EP1545202A2 - Dispositif de stockage de biofilms fabriques - Google Patents

Dispositif de stockage de biofilms fabriques

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Publication number
EP1545202A2
EP1545202A2 EP03796341A EP03796341A EP1545202A2 EP 1545202 A2 EP1545202 A2 EP 1545202A2 EP 03796341 A EP03796341 A EP 03796341A EP 03796341 A EP03796341 A EP 03796341A EP 1545202 A2 EP1545202 A2 EP 1545202A2
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EP
European Patent Office
Prior art keywords
film
storage device
viral
fabricated
phage
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03796341A
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German (de)
English (en)
Inventor
Angela M. Belcher
Seung-Wuk University of Texas LEE
Brent L. University of Texas IVERSON
Soo-Kwan University of Texas LEE
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University of Texas System
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University of Texas System
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Publication date
Application filed by University of Texas System filed Critical University of Texas System
Publication of EP1545202A2 publication Critical patent/EP1545202A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details

Definitions

  • the present invention is directed to the field of molecular storage devices in general, and specifically, toward the storage and preservation of fabricated biofilms for input and output of high-density information.
  • biological materials to process the next generation of microelectronic devices provides a possible solution to resolving the limitations of traditional processing and memory methods.
  • the critical factors in this approach towards the successful development of so-called organic-inorganic hybrid materials are identifying the appropriate compatibilities and combinations of biologic and inorganic materials, the synthesis and application of the appropriate materials, and the long-term storage of these biologic storage devices.
  • the appropriate long-term storage of biologic materials is of enormous economic benefit, especially when it reduces weight and storage space and increases or preserves material stability.
  • Biologic materials in general, are highly sensitive to their environment and require highly specific and often costly materials to ensure their stability, activity, and longevity. Few biologic materials are stable at room temperature for extensive periods of time. In fact, biologic materials are often considered unstable at room temperature. Viruses and bacteria, for example, are temperature and metabolite sensitive, require continuous feedings and appropriate air (gas) conditions to maintain activity, and must be frequently monitored for changes in growth and density.
  • Low temperature storage methods or freeze drying e.g., suspending the materials in 10% glycerol at temperatures as low as -20 to -80 degrees Centigrade
  • a poly (ethylene) glycol-modification technique are generally used.
  • Dessication is another options that offers both advantages and disadvantages. While dessication is not as costly, it does not allow for large-scale preparations (i.e., industrial quantities) .
  • Freeze drying may be used for large-scale production; however, the process is extremely damaging to sensitive biologic materials. Freeze drying is also very inconvenient, cannot ensure sterility and is very cost ineffective, as it requires that expensive agents (e.g., dry ice or other cooling agents) be used even when transferring materials from one facility to another.
  • the subject matter of the present invention includes the storage of variable density organic and inorganic information as a fabricated film that may be specifically engineered and custom designed.
  • biologic material film fabrication also referred to as biofilms, may be used to store both organic and inorganic information from one or more biologic materials, wherein one or more biologic materials may be further bound to other organic or inorganic molecules .
  • Applications of the present invention extend to medicine, engineering, computer technology and optics.
  • the stored information may be biologic, electrical, magnetic, optical, microelectronic, mechanical and combinations thereof.
  • the present invention is a fabricated biofilm storage device comprising a substrate coated with a biologic material applied to a contacting surface to form a stable film.
  • Another form of the present invention is a method of fabricating a biofilm storage device that includes the steps of applying a biologic material to a substrate with a contacting surface that promotes uniform alignment of the biologic material on the contacting surface and allows the formation of a stable film.
  • the present invention is a kit for fabrication a biofilm storage device comprising a substrate with a surface and a biologic material capable of binding specifically to the surface to form a dry thin film.
  • a hybrid fabricated film storage device comprising a substrate comprising an inorganic material with a surface and a biologic material applied to the surface to form a stable and thin film, wherein the film may be biologically active or interact with biologic components.
  • FIGURE 1 depicts (A) photograph of the biofilm, (B) polarized optical micrograph (POM) image of the biofilm, (C) atomic force microscopy (AFM) image of the individual M13 bacteriophage on the mica surface (contacting surface) , and (D) surface morphology of the biofilm contacting surface in accordance with the present invention;
  • POM polarized optical micrograph
  • AFM atomic force microscopy
  • FIGURE 2 depicts the relationship between the titer number and days showing the log plot of titer number and days since fabrication of the biofilm in accordance with the present invention
  • FIGURE 3 depicts selected random amino acid sequences in accordance with the present invention.
  • FIGURE 4A-C depict XPS spectra of structures in accordance with the present invention
  • FIGURE 5A-E depicts phage recognition of heterostructures in accordance with the present invention
  • FIGURES 6-10 depict specific amino acid sequences in accordance with the present invention.
  • FIGURES 11 (A) and (B) depict schematic diagrams of the smectic alignment of M13 phages in accordance with the present invention
  • FIGURES 12A-F depict the A7-ZnS suspensions: (A) and (B) POM images, (C) AFM image, (D) SEM image, (E) TEM image and (F) TEM image (with electron diffraction insert) ;
  • FIGURES 13A-F depict images of the M13 bacteriophage nanoparticle biofilm, including (A) photograph of the film, (B) schematic diagram of the film structure, (C) AFM image, (D) SEM image, (E) and (F) TEM images along the x-z and z-y planes;
  • FIGURE 14 depicts the effect of glucose/sucrose and phage on ⁇ -galactosidase activity during storage at room temperature after (A) drying in desiccator, and (B) freeze-drying, where
  • FIGURE 15 illustrates confocal microscopy images of fluorescent GFPuv viral film one day after fabrication with GFPuv and phage, wherein variations in glucose : sucrose are (A) 5mg/mL:50mg/mL, (B) 2.5mg/mL:25mg/mL, and (C) no glucose or sucrose .
  • FIGS 16A-C (A) Photograph of M13 virus film.
  • Fig. 17A-E Chiral smectic C structure of the viral film from sample 1 (9.93 mg/ml) .
  • A POM image showing the dark and bright stripe patterns (36.8 ⁇ m) (scale bar: 100 ⁇ m; cross represents the direction of analyzer (A) and polarizer (P) )
  • B SEM image of viral film showing zig-zag pattern dechiralization defects on the surface (scale bar: 50 ⁇ m) .
  • C AFM image of the viral film surface that shows the smectic C alignment, (scale bar: 1 ⁇ m) , (D) TEM image of M13 virus (scale bar: 100 nm) , and (E) a laser light diffraction pattern from the viral film.
  • FIG. 18A-D POM and AFM images showing distortion of the smectic structures and phase transitions from sample 1.
  • A POM image showing the distorted dark and bright stripe patterns (scale bar: 100 ⁇ m)
  • B POM image showing the phase transition
  • C POM image showing the phase transition
  • D AFM images corresponded to POM image (A) and (B) respectively.
  • FIG 19A-E POM images of sample 7 that showed texture changes from a vertical stripe (A) pattern to horizontal stripe patterns (B) .
  • C POM image showing the vertical stripe patterns (62.4 ⁇ m) (lOx scale bar: 100 ⁇ m)
  • D AFM image of the viral film surface showing the smectic A alignment, (scale bars: 1 ⁇ m)
  • E SEM images of viral film surface showing the chevron-like cracked patterns and a high-resolution SEM image in the inset.
  • FIG. 20A-B Nematic morphologies of the viral film (sample 11) .
  • A POM image showing the crooked schlieren dark brush patterns (scale bar: 100 ⁇ m)
  • B AFM images of viral film surface showing the nematic ordering of the smectic domains.
  • Figure 21 A schematic diagram illustrating alignment of nanomaterials using an anti-streptavidin M13 virus and a streptavidin linker.
  • Figure 22A-D Photograph of virus pellet (i) , streptavidin conjugated gold nanoparticles suspension (ii) , and gold nanoparticle conjugated with virus (Au-virus) suspension (iii) .
  • B POM image of Au-virus suspension.
  • C TEM image of a virus that bound to a 10 nm gold nanoparticle (scale bar: 100 nm) and a lattice fringe image and a fast Fourier transformation image of gold nanoparticle from the same TEM grids (insets, scale bar: 5 nm) .
  • D TEM image of Au- virus aggregations (scale bar: 500 nm) .
  • Figure 23A-G (A) Photograph of Au-virus film.
  • B POM image of Au-virus film (scale bar: 20 ⁇ m)
  • C SEM image of the Au- virus film surface morphology that shows the long range zigzag patterns (scale bar: 5 ⁇ m) .
