阅读说明：本技术 膀胱癌检测装置和方法 (Bladder cancer detection device and method ) 是由 克拉西米尔·阿塔纳索夫·瓦西里夫 梅勒妮·麦格雷戈 乔纳森·格利德尔 乔丹·李 于 2018-04-12 设计创作，主要内容包括：提供了一种用于从尿液或者源自尿液的流体中选择性捕获目标膀胱癌细胞的装置。所述装置包括具有一个或者多个细胞捕获表面的基底,每一细胞捕获表面包含在所述基底上的官能化膜和共价结合到所述官能化膜上的一种或者多种目标膀胱癌细胞选择性结合试剂。(devices for selectively capturing targeted bladder cancer cells from urine or urine-derived fluids are provided, the devices comprising a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more targeted bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane.)

1, a device for selectively capturing targeted bladder cancer cells from urine or a fluid derived from urine, the device comprising a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more targeted bladder cancer cell-selective binding agents covalently bound to the functionalized membrane.

2, a microfluidic device for selectively capturing targeted bladder cancer cells from urine or a fluid derived from urine, the device comprising a substrate having or more cell capture microchannels, each cell capture microchannel comprising a functionalized membrane on its surface and or more targeted bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane.

3. The device of any of claims 1 and 2, wherein the target bladder cancer cells are selected from or more of urothelial cancer cells, squamous cell carcinoma cells, adenocarcinoma cells, small cell carcinoma cells, and sarcoma urine cells.

4. The device of claim 3, wherein the target bladder cancer cell is a urothelial cancer cell.

5. The device of any of claims 1-4, wherein the functionalized membrane is a plasma polymerized polyoxazoline.

6. The device of any of claims 1-5, wherein the targeted bladder cancer cell-selective binding reagent is an immobilized functional antibody capable of selectively capturing targeted bladder cancer cells.

7. The device of any of claims 1-6, wherein the antibody is an anti-epithelial cell adhesion molecule (anti-EpCAM).

A method of for selectively capturing target bladder cancer cells from urine or a fluid derived from urine, the method comprising:

providing a sample of urine or a fluid derived from urine;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells (if present) from the urine bind to the cell capture surfaces.

9, A method of immobilizing target bladder cancer cells on a basal surface in a fluid derived from urine, the method comprising:

providing a sample of urine or a fluid derived from urine;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells (if present) from the urine bind to the cell capture surfaces.

10, methods of diagnosing or monitoring bladder cancer in a mammal, the method comprising:

providing a sample of urine or urine-derived fluid obtained from said mammal;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells from the urine, if present, bind to the cell capture surfaces, and

analyzing the target bladder cancer cells bound to the cell capture surface.

11. The method of any of claims 8-10, wherein the target bladder cancer cells are selected from or more of urothelial cancer cells, squamous cell carcinoma cells, adenocarcinoma cells, small cell carcinoma cells, and sarcoma urine cells.

12. The method of claim 11, wherein the target bladder cancer cell is a urothelial cancer cell.

13. A method in accordance with any of claims 8 to 12, wherein the functionalised film is a plasma polymerised polyoxazoline.

14. The method of any of claims 8-13, wherein the targeted bladder cancer cell-selective binding agent is an immobilized functional antibody capable of selectively capturing targeted bladder cancer cells.

15. The method of any of claims 8-14, wherein the antibody is an anti-epithelial cell adhesion molecule (anti-EpCAM).

16. Apparatus substantially as herein described with reference to the accompanying examples.

17. A method substantially as herein described with reference to the accompanying examples.

Technical Field

The present disclosure relates to devices and methods for selectively capturing and/or detecting bladder cancer cells, or biomarkers thereof, from urine. The apparatus and method may be particularly applied in point of care (point of care) diagnostic apparatus and methods.

