Abstract:
A device for isolating a microbe or a virion includes a semiconductor substrate; and a trench formed in the semiconductor substrate and extending from a surface of the semiconductor substrate to a region within the semiconductor substrate; wherein the trench has dimensions such that the microbe or the virion is trapped within the trench.

Description:
DOMESTIC PRIORITY 
       [0001]    This application is a divisional of and claims priority to U.S. patent application Ser. No. 14/988,887, filed on Jan. 6, 2016, entitled “SEMICONDUCTOR MANUFACTURED NANO-STRUCTURES FOR MICROBE OR VIRUS TRAPPING OR DESTRUCTION”, the entire contents of which are incorporated herein by reference. 
     
    
     BACKGROUND 
       [0002]    The present invention relates to semiconductors, and more specifically, to semiconductor nanostructures. 
         [0003]    Semiconductor materials have an electrical conductivity value that falls between that of a conductor, such as copper, and an insulator, such as glass. Semiconductor materials are used in many modern electronics. Semiconductor materials may be elemental materials or compound materials. Silicon, germanium, and alloys thereof, are two types of semiconductor materials used in many semiconductor devices. 
         [0004]    Complementary metal oxide semiconductor (CMOS) technology is used for constructing integrated circuits. Semiconductor manufacturing techniques include various precise methods for forming nanoscale structures. CMOS technology is used in microprocessors, microcontrollers, static RAM, and other digital logic circuits. CMOS designs may use complementary and symmetrical pairs of p-type and n-type metal oxide semiconductor field effect transistors (MOSFETs) for logic functions. 
       SUMMARY 
       [0005]    According to an embodiment, a device for isolating a microbe or a virion includes a semiconductor substrate; and a trench formed in the semiconductor substrate and extending from a surface of the semiconductor substrate to a region within the semiconductor substrate; wherein the trench has dimensions such that the microbe or the virion is trapped within the trench. 
         [0006]    According to another embodiment, a device for isolating a microbe or a virion includes a first semiconductor layer having a first trench extending from a first surface to a second surface of the first semiconductor layer; a second semiconductor layer having a second trench extending from a first surface to a second surface of the second semiconductor layer, the second trench having a diameter that is smaller than the first trench; and an elongated gap positioned between portions of the first semiconductor layer and the second semiconductor layer. 
         [0007]    Yet, according to another embodiment, a device for damaging or destroying a microbe or a virion includes a semiconductor substrate; and an array of protrusions having nanoscale dimensions extending from the semiconductor substrate; and wherein the microbe or the virion is damaged or destroyed after being disposed on the array of protrusions. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]    The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the claims at the conclusion of the specification. The foregoing and other features, and advantages of the invention are apparent from the following detailed description taken in conjunction with the accompanying drawings in which: 
           [0009]      FIGS. 1-4B  illustrate structures and methods of trapping microbes according to an embodiment, in which: 
           [0010]      FIG. 1  illustrates a method for using holes (trenches) in a semiconductor substrate to trap a microbe/virion; 
           [0011]      FIGS. 2A, 2B, and 2C  illustrate patterned hole arrays of different dimensions; 
           [0012]      FIGS. 3A and 3B  illustrate a method of using a patterned hole array to determine microbe/virion concentration; 
           [0013]      FIGS. 4A and 4B  illustrate using a roughened nanosurface for trapping a microbe/virion; 
           [0014]      FIG. 5  illustrates a method for size filtering microbes/virions according to another embodiment; 
           [0015]      FIGS. 6A-9  illustrate methods of making nanoscale protrusions to damage/destroy a microbe/virion according to embodiments, in which: 
           [0016]      FIGS. 6A, 6B, 6C, and 6D  epitaxial growth on fins to create an array of nanospikes/needles; 
           [0017]      FIG. 7A  illustrates a small dimension array of nanospikes; 