  • D AFM image of the Au-virus film (scale bar : 1 ⁇ m) .
  • E DIC image of Fluorescein-virus (F-virus) cast film (scale bar: 10 ⁇ m)
  • F Fluorescence images of virus conjugated with fluorescein (F-virus) and
  • G phycoerythrin (P-virus) cast films that show one micrometer fluorescent striped patterns (scale bars: 10 ⁇ m) .
  • biological material refers to a virus, bacteriophage, bacteria, peptide, protein, amino acid, steroid, drug, chromophore, antibody, enzyme, single-stranded or double-stranded nucleic acid, vaccine, and any chemical modifications thereof.
  • the biologic material may self- assemble to form a dry thin film on the contacting surface of a substrate. Dry thin films can be either substantially free of solvent so they are completely dry within conventional detection limits for dryness, or can be retaining residual solvent from the drying process so that the film is solid-like and self-supporting but still has residual wetness from solvent. In many cases, films can be left in a partially hydrated state, and the state of hydration can be optimized for a given application.
  • Self-assembly may permit and random or uniform alignment of the biologic material on the surface.
  • the biologic material may form a dry thin film that is externally controlled by solvent concentration, application of an electric and or magnetic field, optics, or other chemical or field interactions .
  • inorganic molecule refers to compounds such as, e.g., indium tin oxide, doping agents, metals, minerals, radioisotope, salt, and combinations, thereof.
  • Metals may include Ba, Sr, Ti, Bi, Ta, Zr, Fe, Ni, Mn, Pb, La, Li, Na, K, Rb, Cs, Fr, Be, Mg, Ca, Mb, Tl, Hg, Cu, Co, Rh, Sc, or Y.
  • Inorganic compounds may include, e.g., high dielectric constant materials (insulators) such as barium strontium titanate, barium zirconate titanate, lead zirconate titanate, lead lanthanum titanate, strontium titanate, barium titanate, barium magnesium fluoride, bismuth titanate, strontium bismuth tantalite, and strontium bismuth tantalite niobate, or variations, thereof, known to those of ordinary skill in the art .
  • insulators such as barium strontium titanate, barium zirconate titanate, lead zirconate titanate, lead lanthanum titanate, strontium titanate, barium titanate, barium magnesium fluoride, bismuth titanate, strontium bismuth tantalite, and strontium bismuth tantalite niobate, or variations, thereof, known to those of ordinary skill in the art .
  • organic molecule refers to compounds containing carbon alone or in combination, such as nucleotides, polynucleotides, nucleosides, steroids, DNA, RNA, peptides, protein, antibodies, enzymes, carbohydrate, lipids, conducting polymers, drugs, and combinations, thereof.
  • a drug may include an antibiotic, antimicrobial, anti- inflammatory, analgesic, antihistamine, and any agent used therapeutically or prophylactically against mammalian pathologic (or potentially pathologic) conditions.
  • a "substrate” may be a icrofabricated solid surface to which molecules attach through either covalent or non-covalent bonds and includes, e.g., silicon, Langmuir-Bodgett films, functionalized glass, germanium, ceramic, , a semiconductor material, PTFE, carbon, polycarbonate, mica, mylar, plastic, quartz, polystyrene, gallium arsenide, gold, silver, metal, metal alloy, fabric, and combinations thereof capable of having functional groups such as amino, carboxyl, thiol or hydroxyl incorporated on its surface.
  • the substrate may be an organic material such as a protein, mammalian cell, organ, or tissue with a surface to which a biologic material may attach.
  • the surface may be large or small and not necessarily uniform but should act as a contacting surface (not necessarily in monolayer) .
  • the substrate may be porous, planar or nonplanar.
  • the substrate includes a contacting surface that may be the substrate itself or a second layer (e.g., substrate or biologic material with a contacting surface) made of organic or inorganic molecules and to which organic or inorganic molecules may contact.
  • the substrate can be cylindrical or non-flat.
  • Substrates can be supported to improve their mechanical strength or surface to volume ratio. Arrays can be made. Macroporous beads can be used including glass and polystyrene beads. Dense packed pins can be used.
  • Substrate surfaces can be grooved, micromachined, or otherwise made non- flat.
  • the biofilm is created by applying a biologic material to the contacting surface of a substrate.
  • the contact may be through a self-assembly of the biologic material or may be controlled by the surface itself or by external conditions such as solvent concentration, magnetic field, electric field, optics, and combinations thereof.
  • the substrate itself may serve as the contacting surface and may also control the nature and amount of biologic material contact.
  • the contacting surface may be a second substrate that may include one or more organic and or inorganic molecules applied to the contacting surface and to which the biologic material will be in contact.
  • solvent as used herein includes solutions of appropriate ionic strength to encourage high-density arrays or arrangements of the biologic material .
  • the arrays may be ordered or random.
  • the solvent (with or without external control) concentration may be such to promote liquid crystal formation of the biologic material.
  • the biologic material may be preincubated with the contacting surface and or with one or more organic or inorganic molecules. The preincubation may promote formation of particles in the nanometer scale. This preincubation may be further controlled by external conditions such as those described above.
  • the present invention provides cost-effective, long-term storage devices composed of soft mixed materials.
  • the present invention includes several effects not readily resolved in earlier work.
  • the dry thin film fabrication method requires few resources that are of minimal expense.
  • the films are easy to store at they require little space and are amenable to room temperature conditions, and therefore i's especially cost-effective.
  • the films require little effort to manufacture in large scale with little loss over time of activity, structure or other important properties.
  • thin film fabrication of the present invention is a high-capacity storage device.
  • the biofilm fabricated with bacteriophage can store over 4 x 10 13 viruses in a square centimeter of film.
  • the thickness of the thin film is not particularly limited but can be, for example, about 100 nm to about 100 microns, and more particularly about 500 nm to about 50 microns, and more particularly, about one micron to about 25 microns .
  • biologic materials such as peptides and bacteriophage can bind to semiconductor materials.
  • These biologic materials were developed into nucleating nanoparticles that may direct their self-assembly with an ability to recognize and bind other organic and inorganic materials with face specificity, to nucleate size-constrained crystalline semiconductor materials, and to control the crystallographic phase of nucleated nanoparticles (Lee S-W, Mao C, Flynn CE, Belcher AM. Ordering of Quantum Dots Using Genetically Engineered Viruses. 2002 Science 296 -.
  • biofilm This binding of a biologic material to a surface or thin substrate (e.g., semiconductor material) forming an equally thin layer of the biologic material is referred to as a biofilm.
  • a biofilm of the present invention may contain both organic and/or inorganic materials (or molecules) . It may comprise a substrate, an organic layer, a second organic layer, and an inorganic layer or various combinations thereof. Each organic layer may comprise one or more different types of biologic and/or organic materials; similarly, each inorganic layer may comprise one or more different type of inorganic materials.
  • the biofilm surface is well-ordered and may offer biologic, electrical, magnetic, and/or optical properties to the film enabling it to hold and store biologic, electrical, magnetic, and/or optical information.
  • biofilms have been defined as communities of biologic materials or microorganisms attached to a surface. Biofilm growth depends on the age of the biologic material or microorganism (e.g., culture), the build-up of potentially harmful (toxic) by-products or metabolites, and the consumption or use of other materials or nutrients for growth, stability or maintenance. Biofilms may be composed of natural or genetically engineered biologic materials. Of special interest is the use of biologic materials that self assemble. For example, bacteriophage that are genetically engineered to bind to other materials (e.g., semiconductor materials) also organize into well-ordered structures.
  • the self-assembling biological materials may be selected based on specific binding properties to particular surfaces and used to create well- ordered structures of the materials selected. These well- ordered structures may be further used to form layers and/or to support biologic, magnetic, optical, or electrical properties to the film.
  • the biofilm may serve as an information storage device or optical storage media for memory, either of which may be used to store and read bits of data—data that is biologic, magnetic, optical, electrical and combinations thereof.
  • the present invention becomes a biologic material storage device with specific alignment properties.
  • an M13 bacteriophage that has specific binding properties is used to create a biofilm storage device in one of three liquid crystalline phases, a directional order in the nemetic phase, a twisted nemetic structure in the cholesteric phase, and both directional and positional order in smectic phase.
  • the well-ordered biofilm storage device is, thus, created with biologic material alone or in combination with other organic or inorganic molecules (materials) to create, e.g., a type of thin film transistor.
  • a bacteriophage or any virus or other biologic material of interest
  • a bacteriophage is one type of natural "biopolymer” that can stick cohesively to itself and form a type of thin film surface.
