characteristic for neoplasia.
In addition, it is a discovery of the present inventor that iPSCs may possess invasion and migration capability common to malignant neoplastic cells and, thus, may also be used for screening agents against the invasion, migration and metastasis of neoplastic cells.
Invention: Using iPSCs for Screening Anti-Neoplastic Agents for Broad-Spectrum Anti-Tumor/Anti-Cancer Drugs
Based on the discovery of induced pluripotent stem cells (iPSCs) as man-made cancer cells an invention was made that iPSCs can be used for screening anti-neoplastic agents, compounds and drugs that are effective in inhibiting metabolic mutation which serves as a root for neoplastic transformation of normal cells into tumor/cancer cells.
This "odd" invention is apparently against the current mainstream thinking which regards iPSCs as "ethical" replacements for ESCs and "cancer-free" and, thus, even "safe" for cell therapy and regenerative medicine. However, it is right from those reports making "cancer-free" claims for iPSCs that the inventor of this patent application found evidence of the cancer risk for iPSCs [48]. With more detailed reasoning disclosed here for linking iPS reprogramming with neoplastic transformation, iPS researchers should come to a reality that their iPSCs may find a better utility: serving as serendipitous cancer cells for screening anti-neoplastic agents.
The Nonobvious Nature of the Invention
It should be pointed out that the present invention of using iPSCs for screening anti-neoplastic agents is fundamentally different from those inventions of using iPSCs as non-cancerous cells for modeling other diseases [40-41] and screening drugs against those diseases [42-43]. As a matter of fact, iPS researchers have been focused on inventing methods for making iPSCs [44] because human iPSCs have been perceived as "less complicated" "human pluripotent cells" than "embryonic stem cells" (ESCs) and thus are "potentially useful in therapeutic applications in regenerative medicine" [44]. More significantly, claims of generating "cancer"-free "safe" iPSCs suitable for clinical applications are being made repeatedly [28, 45-47], despite the criticisms against the hype contained in these claims [36, 48]. A very recent publication [49] describes acquisition of iPSCs by selecting those cells with transgenes integrated into the so-called "safe harbors," the genomic regions outside positions known for integration mutation. Even though the iPSCs are still made with the already known oncogenes, a claim of "out of harm's way" was still made [50]. This demonstrates the lack of understanding with regard to how iPS reprogramming results in neoplastic transformation and, thus, how similar iPSCs are to cancer cells than to ESCs.
It should be further pointed out that, even after the publication (on Aug. 2, 2012) of the parent patent application [125] of this patent application, the mainstream of iPSCs research still overlooked the discovery of epigenetic-level metabolic changes or metabolic mutations as the basis for the neoplastic nature of iPS reprogramming and the cancerous nature of the iPSCs. Most researchers are still hoping of creating "safe" iPSCs by eliminating genome-integration of the exogenous genes and removing integration remains of virus-vehicle [126-128]. New claims of creating "cancer-free" iPSCs or even "safer" iPSCs are continuously made [129-130]. At present time. mainstream iPS researchers and top journals are still rejecting the discovery of iPSCs as man-made cancer stem cells (mmCSCs) [37-38] which identifies some cancer risks for various "cancer-free" iPSCs [23-24, 26, 51-53]. Strong efforts are still being made in promoting iPSCs as "ethical" and "safe" ESC replacements for cell therapy and regenerative medicine [28, 54-58]. A recent publication reporting generating iPSCs from dermal fibroblasts of a patient suffering from Hutchinson-Gilford Progeria syndrome (HGPS) [59] has even been regarded as to "lead to novel insights into mechanisms of aging" [60], even though these HGPS-iPSCs are merely some cancerous cells carrying the mutations for HGPS.
Thus, even though some recent publications have noticed the "similarity" between iPSCs and cancer cells [61-62], or the common path between the generation of iPSCs and CSCs [63], the authors of these publications are still contributing the intriguing "parallel" as a result of partial [28] or incomplete [47] reprogramming. At the end, the intrinsic cancer risk of iPSCs has been neglected even in the "comprehensive review [28] or "straight talk" [47] by the leading iPS researchers. Arguments have also been made that "although there are common pathways activated during reprogramming and tumorigenesis, there are fundamental differences between iPS and transformed cancer stem cells" [63].
The awarding of a Nobel Prize in Physiology or Medicine to Yamanaka [131] is a strong indication that even the top scientists in the world still lack a minimal appreciation for the discovery of iPSCs as "man-made cancer stem cells" (mm-CSCs) [36-39, 125], especially when this discovery was presented to the Nobel Prize selection committee [132]. Thus, it would not have been obvious at the time the present invention was made that iPSCs could be used for screening anti-cancer drugs when they are still considered as safe "replacements" of ESCs for cell therapy and even for regenerative medicine [133].
The non-obviousness of the deep insight on the intrinsic oncogenic and neoplastic transformation enabled with the iPS reprogramming is further evidenced by the rejection of the mainstream against the publication of the unique view by the inventor (see note in [39]) and the neglect of the prior art disclosed in the published patent application [125] by the mainstream. Some researchers in the field still tried to distinguish iPS reprogramming from oncogenesis and neoplastic transformation [134]. The fact is, there is no report of using iPSCs for screening anti-cancer drugs even after the publication of the patent application disclosing the invention of using iPSCs for screening anti-neoplastic agents, despite the strong need for developing anti-cancer drugs. Thus, even as today, the invention of using iPSCs for screening anti-neoplastic agents remains non-obvious even to the leading scientists in the related technical fields.
For example, a recent review published in Stem Cells and Development, which published a critical review on iPS cells in 2008, but later in 2009 rejected the submission of a manuscript describing cancer cell formation by iPS reprogramming (see note in [39]), considers "induced pluripotency and oncogenic transformation are related processes" [135]. It emphasizes more the "distinctions" between induced pluripotent stem cells (iPSCs) and oncogenic foci (OF) by stating that "iPSCs and OF shared a limited number of genes that were upregulated relative to parental fibroblasts" and "iPSCs and OF were distinct in that only iPSCs activated a host of pluripotency-related genes, while OF activated cellular damage and specific metabolic pathways" [135]. This review concludes that "OF and iPSCs are related, yet distinct cell types, and in which induced pluripotency and induced tumorigenesis are similar processes" [135].
The above view of treating iPSCs and OF as distinct cell types and separating iPS reprogramming from oncogenic transformation is incorrect because some of the differences between OF and iPSCs may be caused by the heterogeneous nature of OF due to the inherent variations in the differentiation status of the composing cells and the high purity of iPSCs as they are selected out based on some stemness markers [36]. However, that difference should not rule out the same oncogenic tumorigenesis or neoplastic transformation pathway used for forming OF and iPSCs. Indeed, using Nanog, a stemness marker and also a reprogramming factor used in some iPS reprogramming, reprogrammed oncogenic foci (ROF) with iPSC-like cells have been generated [135].
Evidences Supporting the Discovery and the Invention
Nevertheless, it is at least admitted by Yamanaka that iPSCs generated with his patented method have undeniable cancer risk as he recognized in his correction [137] to his Science publication [138] after thanking criticisms made by the inventor [139] of this invention [125]. Such risk was also confirmed by many later studies, including Yamanaka's own studies [87-89] The cancer risk may still exist for iPSCs generated with integration-free reprogramming methods [140, 141]. More convincingly, a Letter-to-the-Editor published in Stem cells showed that human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection [142].
Interestingly, the unavoidable cancer risk associated with the prototype reprogramming method [136] has forced Yamanaka to file additional patent applications to claim "inventions" of selecting "safe" iPSCs [143,144]. However, these "inventions" are obvious responses to the public criticisms [36-39, 90-92, 125, 139, 141, 142, 168] made by the inventor of this patent application and actual implementations of some suggestions made in those public criticisms.
After all, the claims made in these patent applications may be invalid as the methods disclosed really lack any effective measure to overcome the intrinsic neoplastic nature of the iPS reprogramming and, thus, are really incapable of selecting cancer-free safe iPSCs [145].
Oncogenic Metabolism Resulted from Epigenetic Changes via iPS Reprogramming
The "root" cause for the neoplastic transformation via the iPS reprogramming is actually not the mutagenesis conventionally associated with random integration of exogenous genes and their vectors. It is some specific epigenomic alterations associated with the elevation of some specific reprogramming factors, as a result of introducing their genes, proteins, or RNAs, or even their metabolites or similar chemicals, into the recipient cells. The reprogramming factors have potent capability of activating the cell growth and reproduction of the recipient cells and, thus, make them highly proliferative. When such rapidly multiplying cells were selected out based upon some stemness markers, the so-called "induced pluripotent stem cells" (iPSCs) are obtained [36-39, 90-92, 125, 139, 141, 142, 168].
However, by clearly identifying the various linkages between iPS reprogramming and neoplastic transformation, it is hoped that the present application will establish a solid foundation for arguing iPSCs as a kind of "neoplastic" cells very similar to "natural" cancer cells and, thus, justifying their use as serendipitous "replacements" for cancer cells in methods for screening agents against neoplastic cells.
c-Myc and Lin28
It turns out that Myc (c-Myc), a very important iPS reprogramming factor, is a notorious oncogene and a master transcription factor that integrates cell proliferation with metabolism through its regulation of thousands of genes including microRNAs (miRNA) [64]. In addition to its known function in regulating the cell cycle and glucose metabolism [65], Myc also stimulates glutamine catabolism [66] through the repression of miRNAs miR-23a and miR-23b [67].
More significantly c-Myc enhances the expression of poly-pyrimidine tract binding protein PTB (also known as hnENPI), hnRNA1 and hnRNA2, and leads to selective expression of pyruvate kinase isoform 2 (PKM2) [65]. PKM2 is the M2 splice isoform of pyruvate kinase (PK) [68] which is a key enzyme for aerobic glycolysis [69], as compared with the M1 splice isoform of pyruvate kinase (PKM1) which is a key enzyme for oxidative phosphorylation. Thus, the selective expression of specific isoform of PK serves as a toggle switch for shifting mass-energy metabolism between an energy production-efficient oxidative phosphorylation and a mass production-efficient aerobic glycolysis.
It is interesting to notice that PKM2 is the dominant form of PK in "embryonic cells" and PKM1 is the dominant form of PK in the "adult cells" [68-69]. This age-specific expression of different isoforms of the same enzyme reflects the physiological need as PKM2 is needed for glycolytic anabolism supporting mass increase in the growth stage of the life, and PKM1 is needed for allowing established cells to perform more energy-consuming functions at the grown up stage of life. Thus, a change in the expression of different isoforms of the same enzyme leads to different modes of metabolism in different life stages. Overgrowth of cell mass, such as neoplasm, is not a desired shift.
