JP2006506059A - Artificial biofilm storage device - Google Patents

Abstract

The present invention includes methods and compositions for storing and preserving biofilms for inputting and outputting high density information. One form of the present invention is a biofilm storage device made with a biological material applied to a substrate, for example to form a dry film that is stable at room temperature for a very long time. Yet another aspect of the present invention is a method of making a biofilm storage device in which biological material is applied to a substrate under conditions that facilitate alignment of the biological material on the substrate. The compositions, methods and kits of the present invention have a wide range of applications in biology, magnetics, optics and microelectronics.

Description

Related Applications This patent application claims priority to US Provisional Application No. 60 / 413,081 to Lee et al., Which is hereby incorporated by reference in its entirety.

Statement on federal research support The United States federal government agrees that the present invention is consistent with the terms of the invention in accordance with the terms of the National Science Foundation (NSF) and the US Army Research Office (ARO) (Grant No. DA 10-01-0456). Has ownership.

The present invention is generally directed to the field of molecular storage devices, and in particular to the storage and preservation of artificial biofilms for inputting and outputting high density information.

  Nucleotide and / or amino acid sequence listings are incorporated by reference with material in computer readable form.

BACKGROUND OF THE INVENTION The use of “biological” materials to fabricate next generation microelectronic devices provides a solution that allows the limitations of conventional processing and storage methods to be removed. A crucial element in this approach aimed at the successful development of so-called organic-inorganic hybrid materials is the identification of the appropriate compatibility and combination of biological and inorganic materials, the synthesis and application of appropriate materials, and their organisms. The long-term storage of the storage device. If the biological material can be stored properly for long periods of time, it is extremely economically beneficial, especially if it reduces weight and storage space and further increases or maintains material stability.

  Modern technologies used to store biological materials such as viruses and their products (eg, DNA and proteins), or other biological materials, are expensive and / or extensively Requires unwieldy chemical modification techniques. Since biological materials are generally highly sensitive to their environment, highly specific and often expensive materials are required to guarantee their stability, activity, and lifetime. Few biological materials are stable at room temperature over a much longer period. In fact, biological materials are often considered unstable at room temperature. For example, viruses and bacteria are highly sensitive to temperature and metabolites, require sustained nutrition and appropriate air (gas) conditions to maintain activity, and are highly resistant to growth and density changes. Must be monitored at a frequency.

  There are several methods for storing and preserving biological material. Generally, cold storage or freeze-drying methods (eg, suspending the material in 10% glycerol under low temperature such as -20 ° C to -80 ° C) or polyethylene glycol modification methods are used. Another option is the drying method, which has both advantages and disadvantages. Drying methods do not require high costs, but do not allow mass production (ie, production in industrial quantities). On the other hand, freeze-drying can be used for mass production. However, this process severely damages sensitive biological materials. In addition, freeze-drying is very inconvenient and sterilizable because it requires the use of expensive substances (eg, dry ice or other coolants) even when transferring material from one facility to another. It cannot be guaranteed and is not very cost effective.

  Current methods used to store and store biological material have several limitations. Current methods are not durable for long periods of time, the recovery rate of biological material after storage is often extremely low, and the quality and activity of the recovered biological material is generally reduced. Thus, there is a need to provide a long-term and cost-effective method for storing and preserving biological material while maintaining the stability and activity of the material and without losing large quantities of the material or its activity Still exists. Proper long-term storage is essential, especially when biological materials are used as replacements for semiconductors, optical storage devices, and other microelectronic devices.

SUMMARY OF THE INVENTION The subject matter of the present invention includes the storage of various densities of organic and inorganic information as artificial membranes that can be specifically designed and manufactured by engineering techniques. A “biological material film construct” as used herein, also referred to as a biofilm, can be used to store both organic and inorganic information from one or more biological materials, Multiple biological materials can be further bound to other organic or inorganic molecules. Applications of the present invention extend to medicine, engineering, computer technology and optics. Further, the stored information may be biological information, electrical information, magnetic information, optical information, microelectronic information, mechanical information, and combinations thereof.

  In one form, the present invention is an artificial biofilm storage device that includes a substrate coated with a biological material applied to a contact surface to form a stable membrane.

  Yet another aspect of the present invention is the step of applying the biological material to a substrate with a contact surface that facilitates uniform alignment of the biological material on the contact surface to enable the formation of a stable membrane. A biofilm storage device comprising:

  In yet another form, the present invention is for making a biofilm storage device comprising a substrate comprising a surface and a biological material that can specifically bind to the surface to form a dry film. It is a kit.

  Yet another aspect of the present invention is a hybrid artificial membrane storage device comprising a substrate comprising an inorganic material comprising a surface and a biological material applied to the surface to form a stable thin film. The membrane may be biologically active or may interact with biological components.

DETAILED DESCRIPTION OF THE INVENTION This patent application is hereby incorporated by reference into US Patent Provisional Application No. 60 to Lee et al., Which is hereby incorporated by reference in its entirety, including detailed descriptions, drawings, examples, and claims. Insist on / 413,081 priority.

  In addition, US patent application Ser. No. 10 / 157,775 filed May 29, 2002 to Belcher et al. And provisional priority patent application No. 60/90 filed Oct. 2, 2001. No. 326,583 is hereby incorporated herein by reference in its entirety. Specifically, Example II relating to biofilm preparation and characterization is incorporated herein by reference.

  Although the methods of making and using the various aspects of the present invention will be discussed, it should be understood that the present invention provides a number of inventive concepts that may be implemented in a wide variety of specific situations. The specific embodiments discussed herein are merely illustrative of methods for making and using the invention and are in no way intended to limit the scope of the invention.

  Terms used herein have meanings as commonly understood by a person of ordinary skill in the areas to which the present invention pertains. Terms such as “a” and “that” are not intended to relate only to a single entity, but include a general class that can be used as a specific example. Although the terminology herein is used to describe specific embodiments of the invention, their use does not limit the invention except as set forth in the claims. As used throughout this specification, the terms “membrane” and “biofilm” are used interchangeably.

  As used herein, the term “biological material” refers to viruses, bacteriophages, bacteria, peptides, proteins, amino acids, steroids, drugs, chromophores, antibodies, enzymes, single-stranded or double-stranded nucleic acids, vaccines. , And chemical modifications thereof. The biological material can self-assemble to form a dry film on the contact surface of the substrate. The dry film is substantially free of solvent so that it is completely dry within the limits of detection of conventional drying, or the film is solid-like and self-supporting but still has a residual wetness from the solvent. The residual solvent from the drying process is maintained to have either In many cases, the membrane may remain in a partially hydrated state, which can be optimized for a given application. Self-assembly allows for a random arrangement or uniform alignment of biological material on the surface. Furthermore, the biological material can form a dry film that is controlled externally by solvent concentration, application of electric and / or magnetic fields, optical instruments, or chemical or electric or magnetic field interactions.

  The term “inorganic molecule” or “inorganic compound” means a compound such as, for example, indium tin oxide, doping agents, metals, minerals, radioisotopes, salts, and combinations thereof. Metals include Ba, Sr, Ti, Bi, Ta, Zr, Fe, Ni, Mn, Pb, La, Li, Na, K, Rb, Cs, Fr, Be, Mg, Ca, Nb, Tl, Hg, Cu, Co, Rh, Sc, or Y may be included. Inorganic compounds include, for example, barium strontium titanate, barium zirconate titanate, lead titanate zirconate, lead lanthanum titanate, strontium titanate, barium titanate, barium fluoride magnesium, bismuth titanate, strontium bismastantarite, And strontium bismastantalite niobate, or variations thereof known to those skilled in the art.

  The term “organic molecule” or “organic compound” refers to carbon alone or nucleotides, polynucleotides, nucleosides, steroids, DNA, RNA, peptides, proteins, antibodies, enzymes, carbohydrates, lipids, conducting polymers, drugs, and It means a compound contained in combination with such a combination. Drugs include antibiotics, antibacterial drugs, anti-inflammatory drugs, analgesics, antihistamines, and drugs used to treat or prevent pathological (or potentially pathological) conditions in mammals Good.

  The term “substrate” as used herein may be a microfabricated solid surface to which molecules are attached, either covalently or non-covalently, such as silicon, Langmuir-Blodgett film, functional glass, germanium. , Ceramics, semiconductor materials, PTFE, carbon, polycarbonate, mica, mylar, plastic, quartz, polystyrene, gallium arsenide, gold, silver, metals, alloys, fabrics, as well as amino groups, carboxyl groups incorporated on its surface, It may be a combination thereof that may have a functional group such as a thiol group or a hydroxyl group. Similarly, the substrate can be an organic material such as a protein, mammalian cell, organ, or tissue with a surface to which biological material can adhere. The surface can be large or small and need not be uniform, but must function as a contact surface (not necessarily a single layer). The substrate can be porous, planar or non-planar. Substrates include the contact surface, which can be the substrate itself or a second layer (eg, a substrate or biological material with a contact surface) made from and contactable by organic or inorganic molecules. . The substrate may be cylindrical or non-planar. Substrates may be supported to improve their mechanical strength or surface area to volume ratio. An array may be made. Porous beads including glass beads and polystyrene beads may be used. High density filled pins may be used. The substrate surface may be grooved, microfabricated, or otherwise made non-planar.

  In general, biofilms are made by applying biological material to the contact surface of a substrate. This contact may be by self-assembly of biological material or may be controlled by external conditions such as the surface itself or solvent concentration, magnetic field, electric field, optical elements, and combinations thereof. In some cases, the substrate itself can function as a contact surface and can further control the nature and amount of contact of the biological material. In other embodiments, the contact surface may be a second substrate that may include one or more organic and / or inorganic molecules applied to the contact surface to which the biological material contacts.

  As used herein, the term “solvent” includes solutions of appropriate ionic strength to facilitate high density arrays or arrangements of biological materials. These arrays may be ordered or random. When ordered, the solvent (with or without external control) concentration may be a concentration that promotes liquid crystal formation of the biological material. The biological material may be preincubated with the contact surface and / or one or more organic or inorganic molecules. Preincubation can promote microparticle formation on the nanometer scale. This preincubation can be further controlled by external conditions as described above.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials that are commonly used are described below.

  Building and preserving well-ordered and well-controlled two-dimensional and three-dimensional structures at the nano-length scale will enable next-generation optical materials, electronic materials and magnetic materials and optical devices, electronic devices and magnetic devices. The main goal to build. Many researchers and companies have focused on building such structures using only conventional materials (eg, inorganic compounds). As disclosed herein, the inventors have shown that soft materials (eg, organic materials and biological materials) can function as self-organized bodies that assemble both organic and inorganic materials at the nanoscale level. certified. However, the storage of these soft (organic and inorganic) mixed materials has proven to be a challenging task.

The present invention provides a cost-effective long-term storage device composed of a soft mixed material. There are several advantages to using the present invention in medical, engineering, material chemistry and optical applications. The present invention includes several effects that were not easily solved by previous studies. First, the dry thin film preparation method can be carried out at a minimum cost without requiring a large amount of funds. Furthermore, these membranes can be easily stored due to their small space requirements, are adaptable to room temperature conditions and are therefore particularly cost effective. In addition, these membranes require little effort for mass production, with little loss of activity, structure or other important properties over time. Finally, the thin film product of the present invention is a high capacity storage device. For example, biofilms made using bacteriophage can store more than 4 × 10 ¹³ viruses in a square centimeter membrane.

  The thickness of the thin film is not particularly limited, but may be, for example, preferably from about 100 nm to about 100 microns, more preferably from about 500 nm to about 50 microns, and even more preferably from about 1 micron to about 25 microns.

  We have previously demonstrated that biological materials such as peptides and bacteriophages can bind to semiconductor materials. These biological materials use appearance specificity to recognize and bind other organic and inorganic materials, nucleate into size-limited crystalline semiconductor materials, and control the crystalline phase of nucleating nanoparticles (See Lee SW, Mao C, Flynn CE, Belcher AM. Ordering of Quantum Dots Using Genetically Engineered Viruses. 2002 Science 296: 892-895.) Includes descriptions of self-supporting polymer membranes and storage of viral membranes at room temperature for at least 7 months without loss of ability to infect bacterial hosts and with little loss of titer The relevant parts of which are incorporated herein by reference). Furthermore, since the aspect ratio of the nanoparticles can be controlled, the electrical, magnetic and optical properties can therefore be controlled. A biological material bound to one surface or equivalently a thin substrate (eg, a semiconductor material) that forms a thin layer of biological material is called a biofilm.