  • biopolymers are those for which size and chemical composition may be controlled exactly, where one method of control is by genetic engineering. Controlled biopolymers offer precise known structure and composition. As a result, fabrication of the film using the controlled biopolymer may be specifically designed as needed.
  • Bacteriophage for example, are filamentous in shape (880 nm in length and 6.6 nm in width) with a surface covered by 2,700 copies of major protein units (known as pVIII) .
  • pVIII major protein units
  • the viral film was fabricated on the liquid/solid interfaces with gradient decrease of the liquid phase by evaporation of the solvent in a dessicator. As the solvent is gradually removed, the phage particles formed epitaxial layer domains on the surface of a solid substrate.
  • Polarized optical micrograph (POM) data of the phage layer that was formed showed in approximately 34 ⁇ m repeating patterns that continued to the centimeter scale.
  • FIGURE IB and C show the POM image of the viral film and AFM image of the individual phage particles, respectively.
  • Figure ID shows an AFM image of the structure of the ordered viruses when assembled into a film.
  • phage particles form an approximately 500 nm domain.
  • the phage particles are laterally stacked on each other. These lateral stacks form micro-domains that are packed to form a lamellar-like layer in the bulk film (see FIGURE ID) . Sequences obtained from these particles are shown in TABLE 1.
  • the viral film preserves the original phage library in its entirety without losing its ability to infect. This is illustrated by resuspending the viral film and using it to biologically pan (biopan) for the streptavin target—a target known to have specific binding motifs, such as His-Pro-Gln. After the second round of sequencing the results show that the His-Pro-Gin sequence appears at the end of the pill units. After the fourth round screening, all peptide sequences are found to exhibit the consensus sequence, His-Pro-Gin.
  • the time-to-infection (time-dependent infecting ability) of the dried phage in film is discussed below.
  • Ten small-size films were fabricated to compare the time dependent titer numbers. In the comparison, 1 ⁇ L of the above-described suspension was dried on the sterilized surface of an eppendorff tube in a dessicator for about one day. Titer numbers for each film were measured after suspending each 1 ⁇ L film in 1 mL TBS buffer solution (pH 7.5) on a different day over a five-month period. The titer numbers were measured and showed little change for at least seven weeks (FIGURE 2) .
  • the titer number decreased to 10% as compared to the number obtained from a one-day-old film suspension. Elongation and/or optimized infection times may be readily maximized for any biofilm without undue experimentation to those of ordinary skilled in the art.
  • the biopanning results including the continued ability of dried phage on film to infect, show that the film fabrication method is a highly efficient storage device of molecular information.
  • the film readily stores high-density engineered DNA and protein information over an extended period of time.
  • the viral components may be replicated easily at any time.
  • the biofilm may serve to functionalize one or more different types of virus and/or its components and may also be used to express a particular protein or protein unit.
  • the medicinal applications of this technique are extensive as the biofilms can be used in a number of therapeutic avenues including drug discovery, high throughput screening, diagnosis one or more pathologic conditions, and for optimizing disease therapies .
  • Phage film (FIGURE 1A) was fractured at or about a dimension of 1 cm x 1 cm and suspended in 1 mL of TBS buffered solution. The suspension
  • the integrity of the dry thin film of phage is extremely high.
  • the thin film stores at least 4 x 10 13 phage per square centimeter.
  • the number of protein units that may be stored is greater than 7200 times 4 x 10 13 phage.
  • the dry film fabrication method presents an inexpensive and optimal way to store extremely large volumes of biologic material, such as DNA, peptides and proteins, as examples, in a highly organized manner over long periods of time .
  • an engineered viral library may be created, preserved, and reused by fabricating a dry thin film.
  • a genetically engineered M13 phage library was made in a film form from highly concentrated suspension. When the biofilm was suspended again in an appropriate solution, M13 phage remain active and were able to infect a bacterial host.
  • a specific biologic material is preserved, stable, and still active in film form. The biofilm remains stable for more than seven months and retains its activity as shown by its ability to be greater than 95% infectious for at least 5 months.
  • biopanning results indicate that most of the 10 9 phage library information was preserved on the film.
  • fabrication of the biofilm is a reversible process with a readily useable application for the storage of high-density engineered molecular information (e.g, DNA, peptide or protein) .
  • three-dimensional memory may be formed that has up to three spatial dimensions.
  • Multiple bit information may be "read” (output) as data that is biologic, optical (such as color wavelengths) , magnetic, or electrical depending on the characteristics of the biologic material and or the inorganic compound or nanoparticles in combination with the biologic material.
  • Data is also "written” (input) to the biofilm by creating a chemical, optical, magnetic, or electrical reaction at a specific (e.g., nanoparticle) location.
  • one or more phage additives may be designed to create a film with very specific binding and or sequence patterns.
  • the resulting film serves as a storage device for input and output of information (as bits of data) with unique optical, electrical, and/or magnetic properties, as further described below.
  • information bits of data
  • the biological material is porous, such as in a hydrogel state, for example, reading and writing can be carried out with dissolved labels.
  • Engineered biologic materials such as viruses or bacteriophage (phage) are often able to recognize one or more specific contacting surfaces that help order their appearance on the contacting surface. For bacteriophage, for example, this is through the selection of combinatorial phage display.
  • the contacting surface recognition results in the ordering of the phage into a self-supporting biofilm that may or may not contain additional inorganic molecules or nanoparticles such as zinc sulfide (ZnS) .
  • ZnS zinc sulfide
  • the presence of the nanoparticles offers additional advantages that help the phage alignment to be magnetically and electrically controlled.
  • Phage recognition of a substrate's contacting surface may also be controlled by precoating the substrate with a second biologic material such as a peptide recognition moiety.
  • a precoated substrate is, for example, a semiconductor surface precoated with an additional compound such as indium tin oxide (ITO) .
  • ITO indium tin oxide
  • This additional compound may or may not be inorganic.
  • some substrates e.g., glass
  • an organic compound e.g., a conducting polymer
  • an external control e.g., electric and or magnetic field
  • an external control may also be used to encourage the ordered alignment of biologic material and to create a highly uniform biofilm, where uniformity includes a nonrandom ordering the biologic material on the contacting surface (or substrate) .
  • the present invention has been used to demonstrate that such biofilms of the present invention may be stored for more than six months without loss of stability, activity or ability of phage to infect a host. Further examples of the process involved in ordering the biologic material are described below, including examples of methods used to prepare the biologic material .
  • Phage-display Library One method of providing a random organic layer is using a Phage-display library, based on a combinatorial library of random peptides containing between 7 and 12 amino acids fused to the pill coat protein of M13 coliphage, provided different peptides were reacted with crystalline semiconductor structures. Five copies of the pill coat protein are located on one end of the phage particle, accounting for 10-16 nm of the particle.
  • the phage-display approach provided a physical linkage between the peptide substrate interaction and the DNA that encodes that interaction.
  • Protein sequences that bond successfully to the specific crystal were eluted from the surface, amplified by, e.g., a million-fold, and reacted against the substrate under more stringent conditions. This binding procedure was repeated five times to select the phage in the library with the most specific binding. After, e.g., the third, fourth and fifth rounds of phage selection, crystal-specific phage were isolated and their DNA sequenced. Peptide binding has been identified that is selective for the crystal composition (for example, binding to GaAs but not to Si) and crystalline face (for example, binding to GaAs (100), but not to GaAs (111) B).
  • FIGURE 3 The partial peptide sequences of the modified pill or pVIII protein are shown in FIGURE 3 (SEQ ID NO: 1-11) , revealing similar amino-acid sequences among peptides exposed to GaAs. With increasing number of exposures to a GaAs surface, the number of uncharged polar and Lewis-base functional groups increased. Phage clones from third, fourth and fifth round sequencing contained on average 30%, 40% and 44% polar functional groups, respectively, while the fraction of Lewis- base functional groups increased at the same time from 41% to 48% to 55%.
  • Lewis bases which should constitute only 34% of the functional groups in random 12-mer peptides from the library used, suggests that interactions between Lewis bases on the peptides and Lewis- acid sites on the GaAs surface may mediate the selective binding exhibited by these clones.
  • the expected structure of the modified 12-mers selected from the library may be an extended conformation, which seems likely for small peptides, making the peptide much longer than the unit cell (5.65 angstroms) of GaAs. Therefore, only small binding domains would be necessary for the peptide to recognize a GaAs crystal.
  • These short peptide domains highlighted in FIGURE 3, contain serine- and threonine-rich regions in addition to the presence of amine Lewis bases, such as asparagine and glutamine.
