WO2011009052A2 - Peptides électroactivés et biodétecteurs - Google Patents

Peptides électroactivés et biodétecteurs Download PDF

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Publication number
WO2011009052A2
WO2011009052A2 PCT/US2010/042280 US2010042280W WO2011009052A2 WO 2011009052 A2 WO2011009052 A2 WO 2011009052A2 US 2010042280 W US2010042280 W US 2010042280W WO 2011009052 A2 WO2011009052 A2 WO 2011009052A2
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Prior art keywords
substrate
peptide
peptides
microbes
electric field
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PCT/US2010/042280
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WO2011009052A3 (fr
Inventor
Laurie B. Gower
Chih-Wei Liao
Ya-Wen Weh
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University Of Florida Research Foundation, Inc.
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Publication of WO2011009052A2 publication Critical patent/WO2011009052A2/fr
Publication of WO2011009052A3 publication Critical patent/WO2011009052A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • Biosensors can be used to detect various biological compounds. There exists a need in the art for an improved biosensor. There also exists a need in the art for improved, customizable, and easily-manipulated surfaces for dynamic surface functionality and patterning. BRIEF SUMMARY OF THE INVENTION
  • a biopanning process can be used to search for peptides that do not necessarily have a strong binding affinity for a surface, but instead can have a reversible binding affinity.
  • the reversibility of the binding affinity can be regulated on a fairly precise level. Accordingly, a "smart" surface can be designed and selected, depending on the desired application.
  • a biopanning process can be used to find electroactivated peptides which can be electrically triggered to adsorb, desorb, or both from an electronic material's surface when an electric field is applied. In one embodiment, this can be done on a large scale, in order to remove a coating with temporal control. In another embodiment, this process can be done on a fine scale by utilizing the precise spatial control offered by microelectronics processing capabilities.
  • clcctroac ⁇ vated peptides can be used with a device surface that is self-cleaning by electrically-triggered release of a coating.
  • Embodiments of the subject invention include a microsystem that can reduce or eliminate a biofilm.
  • a system can be provided where a device is electronically stimulated to release spent receptors. Then, a flow-through system that contains a new batch of fresh receptors can be provided. The fresh receptors can bind and assemble on the surface once it is returned to the de-activated (or activated binding) configuration. Accordingly, embodiments of the subject invention can provide systems that
  • I O can be used for long-term continuous biosensing by replenishing on an automated basis.
  • Such continuous biosensing can be advantageous for a wide range of applications, including, for example, biosensors for soldiers in battle, point-of-care diagnostics in personal healthcare, and testing of animal/plant food products in the agricultural arena.
  • an electroactive peptide is provided, wherein the electroactive
  • 15 peptide can be reversibly bound to a substrate upon application of an electric field.
  • an electro-release method for manufacturing electroactive peptides can comprise the steps of incubating peptide-cxpressing microbes (e.g. phage) with a substrate; washing the substrate to remove any unbound microbes; cluting the remaining microbes; and amplifying the remaining microbes, wherein the washing, eluting, and 0 amplifying steps are each performed repeatedly (e.g. at least two times) to obtain the microbes that bind strongly to the selected target material.
  • This set of strong binding microbes are then applied to a device containing a surface with the target material of interest, and upon application of an electric field, the microbes expressing electroactive peptides are released, collected, and amplified.
  • the electroactive peptides can be 5 synthesized in isolated form using known techniques.
  • an electro-release method for manufacturing electroactive peptides can comprise the steps of incubating peptide-expressing microbes (e.g. phage) with a target substrate; washing the substrate to remove any unbound peptide-expressing microbes; eluting the remaining peptide-expressing microbes; and amplifying the remaining peptide-expressing 0 microbes, wherein the washing, eluting, and amplifying steps are each performed repeatedly (e.g.
  • the method further comprises the steps of binding the obtained microbes, that bind to the target substrate, to a second substrate which is the same material as the target substrate; applying an electric field to the second substrate to release a subset of bound microbes which express clectroactivc peptides; and collecting and amplifying the released microbes expressing the electroactive peptides.
  • an clectroelution method for manufacturing electroactive peptides can comprise the steps of incubating pcptidc-cxpressing microbes (e.g. phage) with a substrate; washing the substrate to remove any unbound microbes; applying a voltage to the microbes to elutc the remaining microbes; and amplifying the remaining microbes, wherein the washing, eluting, and amplifying steps are each performed repeatedly (e.g. at least two times) to obtain the electroactive peptides that are expressed by the electroeluted microbes.
