Patent Application
20210285974
This disclosure relates generally to a method for polymerizing polyphenols, such as dopamine and its derivatives. In certain embodiments, the present disclosure relates to a method for depositing a polyphenol polymer (e.g., polydopamine) on a surface by polymerizing a polyphenol (e.g., dopamine or a dopamine derivative), to a method for detecting an analyte by polymerizing a polyphenol (e.g., dopamine or a dopamine derivative), and to an assay kit comprising a polyphenol (e.g., dopamine or a dopamine derivative).
As recent advances in medicine rapidly unravel the genomic and proteomic signatures of disease development, progression, and response to therapy, sensitive and quantitative analysis of disease biomarkers (e.g., DNA, RNA, and proteins) has become increasingly important in the era of precision medicine where diagnostic and therapeutic decisions are tailored towards individual patients. In parallel, to address the challenge in sensitive and multiplexed biomarker analysis, a large variety of exquisitely designed imaging and detection technologies have also been developed in the past decade. These enabling technologies, often leveraging the unique properties of colloidal nanostructures (e.g., quantum dots, magnetic nanoparticles, and plasmonic nanoparticles) and precisely engineered sensor devices (e.g., nanowire sensors, cantilevers, and microfluidic channels) are so sensitive that their detection limits are commonly seen at the single-molecule level, where low-abundance targets such as circulating oligonucleotides, proteins, viruses, and cells can be enumerated with polymerase chain reaction (PCR)-like sensitivity. Despite these remarkable achievements in biotechnology laboratories, broad adoption of these technological innovations by biological and clinical laboratories, and consequently, the impact thereof, has been limited. Resistance to adoption stems from multiple factors, including complex protocols and specialized reagents and equipment. Moreover, these technologies require new infrastructure, which increases up-front adoption costs, and reduces persistent output and cross-laboratory cross-platform consistency.
Accordingly, there remains a need for high-sensitivity detection methods that avoid specialized reagents or equipment, and/or can be performed with minimal alteration to existing laboratory infrastructure.
One aspect of the disclosure is a method for polymerizing a polyphenol, including:
Another aspect of the disclosure is a method for depositing a polyphenol polymer on a surface, the method including
Another aspect of the disclosure is a method for detecting an analyte, the method including
Another aspect of the disclosure is an assay kit, including
Other aspects of the disclosure will be evident from the disclosure herein.
The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. Thus, before the disclosed processes and devices are described, it is to be understood that the aspects described herein are not limited to specific embodiments, apparati, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.
The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.
As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. As used herein, the transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Some embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the cited references and printed publications are individually incorporated herein by reference in their entirety.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
In various aspects and embodiments, the disclosure relates to a method for polymerizing a polyphenol, including providing a polyphenol, providing an enzyme having a peroxidase-like activity, contacting the polyphenol and an oxidant with the enzyme having peroxidase-like activity, under conditions sufficient to polymerize the polyphenol. The present inventors have determined that enzymes with peroxidase-like activity (such as peroxidases, phosphatases, and ribozymes) can greatly speed the rate of polymerization of polyphenols such as dopamine, providing polyphenol polymers such as polydopamine at fast rates.
As described in detail below, this discovery allows for the targeted deposition of polyphenol polymers at a surface. But, in other embodiments, the polymerization can be performed using aqueous solution-phase chemistry to provide polyphenol polymer. In many embodiments, the polyphenol polymer will precipitate from aqueous solution to form a solid polymer, which can be collected for use in a separate process, or can be allowed to deposit on a surface in contact with the aqueous solution (e.g., in a non-targeted manner). Accordingly, the methods described herein can be used to form surface coatings of polyphenol polymers on a variety of surfaces, or to form polymer that is collected and used in a further process. The person of ordinary skill in the art will determine appropriate reaction conditions based on the disclosure herein. For example, in certain embodiments as otherwise described herein, the polyphenol is present in the reaction mixture in a concentration in the range of 1-100 mg/mL, e.g., 1-75 mg/mL, 1-50 mg/mL, 1-25 mg/mL, 5-100 mg/mL, 5-75 mg/mL, 5-50 mg/mL, 5-25 mg/mL, 10-100 mg/mL, 10-75 mg/mL or 10-50 mg/mL. In certain embodiments as otherwise described herein, the oxidant is present in the reaction mixture in an amount in the range of 0.005-2 M, e.g., in the range of 0.005-1 M, or 0.005-0.5 M, or 0.005-0.1 M, or 0.01-2 M, or 0.01-1M, or 0.01-0.5 M, or 0.01-0.1 M. The reaction can be conducted at a variety of pH values, e.g., in the range of 1-11, or 4-11, or 7-11, or 7-9.
In one aspect, the disclosure relates to a method for depositing a polyphenol polymer (e.g., polydopamine) on a surface. The method includes providing, at a target site, an enzyme having peroxidase-like activity immobilized at the surface, and polymerizing, at the target site, a polyphenol (e.g., dopamine or a dopamine derivative) in the presence of an oxidant and the enzyme to provide the polyphenol polymer, deposited on the surface. The disclosure demonstrates that such a method provides for rapid deposition of commonly available materials onto a surface.
While the examples described below focus on the use of dopamine and derivatives thereof (including conjugates thereof), based on the present disclosure the person of ordinary skill in the art will understand that the methods described herein can be used to polymerize a variety of polyphenols. As used herein, a “polyphenol” is a compound having a polyhydroxyphenyl moiety, e.g., a dihydroxyphenyl moiety or a trihydroxyphenyl moiety (e.g., as a substituent or fused as part of a ring system). Examples of polyphenols include dopamine and dopamine derivatives as described below. Other examples of polyphenols include elegeic acid, theaflavin-3-gallage, gallic acid, tannic acid, pyrogallol, catechol, catechin, epigallocatechin, epigallocatechin, quercetin, morin, naringenin, rutin, naringin, phloroglucinol, hydroquinone, resorcinol, hydroxyhydroquinone, resveratrol, as well as derivatives of these materials (such as conjugates thereof). The person of ordinary skill in the art will appreciate that derivatives of polyhydroxyphenyl-bearing compounds can include any modified that is capable of polymerizing to provide a polyphenol polymer. The methods can be used, for example, with extracts of materials such as green tea, black tea, cacoa bean, and red wine. In certain embodiments, the polyphenol has a molecular weight of no more than 1000 g/mol, e.g., no more than 800 g/mol, or even no more than 500 g/mol. As used herein, a polyphenol polymer is a polymer of a polyphenol, e.g., a homopolymer of a single polyphenol or a copolymer of a plurality of different polyphenols.
In certain embodiments of the disclosure, the polyphenol is dopamine or a derivative thereof. As described in more detail below, a polyphenol polymer formed by polymerization of dopamine or a derivate thereof (i.e., a “polydopamine”) can have a high optical density at certain wavelengths, which can advantageously allow for optical detection of the degree of polymerization. As used herein, the term “dopamine derivative” includes covalently modified dopamine (e.g., ortho or meta to the aminoethyl group), and dopamine otherwise conjugated to a chemical moiety (e.g., a fluorescent tag, biotin, etc.). The person of ordinary skill in the art will appreciate that the dopamine derivatives of the methods described herein may be any modified dopamine compound that is capable of polymerizing to provide a polydopamine.