  • the surfaces were screened with shorter libraries, including 7-mer and disulphide constrained 7-mer libraries. Using these shorter libraries that reduce the size and flexibility of the binding domain, fewer peptide-surface interactions are allowed, yielding the expected increase in the strength of interactions between generations of selection.
  • Phage tagged with streptavidin-labeled 20 nm colloidal gold particles bound to the phage through a biotinylated antibody to the M13 coat protein
  • XPS X-ray photoelectron spectroscopy
  • Some GaAs clones also bound the surface of InP (100) , another zinc-blend structure.
  • the basis of the selective binding, whether it is chemical, structural or electronic, is still under investigation.
  • the presence of native oxide on the substrate surface may alter the selectivity of peptide binding.
  • the preferential binding of the Gl-3 clone to GaAs (100), over the (111) A (gallium terminated) or (lll)B (arsenic terminated) face of GaAs was demonstrated (FIGURES 4B and 4C) .
  • the Gl-3 clone surface concentration was greater on the (100) surface, which was used for its selection, than on the gallium-rich (111) A or arsenic-rich (lll)B surfaces.
  • the intensity of Ga 2p electrons against the binding energy from substrates that were exposed to the Gl-3 phage clone is plotted in FIGURE 4C.
  • the Ga 2p intensities observed on the GaAs (100), (111) A and (lll)B surfaces are inversely proportional to the gold concentrations.
  • the decrease in Ga 2p intensity on surfaces with higher gold-streptavidin concentrations was due to the increase in surface coverage by the phage.
  • XPS is a surface technique with a sampling depth of approximately 30 angstroms; therefore, as the thickness of the organic layer increases, the signal from the inorganic substrate decreases.
  • the Gl-3, G12-3 and G7-4 clones bound to GaAs (100) and InP (100) were imaged using atomic force microscopy (AFM) .
  • the InP crystal has a zinc-blende structure, isostructural with GaAs, although the In-P bond has greater ionic character than the GaAs bond.
  • the 10-nm width and 900-nm length of the observed phage in AFM matches the dimensions of the M13 phage observed by transmission electron microscopy (TEM) , and the gold spheres bound to M13 antibodies were observed bound to the phage (data not shown) .
  • the InP surface has a high concentration of phage.
  • the Gl-3 clone (negatively stained) is seen bound to a GaAs crystalline wafer in the TEM image (not shown) .
  • the data confirms that binding was directed by the modified pill protein of Gl-3, not through non-specific interactions with the major coat protein. Therefore, peptides of the present invention may be used to direct specific peptide-semiconductor interactions in assembling nanostructures and heterostructures (FIGURE 5E) .
  • X-ray fluorescence microscopy was used to demonstrate the preferential attachment of phage to a zinc-blended surface in close proximity to a surface of differing chemical and structural composition.
  • a nested square pattern was etched into a GaAs wafer; this pattern contained 1- ⁇ m lines of GaAs, and 4- ⁇ m Si0 2 spacing in between each line (FIGURES 5A and 5B) .
  • the G12-3 clones were interacted with the GaAs/Si0 2 patterned substrate, washed to reduce non-specific binding, and tagged with an immuno-fluorescent probe, tetramethyl rhodamine (TMR) .
  • TMR tetramethyl rhodamine
  • the tagged phage were found as the three red lines and the center dot, in FIGURE 5B, corresponding to G12-3 binding only to GaAs.
  • the Si0 2 regions of the pattern remain unbound by phage and are dark in color. This result was not observed on a control that was not exposed to phage, but was exposed to the primary antibody and TMR (FIGURE 5A) . The same result was obtained using non-phage bound G12-3 peptide.
  • the GaAs clone G12-3 was observed to be substrate- specific for GaAs over AlGaAs (FIGURE 5C) .
  • AlAs and GaAs have essentially identical lattice constraints at room temperature, 5.66 A° and 5.65 A°, respectively, and thus ternary alloys of AlxGal-xAs can be epitaxially grown on GaAs substrates.
  • GaAs and AlGaAs have zinc-blende crystal structures, but the G12-3 clone exhibited selectivity in binding only to GaAs.
  • a multilayer substrate was used, consisting of alternating layers of GaAs and of lo. 9 sGao. 02 s. The substrate material was cleaved and reacted subsequently with the G12-3 clone.
  • the G12-3 clones were labeled with 20-nm gold- streptavidin nanoparticles.
  • Examination by scanning electron microscopy (SEM) shows the alternating layers of GaAs and Al 0 . 98 Ga 0 .o 2 As within the heterostructure (FIGURE 5C) .
  • X-ray elemental analysis of gallium and aluminum was used to map the gold-streptavidin particles exclusively to the GaAs layers of the heterostructure, demonstrating the high degree of binding specificity for chemical composition.
  • FIGURE 5D a model is depicted for the discrimination of phage for semiconductor heterostructures, as seen in the fluorescence and SEM images (FIGURES 5A-C) .
  • the present invention demonstrates the power use of phage-display libraries to identify, develop and amplify binding between organic peptide sequences and inorganic semiconductor substrates.
  • This peptide recognition and specificity of inorganic crystals has been extended to other substrates, including GaN, ZnS, CdS, Fe 3 0 4r Fe 2 0 3 , CdSe, ZnSe and CaC0 3 using peptide libraries.
  • Bivalent synthetic peptides with two-component recognition (FIGURE 5E) are currently being designed; such peptides have the potential to direct nanoparticles to specific locations on a semiconductor structure.
  • FIGURES 6-10 Examples of specific amino acid sequences (SEQ ID NOS: 12-95) for peptide recognition of CdS (FIGURE 6-9), ZnS (FIGURE 8, 9), and PbS (FIGURE 9-10) crystals, especially after biopanning, are shown in FIGURES 6-10.
  • the phage display or peptide library was contacted with the semiconductor, or other, crystals in Tris-buffered saline (TBS) containing 0.1% TWEEN-20, to reduce phage-phage interactions on the surface. After rocking for 1 h at room temperature, the surfaces were washed with 10 exposures to Tris-buffered saline, pH 7.5, and increasing TWEEN-20 concentrations from 0.1% to 0.5% (v/v). The phage were eluted from the surface by the addition of glycine-HCl (pH 2.2) 10 minute, transferred to a fresh tube and then neutralized with Tris-HCl (pH 9.1) . The eluted phage were titered and binding efficiency was compared.
  • TBS Tris-buffered saline
  • the phage eluted after third-round substrate exposure were mixed with their Escherichia coli ER2537 host and plated on LB XGal/lPTG plates. Since the library phage were derived from the vector M13mpl9, which carries the lacZ ⁇ gene, phage plaques were blue in color when plated on media containing Xgal (5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside) and IPTG (isopropyl- ⁇ -D-thiogalactoside) . Blue/white screening was used to select phage plaques with the random peptide insert . Plaques were picked and DNA sequenced from these plates. Substrate Preparation .
  • Substrate orientations were confirmed by X-ray diffraction, and native oxides were removed by appropriate chemical specific etching.
  • the following etches were tested on GaAs and InP surfaces: NH 4 OH:H 2 0 (1:10), HC1:H 2 0 (1:10), H3P0 4 :H 2 ⁇ 2 :H 2 0 (3:1:50) at 1 minute and 10 minute each time.
  • the best element ratio and least oxide formation (using XPS) for GaAs and InP etched surfaces was achieved using HC1:H 2 0 for 1 minute followed by a deionized water rinse for 1 minute .
  • An ammonium hydroxide etch was used for GaAs in the initial screening of the library.
  • This etch may also be used for all other GaAs substrate examples, however, those of skill in the art will recognize etches may be used.
  • Si (100) wafers were etched in a solution of HF:H 2 0 (1:40) for one minute, followed by a deionized water rinse. The surfaces may be taken directly from the rinse solution and immediately introduced to the phage library. Surfaces of control substrates, not exposed to phage, were characterized and mapped for effectiveness of the etching process and morphology of surfaces by AFM and XPS.
  • Multilayer substrates of GaAs and of Al 0 . 98 Ga 0 .o2 As were grown by molecular beam epitaxy onto GaAs (100) .
  • the epitaxially grown layers were Si-doped (n-type) at a level of 5 x 10 17 cm "3 .
  • XPS X-ray Photoelectron Spectroscopy
  • the XPS instrument used was a Physical Electronics Phi ESCA 5700 with an aluminum anode producing monochromatic 1,487 -eV X-rays. All samples were introduced to the chamber immediately after gold-tagging the phage (as described above) to limit oxidation of the GaAs surfaces, and then pumped overnight at high vacuum to reduce sample outgassing in the XPS chamber .
