Patent Application
20230184756
This application is a 371 and claims benefit of European Application No. EP2021/053235, filed Feb. 10, 2021, which claims benefit of Portugal Application No. 116112, filed Feb. 10, 2020, and United Kingdom Application No. 2002343.8, filed Feb. 20, 2020, which applications are incorporated herein by reference in their entirety.
A Sequence Listing is provided herewith as a Sequence Listing text, MEWE-111_SEQLIST_2023-02-28 created on Feb. 28, 2023 and having a size of 1,078 bytes. The contents of the Sequence Listing text are incorporated herein by reference in their entirety.
This invention relates to materials made from aggregated peptides, which are useful for sampling and testing analytes. In particular, the invention relates to biomaterials comprising aggregated peptides, devices comprising those materials, and methods of using the materials and devices for sampling, analysing or detecting analytes.
Amyloid fibrils are originated via a process designated amyloidogenesis, by which peptide or protein monomers spontaneously self-assemble into higher order aggregates namely oligomers, protofibrils and, finally, the mature amyloid fibrils. The first studies of amyloidogenesis were performed mainly in the context of the so-called amyloid diseases, such as Alzheimer's and Parkinson's syndrome, among others1-10. Due to the association with these pathologies, it was initially thought that mature amyloid fibrils are toxic by themselves and just the result of a random misfolding process. However, several studies showed that toxicity is more often associated with precursor aggregates, i.e., oligomers and protofibrils, than with the mature amyloid fibrils and that amyloidogenesis is actually a highly ordered process1-3,11-13. Furthermore, subsequent investigations demonstrated that amyloid species are involved in several physiological processes, performing functional roles such as extracellular matrix materials of fungi and bacteria14-16, as protective envelops of insects and fish eggs17,18, and other roles, as reviewed elsewhere19-23. Remarkably, amyloid-like nanofibrils seem to be the functional structure of stored peptide hormones24,25, being also involved in melanin formation in humans26,27.
This fresh understanding of amyloid fibrils physiological roles sparked a renewed interest in their study, not only in the framework of misfolding studies but also as possible novel biomaterials. This potential has been previously overlooked but, in fact, amyloid fibrils possess key characteristics that make them appropriate biomaterials for nanotechnology applications19,28. A key requirement is stability, allowing for chemical reactions to occur in the immediate vicinity of the biomaterials without affecting them19. Another major requirement is the ability of the biomaterial to be chemically modified for a specific function, without affecting chemical and mechanical stability19. It is also desirable in some applications for a biomaterial that self-assembles in a well-established and ordered manner to produce diverse topographies19-21,28. Amyloid fibrils possess these exact features and are hence becoming attractive tools in nanotechnology, with potential applications across various fields, including the biomedical sciences, as novel ordered nanomaterials19-21,28-31.
One of the most important characteristics of amyloid fibrils is their particular highly ordered structure1,13,32-35. Fibrillar aggregates derived from different amyloidogenic peptide/protein sequences share common features, being composed of β-sheet structure stabilized by hydrogen-bonds between adjacent β-strands that run perpendicular along the fibril axis1,3,13,19,36. The distances between β-strands are mostly determined by the size of the amino acid side chains19. The mature amyloid fibril is then formed by the assembly of the β-strands protofilaments1,19,36-40. A wealth of data on these processes is now available. Due to this, conceiving new amyloid species for special purposes has become viable, by taking advantage of design algorithms based on empirical and theoretical rules governing amylodogenesis8,28,41-46.
Given the above, amyloid fibrils are excellent candidate biomaterials. Short amyloidogenic peptide sequences have structural compatibility, nanoscale dimensions, organized assembly into well-defined nanostructures, low cost and easy of production (of constituent monomers), allowing various technological developments, including for bio-sensing uses 19-21,28,39,47,48, 60-65. For this purpose, it is important to make use of amyloidogenic peptides that form stable amyloid fibrils in physiological conditions of pH and temperature (in which most biologically relevant interactions and processes occur) and that can be easily derivatized with specific chemical moieties (to add them new functions, when needed). Moreover, having different amyloid topologies, to suit different applications, would be desirable.
The invention is based on the surprising development of new biomaterials from simple peptides. The peptides can assembled into a fibril or gel biomaterial. Typically, the peptides are able to self-assemble into the biomaterial under appropriate conditions. The peptides can be functionalised, for example with a biological or chemical molecule, without preventing this assembly into the higher-order structure of the biomaterial. Although the monomeric peptides themselves are typically soluble in aqueous and/or physiological conditions, the resulting aggregated biomaterial is surprisingly able to maintain its structure over hours, days or weeks. The provision of an easily assembled stable biomaterial is therefore particularly advantageous.
The biomaterial can be labelled with multiple reporter molecules, so that a single binding event between the functionalised biomaterial and a target analyte provides multiple reporter signals. This can advantageously be used to amplify the signal from a single binding event. Furthermore, the ability to provide a fully-formed biomaterial, or to allow self-assembly of the components to form the biomaterial where it is needed, provides a highly adaptable material and, for example, allows for signal amplification in situ with minimal sample processing.
A first aspect of the invention provides a functionalised biomaterial comprising aggregated self-assembling peptides, wherein at least a proportion of the self-assembling peptides are functionalised with a biological agent or a chemical agent. The biomaterial may, in certain embodiments, be a fibril or gel. Typically, the self-assembling peptides are able to self-assemble under physiological conditions, preferably the self-assembling peptides are able to self-assemble spontaneously under physiological conditions (for example 20-40° C., atmospheric pressure of 1, pH 6-8). The assembled biomaterial is typically non-toxic.
The self-assembling peptides may be amyloidogenic peptides.
In certain embodiments, the biomaterial is composed of a peptide that comprises, consists of, or consists essentially of: STVIIE, QVQIIE, ISFLIF and/or GNNQQNY. In some embodiments, 1, 2, 3 conservative substitutions may be made to the peptide provided that the self-assembling properties are retained. Conservative substitutions are known in the art and are typically accepted as being a substitution within the same general class of amino acid residue, as summarised in the table below:
1, 2 or 3 insertions or deletions to the STVIIE, QVQIIE, ISFLIF and/or GNNQQNY peptides may also be made, provided that their self-assembling properties are retained.
The peptide may be part of a larger peptide, provided that the self-assembling properties are retained. For example, at least 1, 2, 3, or more additional amino acid residues may be present at one end or at both ends of the STVIIE, QVQIIE, ISFLIF and/or GNNQQNY. Typically, a longer peptide comprising the STVIIE, QVQIIE, ISFLIF and/or GNNQQNY peptide contains a maximum of 20 amino acid residues, for example 15 or fewer amino acid residues, typically 10 or fewer amino acid residues.