Unfortunately, the re-expression of PKM2 [70] activates those "resting" cells and drives them from normally a "quiescent" state into a "hyper-proliferation" state [71]. This adulthood expression of an embryonic enzyme isoform does not lead to the "rejuvenation" of the whole organism, but a formation of some harmful, and even deadly, neoplasm.
More than just contributing to the transformation of "normal cells" into "neoplastic cells," c-Myc coordinately regulates the expression of 13 different "poor-outcome" cancer signatures [17]. In addition, functional inactivation of c-Myc in human breast cancer cells specifically inhibits distance metastasis in vivo and invasive behavior in vitro [17]. So, c-Myc may also contribute to the acquisition of metastatic capability of the "neoplastic cells."
Therefore, iPSCs generated with inducing factors including c-Myc may naturally possess some basic neoplastic features known to natural cancer cells.
The iPSCs generated without c-Myc may also possess oncogenic nature. For example, Lin28, an inducing factor used in place of c-Myc [25], has recently been found in association with cancers [72-74]. More importantly, Lin28 has been shown as a Myc-downstream factor exerting the similar effect as Myc [74-75].
iPS Reprogramming Factors and Cancer Risk
As a matter of fact, iPS reprogramming factors currently employed are more or less associated with various cancers [37-38, 76-77]. Thus, the oncogenic potential is intrinsic for iPS reprogramming, at least for the proto-type iPS reprogramming methods [39]. This intrinsic oncogenic potential is intensified when a tumor-suppressing mechanism is inhibited or knocked out [39]. Unfortunately, many iPS researchers just do not want to face this dark side of iPS reprogramming, and continue at looking at the "bright" side of their discoveries [78]. They emphasize the enhanced "efficiency" of iPS reprogramming and elevated yield of the iPSCs by knocking out the tumor-suppressing mechanisms [79-83] while ignoring the increased risks of cancer potential from these tumor suppression mechanism-jeopardized iPSCs [84-85].
Nevertheless, increasing reports are presenting observations of chromosomal aberrations [86] and cancer-related epigenome changes [87, 146] in iPSCs. There are also reports documenting formation of rhabdomyosarcomas iPSCs [88]. These observations have led to some concerns over the "variation" in the safety of iPSCs [89]. But, claims of generating "transformation-deficient" [45] and, thus, "safe-induced pluripotent stem cells" (safe-iPSCs) with therapeutic potential [46] are still being made. It has been believed that "although there are common pathways activated during reprogramming and tumorigenesis, pluripotent stem cells and tumorigenic cells have important differences" and thus the critical distinctions between true cancer cells and reprogrammed somatic cells may be that reprogrammed cells remain genetically intact" [61]. Thus, despite a clear message arguing the intrinsic distinctions between iPSCs and ESCs [90-92] and some later experimental reports supporting this argument [93-94], leading iPS researchers still reject the intrinsic distinctions between iPSCs and ESCs and the high similarity between iPSCs and cancer cells [95]. The most recent review on iPSCs still claims: "Numerous studies indicate that, at least for some clones, iPSCs are similar if not indistinguishable from ESCs derived from embryo or nuclear transfer experiments" and "somatic cells can be reprogrammed to a pluripotent state, which is molecularly and biologically indistinguishable from that of ESCs" [28] and a straight talk states that there's no reason . . . to think that true bona fide iPSCs cannot function as well as ESCs [47]. This attitude is also reflected by the lack of appreciation of the cancerous nature of iPSCs even by experienced stem cell researchers.
Amazingly, even some cancer researchers apparently still lack an understanding of the cancerous nature of the iPSCs. A recent study has found "a Myc network accounts for similarities between embryonic stem and cancer cell transcription programs" [96]. Even though some iPSCs were also included in this study, the report failed in identifying iPSCs as cancer cells. As a matter of fact, the corresponding author of this report did not even answer the straight question from the present inventor on whether or not iPSCs are cancer cells.
However, it is hoped that, continued dissection of the iPS reprogramming process may not only lead to a comprehensive identification of a sufficient factor set for complete and safe somatic to pluripotent reprogramming [97] but also an increased awareness of the neoplastic nature of iPS reprogramming [39] [125]. More importantly, if the application of this invention is placed into practice, the cancerous nature of iPSCs may be made very apparent, when anti-neoplastic agents, compounds and/or drugs discovered via methods disclosed in this application are also very effective in killing "natural cancer cells."
Genetic and Epigenetic Mutations affecting the Warburg Effect
It is important to point out that iPS reprogramming can turn "normal cells" neoplastic even without any genetic modification and/or a cell reproduction event. This feature will be a key point for the present invention which is focused on discovering anti-neoplastic agents, compounds and drugs that are effective in inhibiting the metabolic mutation serving as a root process for supporting the malicious competition of "neoplastic cells" against "normal cells." In the past, cancer research has been heavily focused on genetic mutations, including mutations in mitochondria, as causes for neoplasia [99-100]. The discovery of Warburg Effect even led some researchers to believe that neoplastic cells have abnormal mitochondria. The outcome of this genetic cancer dogma is the focus of searching anti-cancer drugs that fix the genetic mutations, including mitochondria mutations [101]. However, many drugs targeting the effects of genetic mutations often fail in killing tumor cells, and even succeed in killing normal cells [102].
It turns out that, many times, it is the mitochondrial uncoupling, the abrogation of ATP synthesis by mitochondria, promotes the Warburg Effect in some neoplastic cells and contributes to their resistance to chemotherapy targeting mitochondria [103]. These cancer cells may shift to the oxidation of non-glucose carbon sources to maintain mitochondrial integrity and function [103]. More importantly, increased level of c-Myc in cancer cells causes an increase in level of glutaminase, a protein that helps cells convert amino acid glutamine into an energy source. The breakdown of glutamine provides cancer cells with a carbon source. In fact, glutamine can serve as a major nutrient for cancer cells [104], especially when facing glucose deprivation [105]. Also worth of notice is that mutation in some genes such as KRAS or BRAF often lead to up-regulation of the expression of GLUT1 (encoding glucose transporter-1) and SGLT1 [106]. Thus neoplastic cells often have enhanced glucose uptake and glycolysis, and can survive even at low glucose concentration [107]. Thus, neoplastic cells may still have normal mitochondria despite their abnormal use. Amazingly, drugs targeting mitochondria sometimes kill normal cells more effectively, but exert less or even no harm to neoplastic cells, which use less or even shut down their mitochondria.
Metabolic Mutation/Alteration, Metabolism Switch, and Oncogenic Metabolism
A metabolic mutation or a metabolism switch may be a predominant feature in cancer cell formation. This change may happen at the epigenetic levels and this oncogenic metabolism has attracted more and more research attention recently. A simple RNA splicing which is a modification of an RNA transcript through removing of introns and joining of exons may produce different proteins out of the same gene [108-109]. This epigenetic regulation plays a very important role in normal development as well as neoplastic tumorigenesis [110-111]. Very often, the alternative splicing changes the mode of mass/energy metabolism [112-113] and this alteration in splicing can be influenced by the conditions in which the cells reside [114-115]. A very recent study just confirmed that some cancer-related epigenome changes have been found in iPSCs [87].
It should be pointed out that, although epigenetic changes have been recognized in iPS reprogramming [146], it was not until recently that mainstream journals formally recognized the "shared mechanisms" between induced pluripotency and oncogenesis [147-148]. However, these examinations often focus on the "parallels" between iPS reprogramming and cancer epigenetics in terms of the common transcription factors (TFs) used in the (iPS) "reprogramming" and the (oncogenic)"transformation" [148] but generally failed to realize the common metabolic transformation between iPS reprogramming and neoplastic transformation as discovered by the inventor of this patent application.
Evidence for Metabolic Mutation or Oncogenic Metabolism in iPSCs
Studies have shown that oncogenes such as Myc [116-117] play very important roles in contributing to glycolytic metabolism in cancer cells [70]. Studies also show that glucose deprivation induces oncogenic mutations and thus a switch to oncogenic metabolism [107]. The fact that hypoxia enhances the generation of iPSCs [118] indicates that iPSCs may have switched into a neoplastic glycolysis.
Two reports showed that butyrate promoted generation of iPSCs [149-150]. However, the promotion of iPSCs generation by butyrate was shown as c-Myc-dependent in one study [149] while the other study showed remarkable butyrate stimulation on reprogramming in the absence of KLF4 or MYC transgene [149]. The later study showed enhanced histone H acetylation, promoter DNA demethylation, and expression of endogenous pluripotency-associated gene [150].
To further support the validity of the present invention of using iPSCs for screening anti-neoplastic agents or anti-cancer drugs some additional experimental studies related to the epigenetic landscape of iPSCs [151], especially in comparison to the metabolism of cancer cells [179, 180], are described herein.
On Aug. 3, 2011, a research paper entitled "somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming" was published [152]. This study reported that "[T]emporal sampling demonstrated glycolytic gene potentiation prior to induction of pluripotent markers. Functional metamorphosis of somatic oxidative phosphorylation into acquired pluripotent glycolytic metabolism conformed to an embryonic-like archetype. Stimulation of glycolysis promoted, while blockade of glycolytic enzyme activity blunted, reprogramming efficiency. Metaboproteomics resolved upregulated glycolytic enzymes and downregulated electron transport chain complex I subunits underlying cell fate determination. Thus, the energetic infrastructure of somatic cells transitions into a required glycolytic metabotype to fuel induction of pluripotency." Thus, this study provided a supporting evidence for the theoretic and also logical discovery of glycolysis as a basis for the neoplastic transformation via iPS reprogramming [39, 125].
In 2012, another research article showed metabolic changes from an oxidative state to a glycolytic state during iPS reprogramming and also identified metabolites that differ between iPSCs and ESCs [153]. Interestingly, this study used an Extracellular Flux analyzer manufactured by Seahorse Bioscience (Massachusetts, United States) to measure the basal oxygen consumption rate (OCR) as a measure of mitochondrial respiration and the extracellular acidification rate (ECAR) as a measure of glycolysis-[125] More interestingly, by comparing the effects of some chemicals on the reprogramming efficiency, this study showed that a glycolysis inhibitor, 2-deoxy-D-glucose (2-DG), inhibited the reprogramming efficiency while a glycolysis stimulator, D-fructose-6-phosphate (F6P), increased reprogramming efficiency [153]. These results showed that, by using iPSCs and by measuring the effects of agents on the metabolism associated with neoplastic transformation, anti-neoplastic agents or anti-cancer drugs can be effectively identified. Unfortunately, even though the authors of this study have stated that their "study not only highlights the importance of metabolism in inducing pluripotency, but suggests that understanding the metabolic changes associated with somatic cell reprogramming may also shed light on the metabolic mechanisms regulating cancer" [153], they did not recognize iPSCs as cancer cells.