Background

The bladder is the most common cancer site in the urinary tract (renal tract) and the most common histological type is transitional cell carcinoma (transitional cell carcinoma). patients with bladder cancers are at high risk of developing other urinary tract cancers and have a particularly high rate of recurrence (70%) (Dawam 2012; Youssef and Lotan 272011). therefore, regular and indefinite monitoring of patients with bladder cancer is necessary.

Bladder cancer is currently monitored by invasive, expensive techniques such as cystoscopy (Bryan et al 2014a), which to date is considered the gold standard of care for clinicians. There is a need for cheaper, less invasive precision tests. (Lotan et al 2009; Ploeg et al 2009)

possible non-invasive means for diagnosing bladder cancer is to analyze the urine of a patient the presence of exfoliated tumor cells in the urine is the basis for cytological examinations of urination (Deden 1954; Kiyoshima et al 2016; McGrew 1961; Murphy1990) which is the current non-invasive standard of care of the sole for bladder cancer detection (Deden 1954; Gaston and Pruthi 2004; McGrew 1961; Papanicolaou and Marshall 1945; Zhang et al 2001) however, the efficacy of urine cytological efficacy is controversial, the sensitivity of which is unexpectedly low (40%) (Andersson et al 2014; 40 Mitra and Cote 2010) is due to the low sensitivity of tumor cells, particularly low-grade tumor cells, to similarities with normal cells (e.g. the abundant cytological examinations of Brysson 2014; 40 Mitra and Cote 2010) is based on the common medical expertise of Bryan 2014 et al; the morphological examinations are only hard to distinguish malignant cells; the cause of this study; Bryan et al 2009 is due to the present clinical findings)

Another challenges faced by urine cytologists are obtaining a clear view of cells of interest in urine protein, debris (debris), and other background cells (Bastacky et al 1999) generally, conditions other than cancer induce similar symptoms and result in increased urine cell composition, including but not limited to infection, or kidney disease (Oliveira Arcolino 50 et al 2015) to minimize background, natural urine has to be treated comprehensively, a process that increases sample analysis time and cost (Wrona et al 2014)

It is therefore desirable to provide devices and methods that can selectively and sensitively capture bladder cancer cells from urine, thereby reducing the reliance on expensive and time consuming methods such as urine cytology and cystoscopic monitoring.

Disclosure of Invention

The present disclosure derives from the discovery that tumor cells that fall in the urine of bladder cancer patients can be selectively immunocaptured from untreated urine or natural urine and collected on a biomaterial platform. In particular, we have found that bladder cancer epithelial cancer cells (bladder cancer cells) can be selectively captured on a basal surface functionalized with a biologically active epithelial cell adhesion molecule (EpCAM) antibody. EpCAM was chosen as a specific capture ligand because it is the most well known epithelial cancer (carcinoma) specific antibody. (Bryan et al 2014 b; Patriarca et al 2012)

In an th aspect, provided herein are devices for selectively capturing targeted bladder cancer cells from urine or a fluid derived from urine, the devices comprising a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more targeted bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane.

In a second aspect, provided herein are microfluidic devices for selectively capturing targeted bladder cancer cells from urine or a fluid derived from urine, the devices comprising a substrate having or more cell-capture microchannels, each cell-capture microchannel comprising a functionalized membrane on its surface and or more targeted bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane.

In a third aspect, the present disclosure provides methods of selectively capturing target bladder cancer cells from urine or a fluid derived from urine, the method comprising:

providing a sample of urine or a fluid derived from urine;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells (if present) from the urine bind to the cell capture surfaces.

The method of the third aspect may further comprise step of detecting the bladder cancer cells of interest on the or more cell capture surfaces the captured bladder cancer cells may be detected using a cancer specific fluorescent active compound such as ALA5, hexosaminide valerate (hexaminovalinate) or hypericin.

In a fourth aspect, the present disclosure provides methods of immobilizing target bladder cancer cells on a basal surface in a fluid from urine or derived from urine, the method comprising:

providing a sample of urine or a fluid derived from urine;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells (if present) from the urine bind to the cell capture surfaces.