For example, in certain embodiments, the polyphenol has the structure A or B below
in which X is OH, O(C1-C4 alkyl), (C1-C4 alkyl), preferably OH; Y is NH2, biotin, PEG-linked biotin, or a fluorophore moiety; and Z is COOH, NH2, biotin, PEG-linked biotin, or a fluorophore moiety.
As used herein, the term “polydopamine” refers to a polymer of dopamine or a dopamine derivative, e.g., a homopolymer of polydopamine or a derivative thereof, or a copolymer of a plurality of polyphenols including polydopamine or a derivative thereof. The person of ordinary skill in the art will appreciate that the term “polydopamine” includes the polymerization product of dopamine or a dopamine derivative provided by the methods described herein.
As described above, in one aspect of the methods of the disclosure, the deposition method includes providing, at a target site, an enzyme having peroxidase-like activity immobilized at a surface. In certain embodiments of the methods as otherwise described herein, the enzyme is adsorbed onto the surface. For example, in certain embodiments of the methods as otherwise described herein, the enzyme is absorbed onto a membrane, e.g., a nitrocellulose membrane. In certain embodiments of the methods as otherwise described herein, the enzyme is linked to the surface via a streptavidin-biotin interaction. In certain embodiments of the methods as otherwise described herein, the enzyme is linked to the surface via an antibody-antigen interaction. In certain embodiments of the methods as otherwise described herein, the enzyme is linked to the surface via a silane coupling agent. For example, in certain embodiments of the methods as otherwise described herein, the enzyme is linked to a silica surface via a trialkoxysilane moiety.
Of course, as described above, other embodiments provide polymerization methods in which the enzyme having peroxidase-like activity is not immobilized at a surface. For example, in various embodiments, the enzyme having peroxidase-like activity is in aqueous solution or suspension when it is contacted with the polyphenol and the oxidant.
Another aspect of the disclosure is method for detecting an analyte. In various aspects and embodiments, the disclosure demonstrates the method to be compatible with virtually all common biodetection and bioimaging techniques (see, e.g., Table 16, below), and capable of providing sensitivities that are orders of magnitude higher than those conventional techniques. The method includes providing a sample comprising the analyte and a primary detection reagent, linked to an enzyme having peroxidase-like activity, and incubating the sample in the presence of the primary detection reagent to provide a target site comprising a complex of the analyte and the detection reagent. The method also includes polymerizing, at the target site, a polyphenol (e.g, dopamine or a dopamine derivative) in the presence of an oxidant and the enzyme to provide a polyphenol polymer (e.g., polydopamine), and detecting the presence of the polyphenol polymer (e.g., the polydopamine). In various aspects and embodiments, certain embodiments of the methods as otherwise described herein are referred to as enzyme-accelerated signal enhancement (EASE).
The person of ordinary skill in the art will appreciate that polyphenol polymers such as polydopamines are versatile coating materials in a variety of surface treatment fields. For example, self-adherent polydopamine films have been shown to form spontaneously, but slowly, on a wide range of surfaces using a dip-coating protocol. Advantageously, the present inventors have determined that the rate of polymerization of polyphenols such as dopamine and dopamine derivatives is increased by a factor of hundreds in the presence of an enzyme having peroxidase-like activity (e.g., horseradish peroxidase (HRP): see
As described above, in one aspect of the methods of the disclosure, the detection method includes providing a primary detection reagent, linked to an enzyme having peroxidase-like activity. In certain embodiments of the methods as otherwise described herein, the primary detection reagent comprises an antibody. For example, in certain embodiments of the methods as otherwise described herein, the primary detection reagent comprises a monoclonal antibody, e.g., a monoclonal antibody to another antibody, to a human immunodeficiency virus (HIV) antigen (such as, for example, p24), a corticotrophin releasing factor (CRF) receptor, a Zika virus (ZIKV) antigen, or an immune regulatory antigen (such as, for example, PD-L1). In certain embodiments of the methods as otherwise described herein, the primary detection reagent comprises streptdavidin. In certain embodiments, the primary detection reagent comprises a peptide, an oligonucleotide, or a derivative thereof (e.g., biotin-labeled deriviatives).
In certain embodiments of the methods as otherwise described herein, the primary detection reagent is capable of binding the analyte. In other embodiments of the methods as otherwise described herein, the detection method further comprises providing an intermediate detection reagent capable of binding the analyte. In certain such embodiments, the detection reagent is capable of binding the intermediate detection reagent, and incubation is further in the presence of the intermediate detection reagent, to provide a target site comprising a complex of the analyte, intermediate detection reagent, and primary detection reagent. For example, in certain embodiments of the methods as otherwise described herein, the intermediate detection reagent comprises an antibody. For example, in certain embodiments of the methods as otherwise described herein, the intermediate detection reagent comprises a monoclonal antibody, e.g., a monoclonal antibody to a human immunodeficiency virus (HIV) antigen (such as, for example, p24), a corticotrophin releasing factor (CRF) receptor (such as, for example, CRFR1), a Zika virus (ZIKV) antigen, or an immune regulatory antigen (such as, for example, PD-L1). In certain embodiments of the methods as otherwise described herein, the primary detection reagent comprises a monoclonal antibody, e.g., a monoclonal antibody to another antibody, to a prostate-specific antigen (kallikrein-3 (KLK3)), to a c-reactive protein (CRP), to a vascular endothelial growth factor (VEGF), to a human immunodeficiency virus (HIV) antigen (such as, for example, p24), a corticotrophin releasing factor (CRF) receptor, a zika virus (ZIKV) antigen, or an immune regulatory antigen (such as, for example, PD-L1). In certain embodiments of the methods as otherwise described herein, the intermediate detection reagent comprises a biotin-labeled affinity molecule.
As described above, in one aspect of the methods of the disclosure, the method includes providing a sample comprising the analyte. In certain embodiments of the methods as otherwise described herein, the analyte is immobilized on a cell surface, or localized in a cell compartment (e.g., an immunohistochemistry or immunofluorescence analyte, e.g., Lamin A or heat shock protein (HSP)-90). In certain embodiments of the methods as otherwise described herein, the analyte is bound to a capture reagent, the capture reagent immobilized on a solid support (e.g., a sandwich-assay analyte, e.g., an enzyme-linked immunosorbent assay (ELISA) analyte, e.g., KLK3, CRP, VEGF, p24, CRFR1, a ZIKV antigen, or PD-L1) In certain such embodiments, the capture reagent comprises an antibody, e.g., a monoclonal antibody. In certain such embodiments, the solid support comprises a microsphere.
As described above, in one aspect of the methods of the disclosure, the method includes detecting the presence of the polyphenol polymer (e.g., polydopamine). In certain embodiments of the methods as otherwise described herein, detection comprises measuring the absorption or emission of the polyphenol polymer (e.g., polydopamine). For example, in certain embodiments of the methods as otherwise described herein, measuring the absorption or emission of the polyphenol polymer (e.g., polydopamine) comprises observing the color change of a target site caused by the absorption of the polyphenol polymer (e.g., polydopamine) after polymerization. In another example, in certain embodiments of the methods as otherwise described herein, measuring the absorption or emission of the polyphenol polymer (e.g., polydopamine) comprises quantitatively measuring the emission of the polyphenol polymer (e.g., polydopamine) polymerized from a polyphenol comprising a fluorescent tag (e.g., dopamine conjugated to a fluorescent tag).