Using induced pluripotent stem cells for screening anti-neoplastic agents


Systems and methods for transmitting location-specific information such as wireless network connectivity information are generally disclosed herein. One embodiment includes device configurations and techniques implemented to establish a secondary wireless network connection to exchange the location-specific information between a location-unaware device and a location-unaware device. In other embodiments, the location-specific information exchanged via the secondary wireless network connection includes the transfer of the connectivity information, such as a Service Set Identifier (SSID) or network authentication information, from a primary network-aware device to establish a primary network connection at a primary network-unaware device.

A variety of devices may become unaware of location-specific information needed to perform operations. For example, one non-limiting use of location-specific information includes connectivity information to establish or re-establish a wireless network connection. When a wireless-network device (such as a personal computer (PC)) awakes or resumes from a standby power state, the device will attempt to reconnect to a wireless network. For devices connecting to a wireless network in an unknown location (e.g., a Wi-Fi network operating according to one of the standards of the IEEE 802.11 standards family, such as IEEE standard 802.11-2007), however, the amount of time needed to reconnect to a wireless network may be as much as 2-3 minutes to perform a scan for available wireless networks in vicinity of the device, identify the appropriate network to re-connect to, and establish the connection with the wireless network. Further, the connection process may need additional time for user involvement to authenticate to or select the appropriate wireless network. A significant amount of the connection time process may be consumed by performance of a wireless network scan, even if the user is aware of the wireless network Service Set Identifier (SSID) or has another device connected to the wireless network.

Identification-assisted wireless network connections


The present invention discloses an aluminum alloy being excellent in wear resistance, containing, in mass %, 12.0 to 13.7% of Si, 2.0 to 5.0% of Cu, 0.1 to 1.0% of Mg, 0.8 to 1.3% of Mn, 0.10 to 0.5% of Cr, 0.05 to 0.20% of Ti, 0.5 to 1.3% of Fe, 0.003 to 0.02% of P, and has a Ca content controlled to less than 0.005 mass %, the balance being Al and inevitable impurities; and an aluminum alloy sliding member excellent in wear resistance, which has in mass %, 12.0 to 14.0% of Si, 2.0 to 5.0% of Cu, 0.1 to 1.0% of Mg, 0.8 to 1.3% of Mn, 0.10 to 0.5% of Cr, 0.05 to 0.20% of Ti, 0.5 to 1.3% of Fe, 0.003 to 0.02% of P, and has a Ca content controlled to less than 0.005 mass %, the balance being Al and inevitable impurities, and contains primary crystals of Si having a grain diameter of 20 μm or more in an amount of 20 pieces/mm2 or less. The alloy may contain one or two of 0.0001 to 0.01 mass % of B, and 0.3 to 3.0 mass % of Ni.