The peptides that form the biomaterial may be provided homogenously, or a heterogeneous mix of different peptides. All of the peptides may be peptides of the invention, for example STVIIE, QVQIIE, ISFLIF and/or GNNQQNY peptides, or other peptides may optionally be included. In some embodiments a majority of STVIIE peptides are used, or only STVIIE peptides are used, some or all of which may be functionalised.
Typically, the majority (e.g. >50%, >60%, >70%, >80%, >90% or more) of peptides are functionalised, for example all or substantially all of the peptides are functionalised, although the biomaterial still functions in assays even when only a minority of the peptides (<50%) are functionalised.
In exemplary embodiments, a fibril incorporates 60%-90% unfunctionalised peptide (e.g. STVIIE) with 10% to 40% functionalised peptide (weight/weight), for example 60%-80% unfunctionalised peptide with 20% to 40% functionalised peptide (weight/weight), about 65%-75% unfunctionalised peptide with 25% to 35% functionalised peptide (weight/weight), or about 70% unfunctionalised functionalised with about 30% functionalised peptide (weight/weight). Other amyloidogenic sequences, mixed in similar or different ratios, of free peptide and biotinylated versions, are provided to give a similar result to those demonstrated herein. In certain embodiments, the peptides form amyloid fibrils with a periodic width of between 100 and 150 nm, a ΔX of between 150 nm and 200 nm, a ΔY of between 2 nm and 5 nm and a height of between 10 nm and 15 nm. In certain embodiments, the amyloid fibrils have a periodic width of 118±16 nm, a ΔX of 170±7 nm, a ΔY of 3.2±0.4 nm and a height of 13.5±0.9 nm.
The functionalised biomaterial is typically stable under physiological conditions. In some embodiments the biomaterial forms within six hours, and optionally is then allowed to develop further for another 24 hours to up to 2 weeks before being stable for use. A stable biomaterial retains its structure over the period in which it is needed, typically over a period of hours, days, weeks or months. This may be 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 6 weeks or more, or 8 weeks or more, for example 15 weeks or more. The physiological conditions are usually physiological pH and/or physiological temperature. Physiological pH is typically between 6 and 8, or between 6.5 and 7.5, or is about 7, or is about 7.4. Physiological temperature is between room temperature (e.g. around 20° C.) and human body temperature (about 3TC), for example 25° C. In some embodiments, the peptides of the invention are able to self-assemble at 20° C. in liquid water, or in general aqueous solutions with pH and salt concentrations at physiological levels at temperatures close to room temperature, when added in sufficient quantities and concentration, to form the aggregated biomaterial.
The biomaterial is functionalised. The term “functionalised” is to be given its usual meaning in the art, and relates to the inclusion of an additional functional molecule into the biomaterial. The functional molecule may be a chemical or biological agent. In some embodiments the chemical agent may be a vitamin, enzyme cofactor, reaction substrate, or catalyst. In some embodiments the biological agent may be a protein, nucleic acid or carbohydrate. In some embodiments, the biomaterial is functionalised by having biotin attached to at least a proportion of its component peptides. Biotin is well-known (IUPAC name 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid). Biotin is therefore an example of a suitable functionalising agent. The terms functionalising agent and functionalising element are used interchangeably herein. Other typical functionalising agents are proteins, for example a receptor, a ligand, or an antibody or antigen-binding fragment (e.g. Fab fragment) of an antibody, or a nucleic acid such as DNA or RNA. The functional molecule typically retains its required function when it is part of the biomaterial of the invention. In certain embodiments, the functional molecule in the biomaterial retains at least 50% of its function in free solution, for example at least 75% or at least 90%. In certain embodiments, the functional molecule in the biomaterial has substantially the same level of function as it has in free solution under equivalent conditions. Function may be determined by an appropriate assay depending on the functional molecule. For an enzyme or catalyst, the activity may be measured in enzyme units, katals (or nano- or micro-katals), or KM. For an antibody or other binding molecule, the function may be measured as the binding affinity, for example the dissociation constant as determined by surface plasmon resonance (for example the well-known Biacore assay) or another suitable assay.
The functionalising agent may be attached to a self-aggregating peptide of the invention using a linker. The linker may be rigid or flexible. The linker may be a heterobifunctional or a homobifunctional chemical linker.
The linker may be a glucoside linker, of which a suitable example is a TRIS-derived triglucoside (Tdts). In other embodiments, the linker may be a polyethylene glycol-based linker, for example PEG9 or PEG13.
In some embodiments, the functionalising agent may be attached directly to the peptide of the invention, for example by a covalent or non-covalent bond.
The functionalising agent may be attached (directly, or using a linker) anywhere on the peptide. In some embodiments, the functionalising agent is attached to the N-terminal half or the C-terminal half of the peptide. In some embodiments, the functionalising agent is attached to one of the 3 residues at either the N-terminus or the C-terminus of the peptide. In some embodiments, the functionalising agent is attached to the N-terminal (i.e. first) residue. In other embodiments, the functionalising agent is attached to the C-terminal (i.e. last) residue.
Suitable functionalising agents or molecules may include biotin.
At least one additional element may be connected to the functionalised biomaterial. Typically, a first additional element is referred to herein as a “bridge element”. This bridge element is optionally attached to the functionalising agent and, in this arrangement, the peptide is typically bound (e.g. covalently) to the functionalising agent which is in turn bound (e.g. non-covalently) to the bridge element. The bridge element may be a protein that binds specifically to the functionalising agent, for example it may be an antibody that binds specifically to an epitope on the functionalising agent (or it may comprise an epitope that binds specifically to an antibody functionalising agent). In some embodiments, the functionalising molecule and bridge element are a receptor and its ligand. The receptor and ligand can be in either orientation, such that the functionalising molecule is the receptor and the bridge element is the ligand, or such that the functionalising molecule is the ligand and the bridge element is the receptor. An example of a suitable bridge element is streptavidin, which can bind to a biotin functionalising agent.
The bridge element can be in solution or be immobilised to a surface, for example the surface of an assay chamber.
A reporter molecule can be bound (covalently or non-covalently) to at least one bridge element. A reporter molecule is typically detectable, and may be an optical reporter (such as a fluorescent or other optically-detectable label) or a chemical reporter, such as a catalyst or enzyme, that when present results in a detectable change in the system. An example of an enzyme reporter is horseradish peroxidase (HRP). As shown in the examples, HRP treated with substrate can be imaged via chemiluminescence.
In certain embodiments, a bridge element comprises multiple reporter molecules. In some embodiments, multiple reporter molecules are attached to each of at least 1, 2, 3, 4, 5 or more bridge elements. Multiple reporter molecules can be attached to multiple bridge elements, or attached to at least 50% of the bridge elements, or to all or substantially all of the bridge elements.