  • Atomic Force Microscopy (AFM) .
  • the AFM used was a Digital Instruments Bioscope mounted on a Zeiss Axiovert 100s- 2tv, operating in tip scanning mode with a G scanner. The images were taken in air using tapping mode.
  • the AFM probes were etched silicon with 125-mm cantilevers and spring constants of 20+100 Nm "1 driven near their resonant frequency of 200+400 kHz. Scan rates were of the order of 1+5 mms "1 . Images were leveled using a first-order plane to remove sample tilt.
  • TEM Transmission Electron Microscopy
  • SEM Scanning Electron Microscopy
  • organic- inorganic hybrid materials (those materials that include both organic and inorganic compounds) offer new routes for novel material development. Size controlled structures in the nanoscale range (nanostructures) are especially useful in microeletronics and offer optical, magnetic, and electric- tunable properties to materials such as semiconductors.
  • the biologic material with its organic component may 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 extended lengths of time is critical to its success as a storage tool for information processing, gathering and analysis.
  • Fd virus smectic ordering structures that have both a positional and directional order have been characterized.
  • the smectic structure of Fd virus has potential application in both multi-scale and nanoscale ordering of structures to build 2 -dimensional (2D) and 3 -dimensional (3D) alignment of particles in the nanometer scale (herein referred to as nanoparticles) .
  • Bacteriophage M13 was used because it can be genetically modified, has been successfully selected to have a shape identical to the Fd virus, and has specific binding affinities for II-VI semiconductor surfaces. Therefore, M13 is an ideal source for smectic structure that can serve in multi -scale and nanoscale ordering of nanoparticles.
  • the present inventors have used combinatorial screening methods to find M13 bacteriophage containing peptide "inserts" that are capable of binding to semiconductor surf ces. These semiconductor surfaces included materials such as zinc sulfide, cadmium sulfide and iron sulfide.
  • semiconductor surfaces included materials such as zinc sulfide, cadmium sulfide and iron sulfide.
  • a biologic material such as bacteriophage combinatorial library clones that bind specific semiconductor material surfaces, are used.
  • biologic material is one that is readily available in large quantity or may be amplified readily for large-scale manufacturing.
  • the phage is amplified cloned and amplified up to concentrations high enough for liquid crystal formation.
  • the anisotrophic shape of bacteriophage was exploited as a method to build well-ordered nanoparticle layers by use of biological selectivity and self-assembly.
  • the filamentous bacteriophage, Fd has a long rod shape (length: 880 nm; diameter: 6 . 6 nm) and monodisperse molecular weight (molecular weight: 1.64 x 10 7 ) that results in the bacteriophage' s lyotropic liquid crystalline behavior in highly concentrated solutions.
  • M13 a similar filamentous bacteriophage, was genetically modified to bind nanoparticles such as zinc sulfide, cadmium sulfide and iron sulfide.
  • the monodisperse bacteriophage, M13 was prepared through standard amplification methods.
  • Nano- and Mesoscale Ordering The ordering of ' bacteriophage on the nano- and mesoscale level shows that the biologic material may form nanoscale arrays of nanoparticles. These nanoparticles are further organized into micron domains and into centimeter length scales.
  • the semiconductor nanoparticles show quantum confinement effects, and can be synthesized and ordered within the liquid crystal.
  • M13 bacteriophage that have specific binding properties to semiconductor surfaces were amplified and purified using standard molecular biological techniques.
  • the suspensions were prepared by adding Na 2 S solutions to ZnCl 2 doped A7 phage suspensions at room temperature.
  • the highest concentration of A7-phage suspension was prepared by adding 20 ⁇ L of 1 mM ZnCl 2 and Na 2 S solutions, respectively into the ⁇ 30 mg of phage pellet. The concentration was measured using extinction coefficient of 3.84 mg/mL at 269 nm.
  • nemetic phase that has directional order
  • cholesteric phase that has twisted nemetic structure
  • smectic phase that has directional and positional orders as well
  • FIGURES 11A and 11B are schematic diagrams of the smectic alignment of M13 phages observed using AFM. Additionally, 5 ⁇ L of M13 suspension (concentration: 30 mg/mL) of M13 bacteriophage suspension was dried for 24 hours on the 8 mm x 8 mm mica substrate that was silated by 3-amino propyl triethyl silane for 4 hours in the dessicator. Images were taken in air using tapping mode. Self-assembled ordering structures were observed due to the anisotropic shape of M13 bacteriophage, 880 nm in length and 6.6 nm in width. In FIGURE 12C, M13 phage lie in the plane of the photo and form smectic alignment .
  • TEM Transmission Electron Microscopy
  • SEM Scanning Electron Microscopy
  • Ml3 phage suspensions were characterized by POM. Each suspension was filled to glass capillary tube of 0.7 mm diameter. The highly concentrated suspension (127 mg/mL) exhibited iridescent color [5] under the paralleled polarized light and showed smectic texture under the cross-polarized light as FIGURE 12A.
  • the cholesteric pitches, FIGURE 12B can be controlled by varying the concentration of suspension as shown in TABLE 3. The pitch length was measured and the micrographs were taken 24 hours later from the preparation of samples.
  • Nanoscale bacteriophage alignment of the A7-ZnS film were observed using SEM.
  • the film was cut then coated via vacuum deposition with 2 nm of chromium in an argon atmosphere. Highly close-packed structures were observed throughout the sample (see FIGURE 13D) .
  • the average length of individual phage, 895 nm is reasonable analogous to that of phage, 880 nm.
  • the film showed the smectic like A or C like lamellar morphologies that exhibited periodicity between the nanoparticle and bacteriophage layers .
  • the length of periodicity corresponded to that of the bacteriophage.
  • the average size of nanoparticle is -20 nm analogous to the TEM observation of individual particles.
  • each A7 recognize one nanoparticle (2 ⁇ 3 nm size) and aligned approximately 20 nm in a width and extended to more than two micrometers in length.
  • the two micrometers by 20 nm bands formed in parallel each band separated by -700 nm. This discrepancy may come from the tilted smectic alignment of the phage layers with respect to observation in the TEM.
  • a y-z axis like nanoparticle layer plane was also observed like FIGURE 5F.
  • the SAED patterns of the aligned particles showed that the ZnS particles have the wurzite hexagonal structure .
  • FIGURE 5C the phage were shown to have formed an parallel aligned herringbone pattern that have almost right angle between the adjacent director normal (bacteriophage axis) on most of surface that is named as smectic O.
  • the film showed long range ordering of normal director that is persistent to the tens of micrometers. In some of areas where two domain layers meet each other, two or three multi-length scale of bacteriophage aligned paralleled and persistent to the smectic C ordering structure .
  • Nano and multi-length scale alignment of semiconductor materials using the recognition and self-ordering method and the composition of the present invention enhances the future microfabrication of electronic devices. These devices have the potential to surpass current photolithographic capabilities. Other potential applications of these materials include optoelectronic devices such as light-emitting displays, optical detectors, and lasers, fast interconnects, nano-meter scale computer component and biological sensors.
  • the biofilm storage device of the present invention may be used to store biologic (e.g., organic) materials such as enzymes and antibodies.
  • biologic molecules such as enzymes that retain their biologic activity are stored as a biofilm. The activity is readily monitored over time based on the known properties of the enzyme.
  • ⁇ -galactosidase a reporter enzyme, is prepared in a biofilm and found to retain long-term enzyme stability and activity.
  • storage solutions e.g., sucrose
  • biologic material e.g., enzyme
  • a biologic material used as a biofilm storage device In order to visualize the structure and function of a biologic material used as a biofilm storage device, light properties of the biologic material or light -emitting molecules that attach to the biologic material may be monitored.
  • a green fluorescent protein variant (GFPuv) that emits green light at a maximum emission wavelength of 509nm may be used to attach to the biologic material (e.g., enzyme or antibody).
  • the light emitting properties may be imaged using instruments well known to one of ordinary skill in the art of biologic imaging.
  • an imaging instrument is confocal microscopy.
  • a biologic material used as a storage device is allowed to contact another biologic material .
  • Either biologic material may be modified in whole or in part to customize the biofilm as needed.
  • biofilms including a biologic material such as bacteriophage, may be modified by changing the proteins displayed at the biologic material (e.g., baceriophage) surface or by targeting peptides that specifically attach to the biologic material and may also attach to another target (e.g., biologic material such as protein, antibody, drug, or nucleic acid) or other stabilizer that result in enhanced stability of the biofilm storage device .