In recent years, there has been a strong demand to lighten the weights of vehicles in order to reduce energy consumption, and in order to meet this demand, cast aluminum alloys such as A390 have been used in the compressor parts and oil pump covers of vehicles. These aluminum alloys have been widely used in engines and other wear-resistant parts for excelling in wear resistance.
A390 aluminum alloy has a composition containing 16.0-18.0 mass % of Si, 4.0-5.0 mass % of Cu, 0.45-0.65 mass % of Mg, less than 0.5 mass % of Fe, less than 0.1 mass % of Mn and less than 0.20 mass % of Ti, and is characterized by the addition of large amounts of Si in order to achieve the necessary wear resistance.
However, as the Si content increases, the liquidus temperature of the aluminum alloy rises, thus requiring the aluminum alloy to be melted and cast at a higher temperature than is generally used. As a result, not only must an expensive lining refractory material be used, but there are various other drawbacks such as reduced furnace durability, increased fuel consumption and reduced durability of the casting dice. Additionally, there are problems such as the distribution of primary crystal Si becoming uneven, and casting defects such as voids.
Additionally, hyper-eutectic Al-Si alloys excelling in wear resistance and burn resistance such as die-cast alloy JIS ADC14 are used in a manner similar to the above alloy. Furthermore, the applicant of the present application has also developed aluminum alloys such as those disclosed in JP-A H5-78770 and JP-A H7-252567 as wear-resistant alloys, and these have been patented as Japanese Patent No. 2709663 and Japanese Patent No.3378342.    Patent Document 1: JP-A H5-78770    Patent Document 2: JP-A H7-252567
The above patents are for a cast aluminum alloy excelling in wear resistance, characterized by comprising 14.0-16.0 wt % of Si, 2.0-5.0 wt % of Cu, 0.1-1.0 wt % of Mg, 0.3-0.8 wt % of Mn, 0.1-0.3 wt % of Cr, 0.05-0.20 wt % of Ti, 0.003-0.02 wt % of P, and 1.5 wt % or less of Fe, wherein the Ca content is limited to less than 0.005 wt % and having a uniform dispersion of primary crystal Si with an average grain size of 10-50 μm; and a cast aluminum alloy excelling in wear resistance, characterized by comprising 14.0-16.0 wt % of Si, 2.0-5.0 wt % of Cu, 0.1-1.0 wt % of Mg, 0.3-0.8 wt % of Mn, 0.1-0.3 wt % of Cr, 0.01-0.20 wt % of Ti, 0.003-0.02 wt % of P, and 1.5 wt % or less of Fe, wherein the Ca content is limited to 0.005 wt % or less, and primary crystal Si and Al—Si—Fe—Mn—Cr intermetallic compounds are both dispersed as crystals with a grain size of 5-30 μm.

Aluminum alloy excellent in wear resistance and sliding member using this alloy


A hydraulic actuator is disclosed having a cylinder with a piston that is moved by hydraulic fluid. A light guide in one end of the cylinder directs a laser beam into the cylinder, and off the piston where the beam is reflected. The beam then exits the cylinder through a second light guide. A control unit measures the time of flight of the laser beam and calculates the piston position.

The invention relates to measuring the excursion of hydraulic actuators using laser time-of-flight techniques. More particularly, it relates to linear hydraulic cylinders having laser light pulse emitters and laser light detectors reflecting off moving internal surfaces of the hydraulic cylinder.
With the increased computerization of traditionally mechanical and hydro-mechanical systems, the ability to accurately control and position a wide variety of agricultural and construction equipment has become possible. Using proportional control valves and hydraulic server motors, agricultural and construction vehicles have been designed that can compensate for a wide variety of environmental variables to much more accurately control the motion and positioning of their various arms and linkages.
To provide the ever-more-accurate positioning and control that microprocessors are capable of, one must know ever more accurately the exact position of the various linkages, arms and actuators that comprise these vehicles. Other methods of determining the location of mechanical components of these vehicles included such things as rotary potentiometers and resistors mounted at pivoting joints, linear variable differential transformers coupled to the outside of extendable devices such as hydraulic cylinders that extend and retract, and linear variable resistors.
These older methods of determining the location of various components of the vehicles are now seriously outmoded. In particular, many of these components include several moving parts that are exposed to the elements, and therefore are prone to be broken, bent or otherwise become un-calibrated.
What it need, therefore, is a more robust system of determining the position of vehicle components that reduces the risk of breakage, mis-calibration, and provides a greater positional accuracy than these previous devices. It is an objective of the present invention to provide such a system.
In accordance with a first embodiment of the invention, a fluid actuated cylindrical actuator is provided that includes a cylinder having first and second ends, an end cap fixed to the first end of the cylinder and having a rod opening, a piston disposed in the cylinder, a rod coupled to the piston and extending from inside the cylinder to outside the cylinder and passing through the rod opening, a first light guide extending from inside the cylinder to outside the cylinder and adapted to transmit at least a first beam of laser light at a first frequency from outside the cylinder to inside the cylinder and to bar the passage of the fluid, and second light guide extending from inside the cylinder to outside the cylinder and adapted to transmit the at least a first beam of laser light at the first frequency from inside the cylinder to outside the cylinder and to bar the passage of fluid. The first light guide may be disposed to transmit the first beam of laser light substantially along a longitudinal axis of the cylinder such that the first beam impinges on a reflective portion of the piston over substantially an entire range of piston travel. The second light guide may be disposed to receive the first beam after it has been reflected off the piston. The optical distance between the first light guide and the second light guide may be a function of the degree of extension of the rod outside the cylinder. The first beam of light may be reflected off a first surface inside the cylinder where the first surface is coupled to the rod and configured to move with the rod. The first beam of light may be reflected off a second surface fixed with respect to the cylinder and movable with respect to the rod and a third surface fixed with respect to the rod and movable with respect to the cylinder. The first beam may vary in optical length when the rod is moved with respect to the cylinder an amount equal to at least four times an axial distance the rod travels.
In accordance with a second embodiment of the invention, a hydraulic actuator for an agricultural or construction vehicle is provided, the actuator including a cylinder having a substantially circular internal diameter and a longitudinal cylindrical axis, a piston having a substantially circular outer diameter and configured to be received in and hydraulically sealed against the inner diameter of the cylinder, a piston rod with a substantially circular outer rod diameter that is fixed to the piston and extends from the piston inside the cylinder, through a first end wall of the cylinder to a location outside the cylinder, wherein the first end wall is disposed to enclose and seal a first end of the cylinder and is substantially perpendicular to the longitudinal axis of the cylinder, a second end wall fixed to the cylinder and disposed to seal a second end of the cylinder substantially perpendicular to the longitudinal axis of the cylinder, the second end wall including a first optical path configured to transmit a beam of laser light through the second end wall to a reflective surface fixed to the piston and further including a second optical path configured to transmit the reflective beam of laser light back through the end wall, a first optical fiber optically and mechanically coupled to the second end wall to transmit the beam of laser light from a remote laser light source to the first optical path and a second optical fiber optically and mechanically coupled to the second end wall to transmit the reflected beam of laser light to a remote laser light receiver.
The first and second optical paths may include at least one hermetically sealed fiber optical feed-through or connector extending through the second end wall. At least one fiber optic feed-through or connector includes an adjustable focal length lens disposed in the beam of laser light and configured to adjust a focal length of the beam of laser light within the cylinder. At least one fiber optic feed-through or connector may include a threaded end cap with the adjustable focal length lens fixed to the end cap. The hydraulic actuator may include another hermetically sealed fiber optic feed-through or connector extending through the second end wall. The first and second optical fibers may be multi-modal optical fibers. The actuator may also include a first laser diode configured to emit a beam of laser light at a wavelength in the range of 840 to 980 nanometers. The hydraulic actuator may also include a first photo-diode configured to receive the beam of reflective laser light and generate an electrical signal indicative of at least one characteristic of the beam. The actuator may also include a second laser diode configured to emit the beam of laser light in the range of 430 to 1300 nanometers. The actuator may include a second photo diode configured to receive a beam of laser light and generate an electrical signal indicative of at least one characteristic of the beam.
In accordance with a third embodiment of the invention, a method of determining the position of the piston of the actuator described in the previous paragraph includes the steps of generating the beam of laser light, reflecting the beam of laser light off a surface fixed to move axially with the piston, receiving the reflected beam of laser light, and calculating a time-of-flight. The step of generating the beam may include the step of generating the beam with a wavelength of between 430 and 1300 nanometers. The step of generating the beam may include the step of generating the beam with a wavelength in the range of 840 and 980 nanometers. The step of generating the beam may include the step of generating a sequence of individual pulses of light, and the step of calculating a time-of-flight may include the step of determining the time-of-flight of at least one pulse in the sequence of individual pulses of light.