In a fifth aspect, the present disclosure provides methods of diagnosing or monitoring bladder cancer in a mammal, the method comprising:

providing a sample of urine or urine-derived fluid obtained from said mammal;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells from the urine, if present, bind to the cell capture surfaces, and

analyzing the target bladder cancer cells bound to the cell capture surface.

In an embodiment of the fifth aspect, the step of assaying the target bladder cancer cells bound to the cell capture surface comprises detecting the target bladder cancer cells using a cancer-specific fluorescently active compound. The cancer specific fluorescent active compound may be ALA5, hexosaminide valerate (hexaminoluteinate) or hypericin.

Drawings

Embodiments of the disclosure will be discussed with reference to the drawings herein:

FIG. 1 is schematic diagrams of a non-invasive diagnostic method of the present disclosure;

figure 2 summarizes the results from the western blots analysis, the immunohistological staining analysis, and the FACS analysis, which confirmed the expression of cancer specific EpCAM membrane protein by bladder cancer cell lines;

FIG. 3 shows the results of FACS analysis, which recognizes EpCAM and E-Cadherin (E-Cadherin) as potential cancer-specific membrane markers;

FIG. 4 shows a western blot analysis confirming the EpCAM expression results obtained by FACS;

FIG. 5 shows the results of an immunohistological staining analysis, which confirmed the FACS and western blots results;

figure 6 shows evidence of selective capture of bladder cancer cells into functionalized fluidic microchannels, compared to positive and negative control microchannels, results of captured cell number and percentage, sensitivity and specificity are given from left to right, the left figure shows the number of healthy cells (F001) and bladder cancer (RT4) cells present on the substrate before (seeding) and after (capturing) rinsing of type 3 microchannels, control common PPOx surface, blocking PPOx substrate, EpCAN EpCAM functionalized PPOx substrate, the middle figure shows the percentage of cells captured per visual field for at least three tested surfaces.

FIG. 7 shows cell capture sensitivity and selectivity for low cell numbers incorporated in real urine for two different exemplary bladder cancer cell lines (HDF: left column; HT1376: right column);

FIG. 8 shows grade 3 bladder cancer cells HT1376 captured under flow conditions from 5mL of real urine containing only 1000 incorporated cells;

FIG. 9 shows the results of fluorescence spectroscopy for cancer cell-specific red fluorescence in co-culture of cancer cells and healthy cells, and for optimization studies with the concentration of fluorescent marker in vitro tests;

FIG. 10 shows an example of the cellular makeup of a confirmed bladder cancer patient urine sample, with blue nuclear staining and red cancer-specific fluorescence;

FIG. 11 shows an example of cancer cell capture results from urine samples of patients with confirmed transitional cell carcinoma (transitional cell carcinoma). cancer cells contained blue (nuclear staining) and prominent red fluorescence (BF: left bar; Dapi: middle bar; HexAla: right bar);

FIG. 12 shows an example of a negative control of the results of cancer cell capture from urine samples from healthy patients (BF: left column; Dapi: middle column; HexAla: right column);

FIG. 13 shows a direct comparison between the cancer cell detection device results and results from cytology and cystoscopy for the same samples;

FIG. 14 illustrates criteria for use in automated enumeration software to confirm cancer characteristics of cells captured in a microchannel; and

FIG. 15 shows images of the ideal status of cancer cell lines HT1376 and HT1197 in 50: 50 coculture with healthy Human Foreskin Fibroblasts (HFFs), shown in the fluorescence micrograph on the right, the top image is overlaid with the bright field image to see the presence of non-fluorescent healthy cells, in the data plotted on the left of the figure, the plotted data at each concentration and time point are, from left to right, HFF; HT 1197; HT 1376; EJ 138; RT4 cells.

Detailed Description

In an th aspect, provided herein are devices for selectively capturing targeted bladder cancer cells from urine or a fluid derived from urine, the devices comprising a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more targeted bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane.