  • Storage temperature can be, for example, about room temperature.
  • Storage temperature can be, for example, about 10°C to about 40°C, and more particularly, about 20°C to about 30°C. These storage temperatures can be maintained for any length of time including at least 7 weeks, at least 5 months, at least 6 months, or at least 7 months.
  • ⁇ -galactosidase in phosphate buffered saline (PBS) solution (pH 7.0) was mixed with stock solutions of glucose, sucrose, and M13 phage to obtain concentrations of 0.5 mg/mL ⁇ -galactosidase, 5 mg/mL glucose, 50 mg/mL sucrose, and 1.25 mg/mL phage. Aliquots (20 ⁇ L) of the solution were placed in 1.5 mL Eppendorf tubes, dried in a dessicator for two days, and stored at room temperature. The dried viral films were suspended in 500 ⁇ L of PBS solution (pH 7.0) .
  • PBS phosphate buffered saline
  • o-nitrophenyl galactoside (1.5xl0 -2 M) were combined in a disposable cuvette.
  • the enzyme activities were obtained by monitoring an increase of absorbance of o-nitrophenol (ONP) at 420 nm for 10 minutes with 30 seconds interval.
  • One unit of activity was defined as the amount of enzyme that can catalyze the transformation of 1 ⁇ mol of ONPG into ONP in 1 minute at 25 degrees Centrigrade (pH 7.0) .
  • At least about 10 ⁇ L of the mixture was dispensed on a glass slide and dried in a desiccator for a day.
  • GFPuv-FLAG stability was monitored using confocal fluorescence microscopy. Concentrations of glucose : sucrose were 2.5 and 25 mg/mL.
  • Biofilm storage devices containing ⁇ -galactosidase and stored after freeze-drying or air-drying showed similar enzyme activity.
  • enzyme activity was also improved in the presence of another biologic materials (e.g., bacteriophage) as well as in the presence of a stabilizer (i.e., storage solution).
  • FIGURE 15 illustrates the confocal microscopy images with GFPuv after excitation at 361 nm.
  • the images illustrate that the addition of a stabilizer such as a glucose .-sucrose storage solution improves the biofilm surface and prevents potential deformation of the biologic material during the fabrication (preparation) process.
  • FIGURE 15A shows a strong GFPuv signal and homogenous biofilm surface . In the absence a glucose: sucrose storage solution, the film exhibits numerous deformations at the film surface (FIGURES 15B and 15C) .
  • the biological material when the biological material comprises multiple display sites, the biological material can be genetically engineered so that one or more of these display sites is modified.
  • the M13 bacteriophage can be modified at the pill, P7, p8, or p9 sites to include specific binding peptides.
  • one end of a biological material can be modified to bind specifically to a surface, and the other end of the biological molecule can be modified to bind to a component which is being stored with a goal of stable storage such as a vaccine or a functional protein.
  • the present invention is thus able to store biologically active biologic materials with activity that persists throughout the storage interval .
  • biologic and/or other active properties of the biofilm may be readily manipulated as needed.
  • Activity can be further modified without undue experimentation by changing the biologic surface via altering surface binding properties, through the addition of storage stabilizers and or inhibitors, and by the addition of other organic or inorganic molecules.
  • Storage solutions that stabilize the biologic material include sugar-containing solutions such as glucose, sucrose, glycol, glycerol, polyethylene glycol.
  • the present invention improves biofilm technology by fabricating stable films composed of biologic materials (including one or more organic and or inorganic materials) that may undergo long-term storage while retaining the original information and/or activity.
  • biologic materials including one or more organic and or inorganic materials
  • Engineered materials may be used to fabricate ordered films (biofilms) with long- term activity and stability that hold and store information that is biologic, electric, magnetic, and/or optical. More importantly, the information may be tailored and of extremely high density, thereby serving as an efficient and cost- effective method of storing nanoscale data.
  • the use of these biofilms extends into applications such as medicine, electronics, computer technology and optics, as examples.
  • nano- and multi-length scale alignment of semiconductor materials was achieved using the recognition and self-ordering system described herein.
  • the recognition and self-ordering of semiconductors may be used to enhance micro fabrication of electronic devices that surpass current photolithographic capabilities.
  • Application of these materials include optoelectronic devices such as light emitting displays; optical detectors and lasers; fast interconnects; and nano-meter scale computer components and biological sensors.
  • Other uses of the biofilms created using the present invention include well-ordered liquid crystal displays, organic-inorganic display technology, and films for high-throughput processing, screening and drug discovery, devices for diagnosis, medical testing and analysis; implant surfaces for data storage and specific data recognition, as examples .
  • the films, fibers and other structures developed from the biofilm of the present invention may even include high-density sensors for detection of small molecules including biological toxins.
  • Other uses include optical coatings and optical switches.
  • scaffoldings for medical implants or even bone implants may be constructed using one or more of the materials disclosed herein, in single or multiple layers or even in striations or combinations of any of these, as will be apparent to those of skill in the art .
  • Other uses for the present invention include electrical and magnetic interfaces, or even the organization of 3D electronic nanostructures for high-density storage, e.g., for use in quantum computing.
  • variable-density and stable storage of viruses for medical application may be created with the films and or matrices created with the present invention.
  • Information storage based on quantum dot patterns for identification e.g., department of defense friend or foe identification, may be incorporated in fabric of armor or coding.
  • the present biofilms may even be used to code and identify money.
  • Other applications include drug delivery, including systems such as, for example, Depomed with layered film assemblies in drug capsules; medical device coatings; and controlled release applications such as, for example, breath mints .
  • Embodiment A includes a set of cited references
  • embodiment B includes a set of cited references.
  • M13 phage viruses
  • FAM atomic force microscopy
  • SEM scanning electron microscopy
  • the viral films were determined to have a chiral smectic C structure.
  • ordering of film formation as a function of virus concentration and the formation of bundle-like domain structure found in viral thin films a mechanism of film formation can be suggested.
  • These virus based film structures are organized on multiple length scales, easily fabricated, and allow integration of aligned semiconductor and magnetic nanocrystals .
  • These self-assembled hybrid materials can be used in, for example, in miniaturized self-assembled electronic devices.
  • M13 phage were prepared using standard biological methods of amplification and purification described previously. 5 Twelve different concentrations of M13 phage (800 ⁇ l each) were prepared as shown in table 1. After transferring to ependorff tubes (1 cm in diameter and 4 cm in length) , the suspensions were allowed to dry in a dessicator for three weeks (weight loss in the drying process: ⁇ 100 mg per day) . Cast films were formed on the wall of the ependorff tubes as the solvent evaporated.
  • Polarized optical microscopy POM images were obtained using Olympus polarized optical microscope. Micrographs were taken using SPOT Digital camera (Diagnostic Inc.) . The optical activity was also observed by changing the angles between the polarizer and analyzer. The polarized optical microscope was used to measure the chiral smectic C spacing patterns. Scanning electron microscopy:
  • a scanning electron microscope (LEO1530) was used to observe the surface morphologies of the viral films.
  • the viral films were coated with chromium using a plasma ion beam sputtering machine.
  • the sample holder was tilted -80 degrees from the horizontal plane and mounted to the SEM sample stage.
  • Atomic force microscope (AFM) (Digital Instruments) was used to study the surface morphologies of the viral film. The images were taken in air using tapping mode. The AFM probes were etched silicon with 125 ⁇ m cantilevers and spring constants of 20-100 N/m driven near their resonant frequency of 250-350 kHz. Scan rates were of the order of l-40 ⁇ m /s. Laser light diffraction:
  • Laser beam diffraction He-Ni laser :632.8 nm was used to measure a chiral smectic C pitch of the viral film. The distance between the screen and sample was 200 cm. The diffraction pattern was recorded by Sony Mavica digital camera. Spacing was calculated by measuring the first order Bragg diffraction spot.
  • POM images of the viral film formed from the initial concentration 9.93 mg/ml revealed optically active dark and bright band patterns ( Figure 17A) .
  • Periodic spacing of these patterns was 36.79 +_ 0.95 ⁇ m and the patterns were continued over the centimeter-scale .
  • parallel band patterns smaller than 1 ⁇ m were also observed. These fine band patterns corresponded to the smectic layer structure of M13 virus molecules.
  • the film was determined to be optically active as evident by the change in intensity of the alternating dark and bright band pattern as the angles between the polarizers were rotated.
  • optically active dark and bright band patterns are consistent with a chiral smectic C structure for the viral films.