In certain embodiments of the methods as otherwise described herein, the detection method further comprises incubating the polydopamine in the presence of a secondary detection reagent. For example, in certain embodiments of the methods as otherwise described herein, the secondary detection reagent comprises an enzyme capable of catalyzing the conversion of a chromogenic substrate (e.g., HRP and enzyme conjugates HRP-streptavidin and streptavidin-poly HRP). In certain such embodiments, detection comprises measuring the absorption or emission of the chromogenic substrate (e.g., 3,3′,5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB), or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)). Advantageously, the present inventors have determined that reactivity of a polydopamine towards the amine, sulfhydryl, and phenol groups of polypeptides allows for localization at the target site of a high concentration of the enzyme capable of catalyzing the conversion of a chromogenic substrate. In another example, in certain embodiments of the methods as otherwise described herein, the secondary detection reagent comprises an amine-functionalized tag. Similarly advantageously, the present inventors have determined that reactivity of a polyphenol polymer (e.g., polydopamine) towards amine groups allows for localization at the target site of a high concentration of the amine-functionalized tag. In certain such embodiments, the amine-functionalized tag comprises a quantum dot. In other such embodiments, the amine-functionalized tag comprises an amine-functionalized dye (e.g., a fluorescent dye, e.g., cyanine 3 (Cy3)). In certain such embodiments, detection comprises measuring the absorption or emission of the secondary detection reagent.
In certain embodiments of the methods as otherwise described herein, the detection method comprises providing a sample comprising the analyte, the analyte immobilized on a cell surface or localized in a cell compartment, an intermediate detection reagent (e.g., a monoclonal antibody) capable of binding the analyte, and a primary detection reagent (e.g., a monoclonal antibody) linked to an enzyme having peroxidase-like activity (e.g., HRP). In certain such embodiments, the method further includes incubating the sample in the presence of the intermediate detection reagent, to provide a target site comprising a complex of the analyte and intermediate detection reagent. In certain such embodiments, the method further includes incubating the sample in the presence of the primary detection reagent, to provide a target site comprising a complex of the analyte, the intermediate detection reagent, and the primary detection reagent. In certain such embodiments, the method further includes polymerizing, at the target site, a polyphenol (e.g., dopamine or a dopamine derivative) in the presence of an oxidant (e.g., H2O2) and the enzyme to provide a polyphenol polymer (e.g., polydopamine), and detecting the presence of the polyphenol polymer. In certain such embodiments, detection comprises measuring the absorption or emission of a polyphenol polymer (e.g., polydopamine). In other such embodiments, the method further includes incubating the polyphenol polymer (e.g., polydopamine) in the presence of a secondary detection reagent (e.g., an amine-functionalized tag, e.g., an amine-functionalized quantum dot). In certain such embodiments, detection comprises measuring the absorbance or emission of the secondary detection reagent.
In certain embodiments of the methods as otherwise described herein, the detection method comprises providing a sample comprising the analyte (e.g., an analyte comprising biotin), the analyte bound to a capture reagent (e.g., a monoclonal antibody), the capture reagent immobilized on a microsphere, and a primary detection reagent (e.g., streptavidin) linked to an enzyme having peroxidase-like activity (e.g., HRP). In certain such embodiments, the method further includes incubating the sample in the presence of the primary detection reagent, to provide a target site comprising a complex of the analyte and the primary detection reagent. In certain such embodiments, the method further includes polymerizing, at the target site, a polyphenol (e.g., dopamine or a dopamine derivative) in the presence of an oxidant (e.g., H2O2) and the enzyme to provide a polyphenol polymer (e.g., polydopamine), and detecting the presence of the polyphenol polymer. In certain such embodiments, the method further includes incubating the polyphenol polymer (e.g., polydopamine)in the presence of a secondary detection reagent (e.g., an amine-functionalized tag, e.g., an amine-functionalized quantum dot). In certain such embodiments, detection comprises measuring the absorption or emission of the secondary detection reagent.
In certain embodiments of the methods as otherwise described herein, the detection method comprises providing a sample comprising the analyte, the analyte bound to a capture reagent (e.g., a monoclonal antibody), the capture reagent immobilized on a solid support, and a primary detection reagent (e.g., a monoclonal antibody) linked to an enzyme having peroxidase-like activity (e.g., HRP). In certain such embodiments, the primary detection reagent is capable of binding the analyte. In certain such embodiments, the method further includes incubating the sample in the presence of the primary detection reagent, to provide a target site comprising a complex of the analyte and the primary detection reagent. In certain such embodiments, the method further includes polymerizing, at the target site, a polyphenol (e.g., dopamine or a dopamine derivative) in the presence of an oxidant (e.g., H2O2) and the enzyme to provide a polydopamine, and detecting the presence of polydopamine. In certain such embodiments, the method further includes incubating the polyphenol polymer (e.g., polydopamine) in the presence of a secondary detection agent comprising an enzyme capable of catalyzing the conversion of a chromogenic substrate (e.g., HRP). In certain such embodiments, detection comprises measuring the absorption or emission of the chromogenic substrate (e.g., DAB).
As described above, in one aspect of the methods of the disclosure, the method includes providing a sample comprising the analyte. In certain embodiments of the methods as otherwise described herein, the analyte is a Lamin antigen, e.g., Lamin A. In certain embodiments of the methods as otherwise described herein, the analyte is a heat shock protein (HSP), e.g., HSP-90. In certain embodiments of the methods as otherwise described herein, the analyte is a kallikrein 3 (KLK3) antigen. In certain embodiments of the methods as otherwise described herein, the analyte is a C-reactive protein (CRP). In certain embodiments of the methods as otherwise described herein, the analyte is a vascular endothelial growth factor (VEGF) antigen. In certain embodiments of the methods as otherwise described herein, the analyte is a human immunodeficiency virus (HIV) antigen, e.g., p24. In certain embodiments of the methods as otherwise described herein, the analyte is a corticotrophin releasing factor (CRF) receptor, e.g., CRFR1. In certain embodiments of the methods as otherwise described herein, the analyte is a zika virus (ZIKV) antigen. In certain embodiments of the methods as otherwise described herein, the analyte is an immune regulator antigen, e.g., programmed death-ligand 1 (PD-L1).
As described above, the present inventors have determined that the various aspects and embodiments of the methods described herein are compatible with virtually all common biodetection and bioimaging techniques. For example, in certain embodiments of the methods as otherwise described herein, the sample comprising an analyte bound to a capture reagent, the capture reagent immobilized on a solid support, comprises the capture surface that could otherwise be utilized in a conventional sandwich ELISA method. In another example, in certain embodiments of the methods as otherwise described herein, the sample comprising an analyte bound to a capture reagent, the capture reagent immobilized on a microsphere, comprises the capture surface that could otherwise be utilized in a conventional suspension microarray method. In yet another example, in certain embodiments of the methods as otherwise described herein, the sample comprising an analyte immobilized on a cell surface or localized in a cell compartment comprises the cell sample that could otherwise be utilized in a conventional immunohistochemistry or immunofluorescence assay method. The person of ordinary skill in the art would appreciate that, in such embodiments, the analyte may be any antigen for which a conventional detection method exists, or for which a conventional detection method may be developed.