Laser based reflective beam cylinder sensor


This disclosure is directed to software modification that may be used to prevent software piracy and prevent unauthorized modification of applications. In some embodiments, a software vendor may modify software prior to distribution to a user. The software vendor may extract cutouts from an application to create a modified application. The modified application and the cutouts may be downloaded by a user device. The user device may run the application using the modified application and by executing the cutouts in a secure execution environment that conceals the underlying code in the cutouts.

Application stores are rapidly becoming an important distributor of applications for many platforms such as smart-phones, tablets, and conventional computers (e.g., notebook computers, desktop computers, etc.). Application stores are often web-based stores that enable a user to download software electronically without receiving a physical product. There are several reasons for the success of this distribution mechanism for applications. One reason is that developers can use application stores to enable broad distribution of their applications without a large investment by the developer.
The application store is rapidly establishing itself as the main software distribution channel. Currently, hundreds of thousands of smartphone applications are available for download for popular smartphone and tablet operating systems (OS). In addition, thousands of conventional computer applications for conventional computers that run Windows® operating system (OS) or Apple OS are also available. Application stores are expected to increase in popularity in the future and become a dominant distributor of software.
While the current application store growth to date is impressive, application stores are dealing with many challenges that either threaten to inhibit their future growth or endanger it completely. In general the challenges for these application stores include: 1) software piracy, 2) counterfeit applications that create operational, legal, and security issues for application store operators, 3) inability to attract big name independent software vendors (ISVs), and 4) difficulty in offering "try before you buy" or other shareware versions of the applications through this distribution mechanism.
Software piracy has been problematic for software developers and vendors since mainstream computing has become widely available to users. Particularly, with the ease of access to the Internet and ease of copying and sending data over the Internet, software piracy continues to run rampant despite heavy penalties imposed to those prosecuted for pirating software.

Software modification for partial secure memory processing


A chip package is provided. The chip package includes a semiconductor chip having on a front side a first connecting pad and a second connecting pad, a carrier having a pad contact area and a recess, encapsulation material encapsulating the conductor chip, a first external connection that is free from or extends out of the encapsulation material, an electrically conductive clip, and a contact structure. The semiconductor chip is arranged with its front side facing the carrier with the first connecting pad over the recess and with the second connecting pad contacting the pad contact area. The clip is arranged over a back side of the semiconductor chip covering the semiconductor chip where it extends over the recess. The electrically conductive contact structure electrically conductively connects the first connecting pad with the first external connection.

Packaged surface mount devices (SMD packages) that include semiconductor chip are often used in high current devices. The SMD packages may generate a considerable amount of heat during operation. The heat that is generated in the semiconductor chip may have to be dissipated to an outside of the package as efficiently as possible.
However, at present, all SMD packages (power and standard) rely only on a bottom side cooling, for example by mounting the SMI) package on a printed circuit board (PCB) that has a high thermal conductivity, e.g. an insulated metal substrate (IMS).

Plurality of transistor packages with exposed source and drain contacts mounted on a carrier


method of manufacturing a semiconductor device capable of manufacturing a miniaturized semiconductor device is provided. The method of manufacturing a semiconductor device according to an embodiment includes the steps of: preparing a semiconductor substrate having a first surface and a second surface which is an opposite surface of the first surface; forming a hard mask having an opening on the first surface; forming a gate trench extending toward the second surface on the first surface using the hard mask as a mask; widening the width of the opening; filling the opening with an interlayer insulating film; and forming a contact hole in the interlayer insulating film by removing the hard mask.

The present invention relates to a method of manufacturing a semiconductor device.
The semiconductor device disclosed in Japanese unexamined Patent Application publication No. 2003-318396 includes a semiconductor substrate, a gate oxide film, a gate electrode, an interlayer insulating film, and a contact plug.
The semiconductor substrate has a first surface and a second surface that is an opposite surface of the first surface. An N+−type silicon substrate disposed on the second surface. An N−−type epitaxial layer is formed on the first surface side of the N+−type silicon substrate. An N+−type source layer is formed on the first surface. In the semiconductor substrate, a P-type base layer is formed so as to be sandwiched between an N+−type source layer and an N−−type epitaxial laver.
A first trench extending toward the second surface is formed in the first surface so as to reach the N−−type epitaxial layer.
A gate oxide film is formed on the bottom surface and the side surface of the first trench. A gate electrode is buried in the first trench. The interlayer insulating film is formed on the first surface. Contact holes are formed in the interlayer insulating film and the semiconductor substrate.
The contact hole penetrates the interlayer insulating film and extends toward the second surface so as to reach the P-type base layer. Contact plugs are buried in the contact holes.

Method for manufacturing a semiconductor device


A method of measuring dimensional changes in a transparent substrate includes forming an array of reference markers on a reference plate, forming an array of substrate markers on the transparent substrate, stacking the reference plate and transparent substrate such that the reference markers and substrate markers overlap, measuring coordinates of the substrate markers relative to coordinates of the reference markers before and after processing the transparent substrate, and determining dimensional changes in the transparent substrate from the difference between the measured relative coordinates of the substrate markers before and after processing the transparent substrate.

Transparent substrates such as glass substrates find many uses in devices requiring transmission or detection of light. Examples of these devices include, but are not limited to, flat panel displays, active electronic devices, photovoltaic devices, and biological arrays. Processing of a transparent substrate into a form that is useful for a specific device may induce dimensional changes in the transparent substrate. Examples of such processing include, but are not limited to, cutting the transparent substrate and heating the transparent substrate above room temperature. Cutting of a glass substrate may result in distortion of the glass substrate due to release of internal stress from the glass substrate. Heating a glass substrate above room temperature may result in shrinkage or expansion of the glass substrate. Distortion, shrinkage, expansion and other types of dimensional changes can be problematic in devices such as listed above, in particular, arrayed devices. Manufacturers typically require transparent substrates incorporated in arrayed devices to have dimensional changes within a prescribed limit. As an example, AMLCD (active matrix liquid crystal display) device manufacturers typically require that glass substrates incorporated in AMLCD devices have a distortion less than several microns after processing. These dimensional requirements are expected to become even more stringent as AMLCD technology and manufacturing advances.
Measurement systems based on absolute coordinate system (typically using interferometry) are currently used to measure dimensional changes in a transparent substrate. In absolute measurement, the transparent substrate being measured is marked with a series of reference marks. The reference marks are pre-characterized in X-Y position locations before processing the substrate. After processing the substrate, the substrate is then placed back on the measurement system and the X-Y reference structures are re-characterized. The dimensional changes are indicated by change in X-Y position of the reference structures before processing versus X-Y position of the reference structures after processing. Absolute measurement systems are likely to have large measurement errors as the measured area gets larger.
From the foregoing, a method of measuring dimensional changes in a transparent substrate is desired. Such measurements can be used to ascertain that the transparent substrate has dimensional changes within a prescribed limit. Such measurements can also be used to fine-tune the composition of the transparent substrate as well as the processing steps resulting in dimensional changes in the transparent substrate. As the AMLCD industry gears towards large size substrates, a method of measuring dimensional changes over large substrate areas, e.g., more than 2 m long, with high resolution, e.g., submicron resolution, is also desired.

Method and apparatus for measuring dimensional changes in transparent substrates


A signal generator includes a first voltage generator, a second voltage generator, an operational amplifier, and an oscillator. The first voltage generator generates a first voltage, and the second voltage generator generates a second voltage. The operational amplifier generates an amplified error signal based on the first voltage and the second voltage, and the oscillator generates a periodic signal based on the amplified error signal. The first voltage generator and the second voltage generator are configured to generate their respective voltages based on the periodic signal. As a result, frequency deviation in the periodic signal may be corrected, for example, without increasing the source current of the oscillator or the gain of the operational amplifier. Also, improved phase noise performance may also be achieved through an increase in loop gain.

