WO2018039587A1 - Phi29 nanochannel for early detection of breast cancer biomarkers - Google Patents

Abstract

Disclosed herein is a biosensor for detecting breast cancer biomarkers in a biological sample from a subject. The biosensor is based on a double-stranded DNA bacteriophage DNA-packaging motor connector protein that has been modified to 1) contain an affinity domain for at least one breast cancer biomarker, and 2) to be incorporated into a membrane layer such as a phospholipid bilayer membrane, to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. In these embodiments, binding of the breast cancer biomarker to the affinity domain alters conductance through the aperture, which can be measured as disclosed herein.

Description

PHI29 NANOCHANNEL FOR EARLY DETECTION OF BREAST

CANCER BIOMARKERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 62/379,976, filed August 26, 2016, which is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No. R01 EB012135 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

Detection of cancer in its early stage is a challenging task for oncologists. Breast cancer is generally asymptomatic in the early stages. Current screening tools such as mammography and breast examination miss up to 40% of early breast cancers, are ineffective for young women, or require invasive biopsy. New noninvasive methods are needed to complement current methodologies for early treatment.

SUMMARY

Disclosed herein is a biosensor for detecting breast cancer biomarkers in a biological sample from a subject. The biosensor is based on a double-stranded DNA bacteriophage DNA-packaging motor connector protein that has been modified to 1) contain an affinity domain for at least one breast cancer biomarker (also referred herein as the "analyte molecule"), and 2) to be incorporated into a membrane layer such as a phospholipid bilayer membrane, to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. In these embodiments, binding of the breast cancer biomarker to the affinity domain alters conductance through the aperture, which can be measured as disclosed herein.

Therefore, a fusion protein is disclosed that comprises a) a transmembrane aperture- forming domain of a DNA-packaging motor connector protein and b) an affinity domain that selectively binds a breast cancer biomarker protein or nucleic acid. In particular

embodiments, the DNA-packaging motor connector protein is from bacteriophage phi29. The breast cancer biomarker can in some embodiments be a protein, such as Thomsen-Friedenreich (TF), Tn (TF precursor), Galectin-3 (Gal-3), Urokinase-dependent plasminogen activator (uPA), Plasinogen activator inhibitor (PAI-1), or a combination thereof. The breast cancer biomarker can in some embodiments be a nucleic acid, such as miR-155- 5p, miR-4484, miR-let-7i-5p, miR-92a-3p, or miR-K12-5-5p, or a combination thereof.

Also disclosed is a composition comprising a membrane layer having incorporated therein one or more of the disclosed fusion proteins to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. In particular embodiments, the membrane layer comprises a phospholipid bilayer.

Also disclosed is a method for detecting a breast cancer biomarker in a biological sample from a subject. The method can involve first providing a conductive channel- containing membrane which comprises a membrane layer (such as a phospholipid bilayer) and incorporated therein one or more of the disclosed fusion proteins. The method can then involve applying an electrical potential to the membrane and measuring a first conductance signal that results from the applied electrical potential. Following this, the method can involve contacting the conductive channel-containing with the biological sample form the subject, and then applying an electrical potential to the membrane and measuring a second conductance signal that results from the applied electrical potential. In these embodiments, a change between the first and second conductance signals can indicate binding of a breast cancer biomarker molecule in the biological sample to the connector protein, thereby detecting the presence of the breast cancer biomarker.

In some embodiments, the conductance signal is measured using a microelectrode on which is formed the membrane layer. For example, the microelectrode can be a Ag/AgCI microelectrode.

Also disclose is a system having one or more chambers, each chamber comprising a microelectrode (e.g., Ag/AgCI microelectrode) having formed thereon a conductive lipid bilayer having incorporated therein one or more of the disclosed fusion proteins to form an aperture through which conductance can occur when an electrical potential is applied across the lipid bilayer, wherein conductance through the aperture is detected by the

microelectrode. Therefore, in some embodiments, the system is a "chip."

For example, the system can be an array having at least 4, 8, 16, 32, 64, or more individually controlled chambers. In some cases, the array is a monoplex array, e.g. wherein each chamber comprises the same fusion proteins for detection of the same breast cancer biomarker. In some cases, the array is a multiplex array, e.g. wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chambers comprise distinct fusion proteins for detection of different breast cancer biomarkers.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figures 1A and 1B show concentration of TF (□) and Tn (■) in healthy (Fig. 1A) and cancer NAF (Fig. 1 B), determined by competitive inhibition ELISA.

Figures 2A to 2E show kinetic studies of EpCAM antibody-peptide interactions.

Figures 2A to 2C show current trace (Fig. 2) and Histogram of Ab binding showing τ _on (Fig. 2B) and τ ₀a (Fig. 2C). Figures 2D and 2E show frequency of association (Fig. 2D) and dissociation (Fig. 2E) as a function of antibody concentration yielding k _on and k ₀ . From the two rate constants, the K _D was determined.

Figures 3A and 3B show binding of Ni²⁺-NTA-Nanogold to C-His tagged connector. Figure 3A is an illustration and Figure 3B is a current trace showing step-wise binding of Nanogold (1.8 nm).

Figure 4A shows LNA vs. RNA nucleotide structure. In LNA, the methylene linkage (orange) between the 2'-0 and 4'-C locks the LNA in 3'-endo conformation. Figure 4B illlustrates location of miRNA Seed region as a target for antimiRNA-seed LNA oligos.

Figure 5A shows parallel current recording from 4 chambers housing phi29 motor channels in Orbit16 platform. Each chamber shows the insertion of one connector channel in the membrane. Figures 5B and 5C show raw data (Fig. 5B) and histogram (Fig. 5C) showing detection of EpCAM Ab in the presence of non-specific Ab and serum proteins. Specific Ab binding can be clearly distinguished from non-specific events.

Figures 6A and 6B are an illustration of 16 individually controlled chambers housing connector functionalized probes for Monoplex detection using TF antigen (Fig. 6A) and Multiplex detection of 12 biomarkers and 2 controls in parallel (Fig. 6B).

DETAILED DESCRIPTION

Disclosed herein is a biosensor for detecting breast cancer biomarkers in a biological sample from a subject. The biosensor is based on a double-stranded DNA bacteriophage DNA-packaging motor connector protein that has been modified to 1) contain an affinity domain for at least one breast cancer biomarker (also referred herein as the "analyte molecule"), and 2) to be incorporated into a membrane layer such as a phospholipid bilayer membrane, to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. In these embodiments, binding of the breast cancer biomarker to the affinity domain alters conductance through the aperture, which can be measured as disclosed herein.

In some embodiments, the disclosed biosensor can be used to diagnose and/or prognose breast cancer in a subject suspected of having breast cancer (e.g. based on detection of a lump, presence of cancer biomarkers, family history, etc.). In some cases, the method can be used to confirm the presence of a cancer, the cancer type (e.g. ductal, lobular, medullary, mucinous, etc), as well as size and/or grade of invasiveness.

The term "subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, e.g., physician. The subject can be either male or female.

The term "biological sample" refers to a tissue (e.g., tissue biopsy), organ, cell (including a cell maintained in culture), cell lysate (or lysate fraction), biomolecule derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), or body fluid from a subject. Non-limiting examples of body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid. In preferred embodiments, the biological fluid is nipple aspirate fluid.

The term "breast cancer biomarker" refers to any molecule whose levels in a bodily fluid of a subject are altered based on the presence or stage of breast cancer in the subject. In some cases, the breast cancer biomarker is a protein, such as Thomsen-Friedenreich (TF), Tn (TF precursor), Galectin-3 (Gal-3), Urokinase-dependent plasminogen activator (uPA), or Plasinogen activator inhibitor (PAI-1). In these and other embodiments, the affinity domain can comprise, for example, an antibody (e.g single chain antibody), an aptamer, or a peptide (e.g. phage display peptide or peptidomimetic).

The term "antibody" refers to natural or synthetic antibodies that selectively bind a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen. The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

As used herein, "peptidomimetic" means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include β-alanine, L-a- amino butyric acid, L-v-amino butyric acid, L-a-amino isobutyric acid, L-£-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, Ν-ε-Boc-N-a-CBZ-L-lysine, Ν-ε- Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-a-Boc-N-5CBZ- L-ornithine, Ν-δ-Boc-N-a-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

The term "aptamer" refers to oligonucleic acid or peptide molecules that bind to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamers are capable of adapting unique tertiary structures and recognizing target molecules with high affinity and specificity. A "nucleic acid aptamer" is a DNA or RNA oligonucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of such molecule. A nucleic acid aptamer may be constituted by DNA, RNA, or a combination thereof. A "peptide aptamer" is a combinatorial protein molecule with a variable peptide sequence inserted within a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast dihybrid conditions, which enhances the probability for the selected peptide aptamers to be stably expressed and correctly folded in an intracellular context.

In some cases, the breast cancer biomarker is a nucleic acid, such as a miR-155-5p, miR-4484, miR-let-7i-5p, miR-92a-3p, or miR-K12-5-5p. In these and other embodiments, the affinity domain can comprise, for example, an aptamer or oligonucleotide probe.