In certain embodiments, a bridge element comprises a single reporter molecule. In some embodiments, the biomaterial comprises at least 1, 2, 3, 4, 5 or more singly-labelled bridge elements. Multiple bridge elements can be labelled with a reporter molecule, for example at least 50%, at least 60%, at least 70% or at least 80% of the bridge elements, or in some embodiments all or substantially all of the bridge elements are labelled with a reporter molecule. Having multiple labelled bridge elements allows for amplification of the signal that is provided by the biomaterial.
In certain embodiments, the functionalising molecule or bridge element is specifically recognisable by a recognition element. The recognition element can be a protein, typically an antibody or receptor. The recognition element may also be a nucleic acid or any other molecule capable of selectively binding a target. In addition to recognising the functionalising molecule or bridge element, the recognition element is typically also able to bind specifically to an analyte of interest. Therefore, the recognition element typically has at least two binding sites, a first binding site for binding the functionalising molecule or the bridge element, and a second binding site or region for binding to an analyte. The recognition element may in some embodiments comprise or consist of a fusion protein of two antibodies, a fusion protein of an antibody and another binding protein, a fusion protein of at least two different proteins or protein domains, an antibody conjugate (such as an ADC or equivalent), or a bispecific or multispecific antibody. In some embodiments, the recognition element may be a streptavidin labelled antibody. If the target is for example a cell that expresses an Fc Receptor (e.g. a Natural Killer cell), then the recognition element could be an antibody with CDRs that bind specifically to the functionalising molecule or bridge element, and the Fc region will then engage the target cell.
When at least one bridge element comprises multiple reporter molecules, or when multiple bridge elements are labelled with a reporter molecule, recognition of the bridge element or the functionalising molecule by the recognition element (e.g. antibody) leads to multiple reporter molecules being captured for each single recognition event (e.g. antibody-ligand interaction), resulting in increased signal detection and amplification. In one embodiment set out in the Examples, the functionalising molecule is biotin, the bridge element is streptavidin, the reporter molecule is an enzyme (optionally HRP), and the recognition element is a streptavidin-labelled antibody. Other arrangements comprising 1, 2, 3 or all 4 of the functionalising molecule, bridge element, reporter molecule and recognition element can be prepared as required by the application of the technology.
According to a second aspect of the invention, there is provided the use of a biomaterial according to the first aspect, in a biological or chemical assay, for example an assay for the detection of an analyte.
A third aspect of the invention provides an assay to detect an analyte, wherein the assay comprises contacting the analyte with the biomaterial of the invention. The analyte may be contacted with a pre-formed biomaterial according to the first aspect, or the analyte may be contacted with one or more components of the biomaterial and the aggregation and assembly permitted to occur in situ. An embodiment wherein the separate components of the biomaterial are added to an analyte mixture is depicted in
The assay may in certain embodiments be a diagnostic assay, a biosensing assay, an immunoassay, an immunodiagnostic assay, a dot blot or an ELISA. The biomaterial of the first aspect or its component parts may conveniently be provided as part of a biosensor or analytical apparatus, suitable for use in this assay, and optionally adapted specifically for use in the assay. The analyte that is detected or analysed may in some embodiments be a biological molecule such as a protein, carbohydrate or polynucleotide, a metabolite, a biomarker, a cell, a human cell, an animal cell, a plant cell, a microorganism, a bacteria, or a virus. In some embodiments, the analyte is from a biological sample, a fluid sample or a tissue sample. In certain embodiments, the assays of the second and third aspects may be to detect a biomarker, a diseased cell, or a pathogen. The pathogen may be a microorganism such as a bacteria, fungus, protozoa or worm. The pathogen may typically be detected in a sample from a patient suspected of carrying the pathogen, for example in a bodily fluid or tissue sample from the patient. The patient is typically human. Typical bacteria for detection may be gram negative or gram positive bacteria. The bacteria may be cocci such as Staphylococci, Streptococci (e.g. S. pneumonia) or Neisseriae (e.g. N. gonorrhoeae or N. meningitidis), gram positive bacilli such as Corynebacteria, Bacillus Anthracis, Listeria monocytogenes, gram negative bacilli such as Salmonella spp., Shigella, Campylobacter, Vibrio, Yersinia pestis, Pseudomas spp., Brucella, Haempohilus, Legionella or Bortedella. Other bacteria that can be detected include Mycobacteria such as M. tuberculosis, M. leprae or M. avium, Rickettsia, or Chlamydia.
In some embodiments, the pathogen may be a virus. Typical viruses that can be detected include: DNA viruses such as adenovirus, herpesvirus, poxvirus, parvovirus, papilloma virus or hepatitis, for example hepatitis B; or RNA viruses such as influenza, coronaviruses, paramyxovirus, picornavirus (e.g. polio, coxsackie, hepatitis A, rhinovirus), togaviruses (e.g. rubella), flaviviruses (e.g. causing yellow fever, dengue fever), rhabdoviruses (e.g. rabies), ebolavirus, or retroviruses such as HIV.
Fungi that can be detected include Candida albicans, Aspergillus or Pneumocystis.
Protozoa that can be detected include Leishmania, Plasmodium, Trypanosoma, Toxoplasma gondii or Crytosporidium.
In certain embodiments, the pathogen to be detected is a rhinovirus, coronavirus, influenza virus, adenovirus, or respiratory syncytial virus. These viruses can often cause symptoms known as the “common cold”.
In an exemplary embodiment, the pathogen to be detected is a coronavirus, more typically a human coronavirus such as the 2019-nCOV.
The biomaterial can be adapted to detect an analyte of interest. In certain embodiments, the biomaterial comprises an antigen-binding protein (e.g. an antibody or antigen-binding antibody fragment) that binds specifically to the analyte, thereby allowing for its detection from a mixture. When the analyte is a pathogen, the antibody or antigen-binding antibody fragment part of the biomaterial will typically bind to a component on the surface of the pathogen, that is typically characteristic for that pathogen, for example a surface protein or carbohydrate. When the analyte is an influenza virus, the biomaterial may typically comprise an antibody or fragment that specifically binds to neuraminidase or hemagluttinin. When the analyte is a coronavirus, the biomaterial may typically comprise an antibody or fragment that specifically binds to the spike protein or hemagluttinin-esterase dimer. When the analyte is a bacteria, for example Pneumococcus, the biomaterial may typically comprise an antibody or fragment that specifically binds to one or more cell capsule sugars. The analyte-specific component of the biomaterial (typically an antibody or antibody fragment) may in certain embodiments be incorporated as the functionalising agent that is attached to the self-assembling peptide. In some other embodiments, the analyte-specific component of the biomaterial (typically an antibody or antibody fragment) forms part of the bridge element.