As described above, in various aspects of the methods of the disclosure, the method includes providing an enzyme having peroxidase-like activity (e.g., provided at a target site, the enzyme immobilized at a surface, or provided linked to a primary detection reagent, or in solution or suspension). In certain embodiments of the methods as otherwise described herein, the enzyme having peroxidase-like activity is a polypeptide. For example, in certain embodiments of the methods as otherwise described herein, the enzyme having peroxidase-like activity is a peroxidase, such as horseradish peroxidase (HRP). In other embodiments of the methods as otherwise described herein, the enzyme having peroxidase-like activity is a phosphatase, such as an alkaline phosphatase. In certain embodiments of the methods as otherwise described herein, the enzyme having peroxidase-like activity comprises a ribozyme or deoxyribozyme. The person of ordinary skill in the art will appreciate that other enzymes may provide sufficient peroxidase-like activity to catalyze the oxidative polymerization of polyphenols as described herein.
As described above, in various aspects of the methods of the disclosure, the method includes polymerizing (e.g., at the target site) a polyphenol (e.g., dopamine or a dopamine derivative). In certain embodiments of the methods as otherwise described herein, the polyphenol includes a fluorescent tag (e.g., a dopamine derivative including dopamine linked to a fluorescent tag). For example, in certain embodiments of the methods as otherwise described herein, the polyphenol includes a quantum dot (e.g., a dopamine derivative comprising dopamine linked to a quantum dot). In another example, in certain embodiments of the methods as otherwise described herein, the polyphenol includes a fluorescent dye (e.g., a dopamine derivative includes dopamine linked to a fluorescent dye). In certain embodiments of the methods as otherwise described herein, the polyphenol includes biotin (e.g., a dopamine derivative including dopamine linked to biotin). In certain embodiments of the methods as otherwise described herein, the method includes polymerizing, at the target site, the polyphenol (e.g., dopamine or a derivative thereof).
As described above, in various aspects of the methods of the disclosure, the method includes polymerizing (e.g., at a target site or otherwise), the polyphenol (e.g., dopamine or a dopamine derivative) in the presence of an oxidant. In certain embodiments of the methods as otherwise described herein, the oxidant is a peroxide such as hydrogen peroxide (H2O2). In other embodiments, other oxidants can be used, e.g., percarbonates.
As described above, in various aspects of the methods of the disclosure, the method includes polymerizing, at the target site, a polyphenol (e.g., dopamine or a dopamine derivative) in the presence of peroxide and an enzyme having peroxidase-like activity. In certain embodiments of the methods as otherwise described herein, the polymerization at the target site is further in the presence of a polypeptide (i.e., other than the enzyme having peroxidase-like activity). Without intending to be bound by theory, the present inventors believe that the polypeptide, comprising groups reactive with polyphenols and polyphenol polymers (e.g., dopamine, a dopamine derivative, and/or a polydopamine) serves to further enhance the polymerization and/or deposition rate of polyphenols in the presence of an oxidant and an enzyme having peroxidase-like activity. For example, in certain embodiments of the methods as otherwise described herein, the polymerization at the target site is further in the presence of bovine serum albumin (BSA). In certain embodiments of the methods as otherwise described herein, the polymerization at the target site is further in the presence of copper or iron. Without intending to be bound by theory, the present inventors believe that iron and/or copper serve to further enhance the polymerization rate of polyphenols derivative in the presence of an oxidant and an enzyme having peroxidase-like activity.
As described above, in various aspects of the methods of the disclosure, the method includes polymerizing (e.g., at a target site or otherwise) a polyphenol (e.g., dopamine or a dopamine derivative) in the presence of peroxide and an enzyme having peroxidase-like activity. In certain embodiments of the methods as otherwise described herein, the polymerization is in a buffer solution. For example, in certain embodiments of the methods as otherwise described herein, the polymerization at the target site is in a Tris buffer solution. In another example, in certain embodiments of the methods as otherwise described herein, the polymerization is in phosphate-buffered saline (PBS). In other embodiments, the buffer is a bicine buffer or a borate buffer. The person of ordinary skill in the art will appreciate that a variety of buffers can be used in the practice of the methods described herein. In certain embodiments of the methods as otherwise described herein, the polyphenol (e.g., dopamine or dopamine derivative) is present in the buffer solution in a concentration within the range of about 1 mM to about 200 mM. For example, in certain embodiments of the methods as otherwise described herein, the polyphenol (e.g., dopamine or dopamine derivative) is present in the buffer solution within the range of about 1 mM to about 190 mM, or about 1 mM to about 180 mM, or about 1 mM to about 170 mM, or about 1 mM to about 160 mM, or about 1 mM to about 150 mM, or about 1 mM to about 140 mM, or about 1 mM to about 130 mM, or about 1 mM to about 120 mM, or about 1 mM to about 110 mM, or about 1 mM to about 100 mM, or about 5 mM to about 200 mM, or about 10 mM to about 200 mM, or about 20 mM to about 200 mM, or about 30 mM to about 200 mM, or about 40 mM to about 200 mM, or about 50 mM to about 200 mM, or about 60 mM to about 200 mM, or about 70 mM to about 200 mM, or about 80 mM to about 200 mM, or about 90 mM to about 200 mM, or about 100 mM to about 200 mM.
As described above, in various aspects of the methods of the disclosure, the method includes polymerizing, at the target site, dopamine or a dopamine derivative in the presence of peroxide and an enzyme having peroxidase-like activity. In certain embodiments of the methods as otherwise described herein, the polyphenol polymer (e.g., polydopamine), deposited by the polymerization, has an optical density of at least about 0.05 at a wavelength of 450 nm or 700 nm. For example, in certain embodiments of the methods as otherwise described herein, the polyphenol polymer (e.g., polydopamine), deposited by the polymerization, has an optical density of at least about 0.1, or at least about 0.25, or at least about 0.5, at a wavelength of 450 or 700 nm (e.g., in a sample having a conventional path length). In certain embodiments of the methods as otherwise described herein, the polyphenol polymer (e.g., polydopamine), deposited by the polymerization, comprises an emission intensity of at least about 10 at a wavelength of 480 nm (e.g., at a conventional excitation wavelength).
Another aspect of the disclosure is an assay kit. In various aspects and embodiments, the disclosure demonstrates the kit to be compatible with virtually all common biodetection and bioimaging techniques. In certain embodiments of the kits as otherwise described herein, the kit includes a primary detection reagent linked to an enzyme having peroxidase-like activity, the primary detection reagent capable of binding an analyte, and dopamine or a dopamine derivative. In certain embodiments of the kits as otherwise described herein, the kit includes an intermediate detection reagent, capable of binding an analyte, a primary detection reagent linked to an enzyme having peroxidase-like activity, the primary detection reagent being capable of binding the intermediate detection reagent, and a polyphenol (e.g., dopamine or a dopamine derivative).
In certain embodiments of the kits as otherwise described herein, the polyphenol is linked to a fluorescent tag or biotin (e.g., a dopamine derivative including dopamine linked to a fluorescent tag or biotin). For example, in certain embodiments of the kits as otherwise described herein, polyphenol is linked to a quantum dot (e.g., a dopamine derivative including dopamine linked to a quantum dot). In another example, in certain embodiments of the kits as otherwise described herein, the polyphenol is linked to a fluorescent dye (e.g., a dopamine derivative including dopamine linked to a fluorescent dye). In certain embodiments of the kits as otherwise described herein, the kit further comprises a secondary detection reagent comprising an amine-functionalized tag or an enzyme capable of catalyzing the conversion of a chromogenic substrate. For example, in certain embodiments of the kits as otherwise described herein, the secondary detection reagent comprises an amine-functionalized quantum dot or an amine-functionalized fluorescent dye, e.g., Cy3. In another example, in certain embodiments of the kits as otherwise described herein, the secondary detection reagent comprises a polypeptide, e.g., horseradish peroxidase.