  • the electroactive peptides can be synthesized in isolated form using known techniques.
  • a dynamically functionalized surface can comprise a substrate and at least one electroactive peptide, wherein the electroactive peptide can be reversibly bound to the substrate upon application of an electric field.
  • a biosensor can comprise a substrate; and elcctroactivatcd peptides having a reversible binding affinity for the substrate; wherein at least some of the elcctroactivatcd peptides adsorb to the substrate, desorb from the substrate, or both, upon application of an electric field.
  • electroactive peptides can serve as linkers to attach other molecules of interest, including, but not limited to, proteins, antibodies, DN ⁇ , and nanoparticles.
  • an electro-el uti on biopanning method can comprise the steps of incubating peptides with a substrate; washing the substrate to remove any unbound peptides; eluting the remaining peptides with application of an electric field; and amplifying the remaining peptides. Some or all of these steps can be repeated to evolve to the most optimal electroactive peptides.
  • an electro-release biopanning method can comprise the steps of incubating peptides with a target substrate; washing the substrate to remove any unbound peptides; eluting the remaining peptides by conventional means (e.g., low pi I, high salt); amplifying the remaining peptides; and then screening those peptides with good binding affinity using an electronic device to find those capable of electroactivatcd release. Some or all of these steps can be repeated to evolve a set of peptides with good binding affinity to the target substrate.
  • the target substrate can be, for example, an inorganic substrate used in electronics devices.
  • a method for replenishing a biosensor after analytes have been captured or clogged the binding site of a receptor can include the steps of: a) releasing spent receptors by applying an electric field to the biosensor, or if an electric field is already present, increasing, decreasing, or removing the existing electric field; b) providing fresh receptors to the biosensor; and c) replenishing fresh receptors to the biosensor by altering the electric ( ⁇ eld in the reverse direction of step a).
  • the receptors can be electroactivated peptides, or species attached to the peptides, where the peptide serves as a linker between the receptor and device.
  • step a) can include applying an electric field
  • step c) can include removing the electric field.
  • step a) can include decreasing an existing electric field
  • step c) can include increasing the electric field.
  • Figure 1 shows a schematic representation of an electroactive peptide binding to a pixel after release of a passive linker.
  • Figure 2 shows a schematic representation of receptors releasing and then an inflow of fresh receptors assembling on the surface.
  • the species attached to the peptide need not be receptors, but could be some other species or chemical functionality of interest.
  • Figure 3 shows an indium zinc oxide (IZO) thin film coated on a sapphire substrate.
  • IZO indium zinc oxide
  • Figure 4 shows a schematic of a phage display biopanning protocol, which can be a first step of an electro-releasing protocol according to the subject invention.
  • Figure 5 shows surface coverage percentage of IZO binding phage clones determined through low pH buffer elution.
  • Figure 6 shows an IZO electro-releasing test device.
  • Figure 7 shows optical microscopy images of phage clones adhering to a device surface. Micrographs on the left were imaged with traditional brightfield, and micrographs on the right used fluorescence imaging with FITC-labcled phage clones. The amino acid sequence of the peptides used was FNGRHGTTDHPT + TNPLSSWTFPTY + ASQITHFPRPPW.
  • Figure 8 shows a schematic of a phage display biopanning protocol using device substrates for an electro-el ution protocol according to the subject invention.
  • Figure 9 shows immunofluorescence images of a phage clone bound to different substrates, l he amino acid sequence of the peptide expressed by the phage was Y ⁇ EKTVDITMIP.
  • Figure 10 shows UV absorbance obtained from ELISA technique using phage expressing different 12-mer peptide sequences bound on different substrates.
  • Figure 11 shows immunofluorescence images of a phage clone bound to different substrates. I ' he 12-mer expressed peptide amino acid sequence - MNRP SPP LP LWV.
  • Biopanning procedures use a combinatorial approach to evolve strong binding affinity between a microbe and a target material of interest.
  • Biopanning can provide customizable peptides to serve as molecular linkers at an organic-inorganic interface such as for the attachment of bioreceptor compounds (e.g. antibodies. DNA aptomers, and peptide epitopes) to inorganic transduction elements in a microelectronics device (e.g. semiconductor, metals, inorganic nanowires, carbon nanotubes. and nanocrystals).