Typically, the analyte-specific component of the biomaterial (typically an antibody or antibody fragment) forms part of the recognition element. In some of the examples below, the bridge element comprises streptavidin (to bind the biotin functionalising agent) and the recognition element comprises a analyte-binding antibody that also binds to the functionalising agent (by means of a streptavidin label on the analyte-recognising antibody). Typically, the recognition element recognises the analyte and binds to the biomaterial, thereby linking the biomaterial to the analyte.
In a fourth aspect, the invention provides an amyloidogenic peptide comprising, consisting or consisting essentially of the peptides STVIIE, QVQIIE, ISFLIF and/or GNNQQNY. In certain embodiments, the peptide at 1 mg/ml is soluble in liquid water at 20° C. In further embodiments, the amyloidogenic peptide is as described above for the first aspect, or as described elsewhere herein.
A fifth aspect of the invention provides a method of preparing a biomaterial, comprising providing amyloidogenic peptides according to the fourth aspect in conditions suitable for them to self-assemble, and allowing the peptides to self-assemble to form the biomaterial. In further embodiments, the biomaterial that is produced according to the fifth aspect is as described above for the first aspect, or as described elsewhere herein.
A sixth aspect of the invention provides a kit comprising one or more amyloidogenic peptides according to the fourth aspect and instructions for their self-assembly into a biomaterial. The kit may optionally further comprise:
A seventh aspect of the invention provides a biosensor comprising a functionalised biomaterial according to the first aspect, or an amyloidogenic peptide according to fourth aspect. The biosensor may be used in the assay of the third aspect or provided as part of a kit according to the sixth aspect.
Other aspects and embodiments of the invention are described in more detail below.
Immobilizing such fibrils on membranes enables them to be detected in 1 hour, with (f) different concentration of streptavidin derivatized HRP. Then, (g) detection in 30, 60 or 90 minutes of incubation with 1 μg/mL of functionalized enzyme is achieved. Then it is shown that, using (h) 60 minutes of incubation with 1 μg/mL of functionalized enzyme, that 20 ng of biotin-PEG13-STVIIE immobilized amyloid fibrils are detected. The interaction is specific since, in all conditions, pure fibrils of free STVIIE peptide (i.e., not derivatized with biotin) give no signal. This shows that biotin-PEG13-STVIIE fibrils can be used to detect/amplify the presence of immobilized proteins, as demonstrated by (i) immobilizing and detecting the presence of even 2 ng (faint signal) of streptavin-derivatized anti-GFAP antibody. Moreover, BSA protein (negative control, not derivatized) shows no signal, supporting the approach specificity. Experiments conducted in triplicate.
The invention is based on a detailed study of amyloid-based biomaterials.
The production and use of a new biomaterial is described herein. The biomaterial has rationally designed and desirable physico-chemical and mechanical properties. In various embodiments, these properties can include at least 1, for example 2, 3, 4 or 5, or more of the following:
These properties make the biomaterial suitable for a number of uses. In certain embodiments, the biomaterial is useful as a nanomaterial to be integrated in nanodevices for nanotechnology and microfluidics. The invention also provides a method of identifying peptides useful in the formation of biomaterials. Candidate amyloidogenic peptide monomers are tested, to determine those that form amyloid fibrils in physiologic conditions of pH, temperature and ionic strength (at which most physiological protein-ligand interactions occur). Good candidate peptides are (b) functionalized with bioactive molecules bridge elements, via different linkers, and tested regarding their ability to (c) form stable fibrils, that are bound to functional bioactive molecules, connected via functional bridge elements (and adequate linker). Those fibrils that are functional and bioactive are then tested regarding the ability to hold several reporter molecules, both when (d) free in solution and when (e) immobilized.
In some embodiments, the biomaterial is useful for developing and improving diagnostics technologies, for example for signal amplification in immunoassays. In some embodiments, diagnostics technologies allow for the detection of one or more of (i) specific antibodies against: other antibodies, proteins, viruses, bacteria, toxins, hormones, disease (cancer) biomarkers and/or other biomolecules; (ii) peptides and/or proteins (functionalized or not), that can serve to identify the above mentioned targets; (iii) other biomarkers that can be targeted by labelling them with an appropriate linker molecule, as described hereafter.
In certain embodiments, the biomaterial can be functionalised, for example by attaching enzymes and/or other relevant functionalized biomolecules. The biomaterial can hold multiple copies of a given molecule, such as an enzyme and/or antibody, optionally via a linker, bioactive molecules and bridge elements. The biomaterial can, similarly, simultaneously hold several copies of different biomolecules. This property enables the biomaterial to have multiple functions, for example multiple enzymatic and/or biomolecule recognition functions. This allows the biomaterial to be used for biosensing, by using as recognition elements antibodies, DNA, RNA or any other active biomolecule(s) that can be inserted into it.
Other biological activities can be simultaneously engineered into the biomaterial, via the linker, bioactive molecules and/or bridge elements.
Typically, the biomaterial is active both when immobilized as well as when in solution, being also able to interact with molecules active and in solution.
The biomaterial is based on amyloidogenic peptide sequences i.e. sequences associated with or capable of forming amyloid aggregations, fibrils or deposits.
The examples below provide a study of the amyloidogenic properties in physiological conditions of the peptides GNNQQNY, QVQIIE, ISFLIF and STVIIE. These short peptides were previously known to form amyloid fibril structures only in acidic conditions49-51. Here we show in particular that STVIIE is able to form fibrils, in physiological pH and temperature conditions. Moreover, it is also demonstrated that these peptides can remain amyloidogenic after being derivatized with biotin, a relevant chemical moiety, widely used in conjugation with streptavidin (to which it binds in an almost covalent manner)39,52. Biotinylated peptide (exemplified by STVIIE) forms amyloid-like structures, which are then demonstrated to be able to bind free in solution and immobilized streptavidin labeled molecules. These are then successfully employed in biosensing. All of the above demonstrate that this amyloid-based technology can be used for signal detection and amplification, among other possible applications.
In a particular embodiment, biotinylated peptide derivatized versions, such as biotinylated STVIIE derivatized versions are shown in the Examples to be particularly useful. Similar results can be obtained with other amyloidogenic protein or peptide sequences, making them equally multi-functional.
Amyloidogenic sequences, such as STVIIE amyloidogenic sequences, derivatized with biotin, via an N-terminal linker, for example a PEG linker such as PEG9 or PEG13, are particularly bioactive. Other linkers can be designed and be highly functional, as described herein. Typically, some flexibility is maintained so that the molecule is not unduly constrained.
Mixed fibril preparations, of biotin-PEG-peptide monomers co-incubated with peptide monomers are also highly functional. For example, mixed fibril preparations, of biotin-PEG9-STVIIE or biotin-PEG13-STVIIE monomers co-incubated with STVIIE peptide monomers are highly functional.