  In general, the biofilm of the present invention may contain both organic and / or inorganic materials (or molecules). The biofilm may include a substrate, an organic layer, a second organic layer, and an inorganic layer, or various combinations thereof. Each organic layer may include one or more different types of biological materials and / or organic materials; similarly, each inorganic layer includes one or more different types of inorganic materials. Good. In general, the surface of a biofilm is well-ordered and can provide biological, electrical, magnetic, and / or optical properties to the film, so that the biofilm is biological information, electrical information It is possible to hold and store magnetic information and / or optical information.

  In practice, a biofilm is defined as a population of biological material or microorganisms attached to a surface. Biofilm growth involves the age of biological materials or microorganisms (eg, cultures), the accumulation of potentially harmful (toxic) by-products or metabolites, as well as other materials for growth, stability or maintenance, or Depends on nutrient consumption or use. A biofilm may be composed of natural or genetically engineered biological material. Of particular interest is the use of self-assembling biological materials. For example, bacteriophages that are genetically engineered to bind to other materials (eg, semiconductor materials) organize into a more ordered structure.

  Thus, self-assembling biological materials (eg, bacteriophage) can be selected based on specific binding properties to a particular surface, creating a well-ordered structure of the selected material. Can be used for. Using these well ordered structures can form layers and / or impart biological, magnetic, optical, or electrical properties to the film. So this biofilm can function as an information storage device or an optical storage medium for memory, both of which can be used to store and read several bits of data, the data being biological data, magnetic data, optical data , Electrical data and combinations thereof.

  By supporting magnetic, optical or electrical conditions, the present invention becomes a biological material storage device with specific alignment characteristics. For example, using M13 bacteriophage with specific binding properties, one of three types: directional regularity within the nematic phase, twisted nematic structure within the cholesteric phase, and both directional and positional order within the smectic phase. A biofilm storage device can be produced with the liquid crystal phase. So well-ordered biofilm storage devices are made using only biological materials or in combination with other organic or inorganic molecules (materials), for example to make one type of thin film transistor .

  In terms of chemical composition, a bacteriophage (or any virus or other biological material of interest) can adhere to itself and form a type of thin film surface that can adhere to itself. A type of “molecule”. In general, the best biopolymer is a biopolymer whose size and chemical composition can be precisely controlled, and one control method is a gene manipulation method. Controlled biopolymers provide precisely known structures and compositions. As a result, membrane preparations using controlled biopolymers can be specially designed as needed. For example, a bacteriophage has a surface covered by 2,700 copies of the major protein unit (known as pVIII), and the shape is filamentous (length 880 nm and width 6.6 nm). In the following examples, the biofilm production method, apparatus and kit of the present invention are described.

Example of Biofilm Product Storage Device Ph.D.12mer system obtained from New England Biolab containing 10 ⁹ phage population using previously described method to obtain high concentration virus suspension Was used to amplify virus membranes in large quantities (J. Sambrook, EFFritsch, T. Maniatis, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press: New York, ed. 2,1989). 3.2 mL of phage library suspension (concentration: at least 10 ⁹ phage / μL) and 4 mL overnight culture were added to 400 mL of LB medium and incubated at 37 ° C. for 4 and a half hours. After purification of the phage, approximately 30 mg pellet was obtained. The pellet was resuspended in 1 mL Tris buffer (TBS) (pH 7.5). This high concentration suspension (about 5 mg / mL) was used to make a viral membrane.

  Viral membranes were made on the liquid / solid interface using a liquid phase gradient reduction by evaporation of the solvent in a desiccator. As the solvent was gradually removed, the phage particles formed an epitaxial layer domain on the solid substrate surface.

  Polarized micrograph (POM) data of the formed phage layer showed a repeating pattern of about 34 μm that followed the centimeter scale. FIGS. 1B and C show a POM image of the viral membrane and an AFM image of individual phage particles, respectively. FIG. 1D shows an AFM image of the ordered structure of the virus when assembled on the membrane.

  It is clear that the phage particles form a domain of about 500 nm. Furthermore, the phage particles are stacked side by side. These lateral stacks form filled microdomains and form lamellar layers within the bulk membrane (see FIG. 1D). The sequences obtained from these particles are shown in Table 1.

(Table 1) Sequence results from suspension before screening

  What is important is that the viral membrane fully maintains the original phage library without loss of infectivity. This resuspends the viral membrane and biologically pans it against a streptavidin target, a target known to have a specific binding motif such as His-Pro-Gln. Proven by using for. The results after the second sequencing show that the His-Pro-Gln sequence appears at the end of the pIII unit. After the fourth round of screening, the entire peptide sequence was found to exhibit a His-Pro-Gln consensus sequence.

  In the following, the time required for infection of the dry phage in this membrane (time-dependent infection ability) will be considered. In order to compare the time-dependent titers, 10 small-sized membranes were prepared. For comparison, 1 μL of the above suspension was dried for about 1 day on a sterile surface of an Eppendorf tube in a desiccator. The titer for each membrane was determined after suspending 1 μL of each membrane in 1 mL of TBS buffer (pH 7.5) on different days for 5 months. The titers measured showed little change over at least 7 weeks (Figure 2).

  After 5 months, the titer dropped to 10% compared to the titer obtained from the 1 day old membrane suspension. One of ordinary skill in the art can readily maximize any biofilm extension and / or optimal infection time without undue experimentation.

  The results of biopanning, including the lasting ability to infect dry phage on the membrane, demonstrate that this membrane preparation method is a highly efficient storage device for molecular information. For example, the membrane easily stores high density genetically engineered DNA and protein information over time. Furthermore, viral components can be easily replicated at any time using a bacterial host.

  This biofilm can serve to functionalize one or more different types of viruses and / or components thereof, and can be used to express specific proteins or protein units. Because this biofilm can be used in numerous therapeutic approaches, including new drug discovery, high-throughput screening, diagnosis of one or more pathological conditions, and optimizing treatment of disease, the medical applications of this technology are wide It extends to.

Biopanning against streptavidin target The phage membrane (FIG. 1A) was crushed to approximately 1 cm × 1 cm dimensions and suspended in 1 mL of TBS buffer. This suspension (1.1 × 10 ⁹ PFU) was exposed to a streptavidin-immobilized Petri plate by the method supplied with the Ph.D.12mer system (New England Biolab). After the second round of biopanning, randomly selected plaques began to show the sequence pattern, His-Pro-Gln, which is a specific binding peptide sequence for streptavidin (Table 2).

配列内のイタリック体の文字はストレプトアビジン結合配列モチーフを表している。 Table 2: Sequencing results using streptavidin targets

The italicized letters in the sequence represent the streptavidin binding sequence motif.

Time-dependent infectivity of dry phage in membrane state
1 μL of the suspension was dried on the sterile surface of an Eppendorf tube in a desiccator. Titers were counted after resuspending these 1 μL membranes in 1 mL TBS solution (pH 7.5) on different days over 5 months (FIG. 2).

The integrity of the dried thin film of phage is very high. This film stores 4 × 10 ¹³ phage per square centimeter. Furthermore, the number of protein units that can be stored is more than 7200 times that of 4 × 10 ¹³ phages. As a result, this dry membrane preparation method presents an inexpensive and optimal method for storing very large amounts of biological material such as DNA, peptides and proteins in a long and highly organized manner.

  As described herein, a genetically engineered virus library can be created, stored, and reused by creating a dry film. A genetically engineered M13 phage library was prepared in the form of a membrane from a high concentration suspension. When the biofilm was resuspended in the appropriate solution, the M13 phage remained active and was able to infect the bacterial host. Importantly, by using the present invention, certain biological materials are stored in a membrane, are stable, and are still active. This biofilm remains stable for more than 7 months and maintains its activity for at least 5 months as evidenced by an infectivity of over 95%.

Results of biopanning, the majority of the 10 ⁹ phage library information indicating that stored on the film. Furthermore, the creation of this biofilm is a reversible process with readily usable applications for storing high density genetically engineered molecular information (eg, DNA, peptides or proteins).

  Using the genetically engineered biofilm of the present invention, a three-dimensional memory having up to three spatial dimensions can be formed. Depending on the properties of the biological material and / or inorganic compound or nanoparticle combined with the biological material, multiple bits of information can be converted into biological data, optical data (such as color wavelength), magnetic data, or electrical It can be “read” (output) as data. Data is “written” (input) to the biofilm by creating chemical, optical, magnetic, or electrical reactions at specific (eg, nanoparticle) locations. Using the present invention, one or more phage additives (or other biological materials) can be designed to create membranes with very specific binding and / or sequence patterns. The resulting film functions as a storage device for inputting and outputting information (as data bits) with unique optical, electrical, and / or magnetic properties, as described in detail below. If the biological material is porous, such as in the hydrogel state, it can be read and written using a soluble label.

Examples of biofilm storage devices ordered using nanoparticles Genetically engineered biological materials such as viruses or bacteriophages (phages) are used to help regulate their appearance on the contact surface It is often possible to recognize specific contact surfaces. For example, in the case of bacteriophages, this is done through combinatorial phage display selection. In this example, contact surface recognition results in ordering of phage into self-supporting biofilms that contain or do not contain additional inorganic molecules or nanoparticles such as zinc sulfide (ZnS). Let The presence of nanoparticles provides an additional advantage that helps to control phage alignment magnetically and electrically. This external force control does not necessarily require the presence of additional inorganic molecules; some biological materials can be ordered externally on the contact surface without the aid of inorganic compounds.

  Phage recognition of the substrate contact surface (eg, semiconductor surface) can also be controlled by pre-coating the substrate with a second biological material such as a peptide recognition component. An example of a pre-coated substrate is a semiconductor surface that is pre-pre-coated with an additional compound such as, for example, indium tin oxide (ITO). This additional compound may or may not be inorganic. For example, some substrates (eg, glass) may be pre-coated with organic compounds (eg, conductive polymers) to promote ordered alignment of biological materials. External control applications such as electric and / or magnetic fields can also be used to promote ordered alignment of biological materials and to create highly uniform biofilms, Sex includes non-random ordering of biological material on a contact surface (or substrate). Using the present invention, it has been demonstrated that such biofilms of the present invention can be stored for more than 6 months without losing stability, activity, or the ability of the phage to infect the host. In the following, another example of the steps involved in ordering the biological material will be described, including an example of a method used to prepare the biological material.

Phage Display Library One method of providing a random organic layer is based on a combinatorial library of random peptides containing 7 to 12 amino acids fused to the pIII coat protein of M13 E. coli phage. The phage display library is used on the assumption that it reacts with. Five copies of pIII coat protein are located at one end of a phage particle consisting of 10-16 nm particles. This phage display approach provided a physical association between the peptide-substrate interaction and the DNA encoding the interaction. In the examples described herein, five single crystal semiconductors: GaAs (100), GaAs (111) A, GaAs (lll) B, InP (100), and Si (100) were used as examples. These substrates allowed systematic evaluation of peptide-substrate interactions, confirming the general applicability of the method of the present invention for various crystal structures.

  Protein sequences that successfully bind to specific crystals were eluted from the surface, amplified, for example, 1 million fold, and reacted to the substrate under more stringent conditions. This binding method was repeated 5 times in order to select phage within the library with the most specific binding. For example, after the third, fourth, and fifth phage selections, crystal-specific phages were isolated and sequenced for their DNA. Selective for crystal composition (eg, bonded to GaAs but not Si) and crystal plane (eg, bonded to GaAs (100) but not GaAs (III) B) Peptide bonds were identified.

  To determine the epitope binding domain to the GaAs surface, 20 clones selected from GaAs (100) were analyzed. The partial peptide sequence of the modified pIII or pVIII protein is shown in Figure 3 (SEQ ID NO: 1-11), which clearly shows that the amino acid sequences between the peptides exposed to GaAs are similar. is there. As the number of exposures to the GaAs surface increased, the number of uncharged polar and Lewis base functional groups increased. Phage clones from the 3rd, 4th, and 5th sequencing contained on average 30%, 40%, and 44% polar functional groups, respectively, while the proportion of Lewis base functional groups Increased from 41% to 48% and 55% at the same time. The increase observed in Lewis bases with only 34% functional groups in the random 12mer peptide from the library used was due to the interaction between the Lewis base on the peptide and the Lewis acid site on the GaAs surface. This suggests that it may mediate the selective binding shown by the clones.