  • bioreceptor compounds e.g. antibodies. DNA aptomers, and peptide epitopes
  • inorganic transduction elements e.g. semiconductor, metals, inorganic nanowires, carbon nanotubes. and nanocrystals.
  • molecular biomimetics This approach for molecular construction of nanoassemblies, which can be referred to as "molecular biomimetics. " ' can use heterofunctional peptides that can bind to different materials at each end.
  • a protcin/bioreceptor can be attached to one end, so that when the other end binds to a transducer surface, the receptor can be very close to the surface for improved sensitivity in measuring the receptor-analyte binding event.
  • multiple components can be assembled onto surfaces patterned with different inorganic domains, with site specificity provided by the molecular recognition capabilities of sets of peptide linkers where each provides customizable selectivity for the patterned regions of inorganic materials.
  • a biopanning process can be used to search for peptides that do not necessarily have a strong binding affinity for a surface, but instead can have a reversible binding affinity.
  • the reversibility of the binding affinity can be regulated on a fairly precise level. Accordingly, a "smart "1 surface can be designed and selected, depending on the desired application.
  • the target material of interest to which a microbe can have a binding affinity
  • the target material can be indium /inc oxide (IZO).
  • IZO indium /inc oxide
  • a solution of peptide-expressing microbes (such as phage or bacterial cell surface) can be incubated with the target material. The target material can then be washed to wash away unbound microbes, ' I he strongly bound peptide-expressing microbes can be eluted and amplified. These biopanning steps can be repeated to enrich the binding affinity of the peptides to the target material after multiple rounds (e.g. two, three, four, five, or more rounds).
  • a low pi I elution buffer or a high salt elution buffer can be used for the elution process.
  • the low pi I elution buffer can have a pH of less than 7 and can be, for example, glycine HCL Buffer (pH 2.2),
  • the high salt elution buffer can be, for example. 4M MgSO 4 OHaO. though embodiments arc not limited thereto.
  • an clectro-clution process can be used instead of chemical elution.
  • Peptide-expressing microbes that are released can be located and then examined to find the strongest binders.
  • a solution of peptide-expressing microbes can be incubated with the target material.
  • the target material can then be washed to wash away unbound peptides.
  • the strongly bound peptides can be eluted by application of an electric field, and then amplified. These biopanning steps can be repeated to enrich the binding affinity of the peptides to the target material for multiple rounds (e.g. two, three, four, five, or more rounds).
  • the electro-elution can be by, for example, applying a voltage.
  • the voltage can be in a range of from about 1 mV to about 100 V.
  • the voltage can be in a range of from about 100 mV to about 10 V. In a further embodiment, the voltage can be in a range of from about 1 V to about 2 V. In a specific embodiment, the elution can be by applying a voltage of 1500 mV; the voltage can be applied for a period of time of from one second to about 60 minutes (e.g. about five minutes). In a further embodiment, the elution can be by applying a voltage of less than about 1 V; the voltage can be applied for a period of time of from about 1 second to about 60 minutes (e.g. about five minutes). In embodiments, the period of time for the elution can be from 1 second to about 10 minutes.
  • the period of time for the elution can be from 1 second to about 5 minutes. In a further embodiment, the period of time for the elution can be from 1 second to about 60 seconds. In yet a further embodiment, the period of time for the elution can be from 1 second to about 30 seconds. In yet another embodiment, the period of time for the elution can be from 1 second to about 20 seconds. In yet another embodiment, the period of time for the elution can be from 1 second to about 10 seconds. In an alternative embodiment, the period of time for the elution can be more than 60 minutes. In another embodiment, an electro-release method can be used. ⁇ solution of phages can be incubated with the target material.
  • the target material can then be washed to wash away unbound phages.
  • the strongly bound phages can be eluted and amplified. Referring to Figure 4, these biopanning steps can be repeated to enrich the binding affinity of the phages to the target material for multiple rounds (e.g. two, three, four, five, or more rounds).
  • a low pH elution buffer or a high salt elution buffer can be used for the elution process
  • fhc low pH elution buffer can have a pH of less than 7 and can be, for example, glycine I ICL Buffer (pll 2.2).
  • the high salt elution buffer can be, for example, 4M MgSO 4 OH 2 O, though embodiments are not limited thereto.