In exemplary embodiments, a mixed fibril preparation may incorporate 60%-90% GNNQQNY, QVQIIE, ISFLIF or STVIIE, preferably STVIIE with 10% to 40% biotinylated peptide (weight/weight), for example 60%-80% GNNQQNY, QVQIIE, ISFLIF or STVIIE, preferably STVIIE with 20% to 40% biotinylated peptide (weight/weight), about 65%-75% GNNQQNY, QVQIIE, ISFLIF or STVIIE, preferably STVIIE with 25% to 35% biotinylated peptide (weight/weight), or about 70% GNNQQNY, QVQIIE, ISFLIF or STVIIE, preferably STVIIE, with about 30% biotinylated peptide (weight/weight). Other amyloidogenic sequences, mixed in similar or different ratios, of free peptide and biotinylated versions, are provided to give a similar result to those demonstrated herein.
The assays described here are performed in physiological conditions. However, if desired, the fibrils' stability allows them also to function in in non-physiological conditions.
In some embodiments, amyloidogenic peptide sequences may be sonicated before use in order to promote fibril formation.
As shown in the Examples below, an amyloidogenic derivatized peptides biomaterial can be incorporated into other technologies and applications, for example diagnostics kits.
The ability of using derivatized amyloidogenic peptides to detect and amplify the presence of several immunoglobulin (IgG) is demonstrated as an example of the utility of this material.
Glial fibrilar acidic protein (GFAP), is detectable, in very low amounts, using several types of derivatized amyloidogenic peptide fibrils that effect signal amplification. Salmonella spp. bacteria are also shown to be detectable, both via a newly-developed assay and when inserted into another commercial immunoassay kit. This detection can be enhanced via signal amplification, resulting in the lowering of the thresholds for accurate diagnostics, for example from days to 6 hours.
The assays are demonstrated via reporter molecule, horseradish peroxidase, that enables a redox (enzyme catalyzed) reaction, that provides the signal. Assays using other labelled reporter molecules, e.g. enzymes, will result in a similar improvement, due to the multiple reporter molecules bound per fibril. Using a redox reaction and a horseradish peroxidase enzyme as reporter molecule, detection, signal amplification, and improved diagnostics are demonstrated via:
Detection has been demonstrated with pure samples as well as in tissue extracts (biopsy-like) samples, indicating its clinical applicability.
Given all of the above, the approach and biomaterial(s) are of general applicability to nanotechnology and biomedicine, in the fields of biosensing and beyond.
The biomaterial of the present invention can be used to sample, detect or analyse chemical or biological analytes. A biological analyte may be a biomarker, biological molecule and/or biological fluids. A biological analyte may also be a pathogen, for example a bacteria or virus, as described elsewhere herein.
The biomaterial of invention can be used advantageously to sample biological analytes from a biological sample, such as a biological tissue or a bodily fluid. Biological fluids have typically been excreted or extracted from the body, such as sputum, mucus, saliva, blood, sweat or urine. Other fluids include phlegm, bile, cerebrospinal fluid and amniotic fluid. Ascitic fluid is another typical bodily fluid.
The present invention also provides a method of diagnosing a condition, disease, disorder or irregularity in a subject, said method comprising obtaining a sample of a biological fluid; detecting the presence or absence of a biomarker, biological molecule or metabolite in the sample of biological fluid in an assay using the biomaterial of the invention; and diagnosing the subject based on the presence or absence of the biomarker, biological molecule or metabolite in the biological fluid.
This may be used to detect a biomarker of a disease or disorder, or to detect the presence of a metabolite that is indicative of good or poor health. Alternatively, this method could be used to detect the presence of a narcotic, illicit drug or performance-enhancing drug in the subject.
The analyte may be a hormone or a derived substance thereof. The analyte may be an antibiotic or a derived substance thereof. The analyte may be chemical substance, a narcotic (for example cocaine, heroin, or amphetamine), a performance-enhancing drug (for example a steroid or EPO), an illicit drug, or a pharmaceutical drug.
The analyte may in some embodiments be a toxin, an environmental toxin, a bacterial toxin, or other biologically active molecule.
In some disclosed embodiments, a further step of treating the patient for a diagnosed disease or disorder may be carried out. This may involve a surgical step, or a step of administering a therapeutic agent to a patient in need thereof, at an effective dose.
As used herein, the term “subject” and “patient” includes humans and animals. In certain aspects, the subject is a mammal, for example a rodent, for example a rat, mouse or Guinea pig, a cat, a dog, a goat, a pig, a cow, a horse, or a primate, for example a human. In certain embodiments, the subject is a human. The animal may be a bird. In other embodiments, the subject is a farm animal, for example an ovine animal, a bovine animal, a caprine animal, an equine animal or a bird such as a chicken, turkey, goose or duck. In some embodiments, the analyte to be detected is a biomarker. Biomarkers that are indicative of bacterial infections include cytokines and interleukins. Particular biomarkers include: TNF-related apoptosis-inducing ligand (TRAIL), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin 1-beta (IL-1β), C-reactive protein (CRP), soluble triggering receptor expressed on myeloid cells 1 (sTREM1), pro-adrenomedullin, serum procalcitonin (PCT), soluble urokinase-type plasminogen activator receptor (suPAR), atrial natriuretic peptide (ANP), IL-6, IL-8, IL-27, and CD64.
Further specific biomarkers that are indicative of viral infections include: Interferon-stimulated gene 15 (ISG15), IL-16, oligoadenylate synthetases-like protein (OASL), Adhesion G protein-coupled receptor E5 (ADGRE5).
Specific cells that are indicative of a disease, disorder or infection to be diagnosed include bacterial cells, including gram-negative bacterial cells, gram positive bacterial cells; host cells such as immune cells, such as dendritic cells, lymphocytes including B cells and T cells, macrophages, NK cells, innate lymphoid cells, eosinophils, basophils, mast cells, neutrophils and/or monocytes; host cells such as cancerous or pre-cancerous cells including, but not limited to, cancer of the respiratory tract such as mouth cancer, tongue cancer, nasal and paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, tracheal cancer, oesophageal cancer, lung cancer, bronchial adenoma; cervical cancer; prostate cancer; colon cancer; rectal cancer; ovarian cancer.
The biomaterial of the invention can in some embodiments comprise a functionalising agent, a bridge element that may optionally be labelled with a reporter molecule, and/or a recognition element that is able to detect a biological molecule, biomarker, protein, virus or cell as described herein. Typically, the bridge element and recognition element each bind to the functionalising agent. Typically, the recognition element comprises an antigen-binding protein, such as an antibody.