  The predicted structure of the modified 12mer selected from this library may be large, as is the case with small peptides that make the peptide much longer than the GaAs unit cell (5.655.6) I think that the. Thus, only a small binding domain will be required for this peptide to recognize GaAs crystals. These short peptide domains highlighted in FIG. 3 contain regions rich in serine and threonine in addition to the presence of amine Lewis bases such as asparagine and glutamine. To determine the correct binding sequence, a short library was screened, including 7mer and disulfide restricted 7mer libraries. With these short libraries that reduce the size and flexibility of the binding domain, a smaller number of peptide-surface interactions are tolerated and produce an expected increase in the strength of interaction between selected generations.

  Specific binding was assessed quantitatively using phage (labeled with streptavidin-labeled 20 nm colloidal gold particles conjugated to the phage through a biotinylated antibody against M13 coat protein). Elemental composition determination was performed by X-ray photoelectron spectroscopy (XPS), monitoring phage-substrate interactions through the intensity of the gold 4f-electron signal (FIGS. 4A-C). In the absence of G1-3 phage, this antibody and gold streptavidin did not bind to the GaAs (100) substrate. For this reason, gold-streptavidin binding was specific for phage and was an indicator of phage binding to the substrate. It was also found that G1-3 clones isolated from GaAs (100) using XPS bind specifically to GaAs (100) but not to Si (100) (FIG. 4A). reference). The S1 clone, when screened against the (100) Si surface, showed poor binding to the GaAs (100) surface in a complementary manner.

  Some GaAs clones also bound to the surface of another sphalerite structure, InP (100). Whether the principle of selective bonding is chemical, structural, or electronic is still under investigation. Furthermore, the presence of native oxide on the substrate surface can change the selectivity of peptide bonds.

  Preferential binding of G1-3 to a GaAs (100) surface superior to a (111) A (gallium terminated) or (111) B (arsenic terminated) surface of GaAs has been demonstrated (Figure 4B and 4C). The G1-3 clone surface concentration was higher on the (100) surface used for the selection than on the (111) A surface rich in gallium or the (111) B surface rich in arsenic. Since these different surfaces are known to exhibit different chemical reactivity, it is not surprising that selectivity has been demonstrated in phage binding to various crystal planes. Although the bulk ends of both 111 surfaces give the same geometric structure, the difference between the outermost layers in the surface bilayer with Ga or As atoms becomes more evident when comparing the surface reconstructions. The composition of the oxides on the various GaAs surfaces is also expected to be different and this in turn can affect the nature of the peptide bond.

  The intensity of Ga 2p electrons versus binding energy from the substrate exposed to the G1-3 phage clone is plotted in FIG. 4C. As predicted from the results shown in FIG. 4B, the Ga 2p intensity observed on the GaAs (100) surface, (lll) A surface, and (111) B surface is inversely proportional to the gold concentration. The decrease in Ga 2p intensity on the surface with increasing gold-streptavidin concentration was attributed to increased surface coverage by phage. XPS is a surface technology with a sampling depth of about 30 mm. Thus, as the organic layer thickness increases, the signal from the inorganic substrate decreases. Using this observation, it was confirmed that the strength of gold-streptavidin was actually due to the presence of phage containing crystal specific binding sequences on the surface of GaAs. The same number of specific phage clones were exposed to various semiconductor substrates with comparable surface areas and binding studies correlated with XPS data were performed. Wild-type clones (non-random peptide inserts) did not bind to GaAs (no plaques were detected). For the G1-3 clone, the eluted phage population was 12 times larger from the GaAs (100) surface than from the GaAs (lll) A surface.

  Atomic force microscopy (AFM) was used to delineate G1-3, G12-3 and G7-4 clones bound to GaAs (100) and InP (100). The InP crystal has a zinc blende structure that is the same structure as GaAs, but the In-P bond has a larger ionicity than the GaAs bond. The 10 nm width and 900 nm length of the phage observed with AFM correspond to the size of the M13 phage observed by transmission electron microscopy (TEM), and gold spheres bound to the M13 antibody bind to the phage. (Data not shown). The InP surface had a high concentration of phage. These data suggest that a number of factors, including atomic size, charge, polarity and crystal structure, are involved in substrate recognition (or contact surface recognition).

  In the TEM image, it is clear that the G1-3 clone (negative staining) is bound to the GaAs crystalline wafer. This data confirmed that this binding was directed by the modified pIII protein of G1-3, but not through non-specific interactions with the major coat protein. Thus, using the peptides of the present invention can direct specific peptide-semiconductor interactions in assembling nanostructures and heterostructures (FIG. 5E).

Using X-ray fluorescence microscopy, it was demonstrated that phage preferentially attached to the sphalerite surface very close to the surface of various chemical and structural compositions. A nested square pattern was etched into the GaAs wafer; this pattern contained 1 μm lines of GaAs and 4 μm spacing of SiO ₂ between each line (FIGS. 5A and 5B). The G12-3 clone was interacted with a GaAs / SiO ₂ patterned substrate, washed to reduce non-specific binding, and labeled with the immunofluorescent probe tetramethylrhodamine (TMR). Labeled phage were found as three red lines and a central dot in FIG. 5B, corresponding to G12-3, which only binds to GaAs. The SiO ₂ region of this pattern remains unbound to the phage and is dark. This result was not observed in controls not exposed to phage and exposed to primary antibody and TMR (FIG. 5A). Similar results were obtained when non-phage bound G12-3 peptide was used.

GaAs clone G12-3 was observed to be more substrate specific for GaAs than AlGaAs (FIG. 5C). Since AlAs and GaAs have substantially identical lattice constraints of 5.66 and 5.65 respectively at room temperature, an AlxGal-xAs ternary alloy can then be grown epitaxially on a GaAs substrate. GaAs and AlGaAs have zinc blende crystal structure, but the G12-3 clone showed selectivity by binding only to GaAs. A multilayer substrate consisting of alternating layers of GaAs and Al _0.98 Ga _0.02 As was used. This substrate material was cleaved and subsequently reacted with the G12-3 clone.

The G12-3 clone was labeled with 20 nm gold-streptavidin nanoparticles. Scanning electron microscope (SEM) inspection shows alternating layers of GaAs and Al _0.98 Ga _0.02 As in the heterostructure (FIG. 5C). Using X-ray elemental analysis of gallium and aluminum to map only gold-streptavidin particles to heterostructure GaAs layers, a high degree of binding specificity for chemical composition was demonstrated. In FIG. 5D, a model for identifying phage for semiconductor heterostructures was displayed (FIGS. 5A-C), as seen in fluorescence and SEM images.

The present invention demonstrates that the use of phage display libraries is extremely effective for identifying, generating, and amplifying the binding of organic peptide sequences to inorganic semiconductor substrates. Peptide recognition and specificity of inorganic crystals have been enlarged using peptide libraries _{GaN, ZnS, CdS, Fe 3} O 4, Fe 2 O 3, CdSe, other substrates including ZnSe and CaCO ₃ . Currently, bivalent synthetic peptides with two-component recognition (FIG. 5E) have been designed. Such peptides have the ability to direct nanoparticles to specific locations on the semiconductor structure. These organic and inorganic pairs provide a powerful building block for creating a new generation of complex and sophisticated electronic structures. Specific amino acid sequences (SEQ ID NO: 12-95) for peptide recognition of CdS (Figures 6-9), ZnS (Figures 8, 9), and PbS (Figures 9-10) crystals, especially after biopanning Examples are shown in FIGS.

Peptide generation, isolation, selection, and characterization Peptide selection To reduce phage-to-phage interactions on the surface, phage display or peptide libraries can be added to Tris buffer (TBS) containing 0.1% TWEEN-20. Contact with the semiconductor or other crystals inside. After shaking for 1 hour at room temperature, the surface was washed with 10 exposures to TWEEN-20 with increasing concentrations from Tris buffer (pH 7.5) and 0.1% to 0.5% (v / v). Phages were eluted from the surface over 10 minutes by the addition of glycine-HCl (pH 2.2), then transferred to a new tube and then neutralized with Tris-HCl (pH 9.1). The eluted phage was titrated and the binding efficiency was compared.

  Phages eluted after the third substrate exposure were mixed with their E. coli ER2537 and plated on LB XGal / IPTG plates. Since the library phage was derived from the vector M13mp19 with the lacZα gene, it contains Xgal (5-bromo-4-chloro-3-indoyl-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactoside) When plated on the medium, the phage plaques turned blue. Blue / white screening was used to select phage plaques with random peptide inserts. Plaques were collected and DNA was sequenced from these plates.

Substrate preparation
The orientation of the substrate was confirmed by X-ray diffraction, and the native oxide was removed by appropriate specific chemical etching. About the following etchants: NH ₄ OH: H ₂ O (1:10), HC1: H ₂ O (1:10), H ₃ PO ₄ : H ₂ O ₂ : H ₂ O (3: 1: 50) Tested on the GaAs and InP surfaces after 1 minute and 10 minutes, respectively. Maximum element ratios and minimum oxide formation rates (using XPS) for GaAs and InP etched surfaces are as follows: use HCl: H ₂ O for 1 minute, then clean with deionized water for 1 minute. obtained. In the initial screening of the library, an ammonium hydroxide etchant was used for GaAs. This etchant can be used for all other GaAs substrate specimens, but those skilled in the art will recognize usable etchants. The Si (100) wafer was etched for 1 minute in an HF: H ₂ O (1:40) solution, and then washed with deionized water. The surface can be removed directly from the wash solution and immediately introduced into the phage library. The surface of the control substrate not exposed to the phage was characterized and mapped for the effectiveness of the etching process and the surface morphology by AFM and XPS.

A multilayer substrate of GaAs and a multilayer substrate of Al _0.98 Ga _0.02 As were grown by molecular beam epitaxy on GaAs (100). The epitaxial growth layer was Si-doped (n-type) at a level of 5 × 10 ¹⁷ cm ⁻³ .

Labeling with antibodies and gold
For XPS, SEM and AFM specimens, the substrate was exposed to phage in TBS for 1 hour and then anti-Fd bacteriophage-biotin conjugate (1 in phosphate buffer, which is an antibody to the FIII phage pIII protein). : 500, manufactured by Sigma) for 30 minutes, and then washed in a phosphate buffer. Streptavidin-20 nm colloidal gold label (1: 200 in phosphate buffer (PBS)) was attached to biotin-binding phages by biotin-streptavidin interaction. These surfaces were exposed to the label for 30 minutes and then washed several times with PBS.

X-ray photoelectron spectroscopy (XPS)
To confirm that the gold signal found in XPS was from gold bound to phage and not from non-specific antibody interactions with the GaAs surface, the following control experiment was performed on XPS specimens: . Following the prepared GaAs (100) surface, (1) phage-free antibody and streptavidin-gold label, (2) antibody-free G1-3 phage and streptavidin-gold label, and (3) Exposed to streptavidin-gold label without either G1-3 phage or antibody.

  The XPS equipment used was a Phi ESCA 5700 manufactured by Physical Electronics with an aluminum anode that produces monochromatic 1,487 eV X-rays. All samples are introduced into the chamber immediately after gold labeling of the phage to limit oxidation of the GaAs surface (as described above) and then high vacuum to reduce sample outgassing in the XPS chamber. And evacuated overnight.

Atomic force microscope (AFM)
The AFM used was a Digital Instruments Bioscope mounted on a Zeiss Axiovert 100s-2tv operating in chip scan mode using a G scanner. Images were taken in air using tapping mode. The AFM probes were silicon etched using 125 mm cantilevers and 20 ± 100 Nm ⁻¹ spring constants driven near their 200 ± 400 kHz resonance frequency. The scan speed was approximately 1 ± 5mms- ¹ . The image was leveled using a primary plane to remove sample tilt.

Transmission electron microscopy (TEM)
TEM images were taken using Philips EM208 at 60 kV. Gl-3 phage (dilution ratio 1: 100 in TBS) is incubated with GaAs strips (500 mm) for 30 minutes, centrifuged to separate particles from unbound phage, washed with TBS, and washed in TBS And resuspended. Samples were stained with 2% uranyl acetate.

Scanning electron microscopy (SEM)
G12-3 phage (dilution ratio 1: 100 in TBS) was incubated with the newly cleaved heterostructure surface for 30 minutes and washed with TBS. G12-3 phage was labeled with 20 nm colloidal gold. SEM and elemental mapping images were collected using a Norian detection system mounted on a Hitachi 4700 field emission scanning electron microscope at 5 kV.