  • the inorganic-binding phages that are collected from the above protocol can then be applied to a device that provides some form of application of an electric field, and the phages that are released can be collected for their reversibility characteristics. Electroactivated peptides are present either on the "tails" or the "coat” proteins of the phages, and can be synthesized in isolated form using known techniques.
  • an clcctro-elution process can be used instead of chemical elution.
  • Peptides that can be released can be located and then examined to find the strongest binders.
  • a solution of phages can be incubated with the target material.
  • the target material can then be washed to wash away unbound phages.
  • the strongly bound phages can be eluted and amplified. These biopanning steps can be repeated to enrich the binding affinity of the peptides to the target material for multiple rounds (e.g. two, three, four. five, or more rounds).
  • the elution can be by, for example, applying a voltage.
  • the elution can be by applying a voltage of 1500 mV; the voltage can be applied for a period of time of from about 1 second to about 60 minutes (e.g. about five minutes). In embodiments, the period of time for the elution can be from 1 second to about 10 minutes. In a further embodiment, the period of time for the elution can be from 1 second to about 5 minutes. In a further embodiment, the period of time for the elution can be from 1 second to about 60 seconds. In yet a further embodiment, the period of time for the elution can be from 1 second to about 30 seconds. In yet another embodiment, the period of time for the elution can be from 1 second to about 20 seconds.
  • the period of time for the elution can be from 1 second to about 10 seconds. In an alternative embodiment, the period of time for the elution can be more than 60 minutes.
  • a biopannmg process can be used for electroactivated peptides which can be electrically triggered to adsorb, desorb. or both from an electronic material's surface when an electric field is applied. In one embodiment, this can be done on a large scale, in order to remove a coating with temporal control. In another embodiment, this process can be done on a fine scale by utilizing the precise spatial control offered by microelectronics processing capabilities.
  • the electroactivated peptides adsorb to the material's surface (substrate) upon application of an electric field and desorb from the substrate when no electric field is applied.
  • the electroacthated peptides desorb from the substrate upon application of an electric field and adsorb to the substrate when no electric field is applied.
  • some of the electroactivated peptides desorb from the substrate upon application of an electric field.
  • the electroactivated peptides adsorb to the substrate when no electric field is applied, and some of the electroactivated peptides desorb from the substrate upon application of an electric field.
  • the subject invention can provide a revolutionary advance in the bionanotechnolog) arena because a surface can be dynamically patterned with both temporal and spatial control provided by electrically-activated pixels for binding and debinding the peptide linkers.
  • an electroactive peptide (depicted by the arrow originating at the phrase "electroactive peptide"; the lower portion of the dual functionalized peptide about to bind to the substrate) can bind to an activated device pixel after release of the passive peptide linker (the dual functional i/ed peptide to the right, with small arrows indicating release from the substrate).
  • the passive peptide linker the dual functional i/ed peptide to the right, with small arrows indicating release from the substrate.
  • With rcvcrsibly controlled binding it can be triggered to move to a new location for dynamic patterning of the peptide and its attached cargo (the upper portion of the dual functionalized peptide).
  • the peptides can be inorganic binding peptides. In an alternative embodiment, the peptides can be organic binding peptides.
  • lithographic techniques both soft and hard, can define a permanent pattern.
  • the soft lithography technique of microcontact printing can provide a method for patterning self-assembled monolayers by providing a means for patterning thermally sensitive materials. This can be particularly useful for spatial patterning of biological components.
  • Dynamic patterning provided by electroactivated peptides as in the subject invention, can provide a means for guiding cells in real time, or transporting nanoscopic components via molecular shuttles in lab-on-a-chip configurations.
  • molecular shuttles can be provided that are not constrained by the limitations of a preset pattern in which the "track" that is laid down for the polymerizing microtubule, for example, is permanent.
  • a dynamically functionalized surface can be provided by reversibly binding electroactivated peptides to a substrate.
  • the electroactivated peptides can be obtained by the methods described herein.
  • the electroactivated peptides can adsorb to or desorb from the substrate upon the application of an electric field (or the reversal or removal of an existing electric field).
  • the substrate can be a target material, for example an inorganic target material such as indium zinc oxide, silicon, or silicon dioxide.
  • the electroactivated peptides are capable of reversibly binding to the target material substrate.
  • Such a dynamic surface could be used in, for example, a lab on a chip, a biosensor, or any other application for which a dynamically functionalized surface could be useful.
  • a dynamically functionalized surface of the subject invention can be patterned with different molecules of different functionality, and the electroactive peptides can be reversibly bound or unbound to patterned regions of the substrate upon application of an electric field.