Advantageously, the device of aspect may be used in a point of care (point of care) device capable of selectively capturing bladder cancer cells from urine in the past decades, experimental urine marker tests have become available.(Tetu 0000) the aim of developing these molecular tests was to perfect cytology by detecting soluble cancer-specific biomarkers (Onal et al 2015) however, to date, there have been no timely tests introduced into clinical guidelines because their added value for diagnosing urothelial tumors remains to be identified (a)

Et al 2015) there is currently no need for non-invasive tests with the sensitivity and specificity required to replace cystoscopy and therefore patient-friendly alternatives still exist. (Cheung et al 2013)

The device of aspect is used to selectively capture target bladder cancer cells from urine the bladder wall has several layers, which are made up of different types of cells most bladder cancers begin at the innermost lining of the bladder (i.e., the urothelium or transitional epithelium) which becomes more advanced and potentially more difficult to treat as the cancer progresses into or through other layers of the bladder wall.

Urinary epithelial carcinoma (also known as transitional cell carcinoma), which is by far the most common type of bladder cancer, is known to begin with urinary epithelial cells that are disposed inside the bladder, urinary epithelial cells are also arranged elsewhere in the urethra, such as the renal portion connected to the ureters, the multiple ureters, and the urethra several other types of cancer may begin with the bladder, including squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma (adenocarinoma), small cell carcinoma (small cell carcinoma), and Sarcoma (Sarcoma).

Suitable substrate materials include glass, silicon, ceramics, metals, plastics, polymeric materials, paper laminates, cellulose, carbon fibers, biomaterials, surfaces containing biomolecules, surfaces containing small organic molecules, surfaces containing inorganic molecules, etc. the plastics may be selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyethylene terephthalate, polyethylene naphthalate, tetrafluoroethylene-hexafluoropropylene copolymer, polyvinylidene fluoride (polyvinylidene-difluoride), nylon, polyvinyl chloride, copolymers of the foregoing, and mixtures of the foregoing.

The substrate has or more cell capture surfaces the or more cell capture surfaces can be formed on the surface of the substrate in or more features the or more features can be in the form of wells, such as in the form of 96-well plates, or they can be or more fluid flow paths of any size, geometry or configuration, the or more fluid flow paths can be in the form of or more channels (open or closed), such as channels commonly used in "flow-through" type diagnostic devices, in embodiments the substrate comprises microfluidic features, such as microfluidic channels in a microfluidic device.

Thus, in a second aspect, there is provided microfluidic devices for selectively capturing targeted bladder cancer cells from urine or a fluid derived from urine, the device comprising a substrate having or more cell-capture microchannels, each cell-capture microchannel comprising a functionalized membrane on its surface and or more targeted bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane.

In certain embodiments, the microchannel is as described in U.S. patent application publication No.2011/0294187, the contents of which are incorporated herein by reference in their entirety, in particular, the microchannel may be defined in a three-dimensional (3D) pattern that enables the flow characteristics (flow profile) within the microchannel to be influenced, which in turn improves the interaction between the flowing sample urine and the cell capture surface and thus significantly improves the cell capture efficiency in some embodiments embodiments, the microchannel surface is made of poly (dimethylsiloxane) (PDMS).

In contrast, the selectivity of the devices of aspects and second aspects means that only cancer cells are immobilized, thus overcoming the problems of complexity, extracellular composition (extracellular) and cell morphology.

As discussed in more detail later, the selectivity of the devices of aspects and second aspects is produced by the use of or more binding agents that selectively bind to a cancer cell of interest and, more specifically, a biomarker of the cancer cell of interest as used herein, the term "selective capture" and similar terms when used in connection with capturing a target bladder cancer cell means that, from a heterogeneous mixture (heterologous mixture) containing the target cancer cell, other cancer cells, and other cells, the target cancer cell preferentially binds such that they are retained on the substrate and the non-target cells can be washed away or otherwise removed from the surface.