The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the scope of the disclosure.
All chemicals and biochemicals (unless specified) were purchased from Sigma-Aldrich (St. Louis, Mo.) and used without further purification. 96-well plastic microplates (each microplate consists of twelve removable strips of wells and a frame) were purchased from R&D Systems (Minneapolis, Minn.). Nitrocellulose membranes were purchased from EMD Millipore (Billerica, Mass.uman cervical cancer (HeLa) cell line was purchased from ATCC (Manassas, Va.). Glass-bottom 24-well plates (black wall) were purchased from Greiner Bio-One (Monroe, N.C.). Fetal bovine serum was purchased from FAA laboratories (Dartmouth, Mass.). Casein (5% solution) was purchased from Novagen (Billerica, Mass.). Anti-HSP90 antibody raised in rabbit (LOT: SAB4300541), anti-Lamin A antibody raised in rabbit (LOT: L1293), and anti-GAPDH antibody raised in rabbit (LOT: G9545) were purchased from Sigma-Aldrich (St. Louis, Mo.). CRHR1/CRF1 antibody was purchased from Novas Biologicals (LOT: NLS1778, Littleton, Colo.). Monoclonal rabbit antibodies raised against Ki-67 was purchased from Epitomics (LOT: 42031, Burlingame, Calif.). Monoclonal rabbit antibodies against Cox4 (REF: 4850s), and mouse programmed death ligand-1 expression (PD-L1) (REF: 29122S) were purchased from Cell signaling Technology (Danvers, Mass.). Goat anti-rabbit IgG (H+L) HRP-2′Ab (LOT: RA230590), goat anti-mouse IgG (H+L) HRP-2′Ab (LOT: 31430), nitrocellulose membranes for dot-blotting (0.45 11m pore size) with high binding affinity, MEM culture medium with L-glutamine, Pierce™ DAB Substrate Kit, QDs (525 nm emission) functionalized with secondary Ab fragments (Qdot goat F(ab′)2 anti-rabbit IgG conjugates (H+L)) (LOT: 1738599), amine-functionalized QDs (Qdot® 525 ITK™ Amino (PEG) Quantum Dots) (LOT: 1763984), amine functionalized QDs (Qdot® 605 ITK™ Amino (PEG) Quantum Dots) (LOT: 1630058), streptavidin functionalized QDs (Qdot® 605 Streptavidin Conjugate) (LOT: Q10101MP), and HRP-conjugated streptavidin (LOT: 1012719A) were purchased from ThermoFisher. Cy3 labelled donkey anti-mouse IgG (H+L) (LOT: 715165150) and Cy3 labelled donkey anti-rabbit IgG (H+L) (LOT: 711165152) were purchased from Jackson ImmunoResearch Laboratories (West Grove, Pa.). Fluorescent beads (carboxylic groups on surface) 5 11 m in diameter with three colors (green 480/520nm excitation/emission maxima, yellow 525/565nm, red 660/690 nm) were purchased from Bangs Laboratories (LOT: 11534; 9920; 11376, Fishers, IN). All antibodies were obtained in PBS without carrier proteins or stabilizing reagents. Mouse IgG, HIV p24, KLK3, CRP and VEGF ELISA kits were either purchased from Abeam (REF: ab151276, Cambridge, Mass.) or R&D Systems (LOT: DHP240; DKK300; DCRPOO; DVEOO). Seroconversion plasma samples from HIV infected patients were purchased from SeraCare (LOT: 06000237; 06000230; 06000227; 06000262, Milford, Mass.). Serial bleeds were collected from patients during the development of an HIV infection. All HIV patients' plasma samples were tested and found negative to HBsAg and HCV. Heathy patient plasma samples (age, 25-65) were purchased from Discovery Life Sciences (Los Osos, Calf.). All plasma samples were tested and found negative to HBV, HCV, HIV and RPR.
To quantify the effect of HRP on FDA polymerization rate, the enzyme-accelerated signal enhancement (EASE) process is compared to the reaction conditions in the conventional dip-coating polymerization procedure where HRP is not present and O2 is the oxidant.
Preparation of dopamine solution for EASE. Dopamine hydrochloride powder (15 mg) was dissolved rapidly in tris buffer (10 mM, 3 ml) at pH 8.5, followed by quick addition of H2O2 (1M, 60 μl). The mixture solution was used fresh.
Polydopamine deposition. Small droplets of HRP (0.1 pg) in PBS buffer and/or BSA (15 pg) in PBS buffer were placed on a nitrocellulose membrane and air-dried for 1 hour at room temperature. The membranes were further exposed to the EASE assay for 1 minute and washed with PBS for 30 seconds.
Results. As shown in
Next, it was determined whether the EASE process can be confined to the vicinity of HRP molecules (
The EASE technology was first applied to IHC and IF, robust technologies capable of interrogating gene expressions in single cells and resolving the heterogeneity issues of complex tissue samples, with well-preserved cell and tissue morphology. IHC and IF work well for high-abundance analyte molecules, but lack the sensitivity to detect antigens of low abundance, in particular in clinical tissue specimens where autofluorescence can be overwhelmingly high. To test the suitability of EASE, two model antigens, Lamin A (nuclear envelope) and HSP-90 (cytoplasm) were stained in formalin-fixed HeLa cells because these two antigens represent analytes in different cell compartments (
Cell culture and fixation. HeLa cells were cultured in MEM medium with L-glutamine, 10% fetal bovine serum, and antibiotics (60 μg ml-1 streptomycin and 60 U ml-1 penicillin) in glass-bottom 24-well plates to 60-80% confluency. Before IF staining, cells were rinsed with 1× tris-buffered saline (TBS), fixed with 4% formaldehyde in TBS for 30 minutes, permeabilized with 2% DTAC (dodecyltrimethylammonium chloride)/TBS for 30 minutes followed by 0.25% TritonX-100/TBS for 5 minutes and washed five times with TBS (each time 3 minutes). The fixed cells were stored in 1×PBS at 4′C.
Cell imaging and signal analysis. An Olympus IX-71 inverted fluorescence microscope equipped with a true-color charge-coupled device (QColor5, Olympus), a LSM 510 Meta confocal microscope (Zeiss, Dublin, Calif.) and a hyper-spectral imaging camera (Nuance, 420-720 nm spectral range, CRI, now Advanced Molecular Vision) were used for cell imaging. Low-magnification images were obtained with a 20× objective (NA 0.75, Olympus) and high-magnification with 40× and 100× oil-immersion objectives (NA 1.40, Olympus). Wide UV filter cube (330-385 nm band-pass excitation, 420 nm long-pass emission, Olympus) was used for imaging of all QD probes. All images were acquired with cells attached to the coverslip bottom of the well and immersed in PBS without anti-fading reagents. For quantitative comparisons, the same exposure time and gain were applied during imaging. Nuance image analysis software and lmageJ were used to identify regions of interest that included stained cells and excluded ‘blank’ cell-free areas. Average fluorescence intensity throughout all regions of interest within a single image was recorded. Identical analysis was performed on 4 images (containing ˜40 cells per field of view) taken from different areas of the sample to obtain an overall average staining intensity and assess signal variation.