The term "nucleic acid" refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3' position of one nucleotide to the 5' end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

By "probe" or "oligonucleotide" is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target"). The stability of the resulting hybrid depends upon the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for nucleic acids (e.g. miRNAs) have at least 80%-90% sequence complementarity, preferably at least 91%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the nucleic acid to which they hybridize.

In particular cases, the nucleic acid probe may comprise at least one locked nucleic acid (LNA) residue. A locked nucleic acid residue is a nucleic acid analog that has a chemical shape similar to a naturally occurring nucleic acid residue (e.g., being able to form 2 or 3 hydrogen bonds with a complementary residue), but is not free to rotate in as many dimensions as a naturally occurring nucleic acid residue. For instance, in some cases, a locked nucleic acid residue may contain a 2-0, 4'-C methylene bridge, where the methylene bridge "locks" the ribose in the 3'-endo structural conformation, which is often found in the certain form of DNA or RNA. The locked ribose conformation may enhance residue stacking and/or backbone pre-organization. This can significantly increase the thermal stability (melting temperature) of the nucleic acid sequence in some cases. A nucleic acid probe containing one or more locked nucleic acid residues may be useful in certain embodiments because the locked nucleic acid residue may exhibit increased affinity for association with the target nucleic acid, e.g., due to the restrictions on its ability to internally rotate.

The viral DNA-packaging motor connector protein can be any such protein that can be incorporated into a membrane layer to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. An exemplary unmodified viral DNA-packaging motor connector protein from bacteriophage phi29 has been purified and its three-dimensional structure has been crystallographically characterized (e.g., Guasch et al., 1998 FEBS Lett. 430:283; Marais et al., 2008 Structure 16:1267). DNA- packaging motor connector proteins of other dsDNA viruses (e.g., T4, lambda, P22, P2, T3, T5 and T7), despite sharing little sequence homology with, and differing in molecular weight from, the phi29 connector, exhibit significant underlying structural similarities (e.g., Bazinet et al., 1985 Ann Rev. Microbiol. 39:109-29). Accordingly, a number of preferred embodiments as described herein refer to the phi29 DNA-packaging motor connector protein (e.g., Genbank Acc. No. ACE96033) and/or to polypeptide subunits thereof including fragments, variants and derivatives thereof (e.g., Acc. Nos. gi 29565762, gi 31072023, gi 66395194, gi 29565739, gi 157738604). In certain other embodiments the use of an isolated viral DNA- packaging motor connector protein from other dsDNA viruses is contemplated, including without limitation the isolated viral DNA-packaging motor connector protein from any of phage lambda, P2, P3, P22, T3, T4, T5, SPP1 and T7, or another isolated dsDNA virus

DNA-packaging motor connector protein (e.g., T4 (Acc. No. NP— 049782)(Driedonks et al., 1981 J Mol Biol 152:641), lambda (Acc. Nos. gi 549295, gi 6723246, gi 15837315, gi 16764273)(Kochan et al., 1984 J Mol Biol 174:433), SPP1 (Acc. No. P54309), P22 (Acc. No. AAA72961)(Cingolani et al., 2002 J Struct Biol 139:46), P2 (Acc. No. NP— 046757, P3 (Nutter et al., 1972 J. Viral. 10(3):560-2), T3 (Acc. No. CAA35152)(Carazo et al., 1986 Jl. Ultrastruct Mol Struct Res 94:105), T5 (Accession numbers AAX12078, YP— 006980;

AAS77191 ; AAU05287), T7 (Acc. No. NP— 041995)(Cerritelli et al., 1996 J Mol Biol 285:299; Agirrezabala et al., 2005 J Mol Biol 347:895)).

Without wishing to be bound by theory, it is believed in this regard that like the phi29 DNA-packaging motor connector protein exemplified herein, these and other dsDNA virus packaging motor connector proteins, which have been substantially structurally

characterized, can be modified according to the teachings herein to obtain an isolated DNA- packaging motor connector protein that can be incorporated into a membrane layer to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. Accordingly, disclosure herein with respect to the phi29 connector protein is intended, for certain embodiments, to be illustrative of related embodiments that are contemplated using any of such other isolated dsDNA viral DNA-packaging motor connector proteins, which may be modified for use in such embodiments according to the teachings found herein.

As described in greater detail herein, isolated DNA-packaging motor connector protein polypeptides, including such polypeptides that have been artificially engineered to possess properties of membrane incorporation (e.g., stable transmembrane integration in a membrane layer) and functional electroconductive transmembrane aperture formation, can be used as electroconductive biosensors for breast cancer biomarkers. Briefly and by way of background, the genome of linear dsDNA viruses is packaged into a preformed procapsid (Black, Ann Rev Microbiol 43, 267-292 (1989), Guo, Seminars in Virology (Editor's Introduction) 5(1), 1-3 (1994), Guo et al., Mol. Microbiol. 64, 886-903 (2007), Rao et al., Annu. Rev. Genet. (2008). This entropically unfavorable process is accomplished by an ATP-driven motor (Guo et al. J Mol Biol 197, 229-236 (1987), Chemla et al., Cell. 122, 683-692 (2005), Hwang et al., Biochemistry 35, 2796-2803 (1996),

Sabanayagam et al., Biophys. J 93, L17-L19 (2007)). In bacteriophage phi29, the motor uses one ATP to package 2 bp (Guo et al., 1987) or 2.5 bp of DNA (Moffitt et al., Nature 457, 446-4U2 (2009). The protein hub of this motor is a truncated cone structure, termed a connector that allows dsDNA to enter during maturation and exit during infection (Kochan et al., J Mol Biol 174, 433-447 (1984), Rishovd et al., Virology 245, 11-17 (1998), Simpson et al., Acta Cryst D57, 1260-1269 (2001), Guasch et al., J. Mol. Biol. 315, 663-676 (2002), Agirrezabala et al., J. Mol Biol. 347, 895-902 (2005). The connector has a central channel formed by twelve GP10 protein subunits. While the connector proteins of viruses share little sequence homology and vary in molecular weight, there is significant underlying structural similarity (Bazinet & King, Ann. Rev. Microbiol. 39, 109-129 (1985)). By demonstrating viral DNA packaging and procapsid conversion to infectious virions, phi29 DNA packaging motor was the first to be assembled in vitro in a defined system and remains one of the most well studied (Guo et al., Proc. Natl. Acad. Sci. USA 83, 3505-3509 (1986)). The motor utilizes six pRNA (packaging RNA) molecules (Guo et al., Science 236, 690-694 (1987), Guo et al., Mol. Cell. 2, 149-155 (1998), Zhang, et al., Mol. Cell. 2, 141-147 (1998), Shu et al., EMBO J. 26, 527-537 (2007)) to gear the machine.

As described herein, phi29 and other isolated dsDNA viral DNA-packaging motor protein connectors, including engineered and mutated versions thereof such as fusion proteins that retain their aperture domain and comprise an affinity domain may be usefully incorporated into membrane layers to form apertures permitting their use as conductive channels when an electrical potential is applied across the membrane. Modified isolated double-stranded DNA virus DNA-packaging motor protein connectors such as the phi29 connector may be engineered to have desired structures for use in the presently disclosed embodiments (Jiminez et al., 1986 Science 232:1113: Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science 309: 1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007)), where protein crystallographic structural data are readily available (e.g., Simpson et al., Acta Cryst D57, 1260-1269 (2001), Guasch et al., J. Mol. Biol. 315, 663-676 (2002), Cai et al., Nanomedicine 4, 8-18 (2008), Guo et al., J. Nanosci. Nanotechnol. 5, 856-863 (2005). Furthermore, the procedures for large scale production and purification of phi29 connector have been developed (Guo et al., 2005; Ibanez et al., Nucleic Acids Res. 12, 2351-2365 (1984), Robinson et al., Nucleic Acids Res. 34, 2698-2709 (2006), Xiao et al., ACS Nano 3, 100-107 (2009).