  • the molecular long axis directory: n
  • These tilted layers form a helical rotation (azimuth angle : ⁇ ) from one layer to next layer, which is depicted in Figure 16B. 17 Therefore, the continuous helical change of the orientational orders through the tilted smectic layers cause different interaction with plane polarized light, and exhibit the optically active band patterns.
  • Reflected polarized optical microscopy (RPOM) of the viral film give similar optically active dark and bright band patterns depending on the angles between the polarizer and analyzer. These RPOM images indicate the presence of dechiralization line defects 17,35 on the surface.
  • the dechiralization line defects arising from the interaction between helically ordered bulk structures and surface effects. Due to the surface effect, the helicoidal ordered chiral smectic C structures are unwound near the surface and result in bright and dark band patterns which correspond to the periodic pitch of chiral smectic C structures.
  • the dechiralization line defects of the viral film were characterized using scanning electron microscope (SEM) ( Figure 17B) .
  • SEM scanning electron microscope
  • Figure 17B Zig-zag patterned long-range ordered structures were observed, which corresponded to the dark and bright band patterns in RPOM.
  • the alternating zig-zag band patterns (-37 ⁇ m) showed periodic +45 degrees and -45 degrees changes with respect to the layer normal .
  • the periodic spacing of the zigzag patterns was consistent with the periodic POM and RPOM patterns.
  • the zig-zag type morphologies of the viral film might be induced from surface defects of chiral smectic C structure of the viral film.
  • the chiral smectic C structure has two ordering parameters, a tilted angle (0) with respect to the layer normal and an azimuth angle ( ⁇ ) with respect to a layered plane. 17 If the helicoidal pitch direction of the chiral smectic C layer is parallel with respect to the layer plane, the azimuth angle ( ⁇ ) of the director can be projected to the layered plane. 18 Due to additional higher ordering properties on the surface, the tilted angle (0) on the surface might have higher angles than the sum of the tilted angles and the projected azimuth angle. 19 Therefore, the 180 degrees phase difference of the azimuth angle ( ⁇ ) is projected into the long-range periodic zig-zag patterns like Figure 16C and 17B.
  • Tilted smectic C morphologies on the free surface of the viral films were characterized using AFM ( Figure 17C) .
  • the M13 virus particles made a tilted layer structure that had an average spacing of 620+27 nm.
  • the molecular long-axis of the virus particles were tilted -45 degrees with respect to the layer normal (z) .
  • the number of layers in a chiral smectic C pitch can be estimated to 59.3 layers. Because the azimuth angle changes 360 degrees in a pitch, it can be estimated from the number of the layers in a pitch
  • the azimuth angle ⁇ ) from the viral film sample 1 was -6 degrees.
  • the viral film characteristics which were fabricated from concentration range 6.38-9.70 mg/ml (sample 2-7), were similar to the viral film (sample 1) fabricated from 9.93mg/ml described above.
  • the pitch length gradually decreased from 9.93 to 7.60 mg/ml and increased until 5.09 mg/ml.
  • the smectic C structure made a transition to smectic A structure.
  • a similar expansion of the pitch near the transition point was also observed from the cholesteric phase transition to the smectic phase. 22 Therefore, the chiral smectic C spacing expansion might be involved with a pre-transition phenomena. All of the films showed clear diffraction patterns which were consistent with periodic patterns in POM (table. 2) . Structure transition:
  • POM band patterns were observed from the viral film fabricated from a concentration of 5.09 mg/ml (sample 7) .
  • POM images of sample 7 exhibited periodic vertical bright band patterns which were divided by schlieren stripe lines when the dark lines were parallel with respect to the polarizers.
  • the analyzer angle changed by around five degrees
  • the POM texture intensity changed to slightly darker and brighter stripe patterns similar to the chiral smectic C viral film.
  • the film also exhibited zig-zag patterned lines through the band patterns.
  • the periodicity of these vertical periodic patterns was 97.43 + 2.92 ⁇ m.
  • the low magnification SEM image ( Figure 19C) from sample 10 showed that the film had regularly occurring periodic chevron-like cracked patterns.
  • the higher magnification SEM image (inset of Figure 19C) of these cracked pattern showed that their directions were parallel with respect to the orientation of the directors. Between the interfaces of zigzag patterns, the disclination lines were observed to correspond to the dark vertical schlieren line patterns in the POM images ( Figure 19C) .
  • smectic A ordered structures were observed in the same region ( Figure 19D) .
  • the viral particles formed -980 x 800 nm domain blocks.
  • the virus particle packing pattern was close to the smectic B structure in which molecules are arranged in layers with the molecular center positioned in a hexagonal close-packed array. These domain blocks formed the parallel - aligned and bookshelf-like smectic A structures on the surface.
  • the average spacing between the two layers measured was 977 +25 nm which is slightly larger than the length of M13 virus .
  • POM image of sample 11 showed the disordered schlieren texture lines (Figure 20A) .
  • Crooked black brush line patterns propagated irregularly within 20-30 micrometer domains. The dark and bright patterns were divided by the crooked black brush lines. Both the dark brush lines and the brightness of the patterns were changed by rotating the film indicating that these brush lines were disclination lines.
  • AFM images of these areas showed the nematic ordered structures of smectic A bundle-like domains (-980 nm x 200 nm) ( Figure 20B) . Each smectic A domain formed nematic like ordered structures which oriented through the molecular long axis as the preferred direction.
  • POM images of the viral film showed optically active dark and bright stripe patterns.
  • SEM images showed the dechiralization defects of chiral smectic C structures.
  • AFM images showed the tilted smectic C ordered structures. Based on these microscopic evidences, it was concluded that the viral films have the chiral smectic C structure.
  • Thickness effects of the chiral smectic C structure of the viral film was also observed.
  • the thickness of the film decreases to - 4.3 ⁇ m, which had - 360 viral particle layers (particle to particle distance: 12 nm) 5 , the surface effect seemed to be dominant throughout the bulk film. Therefore, the chiral smectic C structure made a transition to a smectic A like ordered structure.
  • the orientation of the molecular long-axis was almost perpendicular with respect to the smectic layers.
  • the zig-zag like periodic patterns were still observed.
  • the formation of the vertical zig-zag patterns as observed from sample 7 to sample 10 might come from both the helical structure of the bulk and thickness of the film.
  • the mechanism for the self-ordered virus film formation is still under investigation.
  • the nematic ordered structures which showed the disordered smectic A domains, strongly suggested the formation of bundle-like domains in solution prior to the film formation.
  • the isotropic phase of the viral suspension in the meniscus areas slowly made a transition to the nematic phase.
  • viral particles that have the same orientational order began to make bundlelike domain structures.
  • These domain structures are still flexible to modification of their packing structure.
  • These domains initially become the basic building units of the layered structures. After forming layers, these smectic A domains become close-packed as the solvent evaporates. Complete evaporation of the solvent forms the bulk structures of the viral film.
  • the thickness of the virus film has a critical effect on both the bulk and surface structure.
  • the morphologies of the ZnS nanocrystal virus hybrid films were previously reported 5 .
  • the ZnS nanocrystal hybrid viral films have the optically active ⁇ 72 ⁇ m periodic dark and bright stripe POM patterns which were similar to that of 100% M13 virus films.
  • the surface morphologies of the ZnS nanocrystal hybrid viral films have anti-smectic C structures (smectic 0) , which appear in a zig-zag pattern that have -l.O ⁇ m spacing through the layer normal direction. Based on the POM pitch and AFM zig-zag layer spacing, the ZnS nanocrystal hybrid viral films have -72 layers in a pitch and -5 degrees in azimuth angle.
  • the ZnS nanocrystal hybrid viral films have chiral smectic C structures which are composed of interdigitated domains of M13 viruses bound to 20 nm ZnS nanocrystal aggregates.
  • the interdigitated domains can reduce the packing energies of the big head shape of the ZnS nanocrystal hybrid viral films.
  • the anti -smectic C structure was generally only observed on the surface of the film and generally believed to be a surface effect .
  • Using external force such as a magnetic field or an electric field can aid, for example, in building defect free and well ordered miniaturized electronic devices using these genetically engineered virus based films after hybridization of the viruses with semiconductor or magnetic nanocrystals .
  • Homeotropic-aligned magnetic nanocrystals hybrid virus thin films can be used, for example, for self-supporting, flexible, and highly integrated magnetic memory devices.
  • Additional materials were prepared which can be used as films in storage applications, as well as other applications.