In some embodiments, the biomaterial comprises at least the following components:
In some embodiments, the antigen-binding protein (e.g. a secondary antibody) may bind to a primary antibody that binds to a target analyte
The term “antigen-binding protein” refers to a protein that is capable of specifically binding an antigen, e.g. a target or its signaling partner, or that is capable of binding an antigen with a measurable binding affinity. Examples of antigen-binding proteins include antibodies or antigen-binding fragments thereof, peptibodies, polypeptides and peptides, optionally conjugated to other peptide moieties or non-peptidic moieties. Antigens to which an antigen-binding protein may bind include any proteinaceous or non-proteinaceous molecule that is capable of eliciting an antibody response, or that is capable of binding to a polypeptide binding agent with detectable binding affinity greater than non-specific binding. The antigen to which a modulating antigen-binding protein binds may include a target, a signaling partner of a target, and/or a complex comprising the target and its signaling partner.
The term “antibody” is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen (e.g., Fab′, F′(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. An “immunoglobulin” or “tetrameric antibody” is a tetrameric glycoprotein that consists of two heavy chains and two light chains, each comprising a variable region and a constant region. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab′, F(ab′)2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody, chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as one, two, three, four, five or six CDR sequences, as long as the antibody retains the desired biological activity.
The term “antibody fragment” herein refers to an antigen-binding fragment of an antibody which retains at least 50% (e.g. at least 60%, 70%, 80% or 90%) of the binding affinity of the entire antibody. When used in a diagnostic-type assay, the antigen-binding protein or antibody used to detect an analyte should be capable of selectively binding to the analyte with greater affinity for the specific biomarker than other molecules present in the same biological fluid. The term “selective” encompasses groups that have an affinity for their target analyte that is more than 2 times greater than for other analytes present in the same biological fluid. For example, the affinity for the target analyte may be 2-109 times greater than for other molecules. In some aspects, the affinity for the target is more than 10 times, 100 time, 1000 times, 104 times, 105 times, 106 times, 109 times greater for the target than for other molecules in the same biological fluid.
As used herein, a “biomarker” is a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers may be cells, biological molecules such as proteins, lipids, hormones and/or nucleic acids. The term “biomarker” as used herein include biological molecules and metabolites. The biomarker may be a class of biomarkers, such as proteins, lipids, cells, hormones and/or nucleic acids. The functional group may bind selectively to the entire class of biomarker or may bind to a subset of the class of biomarker. For example, when the biomarker is a peptide, the functional group may bind to all peptide, to specific classes of peptides such as interferons, immunoglobulins, or cytokines; or to individual peptides such as interferon α, interferon β, interferon γ, CRP, TRAIL, sTREM-1, procalcitonin, ANP, pro-vasopressin, proadrenomedullin, suPAR, lactoferrin, galectin-9, CD14, CD32, CD35, CD46, CD55, CD59, CD64, CD88, interleukins including IL-1, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-27. In a further example, when the biomarker is a cell, the functional group may bind to all cells, or to a specific class of cells such as: bacterial cells, including gram-negative bacterial cells, gram positive bacterial cells; host cells such as immune cells, such as dendritic cells, lymphocytes including B cells and T cells, macrophages, NK cells, eosinophils, basophils, neutrophils and/or monocytes; host cells such as cancerous or pre-cancerous cells including, but not limited to, cancer of the respiratory tract such as mouth cancer, tongue cancer, oesophageal cancer, lung cancer; cervical cancer; prostate cancer; colon cancer; rectal cancer, ovarian cancer.
The biomaterial also finds particular utility in the detection of pathogens. Typically, the recognition element will be able to bind specifically to a pathogen.
Typical pathogenic bacteria for detection may be gram negative or gram positive bacteria. The bacteria may be cocci such as Staphylococci, Streptococci (e.g. S. pneumonia) or Neisseriae (e.g. N. gonorrhoeae or N. meningitidis), gram positive bacilli such as Corynebacteria, Bacillus Anthracis, Listeria monocytogenes, gram negative bacilli such as Salmonella spp., Shigella, Campylobacter, Vibrio, Yersinia pestis, Pseudomas spp., Brucella, Haempohilus, Legionella or Bortedella. Other bacteria that can be detected include Mycobacteria such as M. tuberculosis, M. leprae or M. avium, Rickettsia, or Chlamydia.
In some embodiments, the pathogen may be a virus. Typical viruses that can be detected include: DNA viruses such as adenovirus, herpesvirus, poxvirus, parvovirus, papilloma virus or hepatitis, for example hepatitis B; or RNA viruses such as influenza, coronaviruses, paramyxovirus, picornavirus (e.g. polio, coxsackie, hepatitis A, rhinovirus), togaviruses (e.g. rubella), flaviviruses (e.g. causing yellow fever, dengue fever), rhabdoviruses (e.g. rabies), ebolavirus, or retroviruses such as HIV.
Pathogenic fungi that can be detected include Candida albicans, Aspergillus or Pneumocystis.
Pathogenic protozoa that can be detected include Leishmania, Plasmodium, Trypanosoma, Toxoplasma gondii or Crytosporidium.
In certain embodiments, the pathogen to be detected is a rhinovirus, coronavirus, influenza virus, adenovirus, or respiratory syncytial virus. In an exemplary embodiment, the pathogen to be detected is a coronavirus, more typically a human coronavirus such as the 2019-nCOV.
Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.
It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.
Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.
All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Detection of Glial Fibrillar Acidic Protein and Other Proteins as an Example of Detection Using Amyloid-Based Biomaterials
Amyloid fibrils are formed via the amyloidogenesis process, by which peptide or protein monomers aggregate into higher order aggregates. Although amyloid fibrils are often associated with human degenerative pathologies (such as Alzheimer's and Parkinson's diseases), they can perform physiological roles. Moreover, amyloids have also been suggested as potential novel biomaterials. Short amyloidogenic peptide sequences that form stable fibrils in physiological conditions are ideal for nanotechnology and nanomedicine, for example as bioactive gels and/or biosensing platforms. For that, amyloidogenic molecules must be able to be functionalized with a relevant chemical group, for example biotin. Biotin binds to streptavidin (and its homologous avidin), forming the strongest protein-ligand non-covalent interaction in Nature. For this reason, this interaction system has been widely used in many applications, both as initial proof-of-concept and in fully mature technologies.
Here we describe a new technology, based in the production, from short amyloidogenic peptides, of stable amyloid fibrils at physiological pH and temperature conditions.
As demonstrated in the non-limiting experiments reported below, these stable fibrils can be derivatized with biotin and are functional and able to bind streptavidin, both when the later is free in solution and when it is immobilized on a surface.
Subsequently, these functional fibrils are employed in a proof-of-concept experiment to detect immobilized Glial Fibrilar Acidic Protein (GFAP) in tissue samples, in a dot blot immunoassay. Briefly, GFAP detection and amplification is enabled by streptavidin-labeled primary antibodies and our biotin-labeled peptide fibrils, to which streptavidin-labeled reporter molecules later bind. After that, a similar approach was applied with streptavidin labeled secondary antibodies, raised against different Immunoglobulin G (IgG) primary antibodies, immobilized in a dot blot assay. Several combinations of animal species as sources of primary and secondary IgG antibodies were used, showing the general applicability of the approach.