The presently disclosed compositions and methods may include in certain

embodiments the practice of measuring electrical conductance, across a membrane layer in which the herein-described isolated viral DNA-packaging motor connector protein is incorporated, by adapting established electrophysiology instrumentation and methodologies. For example, and as described in greater detail below, modifications may be made to patch- clamp or planar membrane techniques for generating transmembrane potentials and measuring conductance across such membranes. Exemplary descriptions of such methodologies may also be found, for example, in Kasianowicz et al., 1996 Proc Nat. Acad. Sci USA 93:13770; Gu et al., 1999 Nature 398:686; Kasianowicz et al., 2001 Anal. Chem. 73:2268; Henrickson et al., 2000 Phys. Rev. Lett. 85:3057; Hromada et al., 2008 Lab Chip. 8:602; Robertson et al., 2007 Proc. Nat. Acad. Sci. USA 104:8207; U.S. Pat. Nos. 6,267,872; 6,746,594; and 6,936,433. Those familiar with the art will appreciate general methodologic approaches from these and similar references, and it will be further understood that advantages as described herein may be derived in part from the present disclosure, of conductive channel-containing membranes that comprise an incorporated isolated viral DNA-packaging motor connector protein that forms an aperture having a large lumen and functions as a conductive channel across a wide range of applied electrical potentials without undesirable interruptions in conductivity due to voltage gating behavior. Thus, for example by way of illustration and not limitation, using known electrophysiology

methodologies such as those referenced above and described herein, conductance occurs in the presently disclosed conductive channel-containing membrane without voltage gating when an electrical potential is applied that may in selected embodiments be between -100 mV and 100 mV, between -400 mV and 400 mV, between -300 mV and 300 mV, between -200 mV and 200 mV, between -150 mV and 150 mV, between -75 mV and 75 mV, between -50 mV and 50 mV, or within another voltage range as may vary according to the particular conductance conditions that are employed, as will be apparent to the skilled person based on the present disclosure.

Further, and according to non-limiting theory, the present conductive channel- containing membranes offer advantageous detection sensitivities that may derive in part from the aperture formed by the present viral DNA-packaging motor protein connectors, and also provide advantageous analyte characterization capabilities that may derive in part from the stable membrane incorporation of a protein conductive channel that can be engineered or mutated to have desired functional properties such as any of a wide variety of analyte- accessible affinity interaction domains by which to engage the disclosed analytes in a specific binding interaction.

For methods of detecting an analyte, these and related embodiments contemplate sensitivity that is obtained by observation of an altered (e.g., increased or decreased in a statistically significant manner) level of conductance through the aperture in the conductive channel-containing membrane across which electrical potential is applied, when the incorporated connector protein is engaged in a specific binding interaction with the analyte, relative to the level of conductance when no such specific binding interaction is present. Accordingly in certain preferred embodiments there is provided a method for detecting presence of an analyte molecule, comprising (a) contacting a test solution containing the analyte molecule with a conductive channel-containing membrane which comprises a membrane layer and incorporated therein one or a plurality of isolated viral DNA-packaging motor connector proteins that are capable of forming an aperture through which

conductance can occur when an electrical potential is applied across the membrane, and that each comprise a homododecamer of viral DNA-packaging motor connector protein polypeptide subunits, wherein each of said subunits comprises (1) an aperture domain that comprises an isolated viral connector protein polypeptide having an amino terminus and a carboxy terminus, (2) at least one affinity domain, under conditions and for a time sufficient for specific binding of the analyte molecule to the affinity domain, and (3) optionally at least one flexibility domain; and (b) determining, at one or a plurality of time points prior to the step of contacting and at one or a plurality of time points after the step of contacting, a

conductance signal that results from the applied electrical potential, wherein an alteration in the conductance signal after the step of contacting relative to the conductance signal prior to the step of contacting indicates binding of the analyte molecule to the connector protein, and therefrom detecting presence of the analyte molecule. In certain related further

embodiments, the alteration in the conductance signal indicates binding of the analyte molecule to the affinity/alignment domain.

By way of illustration and not limitation, the aperture formed by the membrane- incorporated connector protein is believed to be at least partially obstructed or occluded when analyte is present and is bound to the channel-conductive membrane, resulting in altered, and typically decreased, conductance across the membrane. In the absence of bound analyte, no such constraint is placed on conductance through the channel, such that altered levels of conductance may be readily detected when bound and unbound states of the analyte are observed. As shown below in the Examples, the present conductive channel- containing membrane is believed according to non-limiting theory to afford exquisite sensitivity in the detection of analyte by permitting observation of such an analyte binding- associated alteration in conductance at the level of a single analyte molecule binding to a single membrane-incorporated isolated viral DNA-packaging motor connector protein that has formed a conductive transmembrane aperture. In certain other embodiments, conductance signals may be detected from multiple conductive channels formed by multiple transmembrane apertures of the connectors described herein.

In certain conceptually related embodiments, information beyond the mere detection of the absence or presence of an analyte may be obtained, where a conductance signal profile is generated, for instance, by collecting a record that reflects the amplitude of conductance, including altered conductance as described herein at one or a plurality of time points, and the duration of conductance, including altered conductance as described herein, at one or a plurality of timepoints. Such a signal profile may reflect any number of properties of the analyte in the course of its interaction with the connector protein(s), for example, binding affinity and/or binding avidity (e.g., if the analyte is multivalent), and also potentially, physicochemical properties of the analyte such as relative molecular mass, charge, and/or hydrophobicity, which may vary as a function of the particular analyte, the particular connector protein, the membrane composition, the solvent conditions, the applied electrical potential, and other factors.

In certain embodiments the alteration in the conductance signal indicates binding of the analyte molecule to the affinity domain. In any of the above described methods for detecting an analyte, the step of contacting may be repeated one or a plurality of times, for instance, to generate a conductance signal profile.

It will be appreciated that certain preferred embodiments as described herein relate to an isolated DNA-packaging motor connector protein that comprises a dodecamer of chimeric or fusion polypeptide subunits, i.e., a non-naturally occurring polypeptide that is the product of recombinant genetic engineering techniques with which those skilled in the art will be familiar. In these and related embodiments, the fusion polypeptide comprises (i) an aperture domain, which may be all or an aperture-forming portion of a double-stranded DNA virus DNA-packaging motor protein connector polypeptide such as those discussed above and elsewhere herein; and (ii) at least one affinity domain. Exemplary connectors may comprise homododecamers of viral DNA-packaging motor protein connector polypeptide subunits. According to non-limiting theory, isolated DNA-packaging motor connector proteins as disclosed herein may be stabilized for retention and functional incorporation in membrane layers such as phospholipid bilayers, by modifying those portions of the connector polypeptides that interact with, respectively, hydrophilic phospholipid polar head groups and hydrophobic phospholipid fatty acyl chains, in a manner that energetically favors integration of the connector in the membrane to form a transmembrane aperture. Compositions and methods for introducing proteins to membranes, and for determining their incorporation into membranes, and further for ascertaining their disposition in the membranes as integral or transmembrane proteins, are known in the art and exemplified herein, as also are methodologies for determining functional incorporation of such proteins as electroconductive transmembrane channels.

As also noted above, an affinity domain may be any peptide or polypeptide domain that can be fused to the viral DNA-packaging motor protein connector polypeptide subunit sequence, with or without an intervening flexibility domain being present, that provides an affinity interaction domain such as a receptor, ligand, binding site, counter-receptor or the like, which may be used to engage in specific binding of an analyte molecule and/or to encourage analyte interaction with the conductive channel and/or both.

The presently described conductive channel-containing membrane may be formed by incorporating the isolated viral DNA-packaging motor connector protein as provided herein, including connectors comprised of polypeptide subunits which comprise fusion proteins having an aperture domain and an affinity domain as described herein, into a liposomal membrane. The membrane typically comprises amphipathic lipids such as phospholipids (e.g., one or more of phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, 1 ,2-diphytanoyl-sn glycerol-3- phosphocholine, 1 ,2-dioleoyl-sn-glycero-3-phosphocholine, or other phospholipids with which the skilled artisan will be familiar), which tend to form bilayers when exposed under appropriate conditions to an aqueous milieu.

Viral DNA-packaging motor protein connectors may be effectively incorporated into membranes by first providing amphipathic lipids from which solvent (e.g., an organic solvent such as one or more of chloroform, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, pyridine, diisopropyl ether) has been substantially removed (e.g., such that little or no residual solvent can be detected by visual inspection, and preferably such that the lipid preparation is regarded as dry), and then resuspending the dried amphipathic lipids in a solution that comprises an aqueous solvent, an osmotic agent, and a plurality of isolated viral DNA-packaging motor protein connector subunit polypeptides that are capable of self- assembly into homododecameric connector proteins. Without wishing to be bound by theory, it is believed that the inclusion of the osmotic agent advantageously facilitates functional DNA-packaging motor connector incorporation into the membrane as conductive channels, by influencing the formation and size of, and/or intermolecular dynamics within, substantially spherical liposomes, although it is recognized that other methods for producing conductive channel-containing membranes should not be excluded. Non-limiting examples of osmotic agents that may be used in these and related embodiments include sucrose or other disaccharides, glycerol, mannitol and dextran.

In certain embodiments the liposome comprising the conductive channel-containing membrane may be used in liposomal form, for example, as a vehicle for delivery to cells in vitro or in vivo of nucleic acid molecules that have been concentrated or accumulated in the liposome, such as by electric potential-driven translocation, against the nucleic acid concentration gradient that would otherwise limit the nucleic acid concentration that can be achieved according to recognized equilibrium principles, through the connector apertures, to obtain nucleic acid-containing liposomes. Such liposomes may advantageously find use as therapeutic agents, instance in gene therapy and related strategies. A wide range of formulations are available for in vitro and in vivo therapeutic administration of nucleic acid- containing liposomes, and may be modified for use with liposomes produced according to the present disclosure. See, e.g., WO/2002/034236; WO/2002/036767; WO/2003/094963; WO/2005/034979; WO/2005/120461 ; WO/2000/03683; Lasic, Liposomes in Gene Delivery, 1997, CRC Press, Boca Raton, Fla.; WO 96/40964; WO 1998/51278; WO 2009/086558; US 2007/0042031; US 2006/0240093; US 2006/0083780; US 2004/0142025. Multilamellar and unilamellar liposomes are contemplated, with unilamellar liposomes being preferred in certain embodiments.