Creation of Ordered Vibrid Biofilm Storage Devices We have provided that organic-inorganic hybrid materials (hybrid materials containing both organic and inorganic compounds) provide a new means for new material development. Recognized. Size-controlled structures in the nanoscale range (nanostructures) are particularly useful in microelectronics and provide tunable optical, magnetic and electrical properties to materials such as semiconductors. Biological materials with organic components can further modify the inorganic morphology, phase and nucleation direction of the structure, especially at the nanoscale level. This hybrid creates a highly unique microenvironment with location specific information or data. The ability to store this information for a very long time is critical to succeed as a storage tool for processing, collecting and further analyzing information.

  The use of phage as an example reveals that biological materials, which generally have monodisperse properties, provide new materials with a unique new standard for storing various information therein. With the present invention, a highly ordered structure with nanometer scale ordering was constructed. Complex length scale alignment of II-VI semiconductor materials using genetically engineered self-assembling biological molecules (eg, M13 bacteriophage with specific semiconductor surface recognition components) makes it an ideal device for long-term data storage produce. Thus, monodisperse biomaterials with anisotropic shapes are yet another way to build well ordered structures. Nanolength scale and composite length scale alignments of II-VI semiconductor materials were performed using genetically engineered M13 bacteriophages with recognition components (peptides or amino acid oligomers) for specific semiconductor surfaces.

  Fd virus smectic ordered structures with both positional and directional order have been characterized. The smectic structure of the Fd virus includes multiscale and nanoscale ordering of structures to build two-dimensional (2D) and three-dimensional (3D) alignments of particles at the nanometer scale (hereinafter referred to as nanoparticles). There are potential uses in both. Bacteriophage M13 was used because it is genetically engineered, has been successfully selected to have the same shape as the Fd virus, and has specific binding affinity for II-VI semiconductor surfaces. there were. For this reason, M13 is an ideal source for smectic structures that aid in multiscale and nanoscale ordering of nanoparticles.

  The present inventors have used combinatorial screening methods to find M13 bacteriophages containing a peptide “insert” that can bind to a semiconductor surface. These semiconductor surfaces contained materials such as zinc sulfide, cadmium sulfide and iron sulfide. Biological materials such as bacteriophage combinatorial library clones that bind to specific semiconductor material surfaces were used using molecular biology techniques known to those skilled in the art. In general, biological materials are materials that are readily available in large quantities or that can be easily amplified for mass production. Phages were amplified, cloned and amplified to a high enough concentration for liquid crystal formation.

The anisotropic shape of bacteriophage was exploited as a method to construct well ordered nanoparticle layers by using biological selectivity and self-assembly. For example, filamentous bacteriophage Fd is a long rod (length: 880 nm; diameter: 6.6 nm) and monodisperse molecular weight (molecular weight: 1.64 × 10 ⁷ ) that causes the lyotropic liquid crystal behavior of the bacteriophage in highly concentrated solutions. ). In the present invention, M13, a similar filamentous bacteriophage, was genetically engineered to bind to nanoparticles such as zinc sulfide, cadmium sulfide and iron sulfide. A monodispersed bacteriophage, M13, was prepared by standard amplification methods.

Ordering on the nanoscale and mesoscale The ordering of bacteriophages on the nanoscale and mesoscale levels demonstrates that this biological material can form nanoscale arrays of nanoparticles. These nanoparticles are further organized into micron domains and centimeter length scales. Semiconductor nanoparticles exhibit quantum confinement and can be synthesized and ordered in liquid crystals.

Using standard molecular biology techniques, engineered M13 bacteriophages with specific binding properties to semiconductor surfaces were amplified and purified. For mass amplification, 3.2 mL bacteriophage suspension (concentration: phage ~ 10 ⁷ cells / μL) and 4 mL overnight culture were added to 400 mL LB medium. After amplification, ˜30 mg pellet settled. The suspension was prepared by adding Na ₂ S solution at room temperature to the A7 phage suspension doped with ZnCl ₂ . The highest concentration A7-phage suspension was prepared by adding 20 μL of 1 mM ZnCl ₂ and Na ₂ S solution to ˜30 mg of phage pellet each. The concentration was measured using an extinction coefficient of 3.84 mg / mL at 269 nm.

  As the concentration of the isotropic suspension increased, a nematic phase with directional order, a cholesteric phase with a twisted nematic structure, and a smectic phase with similar directional and positional order were observed. These phases have been observed in Fd viruses that do not have nanoparticles. M13 bacteriophage suspensions containing specific peptide inserts were made and characterized. Uniform 2D and 3D ordering of the nanoparticles was observed throughout the sample.

Atomic force microscope (AFM)
The AFM used was the same as previously described. FIGS. 11A and 11B are schematic diagrams of smectic alignments of M13 phage observed using AFM. In addition, 5 μL of M13 bacteriophage suspension (concentration: 30 mg / mL) over 24 hours on 8 mm x 8 mm mica substrate silylated with 3-aminopropyltriethylsilane in a desiccator for 4 hours And dried. Images were taken in air using tapping mode. Due to the anisotropic shape of the M13 bacteriophage with a length of 880 nm and a width of 6.6 nm, self-assembled ordered structures were observed. In FIG. 12C, the M13 phage is in the plane of the photograph and forms a smectic alignment.

Transmission electron microscopy (TEM)
TEM images were taken as previously described.

Scanning electron microscopy (SEM)
Sample preparation and use of SEM are as previously described. A critical point dry sample of bacteriophage and ZnS nanoparticle smectic suspension (concentration of bacteriophage suspension: 127 mg / mL) was prepared. In FIG. 12D, a region rich in nanoparticles and a region rich in bacteriophages were observed. The separation length of nanoparticles and bacteriophage corresponded to the length of bacteriophage. The ZnS wurtzite crystal structure was confirmed by electron diffraction pattern using a diluted sample of smectic suspension using TEM.

Polarization microscopy (POM)
M13 phage suspension was characterized by POM. Each suspension was filled into a glass capillary tube having a diameter of 0.7 mm. The high-concentration suspension (127 mg / mL) showed iridescent [5] under parallel polarization and a smectic texture structure under cross polarization as shown in FIG. 12A. As is apparent from FIG. 12B, the cholesteric pitch can be controlled by changing the concentration of the suspension as shown in Table 3. The pitch length was measured and micrographs were taken 24 hours after sample preparation.

(Table 3) Relationship between cholesteric pitch and concentration

Preparation of nanocrystalline biofilm The bacteriophage pellet was suspended using 400 μL Tris buffer (TBS, pH 7.5) and 200 μL 1 mM ZnCl ₂ supplemented with 1 mM Na ₂ S. After shaking for 24 hours at room temperature, the suspension contained in a 1 mL Eppendorf tube was slowly dried in a desiccator for 1 week. A translucent film having a film thickness of ˜15 μm was formed inside the tube. The membrane shown in FIG. 13A was carefully removed using tweezers.

Observation of nanocrystalline biofilm by SEM
SEM was used to observe the nanoscale alignment of bacteriophages in A7-ZnS films. To perform SEM analysis, the film was cut and coated with 2 nm of chromium by vacuum deposition in an argon atmosphere. A close-packed structure was observed throughout the sample (see FIG. 13D). The average length of individual phages 895 nm is reasonably similar to the average length of phages 880 nm. The film exhibited a smectic A-like or C-like layered morphology that exhibited periodicity between the nanoparticle layer and the bacteriophage layer. The length of the periodicity corresponded to the length of the bacteriophage. The average size of the nanoparticles was ˜20 nm, similar to the observation for individual particles by TEM.

Observation of nanocrystalline biofilm by TEM
TEM was used to investigate ZnS nanoparticle alignment. This membrane was embedded in an epoxy resin (LR white) for 1 day and polymerized by adding 10 μL of accelerator. After curing, thin sections were prepared from the resin using a Leica ultramicrotome. These ˜50 nm sections were floated on distilled water and taken up on a blank gold grid. A small amount of parallel aligned nanoparticles was observed, corresponding to the xz plane in the schematic (FIG. 13E). Since each bacteriophage had 5 copies of the A7 component, each A7 recognized one nanoparticle (size 2-3 nm), aligned at about 20 nm in width, and extended more than 2 μm in length. Bands of 2 μm × 20 nm were formed in parallel, and each band was separated by ˜700 nm. This discrepancy appears to arise from a tilted smectic alignment of the phage layer compared to observations in TEM. The yz-axis-like nanoparticle layer plane was also observed as in FIG. 5F. The SAED pattern of the aligned particles proved that the ZnS particles have a wurtzite hexagonal structure.

Observation of nanocrystalline biofilm by AFM
AFM was used to investigate the surface orientation of the viral membrane. In FIG. 5C, it is shown that the phage form a parallel aligned herringbone pattern with approximately a right angle between adjacent director standards (bacteriophage axis) over most of the surface termed smectic O. This membrane showed a wide range of regularization of standard directors lasting up to tens of μm. In some areas where the two domain layers intersect each other, two or three complex length scale bacteriophages were aligned in parallel and persistently to the smectic C ordered structure.

  The nano-length scale and composite length scale alignment of semiconductor materials using the recognition and self-ordering methods and compositions of the present invention will enhance the microfabrication of future electronic devices. These devices have potential beyond current photolithography capabilities. Other potential uses for these materials include light emitting displays, optical detectors, and optoelectronic devices such as lasers, high speed interconnectors, nanometer scale computer components and biological sensors.

Stabilization of Biofilm Storage Device and Maintenance of Biological Activity The biofilm storage device of the present invention can be used to store biological (eg, organic) materials such as enzymes and antibodies. In one embodiment of the invention, biological molecules such as enzymes that retain their biological activity are stored as a biofilm. This activity is easily monitored over time based on the known properties of the enzyme. In one embodiment, the reporter enzyme β-galactosidase is prepared in a biofilm and found to retain long-term enzyme stability and activity.

  In yet another aspect of the invention, a preservative solution (eg, sucrose) is used to enhance the stability and long-term activity of the biological material (eg, enzyme). Moreover, this example is particularly important when the addition of a preservative solution used as a stabilizer enhances the preservation of the biofilm storage device and when biological activity is a major component of the biofilm. Illustrates the possibility.

  In order to visualize the structure and function of a biological material used as a biofilm storage device, the optical properties of the biological material or luminescent molecules attached to the biological material can be monitored. For example, a green fluorescent protein variant (GFPuv) that emits green light at a maximum emission wavelength of 509 nm can be used to attach to biological material (eg, an enzyme or antibody). In addition, the luminescent properties can be visualized using instruments well known to those skilled in the field of biological imaging. One example of an imaging instrument is a confocal microscope.

  In yet another aspect of the invention, a biological material used as a storage device is contacted with another biological material. Either biological material can be modified in whole or in part to customize the biofilm as needed. For example, a biofilm containing biological material such as bacteriophage attaches specifically to the biological material by altering the protein displayed on the surface of the biological material (eg, bacteriophage), or Further modified by targeting peptides that can be attached to other targets (eg, biological materials such as proteins, antibodies, drugs, or nucleic acids) or other stabilizers that result in enhanced stability of the biofilm storage device it can.

  The storage temperature may be approximately room temperature, for example. The storage temperature may be, for example, from about 10 ° C to about 40 ° C, and more particularly from about 20 ° C to about 30 ° C. These storage temperatures can be maintained for a period that includes at least 7 weeks, at least 5 months, at least 6 months, or at least 7 months.

Preparation of biofilm storage device containing stable enzyme Concentration of 0.5 mg / mL β-galactosidase, 5 mg / mL glucose, 50 mg / mL sucrose, and 1.25 mg / mL phage to obtain phosphate buffer (PBS ) The enzyme β-galactosidase in (pH 7.0) was mixed with glucose, sucrose, and M13 phage stock. An aliquot (20 μL) of this solution was placed in a 1.5 mL Eppendorf tube, dried in a desiccator for 2 days, and stored at room temperature. The dried virus membrane was suspended in 500 μL of PBS solution (pH 7.0). 100 μL of suspension and 700 μL of o-nitrophenyl galactoside (ONPG) (1.5 × 10 ⁻² M) were bound in a disposable cuvette. Enzyme activity (units) was obtained by monitoring the increase in absorbance of o-nitrophenol (ONP) at 420 nm for 10 minutes at 30 second intervals. One unit of activity was defined as the amount of enzyme capable of catalyzing the conversion of 1 μmol of ONPG to ONP in 1 minute at 25 ° C. (pH 7.0).