The target bladder cancer cells that may be captured using the apparatus of aspects and second aspects may be selected from or more of the group consisting of urological epithelial cancer cells, squamous cell carcinoma (squamous cellcancer) cells, adenocarcinoma cells, small cell carcinoma cells, and sarcoma urine cells.

However, as will be appreciated by those skilled in the art, target bladder cancer cells from other organisms may be used in animal models for disease and drug evaluation.

The cell capture surface of the device of the th and second aspects comprises a functionalized membrane on the substrate, the functionalized membrane may comprise any inorganic, organic and/or biological material, molecule or mixture of molecules that can be attached to the surface of the substrate by covalent or ionic bonds and that contains or more functional groups that can be used to covalently or ionically bind to a targeted bladder cancer cell-selective binding agent as discussed in more detail hereinafter, in many cases the targeted bladder cancer cell-selective binding agent will be a biomolecule, such as a peptide, protein or antibody, and thus the or more functional groups of the functionalized membrane are preferably carboxylic acid groups, carboxylate groups, amino groups or amido groups capable of binding to biomolecules.

Examples of chemical entities suitable for covalent attachment of the targeted bladder cancer cell-selective binding agent. Suitable chemicals include oxazolines, epoxides, aldehydes, anhydrides, thiols, EDC/NHS related chemicals, click chemistry (click chemistry), isocyanates/salts, nitriles, and imines.

In certain embodiments, the functionalized membrane is a polymer. The polymer may be formed by classical polymerization techniques. Thus, the polymer may be formed on the surface of the substrate by polymerizing a suitable starting monomer or prepolymer using a suitable polymerization agent, as is known in the art.

In yet other embodiments, the functionalized membrane may be formed by plasma polymerization, thus, in certain embodiments, the functionalized membrane is a plasma polymer formed by plasma polymerization of or more functional starting materials the starting materials comprising oxazoline, epoxy, aldehyde, anhydride, thiol, isocyanate, nitrile, and imine groups may be plasma polymerized using the conditions described herein to form a plasma polymer comprising oxazoline, epoxy, aldehyde, anhydride, thiol, isocyanate, nitrile, and imine groups, which groups may then react with the bladder cancer cell-selective binding reagent to form covalent bonds therewith.

The conditions required to polymerize the or more functional starting materials to form a plasma polymerized functionalized film may include a power of about 10W to about 50W, a deposition time of about 1 minute to about 7 minutes, and/or about 1.1 to about 3x10^-1 some other deposition conditions will be applicable depending on the plasma deposition apparatus design and power coupling efficiency.

Non-limiting examples of starting materials that may be used include 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2, 4-disubstituted oxazolines, 2, 5-disubstituted oxazolines, 4, 5-disubstituted oxazolines, 2,4, 5-trisubstituted oxazolines, propionaldehyde, glycidyl methacrylate, and allyl glycidyl ether.

In certain embodiments, the functionalized membrane is a plasma polymerized polyoxazoline ("PPOx"). As used herein, the term "polyoxazoline" refers to a homopolymer or copolymer formed from at least oxazoline starting materials or monomers, the oxazoline polymer may or may not contain intact oxazoline structures, the polyoxazoline polymer may be a copolymer formed by plasma polymerization of at least oxazoline starting materials or monomers and at least comonomers.

Plasma polymerized polyoxazoline polymers and functionalized films on the surface of the substrate may be prepared by exposing the surface of the substrate to a plasma comprising an oxazoline monomer vapor under conditions such that the oxazoline monomer polymerizes to form the plasma polymerized polyoxazoline polymer on the surface of the substrate.