           [0018]      FIGS. 7B and 7C  illustrate a large microbe/virion positioned on a small dimension array of nanospikes; 
           [0019]      FIG. 7D  is a large dimension array of nanospikes puncturing a large microbe/virion; 
           [0020]      FIG. 7E  is an array with nanospikes having different sizes; 
           [0021]      FIG. 7F  is an array of nanospikes illustrating different spacing and depths; 
           [0022]      FIG. 7G  is an array of blunt nanospikes; 
           [0023]      FIG. 7H  is an array of rod-shaped nanospikes; 
           [0024]      FIGS. 8A-8E  illustrate methods of making nanospikes, in which: 
           [0025]      FIG. 8A  is a cross-sectional side view of a photoresist and hard mask disposed on a substrate; 
           [0026]      FIG. 8B  is a cross-sectional side view after patterning the photoresist; 
           [0027]      FIG. 8C  is a top view of  FIG. 8B ; 
           [0028]      FIG. 8D  is a cross-sectional side view after rotating the substrate and performing a second patterning process; 
           [0029]      FIG. 8E  is a cross-sectional side view after recessing the substrate; 
           [0030]      FIG. 8F  is a cross-sectional side view after performing a crystallographic etch; 
           [0031]      FIG. 8G  is an electron micrograph image of a hydroxide etched substrate; 
           [0032]    and 
           [0033]      FIG. 8H  is a cross-sectional side view after removing the remaining hard mask to form the array of nanospikes; and 
           [0034]      FIG. 9  illustrates a method for forming a flexible tube with an array of nanospikes. 
       
    
    
     DETAILED DESCRIPTION 
       [0035]    Semiconductor manufacturing techniques may be used to create precision-constructed nanostructures on the same scale as pathogenic organisms, for example, viruses and bacteria. By tuning the size and shape, the nanostructures may be used to trap, measure, physically filter, or attack and destroy the pathogens. Using such methods, the pathogens may not be prone to develop resistance. 
         [0036]    Accordingly, various methods for trapping, measuring, filtering, and attacking pathogens are described herein. The disclosed methods reduce the risk for pathogens developing antibiotic resistance. In some embodiments, a size-based trapping/filtering mechanism is described. In other embodiments, a spike-like envelope puncture mechanism is used. Like reference numerals refer to like elements across different embodiments. 
         [0037]    The following definitions and abbreviations are to be used for the interpretation of the claims and the specification. As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. 
         [0038]    As used herein, the articles “a” and “an” preceding an element or component are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore, “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular. 
         [0039]    As used herein, the terms “invention” or “present invention” are non-limiting terms and not intended to refer to any single aspect of the particular invention but encompass all possible aspects as described in the specification and the claims. 
         [0040]    As used herein, the term “about” modifying the quantity of an ingredient, component, or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions. Furthermore, variation can occur from inadvertent error in measuring procedures, differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods, and the like. In one aspect, the term “about” means within 10% of the reported numerical value. In another aspect, the term “about” means within 5% of the reported numerical value. Yet, in another aspect, the term “about” means within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reported numerical value. 
         [0041]    As used herein, the term “microbe” means a microorganism, for example, a bacteria or an archaeon. 
         [0042]    As used herein, the term “virion” means a viral DNA or RNA core with a protein coat, and optionally an external envelope. 
         [0043]    Turning now to the Figures,  FIGS. 1-4B  illustrate structures and methods of trapping (isolating) microbes/virions according to an embodiment.  FIG. 1  illustrates a method for using holes  120  (trenches) in a substrate  101  to trap a small microbe/virion  130 . The substrate  101  may include a semiconductor material, a dielectric material, or any combination thereof. The size of the substrate  101  depends on the particular application and targeted microbe/virion  130 . 