As also described herein, certain embodiments contemplate incorporation of the herein described isolated viral DNA-packaging motor protein connectors into membrane layers to form liposomes, which may then donate membrane-integrated connectors to planar membrane systems, such as planar bilayer membrane (BLM) systems, by way of artificial membrane fusion manipulations according to art-known methodologies. Accordingly, conductive channel-containing membranes as described herein may be configured as desired for a particular purpose and/or to accommodate use with certain instrumentation, including but not limited to liposomal and/or nanoparticle (including carrier particle) delivery, planar membrane layers, microfluidic chambers, micropore and nanopore electroconductivity chambers, patch-clamp apparatus, fluorescence labeled analyte detection/characterization, and any other configuration compatible for use with the instant conductive channel- containing membrane as may be adapted based on the present disclosure and in view of knowledge in the art.

Disclosed are viral DNA-packaging motor connector protein-derived polypeptides and fusion proteins having amino acid sequence regions that are identical or similar to sequences known in the art, or fragments or portions thereof. For example by way of illustration and not limitation, a mutant bacteriophage phi29 viral DNA-packaging motor connector protein [e.g., Genbank Acc. No. ACE96033] or an engineered bacteriophage phi29 viral DNA-packaging motor connector protein-derived polypeptide fusion protein is contemplated for use in the disclosed compositions and methods, as are polypeptides having at least 80%, 90%, or 95% similarity to the herein disclosed polypeptides and to portions of such polypeptides, wherein such portions of a mutant or engineered phi29 viral DNA-packaging motor connector protein-derived polypeptide generally contain at least 150, 175, 200, 225, 250, 275, including at least 240, 260, 280, 285, 290, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311 , 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330 or more amino acids.

In like fashion, certain other embodiments contemplate other mutant double-stranded DNA bacteriophage virus motor connector proteins such as mutated forms of phage T4 DNA-packaging motor connector protein polypeptide, lambda phage DNA-packaging motor connector protein polypeptide (Accession numbers gi549295, gi6723246, gi15837315, gi 16764273, phage SPP1 DNA-packaging motor connector protein polypeptide (Accession number P54309), phage P22 DNA-packaging motor connector protein polypeptide

(Accession number AAA72961), phage P2 DNA-packaging motor connector protein polypeptide (Accession number NP— 046757), phage P3 DNA-packaging motor connector protein polypeptide (Nutter et al., 1972 J. Virol. 10(3):560-2), phage T3 DNA-packaging motor connector protein polypeptide (Accession number CAA35152, phage T5 DNA- packaging motor connector protein polypeptide (Accession numbers AAX12078, YP— 006980; AAS77191; AAU05287), and phage T7 DNA-packaging motor connector protein polypeptide (Accession number NP— 041995).

The terms "fragment," "derivative" and "analog" when referring to viral DNA- packaging motor connector proteins or polypeptides, refers to any mutant viral DNA- packaging motor connector protein-derived polypeptide described herein, or a fusion protein comprising such polypeptide, that retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active viral DNA-packaging motor connector polypeptide, which in preferred embodiments may be incorporated into a membrane layer to form an aperture through which conductance can occur when an electrical potential is applied across the membrane and/or may be capable of self-assembly into a homododecameric viral DNA-packaging motor connector protein such as may form such an aperture to obtain a conductive channel-containing membrane.

A fragment, derivative or analog of a viral DNA-packaging motor connector protein- derived polypeptide described herein, including polypeptides or fusion proteins or domains or portions thereof encoded by the cDNAs referred to herein and for which nucleotide coding sequences may be known to the art and/or can be deduced from the polypeptide sequences disclosed herein, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which additional amino acids are fused to the mutant viral DNA-packaging motor connector protein-derived polypeptide, including amino acids that are employed for detection or specific functional alteration of the mutant or engineered viral DNA-packaging motor connector protein-derived polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide. Fragments or portions of the nucleic acids encoding polypeptides according to the presently disclosed embodiments may be used to synthesize full-length nucleic acids encoding a mutant or engineered viral DNA-packaging motor connector protein-derived polypeptide. As used herein, "% identity" refers to the percentage of identical amino acids situated at corresponding amino acid residue positions when two or more polypeptide are aligned and their sequences analyzed using a gapped BLAST algorithm (e.g., Altschul et al., 1997 Nucl. Ac. Res. 25:3389) which weights sequence gaps and sequence mismatches according to the default weightings provided by the National Institutes of Health/NCBI database (National Center for Biotechnology Information,

Bethesda, Md.).

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal or intact naturally occurring virus is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co- existing materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

The analysis of the secreted antigens in body fluids represents a very promising approach able to detect cancer-related biomarkers. Within the breast ductal/lobular system is a fluid that is continuously secreted and reabsorbed in non-pregnant women. Using a gentle aspiration device, this breast fluid can be extracted noninvasively through the nipple, referred to as Nipple Aspirate Fluid (NAF) that offers a superior fluid for detection of breast cancer than serum since the protein and glycoantigens or microRNAs (miRNA) come specifically from breast tissue. This fluid can be collected from the epithelial cells lining the ductal system of the breast, the same cells that are the source of the vast majority of breast cancers.

Disclosed is a platform based on a unique biological motor with an elegant and elaborate funnel-shaped channel. A motor channel is inserted into a lipid bilayer and the resulting system exhibits robust properties and generates extremely sensitive, stable and reproducible conductance signatures when analytes interact with the channel. This is a highly sensitive device for capture and fingerprinting of chemicals and biopolymers in real time at single molecule level. The motor channel shrinks and exhibits discrete stepwise conformational changes in response to ligand binding to any of its twelve subunits at the C- terminus (the wider end of the funnel). The resulting current signals clearly discriminated specific and nonspecific binding at single molecule level, thus enabling sensitive single molecule detection of specific targets. A simple, sensitive and robust detection platform is disclosed for diagnosing diseases at an asymptomatic stage using a phi29 motor channel for high-throughput multiplexed detection of biomarkers extracted from breast cancer patient nipple aspirate fluid.

The platform involves: (1) a membrane-embedded motor channel serving as a highly sensitive nanopore; (2) anti-miRNA-seed targeting probes containing Locked Nucleic Acid (LNA) for capturing miRNA target, or Phage Display Peptide (PDP) for capturing breast cancer antigens; and (3) ultrasensitive electrophysiological setup for parallel or multiplexed single molecule sensing. Example 1 : Reengineering the motor channel by fusing specific Phage Displaying Peptide (PDP) probes for capturing breast cancer specific antigens as biomarkers in nipple aspirate fluid

Detection of secreted glycoantigens and proteins is a powerful method for analyzing changes in global expression patterns as a function of physiological, and disease processes (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin.Cancer Res 2005 11(19 Pt 1):6868-71 ; Sauter ER, et al. Br. J Cancer 2002 86(9): 1440-3; Kuerer HM, et al. Cancer 2002 95(11):2276-82; Alexander H, et al. Clin.Cancer Res. 2004 (22):7500-10. Common methods for proteomic profiling include: 2D gel electrophoresis4; Mass

spectrometry (MS) based MALDI (Matrix-Assisted Laser Desorption Ionization) (Tessitore A, et al. Int.J Proteomics 2013 2013:125858) and SELDI (Surface-Enhanced Laser Desorption Ionization) (Sauter ER, et al. Br.J Cancer 2002 May 6 86(9): 1440-3; Paweletz CP, et al. Dis.Markers 2001 17(4):301-7); and, 1D and 2D Liquid Chromatography (LC) coupled with UV or MALDI detection (Tessitore A, et al. Int.J Proteomics 2013 2013:125858). Each technique has distinct advantages and disadvantages, relating broadly to sensitivity, specificity, dynamic range, multiplexing capability, precision, throughput, and ease of use

(Chandramouli K, et al. Hum. Genomics Proteomics 2009 2009; Deyati A, et al. Drug Discov. Today 2013 Jul 18(13-14):614-24; Aldred S, et al. Clin.Biochem. 2004 37(11):943-52). The disclosed nanopore platform offers realtime single molecule resolution with high sensitivity and specificity; high-throughput capabilities; automated operations; and, requires low sample volume without any sample amplifications.