Signal generators are used in a wide variety of applications including smartphones, notebook computers, tablets, gaming devices, and other types of electronic devices and systems. Some signal generators use oscillators to form periodic signals for their intended applications. Examples of periodic signals include clock signals for controlling semiconductor devices and carrier waves for transmitting voice and data in communication systems.
One type of oscillator (known as a feedback oscillator) generates periodic signals using a feedback control loop. The control loop processes the oscillator output signal in a predetermined way and inputs an error signal into an operational amplifier which generates an amplified error signal. The error signal is then used to correct deviations in the oscillator signal to exhibit a stable frequency.
However, signal generators which use feedback oscillators have drawbacks. For example, for reducing phase noise, and susceptibility to systematic errors, you want to increase loop gain. However increasing the loop gain of the feedback oscillator produces a proportional increase in current, which, in turn, increases power consumption. Some oscillators add components in an attempt to compensate for these effects, but these components add cost and complexity and increase the overall size of the signal generator, which may be undesirable in many cases.

Feedback oscillator with multiple switched capacitors


A banknote handling apparatus including a receiving unit that receives banknotes to be counted, a recognition unit that recognizes attributes of the banknotes, a storing unit that stores the banknotes sorted based on a recognition result by the recognition unit, an operation unit for selecting a counting mode and a setting for stacking, and a verification counting process control unit that executes a first counting process when the operation unit selects a verification mode as the counting mode, stores the banknotes in stacking units in a designated first setting for stacking, executes a verification counting process to the banknotes stacked in the first setting for stacking, stores the banknotes in stacking units in a designated second setting for stacking that is different from the first setting for stacking, and determines match/mismatch of the number of banknotes of each denomination in the first counting process and the verification counting process.

One of the banknote handling apparatuses that execute various types of processes related to banknotes is a banknote sorting machine that sorts the banknotes by denomination. Some of the banknote sorting machines include a verification counting process mode. The verification counting process mode is a mode for recounting the banknotes a plurality of times, which are once counted, in order to ensure the credibility of the counting result.
The verification counting process mode is highly demanded mainly in the pachinko market in Japan and in the casino market in the United States, and the verification process is required by law in some countries.
An example of the banknote handling apparatus with the verification counting process mode includes a technique shown in Japanese Utility Model Laid-Open No. 5-52970.
The technique disclosed in the Utility Model draws attention of the operator by a flashed display of the number of counted banknotes if there is a difference between the number of banknotes set by means for setting the number of banknotes and the number of banknotes actually counted.


A method for testing an integrated circuit includes scanning test data from an input and an output pin into a first scan chain during a first state of a clock cycle, and scanning test data from the same input and output pins into a second scan chain during a second state of the clock cycle.

Design for Testability (DFT) is an important requirement for today's complex application specific integrated circuit (ASIC) designs. DFT techniques allow one to perform high quality manufacturing tests after a chip has been synthesized, and to sort out good chips from bad ones. However, due to the ever increasing complexity of today's designs, the Automatic Test Equipment (ATE) tools required for testing are quite complex and expensive. As a result, manufacturing test costs have become a major part of the overall manufacturing cost of ASICs. Conventional testing approaches are unable to reduce this cost without sacrificing the test quality.
For example, the use of scan based Automatic Test Pattern Generation (ATPG) is a common DFT methodology that is widely used. Scan logic allows internal sequential elements of an ASIC, such as a flip-flops, to be controlled and observed during testing. The flip-flops are connected into several chains, called scan chains, which are usually accessed through test pins. The test pins are normally shared with the functional chip pins. When testing is performed, the test vector data is applied through the chains to control the sequential state of the circuit to a desired state. After application of a test vector, the test response data is captured by the flip-flops. The response data is shifted out through the scan chains and is compared against the expected response to check if the chip is functioning correctly.
In conventional scan chain design, a pair of I/O, or scan-in (SI) and scan-out (SO), pins is used to drive a scan chain, as shown in FIG. 1, which shows an example of shifting scan data through a regular scan chain using a conventional scan chain. Data through the chains shifts with each rising (or falling) edge of the clock. In each clock cycle, scan-in data is applied at the SI pin, and scan-out data is observed at the SO pin. The clock is strobed or pulsed to shift the data through the chain. This process is repeated based on the length of the chain, to completely flush the data out of the chain while filling the chain with new data at the same time.
The number of scan chains is usually limited to 16 or 32. The limit on the number of scan chains is bounded by the number of available input and output (I/O) pins that are able to access the chains, and by the number of scan-channels on the ATE used to drive the chains. For example, if one input and one output pin is required to access each chain, the number of the chains is limited to one half of the number of I/O pins.
The chains are usually balanced as much as possible to minimize the length of the longest chain. The number of tester cycles required to shift data through a chain is determined by the length of the chain, i.e. by the number of flip-flops in the chain. Therefore, the reduction in testing time is limited by the length of the chains.
Due to the large number of scan flip-flops and the long scan chain lengths, the majority of test application time is spent in shifting test data through scan chains. Increasing the number of scan chains would reduce the maximum length of the scan chains, thus reducing the number of test cycles required to shift data through the chains. This directly impacts the test cost by reducing the test application time.
The conventional approaches fail to efficiently and cost-effectively perform tests, because the constraints based on the number of available test pins for accessing chains, and/or the number of available scan channels on a tester usually limit the number of scan chains to 16 or 32. Therefore, the conventional approaches are inadequate for testing integrated circuits, because the required amount of testing time is inefficient for testing modern circuit designs. With the rising cost of performing manufacturing tests, it is critical to reduce the test application time, thereby reducing the test cost.

Dual scan chain design method and apparatus


Provided is a resin material for forming an underlayer film which is used to form a resist underlayer film used in a multi-layer resist process, the resin material including a cyclic olefin polymer (I), in which a temperature at an intersection between a storage modulus (G′) curve and a loss modulus (G″) curve in a solid viscoelasticity of the resin material for forming an underlayer film which is as measured under conditions of a measurement temperature range of 30° C. to 300° C., a heating rate of 3° C./min, and a frequency of 1 Hz in a nitrogen atmosphere in a shear mode using a rheometer is higher than or equal to 40° C. and lower than or equal to 200°.

In production of a semiconductor device, a multi-layer resist process has been used to achieve a high degree of integration. In this process, typically, a resist underlayer film is firstly formed on a substrate using a material for forming an underlayer film, a resist layer is formed on an upper surface side of the resist underlayer film, and the resist layer is baked and exposed to form a resist pattern. Next, a desired pattern can be obtained by transferring the resist pattern to the resist underlayer film through etching and transferring the resist underlayer film pattern to the substrate.
A shape of a circuit whose dimension size is large is further processed using not only a substrate having a flat shape but also a substrate having an uneven structure preliminarily formed in a large circuit shape in order to form a more complex circuit, and a multi-patterning method for forming a fine circuit is introduced to the most advanced circuit forming process.
Such a resist underlayer film used in the multi-layer resist process is required to have optical characteristics such as an embedding property for an uneven portion of a substrate having an uneven structure, the flatness of a surface on a side where a resist is applied, optical properties such as a moderate refractive index and an extinction coefficient, and characteristics such as excellent etching resistance.
In recent years, pattern refinement has been further promoted in order to increase the degree of integration. In order to deal with such refinement, various examinations have been conducted on structures, functional groups, and the like of compounds used in materials for forming underlayer films (for example, see Patent Document 1).
Meanwhile, in the refinement for pattern formation in order to obtain a high degree of integration, the influence of irregular reflection or standing waves from a semiconductor substrate in a lithography step carried out using an ArF excimer laser which is a current mainstream has been a major problem. In order to solve this problem, materials with an anti-reflection function as a function required for an underlayer film have been widely examined. As a form in which the anti-reflection function is imparted to an underlayer film, a form in which an uneven structure of a surface of a substrate is embedded with a certain kind of a material and a bottom anti-reflective coating (BARC) is provided on a layer having a flatness or a form in which an anti-reflection function is imparted to a material to be embedded with an uneven structure of a surface of a substrate has been examined.
Particularly in the former material for forming the bottom anti-reflective coating, because of ease of use, various examinations have been conducted on an organic bottom anti-reflective coating formed of a polymer and the like having a light absorbing site. For example, Patent Document 2 discloses a resist underlayer film (bottom anti-reflective coating) which is not intermixed with a resist film formed on an upper layer and in which desired optical constants (a refractive index and an extinction coefficient) are obtained and a selection ratio of a large dry etching rate with respect to a resist film is obtained in a case of being exposed to an ArF excimer laser.

This patent is subject to a terminal disclaimer.