         [0044]    Non-limiting examples of semiconductor materials include Si (silicon, including polysilicon), strained Si, SiC (silicon carbide), Ge (germanium), SiGe (silicon germanium), SiGeC (silicon-germanium-carbon), Si alloys, Ge alloys, III-V materials (e.g., GaAs (gallium arsenide), InAs (indium arsenide), InP (indium phosphide), aluminum arsenide (AlAs)), or any combination thereof. Other non-limiting examples of semiconductor silicon-on-insulator (SOI) substrates with buried oxide (BOX) layers. 
         [0045]    Non-limiting examples of dielectric materials include dielectric oxides (e.g., silicon oxide), dielectric nitrides (e.g., silicon nitride), dielectric oxynitrides, or any combination thereof. 
         [0046]    After forming the substrate  101 , optionally, a microbe/virion binding material  110  is disposed on the substrate  101 . The microbe/virion binding material  110  may be a material that has an affinity for the microbe/virion  130  of interest. In some embodiments (not shown), the microbe/virion binding material  110  may be disposed in the holes  120 . 
         [0047]    An additional layer  102  may be disposed on the substrate  101  to form the holes  120 . Or the holes  120  (apertures/trenches) may be formed directly in the substrate  101 . The additional layer  102  may be the substrate  101  material or another semiconductor and/or dielectric material. 
         [0048]    The holes  120  may be formed in the substrate  101  or the additional layer  102  by performing lithography and etch process. The etch process may be a wet etch process or a dry etch process, for example, a reactive ion etch (RIE) process. The size of the holes  120 , for example, the width  111  and depth  112 , may generally vary and depend on the microbe/virion  130  that is targeted. For some microbes/virions  130 , deeper trenches may more easily trap the target. The depths of the holes  120  may be, for example, up to 200 nm. The diameters of the holes  120  may become smaller (more narrow), as the depth increases. 
         [0049]    The microbe/virion  130  may be for example, a bacterium, an archaeon, or other pathogen. The microbe/virion  130  will be trapped within the holes  120  after a solution or sample including the microbes/virions  130  is disposed on the surface of the semiconductor structures comprising the holes  120 . The microbes/virions  130  then become trapped within the trenches. 
         [0050]    The size of the holes  120  may generally vary and depend on the targeted microbe/virion. In some embodiments, the holes  120  have an average diameter in a range from about 70 to about 700 nm. In other embodiments, the holes  120  have an average diameter in a range from about 70 to about 150 nm. 
         [0051]    In an exemplary embodiment, a 150 millimeter (mm) radius wafer may be used as the substrate. The 150 mm wafer has a surface area of about 0.0707 meters 2  (m 2 ), but only about half of the surface area may be sued to trap virions. An array of holes is formed in the substrate to trap a virus, for example, a poliovirus having about a 30 nm diameter. About 1.2e6 virions may be captured per wafer. 
         [0052]    In another exemplary embodiment, the array of holes may be used as a virus strainer with a tuned pore size. Microelectronics processing methods may be used for precise channel size control. 
         [0053]    In yet another exemplary embodiment, perpendicular double patterning may be used to form the holes. Deeper vertices and/or larger vertices within the substrate may allow virions to enter the substrate. 
         [0054]      FIGS. 2A, 2B, and 2C  illustrate patterned trench/hole arrays of different dimensions.  FIG. 2A  shows a microbe/virion  230  trapped within a hole  220  in a substrate having a diameter of about 105 nm.  FIG. 2B  shows a microbe/virion  230  trapped within a hole  220  having a diameter of about 64 nm.  FIG. 2C  shows a microbe/virion trapped within a hole  220  having a diameter of about 42 nm. 
         [0055]      FIGS. 3A and 3B  illustrate a method of using a patterned hole (trench) array to determine microbe/virion concentration. The patterned arrays may be used to quickly and easily determine microbe/virion concentrations.  FIG. 3A  shows arrays  301 ,  302 ,  303 ,  304  of holes  320  having different average sizes/diameters. A sample substrate/slide is prepared with various arrays of holes. The arrays  301 ,  302 ,  303 ,  304  are formed in different sections on a single substrate/slide. Different sized holes formed on different parts of the substrate/slide may be used to study different sized microbes/virions  330 . 