TF (Thomsen-Friedenreich antigen Galactose- -(1→3)-N-acetyl-D-galactosamine); Tn (TF biosynthetic precursor; N-acetyl-D-galactosamine) and Galectin-3 (Gal-3) are secreted preferentially in nipple aspirate fluid from breast cancer patients (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin.Cancer Res 2005 11(19 Pt 1):6868-71) (Fig. 1). Phage display peptides that bind TF, Tn and Gal-3 with high specificity were therefore generated (Table 1) (Peletskaya EN, et al. J Mol Biol 1997 Jul 18 270(3): 374-84; Landon LA, et al. J Protein Chem. 2003 Feb 22(2): 193-204; Glinsky VV, et al. Cancer Res 2000 May 15 60(10):2584-8; Zou J, et al. Carcinogenesis 2005 Feb 26(2):309-18; Kumar SR, et al. J.Nucl.Med. 2008 May 49(5): 796-803).

Approaches for engineering the connector at N- and C-termini to contain functional tags, purifying the connectors with high batch fidelity, inserting the connectors into membranes, and characterizing their electrophysiological properties were generated (Haque F, et al. Nat.Protoc. 2013 8:373-92; Guo Y, et al. J.Nanosci.Nanotechnol. 2005 5:856-63; Xiao F, et al. ACS Nano 2009 3:100-7; Cai Y, et al. Nanomedicine 2008 4:8-18; Wendell D, et al. NatNanotechnol. 2009 Nov 4:765-72).

An 18 a.a. peptide at the C-terminus of the connector was generated for capture and fingerprinting of EpCAM antibodies in the presence of contaminants in the serum (Wang S, et al. ACS Nano 2013 7:9814-22).

Assays for determining the kinetic parameters of EpCAM peptide/antibody interactions at the single molecule level were established (Fig. 2) (Wang S, et al. ACS Nano 2013 7:9814-22).

Selection of breast cancer specific markers for PDP binding

PDPs targeting breast cancer marker TF, Tn and Gal-3: Glycoantigens are potential biomarkers for breast cancer assessment (Kumar SR, et al. Clin.Cancer Res 2005 Oct 1 11(19 Pt 1):6868-71). Epithelial cancer cells exhibit increased cell surface expression of mucin type antigens with aberrant O-glycosylation. Among such antigens, TF and Tn are displayed on cell-surface proteins and lipids in ~90% of breast adenocarcinomas (Springer GF. Science 1984 224(4654): 1198-206; Osinaga E, et al. Breast Cancer Res Treat. 1994 32(2):139-52; Glinsky VV, et al. Cancer Res 2001 61(12):4851-7). Both TF and Tn have been used clinically as prognostic indicators and have been detected immunologically in breast cancer tissues, lymph nodes, and distant metastases (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin.Cancer Res 2005 11(19 Pt 1):6868-71). TF, when associated with lipids and proteins of cancer cells, mobilized Gal-3 secretion from epithelial cells (Glinsky VV, et al. Cancer Res 2003 63(13): 3805-11), thereby triggering tumor cell transformation and metastatic phenotype (Zou J, et al. Carcinogenesis 2005 26(2):309-18; Raz A, et al. Int.J Cancer 1990 46(5):871-7). TF, Tn and Gal-3 are cancer specific and secreted preferentially in nipple aspirate fluids from breast cancer patients compared to healthy individuals (Fig. 1) (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin. Cancer Res 2005 11(19 Pt 1):6868-71), which is consistent with immunohistochemical analyses of these antigens in biopsy samples (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin.Cancer Res 2005 11(19 Pt 1):6868-71) and proteomic analyses (Alexander H, et al. Clin.Cancer Res. 2004 10(22): 7500- 10; Varnum SM, et al. Breast Cancer Res.Treat. 2003 80(1):87-97).

PDPs targeting breast cancer marker uPA (Urokinase-dependent plasminogen activator system) and PAI (Plasminogen activator inhibitor): Serine proteinase uPA and its inhibitor (PAI type-1) are involved in the control of extracellular matrix turnover, tissue remodeling, and cell migration during patho-physiological processes in breast cancer

(Hildenbrand R, et al. Expert Opin.lnvestig. Drugs 2010 May 19(5):641-52; Hildenbrand R, et al. International Journal of Oncology 2009 Jan 34(1): 15-23). Altered expression of uPA-PAI- 1 are associated with a poor prognosis in breast cancer patients, predicting both outcome and resistance to specific therapies (Schmitt M, et al. Expert Rev.Mol Diagn. 2011 Jul 11(6):617-34; Kantelhardt EJ, et al. BMC Cancer 2011 11 :140). These molecules are the only prognostic markers that have reached the highest level of evidence in multi-centered clinical trials (Schmitt M, et al. Expert Rev.Mol Diagn. 2011 Jul 11(6):617-34). Specific PDPs that bind uPA and PAI-1 are available (Gardsvoll H, et al. FEBS Lett. 1998 Jul 17

431(2):170-4; Hansen M, et al. J Biol Chem. 2005 Nov 18 280(46): 38424-37) (Table 1).

Panel of proposed biomarkers for assessment: To be highly effective in diagnostic, prognostic, and clinical approaches, it is critical to establish an optimal panel of biomarkers. Recent studies have shown that uPA is more predictive of breast cancer in pre-menopausal women (Qin W, et al. BMC Cancer 2012 12:52; Qin W, et al. Ann. Surg. Oncol. 2003

10(8): 948-53), while TF is better at predicting breast cancer in postmenopausal women (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin.Cancer Res 2005

11(19 Pt 1):6868-71). TF/uPA combination can predict breast cancer in both pre- and postmenopausal women with ~88% accuracy (Qin W, et al. BMC Cancer 2012 12:52). Triple combination TF/uPA/PAI-1 resulted in ~99% predictive ability in women requiring surgery due to suspicious lesions (Qin W, et al. BMC Cancer 2012 12:52). Therefore, experiments were conducted to simultaneously investigate all five biomarkers in Table 1 to further enhance predictive capabilities.

Construction of connector with PDP fused to its C- terminal end

Methods for the construction of connector with PDP fused to its C-terminus is straightforward (Cai Y, et al. Nanomedicine 2008 4:8-18; Wang S, et al. ACS Nano 2013 7:9814-22; Xiao F, et al. ACS Nano. 2009 Aug 25 3(8) :2163-70). Briefly, the DNA fragment coding for PDP is inserted into the 3'-end of the open reading frame of the gp10 connector gene. A 6*glycine linker is included for end flexibility. To facilitate purification, a 6* His tag is inserted at the N-terminus. The connectors are purified by His-tag column and verified by SDS-PAGE gel. After purification, the channels with PDP assemble with 12 evenly spaced probes in the same plane within the dodecameric channel (Table 1) (Haque F, et al.

NatProtoc. 2013 8:373-92; Guo Y, et al. J.Nanosci.Nanotechnol. 2005 5:856-63; Xiao F, et al. ACS Nano 2009 3:100-7; Cai Y, et al. Nanomedicine 2008 4:8-18; Wendell D, et al. NatNanotechnol. 2009 4:765-72; Robinson MA, et al. Nucleic Acids Res. 2006 34:2698- 709).

Incorporate PDP functionalized channel into planar lipid membrane to serve as a robust nanopore

Procedures for the incorporation of connectors into lipid membranes have been well established (Haque F, et al. NatProtoc. 2013 8:373-92; Wendell D, et al. NatNanotechnol. 2009 4:765-72; Wang S, et al. ACS Nano 2013 7:9814-22; Haque F, et al. Biomaterials 2015 Mar 26 53:744-52; De-Donatis G, et al. Cell Biosci 2014 4:30; Geng J, et al. ACS Nano 2013 Apr 23 7(4):3315-23; Haque F, et al. Nano Today 2013 8:56-74; Shim J.S, et al. Biomedical Microdevices 2012 Jul 7 14:921-8; Haque F, et al. ACS Nano 2012 Mar 29 6:3251-61 ; Fang H, et al. Biophysical Journal 2012 Jan 4 102:127-35; Geng J, et al. Biomaterials 2011 Jul 31 32:8234-42; Jing P, et al. Nano Lett. 2010 Aug 19 10:3620-7; Jing P, et al. Mol.Biosyst. 2010 6:1844-52). Briefly, connectors are reconstituted into liposomes during the rehydration step followed by vesicle fusion with a planar bilayer. The insertion of the channels results in stepwise increase in conductance. Reengineered connectors display uniform conductance, exhibit a perfectly linear Current- Voltage (l-V) relationship under ±100 mV, and are stable at extreme pH and salt environments (Wendell D, et al. NatNanotechnol. 2009 4:765-72; Fang H, et al. Biophysical Journal 2012 102:127-35; Jing P, et al. Nano Lett. 2010 10:3620-7; Jing P, et al. Mol.Biosyst. 2010 6:1844-52). Herein, these assays are used to thoroughly characterize the electrophysiological properties of PDP functionalized connectors.