Resin material for forming underlayer film, resist underlayer film, method of producing resist underlayer film, and laminate


A biometric authentication method and system may be implemented in a client server architecture to provide substantial access control security and ease of administration. Users are enrolled in the system by providing multiple biometric measurements which are stored in a database as part of the user's biometric profile. Upon attempted access of a computer, the biometric authentication engine determines which biometrics are required and what the biometric matching criteria are based on the location of the computer, time of day and other security conditions. If the user is determined not to be authentic, a security policy may cause an action to occur such as revoking the user's access privileges or causing the login attempt to appear to be successful while the authorities are summoned.

Security measures are increasingly needed to protect sensitive data and facilities. Only authentic users should have access to such data and facilities. However, simple passwords or even magnetic type security cards that have conventionally been used to authenticate users can be lost, stolen or duplicated. In such cases the missing or duplicated security cards or passwords allows a thief access to secure areas.
In addition to providing security, a security method and apparatus must be relatively simple and easy to use and administer. For example, armed guards can protect stored data and facilities containing such data, but it is impossible to have armed personnel protect all important data and secure facilities. Even if sufficient personnel were available, the question still remains: how can a guard know who should have access?
Today, businesses generally deploy networks, such as local area networks (LANs) or wide area networks (WANs) in order to make valuable information and resources available immediately to trusted employees. Computer networks are thus very powerful and enable employees to work efficiently together. However, the power and convenience of computer networks also makes theft of information easier to accomplish.
In U.S. Pat. No. 6,256,737, it is proposed to implement one or more biometric devices associated with a client computer to read biometric(s) of a user and control access to a computer network. The patent, however, performs authentication and grants access to the network based solely on biometrics of a user and fixed confidence thresholds.
Accordingly, there is a need for security measures that are not easily susceptible to theft and duplication. There is a further need for security measures for accessing computers and computer networks that may be implemented securely and conveniently within an organization. There is still a further need for security measures that are capable of providing variations in authentication procedures based on events that comprise security risks.

Method and apparatus for biometric authentication


An essentially herbal or herbal extract composition for the treatment and control of breath odors and aiding digestion, and method of treatment or control of breath odors and aiding digestion therewith. The preferred composition contains ginger, licorice, chamomile, parsley seed oil, and sunflower seed oil in a delivery system which is intended to be ingested and swallowed whole for delivery of the ingredients to the stomach and/or lower in the digestive tract.

Problems with so called "bad breath" have plagued many animals, including humans as far as time can recall. Many different products and methods have been utilized to cover up these offensive odors at various points in time. Frequently, aromatic substances were used for local application to the oral cavity as a means to mask these offensive odors. These efforts have given rise to the enormous breath spray and breath lozenge industry. These products typically only mask the odors, and then for only a short time. They generally only act on the local environment of the oral cavity and upper end of the throat.
As more sophistication developed in this field, it was recognized that some of the offensive odors associated with breath were due to bacteria in the oral cavity, or decaying food particles retained in the mouth. An increased emphasis on brushing the teeth, gums, and tongue as well as greater emphasis on flossing, began to address some of the issues that mere breath mints, breath sprays, and mouthwashes and rinses could not address Unfortunately, these methods addressed only the local oral cavity and not any of the issues of more remote sources of the offensive odors.
Still more recently it has been recognized that much of the offense breath odors are actually not generated locally in the mouth and throat, but more remotely in the stomach and other portions of the intestinal tract. This is particularly evident with the breath from someone with heartburn and belching, where the breath may be heavily tainted with the smell of chyme, a particularly offensive odor. Other sources of offensive breath odor have been recognized as the elimination of substances directly through the lungs.
Digestive aids have been known for some time. Many of these are simply antacids, such as alkaline hydroxides or antacid/gas producing products, such as mineral carbonates and bicarbonates. Other digestive aids have been known in the herbal area, such as ginger, licorice, and chamomile. These substances have been known as flavorings (ginger and licorice); as carminatives (ginger and chamomile), as a remedy for indigestion generally (chamomile), as intestinal stimulants and/or laxatives (ginger and licorice) and as emetics (chamomile); These herbal properties show that the herbs are working in many differing areas to ease "gastrointestinal discomfort", which discomfort may be caused by excessive gas production, a slowing and distension of the upper or lower bowel, or retention in the stomach of an irritant or other noxious substance needing removal. (See particularly Remington: The Science and Practice of Pharmacy, 19th Edition, Mack Publishing, Easton, Pa. 1995, pp. 1387, 1391, 1393, & 1394; Pharmacognosy, 6th edition, Claus et al Editors, Lea & Febiger, Philadelphia, 1970, p 11, 112, & 214; and Martindale: the Extra Pharmacopoeia, 27th Edition, The Pharmaceutical Press, London, England, 1977). Carminative properties, which help to expel gases from the gastrointestinal tract, either through release via the oral and nasal cavities or through the lower intestine and rectum would also be expected to promote release of offensive odors originating in the stomach via one's breath This is clearly opposite the present invention's goal of improving or controlling offensive breath odors while simultaneously aiding digestion.

Internal breath freshener and digestive aid


An apparatus and method for selectively concentrating an analyte on an apparatus by evaporating solvent from the analyte. Concentration is performed by heating or by flowing a gas stream over the analyte to evaporate solvent from the analyte. The invention also provides a method to be used in conjunction with the apparatus.