         [0056]    The substrate/slide is exposed to an environment that includes microbes/virions  330  (e.g., using a solution including the microbe/virion), and the microbes/virions  330  are trapped within the holes  320 , as shown in  FIG. 3B . A solution may include different microbes and/or virions. The surface of the substrate/slide may be rinsed to remove excess particles. 
         [0057]    The microbes/virions  330  may be labeled with a fluorescent marker so that a black box and camera may be used to record fluorescence intensity. The fluorescence intensity will be directly proportional to the labeled microbe/virion  330  concentration in the holes  320 . If the concentration is too high (or at the maximum level of detection), a more dilute solution of the microbe/virion  330  may be used until a measurable concentration is achieved. The microbes/virions  330  also may be studies using other analytical methods. Using arrays of holes to conduct assays as described is fast, inexpensive, robust, and size selective. 
         [0058]      FIGS. 4A and 4B  illustrate using a roughened semiconductor nanosurface for trapping a microbe/virion  430 . The roughened nanosurface shown in  FIG. 4B  may be formed by, for example, growing a high % germanium (Ge) epitaxial silicon germanium (SiGe) film on a silicon substrate. The SiGe film is annealed to form nanoscale Ge agglomerates  421 . Elongated trenches  420  are formed between the Ge agglomerates  421  to form the roughened nanosurface. Optionally, the roughened nanosurface may be coated with an anti-microbial material, for example, copper. 
         [0059]    The microbe/virion  430 , shown in  FIG. 4A , is trapped within the elongated trenches  420 , as shown in  FIG. 4B . The roughened nanosurface may be used to trap microbes/virions  430  with elongated shapes. The roughened nanosurface increases interactions between the substrate surface and the microbe/virion  430 , which results in more microbes/virions  430  being trapped (or destroyed). 
         [0060]      FIG. 5  illustrates a method for size filtering/isolating microbes/virions  530  according to another embodiment. Structures for size filtering may be formed using multiple semiconductor layers  501 ,  502  with different sized holes  510 ,  511 . Multiple patterning and etching steps may be used to create holes  510 ,  511  and fluid gaps  550  (elongated gaps/trenches) between the layers  501 ,  502 . Any number of layers and holes of any dimension may be used. The sequential layers may include different sized holes to filter out different sized microbes/virions  530 . 
         [0061]    A solution of the microbes/virions  530  is disposed on the first layer  501  of the semiconductor structure. The first layer  501  includes holes  510  with diameters that are larger than the holes  511  in the second layer  502 . The solution may include different sized microbes/virions  530 . Microbes/virions  530  are large enough to pass through the holes  510  in the first layer  501 , but are too large to pass through the holes  511  in the second layer  502  will be filtered through the fluid gap  550  between the first and second layers  501 ,  502 . The microbes/virions  530  will exit (be expelled) between the layers and can be collected. Purified fluid, or fluid with smaller microbes/virions that may pass through the holes in the next layer (holes  511 ), will travel through the second layer  502  and exit the holes  511  in the second layer  502 . The semiconductor structure may include other layers beneath second layer  502 , as well as a second fluid gap beneath second layer  502  so that other particles are expelled from a different output area in the structure. 
         [0062]    According to another embodiment, spikes (protrusions) of nanometer sized dimensions (nanospikes or nanoneedles) may be used to damage/destroy microbes/virions according to a third embodiment, which is described in  FIGS. 6A through 9  below. The size of the nanospikes (nanoprotrusions) may depend on the size of the targeted microbe/virion. A solution comprising the microbe/virion is disposed on a surface of the array of protrusions to damage/destroy the microbe/virion. The nanospikes may have any size/dimension, and for example, may have sharp/blunt ends. 