Nipple Aspirate Fluid (NAF) collection and processing: From two previous clinical trials (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin.Cancer Res 2005 Oct 1 11(19 Pt 1):6868-71), de-identified NAF samples were banked from healthy and breast cancer patients, which are used in studies. Additional NAF samples are collected in Years 3-4. NAF is collected using automated FDA- approved breast pumps (NeoMatrix). The procedure is non-invasive, inexpensive and can be repeated with minimal discomfort. NAF is de-identified and coded for blind analysis, and stored at -80°C. Since NAF typically are very viscous, the samples are diluted in PBS supplemented with protease and nuclease inhibitors. Soluble antigens are isolated from particulate and a buoyant lipid layer by centrifugation. If necessary, high abundance proteins such as albumin and IgGs are removed using immunoaffinity columns (Life technologies).

Establish parameters for electronic detection of NAF biomarkers

For benchmarking the platform, purified antigens are loaded to the chamber housing the connector harboring the respective PDP probes in Table 1. Typically 10,000+ binding events are evaluated using a custom automated MATLAB program to obtain statistical significance (Haque F, et al. Biomaterials 2015 53:744-52).

Assessment of binding parameters: One parameter is current blockage percentage, calculated as:

is the current when the channel is open, and

is the current observed upon binding by antigen to the PDP probe. Two classes of events are expected: (1) Nonspecific heterogeneous transient binding events due to non-specific hitting of proteins or non-specific antigens to the peptide probe, shown as irregular blockage signals (Wang S, et al. ACS Nano 2013 7:9814-22). (2) Homogeneous specific binding events, as tight binding of specific antigen to the PDP probe when the NAF concentration or the titer of the antigen is low. If NAF concentration or the titer of the specific antigen is high, the signals are observed as discrete stepwise augmentation of current blockage (with significantly longer dwell time) (Wang S, et al. ACS Nano 2013 7:9814-22).

Assessment of kinetic parameters: Another parameter is the dwell time, τ (duration of a blockage event), which are used to determine the docking/undocking kinetics of antigen- PDP interactions, as done with EpCAM antibody-peptide probe interactions (Fig. 2) (Wang S, et al. ACS Nano 2013 7:9814-22). The binding time of the antigen to the PDP is defined as whereas the time between two consecutive blockage events is defined as

Histograms of are fit to a single-exponential decay (Fig. 2B-C). The frequencies

of association and dissociation are plotted as a function of antigen concentration

(Fig. 2D-E) to determine the rate constants and finally the equilibrium

dissociation constant {K_D). This method has the capability of distinguishing specific from non-specific binding with high confidence (Wang S, et al. ACS Nano 2013 7:9814-22). All five proposed biomarkers are secreted in the NAF and are reflective of the breast tumor microenvironment (Deutscher SL, et al. BMC Cancer 2010 10:519; Kumar SR, et al. Clin.Cancer Res 2005 Oct 1 11(19 Pt 1):6868-71; Qin W, et al. BMC Cancer 2012 12:52; Qin W, et al. Ann.Surg.Oncol. 2003 10(8):948-53). large sample population are investigated as well as samples from healthy contralateral breasts from the same patient (as an internal control) to avoid shortcomings and false discovery of biomarkers. There are two kind of signatures, one for low affinity and one for higher affinity probes (Wang S, et al. ACS Nano 2013 7:9814-22). For lower affinity probes, distinct homologous spikes of current blockage are observed due to the shorter dwell time For higher affinity probes, ladder-like discrete step-wise

current blockages (Fig. 3B) are observed with longer dwell time due to superimposition of multiple binding signals. Example 2: Reengineering the motor channel to harbor miRNA Seed-targeting tiny LNA as probe to capture circulating miRNAs in nipple aspirate fluid.

Different cancers have distinct miRNA expression patterns (Bartel DP. Cell 2009 136(2):215-33; Calin GA, et al. Nature Reviews Cancer 2006 6(11):857-66; Garzon R, et al. Annu.Rev.Med. 2009 60:167-79). Circulating miRNAs enveloped inside extracellular vesicles have attracted widespread attention (Mitchell PS, et al. Proc.Natl.Acad.Sci.il. S A 2008

105(30): 10513-8) due to their many features including, rich information content; accessibility in specimen types such as, nipple aspirate fluid, formalin-fixed tissue, and serum; and, ability to detect them non-invasively with high sensitivity have increased the value of certain miRNAs as biomarkers. Several studies have shown that certain miRNAs are important to the phenotype of a particular cancer or are predictive of tumor progression by measuring their relative levels in healthy and tumor samples through miRNA profiling (Mitchell PS, et al. Proc.Natl.Acad.Sci.U.S.A 2008 105(30): 10513-8; Cortez MA, et al. Expert Opin.Biol Ther. 2009 Jun 9(6):703-11; Kosaka N, et al. Cancer Sci. 2010 Oct 101(10):2087-92).

Current methods include, qRT-PCR (gold standard), microarray, Next Generation Sequencing (NGS), and Northern blotting (Chen C, et al. Nucleic Acids Res 2005

33(20):e179; Li W, et al. Anal.Bioanal.Chem. 2009 394(4): 1117-24; Chugh P, et al.

Wiley.lnterdiscip.Rev.RNA. 2012 3(5):601-16; Pritchard CC, et al. Nat Rev.Genet. 2012 13(5):358-69; Hunt EA, et al. Anal.Biochem. 2009 387(1):1-12; Murphy J, et al. Expert Rev.Mol Diagn. 2009 9(2):187-97; Yendamuri S, et al. Transl.Res 2011 157(4):209-15; Hafner M, et al. RNA 2011 17(9): 1697-712). To date, more than 5,000 human miRNAs have been identified. Since miRNA sequence is -22 nt, a high sequence similarity makes it challenging to design primer pairs for qRT-PCR assay to discriminate multiple miRNAs in parallel. Limitations of NGS platforms include high cost, computational infrastructure for data analysis, and biases in preferred miRNA sequence amplification related to enzymatic steps that favor capture of some miRNAs over others (Krichevsky AM, et al. RNA 2003

9(10):1274-81). Similarly, Northern blotting is limited by low throughput and low sensitivity. Other techniques based on colorimetry, luminescence, enzyme turnover, electrochemistry, molecular beacons, and single-molecule fluorescence have been applied for miRNA detection (Hunt EA, et al. Anal.Biochem. 2009 387(1):1-12; Yendamuri S, et al. Transl.Res 2011 157(4):209-15; Krichevsky AM, et al. RNA 2003 9(10): 1274-81 ; Reis PP, et al. BMC Biotechnol. 2011 11:46.; Neely LA, et al. Nat Methods 2006 3(1):41-6). Nanopore platform offers low cost, label-free, amplification-free real-time single molecule resolution;

highthroughput capabilities; and, require low sample volume with no sample modifications. Recently, detection of miRNAs was demonstrated using solid-state (Wanunu M, et al. Nat Nanotechnol. 2010 5(11):807-14) and a-hemolysin protein nanopores (Wang Y, et al. Nat Nanotechnol. 2011 6(10):668-74). Both studies used DNA probes hybridized to target miRNA and measure the signal of translocation. The disclosed approach does not rely on translocation, but rather on conformational changes of the connector induced by capture of miRNA by anti-miRNA-Seed (LNA oligo) probes located at the opening of the pore.

A 6*His-tag was engineered at C-terminal end of the connector and the resulting channel displays robust electro-physiological attributes (Wendell D, et al. Nat. Nanotechnol. 2009 4:765-72; Haque F, et al. ACS Nano 2012 6:3251-61; Jing P, et al. Nano Lett. 2010 10:3620-7; Jing P, et al. Mol.Biosyst. 2010 6:1844-52). There is efficient binding of Ni(ll)- NTA (Nickel(ll)- Nitrilo-triacetic acid) tagged Nanogold to the C-His tagged connector channel (Fig. 3) (Jing P, et al. Nano Lett. 2010 10:3620-7). Cysteine mutant channels were generated for capture of chemicals with reactive sulfhydryl groups (Haque F, et al. ACS Nano 2012 6:3251-61). A oligonucleotide synthesizer was obtained for high yield solid-state synthesis of RNA oligos.

Synthesize miRNA Seed-targeting tiny LNA as detection probes

LNAs are a class of conformational^ restricted nucleotide analogs, in which the ribose ring is constrained by a methylene linkage between the 2'-0 and 4'-C resulting in a locked 3'-endo conformation (Fig. 4). Structural studies have shown that LNA oligos adopt an A-type (RNA-like) duplex conformation (Bondensgaard K, et al. Chemistry 2000

6(15):2687-95; Natsume T, et al. Chem.Phys.Lett. 2007 434:133-8). Detection of miRNAs using LNAs has emerged as a powerful tool in biological assays, such as microarrays, PCR, in situ hybridization, anti-sense inhibition, and Northern blotting (Castoldi M, et al. Nat Protoc. 2008 3(2):321-9; Shu D, et al. ACS Nano. 2015 9(10):9731-40; Obad S, et al. Nat.Genet. 2011 43(4):371-8; Kumar S, et al. Biochemistry 2014 53(10): 1607-15; Hackler L, et al. Invest Ophthalmol.Vis.Sci. 2010 51 (4): 1823-31). LNAs have several favorable attributes: (1) high thermal stability when hybridized to a complementary RNA strand (For each LNA base incorporation, the Tm of the duplex increases by 2-8 °C (Bondensgaard K, et al. Chemistry 2000 6(15): 2687-95). Thus a LNA with 8-nt complementary to the seed sequence of the miRNA is strong enough to bind and capture the miRNA target); (2) superior single nucleotide discrimination compared to DNA, RNA or PNA (Peptide Nucleic Acid) probes; (3) resistant to exo-/endo-nucleases, and therefore stable in patient samples; (4) strand invasion properties enable detection of 'hard to access' samples; and (5) high water solubility.