  • a new platform is presented for organization of a variety of materials including inorganic nanoparticles, small organic molecules and large biomolecules that organize and self-assemble at the nanometer length scale and are continuous into the centimeter length scale.
  • Long- range ordered nano-sized materials (10 nm gold nanoparticles, fluorescein, phycoerythrin protein) were fabricated using a streptavidin linker and anti-streptavidin M13 bacteriophage (virus) .
  • the anti -streptavidin viruses which formed the basis of the self-ordering system, were selected to have a specific recognition moiety for streptavidin using phage display.
  • the nano-sized materials were previously bound to streptavidin. Through the molecular recognition of the genetically selected virus, the nano-size materials were bound and spontaneously evolved into a self-supporting hybrid film. Functionalized liquid crystalline materials can provide various pathways to build well-ordered and well -controlled two and three-dimensional structures for the construction of next generation optical, electronic and magnetic materials and devices . [1"3] It has been demonstrated that several types of rod-shape viruses form well controlled liquid crystalline phases. 14,51 Recently, a self-assembled ordered nanocrystal film fabrication method was reported using nanocrystal- functionalized M13 virus. C3] Through the utilization of genetic engineering techniques, one-end of the M13 virus was functionalized to nucleate or bind to a desired semiconductor material.
  • nanocrystal-functionalized viral liquid crystalline building blocks were grown into ordered hybrid self-supporting films.
  • the resulting nanocrystal hybrid film was ordered at the nanoscale and at the micrometer scale into 72 ⁇ m periodic striped pattern domains.
  • biological selection and further evolution are required for each material prior to aligning the nanoparticles.
  • a novel nanoparticle alignment method is reported using anti-streptavidin viruses, where the virus was first selected to bind streptavidin protein units.
  • the organized hybrid materials presented here are liquid crystalline films of gold nanoparticles, fluorescent molecules
  • fluorescein and large fluorescent proteins (phycoerythrin) .
  • the anti-streptavidin M13 viruses having specific binding moieties for the streptavidin were isolated through the screening of a phage display library (Fig. 21) .
  • Streptavidin has the known specific binding motif His-Pro- Gln.
  • Cs3 His-Pro-Gln sequences were isolated as pill inserts after second round selection of phage for the streptavidin target.
  • His-Pro-Gln binding motif made up 100 % of the pill insert after fourth round selection and sequencing.
  • the dominant binding sequence after the fourth round was TRP ASP PRO TYR SER HIS LEU LEU GLN HIS PRO GLN.
  • This anti- streptavidin M13 virus was amplified to high concentration
  • the individual mesogen units of 10 nm gold nanoparticles bound viruses were visualized using transmission electron microscopy (TEM) prior to staining with 2 % uranyl acetate.
  • TEM transmission electron microscopy
  • Au-virus Au-virus
  • Fig. 22C 0.01 % dilution of the smectic phase suspension
  • Fig. 22D aggregation of Au-virus complex
  • Most mesogen units observed had one virus bound to one 10 nm Au particle at the pill end of virus.
  • both unbound gold nanoparticles and unbound viruses were observed in less than 20 % of mesogen units.
  • Smectic ordered self-supporting Au-virus films (Fig. 23A) were prepared from a dilute Au-virus solution (-6 mg/ml) .
  • the viruses and nanocrystals were agitated for one week prior to the fabrication of the film.
  • the suspension was kept dry in a dessicator for two weeks.
  • the viral nanocrystal hybrid film was slightly pink in color and transparent.
  • the ordered morphologies of the viral film were characterized by POM, scanning electron microscopy (SEM) and atomic force microscopy (AFM) .
  • the thickness of the film was 5.68 + 0.65 ⁇ m.
  • Optical characterization revealed that the films were composed of -10 - ⁇ m dark-grey periodic horizontal striped patterns (Fig. 23B) . These stripes were optically active and changed their bright and dark patterns depending on the angles between a polarizer and an analyzer. These striped patterned POM characteristics are similar to the smectic virus films that were previously reported by our group. [9]
  • the average tilted angle was -54 degrees with respect to the layer normal .
  • the length of the M13 virus is 880 nm. This -100 nm longer spacing observed through the molecular long axis is strong evidence to support an interdigitated structure .
  • the shape of mesogen unit which has a big head (inorganic gold nanoparticle) with a long tail (organic M13 virus) might have lower packing free energy by forming interdigitated structures.
  • the -10 ⁇ m periodic zig-zag patterns observed in POM and SEM images highly indicated that the Au-virus hybrid films also have chiral smectic C structure in the bulk and dechiralization defects on the surface of the hybrid films.
  • Two kinds of organic materials were also fabricated in virus films.
  • the organic materials were chosen to show that this technique is versatile but these materials also allow easy visualization of the approximately one micrometer periodic long ranged ordering because they are fluorescent.
  • Thin cast films of virus bound fluorescein and phycoerythrin were fabricated using streptavidin and anti-streptavidin M13 viruses. Due to the enhanced ordered properties of liquid crystalline materials near the surface and capillary driving force during the drying process, the smectic layer structure was easily observed from drop-cast thin films of fluorescein complex viruses (F-virus) and phycoerythrin complex viruses (P-virus) (Fig. 23E) . The ordering of these liquid crystalline hybrid materials were enhanced by casting thin films of these materials.
  • nematic liquid crystalline materials formed surface stabilized smectic phase due to the surface effects [11] and chiral smectic C structures transitioned into smectic A structures C9] in thin films.
  • Scanning laser microscopy was used to optically section the F- virus thin films (Fig. 23F) . These thin films showed weak stripe patterns which corresponded to a smectic structure.
  • Applying similar analysis to the thin film of fluorescent P- viruses (Fig. 23G) very clear one micrometer stripe patterns were observed. These one micrometer fluorescence patterns indicated that the fluorescent molecules (fluorescein and phycoerythrin) were bound to the viruses by the linkage of streptavidin, then formed the smectic layer structures. Because the fluorescent materials were imposed at the end of the virus, their position was localized between the smectic layer interface boundaries.
  • anti-streptavidin M13 viruses were used to self-assemble various nano-sized materials.
  • the anti- streptavidin M13 viruses provide a convenient method to organize a variety of nano-sized materials into self-assembled ordered structures. Because the modification of the DNA insert allows for controlled modification of the virus length, the spacing in the smectic layer can be genetically controlled.
  • Cl2 By conjugating other nano-sized materials (magnetic nanoparticles, II -VI semiconductor nanoparticles, functional chemicals, etc) with streptavidin, this anti-streptavidin method can align various nano-sized materials at the desired length scale which is defined by the smectic layers.
  • the anti-streptavidin virus was selected by a phage display method using a M13 bacteriophage library (New England Biolab) .
  • the virus was amplified in a large volume (400 ml scale, 7x 10 7 pfu) .
  • the virus suspension was precipitated into a pellet.
  • 20 mg of the virus pellet was suspended with 1.0 ml of 10 nm gold nanoparticle (Abs: 2.5 at 520 nm) , conjugated with a streptavidin colloidal suspension (Sigma Co . ) , and agitated using a rocker for one day.
  • the viruses conjugated with gold nanoparticles (Au-virus) were centrifuged after adding 167 ⁇ l of poly ethylene glycol solution.
  • the red colored pellet was suspended using -20 ⁇ l of tris-buffered saline solution (pH 7.5) to form Au-virus liquid crystalline suspension (virus concentration: 83.2 mg/ml) .
  • the Au-virus suspension was diluted to - 6 mg/ml (400 ⁇ l) and kept dry in a dessicator for two weeks .

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Abstract

La présente invention comprend un procédé et une composition de stockage et de conservation de biofilms pour l'entrée et la sortie d'informations à haute densité. Une forme de la présente invention est un dispositif de stockage de biofilms fabriqués à l'aide d'une matière biologique appliquée sur un substrat pour former, par exemple, un film mince sec stable à température ambiante pendant des périodes prolongées. Une autre forme de la présente invention est un procédé de fabrication d'un dispositif de stockage de biofilms dans lequel une matière biologique est appliquée sur un substrat dans des conditions favorisant l'alignement de la matière biologique sur le substrat. La composition, le procédé et le matériel de la présente invention ont une application universelle en biologie, en magnétisme, en optique et en micro-électronique.
EP03796341A 2002-09-24 2003-09-24 Dispositif de stockage de biofilms fabriques Withdrawn EP1545202A2 (fr)

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WHALEY S.R. ET AL: "Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly", NATURE, vol. 405, 8 June 2000 (2000-06-08), pages 665 - 668, XP002909553, DOI: doi:10.1038/35015043 *

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