The present invention relates to the field of chromatography and more particularly toward an apparatus and method for concentrating an analyte prior to an analytical detector such as a mass spectrometer.
Although analytical instrumentation is becoming increasingly sensitive and analyte detection continues to improve, many chemical analytes require concentration prior to chemical analysis. Typically this is done using bench-top chemical processes specifically developed or tailored to the analytical problem. Representative of these approaches are solvent condensation or evaporation techniques that eliminate the solvent while retaining the analyte (or analytesxe2x80x94in all cases analyte could just as well as be analytes) by exploiting differences in physical properties such as volatility. Nitrogen or inert gas xe2x80x9cblown-downsxe2x80x9d, rotary evaporation, Kuderna-Danish condensers, distillation (steam, etc.), tube heaters, vacuum evaporation, (freeze drying), and related techniques are typical of approaches to pre-concentrate an analyte by removal of a solvent. A number of commercial machines exist specifically for this purpose such as RapidVap(trademark), CentriVap(trademark), SafetyVap(trademark), as examples. Physical techniques such as centrifugation or selective adsorption (solid-phase extraction, column chromatography), selectively separate analytes from a bulk phase prior to redesolving in another solvent prior to injection. It is after these bench-chemistry steps that an analyte has been concentrated to an appropriate degree that makes detection and quantitation possible with an existing detector. These steps are time-consuming, subject to loss, usually require specialized equipment and technical expertise all of which lead to increased expense.
In gas chromatography, large volume injection techniques have been developed with special hardware (pre-column inlets) to allow more sensitive detection by evaporation of solvent while attempting to retain analyte inside the pre-column inlets prior to the analyte being delivered to the analytical column for chromatographic separation and analysis/detection. Typical volumes are less than or equal to 100-xcexcl (by single injection) and the most frequent approach is to inject the solution, either in portion or in entirety, then evaporate the solvent, which is vented from the chromatograph, and transfer the analyte to the analytical column. This approach suffers from a limitation on volume that can be contained inside the pre-column inlet and, therefore, any increases in volume must be obtained by consecutive injections and evaporation cycles that can result in sample losses. These pre-column inlets that thermally program the vaporization of the solvent are called PTVs.
Another similar approach is the cool-on-column solvent venting arrangements that use a long, large diameter of capillary tubing (approximately 1 ml volume) to retain the injected volume. The operation is again the same as the PTV in that the temperature is programmed to vaporize the solvent and retain the analyte. Again the injection volume is fixed by the mechanical configuration of the assorted tubing. In both these approaches the ability to retain analytes is determined by the difference in the boiling point between the solvent and the analyte and the ability of the pre-column inlet and /or analytical column (phase) to selectively capture and retain the analyte.
However, these large-volume pre-column inlets have been considered attractive relative to the standard pre-column inlet such as split/splitless or on-column that only allow less than 4-xcexcl injections and provide no capability for in-situ concentration.
Both the standard volume pre-column inlet arrangements and the existing large volume port technologies are limited in the volume that can be concentrated by the fact that liquid injections vaporize inside the port and mechanical arrangements (namely, the liquid or vapor volume that can be contained) place an upper bound on the concentration factors that can be achieved. It would be desirable to obtain in-situ concentration that is flexible and less constrained in the concentration factors that can be obtained.
In the biochemical and organic chemistry fields there is often a need to concentrate an analyte before it is loaded into an analytical instrument. For instance, some products may need to be concentrated, because they are expensive to derivatize, difficult to extract or synthesize. Small amounts of product or intermediates are produced and need to be characterized and identified accurately before proceeding to future research steps. However, low concentrations of product can be a problem for a researcher, because they may challenge the limits of instrument sensitivities or increase the possibility of inaccurate abundance measurements. In addition, the transfer of these analytes from instrument to instrument or from instrument to storage container can result in significant additional loss of product. For these reasons, simple techniques and instruments for both concentrating and analyzing analytes would be of interest to researchers.
A number of instruments already exist for concentrating analytes. For instance, in the biochemical fields analytes may be concentrated using centrifuges, ultra-centrifuges and filtering. A number of commercial products exist for this purpose. For instance, Amicon(trademark) produces a number of filters that allow for desalting of samples, removal of solvent and concentration of small amounts of analyte. However, these devices often require access to a centrifuge or micro-centrifuge for spinning the analytes and are effective only when concentrating small volumes.
On a larger scale, typical analyte concentration is performed on the bench top using rotary evaporation, K-D, dry nitrogen blow down or in a PTV injection port by depositing the analyte in a liner and then evaporating the solvent by heating the liner. Each of these methods provides for concentration of analyte by removal of solvent. However, most of these methods and instruments suffer from the limitation of possible loss of analyte. In addition, since the concentration of analyte is performed separately, these methods can be time intensive and laborious. It would, therefore, be of particular interest to be able to concentrate an analyte without having to transfer, or resolvate the analyte.
Recent trends in analytical instrumentation include components for concentrating analytes in situ (i.e. directly in the instrument or more preferably before the analytes are introduced onto the pre-column). For instance, quartenary pumps are being used in HPLC instruments to pre-mix analytes before the analyte is injected and separated. Present chromatographic technology, however, requires additional hardware, components or complex instrumentation design to appropriately concentrate analytes. In addition, most of the chromatography instrumentation is designed to concentrate the analyte inside the pre-column just before it is introduced onto the column. A problem with this type of device is that the analyte is too dilute or contains a volatile solvent that significantly lowers the amount of sample concentration. It would, therefore, be desirable to be able to adapt existing chromatography hardware to serve the purpose of concentrating analytes in situ (i.e. on the syringe needle tip). A brief review of pre-column inlets, therefore, is in order to clarify existing technology that may be adapted to serve these purposes.
A number of different pre-column inlets or injectors exist to provide accurate, reproducible and predictable introduction of analytes into gas chromatography columns. Usually the analyte is a liquid and can be injected using a syringe, but other devices are available. For instance, analytes can be introduced onto columns by automatic analyzers or valves. Pre-column inlets can be divided into two major categories including packed-column inlets and capillary-column inlets. In gas chromatography the packed column inlets are fairly popular. A second type of column called a capillary-column inlet is also quite popular. These inlets include: capillary direct or (vaporizing inlet), split/splitless inlets (a vaporizing inlet), programmed temperature vaporizer inlets (vaporizing) and cool-on-column (non-vaporizing) inlets.
Capillary inlets are used with wide-bore capillary columns (I.D.xe2x89xa70.5 mm) and are made by substituting a special insert inside a packed-column inlet. The two types of inlets, including the split and splitless inlets, are quite different in design and operation. The split inlet was the earlier design and is a vaporizing inlet that vents most of the analyte in the split mode and transfers most of it to the column in the splitless mode. The splitless inlets load the analyte directly onto the column whereas the split inlets allow for loading partial amounts of analyte onto the column while at the same time allows venting of a portion of the final analyte. Programmed temperature vaporizer (PTV) inlets combine the benefits of split, splitless and on-column inlets. Analytes are usually injected into a cool liner and no syringe needle discrimination can occur. Inlet temperature is then usually increased to vaporize the analyte. A variety of user programs can then be used to provide determined vent times and temperatures to achieve results similar to split or spiitless transfers. PTV injection provides the most flexibility. Other advantages of PTV inlets include: the ability to trap nonvolatile components in a liner, removal of solvent and low boiling components, use of large injection volumes, no special syringe needed, no syringe needle discrimination, retention time and high reproducibilities similar to cool on column injections. A number of commercial injectors exist for these purposes. For instance, the Apex Prosep(trademark) pre-column inlet is highly versatile and may be used for injecting analytes. The system includes a standard inlet that contains both temperature and vaporizing regulation. However, these systems have only been used to concentrate analytes after they have been ejected from the syringe needle into the pre-column inlet. A number of techniques are used at this point to concentrate, or focus, the injected analtye. The major focusing techniques include stationary phase focusing, solvent focusing and thermal focusing.
Stationary phase focusing is the most frequently used technique and is possible only in temperature-programmed analysis. Generally, in gas chromatography, retention is a function of temperature. For instance, the speed at which the solutes travel down the column is dependent upon the temperature changes. As the temperature is increased, the solutes increase their speed of separation and as the temperature is lowered, they slow their movement. As the vaporizing analyte moves from the inlet to the column, it comes in contact with the stationary phase and is trapped in a defined range. The lower the applied temperature, the more effective the focusing.
Solvent focusing occurs in a different fashion compared to stationary phase focusing. For instance, as a condensed solvent starts to evaporate, solutes with volatility similar to that of the solvent tend to concentrate and focus on the solvent tail. This technique yields very narrow peaks.
Thermal focusing is the last technique and relates to the condensation of gases in a tube or at the head of the column. Peaks narrow as the solute volume is reduced during condensation. This technique is particularly helpful in narrowing bandwidths when the column temperature is approximately 150xc2x0 C. below the boiling point of the solvent. In addition, thermal focusing does not rely on chromatographic processes. It only requires a surface on which vapors can condense. To date, each of these techniques has been applied to a gas chromatography column or pre-column inlets. These techniques are not designed for concentrating the analyte at the pre-column stage. A disadvantage of this is that lower amounts of analyte are loaded onto the column and by the time the separated components reach the final mass analyzer, a low signal to noise ratio is obtained. This negatively affects the overall results. Therefore, there is a substantial need for an apparatus and method that concentrates the analyte before it is injected into the pre-column inlet.
Accordingly, it is an object of the present invention to overcome the above-mentioned disadvantages of the prior art by providing a novel method and apparatus for selectively removing solvent and concentrating an analyte before it is subject to chromatographic analysis.
It is another object of the invention to provide an improved method and apparatus for concentrating an analyte in situ using an instrument pre-column inlet port and syringe.
It is a still further object of the invention to provide an improved method of removing solvent from an analyte before injection into a pre-column inlet port or chromatographic column.
Additional objects, advantages and novel features of the invention set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by the practice of the invention.
In a general aspect, then, the present invention relates to an apparatus for concentrating an analyte at a gas chromatograph (GC). The apparatus has a dispensing means for holding and dispensing an analyte, an injection means for receiving the dispensing means, and a concentration means in contact with the injection means for concentrating the analyte on the dispensing means. The dispensing means may include an auto-sampler having a syringe with a needle and tip or similar type device. The dispensing means may also comprise the syringe needle and tip without the auto-sampler. The injection means includes a pre-column inlet having an inlet port designed for receiving the dispensing means. The concentration means is in contact with the injection means and is designed for evaporating or heating and removing the solvent from the analyte at the dispensing means. The concentration means may be a heater or a gas inlet port.
The invention also includes a method of removing solvent from an analyte and concentrating the analyte in a mass spectrometer. The technique accomplishes the concentration before the analyte is injected into a pre-column inlet and loaded onto a gas chromatograph by first collecting the analyte in the dispensing means. The dispensing means is then inserted into the injection means and the solvent is removed from the analyte through evaporation. The evaporation may occur by heating the analyte or by applying a gas stream. The concentrating means uses a combination of solvent and/or thermal focusing to concentrate the analyte.
Additional objects, advantages and novel features of the invention will be set forth in the part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.