Selection of miRNAs for constructing LNAs: Clinical microarray analysis revealed several miRNAs dysregulated in breast cancer patient nipple aspirate fluids and some of these can serve as robust prognostic or predictive markers for a wide range of breast cancer subtypes (Zhang K, et al. Ann Surg Oncol. 201522 Suppl 3:S536-44; do Canto LM, et al. Cancer Research 2013 Apr 15 73(8):3465). The microarray results were further validated by qRT-PCR analysis with matching sequences in the miRNA databases. Based on these data, we selected a panel of miRNAs for differentiating breast cancer from benign tumors and healthy controls (Table 2). Significant upregulation of oncogenic miRNAs (miR-K12-5-5p, miR-4484, miR-let-7i-5p, miR-92a-3p, miR-155-5p) in nipple aspirate fluid has relevant oncogenic roles by promoting invasion, proliferation and metastases, and poor prognosis in breast cancer patients, and, therefore have high prognostic values (Zhang K, et al. Ann Surg Oncol. 2015 22 Suppl 3:S536-44; do Canto LM, et al. Cancer Research 2013 Apr 15 73(8):3465).

Synthesis and purification of LNA probes: Nucleotides 2-7 of the mature miRNA sequence (Obad S, et al. Nat.Genet. 2011 43(4):371-8) (Fig. 4B) create the 'Seed region' that determines the specificity of binding to LNA. Individual LNA oligo probes for each target miRNA (Table 2) are synthesized by phosphoramidite chemistry: (1) using an oligo synthesizer with Ni(ll)-NTA end modifications for conjugation; or (2) custom-ordered from Exiqon. The LNA oligos are then purified by routine HPLC-based procedures.

Incorporation of LNA oligo probes to the connector

Thr well characterized C-His tagged membrane-embedded connectors (Wendell D, et al. NatNanotechnol. 2009 4:765-72; Haque F, et al. ACS Nano 2012 6:3251-61 ; Jing P, et al. Nano Lett. 2010 10:3620-7; Jing P, et al. Mol.Biosyst. 2010 6:1844-52) are for

conjugating LNA detection probes.

Conjugation strategy: The 6*His-tag at the C-terminal end of the connector is used for Ni(ll)-NTA binding. NTA forms a tetradentate chelate with the Ni(ll) ion. The electron donor groups on histidine imidazole rings readily form coordinate bonds with the Ni(ll)-NTA complex, with K_D = 390 nM (Takahira I, et al. Bioorg.Med.Chem.Lett. 201424(13):2855-8; Khan F, et al. Anal.Chem. 2006 78(9): 3072-9). Since the connector channel has 12 subunits, 12 LNA probes are conjugated to each connector. There was efficient binding of Ni(ll)-NTA tagged Nanogold to C-His tagged connector channel (Fig. 3).

Validate the presence of LNA probes on the C-terminal end of the connector: Upon addition of Ni(ll)-NTA tagged LNA probe, there can be stepwise capture of the LNA probes by the His-tags protruding out of the C-terminal end, similar to the case with Ni(ll)-NTA- Nanogold probes (Fig. 3) (Jing P, et al. Nano Lett. 2010 10:3620-7). The current amplitude for each LNA capture are deduced based on the blockage events with appropriate controls (such as, LNA probe without NTA). To maximize the number of probes bound per connector, the LNA probes are pre-bound to the connector (under saturating conditions) prior to insertion into lipid membrane. The number of probes bound per connector and number of connectors in the membrane are back calculated. Gel retardation assay (Xiao F, et al.

Nucleic Acids Res 2005 33:2640-9; Xiao F, et al. ACS Nano. 2010 Jun 22 4(6):3293-301) or single fluorescence photobleaching assay (Shu D, et al. EMBO J. 2007 26:527-37; Zhang H, et al. RNA 2007 13:1793-802; Zhang H, et al. Methods 2014 Jan 1567:169-76) are used to verify the binding and LNA stoichiometry per connector. Any unbound probes are washed by automated buffer exchange in the chamber. This approach is used to generate 5 unique LNA probe bound connectors designed for specific miRNA targets (Table 2). Establish parameters for electronic detection of synthetic miRNA

To benchmark miRNA detection capabilities, synthesized and purified miRNAs are used as ligands for the assay. miR-155-5p is used to establish the detection parameters and then the parameters extended to the other four miRNAs. Purified miR-155-5p are loaded to the chamber housing the connector conjugated with anti-miR-155-5p LNA probe.

Modulations in current induced by miR-155-5p and anti-miR-155-5p LNA interactions are then evaluated. Typically 10,000+ binding events are evaluated using a custom automated MATLAB program to obtain statistical significance (Haque F, et al. Biomaterials 2015 53:744-52).

Assessment of binding parameters: the current blockage percentage observed upon capture of miRNA by the LNA probe is determined, and parameters for specific detection from non-specific binding signals are established.

Assessment of kinetic parameters: The dwell time distribution is used to determine the docking and undocking kinetics of miRNA-LNA probe interactions, as done with EpCAM antibody - EpCAM antigen probe interactions (Fig. 2) (Wang S, et al. ACS Nano 2013

7:9814-22).

Quantification of synthetic miRNAs: The target miRNA are quantified by measuring the capture rate for the miRNA: LNA duplex at the C-terminus of the pore. A calibration curve showing the capture rate as a function of synthetic miRNA: LNA concentration is constructed. Upon introducing an unknown concentration of miRNA sample to the nanopore, the mean capture rate is calculated and the miRNA concentration are determined from the calibration curve. Based on previous RNA characterization assays (Haque F, et al. Biomaterials 2015 53:744-52; Geng J, et al. ACS Nano 2013 7(4):3315-23; Jing P, et al. Nano Lett. 2010 10:3620-7), miRNAs can be detected in the ng - pg/μί. scale, which is well within the limits of clinical samples (Pritchard CC, et al. Nat Rev.Genet. 2012 13(5): 358-69).

Extraction of miRNAs from patient NAF samples

NAF is collected and processed. Typically, circulating miRNAs are present in exosomes to prevent degradation. RNA Isolation Kits (Exiqon) are used for extracting the miRNAs. Briefly, after thawing, NAF samples are centrifuged at 3000 ^χ g for 5 min to pellet any debris and insoluble components. Exosomes are lysed followed by precipitation of proteins. The supernatant is loaded onto a spin-column. The resin only binds RNA, while residual proteins are removed in the flow-through. Bound RNA are washed to remove any remaining impurities, and purified RNA is eluted in RNase free water.

Electronic detection of miRNA extracted from breast cancer patients and healthy individuals Extracted miRNA samples are divided into two aliquots for the nanopore and qRT- PCR assays, respectively. Herein, the miRNA extracts are loaded into individual nanopore chambers housing different LNA probe bound connectors. We will then deduce the relative expression levels of each of the five miRNAs in breast cancer and healthy individuals using electronic detection parameters established above with additional modifications as noted below.

Normalization strategies: To standardize the miRNA detection system across different individuals, commonly used miRNA normalizer 'RNU6B (U6)' that is stably expressed in both healthy individuals and breast cancer patients is used (Ng EK, et al. Gut 2009 58(10): 1375-81). Combining the 'endogenous' normalizer with a 'spiked-in' control offers an ideal normalization strategy. Synthetic C. elegans miR-39 is spiked-in, which is absent in the human genome as an internal control (Li Y, et al. Anal.Biochem. 2012

431(1):69-75). The frequency of miR-39 events should be independent of the

samples. Therefore the ratio is ^used to normalize the assays. This

frequency ratio in the breast cancer group can be significantly higher than the healthy group.

Cross-validate our miRNA profiling data using qRT-PCR

Obtained data is cross-validated using qRT-PCR to confirm that the phi29 motor channel can indeed be used for accurate assessment of miRNA levels in breast cancer nipple aspirate fluid. Total RNA samples are assayed for expression of the specific human miRNA targets. Reverse transcription is performed on 10 ng of each miRNA sample using the TaqMan® MiRNA Reverse Transcription Kit (Life Technologies) to generate cDNA products, which are then subjected to quantitative real-time PCR analysis using TaqMan® Universal PCR Master Mix (Life Technologies) and Roche LightCycler® 480-II instrument. For both reverse transcription and cDNA amplification, primer sets from target specific TaqMan® MicroRNA Assays are used.