Finally, with another specific target, Salmonella spp., we employed this amyloid-based technology for the target detection in both a dot blot and a standard enzyme linked immunosorbent assay (ELISA) formats, demonstrating its applicability in yet another model system.
All of these experimental results demonstrate, in different detection, amplification and immunodiagnostics assay formats, the general applicability of the technology.
Materials and Methods
Chemicals
The peptides used (STVIIE, GNNQQNY, QVQIIE and ISFLIF) were purchased from JPT Peptide Technologies (JPT Peptide Technologies GmbH, Berlin, Germany) with a purity of 95% while Aβ(1-42) peptide was purchased Phoenix Pharmaceuticals Inc (Phoenix Pharmaceuticals, Inc., California, USA) with a purity over 97%. Congo red was purchased in Sigma (Sigma-Aldrich Quimica, S. L., Sintra, Portugal). N-terminus biotinylated STVIIE were also commercially obtained from JPT Peptide Technologies (JPT Peptide Technologies GmbH, Berlin, Germany). Tris (tris(hydroxymethyl)aminomethane) and EDTA (ethylenediaminetetraacetic acid) were purchased from Merck (Merck KGaA, Darmstadt, Germany) and Sigma (Sigma-Aldrich Quimica, S. L., Sintra, Portugal), respectively.
Sample Preparation
STVIIE, GNNQQNY, QVQIIE and ISFLIF were prepared at the highest final concentration that they were able to be fully dissolved, respectively 1 mg/mL (STVIIE and GNNQQNY), 0.55 mg/mL (QVQIIE) and 0.1 mg/mL (ISFLIF). For all peptide samples the final incubation buffer was 50 mM Tris-HCl pH 7.5, 5 mM EDTA buffer. Briefly, after weighing the peptide, half of the final volume of H2O ultrapure was added. A short vortex of approximately 1500 rpm and 30 seconds and an ultrasound bath of 280 seconds in water/ice were performed two times. The other half of the final volume was then added, containing 100 mM Tris-HCl pH 7.5, 10 mM EDTA buffer was added, with the peptide samples being therefore in the final incubation buffer. Before incubation, two other cycles of vortex and ultrasound bath were performed. The final peptide solution sample was incubated at room temperature.
Circular Dichroism Spectroscopy
Following previous approaches11,53, circular dichroism (CD) measurements were carried out in a JASCO spectropolarimeter J-815 (Tokyo, Japan), using cuvettes of 1.0 mm path length. Spectra were acquired between 195 and 260 nm, at 25.0° C., with data pitch of 0.5 nm, wavelength sampling velocity of 200 nm min−1, data integration time of 1 s and performing at least 3 accumulations. Measurements were conducted in 50 mM Tris-HCl pH 7.5, 5 mM EDTA buffer. In addition to blank subtraction, experimental instrument-related baseline drift was corrected by subtracting to all spectra the average of the signal between 250 and 260 nm. The spectra were normalized to mean residue molar ellipticity (deg cm2 dmol−1 residue−1). All conditions were measured independently and in triplicate.
Fourier Transform Infra Red (FTIR) Spectroscopy
Following previous approaches11,54, peptide samples were applied to the FTIR sample holder at 298.15 K. InfraRed spectra were recorded on a Bruker Tensor 27 infrared spectrophotometer (Bruker Optik GmbH, Ettlingen, Germany) equipped with a Bio-ATR II accessory. The spectrophotometer was continuously purged with dried air. Spectra were recorded at a spectral resolution of 4 cm−1 and 120 accumulations were performed per measurement. The final spectra were corrected to the baseline (the final incubation buffer) and rescaled in the amide I area (˜1600 to ˜1700 cm−1).
Congo Red Assay
In line with previous work55,56, before measurements, peptides were diluted ten times from their stock incubation conditions in 50 mM Tris-HCl pH 7.5, 5 mM EDTA buffer and incubated with κ μM Congo red for 1 hour. The readings were performed using a UV-Vis Spectrophotometer Shimadzu UV-2700 (Shimadzu Corporation, Kyoto, Japan) with a wavelength between 300 nm and 700 nm. Readings were recorded in triplicate. The fibrillation kinetics was recorded in triplicate for a total period of 4 weeks of incubation.
Atomic Force Microscopy
According to experimental procedures at the host lab57-59, the samples were placed in poly-1-lysine slides and dried using a vacuum chamber. When fully dried, the samples were rinsed with ultra-pure water and dried with a gentle N2 air flux. A NanoWizard II atomic force microscope (JPK Instruments, Berlin, Germany), mounted on the top of an Axiovert 200 inverted optical microscope (Zeiss, Jena, Germany), was used for the microscopic experiments. The AFM head is equipped with a 5.85-μm z-range linearized piezoelectric scanner and an infrared laser. For the acquisition of the different AFM images, it was used a ACL-50 tip (Applied NanoStructures, Inc., California, USA) with a spring constant between 20-95 N/m and a frequency between 145-230 kHz. Amyloid fibrils were acquired in intermittent (air) mode with a setpoint of between 0.4-0.5 V, a line rate of 0.7-0.8 Hz, an IGain of 20-50 Hz and a PGain of 0.001-0.004. The images acquired were treated afterwards with the JPKSPM Data Processing (JPK Instruments, Berlin, Germany).
Morphological Characterization of the Amyloid Fibrils
The morphological characterization of the AFM images of ISFLIF amyloid fibrils with and without biotin was carried out in the program Gwyddion 2.31. Using the extract profiles command cross lines were drawn in the fibrils surface allowing the determination of the fibrils height and width. The height and width values determined resulted from the average of 20 individual fibers from at the least three different fibrils AFM images. The height and width values were presented as the mean with the associated standard error of the mean (SEM).
Cell Culture and Cytotoxicity Assays
Human H4 neuroglioma cells were maintained at 37° C. in OPTI-MEM I (Gibco, Invitrogen, Barcelona, Spain) supplemented with 10% fetal bovine serum and seeded onto 24-well plates at a density of 60.000 cells/cm2 24 h prior treatment. Cells were treated with 0.2, 2 and 20 μM of fibrillated STEVIIE, its biotin conjugated species, biotin and vehicle for 6 and 24 hours. Conditioned media of treated cells was collected, and cytotoxicity immediately assessed by measuring the activity of released lactate dehydrogenase (LDH) in a plate reader (Tecan Infinite 200), according to the manufacturer's protocol (Clontech). The maximum activity was determined by lysing the cells with triton X-100 (final concentration 1%).