IHC/IF-EASE single cell imaging. Prior to staining, the endogenous peroxidase activity of cells was quenched by 3% H2O2 solution. Cells were first blocked with 2% BSA/0.1% casein in 1× PBS for 30 minutes. Rabbit anti-Lamin A IgG (LOT: L1293, Sigma-Aldrich) or anti-HSP90 IgG (LOT: SAB4300541, Sigma-Aldrich) (intermediate detection reagent) diluted in PBS buffer containing 6% BSA was added to the cells. After 1-hour incubation, cells were washed three times (5 minutes each) with PBS containing 2% BSA, followed by another 1-hour incubation with goat anti-rabbit IgG (H+L) HRP-2′Ab (LOT: RA230590, ThermoFisher) (primary detection reagent). Unbound antibodies were washed away with PBS with 2% BSA (5 min×3), and fresh enzyme substrate (dopamine or DAB) was added to cells for 15 minutes incubation. The ideal staining result is strong chromogen signal of interested analyte locations with low nonspecific signals in background. To characterize the staining stability after storage, the stained cells were stored in 1×PBS at 4° C., and washed with fresh PBS every four days. Images were captured every three weeks on the same cell subset with the same exposure and gain. For immunofluorescence imaging with a secondary detection reagent (QDs), after the PDA development step, amine-functionalized PEG-coated QDs (10 nM) were incubated with cells for 1 hour.
Results. As shown in
To probe the sensitivity enhancement of EASE, fluorescence probes (secondary detection reagents) were brought into the assay after PDA deposition, taking advantage of PDA's remarkable reactivity to any fluorophores with primary amines and the convenience of quantifying fluorescence signals. Pegylated QDs with terminal amines were used as the fluorophore because of their photostability, which allows for accurate measurement of fluorescence intensity. As shown in
To evaluate the sensitivity quantitatively, staining was first performed on HSP-90. Unlike ELISA assays where analyte molecules can be easily immobilized at various densities, engineering cells with a variety of precisely controlled antigen expression levels is extremely difficult. Instead, the concentration of the intermediate detection reagent (1′Ab) was reduced in a serial fashion to bring down the signal intensity. As shown in
Next, to directly evaluate IF-EASE in imaging low-abundance analytes, the expression of GAPDH in HeLa cells was silenced using RNA interference (RNAi)-mediated gene knockdown. RNA interference. GAPDH expression knock-down was done by transfecting siRNA targeting GAPDH into HeLa cells. Annealed siRNA with 3′-TT overhangs was purchased from IDT (Coralville, Iowa). The sense strand sequence was 5′-CAUCAUCCCUGCCUCUACUTT-3′. HeLa cells were grown in a 10 cm TC-treated dish, trypsinized, and mixed in suspension with culture medium containing 25 nM GAPDH siRNA, together with 0.5 μl per well DharmaFECT-2 transfection reagent (Dharmacon). The cells (500 μl cell suspension per well) were then seeded into a glass-bottom 24-well plate, and incubated for 36 or 60 hours. Following RNAi, the cells were processed for staining using IF-EASE. The intermediate detection reagent (1′Ab) was anti-GAPDH (rabbit, LOT: G9545, Sigma-Aldrich).
Results. As shown in
Suspension microarrays are highly multiplexed genotyping and phenotyping platforms used in molecular biology, drug screening, and disease diagnosis. Compared to planar microarrays that are spatially addressable, suspension microarrays are often fabricated by doping microspheres with combinations of luminescent materials and are decoded with flow cytometers (e.g., Luminex microbeads). To determine whether an unknown analyte is present or not, conventional methodologies such as direct or sandwich hybridization and immuno-recognition are applied. The suspension microarrays offer advantages such as faster binding kinetics, but their detection sensitivities are essentially the same as the planar counterparts.
Preparation of antigen-coated fluorescent beads. IgG purified from mouse and rabbit serum (capture reagent) were covalently linked to the surface of green and yellow fluorescent beads, via 2-step carbodiimide-mediated cross-linking between the carboxylic groups on bead surface and the primary amines on IgG. Briefly, fluorescent beads were first washed and suspended in MES buffer (pH 4.8) with 0.01% Tween-20 at 0.1 w/v% (˜107 beads ml-1) and activated for 15 minutes upon addition of 10 mg ml-1 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 mg ml-1 N-hydroxysulfosuccinimide (sulfa-NHS). The activated beads were washed by centrifugation (5,000 g×2 min) twice using 50 mM borate buffer (pH 8.5) with 0.01% Tween-20 to remove excess crosslinkers and then incubated with IgG (2.5 mg ml-1) in borate buffer with 0.01% Tween-20 for 6 hours. The resulting IgG-coated beads were washed 3 times to remove excess IgG, resuspended in PBS (with 0.5% BSA), and stored at 4° C.
Suspension microarray with EASE. Biotinylated goat anti-mouse and goat anti-rabbit IgGs (model analytes) were captured by the antibody-coated green and yellow beads. PBS containing 0.5% BSA was used as incubation and blocking buffer throughout the experiment. All incubation steps were carried out at room temperature under gentle rotation. All washing steps were done by centrifuging the microbeads at 3,000 g for 2 minutes. Each microbead type was resuspended in 100 μl PBS at a final concentration of 1×106 beads ml−1. The beads were first incubated in the blocking buffer for 30 minutes. Biotinylated anti-mouse or -rabbit IgGs were added to the bead solution, incubated for 30 minutes, washed 3 times with PBS (0.5% BSA), and resuspended in 100 μl buffer. Then HRP-streptavidin probes (primary detection reagent) (1:3000 dilution) were added to the bead solution, incubated for 30 minutes, washed 3 times with PBS (0.5% BSA), resuspended in 100 μl dopamine solution for EASE, followed by 15 min incubation. The microbeads were washed another 3 times in BSA-free PBS, and mixed with amine-functionalized PEG-coated QDs (secondary detection reagent) (1 nM final concentration) for 1 hour incubation. At the end of QD incubation, the beads were washed 5 times with DI water and concentrated in 10 μl water for microscopy examination. A hyper-spectral imaging camera (Nuance, 420-720 nm spectral range, CRI, now Advanced Molecular Vision) and software were used to unmix and quantify fluorescence signal components. False-color composite images were obtained by merging individual channels. For quantitative analysis, Nuance image analysis software was used to automatically identify regions of interest that included QD labelling. Identical analysis was performed on 5 images (containing at least 20 beads per field of view). High-throughput quantitative analysis was achieved on a LSR-II flow cytometer (BD Biosciences). For each sample, at least 5,000 beads were counted. The flow cytometry data was analyzed with FlowJo 9.3.3 (TreeStar).