         [0063]      FIGS. 6A, 6B, 6C, and 6D  show epitaxial growth on fins (protrusions) to create an array of nanospikes. The nanospikes may be formed by, for example, forming epitaxial growth on semiconductor fin structures (e.g., FinFET fins). As shown in  FIG. 6A , fins  601  are patterned in a semiconductor substrate material. The epitaxial growth process may be, for example, chemical vapor deposition (CVD) (liquid phase (LP) or reduced pressure chemical vapor deposition (RPCVD), vapor-phase epitaxy (VPE), molecular-beam epitaxy (MBE), liquid-phase epitaxy (LPE), metal organic chemical vapor deposition (MOCVD), or other suitable processes. The epitaxial growth process creates a diamond-shaped epitaxial growth  602  on surfaces and sidewalls of the fins  601 , as shown in  FIGS. 6A, 6C, and 6D . The epitaxial growth process may be controlled to create spikes of different sizes, as shown in  FIG. 6C . Larger nanospikes may be created by merging two adjacent epitaxial growths, as shown by the large merged epitaxial growth  610 . Larger or smaller nanospikes may be formed, depending on the targeted microbe/virion. 
         [0064]    The dimensions and density of the nanospikes is chosen based on the targeted microbe/virion size. The poliovirus has a diameter of about 30 nm. An  Escherichia coli  is about 0.5 micron×2 microns. A human cell is about 10-100 microns in size. 
         [0065]      FIG. 7A  illustrates an array of nanospikes  702  in which smaller microbes/virions  730  may be positioned between the individual nanospikes.  FIGS. 7B and 7C  illustrate a large microbe/virion  731  positioned on a small dimension array of nanospikes  702 . The small dimension array of nanospikes  702  may not puncture a large microbe/virion  731  and may have a “bed of nails” effect.  FIG. 7D  is a large dimension array of nanospikes  702  puncturing a large microbe/virion  731 . Smaller microbes/virions  730  are not punctured and fall between the nanospikes  702 . Therefore, the nanospikes  702  only damage/destroy a specific size range of microbes/virions. 
         [0066]      FIG. 7E  is an array with nanospikes  702  having different sizes. The fractal pattern of spike sizes may be used for a broad range of microbe/virion size destruction, as shown for small microbe/virions  730  and large microbe/virions  731 . The spacing  710  and depth  711  of the nanospikes  702  may also be altered, depending on the size of the target, as shown in  FIG. 7F . Nanospikes  702  may have different shapes, and may be, for example, blunt nanospikes ( FIG. 7G ) or rod-like (cylindrical) nanospikes ( FIG. 7H ). 
         [0067]      FIGS. 8A-8E  illustrate methods of making nanoprotrusions (nanospikes) according to some embodiments.  FIG. 8A  is a cross-sectional side view of a photoresist  802  and hard mask  803  disposed on a substrate  801 . The substrate  801  may include a semiconductor material. Non-limiting examples of semiconductor materials include Si (silicon, including polysilicon), strained Si, SiC (silicon carbide), Ge (germanium), SiGe (silicon germanium), SiGeC (silicon-germanium-carbon), Si alloys, Ge alloys, III-V materials (e.g., GaAs (gallium arsenide), InAs (indium arsenide), InP (indium phosphide), aluminum arsenide (AlAs)), or any combination thereof. Other non-limiting examples of semiconductor silicon-on-insulator (SOI) substrates with buried oxide (BOX) layers. The substrate  801  may be formed using, for example, chemical vapor deposition (CVD) (liquid phase (LP) or reduced pressure chemical vapor deposition (RPCVD), vapor-phase epitaxy (VPE), molecular-beam epitaxy (MBE), liquid-phase epitaxy (LPE), metal organic chemical vapor deposition (MOCVD), or other suitable processes. 