Monitor biological activity and stability in biofilms
GFPuv encoding DNA (Clontech) was amplified by PCR and subcloned into pFLAG-CTC vector (Sigma) to express GFPuv-FLAG in E. coli. Whole cell extracts were prepared and the expressed GFPuv-FLAG was purified using an anti-FLAG M2 affinity gel column (Sigma). GFPuv-FLAG with a final concentration of 100 μg / mL GFPuv-FLAG, 5 mg / mL phage, 5 mg / mL glucose, and 50 mg / mL sucrose, phage and glucose: sucrose (1:10 (w / w) ) Was prepared. At least about 10 μL of the mixture was dispersed on a glass slide and dried in a desiccator for 1 day. A confocal fluorescence microscope was used to monitor the stability of GFPuv-FLAG. The glucose: sucrose concentrations were 2.5 and 25 mg / mL.

  After storage of the prepared biofilm storage device, it was found that the added activity of β-galactosidase was improved when glucose: sucrose was added as a preservation solution or stabilizer (FIGS. 14A and 14B). The sample used as a control was biological material (eg, β-galactosidase) prepared as described above and dried in a desiccator in the absence of bacteriophage and sugar. Enzyme storage as a biofilm storage device obviously had no effect on enzyme activity. Biofilm storage devices containing β-galactosidase and stored after freeze-drying or air-drying showed similar enzyme activity. Interestingly, enzyme activity was improved in the presence of other biological materials (eg, bacteriophage) as well as in the presence of stabilizers (eg, stock solutions).

  FIG. 15 shows a confocal microscope image using GFPuv after excitation at 361 nm. These images illustrate that the addition of a stabilizer such as glucose: sucrose stock solution improves the biofilm surface and prevents potential deformation of the biological material during the production (preparation) process. FIG. 15A shows a strong GFPuv signal and a homogeneous biofilm surface. In the absence of a glucose: sucrose stock solution, the membrane exhibits numerous deformations at the membrane surface (FIGS. 15B and 15C).

  Furthermore, if the biological material includes multiple display sites, the biological material can be genetically manipulated such that one or more of these display sites are modified. For example, M13 bacteriophage can be modified at the pIII, P7, p8, or p9 sites to include specific binding peptides. For example, one end of the biological material can be modified to specifically bind to the surface, while the other end of the biological material is stored for stable storage, such as a vaccine or functional protein Can be modified to bind to a component.

  Thus, the present invention is capable of storing biologically active biological material with activity that lasts throughout the entire storage period. With additional modifications, the biological properties and / or other activity properties (eg, electrical properties, magnetic properties, optical properties, mechanical properties) of the biofilm can be easily manipulated as needed. Activity is performed unnecessarily without changing the biological surface by changing the surface binding properties, through the addition of storage stabilizers and / or inhibitors, and by the addition of other organic or inorganic molecules. Can be further modified. Stock solutions that stabilize biological materials include sugar-containing solutions such as glucose, sucrose, glycol, glycerol, polyethylene glycol and the like.

  The present invention creates a stable membrane composed of biological materials (including one or more organic and / or inorganic materials) that can be stored for long periods while maintaining initial information and / or activity. Improve biofilm technology. Using genetically engineered materials can create ordered membranes (biofilms) with long-term activity and stability that retain and store biological, electrical, magnetic, and / or optical information . More importantly, the information can be tailored and extremely dense, thereby functioning as an efficient and cost effective way to store nanoscale data. The use of these biofilms extends to applications such as medical, electronics, computer technology and optics, as some examples.

  Using the compositions and methods of the present invention, nano-length and composite length-scale alignment of semiconductor materials has been achieved using the recognition and self-ordering systems described herein. Using semiconductor recognition and self-ordering can enhance the microfabrication of electronic devices beyond current photolithography capabilities. Applications for these materials include optoelectronic devices such as light-emitting displays, optical detectors, and lasers; high-speed interconnects; and nanometer-scale computer components and biological sensors. Other uses of biofilms made using the present invention include, as some examples, well-ordered liquid crystal displays, organic-inorganic display technologies, and high-throughput processing, screening and new drug discovery Membranes, devices for diagnosis, medical examination and analysis; implant surfaces for data storage and specific data recognition.

  Membranes, fibers and other structures developed from the biofilms of the present invention can even include a high density sensor for detecting small molecules including biological toxins. Other uses include optical coatings and optical switches. Optionally, scaffolds or even bone implants for medical implants, as would be apparent to one of ordinary skill in the art, can include one or more of the materials disclosed herein in a single layer or multiple layers, or in a strip or any of these It can be constructed using any combination of these. Other uses of the invention include the organization of electrical and magnetic interfaces, or even 3D electronic nanostructures for high density storage, such as for use in quantum computing. Alternatively, various densities and stable storage of viruses for medical use, such as biologically compatible vaccines, adjuvants and vaccine containers, can be made using the membranes and / or matrices made according to the present invention.

  Information storage based on quantum dot patterns for identification, such as identification of enemies and allies by the Department of Defense, can be incorporated into the fabric or coding of protective clothing. This biofilm can even be used to encode and identify money.

  Other applications include drug delivery including systems such as Depomed with a layered membrane assembly in drug capsules; medical device coatings; and sustained release applications such as bad breath mint.

  Additional descriptions and examples are provided in Aspect A and B below. Aspect A includes a set of references, and Aspect B also includes a set of references.

Additional description and examples (Aspect A)
The paper Lee et al. “Chiral Smectic C Structures of Virus-Based Films” Langmuir, 2003, 19, 1592-1598 provides a summary, drawings, tables, introductions, experimental section, references, results and discussion sections. The entirety of which is incorporated herein by reference.

  Additional materials were prepared that could be used as membranes in storage applications as well as other applications. In these additional experiments, M13 phage (viruses) aligned and assembled using the meniscus phenomenon were used to create a wide range of ordered virus-based membranes. Their ordered structure and morphology were examined and characterized using a polarizing microscope (POM), atomic force microscope (AFM) and scanning electron microscope (SEM). M13 virus particles with a length of 880 nm were the basic building blocks of artificial membranes. Because viruses have a unique micrometer length scale, the smectic ordering of virus particles could be easily visualized using conventional microscopy techniques and compared with theoretical models of chiral liquid crystal structures. . From the test results of POM, AFM and SEM, it was determined that the viral membrane had a chiral smectic C structure. By comparing the ordering of film formation as a function of virus concentration found in virus thin films and the formation of bundle-like domain structures, a mechanism of film formation can be proposed. These virus-based membrane structures are organized on a composite length scale, easily fabricated, and allow for the integration of aligned semiconductor and magnetic nanocrystals. These self-assembling hybrid materials can be used, for example, in microminiature self-assembling electronic devices.

Building well-ordered defect-free 2D and 3D structures on the nanometer scale has become a critical issue for building the next generation of optical, electronic and magnetic materials and devices. ^1-5 . Numerous techniques have been tried to organize nanoparticles and other nanometer-sized objects on a small length scale, including conventional hydrogen bond recognition to newly developed DNA linker systems. It has proved difficult to extend simple patterns to the micrometer scale ^6-7 . The use of biological materials can provide an alternative to conventional fabrication methods for constructing microminiature nanoscale devices ^5,8 . Some desirable functions of biological systems include the ability to organize precise self-assembled structures, highly evolved molecular recognition for both organic and inorganic materials, and the ability to synthesize inorganic materials into hierarchical structures. It is. Several types of biomaterials in nanoscale assembly of complex structures have been developed ^5,8-13. Recently, a novel method for self-assembling quantum dots in ordered nanocrystal films using nanocrystal-functionalized M13 phage has been reported ⁵ . The M13 virus was engineered to nucleate at one end of the M13 virus or to bind to the desired material. These nanocrystalline functionalized viral liquid crystal building blocks were grown into hybrid ordered self-supporting membranes. The resulting nanocrystalline hybrid film was ordered into a 72 μm periodic pattern at the nanometer and micrometer scales. Smectic O structure on the surface of nanocrystal hybrid film and smectic A or C structure in bulk have been reported.

  Here, a very extensive characterization of these virus-based membranes, including the chiral action of the viral building blocks, has been reported, and this is the powerful that these virus-based membranes are organized into a chiral smectic C structure. Provide proof. Viral membranes made from various concentrations provide various other textures depending on the film thickness. Viral membrane results were compared with previously reported ZnS nanocrystal hybrid virus membranes.

This represents a novel example of a widely ordered lyotropic liquid crystal chiral smectic C film. This is further evidence in support of Meyer's theoretical proposal that smectic C structures formed from chiral molecules have a chiral smectic C structure ¹⁴ . Although several microscopy techniques have been used to visualize ordered liquid crystal materials, understanding of the molecular orientation of liquid crystal ordered structures is generally limited by the small size and softness of mesogenic units of conventional liquid crystal materials ^Has been ^{15, 16, 30, 34} . However, using micrometer-scale biomolecules (viruses), surface defects in chiral smectic C structures were easily characterized. Furthermore, in order to create well-ordered and complex structures without defects using virus building blocks, a basic understanding of the surface and bulk structure of these materials is required for semiconductor nanocrystal hybrid virus membranes. It is also important for other uses.

Table 1 Viral membrane thickness as a function of initial bulk concentration

B.POMによって測定した周期的なジグザグのスメクチックAパターン

(Table 2)
A. Chiral smectic C pitch measured by polarizing microscope (POM) and laser light diffraction

B. Periodic zigzag smectic A pattern measured by POM

Experiment (Aspect A)
Preparation of viral membrane
M13 phage was prepared using standard biological methods for amplification and purification described previously ⁵ . As shown in Table 1, 12 concentrations of M13 phage (800 μL each) were prepared. After being transferred to an Eppendorf tube (1 cm in diameter and 4 cm in length), the suspension was allowed to dry in a desiccator for 3 weeks (weight loss during the drying process: ˜100 mg per day). As the solvent evaporated, a cast film was formed on the wall of the Eppendorf tube.

Polarization microscopy
POM images were obtained using an Olympus polarizing microscope. The micrograph was taken using a SPOT digital camera (Diagnostic). Optical activity was also observed by changing the angle between the polarizer and the analyzer. The chiral smectic C spacing pattern was measured using a polarizing microscope.

Scanning Electron Microscope The surface morphology of the viral membrane was observed using a scanning electron microscope (LEO1530). In order to enhance contrast and avoid surface charging effects under the electron beam, the virus film was chromium coated using a plasma ion beam sputtering apparatus. In order to measure the film thickness of the film sample, the sample holder was tilted ~ 80 degrees from the horizontal plane and placed on the SEM sample stage.

Atomic Force Microscopy An atomic force microscope (AFM) (Digital Instruments) was used to examine the surface morphology of the viral membrane. Images were taken in air using tapping mode. The AFM probes were etched silicon with 125 μm cantilevers and 20-100 N / m spring constants driven near their 250-350 kHz resonance frequency. The scan speed was approximately 1-40 μm / s.

Laser light diffraction The chiral smectic C pitch of the virus film was measured using laser light diffraction (He-Ni laser: 632.8 nm). The distance between the screen and the sample was 200 cm. The diffraction pattern was recorded with a Sony Mavica digital camera. The spacing was calculated by measuring the first order Bragg diffraction spot.

Film formation and film thickness
Cast membranes made from initial virus concentrations of 1.79-9.93 mg / mL were self-supporting and could be manipulated with tweezers (FIG. 16A). Under these conditions for this viral material, viral membranes made from concentrations below ˜1 mg / mL were generally too thin to be self-supporting when removed from the substrate. The film thickness measured using SEM is shown in Table 1. In general, the film thickness was proportional to the initial concentration of the bulk suspension.

Chiral Smectic C Ordered Film A POM image of a virus film formed from an initial concentration of 9.93 mg / mL (Sample 1) revealed an optically active light-dark band pattern (FIG. 17A). The periodic spacing of these patterns was 36.79 ± 0.95 μm, and these patterns persisted across the centimeter scale. When using an optical microscope and changing the focus level through the optical axis at higher magnification, parallel band patterns smaller than 1 μm were also observed. These fine band patterns were consistent with the smectic layer structure of the M13 virus molecule. This film was determined to be optically active, as evidenced by changes in the intensity of alternating light and dark band patterns as the angle between the polarizers was rotated.