Results. To demonstrate the compatibility of EASE with suspension microarrays, fluorescent microspheres were coated with immunoglobulin G (IgG) (capture reagent) to detect a model analyte, biotinylated 2′Ab. Presence or absence of the analyte was detected with either streptavidin-QD conjugates (conventional sandwich method) or the EASE technology (primary detection reagent (streptavidin-HRP), PDA, and secondary detection reagent (QD-NH2)) (
To assess the specificity of this ultrasensitive detection assay, two control experiments were conducted. In the first experiment where the analyte molecule was missing, no significant signals were detected with or without the EASE process, confirming the antibody-antigen binding specificity (
To demonstrate the versatility of EASE, it was further applied to ELISA and immuno strip tests, robust and poplar biochemical assays. These assays using antibodies for molecular recognition and enzyme-catalyzed chromogen development for analyte identification are easy to perform, having broad applications in both research and clinical laboratories. On the other hand, their mediocre detection sensitivities are also well acknowledged. Compared to the suspension assays discussed above, a technical feature of these assays is that they are performed on solid supports (flat surfaces or porous membranes), rendering the sample washing steps quick and easy (dip in and out of washing buffer without the need of a centrifuge). This seemingly insignificant feature, combined with the unique bioconjugation capability of FDA allows EASE to be carried over for more than one time. For example, in the first round of amplification, HRP molecules bound to the analyte can catalyze localized deposition of FDA. The FDA layer can in turn capture a large number of HRP molecules that are capable of catalyzing the conversion of chromogenic substrates (
The sensitivity of ELISA-EASE in detecting HIV p24 in plasma was probed by spiking HIV p24 of known concentrations into plasma from healthy donors. For plasma samples from both HIV infected patients and healthy donors, immune complex disruption and neutralization procedures were applied to treat the samples. 20 μl 5% Triton X-100, 90 μl plasma samples, 90 μl glycine reagent (1.5 M) were mixed and incubated for 1 hour at 37° C. 90 pl tris buffer (1.5 M) was then added into the mixed solution and incubated for 10 minutes at room temperature. The plasma samples from HIV-positive groups with high HIV p24 concentration were diluted (10× and 100×) to fit within the ELISA working ranges for measurement.
Results. To probe the sensitivity and specificity of ELISA with or without EASE, a standard sandwich ELISA assay was established to detect mouse IgG (model analyte). Serial dilution of the analyte molecule resulted in gradients of color development that could be easily visualized by naked eye (substrate: tetramethylbenzidine or TMB). As shown in
Building on the remarkable sensitivity enhancement achieved on ELISA plates, the HIV biomarker p24 was further tested using lateral flow strips (
Lateral flow test-EASE. The striper unit, BioDot ZX010 (BioDot), was equipped with 4 frontline dispensers. Reagents (capture antibody) to be striped were aspirated through the end of the frontline dispenser. The nitrocellulose membrane (Sartorius CN95) was placed on the stage of the striper and secured, and then the frontline dispensers were adjusted to the appropriate position above the nitrocellulose membrane. The striper was programed to release the reagents at a rate of 1 μl cm−1. The membrane was placed in a forced air oven at 37° C. for 30 minutes before cooling in a desiccated environment. Once cooled, the membrane was placed on a backing card (DCN MIBA-020), and then the wick (GE Healthcare, CF5) was laid over the nitrocellulose with a 2 mm overlap. The completed card was placed in the staging area of the guillotine strip cutter (Kinbio ZQ200), and cut into 4 mm wide strips before being stored in Mylar bags that are sealed shut after including desiccant packets until use.
HIV p24 was used as a model analyte for the lateral flow test. Capturing antibodies (HIV p24 antibody) were immobilized onto nitrocellulose membrane, The membrane was blocked with 0.5% tween-20/2% BSA in PBS for 30 minutes. The membrane was then exposed to HIV p24 sample solutions (10 min). After washing (3×), the strips were treated with HIV p24 antibody-HRP conjugates (primary detection reagent) for 30 minutes and washed 3 times again. DAB was used as the enzyme substrate for 10 min color development,
Results. As shown in
With the EASE platform validated in the above bioassays, additional biological problems that require much improved detection sensitivity to resolve were addressed. The usefulness of EASE in detection of four biologically significant low-abundance analytes, HIV in blood, in situ protein detection in brain samples, Zika virus (ZIKV) imaging in the placenta, and programmed death-ligand 1 (PD-L1) in tumor, was demonstrated.
Early diagnosis of HIV provides timely access to treatments, thus improving patient outcomes and quality of life. A study of ˜16,000 patients on antiretroviral (ARV) treatments shows substantial numbers of patients beginning ARV later than recommended, due to late diagnosis. For adults, early knowledge of infection also leads to behavioral changes that could reduce 30% of new infections per year. For children and infants, earlier diagnosis is even more important. At this time, over 200,000 children acquire HIV worldwide every year, with most cases due to transmission to infants from their mothers during pregnancy, birth, or breastfeeding. HIV progresses rapidly in infants without treatment they can die within months—but early treatment by ARV greatly improves outcomes, Large-scale programs (e.g., President's Emergency Plan for AIDS Relief (PEPFAR)) have made ARV available, but early diagnosis remains a barrier to treatment.
HIV can be detected in blood or plasma by 1) nucleic acid amplification tests (NAAT), 2) lab based immunoassays (ELISA), or 3) rapid tests (similar to pregnancy tests). In general, NAAT is sensitive, but very expensive, and rapid test is of low performance and cannot be used in infants (false positive due to antibodies from the motherm). For decades, ELISA has been the workhorse laboratory HIV test and is the first test in the Centers for Disease Control and Prevention (CDC) testing algorithm. The sensitivity of ELISA, however, has been a major limitation (even for the most recent generation, detections are made around two weeks after infection). Increasing detection to an earlier time has been a major unmet clinical need.
The ELISA-EASE assay was used to detect p24 antigen, the key protein that makes up most of the viral capsid, in patient sera. Quantitative measurement of its presence in serum is highly valuable to blood screening, diagnosis of infection, and monitoring treatment responses. As recommended by the CDC, HIV p24 antigen detection using ELISA offers a number of advantages such as reduced cost, fast assay times, and applicability in low-resource settings. On the other hand, it is commonly acknowledged that p24 ELISA is an insensitive assay with a LOD of approximately 10 pg ml−1, limiting its use to samples with high viral loads. Incorporating EASE technology, however, can improve the ordinary detection sensitivity of ELISA to extraordinary levels, as shown in the above ELISA studies conducted in buffers.
To demonstrate its ability in clinical diagnosis, sera from 24 donors (obtained from SeraCare, Milford, Mass. and Discovery Life Sciences, Los Osos, Calif.) were assayed with either standard ELISA or ELISA with EASE. Among these samples, four were obtained from HIV-infected patients (PRB 946, PRB 949, PRB 953, and PRB 977) whose viral loads had been determined using PCR (data from SeraCare); and 20 HIV-negative donors were included to exclude biased results due to nonspecific interactions (Table 5). The analytical LOD was determined by spiking HIV p24 antigen of various concentrations into plasma. Results from 9 repeated runs performed on 9 consecutive days showed a highly consistent value (
No positive detection was made using ELISA, ELISA-EASE, or PCR, showing detection specificity across all three methods.
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CRF and its canonical G-protein coupled receptors, corticotrophin releasing factor receptor type 1 (CRFR1) and CRFR2 play an essential role in stress responsiveness regulated by the central nervous system. Alterations in the function of the CRF system and changes in CRF receptor signaling are broadly linked to neuropsychiatric disorders including addiction and depression. The ability to resolve the spatial distribution of CRF receptors in the brain will transform our understanding of how these receptors influence neural circuit function and how alterations in the expression and distribution of these receptors contribute to the disease states. Detection of CRF receptors has been largely limited to in situ hybridization detection on the mRNA level and radio-ligand binding assays, which provide poor spatial resolution. High-resolution localization of these receptors using conventional immunostaining techniques has been limited by the low levels of receptor expression. To test the effectiveness of EASE technology to enhance CRFR1 detection using antibody staining, immunostaining for CRFR1 was performed using conventional methods and EASE.