  • microtubules can be constructed and destructcd from regulatory signals by the cellular machinery.
  • the patterned track can be manipulated in real time with electronic stimulation of a pixilated device surface that can dictate the polymerization path.
  • this path can then respond to feedback from a sensing part of the device which alters the path of the molecular motor to fit the needs of the device, which can be constantly changing in certain circumstances.
  • Such a dynamic response capability can provide for a truly smart and responsive system, and one that can respond on command.
  • the dynamic patterning capabilities of the subject invention provide significant advantages in the field of biosensors.
  • electroactivated peptides can be used with a device surface that is self-cleaning by electrically-triggered release of a coating.
  • Biofouling contributes to a significant number of infections created with implanted biomaterials and devices, and is a significant problem with biosensor arrays as well.
  • Embodiments of the subject invention include a microsystem that can reduce or eliminate a biofilm. Bi ⁇ films are also a problem on non-biomedical materials, such as ship hulls and industrial pipelines.
  • the clcctroactivc peptides of the subject invention can serve as a linker between the target surface and some cargo to be transported.
  • the present invention can also provide advantages in continuous sensing devices by providing a system that can replenish spent receptors that have become clogged upon binding to the analyte or impurities.
  • a system can be provided where the device is electronically stimulated to release the spent receptors. Then, a flow-through system that contains a new batch of fresh receptors can be provided. The fresh receptors can bind and assemble on the surface once it is returned to the de-activated (or activated binding) configuration.
  • embodiments of the subject invention can provide systems that can be used for long-term continuous biosensing by replenishing on an automated basis.
  • Such continuous biosensing can be advantageous for a wide range of applications, including, for example, biosensors for soldiers in battle, point- of-care diagnostics in personal healthcare, and testing of animal/plant food products in the agricultural arena.
  • the electroactive peptide linker (the lower portions, as indicated by the arrows originating at the phrase "electroactive peptide linker) can be triggered to release its binding state.
  • the device can then be refurbished with inflow of fresh receptors that assemble on the de-activated surface (original peptide binding state).
  • the surface can be patterned for multi-components as well.
  • a biosensor can be returned to its original state after analytes are captured.
  • a method for replenishing a biosensor after analytes have been captured can include the steps of: a) releasing spent receptors by applying an electric field to the biosensor, or if an electric field is already present, increasing or decreasing the existing electric field; b) providing fresh receptors to the biosensor: and c) replenishing fresh receptors to the biosensor by altering the electric field in the reverse direction of step a).
  • the receptors can be linked to the device by electroactivated peptides.
  • step a) can include applying an electric field
  • step c) can include removing the electric field.
  • step a) can include decreasing an existing electric field
  • step c) can include increasing the electric field.
  • phage display can be used to biopan for inorganic binding peptides that are reversible upon application of an electric field. This can provide dynamic functionali/ation of microelectronics surfaces, for applications ranging from self cleaning devices to dynamic patterning and micro-transport.
  • strong binding peptides are panned for to the target material of interest.
  • the targe material of interest can be, for example, indium /inc oxide.
  • an electro-releasing device can be used to collect any of the strong binders that also have the rcvcrsibilty properties.
  • an electro-el ution biopanning protocol can be used.
  • the electric field can be used to elute off reversible peptides. Those peptides can then be collected and screened for peptides that also have sufficient binding strength to the target material.
  • Phage displa is a combinatorial approach for selecting peptides with specific binding affinity to inorganic materials.
  • Amorphous indium /inc oxide (IZO) which is a transparent semiconducting oxide, was used as a target material.
  • the phage display peptide Ph.D.-C12C library kit was purchased from New England Biolabs for biopanning the target TZO substrates.
  • This library consisted of randomized 12- mer peptides fused to a minor coat protein (pill) of M13 phage and contained 2.7x10 9 independent clones.
  • the IZO thin film was fabricated by sputtering IZO onto the top and bottom surface of a sapphire plate. IZO-coated sapphire was the substrate used for biopanning.
  • the binding affinities of some phage clones to the IZO thin film were characterized using the immunofluorescence microscopy technique.
  • a single clone was incubated with a fresh IZO thin film and then washed several times with PC buffer containing detergent to remove unbound phage.
  • the bound phages were labeled with Anti-M13 antibody and Anti- mouse IgG-FlTC secondare antibody, and then the phages were visualized through fluorescence microscopy.