Dot-Blot Assays
A vacuum-based Dot Blot 48-sample apparatus is applied in the dot blot technique to immobilize samples onto the membrane. A polyvinylidene difluoride (PVDF) membrane is activated by submersion in methanol for 30 sec and then washed in distilled water and saline buffer to remove methanol. Samples are diluted in a saline buffer and immobilized on the PVDF membrane. Membrane is then blocked with 5% of bovine serum albumin at room temperature for 60 min in the 2D rotator in order to decrease non-specific binding. After blocking, three wash cycles are performed for 10 min each, the first two with a saline buffer plus a mild detergent and the final cycle with saline buffer alone. Depending on the purpose of the dot blot assay, antibodies are incubated with DART technology for further samples detection. The reporter molecule used is HRP conjugated to streptavidin. Finally, the substrate Clarity™ ECL is incubated with the membrane and then metabolized by the enzyme. A dot-shaped visible signal is produced and detected by chemiluminescence, indicating a positive result, using the Bio-Rad ChemiDoc™ XRS+ apparatus.
Results
A peptide that forms fibrils with stable amyloid morphologies in physiological conditions of pH and temperature is preferred, in order to be compatible with most biologically relevant protein-ligand and antibody-ligand interactions. It is important to have a short peptide sequence (to keep costs low) that readily dissolves in water (instead of hydrophobic solvents) at high concentrations. If stable fibrils are formed it is then possible to develop nanotechnology applications, namely biomolecule detection and/or signal amplification. The approach scheme is shown in
The approach starts from ideal peptide sequences (known to be amyloidogenic) and tests their ability to form amyloid fibrils in physiological conditions (
Biotin was used to functionalise amyloid fibrils, streptavidin used as bridge element, horseradish peroxidase (HRP) linked to streptavidin as reporter molecule, and streptavidin-labeled primary antibodies as recognition elements. Variations of this approach may be employed, with other bridge elements, reporter molecules and/or recognition elements (in particular other antibodies and derivatized DNA or RNA detection sequences, to identify and target other specific biomarkers). These results provide proof-of-concept, establishing the feasibility of the technology.
The most promising amyloidogenic peptide is typically selected. For this purpose, atomic force microscopy (AFM) was employed. AFM is a technique that can be very useful in the acquisition of surface images with high resolution and sensibility, being therefore suitable for such studies of amyloid fibrils and employed in this analysis. Representative AFM images typical of each sample are displayed in
STVIIE amyloid fibril formation process in physiological conditions of pH and temperature was further characterized (
Regarding STVIIE amyloid fibrils, it is clear that they display repetitive patterns of specific sizes (
The formation of aggregates by STVIIE quickly leads the larger elements to deposit from solution. This hinders circular dichroism studies of STVIIE secondary structure. For this reason Fourier transform infra-red (FTIR) spectroscopy was used instead (
Cross β-sheet secondary structure is a good indicator of amyloid presence (
With the above in mind, STVIIE was selected for functionalization studies (
Given the above, biotin-PEG9-STVIIE preparations were further studied (
Overall, streptavidin fluorescence emission spectra in the presence of free biotin and of biotinylated peptide reveal similar profiles, demonstrating that the biotin moiety that is linked to the peptide fibrils binds to the streptavidin free in solution, as intended. Moreover, that binding to free streptavidin is almost as large as that of free biotin moieties.
Following, the binding of the biotinylated peptide fibril preparations to immobilized streptavidin was investigated, by observing infrared spectral changes occurring on immobilized streptavidin, as a result of interaction with biotin ligands (
Finally, it was necessary to demonstrate that biotinylated fibrils such as the ones designed can be employed in nanotechnology, for example, in signal amplification assays. This approach was followed, first in the context of antibody-mediated recognition events of a specific ligand, in a dot-blot immunoassays test format (
Therefore, to fully confirm with a relevant biological sample, the approach was used to detect another protein glial fibrillary acidic protein (GFAP), a glial specific protein. First, in a direct (with a single antibody) immunochemistry dot blot assay, GFAP was immobilized on a membrane surface, detected by a specific streptavidin-derivatized anti-GFAP antibody and, then, the biotin labelled amyloid fibrils are used for signal amplification (of single detection events), lowering detection thresholds (
The approach was then tested in a direct blot assay with biopsy-like samples, i.e., tissue extracts from mice, namely liver, where GFAP is mostly absent, and brain, where it is abundant (
At this point, with all the data and information described from
Another variation concerns using mixed preparations of amyloid fibrils (
Salmonella spp. is also detected (namely Salmonella enterica Tiphymurium), via amyloid based signal amplification, in an indirect ELISA test format, as shown in the experiment schematics (
The results of using other amyloidogenic sequences besides STVIEE are also shown here, all incubated in physiological conditions as described above (
Amyloid toxicity, although initially thought to be caused by mature fibrils, has been demonstrated to be mostly associated with the fibrils precursors (oligomers and protofibrils)1-10. This knowledge, alongside with amyloid fibrils chemical and mechanical stability, triggered the interest in amyloid fibrils as biomaterials. For such purposes, short amyloid peptides are better than longer expensive sequences. It is also important that amyloid fibrils that are formed and remain stable in physiological pH and temperature conditions. Moreover, they should be modifiable with chemical moieties to add them new functions if desired.
The data provided herein demonstrate that, among other candidate peptides, STVIIE is able to form stable fibrils in physiological conditions. The data also demonstrate that biotin can be added to the N-terminus of the STVIIE peptide via a PEG9 linker, resulting in mature and well-structured amyloid fibrils. It is also shown that not only does this modified peptide remain able to form amyloid fibrils but also that it acquires a new function, becoming able to bind to free and immobilized streptavidin.
By employing streptavidin labelled antibodies and reporter molecules, the data demonstrate that the biotinylated peptide produced can be employed to detect the glial fibrillar acidic protein at low concentrations, inclusively in cell extracts. Moreover, the inventors have also demonstrated the ability of the biotinylated peptide to bind and detect other immobilized proteins, namely biologically relevant IgG molecules. The data yet further show that biotin-PEG13-STVIIE, as well as mixed preparations, of biotin derivatized and of free peptide, form amyloid fibrils that detect and enable signal amplification in immunoassays.
A bacteria, Salmonella spp., was also detected, namely in an ELISA format, via the amyloid-based nanomaterials. These comprehensive data show the biological activity, usefulness, and applicability of the described biomaterial, enabling nanotechnology applications employing these and similar peptides (modified and functionalized, as described or in similar ways), namely for uses in biosensing and/or signal amplification technologies.
It is understood that the Examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 116112 | Feb 2020 | PT | national |
| 2002343.8 | Feb 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/053235 | 2/10/2021 | WO |