These optically active light and dark band patterns are consistent with the chiral smectic C structure for the viral membrane. In the chiral smectic C structure, the molecular long axis (director: n) has an inclination angle (θ) with respect to the layer standard (z). These graded layers form a helical rotation (azimuth angle φ) from one layer to the next, depicted in FIG. 16B ¹⁷ . For this reason, the continuous helical change of orientational order through the tilted smectic layer causes various interactions with plane polarized light, and further shows an optically active band pattern. Reflection polarization microscopy (RPOM) of this viral membrane produced a similar optically active light-dark band pattern depending on the angle between the polarizer and analyzer. These RPOM images suggest the presence of ^dechiralized line defects ^l7,35 on the surface. Dechiralization line defects arise from the interaction between the helical ordered bulk structure and surface tension effects. The helical ordered chiral smectic C structure is not wound near the surface due to surface tension effects, resulting in a light and dark band pattern corresponding to the periodic pitch of the chiral smectic C structure.

A scanning electron microscope (SEM) was used to characterize defects in the dechiralization line of the viral membrane (FIG. 17B). A wide ordered structure of zigzag pattern was observed, which corresponded to the light and dark band pattern in RPOM. The alternating zigzag band pattern (˜37 μm) showed a periodic change from +45 degrees to −45 degrees compared to the layer standard. The periodic spacing of the zigzag pattern was consistent with the periodic POM and RPOM patterns. The zigzag type morphology of the viral membrane could be introduced from surface defects in the chiral smectic C structure of the viral membrane. The chiral smectic C structure has two regularization parameters: tilt angle (θ) relative to the layer standard and azimuthal angle (φ) relative to the layered plane ¹⁷ . If the helical pitch direction of the chiral smectic C layer is parallel to the layer plane, the director azimuth (φ) can be projected onto the layer plane ¹⁸ . Because of the additional higher ordering properties on the surface, the tilt angle (θ) on the surface may have an angle greater than the sum of the tilt angle and the projected azimuth ¹⁹ . For this reason, the phase difference of 180 degrees of the azimuth angle (φ) is projected onto a wide-range zigzag pattern as shown in FIGS. 16C and 17B.

AFM was used to characterize the tilted smectic C morphology on the free surface of the viral membrane (FIG. 17C). M13 virus particles created a gradient layer structure with an average spacing of 620 ± 27 nm. The molecular long axis of virus particles was tilted ˜45 degrees with respect to the layer standard (z). The spacing (886 ± 36 nm) measured through the director (n) between two adjacent layers corresponded to the length scale (880 nm) of M13 phage particles ²⁰ . Based on the average layer spacing from the AFM image and the chiral smectic C pitch from the POM image, it can be estimated that the number of layers in the chiral smectic C pitch is 59.3 layers. Since the azimuth angle changes 360 degrees at one pitch, it can be estimated from the number of layers in one pitch (59.3 layers). The azimuth angle (φ) from virus membrane sample 1 was ˜6 degrees.

  The helical periodic pitch of the viral membrane was also measured using laser light scattering. A clear diffraction pattern (FIG. 19E) produced a 35.8 μm pitch consistent with the periodic pattern from POM and SEM.

Distortion of chiral smectic C-ordered film A locally distorted texture was observed in a certain region of the film (Fig. 18A). In these distorted regions, the band pattern was parallel to the ordered band pattern described previously. It was observed that the spacing in these regions was irregular and varied. On the bottom part of the membrane (c region in FIG. 16A), a gray band pattern similar to the previously reported Chiral Smectic A texture ²¹ occurred (FIG. 15B). Using AFM, a twisted deformation of the smectic A structure was observed on these distorted band texture regions. An AFM image (FIG. 18C) showed that the smectic layer was twisted, forming a dislocation line indicating an orientational discontinuity. These chiral smectic A POM textures and twisted smectic layer morphology are based on twisted grain boundaries (TGB), where chiral smectic C structures are known to form between the chiral smectic C phase and the isotropic phase. ²¹⁾ suggesting that there is a possibility of metastasis. AFM images collected from the gray POM region (Figure 18B) showed irregularly distorted smectic C domains. However, when a differential interference contrast (DIC) filter was applied to this gray pattern texture region, a periodic band pattern similar to the chiral smectic C periodic pattern was observed. These periodic DIC images and distorted AFM morphology suggest that this gray pattern region may have a chiral smectic C structure in the bulk and that the strain may be localized in the surface region. It was.

The characteristics of the virus membrane made from the concentration range of 6.38-9.70 mg / mL (samples 2-7) were similar to the virus membrane made from the above 9.93 mg / mL concentration (sample 1). The pitch length gradually decreased from 9.93 to 7.60 mg / mL and increased to 5.09 mg / mL. At this concentration (5.09 mg / mL), the smectic C structure transitioned to the smectic A structure. From transition to smectic phase from the cholesteric phase, the expansion of similar pitch near the transition point was observed ^22. Therefore, it seems that the expansion of chiral smectic C interval is related to the transition precursor. All of these films showed a clear diffraction pattern consistent with the periodic pattern in POM (Table 2).

Structural transition Various POM band patterns (upper part of FIG. 4A) were observed from virus membranes made from a concentration of 5.09 mg / mL (sample 7). The POM image of Sample 7 showed a vertical periodic bright band pattern divided by schlieren stripe lines when the dark line was parallel to the polarizer. When the analyzer angle was changed about 5 degrees, the intensity of the POM texture changed to a slightly darker and lighter stripe pattern similar to the chiral smectic C virus membrane. The film also showed zigzag pattern lines throughout the entire band pattern. The periodicity of these vertical periodic patterns was 97.43 ± 2.92 μm. When the sample was rotated through the optical axis, the bright band pattern changed to an alternating bright and dark stripe pattern. The intensity dependence on both the angular change between polarizers and the sample rotation suggests that there is a periodic change in orientation on the film surface.

  On the central part (b region in FIG. 16A) of the sample surface (5.09 mg / mL), a step change of the POM texture (the lower part of FIG. 4A and the upper part of FIG. 19B) was observed. In Samples 1 to 6, the vertical stripe pattern gradually changed to a parallel light and dark stripe pattern (lower part of FIG. 19B). These parallel stripe patterns had a periodicity of 41.04 ± 2.18 μm. Unwinding defects in the chiral smectic C structure were observed where the vertical stripes intersected the parallel stripes. The schlieren line texture spread parallel to the direction of the meniscus force. The sample area near the lower part of the membrane showed the gray texture observed in Sample 1.

Smectic A ordered film Samples 8-10 (4.39-2.59 mg / mL) POM images showed the same bright vertical band pattern (FIG. 19C) as observed for sample 7. However, the spacing between the two vertical dark lines varied as shown in Table 2. A wide range periodic zigzag pattern on the surface was also characterized using SEM.

  A low magnification SEM image from sample 10 (FIG. 19C) showed that this film had a regularly occurring chevron-like crack pattern. High magnification SEM images of these crack patterns (inset in FIG. 19C) showed that their direction was parallel to the director orientation. Between the interfaces of the zigzag pattern, disclination lines corresponding to the vertical dark schlieren line pattern in the POM image (FIG. 19C) were observed. Using AFM, a smectic A ordered structure was observed in the same region (FIG. 19D). Viral particles formed a domain block of ˜980 × 800 nm. In the smectic domain, the packing pattern of virus particles was close to a smectic B structure where the molecules were arranged in a layer with molecular centers arranged in a hexagonal close-packed crystal array. These domain blocks formed a bookshelf-like smectic A structure aligned in parallel on the surface. The average spacing measured between these two layers was 977 ± 25 nm, slightly larger than the length of the M13 virus.

Nematic Ordered Film The POM image of Sample 11 showed a disordered schlieren line texture (Figure 20A). The bent black brush line pattern spread irregularly within the 20-30 μm domain. The light and dark pattern was divided by a bent black brush line. The brightness of the black brush lines and these patterns changed with the rotation of the film, suggesting that these brush lines are disclinations. AFM images of these regions showed a nematic ordered structure of smectic A bundle-like domains (˜980 nm × 200 nm) (FIG. 20B). Each smectic A domain formed a nematic-like ordered structure oriented through the molecular long axis as the preferred direction.

The proposed for chiral smectic C structure first Study chirality (optical isomers) it was Meyer ^14. He predicted that when a smectic C structure was formed from a chiral molecule, the resulting structure was a chiral smectic C structure. ^L7,23,24 many chiral thermotropic liquid crystal material having a chiral smectic C structures have been synthesized. However, since the orientation of the lyotropic liquid crystal is not uniform, it has been difficult to test the chirality of the lyotropic smectic structure compared to the thermotropic liquid crystal. The chirality of lyotropic smectic liquid crystals has been reported ^20,25,26 . A twisted grain boundary layer of Fd virus was observed ²⁰ . Evidence from light microscopy of the chiral smectic phase (SmC ^* , SmI ^* , SmF ^* ) of filamentous actin (F-actin) has been reported, but because F-actin has a polydisperse nature, F- Extensive ordering of actin's chiral smectic C structure could not be observed ²⁵ . Furthermore, it has proven difficult to fabricate a broadly ordered lyotropic liquid crystal structure without using an external field. Widely ordered samples such as virus fibers and virus suspensions can be prepared from external field ^effects27,28 . However, these samples lost their chiral properties in response to external fields. Viral membranes made from monodisperse M13 phage tested in this paper utilized meniscus force to create a broadly ordered chiral smectic C structure up to several centimeters long without using an external field. The POM image of the viral membrane showed an optically active light and dark stripe pattern. SEM images showed dechiralization defects in the chiral smectic C structure. AFM images showed a tilted smectic C ordered structure. Based on these microscopic evidence, it was concluded that these viral membranes have a chiral smectic C structure.

The effect of film thickness on the chiral smectic C structure of the virus membrane was also observed. When the film thickness that had ˜360 virus particle layers (inter-particle distance: 12 nm) ⁵ decreased to ˜4.3 μm, the surface tension effect seemed to prevail in the whole bulk membrane. For this reason, the chiral smectic C structure was transformed into a smectic A-like ordered structure. The orientation of the molecular long axis was almost perpendicular to the smectic layer. However, a zigzag-like periodic pattern was also observed. The formation of the vertical zigzag pattern observed in Sample 7 to Sample 10 appears to arise from both the bulk helical structure and the film thickness. Due to the effect of film thickness, relatively thin viral films (film thickness 2-4 μm) are aligned in a smectic A pattern, which is similar to a thin nematic film with a smectic-like ordered structure ¹⁹ . The intrinsic chiral properties of the stratifying virus appear to stabilize the zigzag patterned smectic A structure instead of the bookshelf-like smectic A patterned structure.

The mechanism for self-regulated virus film formation is still under investigation. The nematic ordered structure showing a distorted smectic A domain strongly suggested that bundle-like domains were formed in solution before film formation. The isotropic phase of the virus suspension in the meniscus region slowly transitioned to the nematic phase. However, virus particles with the same orientational order began to make bundle-like domain structures. These domain behaviors are still flexible to the modification of their filling structure. These domains initially become the basic building units of layered structures. After forming the layer, these smectic A domains become dense as the solvent evaporates. When the solvent is completely evaporated, the bulk structure of the viral membrane is formed. Viral film thickness has a crucial effect on both bulk and surface structure. Surface tension is dominant in the formation of thin virus films. These interactions drive the bundle-like domains to align with the smectic A pattern. However, for thick virus membranes (with more than 360 virus layers), the surface morphology is affected by both surface tension and bulk chiral structure. For this reason, in the thin film sample, the smectic C pattern is dominant compared to the smectic A form. In experiments involving liquid crystal cast films, bundle formation has also been observed ^29-31 . From the M13 virus membranes formed on mica, SiO ₂ , and silicon substrates, M13 bundles are frequently observed at the beginning of membrane formation and serve as nucleation centers for directional deposition of viruses on these substrates ²⁹ seemed to work.

The morphology of the ZnS nanocrystal virus hybrid membrane has been previously reported ⁵ . The ZnS nanocrystal hybrid film has an optically active ˜72 μm periodic light and dark stripe POM pattern similar to 100% M13 virus film. However, the surface morphology of the ZnS nanocrystal hybrid virus film has an anti-smectic C structure (smectic O), which appears in a zigzag pattern with ~ 1.0 μm spacing through the layer normal direction. Based on the POM pitch and the AFM zigzag layer spacing, the ZnS nanocrystal hybrid virus film has ~ 72 layers and ~ 5 degrees azimuth in one pitch. Based on the surface morphology found from these 100% M13 virus control membranes and ZnS nanocrystal hybrid virus membranes, ZnS nanocrystal hybrid virus membranes can interact with each other of M13 viruses bound to 20 nm ZnS nanocrystal aggregates. It can be concluded that it has a chiral smectic C structure composed of meshing domains. Interdigitated domains can reduce the packing energy of the large head shape of the ZnS nanocrystal hybrid virus membrane. The anti-smectic C structure was generally observed only on the surface of the film and was generally considered to be a surface tension effect.