Example 3: Incorporate probe-functionalized motor channels into lonera Orbit16 equipment for high throughput detection of breast cancer biomarkers for clinical translation

For clinical translation, the platform can be integrated into a user-friendly machine that can be used in a hospital for automated multiplexed high-throughput detection.

Incorporating the device into routine practice can enable physicians and patients to monitor cellular changes within breast ducts earlier, before they develop into larger, potentially cancerous lesions. Therefore, a robust multichannel platform was developed for single pore sensing. For analytical tasks that require low-noise and high-frequency bandwidth recording, low- capacitance micro-bilayer on smaller-than-standard (100 m) apertures are required, which are not readily produced in a microarray format. A major challenge is to provide a high- density microarray platform for parallel bilayer recording without compromising the precision of state-of-the-art single micro-bilayer nanopore experiments, lonera's vOrbit16 platform overcomes this challenge (Del Rio Martinez JM, et al. Small. 2015 11(1):119-25; Baaken G, et al. ACS Nano 2011 5(10):8080-8; Baaken G, et al. Lab Chip. 2008 8(6):938-44).

lonera's parallel bilayer microarray device involves a 4x4 array of microcavities with integrated Ag/AgCI electrodes in a polymer MECA (MicroElectrode Cavity Array) substrate directly interfaced with the circuit electronics. The platform combined with the disclosed robust nanopore has several advantages (Del Rio Martinez J M, et al. Small. 2015 11(1):119- 25; Baaken G, et al. ACS Nano 2011 5(10):8080-8; Baaken G, et al. Lab Chip. 2008

8(6):938-44) for biomarker profiling: (1) automated formation of 16 bilayers housing multiple connectors; (2) 16 simultaneous measurements for multiplexed high-throughput analysis; (3) low noise and high bandwidth; (4) automated perfusion for rapid solution exchange; (5) integrated bilayer membranes that display long-term stability, and remarkable resistance to mechanical stress (requiring no vibration insulation and tolerating repeated solution exchanges and non-stop electrical forces); (6) reusable chips; (7) various chip configurations for experimental flexibility and output; (8) cost-efficient batch production of chips; and (9) allows integration of robotic arms for unattended operation.

A Poly(Di-Methyl-Siloxane) (PDMS) based Lab-On-a-Chip (LOC) device was developed for high throughput screening (Shim J.S, et al. Biomedical Microdevices 2012 14:921-8). By capturing a small amount of the lipid solvent at the junction of the

microchannel, multiple lipid bilayers inside the array were reliably created within the microchannel. Each microchannel was independently connected to an Ag/AgCI electrode array, allowing simultaneous recording of multiple lipid bilayers during the translocation of DNAs through the phi29 connector in real time. Connectors were successfully incorporated into an Orbit 16 device. Individual chambers can be monitored in parallel (Fig. 5A). A custom MATLAB-based algorithm was devloped for quantitative ultra-fast processing of nanopore derived data (Haque F, et al. Biomaterials 2015 53:744-52).

Incorporate probe functionalized motor channels into Orbit16

The bilayer in Orbit16 platform is automatically formed by remotely actuated painting followed by fusion of proteo-liposomes to generate membrane embedded nanopores.

Recordings were conducted on the MECA 16 Chip, which demonstrates the feasibility of using the Orbit16 (Fig. 5B). Parallel recordings were done from 16 chambers with a Tecella- Triton multichannel amplifier integrated in Orbit16. Each of the 16 chambers (cavity: 5 μιη) in the chip can house hundreds of phi29 channels (diameter ~14 nm) while retaining robust substrate stability. The membrane insertion efficiency, signal stability, current homogeneity, and membrane durability are characterized using established assays (Wendell D, et al.

Nat.Nanotechnol. 2009 4:765-72; Shim J.S, et al. Biomedical Microdevices 2012 14:921-8; Jing P, et al. Nano Lett. 2010 10:3620-7; Jing P, et al. Mol.Biosyst. 20106:1844-52).

Generate MECA chips housing one kind of probe-functionalized connectors: For detecting antigens with low titer, it is ideal to develop one MECA chip harboring one kind of probe functionalized connectors in each of the 16 chambers. A total of 10 unique MECA chips are developed for each of the five PDP (from Table 1) and 5 anti-miRNA probes (from Table 2) (Fig. 6A). Each chamber can further house potentially hundreds of connectors. Due to multi-fold increase in the event number as multiple nanopores participate in recording, targets can be detected at ultra-low concentrations. Nipple aspirate fluids are added to the chambers for evaluation of the target biomarker following the detection parameters outlined above. This method is very sensitive at reaching single molecule level, and the method is specific for each antigen, since the current blockage rate can distinguish the specific binding from the non-specific binding (Fig. 5B-C) (Wang S, et al. ACS Nano 2013 7:9814-22).

After benchmarking the detection limits of the system, a multiplexed platform harboring one unique connector in each of the 16 chambers is developed for parallel detection of 16 biomarkers using a single chip (Fig. 6B). The MECA chip design is also exteded to an 8x8 array (64 unique biomarkers) for providing a very comprehensive and quantitative biomarker profile.

Development of computer program for automated data analysis

Typically ~10000 events need to be analyzed to ensure the result is within statistical significance, a simple automated MATLAB algorithm is developed for quantitative ultra-fast processing of thousands of nucleic acid translocation events, as well as single molecule binding events (Haque F, et al. Biomaterials 2015 53:744-52). Analysis of samples from breast cancer patients can involve further modification of the algorithm so that one can (a) identify all non-specific and specific binding events using the outlined set of parameters; (b) quantify identified events; and (c) further discriminate between 'contaminative' signals and true capture events.

Correlate data with clinical associations

The data is analyzed to establish the ability to correlate the acquired data with clinical breast cancer classifications based on: tumor type (ductal, lobular, medullary, mucinous, etc), size of invasive component, grade of the invasive component, expression of hormone receptors including Estrogen Receptor (ER) and Progesterone Receptor (PR), expression of the growth factor receptor HER2/neu (ERBB2), mutational status of breast cancer susceptibility genes (BRAC1 and BRAC2), menopausal status and age, the presence and number of lymph node metastases.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A fusion protein comprising a) a transmembrane aperture-forming domain of a DNA- packaging motor connector protein and b) an affinity domain that selectively binds a breast cancer biomarker protein or nucleic acid.

2. The fusion protein of claim 1, wherein the DNA-packaging motor connector protein is from bacteriophage phi29.

3. The fusion protein of claim 1 or 2, wherein the breast cancer biomarker protein comprises Thomsen-Friedenreich (TF), Tn (TF precursor), Galectin-3 (Gal-3), Urokinase- dependent plasminogen activator (uPA), Plasinogen activator inhibitor (PAI-1), or a combination thereof.

4. The fusion protein of claim 1 or 3, wherein the breast cancer biomarker nucleic acid comprises a miR-155-5p, miR-4484, miR-let-7i-5p, miR-92a-3p, or miR-K12-5-5p, or a combination thereof.

5. A composition comprising a membrane layer having incorporated therein one or more fusion proteins of any one of claims 1 to X to form an aperture through which conductance can occur when an electrical potential is applied across the membrane.

6. The composition of claim 5, wherein the membrane layer comprises a phospholipid bilayer.

7. A method for detecting a breast cancer biomarker in a biological sample from a subject, comprising

(a) providing a conductive channel-containing membrane which comprises a membrane layer and incorporated therein one or more fusion proteins of any one of claims 1 to 4;

(b) applying an electrical potential to the membrane and measuring a first conductance signal that results from the applied electrical potential;

(c) contacting the conductive channel-containing with the biological sample form the subject,

(d) applying an electrical potential to the membrane and measuring a second conductance signal that results from the applied electrical potential,

wherein a change between the first and second conductance signals indicates binding of a breast cancer biomarker molecule in the biological sample to the connector protein, thereby detecting the presence of the breast cancer biomarker.

8. The method of claim 7, wherein the membrane layer comprises a phospholipid bilayer.

9. The method of claim 7 or 8, wherein the conductance signal is measured using a microelectrode on which is formed the membrane layer.

10. The method of claim 9, wherein the microelectrode comprises a Ag/AgCI microelectrode.

11. A system, comprising one or more chambers, each chamber comprising a microelectrode having formed thereon a conductive lipid bilayer having incorporated therein one or more fusion proteins of any one of claims 1 to 4 to form an aperture through which conductance can occur when an electrical potential is applied across the lipid bilayer, wherein conductance through the aperture is detected by the microelectrode.

12. The system of claim 11, comprising at least 16 individually controlled chambers.

13. The system of claim 11 or 12, wherein each chamber comprises the same fusion proteins for detection of the same breast cancer biomarker.

14. The system of claim 11 or 12, wherein at least two chambers comprise different fusion proteins for detection of different breast cancer biomarkers.

15. The system of any one of claims 11 to 14, wherein the microelectrode comprises a Ag/AgCI microelectrode.