         [0068]    The hard mask  802  may be an insulating material, silicon nitride (SiN), SiOCN, SiBCN, or any combination thereof. The hard mask  802  may be formed using a deposition method, for example, a CVD method or a physical vapor deposition (PVD) method. The photoresist  803  may be, for example, a polymeric spin-on material or other polymeric material. 
         [0069]      FIG. 8B  is a cross-sectional side view after patterning the hard mask  802  and removing the photoresist  803 . The photoresist is exposed to a desired pattern of radiation, and the exposed photoresist is developed with a resist developer to provide a patterned photoresist. At least one etch is employed to transfer the pattern from the patterned photoresist  803  into the hard mask  802  and to form gaps  810  (trenches) between hard mask pillars  811 . The etch process may be a dry etch and/or wet etch process. After transferring the pattern, the patterned photoresist  803  is removed utilizing resist stripping processes, for example, ashing.  FIG. 8C  is a top view of  FIG. 8B , showing that elongated “stripes” of the hard mask  802  are formed. 
         [0070]      FIG. 8D  is a cross-sectional side view after rotating the substrate  801  about 90 degrees and performing a second patterning and etch process, as described above for  FIGS. 8A-8C . A second photoresist layer may be used, but is not necessary. A single photoresist may be used for the first and second patterning processed, for example, by a single resist double lithographic (or patterning) process. The second patterning process forms an array of individual scattered cube-like structures of the hard mask  802 . Additional patterning (e.g., sidewall image transfer) and etching processes may be employed to form cube-like structures having smaller dimensions. 
         [0071]      FIG. 8E  is a cross-sectional side view after, optionally, recessing the substrate  801  to form trenches  820  beneath the hard mask  802 . Recessing the substrate  801  creates will create a sharper tip in the final nanospike. 
         [0072]      FIG. 8F  is a cross-sectional side view after performing a crystallographic etch on the substrate trenches  820  to form inverse pyramid-shaped trenches  821 . The crystallographic etch may be, for example, a crystallographic hydroxide etch. The hydroxide etch may include, for example, NH 4 OH, KOH, tetramethylammonium hydroxide (TMAH), tetraethylammonium hydroxide (TEAH), or a combination thereof. The etch process may be a timed etch process or an over-etch process. 
         [0073]    The inverse pyramid-shaped trenches  821  are formed due to different etch rates on different crystal planes in the substrate  801 .  FIG. 8G  is an electron micrograph image of a hydroxide etched substrate having inverse pyramid-shaped trenches  821 . 
         [0074]      FIG. 8H  is a cross-sectional side view after removing the remaining hard mask  802  material to form the array of nanospikes  840  in the substrate  801 . 
         [0075]      FIG. 9  illustrates a method for forming a flexible tube  910  with an array of nanospikes. The array of nanospikes extends into the center of the tube. A substrate sheet of nanospikes  901  is converted to a flexible film  902 . The flexible film may be formed by, for example, performing a spalling process, for example, with nickel (Ni). The flexible film  902  may be inserted into another tube or capillary array to form a lining. Force, demonstrated by arrows  920 , may be applied to constrict the passage between the flexible films  902  and bring more microbes/virions per unit volume into contact with the nanospikes. In other embodiments (not shown), the flexible film  902  may be rolled into the shape of a tube. 
         [0076]    As described above, various embodiments provide methods for isolating, trapping, measuring, filtering, and attacking pathogens are described herein. The disclosed methods reduce the risk for pathogens developing antibiotic resistance. In some embodiments, a size-based trapping/filtering mechanism is described. In other embodiments, a spike-like envelope puncture mechanism is used. 
         [0077]    The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, element components, and/or groups thereof. 
         [0078]    The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated. 
         [0079]    The diagrams depicted herein are just one example. There may be many variations to this diagram or the steps (or operations) described therein without departing from the spirit of the invention. For instance, the steps may be performed in a differing order or steps may be added, deleted or modified. All of these variations are considered a part of the claimed invention. 
         [0080]    The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.