The observed morphology of M13 virus membrane and ZnS nanocrystal hybrid virus membrane is approximately 1/1000 the size of rod-like polymer (poly (γ-benzyl α, L-glutamic acid), (PBLG)) and viral system ^4,32,33 very similar to the form of the rod-coil block copolymer. Monodispersed rod polymers are known to form smectic film structures ³² . A high proportion of rod-coil (f _rod-coil > 0.96) block copolymers show interdigitated morphology of bilayers exhibiting smectic C and O structures ⁴ . TGB structure of PBLG membrane prepared from monodisperse PBLG is reported to have a chiral smectic structure ^33. The same membrane formed using the technique of the present invention can produce a chiral smectic C structure and therefore also supports Meyer's prediction.

  Using external forces such as magnetic or electric fields, after hybridization of the virus with semiconductors or magnetic nanocrystals, membranes based on these genetically engineered viruses can be used, for example, with well-ordered ultra-thickness without defects. Helps build small electronic devices. Homeotropically aligned magnetic nanocrystal hybrid virus thin films can be used, for example, for self-supporting flexible and highly integrated magnetic memory devices.

References for additional explanation and examples (Aspect A)

Additional description and examples (Aspect B)
The paper Lee et al. “Virus-Based Alignment of Inorganic, Organic, and Biological Nanosized Materials” Advanced Materials, 2003, 15, 9, 689-692 is hereby incorporated by reference in its entirety, including drawings, experiments, and results and discussion. Incorporated into the book.

  Additional materials were prepared that could be used as membranes in storage applications as well as other applications. In an additional aspect, a novel platform for organizing various materials including inorganic nanoparticles, organic small molecules and biopolymers that are organized and self-assembled on a nanometer length scale and follow the centimeter length scale Is presented. Widely ordered materials (10 nm gold nanoparticles, fluorescein, phycoerythrin protein) were made using a streptavidin linker and anti-streptavidin M13 bacteriophage (virus). Anti-streptavidin virus that forms the basis of a self-regulating system with a specific recognition component for streptavidin was selected using phage display. The nano-sized material was previously bound to streptavidin. Through molecular recognition of genetically selected viruses, nano-sized materials were combined and evolved naturally into self-supporting hybrid membranes.

Functionalized liquid crystal materials to build well-ordered and well-controlled 2D and 3D structures to build next generation optical, electronic, and magnetic materials and devices ^[1-3] can provide various means. Several types of rod-shaped viruses have been shown to form well-controlled liquid crystal phases ^[4,5] . In recent years, a self-assembled ordered nanocrystalline film fabrication method using nanocrystal functionalized M13 virus has been reported ^[3] . By utilizing genetic engineering techniques, one end of the M13 virus was functionalized to nucleate or bind to the desired semiconductor material. These nanocrystal functionalized viral liquid crystal building blocks were grown into ordered hybrid self-supporting membranes. The resulting nanocrystalline hybrid film was ordered into 72 μm periodic stripe pattern domains at the nanoscale and micrometer scale. Previous systems could easily nucleate and align nanoparticles to II-VI semiconductor materials via a one-pot synthesis route. Aligning other materials, including metals and electro-optic materials, requires biological selection and other evolution for each material before aligning the nanoparticles. Here, a novel nanoparticle alignment method using anti-streptavidin virus has been reported in which the virus that binds to the streptavidin protein unit is first selected. This allowed general handling of the virus to pick up the material covalently bound to streptavidin. Therefore, the hybrid material can be organized by utilizing the self-assembling property of the anti-streptavidin virus. The organized hybrid material presented here is a liquid crystal film of gold nanoparticles, fluorescent molecules (fluorescein) and large fluorescent protein (phycoerythrin).

Anti-streptavidin M13 virus with a specific binding component for streptavidin was isolated through screening of a phage display library (Figure 21) ^[6,7] . Streptavidin has a known specific binding motif His-Pro-Gln ^[6] . The His-Pro-Gln sequence was isolated as a pIII insert after the second round of selection of phage against the streptavidin target. After the fourth round of selection and sequencing, the His-Pro-Gln binding motif constituted up to 100% of the pIII insert. The dominant binding sequence after the fourth round selection was TRP ASP PRO TYR SER HIS LEU LEU GLN HIS PRO GLN. The anti-streptavidin M13 virus was amplified to a high concentration (˜10 ¹² pfu) and reacted with 10 nm gold nanoparticles (FIG. 2A), fluorescein and phycoerythrin previously conjugated with streptavidin. These highly concentrated suspensions showed liquid crystal properties.

  High concentration Au-viral liquid crystal suspension (˜83 mg / mL) showed iridescent birefringence texture when analyzed using polarized light microscope (POM) (FIG. 2B). This iridescent birefringent texture corresponded to the smectic liquid crystal phase structure. Cholesteric fingerprint texture (76-20 mg / mL) and nematic texture (14 mg / mL) were observed when the suspension was systematically diluted.

Individual mesogenic units of 10 nm gold nanoparticles bound to the virus were visualized using transmission electron microscopy (TEM) before staining with 2% uranyl acetate. These individual Au and virus complexes (Au-virus) were isolated from a 0.01% dilution of a smectic phase suspension (FIG. 22C). Aggregation of Au-virus complex was observed in the 0.1% dilution (FIG. 22D). The majority of mesogenic units observed had one virus bound to one 10 nm Au particle at the pIII end of the virus. However, both unbound gold nanoparticles and unbound virus were observed in less than 20% of the mesogenic units. In addition, two gold nanoparticles bound to one virus and one gold nanoparticle bound to two viruses were also observed (less than ˜5%). These undesirable binding behaviors between virus-bound gold nanoparticles and streptavidin-bound gold nanoparticles can be caused by mismatches in the number of recognition groups between the virus and streptavidin. The M13 virus has five pIII streptavidin recognition units at the end of the virus, and streptavidin is known to have four binding sites for biotin ^[8] . Due to empirical stoichiometric control and positional effects, the majority of the population was able to build mesogenic units containing one virus with one Au nanoparticle.

  A smectic ordered self-supporting Au-virus membrane (FIG. 23A) was prepared from diluted Au-virus solution (˜6 mg / mL). Virus and nanocrystals were agitated for 1 week prior to membrane preparation. This suspension was allowed to dry in a desiccator for 2 weeks. The virus nanocrystal hybrid film was transparent with a faint pinkish color. The ordered morphology of the viral membrane was characterized by POM, scanning electron microscope (SEM) and atomic force microscope (AFM). The film thickness was 5.68 + 0.65 μm.

Optical characterization revealed that the film was composed of ˜10 μm dark gray periodic horizontal stripe patterns (FIG. 23B). These stripes were optically active and their light and dark patterns varied depending on the angle between the polarizer and the analyzer. The characteristics of the stripe pattern revealed by these POMs are similar to the smectic virus membranes previously reported by the inventors ^[9] .

The surface morphology of these stripe patterns was characterized using SEM. The SEM image (FIG. 23C) showed that the Au-virus hybrid membrane has a broadly ordered zigzag periodic morphology composed of 10-12 smectic layers in a periodic pattern. The average spacing of the zigzag periodic bands corresponded to one chiral smectic C pitch of a typical viral membrane ^[9] , 9.34 ± 0.78 μm. AFM images (Figure 23D) demonstrated that the hybrid membrane has a smectic C structure. The average layer spacing between two adjacent layers was 833 ± 12 nm. The layer spacing measured through the molecular long axis was 977 ± 65 nm. The average tilt angle was ~ 54 degrees with respect to the layer standard. The length of the M13 virus is 880 nm. This spacing of ~ 100 nm observed through the molecular long axis is strong evidence to support interdigitated structures ^[10] . The shape of a mesogenic unit with a large head (inorganic gold nanoparticles) with a long tail (organic M13 virus) may have low filling free energy by forming an interdigitated structure. Furthermore, the ~ 10 μm periodic zigzag pattern observed in POM and SEM images strongly indicates that the Au-virus hybrid film also has a chiral smectic C structure in the bulk and a dechiralization defect on the surface of the hybrid film. It was.

Two more organic materials were made in the virus membrane. These organic materials were selected because this technology is versatile, but because these materials are fluorescent, it is likely to allow easy visualization of a periodic wide range of regularization of one micrometer It was to prove that. Streptavidin and anti-streptavidin M13 virus were used to make thin cast membranes of virus bound to fluorescein and phycoerythrin. Near the surface, the ordering properties of the liquid crystal material are enhanced, and the capillary driving force acts during the drying process, so the drop cast films of fluorescein complex virus (F-virus) and phycoerythrin complex virus (P-virus) A smectic layer structure was easily observed (FIG. 23E). The ordering of these liquid crystal hybrid materials was enhanced by casting thin films of these materials. In a similar phenomenon, nematic liquid crystal materials formed a surface-stabilized smectic phase due to the surface tension effect ^[11] , and the chiral smectic C structure transitioned to a smectic A structure ^{[9] in the} thin film. Optical sections were prepared from F-virus thin films using a scanning laser microscope (FIG. 23F). These thin films showed a weak stripe pattern corresponding to the smectic structure. When a similar analysis was applied to a thin film of fluorescent P-virus (FIG. 23G), a very clear 1 μm stripe pattern was observed. These 1 μm fluorescence patterns suggested that fluorescent molecules (fluorescein and phycoerythrin) were bound to the virus by binding streptavidin and then formed a smectic layer structure. As fluorescent material was added at the edge of the virus, their location was localized between smectic layer interface boundaries.

In the present invention, anti-streptavidin M13 virus was used to self-assemble various nano-sized materials. This anti-streptavidin M13 virus provides a convenient way to organize various nano-sized materials into self-assembled ordered structures. Since modification of the DNA insert allows for controlled modification of the viral length, the spacing in the smectic layer can be controlled by genetic manipulation ^[12] . By combining other nano-sized materials (magnetic nanoparticles, II-VI semiconductor nanoparticles, functional chemicals, etc.) with streptavidin, this anti-streptavidin method can achieve the desired length scale defined by the smectic layer. Can align various nano-sized materials.

Experimental Part Anti-streptavidin virus was selected by phage display method using M13 bacteriophage library (New England Biolab). This virus was amplified in large quantities (400 mL scale, 7 × 10 ⁷ pfu). This virus suspension was allowed to settle into the pellet. Suspend 20 mg of virus pellet using 1.0 mL of 10 nm gold nanoparticles (Abs: 2.5 at 520 nm), bind with colloidal suspension of streptavidin (Sigma) and use rocker for 1 day Stir. Virus (Au-virus) bound to gold nanoparticles was centrifuged by adding 167 μL of polyethylene glycol solution. Suspension of the red pellet using -20 μL of Tris buffer (pH 7.5) produced an Au-viral liquid crystal suspension (virus concentration: 83.2 mg / mL). To make the Au-virus membrane, the Au-virus suspension was diluted to ~ 6 mg / mL (400 μL) and allowed to dry in a desiccator for 2 weeks.

Preparation of fluorescein-virus cast membrane
20 μL of virus suspension (1.9 × 10 ⁻⁷ M in Tris-HCl buffer (pH 7.5)) to 20 μL of 0.01 mg / mL fluorescence-streptavidin suspension (1.9 × 10 ⁻⁷ M, MW: 53,200). 1 μL of the suspension was cast and dried on a glass substrate. The molar concentration of the virus suspension was measured using a UV-Vis spectrometer (absorption coefficient: 1.2 × 10 ⁸ M ⁻¹ cm ^{−1 at} 268 nm) ^[13] .

Preparation of phycoerythrin-virus and cast membrane
20 μL of virus suspension (˜6 mg / mL, 1.9 × 10 ⁻⁷ M, MW: 292,800 in Tris-HCl buffer (pH 7.5)) 20 μL of 0.05 mg / mL R-phycoerythrin-streptavidin ( Mixed with 1.7 × 10 ⁻⁷ M) in Tris-HCl buffer (pH 7.5) containing 5% sucrose. 1 μL of the suspension was cast and dried on a glass substrate.