Histology preparation of brain tissues for CRFR1 staining. Mice were deeply anesthetized with 50 mg/kg of Beuthanasia-D and transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. Whole brain tissue was dissected, fixed overnight in 4% paraformaldehyde, and cryoprotected by soaking in a 30% sucrose solution for 48 hours. The brains were flash frozen in OCT and stored at −80° C. The frozen brains were then cryosectioned to 30 μm-thick sections and stored in lx PBS with 0.1% NaAz prior to immunostaining.
CRFR1 IF staining in brain sections. Coronal 30 μm sections were selected based on a reference atlas (Franklin and Paxinos) and analyzed for protein expression. Primary antibody against CRFR1 (Nevus Biologicals, cat. No. NLS1778) (intermediate detection reagent) was diluted 1:100. Cy3- or HRP-labeled secondary antibodies (donkey anti-rabbit, Jackson Immunolabs, and goat anti-rabbit) (conventional reagent or primary detection reagent, respectively) were diluted 1:250. Sections were incubated in 3% hydrogen peroxide 1×TBS buffer (10 min) to quench the intrinsic peroxide in tissue, washed with 1× TBS for 10 minutes, and blocked with 1× TBST (TBS+0.3% TritonX 100) with 3% donkey serum for 60 minutes. The blocked sections were stained with the primary antibody diluted in the blocking buffer overnight, washed three times in lx TBS for 10 minutes, and incubated in Cy3- or HRP-conjugated secondary antibodies for 1 hour at room temperature. IF-EASE was applied as described in the Examples above (amine-Cy3, a secondary detection reagent, was used as the reactive fluorophore). The sections were washed three more times in 1× TBS and mounted.
Results. Analysis of CRFR1 detection revealed only a small number of CRFR1-positive cells in the cerebral cortex of the mouse brain using conventional immunostaining (
Zika is a mosquito-borne flavivirus initially identified in the 1950s' in monkeys. Its recent outbreak in Brazil has been correlated with cases of fetal microcephaly as well as Guillian Barré, raising major global concerns. While there is now scientific consensus, including our own work, that ZIKV indeed causes fatal brain injury, the mechanism of how it occurs is largely unknown. qPCR and deep sequencing are capable of identifying ZIKV in the placenta, but cannot elucidate the means by which ZIKV crossed the placental barrier due to their inability to track ZIKV through conventional immunohistologic analysis.
Immunostaining of ZIKV-infected placenta. Placental samples were collected from pregnant pigtail macaques (Macaca nemestrina), who were inoculated with ZIKV (strain FSS13025, Cambodia 2010) or from a normal pregnancy. Formaldehyde-fixed sections of frozen placental chorionic villi were stained using both conventional IF and IF-EASE. The primary antibody (ZIKV E-protein Clone ZV-13, Diamond lab) (intermediate detection reagent) was diluted 1:200. Other reagents such as the primary detection reagent as well as the staining protocol were the same as that described in the CRFR1 experiments. A healthy control was used for studying the specificity of IF-EASE. Adjacent tissue slides were used for all staining conditions.
Results. The EASE technology enabled direct visualization of ZIKV-infected cells within the placental chorionic villus core of pregnant nonhuman primates. As shown in
PD-L1 also known as CD-274 or B7-H1, is a cell surface ligand, which binds and triggers PD-1, a potent immune-inhibitory receptor on T cells49. Monoclonal antibodies which block this interaction, by binding either PD-L1 or PD-1, have proven to be efficacious immune-oncology agents in a variety of tumor types. Immunohistochemical assays for detecting PD-L1+ cells within tumors have also been approved as companion diagnostic tests for patient selection in limited therapeutic indications, but broader application of anti-PDL1 IHC is limited by both biologic and technical factors. PD-L1 expression vary broadly across a wide range and levels below the detection thresholds of current IHC assays still have biologic significance. Therefore, it was determined whether EASE can be used to detect low-level PD-L1 signals while preserving good signal-to-noise ratios, an unmet clinical need for immunotherapy. Clinical formalin-fixed paraffin-embedded (FFPE) pancreatic tumor specimens with low PD-L1 expression were used to test the performance of IF-EASE with conventional IF.
PD-L1 immunostaining of pancreatic tumor specimens. The FFPE pancreatic tumor tissue specimens from two patients (SU-09-21157; SU-10-26808) were deparaffinized by washing the slides with xylene (7 min, 3 times), 100% ethanol (2 min, twice), 95% ethanol (2 min, twice), 70% ethanol (2 min, twice) and DI water (2 min). The sections were then incubated in 3% hydrogen peroxide in 1× TBS buffer (30 min) to quench the intrinsic peroxide. Antigen retrieval was performed by incubating the sections with the Trilogy antigen retrieval buffer under high pressure (15 min), cooling down (20 min), and washing with ix TBS (5 min, 2 times). The sections were subsequently stained using both conventional IF and IF-EASE. The protocols are the same as the ones described immediately above, except the primary antibody (intermediate detection reagent) is mouse anti-PD-L1 (1:150 dilution, Cell signaling Technology, REF: 29122S). Adjacent tissue slides were used for all staining conditions.
Results. As shown in
HRP can speed up PDA polymerization by approximately 300 times. More importantly, due to the excellent reactivity of PDA to primary amines, the polymer chains quickly crosslink with nearby biomolecules (rich in many reactive chemical groups including NH2), forming a localized network for immobilization of a large number of reporter molecules and nanoparticles (having accessible amine groups) for signal enhancement, while preserving the spatial information. This technology, dubbed EASE, is useful in a number of contexts including immunohistochemistry and immunofluorescence for single cell imaging, ELISA, lateral flow strips, and suspension microarrays, as highlighted below in Table 16, summarizing the assays of Examples 2-8. Consistently, it improves bio-imaging and—detection sensitivity by at least 2-3 orders of magnitude, regardless of the assay format. Most significantly, EASE achieves this remarkable sensitivity without changing the design of common assay formats, or requiring specialized equipment and reagents, in contrast to most ultrasensitive detection technologies invented in the past 10-20 years. Therefore, EASE can be directly incorporated into the current biological and clinical infrastructure for immediate impact.
The flexibility of this general technology has been demonstrated to be useful in a number of real biological problems that cannot be solved (or are at least extremely difficult to solve) using conventional bioassays. EASE was applied to ELISA-based detection of HIV infection in patient blood samples. For comparison, the measurements were benchmarked against the gold-standard assays, standard ELISA and PCR. The EASE-enabled ELISA outperformed the standard ELISA by >1,000 times in sensitivity, which translates into detection of 2-3 viruses per 100 μl of blood. This sensitivity is similar to that of PCR-based approaches allowing HIV detection 1-2 weeks earlier, yet ELISA is faster and cheaper to perform, and compatible with point-of-care (POC) applications (rending equipment such as a costly thermocycler unnecessary). Furthermore, EASE is a robust process that can be applied to a variety of real biological and clinical problems, such as brain biology, in situ virus imaging in placenta, and PD-L1 imaging for immunotherapy.
This application claims the benefit of priority of U.S. Provisional Patent Application no. 62/414,117, filed Oct. 28, 2016, and U.S. Provisional Patent Application No. 62/504,995, filed May 11, 2017, each of which is hereby incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. R21 CA192985, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/059131 | 10/30/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62414117 | Oct 2016 | US | |
| 62504995 | May 2017 | US |