The conditions required to polymerize the oxazoline monomer to form a plasma polymerized polyoxazoline polymer may include a power of from about 10W to about 50W, a deposition time of from about 1 minute to about 7 minutes, and/or from about 1.1 to about 3x10^-1A monomer pressure of mbar, a power of greater than 30W for a time of greater than 5 minutes is a particularly suitable condition for polymerizing the oxazoline monomer to form a plasma polymerized polyoxazoline polymer, as they provide a stable plasma polymerized polyoxazoline polymer film having a thickness of greater than about 30nm, however, coatings having a thickness of greater than 1nm and deposited under other conditions of the series may also be suitable.

One skilled in the art will appreciate that the plasma is an ionized gas or gases that are electrically activated, which when activated (e.g., ignited) forms a highly reactive environment that can alter direct exposure to a plasma discharge, the plasma deposition step can be operated at a wide range of pressures (e.g., 10mTorr to above atmospheric pressure (e.g., 10 times atmospheric pressure or higher)), the plasma can be formed from inert gases (e.g., helium, neon, argon, krypton, xenon, radon) and the oxazoline monomer (or other suitable monomer composition as desired according to the chemistry of the functionalized film), the plasma can be formed at a range of alternating current frequencies (AC) and a constant frequency (DC) at a range of RF frequencies (AC 84), a constant frequency (DC), and a low frequency (DC) pulse.

The above plasma polymerization conditions can also be used to form plasma polymers from other functional starting materials as desired.

The oxazoline monomer may be a substituted oxazoline having a substituent at any position of the 2-, 4-, or 5-position of the oxazoline ring or a combination of such substituents any of these oxazoline species may be used to form the plasma polymerized polyoxazoline polymer provided that they are a vapor under the plasma deposition conditions used in embodiments the oxazoline monomer is selected from the group consisting of 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2, 4-disubstituted oxazolines, 2, 5-disubstituted oxazolines, 4, 5-disubstituted oxazolines, and 2,4, 5-trisubstituted oxazolines or more substituents on the oxazoline ring may be selected from the group consisting of halogen, OH, NO₂、CN、NH₂Optionally substituted C₁-C₁₂Alkyl, optionally substituted C₂-C₁₂Alkenyl, optionally substituted C₂-C₁₂Alkynyl, optionally substituted C₂-C₁₂Heteroalkyl, optionally substituted C₃-C₁₂Cycloalkyl, optionally substituted C₂-C₁₂Heterocycloalkyl, optionally substituted C₂-C₁₂Heterocycloalkenyl, optionally substituted C₆-C₁₈Aryl, optionally substituted C₁-C₁₈Heteroaryl, optionally substituted C₁-C₁₂Alkoxy, optionally substituted C₂-C₁₂Alkenyloxy, optionally substituted C₂-C₁₂Alkynyloxy, optionally substituted C₂-C₁₂Heteroalkoxy, optionally substituted C₃-C₁₂Cycloalkoxy, optionally substituted C₃-C₁₂Cycloalkenyloxy, optionally substituted C₁-C₁₂Heterocycloalkoxy, optionally substituted C₂-C₁₂Heterocyclic alkenyloxy, optionally substituted C₆-C₁₈Aryloxy, optionally substituted C₁-C₁₈Heteroaryloxy, optionally substituted C₁-C₁₂Alkylamino, SO₃H、SO₂NH₂、SO₂R、SONH₂、SOR、COR、COOH、COOR、CON RR'、NRCOR'、NRCOOR'、NRSO₂R ', N RCON R ' R ", and NRR ' in embodiments, the oxazoline monomer comprises a 2-substituted oxazoline in particular embodiments, the oxazoline monomer is a 2-alkyl-2-oxazoline, the alkyl substituent may be C₁-C₁₂Alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and the like.

In particular embodiments, the oxazoline monomer is selected from the group consisting of: 2-alkyl-2-oxazolines and 2-aryl-2-oxazolines. The alkyl substituent of the 2-alkyl-2-oxazoline group may be C₁-C₁₀Alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and the like. The alkyl substituent may be optionally substituted. The aryl substituent of the 2-aryl-2-oxazoline group may be C₅-C₁₀Aryl, e.g. optionally substituted phenyl, optionally substituted naphthyl, optionally substituted thienylOptionally substituted indolyl, etc.