Microscopic examination
POM images were obtained using an Olympus polarizing microscope. The micrograph was taken using a SPOT digital camera (Diagnostic). Scanning laser microscope images were obtained using a Leica TCS 4D, and SEM images were obtained using a LEO 1530 operating at an acceleration voltage of 1 KV. TEM images were obtained using Philips 208 operating at an acceleration voltage of 80 kV and JEOL 2010F operating at an acceleration voltage of 200 kV. AFM images were taken in air using a tapping mode (Digital Instruments). The AFM probes were silicon etched using spring constants of 20-100 N / m when driven near 125 μm cantilevers and their 250-350 kHz resonant frequency. The scan speed was approximately 1-40 μm / s.

References for additional explanation and examples (Aspect B)

  While this invention has been described with reference to exemplary embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of the exemplary aspects of the invention, as well as other aspects, will be apparent to those of skill in the art upon reviewing the description of the invention. Thus, it is intended that the appended claims include such modifications or embodiments.

  Reference to references in this specification is not an admission that these references are prior art to the present invention.

The description and other advantages of the present invention may be more clearly understood by referring to the following description in conjunction with the accompanying drawings, in which corresponding numerals in the various drawings represent corresponding parts.
(FIG. 1) According to the present invention, (A) a photograph of a biofilm, (B) a polarizing microscope (POM) image of the biofilm, (C) an atomic force microscope of individual M13 bacteriophages on the mica surface (contact surface) ( (AFM) image, and (D) surface morphology of biofilm contact surface.
(FIG. 2) A graph showing the relationship between titer number and elapsed days showing a logarithmic plot of titer number and elapsed days after biofilm production according to the present invention.
FIG. 3 is a diagram of random amino acid sequences selected according to the present invention.
FIG. 4 is an XPS spectrum diagram of a structure according to the invention.
FIG. 5 shows the phage recognition of heterostructure according to the present invention.
(FIGS. 6 to 10) Specific amino acid sequences according to the present invention.
FIG. 11 is a schematic representation of smectic alignment of M13 phage according to the present invention.
(FIG. 12) A diagram showing an A7-ZnS suspension. (A) and (B) POM images, (C) AFM images, (D) SEM images, (E) TEM images, and (F) TEM images (using electron diffraction diffraction inserts).
(FIG. 13) includes (A) a photograph of the film, (B) a schematic representation of the film structure, (C) an AFM image, (D) an SEM image, (E) and (F) TEM images along the xz plane and zy plane , M13 bacteriophage nanoparticle biofilm image.
FIG. 14 shows the effect of glucose / sucrose and phage on β-galactosidase activity during storage at room temperature after (A) drying in a desiccator and (B) after freeze drying. (■ -black square) is β-galactosidase dried with sugar plus phage, (▲ -black triangle) is β-galactosidase dried with sugar, (● -black circle) ) Is β-galactosidase dried with phage, and (▼ -black inverted triangle) is β-galactosidase dried without additives, day 0 after freeze-drying or in desiccator Represents the activity recovered after drying.
FIG. 15 is a confocal microscope image of a fluorescent GFPuv virus membrane one day after production using GFPuv and phage. Variations in glucose: sucrose are (A) 5 mg / mL: 50 mg / mL, (B) 2.5 mg / mL: 25 mg / mL, and (C) no glucose or sucrose.
(FIG. 16) (A) Photograph of M13 virus membrane. (B) Schematic representation of the M13 virus membrane structure in the bulk with chiral smectic C ordered structure (z: director (molecular long axis); n: layer standard; θ: tilt angle; φ: azimuth rotation angle). (C) Schematic representation of the surface morphology of the M13 virus membrane with its helical ordered structure unraveled and forming a zigzag pattern due to surface tension effects. The dotted line represents the disclination line, and the spacing between two adjacent disclination lines corresponds to the half pitch (1 / 2P) of the chiral smectic C helical pattern.
FIG. 17 is a diagram of the chiral smectic C structure of the viral membrane from sample 1 (9.93 mg / mL). (A) POM image showing light and dark stripe pattern (36.8μm) (scale bar: 100μm; x indicates the direction of analyzer (A) and polarizer (P)), (B) removal of zigzag pattern on surface SEM image of a viral membrane showing a chiralization defect (scale bar: 50 μm). (C) AFM image (scale bar: 1 μm) of the viral membrane surface showing smectic C alignment, (D) TEM image of M13 virus (scale bar: 100 nm), and (E) Laser diffraction pattern from the viral membrane.
(FIG. 18) POM and AFM images showing strain and phase transition of smectic structure from sample 1. (A) POM image showing a distorted light and dark stripe pattern (scale bar: 100 μm), (B) POM image showing phase transition, (C), (D) POM of (A) and (B) respectively AFM image corresponding to the image.
(FIG. 19) POM image of Sample 7 showing the change in texture from the vertical stripe pattern (A) to the horizontal stripe pattern (B). Sample 10 smectic A form. (C) POM image (62.4 μm) showing a vertical stripe pattern (10 × scale bar: 100 μm), (D) AFM image of the viral membrane surface showing smectic A alignment. (Scale bar: 1 μm), (E) SEM image of the chevron-shaped crack pattern and high resolution SEM image in the inset.
(FIG. 20) Nematic morphology of the viral membrane (sample 11). (A) POM image (scale bar: 100 μm) showing bent dark schlieren brush pattern, (B) AFM image of viral membrane surface showing nematic ordering of smectic domains.
FIG. 21 is a schematic illustrating the alignment of nanomaterials using anti-streptavidin M13 virus and streptavidin linker.
(FIG. 22) (A) Photograph of virus pellet (i), streptavidin-bound gold nanoparticle suspension (ii), and gold nanoparticle suspension (iii) bound to virus (Au-virus). (B) POM image of Au-virus suspension. (C) TEM image of virus bound to 10 nm gold nanoparticles (scale bar: 100 nm) and striped and fast Fourier transform images of gold nanoparticles from the same TEM grid (inset, scale bar: 5 nm). (D) TEM image of Au-virus aggregates (scale bar: 500 nm).
(FIG. 23) (A) Au-virus membrane photograph. (B) POM image of Au-virus membrane (scale bar: 20 μm), (C) SEM image of Au-virus membrane surface morphology showing a wide zigzag pattern (scale bar: 5 μm). (D) AFM image of Au-virus membrane (scale bar: 1 μm). (E) DIC image of fluorescein-virus (F-virus) cast membrane (F-virus) (scale bar: 10 μm), (F) fluorescein-bound virus (F-virus) cast membrane, and (G) 1 μm Fluorescence image of a virus (P-virus) cast membrane bound to phycoerythrin showing a fluorescent stripe pattern (scale bar: 10 μm).

[Sequence Listing]

Claims (35)

An artificial biofilm storage device for long-term storage of biological material,
Optionally, a substrate having a contact surface;
An artificial biofilm storage device comprising a biological material on any contact surface and forming a stable membrane, the membrane being stable at room temperature for at least 7 weeks.   Substrate is present and the substrate is Langmuir-Blodgett film, functional glass, germanium, silicon, semiconductor material, PTFE, polycarbonate, mica, mylar, protein film, plastic, quartz, polystyrene, gallium arsenide, gold 2. The artificial biofilm storage device of claim 1, selected from the group consisting of: silver, metal, alloy, fabric, mammalian tissue, and combinations thereof.   2. The artificial biofilm storage device of claim 1, wherein the stable membrane is self-supporting.   2. The artificial biofilm storage device of claim 1, wherein the stable membrane comprises one or more organic or inorganic molecules in addition to the biological material.   An organic molecule is present and the organic molecule is carbon, single-stranded nucleic acid, double-stranded nucleic acid, peptide, protein, antibody, enzyme, steroid, drug, chromophore, conductive polymer, vaccine, and combinations thereof 5. The artificial biofilm storage device of claim 4, selected from the group consisting of:   5. The artificial biofilm storage device of claim 4, wherein inorganic molecules are present and the inorganic molecules are selected from the group consisting of indium tin oxide, dopants, metals, alloys, minerals, semiconductors, and combinations thereof. .   Biological materials are viruses, bacteriophages, bacteria, peptides, proteins, antibodies, enzymes, amino acids, steroids, drugs, carbohydrates, lipids, chromophores, single- or double-stranded nucleic acids, vaccines, and their chemistry 2. The artificial biofilm storage device according to claim 1, which is selected from the group consisting of modifications.   The artificial biofilm storage device of claim 1, wherein the biological material further comprises a vaccine.   2. The artificial biofilm storage device of claim 1, wherein the membrane exhibits biological properties, optical properties, electrical properties, magnetic properties, or combinations thereof.   2. The artificial biofilm storage of claim 1, wherein the stable membrane is used for diagnosis, screening, analysis, testing, information collection, data processing, new drug discovery, microelectronics, optical equipment, data storage, research, or a combination thereof. apparatus. A method of making a biofilm storage device comprising:
Applying a biological material to a substrate comprising a contact surface, wherein the contact surface facilitates uniform alignment of the biological material on the contact surface;
Allowing the formation of a stable film that is stable at room temperature for at least 7 weeks.   12. The method of claim 11, wherein the biological material is a combinatorial library.   12. The method of claim 11, wherein the step of making the biofilm storage device is reversible.   Biological materials are viruses, bacteriophages, bacteria, peptides, proteins, antibodies, enzymes, amino acids, steroids, drugs, carbohydrates, lipids, chromophores, single- or double-stranded nucleic acids, vaccines, and their chemistry 12. The method of claim 11, wherein the method is selected from the group consisting of modifications.   12. The method of claim 11, wherein at least two biological materials are applied.   12. The method of claim 11, wherein the biological material is layered with organic compounds, inorganic compounds, and combinations thereof. A kit for making a biofilm storage device,
A container;
A storage membrane comprising biological material that is stable at room temperature for at least 7 weeks.   18. The kit of claim 17, wherein the thin film stores high density information at room temperature.   19. The kit of claim 18, wherein the high density information is used for diagnosis, screening, analysis, testing, information collection, data processing, microelectronics, optical instruments, research, or combinations thereof. A hybrid artificial membrane storage device,
A substrate comprising a surface;
A hybrid artificial membrane storage device comprising a biological material applied to a surface to form a biologically stable thin film, the membrane further comprising an inorganic material.   21. The hybrid artificial membrane storage device of claim 20, wherein the inorganic material is selected from the group consisting of indium tin oxide, doping agents, metals, alloys, minerals, or combinations thereof.   21. The hybrid artificial membrane storage device of claim 20, wherein the one or more organic or inorganic molecules are preincubated with the biological material.   The biological material is selected from the group consisting of viruses, bacteriophages, bacteria, peptides, proteins, amino acids, steroids, drugs, chromophores, single- or double-stranded nucleic acids, vaccines, and chemical modifications thereof. 21. The hybrid artificial membrane storage device according to claim 20, wherein   A viral membrane made for use as a storage device comprising phage particles in a stable membrane, wherein the membrane is stable at room temperature for at least 7 weeks.   25. The viral membrane of claim 24 further comprising an inorganic material in combination with phage particles.   25. The membrane comprises phage particles of a phage display library, the phage particles are selected to provide specific binding to a biological molecule, and the phage particles are bound to a biological molecule. Viral membranes. A method of forming a viral membrane comprising:
Preparing a concentrated suspension of virus phage particles in a solvent;
Removing the solvent such that the phage particles form a membrane under conditions where the membrane is stable at room temperature for at least 7 weeks.   28. The method of claim 27, wherein the membrane further comprises an inorganic compound in combination with phage particles.   A self-supporting membrane for use as a storage device comprising one or more biological materials, wherein the membrane is stable for at least 6 months.   A method for improving the stability and long-term activity of a biofilm storage device, comprising the step of including in the biofilm storage device a preservation solution that improves the stability and long-term activity of the biofilm storage device.   32. The method of claim 30, wherein the storage device comprises an enzyme and a virus.   A method of visualizing the structure and function of a biological material used as a biofilm storage device, comprising monitoring the optical properties of the biological material.   35. The method of claim 32, wherein the luminescent molecule is fluorescent.   A method of forming a virus film for a storage device that maintains the ability of virus particles to infect bacterial hosts, wherein the solvent is removed from the concentrated suspension of virus particles to form a virus film on a substrate. Wherein the viral particles retain their ability to infect bacterial hosts based on titer measurements after at least 7 weeks.   35. The method of claim 34, wherein the viral particle comprises a genetically engineered phage library prior to membrane formation and the library information is stored in a membrane format.

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