  • Program Image J was utilized to calculate the surface coverage of each clone on the IZO thin film from the corresponding fluorescence image. Referring to Table 3 and Figure 5, coverage was qualitatively defined as follows: between 80-100% as strong binder, 60-80% as moderate binder, and lower than 60% as weak binder.
  • Table 3 The sequence of IZO binding phages and their binding affinity following low pH buffer elution. (Binding affinity- S: strong binding, M: moderate binding, W: weak binding) Sample code Sequence Binding % Affinity
  • a small device was designed which was coated with the target material IZO.
  • photolithography was applied in the clean-room at the Nano scale Research Facilities (NRF) at the University of Florida.
  • Figure 6a shows a cross section of the device
  • Figure 6b shows a schematic of a surface view of the device which shows the bending channel location of the electric field placed across the channel of lOO ⁇ m width.
  • Figure 6c is a photograph of the surface view of the device processed on a 5 mm x 10 mm silicon wafer substrate and a large surface area of the bending region.
  • SiO 2 ZSi wafers were used as substrates, cleaned with trichloroethylene, acetone, and methanol, and dried by nitrogen gas to remove organic and inorganic contaminations.
  • the Headway spinner was used to coat Shipley S 1813 positive photoresist (PR), and the PR- coated substrate was baked on a hotplate. Then the PR-covered substrates were exposed to light by Karl Suss M ⁇ -6 Contact aligner System, and developed by MF-300 Developer.
  • KJL CMS-18 Multi-source Sputtering System was used to make metal electrodes, which are composed of I i, ⁇ u, and Ti layers.
  • the IZO thin films were deposited in the Ar plasma by rf-magnetron sputtering, and the thickness of the IZO layer was controlled to around 200 nm, followed by connecting gold wires to metal electrodes.
  • EXAMPLE 3 Electro-releasing Test
  • phage clones with strong binging affinity to IZO thin film were selected and tested to determine if the clones could be released by applying an electric field.
  • Three phage clones were mixed together with amino acid sequences of their expressed peptides as follows: FNGRHGl TDHPT, TNPLSSWTFPTY, and ASQITHFPRPPW, and incubated with the IZO device. After washing away unbound phage by PC buffer containing 0.3% detergent and labeled with antibodies, optical brightfield images ( Figure 7a) and fluorescent images ( Figure 7b) were obtained without triggering the voltage Lo locate the bending channels and to see the phage binding coverage respectively.
  • FIGS 7c and 7d are the optical image and fluorescent image, respectively after electro-releasing.
  • the releasing bending channels have less fluorescent signal of the labeled bound phages after electro-releasing as compared to the prior image without applying electro field, which indicates that some phages may be released by the triggering of an electric field.
  • an electro-elution process can be used instead of chemical elution.
  • the initial experiment seeks peptides that can be released, and then they are examined to find the strongest binders.
  • the 10 ⁇ L phage display peptide Ph.D.-C12C library kit of Example 1 was incubated with cleaned IZO coated device in PC buffer without detergent, and the device was then washed several times with PC buffer with 0.1% detergent to remove unbound phage.
  • the strongly bound phages were eluted b> applying a voltage at 1500 mV for 5 minutes and amplified by infecting the host bacterial strain ER2738.
  • a phage clone was obtained with amino acid sequence: YAEKTVD1TMIP.

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Abstract

L'invention porte sur des peptides électroactifs et sur des surfaces dynamiquement fonctionnalisées. Une surface peut avoir des motifs obtenus dynamiquement avec à la fois une commande temporelle et spatiale fournie par des pixels électriquement activés pour une liaison et une déliaison de liants peptidiques. L'invention porte également sur des peptides électroactifs qui peuvent être utilisés pour des surfaces dynamiquement fonctionnalisées, fournissant une capacité d'auto-nettoyage de surfaces encrassées. Deux procédés de fabrication de peptides électroactifs sont fournis, un protocole de bioadhérence (biopanning) à électro-libération et un protocole de bioadhérence à électro-élution.
PCT/US2010/042280 2009-07-17 2010-07-16 Peptides électroactivés et biodétecteurs WO2011009052A2 (fr)

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Cited By (1)

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CN112898032A (zh) * 2021-02-04 2021-06-04 江西理工大学 一种凝胶注模陶瓷生坯热脱脂速率控制方法

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