The surface of the substrate may be treated prior to deposition of the plasma polymerized polyoxazoline polymer. For example, the surface may be treated by cleaning with a detergent, water or a suitable solvent. Alternatively, or additionally, the surface may be treated by exposing the surface to air within the plasma chamber, thereby activating the surface.

The plasma polymerized functionalized membrane may have a thickness greater than 30nm, for example, a thickness of about 30nm, 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, or about 100 nm.

The reactivity of oxazoline rings present at the omega-terminus of polyoxazolines has been used for conjugation with proteins and drugs in solution. The reactivity of the oxazoline ring is believed to result in the formation of covalent amide linkages by reaction with carboxylic acid functional groups. The plasma deposited coating enables the intact oxazoline rings to remain on the surface, however this is not typically the case when other techniques of surface preparation are used. Maintaining the reactive chemical functionality then enables convenient and rapid covalent coupling of proteins, antibodies, and the like.

The targeted bladder cancer cell-selective binding agent can be any molecule that selectively binds to the targeted bladder cancer cell. The targeted bladder cancer cell-selective binding agent can be a biomolecule. The biomolecule may, for example, be selected from amino acids, peptides, proteins, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents, antithrombotic agents (antithrombogenic agents), and cell attachment proteins. The biomolecule may be in the form of a particle or nanoparticle comprising the biomolecule.

The reaction between the or more functional groups on the functionalized membrane and the targeted bladder cancer cell-selective binding reagent may be facilitated by a coupling reagent such as a carbodiimide coupling reagent (e.g., EDC, DCC, DIC), a triaminophosphonium coupling reagent (e.g., BOP, PyBOP, PyBrOP) or a tetramethylammonium/tetramethyluronium coupling reagent (e.g., HATU, HBTU, HCTU), for example.

A crosslinker structure can be used between the functionalized membrane and the targeted bladder cancer cell binding agent.

The target bladder cancer cell-selective binding agent can be any inorganic, organic, and/or biological molecule that allows the device to selectively capture the bladder cancer cells.

The selection of the appropriate targeted bladder cancer cell-selective binding reagent is selection matters depending on the particular targeted bladder cancer cell population of interest.

The second functional group of the targeted bladder cancer cell-selective binding agent is capable of binding to a cell. The binding between the second functional group and the cell may be direct or indirect. For direct binding to a cell, the second functional group itself comprises a reactive group capable of recognizing and capturing a cell. The reactive group may be specifically selected to recognize and capture a particular cell type of interest, and cell recognition and capture may be achieved by any means known in the art. For example, cell recognition may be based on chemical or biological reactions, including, but not limited to, peptide recognition, nucleic acid recognition, and/or chemical recognition. Cell identification may also be based on non-chemical or non-biological reactions, such as, but not limited to, electrokinetic identification or size-based classification.

The cell binding agent may be in the form of a single component or a complex comprising two or more components, so long as at least of the components are capable of binding the target cell.

For example, the targeted bladder cancer cell-selective binding agent, or components of the targeted bladder cancer cell-selective binding agent, can be an antibody, a lymphokine, a hormone, a growth factor, or any other cell-binding molecule or substance that specifically binds to targeted bladder cancer cells.

In certain embodiments, the selective binding agent for bladder cancer cells of interest is or more antibodies, or fragments thereof, which may be selected from the group consisting of polyclonal or monoclonal antibodies, including fully human antibodies, single chain antibodies (polyclonal and monoclonal), fragments of antibodies (polyclonal and monoclonal) such as Fab, Fab ', F (ab')₂And Fv; chimeric antibodies and antigen-binding fragments thereof; and domain antibodies (dAbs) and antigen binding fragments thereof, including camelid antibodies. In certain embodiments, the targeted bladder cancer cell-selective binding agent is a monoclonal antibody. In certain other embodiments, the selective binding agent for bladder cancer cells of interest is a polyclonal antibody.

The antigen of interest may include whole bladder cancer cells of interest, antigens isolated from the cancer cells of interest, whole viruses, attenuated whole viruses, or viral proteins such as viral capsid proteins.

The targeted bladder cancer cell-selective binding reagent can also be a combination of two or more different types of antibodies.

Thus, in certain embodiments, the devices of the and second aspects can include immobilized functional antibodies capable of selectively capturing targeted bladder cancer cells, in certain embodiments, the antibodies are anti-epithelial cell adhesion molecules (anti-EpCAM) that specifically bind to EpCAM-expressing cancer cells in urine, urological epithelial cancers are such EpCAM-expressing cancer cell types other EpCAM-expressing cancer cell types include those corresponding to HT1197, HT1376, RT4, and EJ138 bladder cancer cell lines HTC116 cells are epithelial cancer cell types with particularly high expression levels of EpCAM as an example other antibodies that can be used include E-cadherin (E-cadherin), CA19-9, CD146, CD147, CD10, CD44, CD24, CD133, CD166, mucins (e.g., MUC1 and MUC4), cadherin (CDH 1-28), urolytic proteins, and Lewis antigen antibodies.

In the example provided herein, the substrate is step optimized to separate cancer cells added in a biologically relevant medium from physiological buffers by artificial urine and ultimately by authentic patient urine.

Non-antibody molecules can also be used to target specific targeted bladder cancer cell populations.

once captured on the cell capture surface of the substrate, target bladder cancer cells can be separated from urine and other components of urine by washing, for example with PBS.

The captured target bladder cancer cells can be detected by fluorescent (fluorescent) or luminescent (luminescent) markers.

Advantageously, the captured bladder cancer cells can be distinguished from healthy cells in vitro using a cancer-specific fluorescently active compound. For example, ALA5 is a compound that is metabolized more rapidly by cancer cells, resulting in luminescence that can be used to distinguish captured cancer cells. Other cancer specific fluorescently active compounds that can be used include hexosaminide valerate (hexaminolevulinate) and hypericin (hypericin).

If desired, the captured cancer cells can be selectively released from the capture surface of the substrate.

In a third aspect, the present disclosure provides methods of selectively capturing target bladder cancer cells from urine or a fluid derived from urine, the method comprising:

providing a sample of urine or a fluid derived from urine;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells (if present) from the urine bind to the cell capture surfaces.

The method of the third aspect may further include detecting the target bladder cancer cell on the or more cell capture surface.

In a fourth aspect, the present disclosure provides methods of immobilizing target bladder cancer cells on a basal surface in a fluid from urine or derived from urine, the method comprising:

providing a sample of urine or a fluid derived from urine;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells (if present) from the urine bind to the cell capture surfaces.

In a fifth aspect, the present disclosure provides methods of diagnosing or monitoring bladder cancer in a mammal, the method comprising:

providing a sample of urine or urine-derived fluid obtained from said mammal;

providing a substrate having or more cell capture surfaces, each cell capture surface comprising a functionalized membrane on the substrate and or more target bladder cancer cell-selective binding reagents covalently bound to the functionalized membrane;

contacting a sample of the urine or urine-derived fluid with the or more cell capture surfaces under conditions such that at least portions of the target bladder cancer cells from the urine, if present, bind to the cell capture surfaces, and

analyzing the target bladder cancer cells bound to the cell capture surface.

The devices and methods described herein provide a rapid and selective method for capturing targeted bladder cancer cells from urine.current diagnostic tests for urine for bladder cancer are expensive and have limited sensitivity and specificity.the devices and methods described herein provide a generation specific urine sample test for detecting cancer cells in urine.the unique reactivity of plasma deposited polyoxazoline is used to covalently bind cancer specific antibodies in microchannels.cancer cells dispersed in patient urine are successfully captured with up to 99% selectivity and 100% sensitivity over a wide range of cell concentrations.